The role of the complement system in ischemic stroke and neural plasticity       Anna  Stokowska   Centre  for  Brain  Repair  and  Rehabilitation,     Department  of  Clinical  Neuroscience  and  Rehabilitation,   Institute  of  Neuroscience  and  Physiology,   The  Sahlgrenska  Academy  at  University  of  Gothenburg,   Sweden   Göteborg  2013     Cov e r ill u s t r a t i o n : Complement in stroke and neural plasticity, by Anna Stokowska, using the following images: - crystal structures of complement C3 and C3a - retrieved from RSCB.org (PDB ID: 2A73) and protepedia.org (ID: 4i6o), respectively - ischemic stroke, due to right MCA occlusion, a CT scan - by Lucien Monfils, retrieved from commons.wikimedia.org, under CC:BY 2.5) - neuroblasts (red) migrating towards the infarct site – image by Anna Stokowska - dendritic spines of a hippocampal neuron (green) - image by Marta Perez Alcazar                         The role of the complement system in ischemic stroke and neural plasticity © Anna Stokowska 2013 anna.stokowska@neuro.gu.se annsto@gmail.com ISBN 978-91-628-8804-6 ISBN 978-91-628-8805-3 (electronic version) Printed in Bohus, Sweden 2013 Ale Tryckteam AB   To my family   Cov e r ill u s t r a t i o n : Complement in stroke and neural plasticity, by Anna Stokowska, using the following images: - crystal structures of complement C3 and C3a - retrieved from RSCB.org (PDB ID: 2A73) and protepedia.org (ID: 4i6o), respectively - ischemic stroke, due to right MCA occlusion, a CT scan - by Lucien Monfils, retrieved from commons.wikimedia.org, under CC:BY 2.5) - neuroblasts (red) migrating towards the infarct site – image by Anna Stokowska - dendritic spines of a hippocampal neuron (green) - image by Marta Perez Alcazar                         The role of the complement system in ischemic stroke and neural plasticity © Anna Stokowska 2013 anna.stokowska@neuro.gu.se annsto@gmail.com ISBN 978-91-628-8804-6 ISBN 978-91-628-8805-3 (electronic version) Printed in Bohus, Sweden 2013 Ale Tryckteam AB   To my family  A BSTRACT Evidence from experimental animal studies suggests that complement activation in the brain is a “double -edged sword” as it exerts beneficial or detrimental effects depending on the context. Here, we assessed whether complement activation in the systemic circulation could be a predictive biomarker of functional outcome after stroke. Further, we studi ed the role of the complement system in brain plasticity and recovery after ischemic stroke. We found that acute and delayed phase plasma levels of C3 and C3a differ substantially among patients suffering from ischemic stroke of different etiology, and the association of plasma C3 and C3a levels with case/control status and with functional outcome is ischemic stroke subtype-dependent. In large vessel disease and cardioembolic stroke patients, C3 levels at 3-month follow up were associated with an unfavorable functional outcome at both 3 months and 2 years after stroke. However, in cardioembolic stroke patients moderate increase in plasma C3a/C3 ratio predicted favorable outcome after 2 years (Paper I and II). Furthermore, two single nucleotide polymorphisms (SNPs) in the C 3 gene were found to be associated with ischemic stroke independently of traditional risk factors and one of these SNPs was associated with cryptogenic stroke (Paper III). Also, two SNPs were associated with plasma C3a or C3 levels independently of age, sex and case/control status. Taken together, the role of the complement system in ischemic stroke is strongly dependent on stroke etiology. We have also found that C3a overexpression in mice increased, whereas C3a receptor (C3aR) deficiency decreased the number of post-stroke-born neurons in the peri-infarct cortex without affecting the infarct size. Furthermore, the density of pre -synaptic puncta and GAP43 -positive axonal growth cones in the cortex surrounding the infarct were lower in the C3aR-deficient compared to control mice, while in the C3a-overexpressing mice post-stroke axonal plasticity response was increased. Mice lacking C3aR showed a more pronounced sensorimotor functional deficit as assessed by behavioral testing (Paper IV). These results indicate that C3aR signaling should be considered as a target when designing therapeutic strategies to improve functional recovery after ischemic stroke. To study complement-related neural plasticity in a non-pathological context, we performed electrophysiological recordings in the CA1 region of live hippocampal slices of young mice lacking C3 and control mice. We found that the C3 -deficient mice had a decreased neurotransmitter release probability but dendritic spine density, and frequency and amplitude of miniature excitatory postsynaptic potentials were comparable in both groups of mice. Behavioral testing using the IntelliCage platform revealed that the C3-deficient mice performed better in the place and reversal learning tasks (Paper V). These findings may have implications for the management of disorders involving synapse elimination, such as Alzheimer’s diseases, autism or multiple sclerosis. Key w o r d s : ischemic stroke, complement system, neurogenesis, synaptic plasticity, hippocampus, learning and memory, functional outcome I S B N 97 8 - 9 1 - 6 2 8 - 8 8 0 4 - 6 IS B N 97 8 - 9 1 - 6 2 8 - 8 8 0 5 - 3 (e l e c t r o n i c ve r s i o n )  A BSTRACT Evidence from experimental animal studies suggests that complement activation in the brain is a “double- edged sword” as it exerts beneficial or detrimental effects depending on the context. Here, we assessed whether complement activation in the systemic circulation could be a predictive biomarker of functional outcome after stroke. Further, we studi ed the role of the complement system in brain plasticity and recovery after ischemic stroke. We found that acute and delayed phase plasma levels of C3 and C3a differ substantially among patients suffering from ischemic stroke of different etiology, and the association of plasma C3 and C3a levels with case/control status and with functional outcome is ischemic stroke subtype-dependent. In large vessel disease and cardioembolic stroke patients, C3 levels at 3-month follow up were associated with an unfavorable functional outcome at both 3 months and 2 years after stroke. However, in cardioembolic stroke patients moderate increase in plasma C3a/C3 ratio predicted favorable outcome after 2 years (Paper I and II). Furthermore, two single nucleotide polymorphisms (SNPs) in the C 3 gene were found to be associated with ischemic stroke independently of traditional risk factors and one of these SNPs was associated with cryptogenic stroke (Paper III). Also, two SNPs were associated with plasma C3a or C3 levels independently of age, sex and case/control status. Taken together, the role of the complement system in ischemic stroke is strongly dependent on stroke etiology. We have also found that C3a overexpression in mice increased, whereas C3a receptor (C3aR) deficiency decreased the number of post-stroke-born neurons in the peri-infarct cortex without affecting the infarct size. Furthermore, the density of pre -synaptic puncta and GAP43- positive axonal growth cones in the cortex surrounding the infarct were lower in the C3aR-deficient compared to control mice, while in the C3a-overexpressing mice post-stroke axonal plasticity response was increased. Mice lacking C3aR showed a more pronounced sensorimotor functional deficit as assessed by behavioral testing (Paper IV). These results indicate that C3aR signaling should be considered as a target when designing therapeutic strategies to improve functional recovery after ischemic stroke. To study complement-related neural plasticity in a non-pathological context, we performed electrophysiological recordings in the CA1 region of live hippocampal slices of young mice lacking C3 and control mice. We found that the C3 -deficient mice had a decreased neurotransmitter release probability but dendritic spine density, and frequency and amplitude of miniature excitatory postsynaptic potentials were comparable in both groups of mice. Behavioral testing using the IntelliCage platform revealed that the C3-deficient mice performed better in the place and reversal learning tasks (Paper V). These findings may have implications for the management of disorders involving synapse elimination, such as Alzheimer’s diseases, autism or multiple sclerosis. Key w o r d s : ischemic stroke, complement system, neurogenesis, synaptic plasticity, hippocampus, learning and memory, functional outcome I S B N 97 8 - 9 1 - 6 2 8 - 8 8 0 4 - 6 IS B N 97 8 - 9 1 - 6 2 8 - 8 8 0 5 - 3 (e l e c t r o n i c ve r s i o n )  POPULÄ RVETENSKAPLIG SAMMA NFATTNING Stroke, eller slaganfall, är den ledande orsak en till handikapp hos vuxna i västvärlden och leder oftast till e tt permanent assistansberoende. Ischemisk stroke, hjärninfarkt , är den vanligaste formen av slaganfall och orsaka s av en blodpropp som förhindrar blodflöde till en del av hjärna. Som följd, på grund av syr e- och näringsbrist, dör nervceller i det påverkade området . Detta leder också till inflammation och ytterligare skador på hjärn an. Idag är den enda behandlingen för stroke en upplösning av blodproppen genom en intravenös injektion av ett enzym som kallas tPA. Ett problem är dock att få patienter kvalificerar sig för denna behandling , då den bara är effektiv och relativt säker under en kort tidsperiod (4.5 timmar) från det att symptomen har börjat. För att kunna hjälpa fler patienter är det d ärför viktigt att utveckla nya behandlingar som kan användas i samband me d en långsiktig rehabilitering. Hjärnan har en begränsad förmåga att läka de områden som skadats svårt vid stroken. Dock har hjärnan har en fantastisk förmåga att anpassa sig till den nya situationen genom att skapa nya cellkontakter, synapser, och genom att ändra egenskaperna hos de fungerande cellkopplingarna så att de kan ta över funktioner som de förstörda kopplingar tidigare utförde. Detta kallas för ”neuroplasticitet” . Detta begrepp inkluderar även andra omformande processer i den friska hjärnan, t.ex. de som sker vid inlärning. Immunförsvaret skyddar vår kropp från de skadliga effekterna av mikroorganismer (bakterier, virus m.m.) och förändrade celler som annars kan omvandlas till cancerceller. Komplementsystmet är en viktig del av det ospecifik a immunförsvaret och består av mer än 30 proteiner (äggviteämnen) som verkar i kaskadform inne i blodet och andra kroppsvätskor och vävnader. Komplementsystmet är också involverat i de inflammatoriska reaktioner som sker i hjärnan efter en stroke. Dessa inflam matoriska reaktioner, om de blir okontrollerade,tror man är en bidragande faktor till att hjärnskadorna kan öka efter en stroke. L evern är den huvudsakliga källan av komplementproteiner men överraskande nog har det visat sig att även hjärnans celler producerar komplementproteiner. Detta pekade på någon annan icke -immunologisk roll för komplement systemet i hj ä rnan. Eftersom det inte finns många studier kring de långsiktiga effekterna av komplementsystemets inverkan efter stroke, försökte vi förstå komplement - systemsreaktion hos strokepatienter. För att studera komplementsystemets roll i neuroplasticitet jämförde vi möss med förändrat komplementsystemet med normala möss och analyserade förändringar i deras hjärnor under normal utveckling och efter stroke. Vå ra kliniska studier (på människor) visade att komplementnivå erna i blodet är förhöjd till olika grad i de olika ischemiska subtyperna av slaganfall efter stroke. Denna förhöjning var karaktäristisk för strokepatienter na och berodde inte på de traditionella riskfaktorerna (ålder, kön, hög blodtryck, sockersjuka, rökning och höga blodfettnivåer ). Viktig nog höga blodn ivåer av komplementproteinet C3 tre månader efter stroke korrelerade medmed högre grad av handikapp, men bara hos patienter med visa subtyper av stroke. Dock verkade måttlig aktivering av komplement systemet (C3a/C3 kvot) ha ett något positiv effekt eftersom den associerades med mindre grad av handikapp efter   slaganfallet för patienter med kardioembolisk stroke (stroke orsakades av en vandrade blodpropp som formats i hjärtan) . Vi fann även e tt samband mellan några genetiska varianter av C3-genen och förekomst en av stroke, särskil t kryptogen stroke, den vars orsak inte kan identifieras trots extensiv utredning. Då rätt prognos kan underlätta specialanpassning av patientens rehabilitering, samt andra behandlingar, och bidra till en bättre återhämtning, så kan mätning av komplement systemskomponenter i blodet vara ett bra diagnostiskt verktyg, åtminstone i vissa typer av ischemisk stroke. I våra experim entella strokestudier har vi upptäckt att genetiskt förändrade möss som producerar komplementsystemspeptiden C3a i hjärnan i samband med ischemisk stroke har fler nyföd da nervceller och fler växande nervutskott i området runt den skadade regionen av hjärna n. I motsats till detta så har möss som saknar receptor , mottagare, för C3a färre nya nervceller och färre och mindre växande nervutskott runt strokeområdet. Dessa möss har också större funktionsnedsättning. Tillsammans pekar detta mot att C3a är viktigt fö r olika typer av neuroplastiska mekanismer som är involverade i återhämtning efter stroke . För att fördjupa vår förståelse av komplement systemets roll i hjärnan har vi också studerat neurologiska funktioner i hippocampus - en del av hjärnan som är viktig f ör inlärning och minne. Våra studier på möss visar att komplementsystemet spelar roll vid en typ av neuroplasticitet som är viktig för normala funktioner av hjärnan. Med hjälpen av elektrofysiologiska metoder fann vi att unga möss, som saknar det viktigaste komplementsystemsproteinet C3 har ökad synaptisk funktion i hippocampus. Detta beror troligen på att de har fler synapser i hippocampus . Som följd av detta, är dessa möss bättre på att lära sig att utföra spatialminne sberoende uppgifter. Dessa resultat kan vara av betydelse för behandling av nervskadesjukdomar som orsakas av synapsförlust såsom Alzheimers sjukdom, autism och multipel skleros (MS). Sammanfattningsvis visar våra resultat att komplementsyste met är en viktig vid stroke och att det är involvera t både vid skadliga inflammationsprocesser och reparationsprocesser. Dessutom påverkar komplementsystemet även plasticitet en hos en frisk hjärna . Detta styrker åsikten att denna del av immunsystemet även är involverat i processer som inte är immunförsvarsr elaterade  POPULÄ RVETENSKAPLIG SAMMA NFATTNING Stroke, eller slaganfall, är den ledande orsak en till handikapp hos vuxna i västvärlden och leder oftast till e tt permanent assistansberoende. Ischemisk stroke, hjärninfarkt , är den vanligaste formen av slaganfall och orsaka s av en blodpropp som förhindrar blodflöde till en del av hjärna. Som följd, på grund av syr e- och näringsbrist , dör nervceller i det påverkade området . Detta leder också till inflammation och ytterligare skador på hjärn an. Idag är den enda behandlingen för stroke en upplösning av blodproppen genom en intravenös injektion av ett enzym som kallas tPA. Ett problem är dock att få patienter kvalificerar sig för denna behandling , då den bara är effektiv och relativt säker under en kort tidsperiod (4.5 timmar) från det att symptomen har börjat. För att kunna hjälpa fler patienter är det d ärför viktigt att utveckla nya behandlingar som kan användas i samband me d en långsiktig rehabilitering. Hjärnan har en begränsad förmåga att läka de områden som skadats svårt vid stroken. Dock har hjärnan har en fantastisk förmåga att anpassa sig till den nya situationen genom att skapa nya cellkontakter, synapser, och genom att ändra egenskaperna hos de fungerande cellkopplingarna så att de kan ta över funktioner som de förstörda kopplingar tidigare utförde. Detta kallas för ”neuroplasticitet” . Detta begrepp inkluderar även andra omformande processer i den friska hjärnan, t.e x. de som sker vid inlärning. Immunförsvaret skyddar vår kropp från de skadliga effekterna av mikroorganismer (bakterier, virus m.m.) och för ändrade celler som annars kan omvandlas till cancerceller. Komplementsystmet är en viktig del av det ospecifik a immunförsvaret och består av mer än 30 proteiner (äggviteämnen) som verkar i kaskadform inne i blodet och andra kroppsvätskor och vävnader. Komplementsystmet är också involverat i de inflammatoriska reaktioner som sker i hjärnan efter en stroke. Dessa inflammatoriska reaktioner, om de blir okontrollerade,tror man är en bidragande faktor till att hjärnskadorna kan öka efter en stroke. L evern är den huvudsakliga källan av komplementproteiner men överraskande nog har det visat sig att även hjärnans celler producerar komplementproteiner. Detta pekade på någo n annan icke-immunologisk roll för komplementsystemet i hj ä rnan. Eftersom de t inte finns många studie r kring de långsiktiga effekte rna av komplementsystemets inverkan efter stroke, för sökte vi förstå komplement- systemsreaktion hos strokepatienter. För att studera komplement systemets roll i neuroplasticitet jämförde vi möss med förändrat komplementsystemet med normala möss och analyserade för ändringar i deras hjärn or under normal utveckling och efter stroke. V å ra kliniska studier (på människor) visade att komplementnivå erna i blodet är förhöjd till olika grad i de olika ischemiska subtyperna av slaganfall efter stroke. Denna förhöjning var karaktäristisk för strokepatienterna och berodde inte på de traditionella riskfaktorerna (ålder, kön, hög blodtryck, sockersjuka, rökning och höga blodfettnivåer ). Viktig nog höga blodn ivåer av komplementproteinet C3 tre månader efter stroke korrelerade medmed högre grad av handikapp, men bara hos patienter med visa subtyper av stroke. Dock verkade måttlig aktivering av komplement systemet (C3a/C3 kvot) ha ett någo t positiv effekt eftersom den associerades med mindre grad av handikapp efter   slaganfallet för patienter med kardioembolisk stroke (stroke orsakades av en vandrade blodpropp som formats i hjärtan). Vi fann även ett samband mellan några genetiska varianter av C3-genen och förekomst en av stroke, särskilt kryptogen stroke, den vars orsak inte kan identifieras trots extensiv utredning. Då rätt prognos kan underlätta specialanpassning av patientens rehabilitering, samt andra behandlingar, och bidra till en bättre återhämtning, så kan mätning av komplement systemskomponenter i blodet vara ett bra diagnostiskt verktyg, åtminstone i vissa typer av ischemisk stroke. I våra experim entella strokestudier har vi upptäckt att genetiskt förändrade möss som producerar komplementsystemspeptiden C3a i hjärnan i samband med ischemisk stroke har fler nyföd da nervceller och fler växande nervutskott i området runt den skadade regionen av hjärna n. I motsats till detta så har möss som saknar receptor, mottagare, för C3a färre nya nervceller och färre och mindre växande nervutskott runt strokeområdet. Dessa möss har också större funktionsnedsättning. Tillsammans pekar detta mot att C3a är viktigt fö r olika typer av neuroplastiska mekanismer som är involverade i återhämtning efter stroke . För att fördjupa vår förståelse av komplementsystem ets roll i hjärnan har vi också studerat neurologiska funktioner i hippocampus - en del av hjärnan som är viktig f ör inlärning och minne. Våra studier på möss visar att komplementsystemet spelar roll vid en typ av neuroplasticitet som är viktig för normala funktioner av hjärnan. Med hjälpen av elektrofysiologiska metoder fann vi att unga möss, som saknar det viktigast e komplementsystemsproteinet C3 har ökad synaptisk funktion i hippocampus. Detta beror troligen på att de har fler synapser i hippocampus . Som följd av detta, är dessa möss bättre på att lära sigatt utföra spatialminne sberoende uppgifter. Dessa resultat kan vara av betydelse för behandling av nervskadesjukdomar som orsakas av synapsförlust såsom Alzheimers sjukdom, autism och multipel skleros (MS). Sammanfattningsvis visar våra resultat att komplementsyste met är en viktig vid stroke och att det är involvera t både vid skadliga inflammationsprocesser och reparationsprocesser. Dessutom påverkar komplementsystemet även plasticitet en hos en frisk hjärna . Detta styrker åsikten att denna del av immunsystemet även är involverat i processer som inte är immunförsvarsr elaterade. Anna Stokowska   i LIST OF PAPERS This thesis is based on the following studies, referred to in the text by their Roman numerals. I. S t o k o w s k a , A , Olsson, S, Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M. Plasma C3 and C3a levels in cryptogenic and large vessel disease stroke: associations with outcome. Cerebrovasc. Dis. 2011; 32:114 -122 II. S t o k o w s k a , A , Olsson, S, Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M. Cardioembolic and small vessel disease stroke show differences in associations between systemic C3 levels and outcome. PLoS One . 201 3 ; 8( 8 ) : e721 3 3 . III. Olsson, S, S t o k o w s k a , A , Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M . Genetic variation in complement component C3 shows associations with ischemic stroke. Eur. J. Neurol . 201 1 ; 18: 1272-1274. IV. S t o k o w s k a , A , Atkins, AL, Barnum, SR, Wetsel, RA, Dragunow, M, Pekna, M. Receptor for complement peptide C3a stimulates neural plasticity after experimental brain ischemia. Manuscript V. Perez -Alcazar, M, Daborg, J, S t o k o w s k a , A , Wasli ng, P, Björefeldt, A, Kalm, M, Zetterberg, H, Carlström, K, Blomgren, K, Clementson Ekdahl, C, Hanse, E, Pekna, M. Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3. Manuscript submitted to Hippocampus 2013 Anna Stokowska   i LIST OF PAPERS This thesis is based on the following studies, referred to in the text by their Roman numerals. I. S t o k o w s k a , A , Olsson, S, Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M. Plasma C3 and C3a levels in cryptogenic and large vessel disease stroke: associations with outcome. Cerebrovasc. Dis. 2011; 32:114 -122 II. S t o k o w s k a , A , Olsson, S, Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M. Cardioembolic and small vessel disease stroke show differences in associations between systemic C3 levels and outcome. PLoS One. 201 3 ; 8( 8 ) : e721 3 3 . III. Olsson, S, S t o k o w s k a , A , Holmegaard, L, Jood, K, Blomstrand, C, Jern, C, Pekna, M . Genetic variation in complement component C3 shows associations with ischemic stroke. Eur. J. Neurol . 201 1 ; 18: 1272- 1274. IV. S t o k o w s k a , A , Atkins, AL, Barnum, SR, Wetsel, RA, Dragunow, M, Pekna, M. Receptor for complement peptide C3a stimulates neural plasticity after experimental brain ischemia. Manuscript V. Perez- Alcazar, M, Daborg, J, S t o k o w s k a , A , Wasling, P, Björefeldt, A, Kalm, M, Zetterberg, H, Carlström, K, Blomgren, K, Clementson Ekdahl, C, Hanse, E, Pekna, M. Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3. Manuscript submitted to Hippocampus 2013 Comple m ent in stroke and neural plasticity   ii TABLE OF CONTETNS BA C K G R O U N D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ST R O K E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 C l inical Background ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Etiological subtypes of ischemic stroke ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Risk factors for ischemic stroke ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Pathob iology of ische m i c brain damage .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Glutamate toxicity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Oxidative stress .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Post- ischemic inflammation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 T H E C O M P L E M E N T SY S T E M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 T h e th i r d co m p l e m e n t co m p o n e n t (C 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 A c t i v a t i o n of th e co m p l e m e n t ca s c a d e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 Classical pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Lectin pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Alternative pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Terminal pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 C o n t r o l of th e co m p l e m e n t sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 C o m p l e m e n t re c e p t o r s ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 N o n - i m m u n o l o g i c a l fu n c t i o n s of th e co m p l e m e n t sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 Tissue regeneration ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Regulation of stem cell translocation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 C o m p l e m e n t in th e ce n t r a l ne r v o u s sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 Complement in unchallenged CNS .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Complement in ischemic brain injury ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 N E U R A L P L A S T I C I T Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 M e c h a n i s m s of ne u r a l pla s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Functional synaptic plasticit y ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Structural plasticity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Forms of neural plasticity in recovery of function after ischemic stroke ..... . . . . . . . . . . . . 27 T h e ro l e of ne u r o g e n e s i s in ne u r a l pla s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0 T h e i m m u n e sy s t e m an d b r a i n p la s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Microglia .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Macrophages .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Astrocytes .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 T lym p hocytes .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Pro- inflammatory cytokines .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Emerging roles of complement in neural plasticity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 A I M S O F THE THES I S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 Anna Stokowska   iii METHODS ................................ ................................ ....................... 41 H u m a n su b j e c t s (I , II , II I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 M ic e (I V , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 E L I S A (I , II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 G e n o t y p i n g (I I I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 E x p e r i m e n t a l st r o k e m o d e l (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6 B r d U ad m i n i s t r a t i o n (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7 Im m u n o h i s t o c h e m i s t r y an d flu o r e s c e n t - d y e ne u r o n lo a d i n g (I V , V ) . . . . . . . . . . . . . . . . . . . . . . 4 7 Tissue preparation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Im munofluorecent staining ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 E v a l u a t i o n of th e in f a r c t vo l u m e (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0 Q u a n t i t a t i v e an a l y s i s of im m u n o s t a i n i n g s (V I , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 Cell Counting (IV , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Peri- infarct synaptogenesis and axonal sprouting (IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Dendritic spines (V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 E l e c t r o p h y s i o l o g y (V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 Acute slices preparation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Extracellular field recordings .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Intracellular recordings ............................................................................................ 5 5 In vivo ele c t r o e n c e p h a l o g r a p h y (V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 Beh a v i o r a l te s t i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 E valuati on of sensorimotor deficits (IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Evaluation of hippocampal - dependent cognitive performance (V) . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Sta t i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 R E S U LTS AN D D IS C U S S I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0 S y s t e m i c co m p l e m e n t re s p o n s e dif f e r s be t w e e n isc h e m i c st r o k e su b t y p e s (P a p e r I an d II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0 P la s m a C 3 an d C 3 a le v e l s sh o w et i o l o g y - d e p e n d e n t as s o c i a t i o n s wit h fu n c t i o n a l ou t c o m e af t e r is c h e m i c st r o k e (P a p e r I an d II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 G e n e t i c va r i a t i o n in co m p l e m e n t co m p o n e n t C 3 sh o w s as s o c i a t i o n wit h is c h e m i c st r o k e (P a p e r II I ) as w e l l as C 3 an d C 3 a le v e l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 R e c e p t o r fo r co m p l e m e n t pe p t i d e C 3 a st i m u l a t e s ne u r a l pla s t i c i t y aft e r ex p e r i m e n t a l br a i n is c h e m i a (P a p e r IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 A lt e r e d co g n i t i v e pe r f o r m a n c e an d sy n a p t i c fu n c t i o n in th e hip p o c a m p u s of m ic e la c k i n g C 3 (P a p e r V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 7 M A I N C O N C L U S I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 C O N C L U D I N G R E M A R K S A N D FUTUR E D IR E CTIO N S . . . . . . . . . . . . . . . . . . . . . . . . 7 2 A C K N O W L E D G E M E NTS ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 R E F E R E N C E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 P A P E R S I - V Comple m ent in stroke and neural plasticity   ii TAB LE OF CONTETNS BA C K G R O U N D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ST R O K E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 C l inical Background ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Etiological subtypes of ischemic stroke ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Risk factors for ischemic stroke ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Pathob iology of ische m i c brain damage .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Glutamate toxicity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Oxidative stress .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Post- ischemic inflammation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 T H E C O M P L E M E N T SY S T E M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 T h e th i r d co m p l e m e n t co m p o n e n t (C 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 A c t i v a t i o n of th e co m p l e m e n t ca s c a d e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 Classical pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Lectin pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Alternative pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Terminal pathway .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 C o n t r o l of th e co m p l e m e n t sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 C o m p l e m e n t re c e p t o r s ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 N o n - i m m u n o l o g i c a l fu n c t i o n s of th e co m p l e m e n t sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 Tissue regeneration ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Regulation of stem cell translocation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 C o m p l e m e n t in th e ce n t r a l ne r v o u s sy s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 Complement in unchallenged CNS .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Complement in ischemic brain injury ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 N E U R A L P L A S T I C I T Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 M e c h a n i s m s of ne u r a l pla s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Functional synaptic plasticit y ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Structural plasticity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Forms of neural plasticity in recovery of function after ischemic stroke ..... . . . . . . . . . . . . 27 T h e ro l e of ne u r o g e n e s i s in ne u r a l pla s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0 T h e i m m u n e sy s t e m an d b r a i n p l a s t i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Microglia .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Macrophages .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Astrocytes .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 T lym p hocytes .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Pro- inflammatory cytokines .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Emerging roles of complement in neural plasticity ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 A I M S O F THE THES I S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 Anna Stokowska   iii METHODS ....................................................................................... 41 H u m a n su b j e c t s (I , II , II I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 M ic e (I V , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 E L I S A (I , II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 G e n o t y p i n g (I I I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 E x p e r i m e n t a l st r o k e m o d e l (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6 B r d U ad m i n i s t r a t i o n (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7 Im m u n o h i s t o c h e m i s t r y an d flu o r e s c e n t - d y e ne u r o n lo a d i n g (I V , V ) . . . . . . . . . . . . . . . . . . . . . . 4 7 Tissue preparation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Im munofluorecent staining ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 E v a l u a t i o n of th e in f a r c t vo l u m e (I V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0 Q u a n t i t a t i v e an a l y s i s of im m u n o s t a i n i n g s (V I , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 Cell Counting (IV , V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Peri- infarct synaptogenesis and axonal sprouting (IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Dendritic spines (V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 E l e c t r o p h y s i o l o g y (V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 Acute slices preparation ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Extracellular field recordings .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Intracellular recordings ............................................................................................ 5 5 In vivo ele c t r o e n c e p h a l o g r a p h y (V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 Beh a v i o r a l te s t i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 E valuati on of sensorimotor deficits (IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Evaluation of hippocampal - dependent cognitive performance (V) . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Sta t i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 R E S U LTS AN D D IS C U S S I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0 S y s t e m i c co m p l e m e n t re s p o n s e dif f e r s be t w e e n isc h e m i c st r o k e su b t y p e s (P a p e r I an d II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0 P la s m a C 3 an d C 3 a le v e l s sh o w et i o l o g y - d e p e n d e n t as s o c i a t i o n s wit h fu n c t i o n a l ou t c o m e af t e r is c h e m i c st r o k e (P a p e r I an d II ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 G e n e t i c va r i a t i o n in co m p l e m e n t co m p o n e n t C 3 sh o w s as s o c i a t i o n wit h is c h e m i c st r o k e (P a p e r II I ) as w e l l as C 3 an d C 3 a le v e l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 R e c e p t o r fo r co m p l e m e n t pe p t i d e C 3 a st i m u l a t e s ne u r a l pla s t i c i t y aft e r ex p e r i m e n t a l br a i n is c h e m i a (P a p e r IV ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 A lt e r e d co g n i t i v e pe r f o r m a n c e an d sy n a p t i c fu n c t i o n in th e hip p o c a m p u s of m ic e la c k i n g C 3 (P a p e r V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 7 M A I N C O N C L U S I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 C O N C L U D I N G R E M A R K S A N D FUTUR E D IR E CTIO N S . . . . . . . . . . . . . . . . . . . . . . . . 7 2 A C K N O W L E D G E M E NTS ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 R E F E R E N C E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 P A P E R S I - V Comple m ent in stroke and neural plasticity   iv A BBREVIATIONS AM P A α-amino-3-hydroxy -5-methyl-4-isoxazolepropionic acid BBB blood brain barrier b F G F basic fibroblast growth factor BN D F brain-derived neurotrophic factor B r d U 5-bromo-2' -deoxyuridine CE cardioembolism C N S central nervous system C R P C-reactive protein C R 1 - 4 complement receptors 1-4 C S T corticospinal tract C 3 a R C3a receptor C 5 a R C5a receptor C 5 L 2 C5a-like receptor 2 D A F decay accelerating factor D G dentate gyrus E L I S A enzyme -linked immunosorbent assay E P S P excitatory postsynaptic potential EPS C excitatory postsynaptic current G A B A γ-amminobutyric acid G A P - 4 3 growth-associated protein 43 GA T GABA transporter G D N F glial cell line-derived neurotrophic factor G F A P glial fibrillary acidic protein G L A S T glutamate aspartate transporter GLT - 1 glilal glutamate transporter 1 GPC R G- protein-coupled receptor I F N γ interferon γ I G F - 1 insulin-like growth factor 1 I L interleukine i . p . intraperitoneally K O knock-out L P S lipopolysaccharide Anna Stokowska   v L T P long-term potentiation L V D large vessel disease M A C membrane attack complex M A S P MBL -associated serine proteases M B L mannose binding lectin M C A o middle cerebral artery occlusion M C P membrane co-factor protein m G l u R metabotropic glutamate receptor m R S modified Rankin Scale N G F nerve growth factor N S C s neural stem cells NSP C s neural stem/progenitors cells N M D A N-methyl-D-aspartate N T - 3 neurotrophin-3 O B olfactory bulb O R odds ratio P B S phosphate buffered saline RO S reactive oxygen species R T room temperature SA H L S I S the Sahlgrenska Academy Study on Ischemic Stroke SG Z subgranular zone SN P single nucleotide polymorphism SSS Scandinavian stroke scale S V D small vessel disease S V Z subventricular zone T N F α tumor necrosis factor α T O A S T Trial of Org 10172 in Acute Stroke Treatment V E G F vascular endothelial growth factor W T wild type Comple m ent in stroke and neural plasticity   iv A BBREVIATIONS AM P A α-amino-3-hydroxy -5-methyl-4-isoxazolepropionic acid BBB blood brain barrier b F G F basic fibroblast growth factor BN D F brain-derived neurotrophic factor B r d U 5-bromo-2' -deoxyuridine CE cardioembolism C N S central nervous system C R P C-reactive protein C R 1 - 4 complement receptors 1-4 C S T corticospinal tract C 3 a R C3a receptor C 5 a R C5a receptor C 5 L 2 C5a-like receptor 2 D A F decay accelerating factor D G dentate gyrus E L I S A enzyme -linked immunosorbent assay E P S P excitatory postsynaptic potential EPS C excitatory postsynaptic current G A B A γ-amminobutyric acid G A P - 4 3 growth-associated protein 43 GA T GABA transporter G D N F glial cell line-derived neurotrophic factor G F A P glial fibrillary acidic protein G L A S T glutamate aspartate transporter GLT - 1 glilal glutamate transporter 1 GPC R G -protein-coupled receptor I F N γ interferon γ I G F - 1 insulin-like growth factor 1 I L interleukine i . p . intraperitoneally K O knock-out L P S lipopolysaccharide Anna Stokowska   v L T P long-term potentiation L V D large vessel disease M A C membrane attack complex M A S P MBL -associated serine proteases M B L mannose binding lectin M C A o middle cerebral artery occlusion M C P membrane co-factor protein m G l u R metabotropic glutamate receptor m R S modified Rankin Scale N G F nerve growth factor N S C s neural stem cells NSP C s neural stem/progenitors cells N M D A N- methyl-D-aspartate N T - 3 neurotrophin-3 O B olfactory bulb O R odds ratio P B S phosphate buffered saline RO S reactive oxygen species R T room temperature SA H L S I S the Sahlgrenska Academy Study on Ischemic Stroke SG Z subgranular zone SN P single nucleotide polymorphism SSS Scandinavian stroke scale S V D small vessel disease S V Z subventricular zone T N F α tumor necrosis factor α T O A S T Trial of Org 10172 in Acute Stroke Treatment V E G F vascular endothelial growth factor W T wild type Anna Stokowska   1 BA CKGROUND ST R O K E C l inical background Stroke is currently the second most common cause of death and a leading cause of disability in adults worldwide (WHO, 2010 ). In Sweden more than 30 000 cases are diagnosed each year. Stroke is defined as a condition with sudden neurological symptoms of greater than 24 hours duration, occurring due to the interruption of blood supply to the brain and the subsequent shortage of oxygen and nutrients. If this situation is prolonged, it leads to metabolic breakdown, accumulation of toxic products, and brain cell death (infarction). The lack of blood supply can be caused by the obstruction of a blood vessel (ischemic stroke) or its rupture (hemorrhagic stroke). These two situations can also occur one after another in the condition termed hemorrhagic transformation of ischemic stroke. Ischemic stroke is the most common form of stroke and constitutes about 85% of all strokes, while hemorrhagic stroke accounts for about 15% (Donnan et al., 2008). Artery occlusion in ischemic stroke is most often due to a thrombus formation. The source of the thrombus may be local, when it is formed at the site of the occlusion, or remote, such as the heart or the surface of atherosclerotic plaque in large artery. I n the latter case, the circulating clot is referred to as an embolus. Currently, the only available treatments for ischemic stroke patients are early thrombolysis or thrombectomy. Intravenous administration of recombinant tissue plasminogen activator (rtPA) within 4.5 hrs from the ischemia onset has been found successful in improving the outcome of eligible stroke patients (Cronin, 2010). The rtPA works by converting plasminogen to plasmin that in turn degrades fibrin, resulting in clot dissolution. Unfortunately, this therapy is available only for a limited group of patients due to the narrow time window for administration (which is often missed). Age and symptom severity limits as well as restrictions on co-morbidities are additional reasons for exclusion. Therefore, large efforts are currently being made to develop therapeutic strategies that would be applicable in the later stage after ischemic stroke and could promote recovery of function. Such strategies include adjuvant therapies which stimulate neural plasticity, especially when applied in conjunction with relevant neurorehabilitation (reviewed in Pekna et al., 2012). Anna Stokowska   1 BA CKGROUND ST R O K E C l inical background Stroke is currently the second most common cause of death and a leading cause of disability in adults worldwide (WHO, 2010) . In Sweden more than 30 000 cases are diagnosed each year. Stroke is defined as a condition with sudden neurological symptoms of greater than 24 hours duration, occurring due to the interruption of blood supply to the brain and the subsequent shortage of oxygen and nutrients. If this situation is prolonged, it leads to metabolic breakdown, accumulation of toxic products, and brain cell death (infarction). The lack of blood supply can be caused by the obstruction of a blood vessel (ischemic stroke) or its rupture (hemorrhagic stroke). These two situations can also occur one after another in the condition termed hemorrhagic transformation of ischemic stroke. Ischemic stroke is the most common form of stroke and constitutes about 85% of all strokes, while hemorrhagic stroke accounts for about 15% (Donnan et al., 2008). Artery occlusion in ischemic stroke is most often due to a thrombus formation. The source of the thrombus may be local, when it is formed at the site of the occlusion, or remote, such as the heart or the surface of atherosclerotic plaque in large artery. I n the latter case, the circulating clot is referred to as an embolus. Currently, the only available treatments for ischemic stroke patients are early thrombolysis or thrombectomy. Intravenous administration of recombinant tissue plasminogen activator (rtPA) within 4.5 hrs from the ischemia onset has been found successful in improving the outcome of eligible stroke patients (Cronin, 2010). The rtPA works by converting plasminogen to plasmin that in turn degrades fibrin, resulting in clot dissolution. Unfortunately, this therapy is available only for a limited group of patients due to the narrow time window for administration (which is often missed). Age and symptom severity limits as well as restrictions on co-morbidities are additional reasons for exclusion. Therefore, large efforts are currently being made to develop therapeutic strategies that would be applicable in the later stage after ischemic stroke and could promote recovery of function. Such strategies include adjuvant therapies which stimulate neural plasticity, especially when applied in conjunction with relevant neurorehabilitation (reviewed in Pekna et al., 2012). Comple m ent in stroke and neural plasticity   2 Etiological subtypes of ischemic stroke The clinical presentation of ischemic stroke may differ depending on the location and size of the infarction , factors which also influence the prognosis. These differences are determined to a large degree by the heterogeneity of underlying pathophysiology, which forms the basis for etiological classification of ischemic stroke. The most commonly used classification criteria are derived from Trial of Org. 10172 in Acute Stroke Treatment and define the following subtypes: large vessel disease (LVD), small vessel disease (SVD), cardioembolism (CE), other determined cause of stroke and stroke of undetermined etiology (Adams et al., 1993). As for the purpose of this thesis the stratification of patients according to the etiology is of interest, a short characterization of the ischemic stroke subtypes is given below (summarized in Table 1). Ischemic stroke due to large vessel disease (LVD) LVD is the cause of around 15 -20% of ischemic strokes although the proportion in the population may vary depending on age, sex and ethnicity (Kirshner, 2009) . This type of stroke is diagnosed by identifying a significant stenosis or occlusion of a large or medium-sized pre -cerebral or cerebral artery due to atherosclerosis. As atherosclerotic plaques predominantly develop near the branching point of arteries, artery -to-artery embolization is the most common cause of ischemic stroke in this patients group. In terms of diagnosis, the mere presence of plaques is not sufficient for assigning LVD pathology as a cause of stroke and other clinical findings need to be consistent with the location of the atherosclerotic lesion, while a cardiac source of embolus needs to be excluded (Rovira et al., 2005). Ischemic stroke due to small vessel disease (SVD) SVD constitutes around 25% of all ischemic stroke causes. It manifests itself as the occlusion of end-arteries supplying deep brain structures such as basal ganglia, thalamus, brain stem and deep white matter, resulting usually in a small infarct (<15 mm on magnetic resonance image). Although SVD pathogenesis is not entirely clear, microatheroma and lipohyalinosis have been found to be associated with this type of ischemic lesions (de Jong et al., 2002 ). Clinically, SVD strokes present themselves as a Anna Stokowska   3 so called “lacunar syndrome”, characterized by the absence of co rtical symptoms or visual field deficits and include pure motor stroke, pure sensory stroke, ataxic hemiparesis or somatosensory stroke (Bamford et al., 1987). Other conditions that could also cause occlusion of a small brain vessel such as vasculitis, hematological diseases or embolism (from heart or large extracranial artery) need to be ex cluded for correct diagnosis (Arboix and Marti -Vilalta, 2004 ). Ischemic stroke due to cardioembolism (CE) About 25% of all ischemic strokes are caused by emboli originating from the heart. Infarcts in this stroke subtype are fairly large thus causing severe disabilities and the ischemic events are prone to recurrence. Atrial fibrillation is the major contributor to cardiac embolus formation by leading to atrial stasis that is associated with increased prothrombotic state (Rovira et al., 2005). Other major risk factors for cardioembolism include recent myocardial infarction, ventricular thrombosis, prosthetic valve endocarditis and patent foramen ovale although that latter cause is highly controversial (Ferro, 2003 ; Freeman and Aguilar, 2011 ). Ischemic stroke of undetermined etiology This category can be subdivided into two subcategories. If despite extensive investigation the cause of ischemic stroke remains undefined the stroke is classified as cryptogenic. This subtype constitutes about 30% of all ischemic strokes, however this number varies depending on the extent of investi gation. Patients presenting with cryptogenic stroke are typically younger as compared to other etiologic subtypes. It has been suggested that this stroke subtype is in itself heterogeneous (Guercini et al., 2008 ; Jickling et al., 2012 ). In some cases, more than one possible cause is identified or the investigation is cursory and then the stroke is classified as ischemic stroke due to an undetermined cause. Stroke of other determined etiology In addition to the major classes of ischemic stroke, other cause of ischemic stroke may be identified. The rare determined causes are: arterial dissection, vasculitis, Comple m ent in stroke and neural plasticity   2 Etiological subtypes of ischemic stroke The clinical presentation of ischemic stroke may differ depending on the location and size of the infarction , factors which also influence the prognosis. These differences are determined to a large degree by the heterogeneity of underlying pathophysiology, which forms the basis for etiological classification of ischemic stroke. The most commonly used classification criteria are derived from Trial of Org. 10172 in Acute Stroke Treatment and define the following subtypes: large vessel disease (LVD), small vessel disease (SVD), cardioembolism (CE), other determined cause of stroke and stroke of undetermined etiology (Adams et al., 1993). As for the purpose of this thesis the stratification of patients according to the etiology is of interest, a short characterization of the ischemic stroke subtypes is given below (summarized in Table 1). Ischemic stroke due to large vessel disease (LVD) LVD is the cause of around 15 -20% of ischemic strokes although the proportion in the population may vary depending on age, sex and ethnicity (Kirshner, 2009 ). This type of stroke is diagnosed by identifying a significant stenosis or occlusion of a large or medium-sized pre -cerebral or cerebral artery due to atherosclerosis. As atherosclerotic plaques predominantly develop near the branching point of arteries, artery -to-artery embolization is the most common cause of ischemic stroke in this patients group. In terms of diagnosis, the mere presence of plaques is not sufficient for assigning LVD pathology as a cause of stroke and other clinical findings need to be consistent with the location of the atherosclerotic lesion, while a cardiac source of embolus needs to be excluded (Rovira et al., 2005). Ischemic stroke due to small vessel disease (SVD) SVD constitutes around 25% of all ischemic stroke causes. It manifests itself as the occlusion of end-arteries supplying deep brain structures such as basal ganglia, thalamus, brain stem and deep white matter, resulting usually in a small infarct (<15 mm on magnetic resonance image). Although SVD pathogenesis is not entirely clear, microatheroma and lipohyalinosis have been found to be associated with this type of ischemic lesions (de Jong et al., 2002 ). Clinically, SVD strokes present themselves as a Anna Stokowska   3 so called “lacunar syndrome”, characterized by the absence of co rtical symptoms or visual field deficits and include pure motor stroke, pure sensory stroke, ataxic hemiparesis or somatosensory stroke (Bamford et al., 1987). Other conditions that could also cause occlusion of a small brain vessel such as vasculitis, hematological diseases or embolism (from heart or large extracranial artery) need to be excluded for correct diagnosis (Arboix and Marti -Vilalta, 2004 ). Ischemic stroke due to cardioembolism (CE) About 25% of all ischemic strokes are caused by emboli originating from the heart. Infarcts in this stroke subtype are fairly large thus causing severe disabilities and the ischemic events are prone to recurrence. Atrial fibrillation is the major contributor to cardiac embolus formation by leading to atrial stasis that is associated with increased prothrombotic state (Rovira et al., 2005). Other major risk factors for cardioembolism include recent myocardial infarction, ventricular thrombosis, prosthetic valve endocarditis and patent foramen ovale although that latter cause is highly controversial (Ferro, 2003; Freeman and Aguilar, 2011 ). Ischemic stroke of undetermined etiology This category can be subdivided into two subcategories. If despite extensive investigation the cause of ischemic stroke remains undefined the stroke is classified as cryptogenic. This subtype constitutes about 30% of all ischemic strokes, however this number varies depending on the extent of investigation. Patients presenting with cryptogenic stroke are typically younger as compared to other etiologic subtypes. It has been suggested that this stroke subtype is in itself heterogeneous (Guercini et al., 2008 ; Jickling et al., 2012 ). In some cases, more than one possible cause is identified or the investigation is cursory and then the stroke is classified as ischemic stroke due to an undetermined cause. Stroke of other determined etiology In addition to the major classes of ischemic stroke, other cause of ischemic stroke may be identified. The rare determined causes are: arterial dissection, vasculitis, Comple m ent in stroke and neural plasticity   4 hypercoagulable states, hematological disorders or rare monogenic diseases (Levine, 2005; Ballabio et al., 2007; Ferro et al., 2010 ). Table 1. Features of TOAST classification of ischemic stroke subtypes . Adapted from Adams et al. (1993). Risk factors for ischemic stroke Several risk factors have been identified to be associated with ischemic stroke and they can be divided into non-modifiable and modifiable. The most important non- modifiable risk factors are old age, male sex, ethnicity, and family history of stroke, whereas some modifiable factors are hypertension, atrial fibrillation, diabetes mellitus, smoking, alcohol consumption, obesity and physical inactivity (Kirshner, 2009; Kokubo, 2012). In the recent years, factors such as chronic stress, increase in blood inflammatory and hemostatic markers as well as genetic polymorphism have been shown to be associated with increased risk of ischemic stroke, although causal role of these factors Features LVD stroke SVD stroke CE stroke Other cause Cryptogenic stroke Clinical Cortical or cerebellar dysfunction + - + +/- +/- Lacunar syndrome - + - +/- +/- Imaging Cortical, cerebellar, brainstem, or subcortical infarct >15 mm + - + +/- +/- Subcortical or brainstem infarct <15 mm - + - +/- - Tests Stenosis of an appropriate extracranial or intracranial artery + - - - - Cardiac source of emboli - - + - - Other abnormality on tests - - - + - Anna Stokowska   5 remains to be determined (Hankey, 2006 ). Due to the already mentioned heterogeneity of pathophysiological mechanisms in ischemic stroke, it is conceivable that different stroke subtypes have different profiles of risk factors. Pathobiology of ische m i c brain damage The brain requires a constant supply of oxyge n and glucose to maintain its normal function, therefore if these demands cannot be met, the cellular “ ischemic cascade” is initiated. These events produce an irreversibly damaged ischemic core and a potentially salvageable, hypoperfused adjacent region ca lled penumbra (Hossmann, 1994 ). Cell death in the core is rapid (occurring within minutes) while the damage in penumbra develops more slowly owing to the collateral blood flow provided by anastomoses within the circle of Willis and leptomeninges (Ringelstein et al., 1992). However, if the normal levels of oxygen and glucose are not restored, tissue in that region will eventually die. The so called “ischemic cascade” consists of events that are secondary to the widespread death of neurons and glial cells and include mainly glutamate toxicity, oxidative stress and inflammation (Fi gure. 1). Glutamate toxicity As a consequence of oxygen and glucose deprivation following ischemic stroke, a sharp decline in cellular ATP levels results in a dysfunction of membrane ion pumps. This causes a cellular efflux of K + and a consequent influx of Na+ , Ca2+ , and water (Martin et al., 1994). Increased intracellular levels of Ca2+ lead to depolarization of the affected neuronal membrane and the release of excitatory neurotransmitter glutamate. Glutamate binds to and activates N -methyl-D-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mG luRs) on the neighboring neurons. Accompanying malfunction of glial amino acid transporters leads to failure in the re-uptake of extracellular glutamate and results in even further Ca 2+ influx to the neurons (Manev et al., 1989). This accumulation of calcium ions leads to the activation of intracellular lipases and proteases causing cell damage and cell death. This series of events is referred to as excitotoxicity and has a critical role in the pathogenesis of ischemic stroke. Comple m ent in stroke and neural plasticity   4 hypercoagulable states, hematological disorders or rare monogenic diseases (Levine, 2005; Ballabio et al., 2007; Ferro et al., 2010 ). Table 1. Features of TOAST classification of ischemic stroke subtypes . Adapted from Adams et al. (1993). Risk factors for ischemic stroke Several risk factors have been identified to be associated with ischemic stroke and they can be divided into non-modifiable and modifiable. The most important non- modifiable risk factors are old age, male sex, ethnicity, and family history of stroke, whereas some modifiable factors are hypertension, atrial fibrillation, diabetes mellitus, smoking, alcohol consumption, obesity and physical inactivity (Kirshner, 2009 ; Kokubo, 2012). In the recent years, factors such as chronic stress, increase in blood inflammatory and hemostatic markers as well as genetic polymorphism have been shown to be associated with increased risk of ischemic stroke, although causal role of these factors Features LVD stroke SVD stroke CE stroke Other cause Cryptogenic stroke Clinical Cortical or cerebellar dysfunction + - + +/- +/- Lacunar syndrome - + - +/- +/- Imaging Cortical, cerebellar, brainstem, or subcortical infarct >15 mm + - + +/- +/- Subcortical or brainstem infarct <15 mm - + - +/- - Tests Stenosis of an appropriate extracranial or intracranial artery + - - - - Cardiac source of emboli - - + - - Other abnormality on tests - - - + - Anna Stokowska   5 remains to be determined (Hankey, 2006 ). Due to the already mentioned heterogeneity of pathophysiological mechanisms in ischemic stroke, it is conceivable that different stroke subtypes have different profiles of risk factors. Pathobiology of ische m i c brain damage The brain requires a constant supply of oxygen and glucose to maintain its normal function, therefore if these demands cannot be met, the cellular “ ischemic cascade” is initiated. These events produce an irreversibly damaged ischemic core and a potentially salvageable, hypoperfused adjacent region ca lled penumbra (Hossmann, 1994) . Cell death in the core is rapid (occurring within minutes) while the damage in penumbra develops more slowly owing to the collateral blood flow provided by anastomoses within the circle of Willis and leptomeninges ( Ringelstein et al., 1992). However, if the normal levels of oxygen and glucose are not restored, tissue in that region will eventually die. The so called “ischemic cascade” consists of events that are secondary to the widespread death of neurons and glial cells and include mainly glutamate toxicity, oxidative stress and inflammation (Fig ure 1). Glutamate toxicity As a consequence of oxygen and glucose deprivation following ischemic stroke, a sharp decline in cellular ATP levels results in a dysfunction of membrane ion pumps. This causes a cellular efflux of K + and a consequent influx of Na + , Ca2+ , and water (Martin et al., 1994). Increased intracellular levels of Ca2+ lead to depolarization of the affected neuronal membrane and the release of excitatory neurotransmitter glutamate. Glutamate binds to and activates N -methyl-D-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mG luRs) on the neighboring neurons. Accompanying malfunction of glial amino acid transporters leads to failure in the re-uptake of extracellular glutamate and results in even further Ca 2+ influx to the neurons (Manev et al., 1989). This accumulation of calcium ions leads to the activation of intracellular lipases and proteases causing cell damage and cell death. This series of events is referred to as excitotoxicity and has a critical role in the pathogenesis of ischemic stroke. Comple m ent in stroke and neural plasticity   6 Oxidative stress O xidative stress is defined as the condition occurring when the physiological balance between oxidants and antioxidants is disrupted in favor of the former with potential damage for the organism. Oxidative stress leading to ischemic cell death involves the formation of reactive oxygen species (ROS) and reactive nitrogen species through multiple injury mechanisms, such as mitochondrial inhibition, Ca 2+ overload, reperfusion injury, and inflammation (Coyle and Puttfarcken, 1993). Brain ischemia generates superoxid e (O 2 -), which is the primary radical from which hydrogen peroxide, the source of hydroxyl radical, is formed. Ischemia causes an increase in nitric oxide synthase Figure 1. S c h e m a t i c s of th e is c h e m i c ca s c a d e . Due to energy shortage, ion imbalance results in depolarization of presynaptic neurons and uncontrolled release of glutamate which is not recycled by astrocytes, leading to glutamate “spill -over” to the perisynaptic space. Upon binding of glutamate to its receptors, mobilization of calcium i ons from the intracellular storage sites occurs in post-synaptic neurons, leading to the release of toxic metabolites. Resulting neuronal cell death activates microglia which, through the release of inflammatory mediators, recruit leukocytes and promote their extravasation. This in turn can exacerbate inflammation and cause secondary tissue damage. Glu –glutamate. Anna Stokowska   7 (NOS) type I and III activity in neurons and vascular endothelium, respectively. At a later stage, elevated NOS type II (iNOS) activity occurs in a range of cells including glia and infiltrating neutrophils (Lakhan et al., 2009 ). Large numbers of ROS ar e generated during an acute ischemic stroke and there is considerable evidence that oxidative stress is an important mediator of tissue injury in acute ischemic stroke (Cuzzocrea et al., 2001 ). Post- ischemi c inflammation Inflammation is a physiological process that helps the body to eliminate pathogens and activate tissue regeneration. However, when uncontrolled, inflammatory processes can become excessive or chronic and can exacerbate tissue damage , and prevent recovery. Inflammation following cerebral ischemia is characterized by the accumulation of inflammatory cells and mediators in the ischemic brain. The first cells responding to ischemic insult are microglia which are the resident immunocompetent cells of the brain. Once activated, microglia undergo morphological transformation from the resting ramified state to the ameboid one (virtually indistinguishable from blood-derived macrophages) and they migrate to the site of injury to phagocyt ose apoptotic and necrotic cellular debris (Streit et al., 1999). Microglia contribute to brain tissue damage by releasing inflammatory mediators such as ROS, proteases and pro -inflammatory cytokines such as interleukin (IL) -1, IL -6 and tumour necrosis factor α (TNF α). These cytokines upregulate the expression of cell - adhesion molecules (CAMs) on endothelium of cerebral blood vessels (Wang and Feuerstein, 1995 ). This in turn promotes the adherence of circulating leukocytes to vessel walls followed by their migration into brain parenchyma with subsequent release of additional pro-inflammatory mediators and secondary injury . Neutrophils are the first white blood cells recruited from the periphery to the ischemic tissue (Stevens et al., 2002). They are followed by monocytes/macrophages and finally lymphocytes, which are believed to contribute to the delayed brain tissue damage mainly through the release of directly neurotoxic interferon γ (IFN γ) (Lambertsen et al., 2004 ). Oxidative stress and the inflammatory cascade alter the permeability of the blood-brain barrier (BBB). The activation of matrix metalloproteinases (MMPs) and the expression of various other proteases lead to BBB breakdown which exacerbates leukocyte extravasation. Comple m ent in stroke and neural plasticity   6 Oxidative stress O xidative stress is defined as the condition occurring when the physiological balance between oxidants and antioxidants is disrupted in favor of the former with potential damage for the organism. Oxidative stress leading to ischemic cell death involves the formation of reactive oxygen species (ROS) and reactive nitrogen species through multiple injury mechanisms, such as mitochondrial inhibition, Ca 2+ overload, reperfusion injury, and inflammation (Coyle and Puttfarcken, 1993). Brain ischemia generates superoxid e (O 2 -), which is the primary radical from which hydrogen peroxide, the source of hydroxyl radical, is formed. Ischemia causes an increase in nitric oxide synthase Figure 1. S c h e m a t i c s of th e is c h e m i c ca s c a d e . Due to energy shortage, ion imbalance results in depolarization of presynaptic neurons and uncontrolled release of glutamate which is not recycled by astrocytes, leading to glutamate “spill -over” to the perisynaptic space. Upon binding of glutamate to its receptors, mobilization of calcium i ons from the intracellular storage sites occurs in post-synaptic neurons, leading to the release of toxic metabolites. Resulting neuronal cell death activates microglia which, through the release of inflammatory mediators, recruit leukocytes and promote their extravasation. This in turn can exacerbate inflammation and cause secondary tissue damage. Glu –glutamate. Anna Stokowska   7 (NOS) type I and III activity in neurons and vascular endothelium, respectively. At a later stage, elevated NOS type II (iNOS) activity occurs in a range of cells including glia and infiltrating neutrophils (Lakhan et al., 2009 ). Large numbers of ROS ar e generated during an acute ischemic stroke and there is considerable evidence that oxidative stress is an important mediator of tissue injury in acute ischemic stroke (Cuzzocrea et al., 2001 ). Post- ischemi c inflammation Inflammation is a physiological process that helps the body to eliminate pathogens and activate tissue regeneration. However, when uncontrolled, inflammatory processes can become excessive or chronic and can exacerbate tissue damage , and prevent recovery. Inflammation following cerebral ischemia is characterized by the accumulation of inflammatory cells and mediators in the ischemic brain. The first cells responding to ischemic insult are microglia which are the resident immunocompetent cells of the brain. Once activated, microglia undergo morphological transformation from the resting ramified state to the ameboid one (virtually indistinguishable from blood-derived macrophages) and they migrate to the site of injury to phagocyt ose apoptotic and necrotic cellular debris (Streit et al., 1999). Microglia contribute to brain tissue damage by releasing inflammatory mediators such as ROS, proteases and pro -inflammatory cytokines such as interleukin (IL) -1, IL -6 and tumour necrosis factor α (TNF α). These cytokines upregulate the expression of cell - adhesion molecules (CAMs) on endothelium of cerebral blood vessels (Wang and Feuerstein, 1995 ). This in turn promotes the adherence of circulating leukocytes to vessel walls followed by their migration into brain parenchyma with subsequent release of additional pro-inflammatory mediators and secondary injury. Neutrophils are the first white blood cells recruited from the periphery to the ischemic tissue (Stevens et al., 2002). They are followed by monocytes/macrophages and finally lymphocytes, which are believed to contribute to the delayed brain tissue damage mainly through the release of directly neurotoxic interferon γ (IFN γ) (Lambertsen et al., 2004 ). Oxidative stress and the inflammatory cascade alter the permeability of the blood-brain barrier (BBB). The activation of matrix metalloproteinases (MMPs) and the expression of various other proteases lead to BBB breakdown which exacerbates leukocyte extravasation. Comple m ent in stroke and neural plasticity   8 Astrocytes, similar to microglia, are capable of secreting inflammatory factors such as cytokines, chemokines, and nitric oxide (Swanson et al., 2004). Chemokines are a class of cytokines that guide the migration of blood borne inflammatory cells, such as neutrophils and macrophages, towards the source of the chemokine. Consequently, they play important roles in cellular communication and inflammatory cell recruitment (Lakhan et al., 2009) . Expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and fractalkine following focal ischemia and the resulting increase in leukocyte infiltration is thought to have a deleterious effect ischemic brain (Kim et al., 1995 ). One of the inflammatory mediators which also seems to play a major role after cerebral ischemia is the complement system. The activated complement cascade generates peptides with pro-inflammatory and chemotactic properties which among others functions upregulate the expression of CAMs thus promoting recruitment of inflammatory cells. Anna Stokowska   9 T H E C O M P L E M E N T SY S T E M The complement system was first discovered in the end of 19th century by Jules Bordet as a heat-sensitive factor in fresh serum that “complements” the effect of a specific antibody in the lysis of bacteria and red blood cells. Its importance as an effector of humoral immunity was extended with the early observations of opsonisation and participation in cellular immunity. The complement system is a general term attributed to a constellation of more than 30 soluble plasma and body fluid proteins and a number of cell receptors and control proteins found in the blood and tissues (Janeway et al., 2005 ). Their roles in innate immunity include the opsonisation and lysis of pathogens, elimination of soluble immune complexes, release of anaphylatoxins, stimulation of leukocytes chemotaxis and release of inflammatory cytokines. Complement affects adaptive immunity by regulating B and T lymphocyte function (Carroll, 2004). Complement activation provides a rapid and effective defense barrier against bacteria, viruses, virus-infected cells, parasites, and tumor cells. The predominant site of peripheral complement protein synthesis is the liver, where hepatocytes constantly produce and replenish circulating complement factors (Alper et al., 1969). Also monocytes and macrophages have been found to produce the majority of complement components especially upon stimulation with pro-inflammatory cytokines (Einstein et al., 1977; Cole et al., 1983). Activation of these circulating complement proteins in response to an injury or an infectious challenge results in a self -amplifying cascade of proteolytic reactions through one of the three major identified pathwa ys, namely the classical, lectin or alternative pathways (Figure 2). T h e th i r d co m p l e m e n t co m p o n e n t (C 3 ) C3 is the central element of complement system and also the most abundant complement protein in plasma. The physiological concentration of C3 in human plasma is up to 1 mg/ml and increases during inflammatory states as C3 belongs to the acute phase proteins. The main source of C3 in the periphery is the liver, although it is also produced in other tissues. C3 is a large glycoprotein, consisting of α and β chains connected by a disulphide bridge. The α chain contains also an internal thioesther bond, which is hidden inside the inactive C3 molecule. The C3 α chain can be cleaved by a C3 Comple m ent in stroke and neural plasticity   8 Astrocytes, similar to microglia, are capable of secreting inflammatory factors such as cytokines, chemokines, and nitric oxide (Swanson et al., 2004). Chemokines are a class of cytokines that guide the migration of blood borne inflammatory cells, such as neutrophils and macrophages, towards the source of the chemokine. Consequently, they play important roles in cellular communication and inflammatory cell recruitment (Lakhan et al ., 2009). Expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and fractalkine following focal ischemia and the resulting increase in leukocyte infiltration is thought to have a deleterious effect ischemic brain (Kim et al., 1995 ). One of the inflammatory mediators which also seems to play a major role after cerebral ischemia is the complement system. The activated complement cascade generates peptides with pro-inflammatory and chemotactic properties which among others functions upregulate the expression of CAMs thus promoting recruitment of inflammatory cells. Anna Stokowska   9 T H E C O M P L E M E N T SY S T E M The complement system was first discovered in the end of 19th century by Jules Bordet as a heat-sensitive factor in fresh serum that “complements” the effect of a specific antibody in the lysis of bacteria and red blood cells. Its importance as an effector of humoral immunity was extended with the early observations of opsonisation and participation in cellular immunity. The complement system is a general term attributed to a constellation of more than 30 soluble plasma and body fluid proteins and a number of cell receptors and control proteins found in the blood and tissues (Janeway et al., 2005 ). Their roles in innate immunity include the opsonisation and lysis of pathogens, elimination of soluble immune complexes, release of anaphylatoxins, stimulation of leukocytes chemotaxis and release of inflammatory cytokines. Complement affects adaptive immunity by regulating B and T lymphocyte function (Carroll, 2004). Complement activation provides a rapid and effective defense barrier against bacteria, viruses, virus-infected cells, parasites, and tumor cells. The predominant site of peripheral complement protein synthesis is the liver, where hepatocytes constantly produce and replenish circulating complement factors (Alper et al., 1969). Also monocytes and macrophages have been found to produce the majority of complement components especially upon stimulation with pro-inflammatory cytokines (Einstein et al., 1977; Cole et al., 1983). Activation of these circulating complement proteins in response to an injury or an infectious challenge results in a self -amplifying cascade of proteolytic reactions through one of the three major identified pathwa ys, namely the classical, lectin or alternative pathways (Figure 2). T h e th i r d co m p l e m e n t co m p o n e n t (C 3 ) C3 is the central element of complement system and also the most abundant complement protein in plasma. The physiological concentration of C3 in human plasma is up to 1 mg/ml and increases during inflammatory states as C3 belongs to the acute phase proteins. The main source of C3 in the periphery is the liver, although it is also produced in other tissues. C3 is a large glycoprotein, consisting of α and β chains connected by a disulphide bridge. The α chain contains also an internal thioesther bond, which is hidden inside the inactive C3 molecule. The C3 α chain can be cleaved by a C3 Comple m ent in stroke and neural plasticity   10 convertase to generate two fragments, the small C3a and the larger C3b, which consists of the remaining part of the parental molecule (Sahu and Lambris, 2001 ). The cleavage causes a conformational change of C3b leading to the exposure of the thioesther bond that is susceptible to attack by electron donors such as hydroxyl or amine groups (Janssen et al., 2006). This reaction allows for the interaction of C3b with proteins and carbohydrates on the target surface by forming covalent ester and amide bonds. As a free thioesther bond is rapidly inactivated on contact with water, binding of C3b occurs only when it is generated in close proximity of the activating surface ( Sahu and Lambris, 2001). C3b binds to the C3 convertase complex forming the C5 convertase (Figure 2). Apart from being a subunit of complement-related convertase, C3b is an opsonin and coats the circulating immune complexes recognized by the C3b receptor (CR3) on erythrocytes for their transport and neutralization in the spleen. C3b binds also directly to foreign or altered cells and thus promotes clearance and neutralization of pathogens apoptotic debris. C3a participates in the recruitment and activation of leukocytes to produce and release inflammatory mediators and substances such as histamine which mediate the development of allergic reactions and it is therefore regarded as an anaphylatoxin ( Klos et al., 2009). Unless it is bound to its receptor, C3aR, C3a is rapidly inactivated by plasma carboxypeptidases through the removal of C -terminal arginine, which results in the formation of C3adesArg. This truncated molecule does no longer bind or activate C3aR but it is a ligand for C5a-like receptor 2 (C5L2) (Kalant et al., 2003 ). C3adesArg is more commonly known under the name acylation stimulating protein (ASP) due to its marked stimulating action on triacylglycerol synthesis in human adipocytes and skin (Cianflone et al., 1989). It has therefore been linked to the pathogenesis of obesity, insulin resistance and metabolic syndrome (Zimmet et al., 1999 ; Sniderman et al., 2000; Wamba et al., 2008; Mamane et al., 2009). A c t i v a t i o n of th e co m p l e m e n t ca s c a d e Classical pathway The classical pathway was the first activation pathway of the complement system to be discovered and it involves complement components C1, C2 and C4. It is initiated by Anna Stokowska   11 Figure 2 . A c t i v a t i o n an d re g u l a t i o n of th e co m p l e m e n t sy s t e m . The three activation pathways converge at the level of C3 convertase which together with coagulation factors, activate the terminal pathway. Inhibitors and their interaction points are indicated in gray. Anaphylatoxins are marked with asterisks. Ag-Ab – antigen-antibody complex es; GlcNAc – N-acetyl-glucose, LPS – lipopolysaccharide. the C1 complex consisting of recog nition protein C1q, which bind s primarily to antigen–antibody complexes, a pair of C1r and a pair of C1s molecules. C1q is a homohexamer with subunits consisting of C -terminal globular head domain and N-terminal collagenous tail. At least two out of the six globular heads need to be bound to the immune complexes through the Fc region of type G or M immunoglobulin for the classical pathway to be fully activated (Janeway et al., 2005 ). This binding leads to conformational change in the C1q molecule and subsequent activation of C1r and C1s molecules. Activation of C1r and C1s, with generation of C1s esterase, is followed by cleavage of C4 and C2. This cleavage releases small peptides and allows the assembly of the C3 convertase, C4bC2a complex (Figure 2). Comple m ent in stroke and neural plasticity   10 convertase to generate two fragments, the small C3a and the larger C3b, which consists of the remaining part of the parental molecule (Sahu and Lambris, 2001 ). The cleavage causes a conformational change of C3b leading to the exposure of the thioesther bond that is susceptible to attack by electron donors such as hydroxyl or amine groups (Janssen et al., 2006). This reaction allows for the interaction of C3b with proteins and carbohydrates on the target surface by forming covalent ester and amide bonds. As a free thioesther bond is rapidly inactivated on contact with water, binding of C3b occurs only when it is generated in close proximity of the activating surface (Sahu and Lambris, 2001). C3b binds to the C3 convertase complex forming the C5 convertase (Figure 2). Apart from being a subunit of complement-related convertase, C3b is an opsonin and coats the circulating immune complexes recognized by the C3b receptor (CR3) on erythrocytes for their transport and neutralization in the spleen. C3b binds also directly to foreign or altered cells and thus promotes clearance and neutralization of pathogens apoptotic debris. C3a participates in the recruitment and activation of leukocytes to produce and release inflammatory mediators and substances such as histamine which mediate the development of allergic reactions and it is therefore regarded as an anaphylatoxin (Klos et al., 2009). Unless it is bound to its receptor, C3aR, C3a is rapidly inactivated by plasma carboxypeptidases through the removal of C -terminal arginine, which results in the formation of C3adesArg. This truncated molecule does no longer bind or activate C3aR but it is a ligand for C5a-like receptor 2 (C5L2) (Kalant et al., 2003 ). C3adesArg is more commonly known under the name acylation stimulating protein (ASP) due to its marked stimulating action on triacylglycerol synthesis in human adipocytes and skin (Cianflone et al., 1989). It has therefore been linked to the pathogenesis of obesity, insulin resistance and metabolic syndrome (Zimmet et al., 1999 ; Sniderman et al., 2000; Wamba et al., 2008; Mamane et al., 2009). A c t i v a t i o n of th e co m p l e m e n t ca s c a d e Classical pathway The classical pathway was the first activation pathway of the complement system to be discovered and it involves complement components C1, C2 and C4. It is initiated by Anna Stokowska   11 Figure 2 . A c t i v a t i o n an d re g u l a t i o n of th e co m p l e m e n t sy s t e m . The three activation pathways converge at the level of C3 convertase which together with coagulation factors, activate the terminal pathway. Inhibitors and their interaction points are indicated in gray. Anaphylatoxins are marked with asterisks. Ag -Ab – antigen-antibody complex es; GlcNAc – N-acetyl-glucose, LPS – lipopolysaccharide. the C1 complex consisting of recognition protein C1q, which bind s primarily to antigen–antibody complexes, a pair of C1r and a pair of C1s molecules. C1q is a homohexamer with subunits consisting of C -terminal globular head domain and N-terminal collagenous tail. At least two out of the six globular heads need to be bound to the immune complexes through the Fc region of type G or M immunoglobulin for the classical pathway to be fully activated (Janeway et al., 2005 ). This binding leads to conformational change in the C1q molecule and subsequent activation of C1r and C1s molecules. Activation of C1r and C1s, with generation of C1s esterase, is followed by cleavage of C4 and C2. This cleavage releases small peptides and allows the assembly of the C3 convertase, C4bC2a complex (Figure 2). Comple m ent in stroke and neural plasticity   12 The classical pathway is also activated by antibody-independent mechanisms involving direct binding of C1q to viral envelopes, cell walls of Gram -negative bacteria, C-reactive protein (CRP), intermediate filaments and central nervous system (CNS) myelin (Janeway et al., 2005 ). Lectin pathway The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) and ficolins to carbohydrate groups on the surface of bacterial cells (Fujita et al., 2004 ) . MBL and ficolins are typical pattern-recognition molecules, which serve to attach the MBL - associated serine proteases (MASP) 1, 2, and 3, thus activating MASP esterase activity. Upon activation, MASPs cleave and activate C4 and C2, thus generating the C3 convertase, C4bC2a, similar to the classical pathway (Petersen et al., 2000; Dahl et al., 2001). MBL can also bind to apoptotic cells thus serving as an opsonin (Nauta et al., 2003). Alternative pathway The alternative pathway of complement activation is initiated by a constant, low level spontaneous hydrolysis of thioesther bonds in C3 in plasma, leading to the formation of C3(H2O) molecule ( Pangburn et al., 1981). C3(H 2O) formation involves conformational “tick -over” of C3, which exposes neo -epitope in a conformation very similar to C3b. Activation of a serine protease, factor D, cleaves factor B into Ba and Bb when factor B is complexed with C3b or C3(H 2O). The C3(H 2O) Bb complexes generate C3b as they cleave circulating C3. H owever, C3b is short-lived while remaining in the fluid phase. Upon binding to an activating non -self surface, C3b builds a complex with Bb, generating a solid phase C3 convertase of the alternative pathway, C3bBb the stability of which is promoted by a protein called properdin. Binding of additional C3b fragment to the deposited C3-convertase complexes makes up alternative pathway C5 -convertase (C3bBbC3b). Spontaneous low-grade activation of the alternative pathway is believed to be a surveillance mechanism which becomes amplified when blood comes in contact with an activating surface. Among the alternative pathway activators are bacterial or microbial fragments, tumor cells, virus envelopes, plastic surfaces, peripheral nerve myelin, and intracellular organelles. These activators act by protecting the C3 convertase from Anna Stokowska   13 inactivation by complement regulators that cause C3b cleavage and Bb decay (Xu et al., 2001) . Termin al pathway As already mentioned, binding of an additional C3b fragment to the C3 convertase brings about C5 convertase properties to the complex and allows for cleaving parental C5 protein to C5a and C5b fragments. In the recent years, an alternative mechanism for complement activation that bypasses the convertase stages has been identified. It involves direct cleavage of C3 and C5 mainly by coagulation cascade proteases (factors IXa, Xa and XIa, thrombin and plasmin) and is part of the so called extrinsic pathway (Huber - Lang et al., 2006 ; Amara et al., 2010). C5a is the most potent anaphylatoxin in the body while C5b initiates formation of the membrane attack complex (MAC) on the activated surface. C5b remains attached to the convertase complex but allows for the binding o f components C6 and C7. Upon attachment of C8 to the C5bC6C7 complex, insertion in to the target membrane is possible due to the exposition of hydrophobic surfaces of the complex. The following attachment of multiple C9 molecules leads to formation of MAC, a pore-like structure in the membrane leading to lysis of the target cell (Janeway et al., 2005). C o n t r o l of th e co m p l e m e n t sy s t e m Considering the lack of active discrimination between self and non-self particles and its potentially destructive effect also towards host cells, the complement system has to be tightly regulated. Complement regulation is achieved by the presence of soluble and membrane-bound inhibitors on self-cells, short half-life of convertases (unless they are stabilized by binding to relevant target surface) and transient presence of active proteases, such as factor D (Turnberg and Botto, 2003). Regulation can occur on different levels of the complement cascade (Fig 2). For example, C1 complex inhibitor (C1 -INH) works by causing dis sociation of serine protease subunits C1r and C1s, MASP 1 and MASP 2 from the recognition molecules C1q and MBL, respectively, thereby inhibiting the activation of classical and lectin pathways. The main complement regulators are the inhibitors of crucial enzymatic Comple m ent in stroke and neural plasticity   12 The classical pathway is also activated by antibody-independent mechanisms involving direct binding of C1q to viral envelopes, cell walls of Gram -negative bacteria, C-reactive protein (CRP), intermediate filaments and central nervous system (CNS) myelin (Janeway et al., 2005 ). Lectin pathway The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) and ficolins to carbohydrate groups on the surface of bacterial cells (Fujita et al., 2004 ) . MBL and ficolins are typical pattern-recognition molecules, which serve to attach the MBL - associated serine proteases (MASP) 1, 2, and 3, thus activating MASP esterase activity. Upon activation, MASPs cleave and activate C4 and C2, thus generating the C3 convertase, C4bC2a, similar to the classical pathway (Petersen et al., 2000; Dahl et al., 2001). MBL can also bind to apoptotic c ells thus serving as an opsonin (Nauta et al., 2003). Alternative pathway The alternative pathway of complement activation is initiated by a constant, low level spontaneous hydrolysis of thioesther bonds in C3 in plasma, leading to the formation of C3(H2O) molecule (Pangburn et al., 1981). C3(H 2O) formation involves conformational “tick -over” of C3, w hich exposes neo -epitope in a conformation very similar to C3b. Activation of a serine protease, factor D, cleaves factor B into Ba and Bb when factor B is complexed with C3b or C3(H 2O). The C3(H 2O) Bb complexes generate C3b as they cleave circulating C3. H owever, C3b is short-lived while remaining in the fluid phase. Upon binding to an activating non -self surface, C3b builds a complex with Bb, generating a solid phase C3 convertase of the alternative pathway, C3bBb the stability of which is promoted by a protein called properdin. Binding of additional C3b fragment to the deposited C3-convertase complexes makes up alternative pathway C5 -convertase (C3bBbC3b). Spontaneous low-grade activation of the alternative pathway is believed to be a surveillance mechanism which becomes amplified when blood comes in contact with an activating surface. Among the alternative pathway activators are bacterial or microbial fragments, tumor cells, virus envelopes, plastic surfaces, peripheral nerve myelin, and intracellular organelles. These activators act by protecting the C3 convertase from Anna Stokowska   13 inactivation by complement regulators that cause C3b cleavage and Bb decay (Xu et al., 2001) . Termin al pathway As already mentioned, binding of an additional C3b fragment to the C3 convertase brings about C5 convertase properties to the complex and allows for cleaving parental C5 protein to C5a and C5b fragments. In the recent years, an alternative mechanism for complement activation that bypasses the convertase stages has been identified. It involves direct cleavage of C3 and C5 mainly by coagulation cascade proteases (factors IXa, Xa and XIa, thrombin and plasmin) and is part of the so called extrinsic pathway (Huber - Lang et al., 2006 ; Amara et al., 2010). C5a is the most potent anaphylatoxin in the body while C5b initiates formation of the membrane attack complex (MAC) on the activated surface. C5b remains attached to the convertase complex but allows for the binding o f components C6 and C7. Upon attachment of C8 to the C5bC6C7 complex, insertion in to the target membrane is possible due to the exposition of hydrophobic surfaces of the complex. The following attachment of multiple C9 molecules leads to formation of MAC, a pore-like structure in the membrane leading to lysis of the target cell (Janeway et al., 2005). C o n t r o l of th e co m p l e m e n t sy s t e m Considering the lack of active discrimination between self and non-self particles and its potentially destructive effect also towards host cells, the complement system has to be tightly regulated. Complement regulation is achieved by the presence of soluble and membrane-bound inhibitors on self-cells, short half-life of convertases (unless they are stabilized by binding to relevant target surface) and transient presence of active proteases, such as factor D (Turnberg and Botto, 2003). Regulation can occur on different levels of the complement cascade (Fig . 2). For exam ple, C1 complex inhibitor (C1 -INH) works by causing dis sociation of serine protease subunits C1r and C1s, MASP 1 and MASP 2 from the recognition molecules C1q and MBL, respectively, thereby inhibiting the activation of classical and lectin pathways. The main complement regulators are the inhibitors of crucial enzymatic Comple m ent in stroke and neural plasticity   14 complement complexes (C3 - and C5-convertases) acting either by accelerating the decay of these complexes or by promoting the degradation of C3b and C4b fragments. Although these active complexes have an intrinsically short half -life of a few minutes, their dissociation is further promoted by C4-binding protein (C4BP) and factor H for the classical/lectin and alternative pathway, respectively. C4BP fulfills its function by accelerating the decay of the C4bC2a complex. Factor H has affinity to neutral or anionic polysaccharides and siaylic acid present on the surface of mammalian cells. By binding C3b, factor H prevents its deposition on host cells. and enables C3b cleavage by factor I (Janeway et al., 2005 ). C3b and C4b fragments released from the dissociated convertase complexes become inactivated by a step-wise cleavage. Plasma component factor I cleaves them to iC3b and iC4b fragments, but only in the presence of such co-factors as C4BP, factor H, cell- bound membrane co-factor protein (MCP) or complement receptor 1 (CR1). iC3b is further cleaved by factor I to soluble C3c and membrane bound C3dg. Next, plasma proteases cleave off the free C3g fragment leaving C3d on the cell surface (Janeway et al., 2005 ). Membrane-bound inhibitors with decay accelerating activity include the decay acceleration factor (DAF, CD55), membrane co -factor protein (MCP, CD46) and CR1 (CD35). Both DAF and CR1 promote the dissociation of both classical and alternative pathway C3-convertases, while MCP is a co-factor for factor I-mediated cleavage of C3b and C4b but does not dissociate the C3-convertase (Liszewski et al., 2000 ). Also the terminal pathway is tightly controlled to prevent damaging the host cells. MAC formation is controlled mainly by glycosylphosphatidilinositol (GPI) -anchored membrane protein CD59 (protectin), which blocks the accumulation of C9 molecules in the proximity of C5b -C8 complexes. Also soluble factors such as vitronectin (S -protein) and clusterin are involved in the control of undesirable MAC formation (Turnberg and Botto, 2003). Anna Stokowska   15 C o m p l e m e n t re c e p t o r s There are three groups of receptors for complement components: receptors for cleavage products of C3, receptors for C1q molecules, and receptors for the anaphylatoxic peptides. Stimulation of these receptors evokes a variety of biological responses involving for example the removal of immune complexes and apoptoti c cells, stimulation of phagocytosis and complement inhibition. The C3d-binding CR2 receptor bridges innate and adaptive immune responses by increasing the synthesis of antibodies by B lymphocytes. Moreover complement receptors 1-4 (CR1-CR4) with affinity for C3- derived opsonins are crucial for phagocytosis and thus contribute to efficient antigen presentation (reviewed in Carroll, 2004). Receptor for the collagenous tail of C1q (cC1qR, calreticulin) is expressed on phago cytes and together with CR1 mediates clearance of C1q -coated pathogens, apoptotic cells and immune complexes (Peterson et al., 1997). There are three more C1q r eceptors (gC1qR, C1qRp and CD91), which are believed to stimulate phagocytosis and interact with integrins to promote cell adhesion (Nayak et al., 2012). Three 7-transmembrane domain G -coupled receptors, C3aR, C5aR and C5L2 have differential affinity for the anaphylatoxins C3a, C4a, C5a and their derivatives. C3aR binds C3a but not C3adesArg, while C5aR binds C5a with high- and C5adesArg with somewhat lower affinity (Klos et al., 2009 ). The third anaphylatoxin, C4a seems to be structurally related to C3a but evokes markedly weaker responses and its functions seem to be species-specific. C4a is a potent agonist of guinea pig but not human C3aR and no other definite human receptor for C4a has been identified (Lienenklaus et al., 1998 ). The G -protein-coupled receptors (GPCRs) that detect C3a or C5a are linked with several well-defined intracellular signaling pathways (Fig. 3). Upon C3a binding to C3aR, intracellu lar signal transduction is promoted via heterotrimeric G -proteins that mobilize calcium fluxes from the extracellular compartment. Downstream signaling events include activation of protein kinase C by phospholipase C, and mitogen activated protein kinases (MAPKs) Erk1 and Erk2. In some cell types, signaling pathways require activation of phosphatidylinositol - Comple m ent in stroke and neural plasticity   14 complement complexes (C3 - and C5-convertases) acting either by accelerating the decay of these complexes or by promoting the degradation of C3b and C4b fragments. Although these active complexes have an intrinsically short half -life of a few minutes, their dissociation is further promoted by C4-binding protein (C4BP) and factor H for the classical/lectin and alternative pathway, respectively. C4BP fulfills its function by accelerating the decay of the C4bC2a complex. Factor H has affinity to neutr al or anionic polysaccharides and siaylic acid present on the surface of mammalian cells. By binding C3b, factor H prevents its deposition on host cells. and enables C3b cleavage by factor I (Janeway et al., 2005 ). C3b and C4b fragments released from the dissociated convertase complexes become inactivated by a step-wise cleavage. Plasma component factor I cleaves them to iC3b and iC4b fragments, but only in the presence of such co-factors as C4BP, factor H, cell -bound membrane co-factor protein (MCP) or complement receptor 1 (CR1). iC3b is further cleaved by factor I to soluble C3c and membrane bound C3dg. Next, plasma proteases cleave off the free C3g fragment leaving C3d on the cell surface (Janeway et al., 2005 ). Membrane-bound inhibitors with decay accelerating activity include the decay acceleration factor (DAF, CD55), membrane co -factor protein (MCP, CD46) and CR1 (CD35). Both DAF and CR1 promote the dissociation of both classical and alternative pathway C3-convertases, while MCP is a co-factor for factor I-mediated cleavage of C3b and C4b but does not dissociate the C3-convertase (Liszewski et al., 2000 ). Also the terminal pathway is tightly controlled to prevent damaging the host cells. MAC formation is controlled mainly by glycosylphosphatidilinositol (GPI) -anchored membrane protein CD59 (protectin), which blocks the accumulation of C9 molecules in the proximity of C5b -C8 complexes. Also soluble factors such as vitronectin (S -protein) and clusterin are involved in the control of undesirable MAC formation (Turnberg and Botto, 2003). Anna Stokowska   15 C o m p l e m e n t re c e p t o r s There are three groups of receptors for complement components: receptors for cleavage products of C3, receptors for C1q molecules, and receptors for the anaphylatoxic peptides. Stimulation of these receptors evokes a variety of biological responses involving for example the removal of immune complexes and apoptoti c cells, stimulation of phagocytosis and complement inhibition. The C3d-binding CR2 receptor bridges innate and adaptive immune responses by increasing the synthesis of antibodies by B lymphocytes. Moreover complement receptors 1-4 (CR1-CR4) with affinity for C3- derived opsonins are crucial for phagocytosis and thus contribute to efficient antigen presentation (reviewed in Carroll, 2004). Receptor for the collagenous tail of C1q (cC1qR, calreticulin) is expressed on phagocytes and together with CR1 mediates clearance of C1q -coated pathogens, apoptotic cells and immune complexes (Peterson et al., 1997). There are three more C1q receptors (gC1qR, C1qRp and CD91), which are believed to stimulate phagocytosis and interact with integrins to promote cell adhesion (Nayak et al., 2012). Three 7-transmembrane domain G -coupled receptors, C3aR, C5aR and C5L2 have differential affinity for the anaphylatoxins C3a, C4a, C5a and their derivatives. C3aR binds C3a but not C3adesArg, while C5aR binds C5a with high- and C5adesArg with somewhat lower affinity (Klos et al., 2009 ). The third anaphylatoxin, C4a seems to be structurally related to C3a but evokes markedly weaker responses and its functions seem to be species-specific. C4a is a potent agonist of guinea pig but not human C3aR and no other definite human receptor for C4a has been identified (Lienenklaus et al., 1998 ). The G- protein-coupled receptors (GPCRs) that detect C3a or C5a are linked with several well-defined intracellular signaling pathways (Figure 3). Upon C3a binding to C3aR, intracellular signal transduction is promoted via heterotrimeric G -proteins that mobilize calcium fluxes from the extracellular compartment. Downstream signaling events include activation of protein kinase C by phospholipase C, and mitogen activated protein kinases (MAPKs) Erk1 and Erk2. In some cell types, signaling pathways require activation of phosphatidylinositol - Comple m ent in stroke and neural plasticity   16 bisphosphate-3-kinase (PI3K γ) and subsequent phosphorylation of Akt as well as Erk1 and Erk2 ( Klos et al., 2009 ). C5a binding to C5aR causes calcium fluxes from intracellular stores as well as the extracellular compartment, and receptor internalization via clathrin coated pits. C5aR activation leads to downstream activation of several components of different signaling pathways such as PI3K γ kinase, phospholipase C and D, Raf-1/B -Raf mediated activation of MEK -1, and Wiskott -Aldrich syndrome protein (WASP). WASP is a multifunctional protein that regulates actin dynamics and therefore may play an important role in the C5a dependent chemotaxis (Klos et al., 2009 ). Fi gure 3. I n t r a c e l l u l a r sig n a l i n g of th e an a p h y l a t o x i n re c e p t o r s . Schematic structure of the receptors is given together with an overview of the major downstream signaling pathways and their cellular effects. In contrast to C3aR and C5aR, C5L2 does not bind G proteins but acts via binding β -arrestin and interaction with C5aR (see text for details). G α, β, γ – subunits of G protein; PLC/PLD - phospholipase C/D; DAG – diacylglycerol; PIP 3 - phosphatidylinositol triphosphate; IP 3 – inositol triphosphate; PKC - protein kinase C; GEFs – guanine exchange factors; protein; JAK – Janus kinase; STAT - Signal Transducer and Activator of Transcription; [Ca 2+ ] i – intracellular calcium storage. Anna Stokowska   17 Activation of C5aR leads to chemoattraction and activation of neutrophils, basophils, eosinophils and macrophages as well as endothelial cells activation and smooth muscle contraction. Upon activation with C5a, eosinophils release major basic protein, monocytes/macrophages produce inflammatory cytokines and mast cells release the content of their granules including histamine, which in turn causes vasodilation and increases vascular permeability. C3aR stimulation also attracts eosinophils and mast cells but not neutrophils and macrophages, and in addition promotes the secretion of lysosomal enzymes from leukocytes and histamine from mast cells, and smooth muscle contraction (Klos et al., 2009 ). The function of the third and most recently discovered anaphylatoxin receptor, C5L2 (also known as C5a2R or GPR77) is much less defined. This molecule can be foun d on a similar repertoire of cell types as the canonical C5aR and the expression of these two receptors predominantly occurs in tandem, suggesting a functional link between them (Li et al., 2013). Unlike C5aR and the majority of GPCRs, C5L 2 is expressed predominantly intracellularly. Interestingly, it is capable of binding C5a, C5adesArg (with higher affinity than C5aR does) and according to some studies, it also interacts with C3a and C3adesArg (Cain and Monk, 2002; Cui et al., 2009). Due to deviations from the conserved sequence and structure in regions required for G -protein coupling, C5L2 has been pr oposed to be a non-signaling decoy receptor for C5aR, although this hypothesis has been challenged by subsequent studies pointing to a much more complex role of this molecule. Although the above described functions support the traditional view of anaphylatoxin receptors as pro-inflammatory immune regulators, in recent years, a large body of evidence has emerged that points to their role in limiting the inflammatory responses in a context -dependent manner. Specifically, the function of C3aR seems to be more selective and anti-inflammatory than C5aR as C3a has been shown to reduce LPS -induced pro- inflammatory cytokine release from peripheral blood mononuclear cells and lymphocytes (Takabayashi et al., 1998; Fischer et al., 1999 ). Studies of C3aR-deficient mice in endotoxic shock and gut ischemia/reperfusion injury models support the in vitro findings (Kildsgaard et al., 2000 ; Boos et al., 2005; Wu et al., 2013 ). Fur thermore, the biological role of C5L2 appears to be to decrease the pro -inflammatory response conveyed by C5aR, at least in studies of non-disease models. However, based on the observations of Comple m ent in stroke and neural plasticity   16 bisphosphate-3-kinase (PI3K γ) and subsequent phosphorylation of Akt as well as Erk1 and Erk2 (Klos et al., 2009 ). C5a binding to C5aR causes calcium fluxes from intracellular stores as well as the extracellular compartment, and receptor internalization via clathrin coated pits. C5aR activation leads to downstream activation of several components of different signaling pathways such as PI3K γ kinase, phospholipase C and D, Raf-1/B -Raf mediated activation of MEK -1, and Wiskott -Aldrich syndrome protein (WASP). WASP is a multifunctional protein that regulates actin dynamics and therefore may play an important role in the C5a dependent chemotaxis (Klos et al., 2009 ). Fi gure 3. I n t r a c e l l u l a r sig n a l i n g of th e an a p h y l a t o x i n re c e p t o r s . Schematic structure of the receptors is given together with an overview of the major downstream signaling pathways and their cellular effects. In contrast to C3aR and C5aR, C5L2 does not bind G proteins but acts via binding β-arrestin and interaction with C5aR (see text for details). G α, β, γ – subunits of G protein; PLC/PLD - phospholipase C/D; DAG – diacylglycerol; PIP 3 - phosphatidylinositol triphosphate; IP 3 – inositol triphosphate; PKC - protein kinase C; GEFs – guanine exchange factors; protein; JAK – Janus kinase; STAT - Signal Transducer and Activator of Transcription; [Ca 2+ ] i – intracellular calcium storage. Anna Stokowska   17 Activation of C5aR leads to chemoattraction and activation of neutrophils, basophils, eosinophils and macrophages as well as endothelial cells activation and smooth muscle contraction. Upon activation with C5a, eosinophils release major basic protein, monocytes/macrophages produce inflammatory cytokines and mast cells release the content of their granules including histamine, which in turn causes vasodilation and increases vascular permeability. C3aR stimulation also attracts eosinophils and mast cells but not neutrophils and macrophages, and in addition promotes the secretion of lysosomal enzymes from leukocytes and histamine from mast cells, and smooth muscle contraction (Klos et al., 2009 ). The function of the third and most recently discovered anaphylatoxin receptor, C5L2 (also known as C5a2R or GPR77) is much less defined. This molecule can be found on a similar repertoire of cell types as the canonical C5aR and the expression of these two receptors predominantly occurs in tandem, suggesting a functional link between them (Li et al., 2013). Unlike C5aR and the majority of GPCRs, C5L2 is expressed predominantly intracellularly. Interestingly, it is capable of binding C5a, C5adesArg (with higher affinity than C5aR does) and according to some studies, it also interacts with C3a and C3adesArg (Cain and Monk, 2002; Cui et al., 2009). Due to deviations from the conserved sequence and structure in regions required for G -protein coupling, C5L2 has been proposed to be a non-signaling decoy receptor for C5aR, although this hypothesis has been challenged by subsequent studies pointing to a much more complex role of this molecule. Although the above described functions support the traditional view of anaphylatoxin receptors as pro-inflammatory immune regulators, in recent years, a large body of evidence has emerged that points to their role in limiting the inflammatory responses in a context -dependent manner. Specifically, the function of C3aR seems to be more selective and anti-inflammatory than C5aR as C3a has been shown to reduce LPS -induced pro- inflammatory cytokine release from peripheral blood mononuclear cells and lymphocytes (Takabayashi et al., 1998; Fischer et al., 1999 ). Studies of C3aR-deficient mice in endotoxic shock and gut ischemia/reperfusion injury models support the in vitro findings (Kildsgaard et al., 2000 ; Boos et al., 2005; Wu et al., 2013 ). Fur thermore, the biological role of C5L2 appears to be to decrease the pro -inflammatory response conveyed by C5aR, at least in studies of non-disease models. However, based on the observations of Comple m ent in stroke and neural plasticity   18 C5L2 function in septic animals, an alternative pro- inflammatory role for this receptor has been suggested, rendering it the most enigmatic complement receptor (Li et al., 2013). Also, owing to their expression on a wide variety of cell types, several non - immunological functions have been assigned to the anaphylatoxin receptors. N o n - i m m u n o l o g i c a l fu n c t i o n s of th e co m p l e m e n t sy s t e m In the last two decades, novel functions of the complement system have been identified which suggest it has roles far beyond immunity. Among them are tissue regeneration, stem cell chemoattraction and regulation of their migration well as various non-immune functions in CNS. Tissue regeneration First evidence for a non -immune function of the complement system came from a study that found C3 mRNA in blastema cell layer of regenerating limbs of salamander (Del Rio-Tsonis et al., 1998). Next, both C3 and C5 were detected in regenerating but not intact limbs and lens of newt (Kimura et al., 2003 ). Soon it became apparent that complement involvement in tissue regeneration is not restricted to lower vertebrates, known for their remarkable regeneration capabilities. Mice deficient in C3 or C5 showed reduced liver regeneration in response to toxic liver injury or hepatectomy (Mastellos et al., 2001; Strey et al., 2003; Daveau et al., 2004; Markiewski et al., 2004). The liver regeneration in these studies has been shown to be dependent on C3a and C5a signaling through their canonical receptors (Mastellos et al., 2001; Markiewski et al., 2004), by priming and stimulation of hepatocyte proliferation. Further, C3 -deficient mice showed delayed removal of damaged and apoptotic liver cells following toxic liver injury. Regulation of stem cell translo cation Human mesenchymal stem cells (MSC), which are involved in the repair of various tissues, have also been found to express complement peptide receptors and to be chemoattracted by C3a and C5a (Schraufstatter et al., 2009). Furthermore, C5aR has been shown to control osteoblast migration during fracture healing and efficient osteoclast Anna Stokowska   19 differentiation required local complement activation (Ignatius et al., 2011b; Ignatius et al., 2011a). The complement cascade has in recent years also emerged as an important and up to now underappreciated modulator of trafficking of hematopoietic stem/progenitor cells (HSPCs) (reviewed in Ratajczak et al., 2008 ). It has been reported that the complement becomes activated in bone marrow during mobilization of hematopoietic stem cells to the periphery. C3a increases the responsiveness of HSPCs to stromal derived factor -1 (SDF - 1) gradient, which is the primary factor regulating trafficking of these cells between marrow stroma and peripheral blood (Reca et al., 2003). Also C5a and soluble MACs regulate the homing of stem cells back to bone marrow (Lee et al., 2009 ; Kim et al., 2011). C o m p l e m e n t in th e ce n t r a l ne r v o u s sy s t e m For decades, the brain has been considered an immune-privileged organ owing to the existence of the BBB, formed by endothelial cells, pericytes and astrocytes. This tight barrier prevents plasma proteins, including complement components, from entering the intact brain. However, complement proteins are produced locally in the CNS and complement is considered to provide local defense mechanisms against invading pathogens (Morgan and Gasque, 1996 ). Both murine and human neurons and glia collectively produce nearly all of the complement proteins in vitro , especially upon stimulation with pro-inflammatory cytokines (Barnum, 1995; Thomas et al., 2000). Complement in unchallenged CNS The presence of C3aR, C5aR or C5L2 on glia was somewhat expected owing to the monocytic origin of microglia and the fact that astrocytes also are the immunocompetent cells that participate in antigen presentation, and can produce a number of cytokines, chemokines and complement components (Weber et al., 1994 ; Morgan and Gasque, 1996; Gavrilyuk et al., 2005 ). However, the discovery of these receptors on neuron s was surprising and led to speculations that complement may play a role in CNS homeostasis and modulation of neuronal functions. (Davoust et al., 1999; O'Barr et al., 2001 ; Gavrilyuk et al., 2005 ). Hippocampal and cortical neurons express C3aR and C5aR most Comple m ent in stroke and neural plasticity   18 C5L2 function in septic animals, an alternative pro -inflammatory role for this receptor has been suggested, rendering it the most enigmatic complement receptor (Li et al., 2013). Also, owing to their expression on a wide variety of cell types, several non - immunological functions have been assigned to the anaphylatoxin receptors. N o n - i m m u n o l o g i c a l fu n c t i o n s of th e co m p l e m e n t sy s t e m In the last two decades, novel functions of the complement system have been identified which suggest it has roles far beyond immunity. Among them are tissue regeneration, stem cell chemoattraction and regulation of their migration well as various non-immune functions in CNS. Tissue regeneration First evidence for a non -immune function of the complement system came from a study that found C3 mRNA in blastema cell layer of regenerating limbs of salamander (Del Rio-Tsonis et al., 1998). Next, both C3 and C5 were detected in regenerating but not intact limbs and lens of newt (Kimura et al., 2003 ). Soon it became apparent that complement involvement in tissue regeneration is not restricted to lower vertebrates, known for their remarkable regeneration capabilities. Mice deficient in C3 or C5 showed reduced liver regeneration in response to toxic liver injury or hepatectomy (Mastellos et al., 2001; Strey et al., 2003; Daveau et al., 2004; Markiewski et al., 2004). The liver regeneration in these studies has been shown to be dependent on C3a and C5a signaling through their canonical receptors (Mastellos et al., 2001; Markiewski et al., 2004), by priming and stimulation of hepatocyte proliferation. Further, C3 -deficient mice showed delayed removal of damaged and apoptotic liver cells following toxic liver injury. Regulation of stem cell translo cation Human mesenchymal stem cells (MSC), which are involved in the repair of various tissues, have also been found to express complement peptide receptors and to be chemoattracted by C3a and C5a (Schraufstatter et al., 2009). Furthermore, C5aR has been shown to control osteoblast migration during fracture healing and efficient osteoclast Anna Stokowska   19 differentiation required local complement activation (Ignatius et al., 2011b; Ignatius et al., 2011a). The complement cascade has in recent years also emerged as an important and up to now underappreciated modulator of trafficking of hematopoietic stem/progenitor cells (HSPCs) (reviewed in Ratajczak et al., 2008 ). It has been reported that the complement becomes activated in bone marrow during mobilization of hematopoietic stem cells to the periphery. C3a increases the responsiveness of HSPCs to stromal derived factor -1 (SDF - 1) gradient, which is the primary factor regulating trafficking of these cells between marrow stroma and peripheral blood (Reca et al., 2003). Also C5a and soluble MACs regulate the homing of stem cells back to bone marrow (Lee et al., 2009 ; Kim et al., 2011). C o m p l e m e n t in th e ce n t r a l ne r v o u s sy s t e m For decades, the brain has been considered an immune-privileged organ owing to the existence of the BBB, formed by endothelial cells, pericytes and astrocytes. This tight barrier prevents plasma proteins, including complement components, from entering the intact brain. However, complement proteins are produced locally in the CNS and complement is considered to provide local defense mechanisms against invading pathogens (Morgan and Gasque, 1996 ). Both murine and human neurons and glia collectively produce nearly all of the complement proteins in vitro , especially upon stimulation with pro-inflammatory cytokines (Barnum, 1995; Thomas et al., 2000). Complement in unchallenged CNS The presence of C3aR, C5aR or C5L2 on glia was somewhat expected owing to the monocytic origin of microglia and the fact that astrocytes also are the immunocompetent cells that participate in antigen presentation, and can produce a number of cytokines, chemokines and complement components (Weber et al., 1994 ; Morgan and Gasque, 1996; Gavrilyuk et al., 2005) . However, the discovery of these receptors on neuron s was surprising and led to speculations that complement may play a role in CNS homeostasis and modulation of neuronal functions. (Davoust et al., 1999; O'Barr et al., 2001 ; Gavrilyuk et al., 2005 ). Hippocampal and cortical neurons express C3aR and C5aR most Comple m ent in stroke and neural plasticity   20 abundantly of all cell types in the unchallenged CNS. Additionally, spinal cord motor neurons express relatively high levels of C5aR and C5L2 ( Nataf et al., 1998; Woodruff et al., 2008). In the recent years, C3aR and C5aR have been found to be expressed on neural stem/progenitor cells (NSPCs) in murine neurogenic brain regions (Rahpeymai et al., 2006). In line with the surprisingly widespread role of anaphylatoxic peptides and their receptors in regulating the migration of stem cells, C3a has been identified as an organizer of the migration of neural crest cells and (together with C5a) of cerebellar neuronal precursors during development (Benard et al., 2008). Furthermore, C5a has been found to be mitogenic for human undifferentiated neuroblastoma cells and neuroprotective for mature neurons (O'Barr et al., 2001 ). A group of proteins related to C1q, namely cerebellins (Cbln) and C1q -like molecules (C1ql) have been found to be expressed in the cerebellum, as well as other regions of the developing and mature brain (Yuzaki, 2008 ; Bolliger et al., 2011). Furthermore, half of the more than 50 genes encoding putative complement regulators predicted in the mouse genome are expressed in the CNS . These molecules are predicted to be involved in synapse organization and plasticity (Gendrel et al., 2009 ; Yuzaki, 2010). Complement during ischemic brain injury The diverse roles played by the complement system in acute brain disorders are not fully elucidated, however a growing body of evidence suggests a role for the complement system in secondary brain damage (Stahel et al., 1998). During stroke, BBB integrity is disrupted allowing for influx of plasma proteins, including complement components. Investigation of brain tissue of patients with ischemic stroke revealed deposition of C1q, C3c and C4d in all ischemic lesions, suggesting activation of the classical pathway. In necrotic zones of the brains from the same patients, C9, C -reactive protein and IgM were found (Pedersen et al., 2009). The possibility of harmful uncontrolled complement activation following ischemic insults was underlined by the findings of virtual absence of CD59 and CD55 in ischemic lesions. Moreover, systemic activation of the complement cascade is evident in stroke patients as judged by elevated plasma levels of C3, C3a, C4d and soluble terminal complexes C5b -9 (Pedersen et al., 2004; Tamam et al., 2005; Mocco Anna Stokowska   21 et al., 2006a; Szeplaki et al., 2009 ). Consistent with the above, a number of complement inhibition strategies were found to be beneficial in terms of limiting the infarct volume and improving behavioral outcome following experimental cerebral ischemia with reperfusion (Huang et al., 1999 ; De Simoni et al., 2003; Costa et al., 2006; Mocco et al., 2006b). However, in the animal model of permanent ischemia, g enetic C3 deficiency led to increased infarct volume suggesting potentially beneficial effects of complement activation in the course of stroke (Rahpeymai et al., 2006). Moreover, both C3a and C5a were found to be neuroprotective against excitotoxicity, although through two distinct mechanisms (Mukherjee and Pasinetti, 2001; van Beek et al., 2001). This function of C3a appears to be mediated by astrocytes, whereas C5a acts through the regulation of the caspase cascade and glutamate receptor subunit 2 (GluR2) expre ssion in neurons (Mukherjee et al., 2008 ). These two protective mechanisms could be relevant for the ischemic brain injury, glia and neurons highly upregulate their C3aR and C5aR expression in response to permanent focal cerebral ischemia (Van Beek et al., 2000 ). In support of that, a recent report revealed marked neuroprotective properties both of C3a overexpression in the brain and of administration of exogenous C3a peptide following neonatal ischemic brain injury (Järlestedt et al., 2013 ) Taken together, these findings indicate that the role of complement activation in the ischemic brain is complex and can be viewed as a double -edge sword (similar to inflammation in general) which is detrimental or beneficial depending on the context. Comple m ent in stroke and neural plasticity   20 abundantly of all cell types in the unchallenged CNS. Additionally, spinal cord motor neurons express relatively high levels of C5aR and C5L2 (Nataf et al., 1998; Woodruff et al., 2008). In the recent years, C3aR and C5aR have been found to be expressed on neural stem/progenitor cells (NSPCs) in murine neurogenic brain regions (Rahpeymai et al., 2006). In line with the surprisingly widespread role of anaphylatoxic peptides and their receptors in regulating the migration of stem cells, C3a has been identified as an organizer of the migration of neural crest cells and (together with C5a) of cerebellar neuronal precursors during development (Benard et al., 2008). Furthermore, C5a has been found to be mitogenic for human undifferentiated neuroblastoma cells and neuroprotective for mature neurons (O'Barr et al., 2001 ). A group of proteins related to C1q, namely cerebellins (Cbln) and C1q -like molecules (C1ql) have been found to be expressed in the cerebellum, as well as other regions of the developing and mature brain (Yuzaki, 2008 ; Bolliger et al., 2011). Furthermore, half of the more than 50 genes encoding putative complement regulators predicted in the mouse genome are expressed in the CNS . These molecules are predicted to be involved in synapse organization and plasticity (Gendrel et al., 2009 ; Yuzaki, 2010). Complement during ischemic brain injury The diverse roles played by the complement system in acute brain disorders are not fully elucidated, however a growing body of evidence suggests a role for the complement system in secondary brain damage (Stahel et al., 1998). During stroke, BBB integrity is disrupted allowing for influx of plasma proteins, including complement components. Investigation of brain tissue of patients with ischemic stroke revealed deposition of C1q, C3c and C4d in all ischemic lesions, suggesting activation of the classical pathway. In necrotic zones of the brains from the same patients, C9, C -reactive protein and IgM were found (Pedersen et al., 2009). The possibility of harmful uncontrolled complement activation following ischemic insults was underlined by the findings of virtual absence of CD59 and CD55 in ischemic lesions. Moreover, systemic activation of the complement cascade is evident in stroke patients as judged by elevated plasma levels of C3, C3a, C4d and soluble terminal complexes C5b -9 (Pedersen et al., 2004; Tamam et al., 2005; Mocco Anna Stokowska   21 et al., 2006a; Szeplaki et al., 2009 ). Consistent with the above, a number of complement inhibition strategies were found to be beneficial in terms of limiting the infarct volume and improving behavioral outcome following experimental cerebral ischemia with reperfusion (Huang et al., 1999; De Simoni et al., 2003; Costa et al., 2006; Mocco et al., 2006b). However, in the animal model of permanent ischemia, genetic C3 defici ency led to increased infarct volume suggesting potentially beneficial effects of complement activation in the course of stroke (Rahpeymai et al., 2006). Moreover, both C3a and C5a were found to be neuroprotective against excitotoxicity, although through two distinct mechanisms (Mukherjee and Pasinetti, 2001; van Beek et al., 2001). This function of C3a appears to be mediated by astrocytes, whereas C5a acts through the regulation of the caspase cascade and glutamate receptor subunit 2 (GluR2) expre ssion in neurons (Mukherjee et al., 2008 ). These two protective mechanisms could be relevant for the ischemic brain injury, glia and neurons highly upregulate their C3aR and C5aR expression in response to permanent focal cerebral ischemia (Van Beek et al., 2000 ). In support of that, a recent report revealed marked neuroprotective properties both of C3a overexpression in the brain and of administration of exogenous C3a peptide following neonatal ischemic brain injury (Järlestedt et al., 2013 ). Taken together, these findings indicate that the role of complement activation in the ischemic brain is complex and can be viewed as a double -edge sword (similar to inflammation in general) which is detrimental or beneficial depending on the context. Comple m ent in stroke and neural plasticity   22 N E U R A L P L A S T I C I T Y It was long thought that the brain only changed during development and that the adult brain was fixed in its functional organization, with specific areas allocated to and hard-wired for specific functions. Today, there is no doubt that the brain is reorganizing itself constantly, for example every time new knowledge is stored or a new motor skill learned. This ability of the brain to change and adapt due to experience is called “neural plasticity”. The te rm was introduced by the Polish neuroscientist Jerzy Konorski in 1948 with the formal hypothesis embodying these ideas advanced shortly thereafter by the Canadian neuroscientist Donald Hebb (Konorski, 1948; He bb, 1949). Neural plasticity refers to a variety of structural and functional changes in neural pathways and synapses (connections between neurons, through which information is transmitted from one neuron to another), which occur in response to changes in behavior, environment and neural processes, as well as changes resulting from injury. The study of mechanisms underlying brain plasticity during development and learning provides a good basis for the understanding of functional reorganization of the brain after injury such as stroke. Therefore, physiology of plastic changes is most often studied using model systems such as organotypic slice cultures of hippocampus, a brain structure indispensable for memory formation and learning. M e c h a n i s m s of pla s t i c i t y Plastic changes of the brain can be either short-lived or long-lasting and depend on several mechanisms, which constitute the basis for functional and structural reorganization associated with both learning and recovery after CNS injury. Functional synap tic plasticity Neurons communicate with each other primarily through fast chemical synapses. At such synapses, an action potential generated near the cell body of the presynaptic cell propagates down the axon where it opens voltage -gated Ca2+ channels. Ca2+ ions entering nerve terminals trigger the rapid release of vesicles containing a neurotransmitter, which is ultimately detected by receptors on the postsynaptic cell. Virtually all types of synapses are regulated by a variety of short-lived or long-lasting processes. Anna Stokowska   23 Short-term synaptic plasticity Short-term synaptic plasticity is the modulation of synaptic strength following repetitive synaptic activity that occurs within milliseconds (ms) to a few minutes (reviewed in Zucker and Regehr, 2002 ). Synaptic strength, or in other words efficacy, is determined by the number of functional neurotransmitter release sites (n), the release probability of these sites (p), and the quantal size i.e . the magnitude of post-synaptic response to the release of single presynaptic vesicle (q). Therefore, the synaptic efficacy equals n*p*q and it is represented by the mean amplitude of the evoked synaptic response (Korn and Faber, 1991 ). The change of synaptic efficacy follows Hebbian rules (discussed in the next section) and is one of the fundamental principles of cortical plasticity (Hebb, 1949 ). Synaptic efficacy and the threshold for activation can be influenced by the temporal structure and synchronization of impulse arrival and neuronal firing. Short-term synaptic plasticity has a pre-synaptic nature and may involve both enhancement and decrease of synaptic transmission. Synaptic enhancement lasting ~100 ms is referred to as facilitation and it can be observed with pairs of brief stimuli. Due to residual Ca2+ in the pre-synaptic terminal following the first discharge, the release probability of the second discharge increases up to several times the size of the first one. The reverse phenomenon is called depression. In synapses with a high initial release probability the large initial discharge causes a depletion of neurotransmitter vesicles available for release from the pre-synaptic terminal, rendering the second post-synaptic response smaller. A third phenomenon, short-term potentiation, occurs with tetanic stimulation which is a series of high-frequency stimuli, and results in postsynaptic potentials of increasing size. Repeated high -frequency stimulation causes calcium ions to accumulate in the pre-synaptic terminal which increases the release probability (p) and/or, via fusion of vesicles, the quantal size (q) (Figure 4) . The degree of enhancement of each potential is proportional to the number of preceding high-frequency stimuli. Likewise, when stimulation returns to baseline frequency the time taken for the post - synaptic potential to return to the original magnitude is proportional to the number of rapid stimuli in the tetanic phase and can be up to a few minutes in length. (Zucker and Regehr, 2002). Comple m ent in stroke and neural plasticity   22 N E U R A L P L A S T I C I T Y It was long thought that the brain only changed during development and that the adult brain was fixed in its functional organization, with specific areas allocated to and hard-wired for specific functions. Today, there is no doubt that the brain is reorganizing itself constantly, for example every time new knowledge is stored or a new motor skill learned. This ability of the brain to change and adapt due to experience is called “neural plasticity”. The te rm was introduced by the Polish neuroscientist Jerzy Konorski in 1948 with the formal hypothesis embodying these ideas advanced shortly thereafter by the Canadian neuroscientist Donald Hebb (Konorski, 1948 ; He bb, 1949). Neural plasticity refers to a variety of structural and functional changes in neural pathways and synapses (connections between neurons, through which information is transmitted from one neuron to another), which occur in response to changes in behavior, environment and neural processes, as well as changes resulting from injury. The study of mechanisms underlying brain plasticity during development and learning provides a good basis for the understanding of functional reorganization of the brai n after injury such as stroke. Therefore, physiology of plastic changes is most often studied using model systems such as organotypic slice cultures of hippocampus, a brain structure indispensable for memory formation and learning. M e c h a n i s m s of pla s t i c i t y Plastic changes of the brain can be either short-lived or long-lasting and depend on several mechanisms, which constitute the basis for functional and structural reorganization associated with both learning and recovery after CNS injury. Functional synap tic plasticity Neurons communicate with each other primarily through fast chemical synapses. At such synapses, an action potential generated near the cell body of the presynaptic cell propagates down the axon where it opens voltage -gated Ca2+ channels. Ca2+ ions entering nerve terminals trigger the rapid release of vesicles containing a neurotransmitter, which is ultimately detected by receptors on the postsynaptic cell. Virtually all types of synapses are regulated by a variety of short-lived or long-lasting processes. Anna Stokowska   23 Short-term synaptic plasticity Short-term synaptic plasticity is the modulation of synaptic strength following repetitive synaptic activity that occurs within milliseconds (ms) to a few minutes (reviewed in Zucker and Regehr, 2002 ). Synaptic strength, or in other words efficacy, is determined by the number of functional neurotransmitter release sites (n), the release probability of these sites (p), and the quantal size i.e . the magnitude of post-synaptic response to the release of single presynaptic vesicle (q). Therefore, the synaptic efficacy equals n*p*q and it is represented by the mean amplitude of the evoked synaptic response (Korn and Faber, 1991) . The change of synaptic efficacy follows Hebbian rules (discussed in the next section) and is one of the fundamental principles of cortical plasticity (Hebb, 1949 ). Synaptic efficacy and the threshold for activation can be influenced by the temporal structure and synchronization of impulse arrival and neuronal firing. Short-term synaptic plasticity has a pre-synaptic nature and may involve both enhancement and decrease of synaptic transmission. Synaptic enhancement lasting ~100 ms is referred to as facilitation and it can be observed with pairs of brief stimuli. Due to residual Ca2+ in the pre-synaptic terminal following the first discharge, the release probability of the second discharge increases up to several times the size of the first one. The reverse phenomenon is called depression. In synapses with a high initial release probability the large initial discharge causes a depletion of neurotransmitter vesicles available for release from the pre-synaptic terminal, rendering the second post-synaptic response smaller. A third phenomenon, short-term potentiation, occurs with tetanic stimulation which is a series of high-frequency stimuli, and results in postsynaptic potentials of increasing size. Repeated high -frequency stimulation causes calcium ions to accumulate in the pre-synaptic terminal which increases the release probability (p) and/or, via fusion of vesicles, the quantal size (q) (Figure 4) . The degree of enhancement of each potential is proportional to the number of preceding high-frequency stimuli. Likewise, when stimulation returns to baseline frequency the time taken for the post - synaptic potential to return to the original magnitude is proportional to the number of rapid stimuli in the tetanic phase and can be up to a few minutes in length (Zucker and Regehr, 2002). Comple m ent in stroke and neural plasticity   24 Long term synaptic plasticity The second and extensively studied mechanism of synaptic plasticity is long -term potentiation (LTP). This process is assumed to be the basis for the acquisition of knowledge and memory formation. It relies on specific patterns of synaptic activity which lead to the strengthening of connections between the synapses of neighboring neurons which fire together, thus providing physiological substrate for a lasting association. This concept was proposed in 1949 by Donald Hebb and was confirmed experimentally twenty years later using hippocampal circuits as a model system (Bliss and Lomo, 1973) . LTP requires activation of NMDAR, which acts as a coincidence detector, and an increased intracellular calcium concentration (Wigstrom and Gustafsson, 1986). Figure 4. S h o r t - t e r m sy n a p t i c pla s t i c i t y an d th e re l a t e d pr e s y n a p t i c m e c h a n i s m s . Postsynaptic facilitation in response to the second stimulus occurs due to residual calcium ions in the presynaptic terminal (gray shading) that increase neurotransmitter release probability. Postsynaptic depression results from depletion of neurotransmitter-containing vesicles in the active zone following previous large discharge. Repeated high frequency stimulation (tetanic stimulus) causes extended potentiation of the postsynaptic response due to accumulation of calcium ions in the presynaptic terminal (gray shading) leading to increased release probability and/or increased quantal size due to fusion of vesicles. Anna Stokowska   25 NMDAR is a voltage- and ligand-gated ion channel, which opens only when the glutamate release from a presynaptic vesicle to the synaptic cleft coincides with postsynaptic membrane depolarization, i.e. when both pre- and postsynaptic neurons are activated (Figure 5). Post -synaptic depolarization is evoked by initial activation of glutamate-gated α-amino-3-hydroxyl -5-methyl-4-izoxazolepropionic receptors (AMPARs), an event that is required for expelling the Mg 2+ ion that blocks the NMDAR ion channel (Nicoll et al., 1988). Opened NMDAR allows for the influx of calcium, an intracellular second messenger that is bound by calcium-dependent kinases and induces a cascade of intracellular signaling. This activation initiates several pathways which lead to the induction of transcription factors and thus protein synthesis. The latter is the basis for lasting changes in properties of activated neurons (Kauer et al., 1988 ). These effector events might differ between different types of synapses but in the model hippocampal CA1-CA3 synapse, LTP causes potentiation of AMPAR conductance as well as recruitment of additional AMPARs from the peri-synaptic pool to the postsynaptic membrane (Malinow, 2003). As mentioned above, LTP has been described originally in the hippocampus but it is observed in many brain structures such as cerebral cortex, cerebellum and amygdala (Clugnet and LeDoux, 1990 ; Hess and Donoghue, 1994 ). Figure 5. L T P in hip p o c a m p a l cir c u i t r y ca u s e d by sy n c h r o n i z e d pr e - an d po s t s y n a p t i c ac t i v i t y . Single stimulus applied to Schaffer collateral synaptic input evokes normal excitatory postsynaptic potentials ( EPSPs ) in the postsynaptic CA1 neuron. However, if the CA1 neuronal membrane is briefly depolarized (by applying current pulses through the recording electrode) in conjunction with Schaffe r collateral stimulation, a persistent increase in the EPSP amplitude occurs (synapse undergoes LTP). Modified from (Purves et al., 2008) Comple m ent in stroke and neural plasticity   24 Long term synaptic plasticity The second and extensively studied mechanism of synaptic plasticity is long -term potentiation (LTP). This process is assumed to be the basis for the acquisition of knowledge and memory formation. It relies on specific patterns of synaptic activity which lead to the strengthening of connections between the synapses of neighboring neurons which fire together, thus providing physiological substrate for a lasting association. This concept was proposed in 1949 by Donald Hebb and was confirmed experimentally twenty years later using hippocampal circuits as a model system (Bliss and Lomo, 1973 ). LTP requires activation of NMDAR, which acts as a coincidence detector, and an increased intracellular calcium concentration (Wigstrom and Gustafsson, 1986) . Figure 4. S h o r t - t e r m sy n a p t i c pla s t i c i t y an d th e re l a t e d pr e s y n a p t i c m e c h a n i s m s . Postsynaptic facilitation in response to the second stimulus occurs due to residual calcium ions in the presynaptic terminal (gray shading) that increase neurotransmitter release probability. Postsynaptic depression results from depletion of neurotransmitter-containing vesicles in the active zone following previous large discharge. Repeated high frequency stimulation (tetanic stimulus) causes extended potentiation of the postsynaptic response due to accumulation of calcium ions in the presynaptic terminal (gray shading) leading to increased release probability and/or increased quantal size due to fusion of vesicles. Anna Stokowska   25 NMDAR is a voltage- and ligand-gated ion channel, which opens only when the glutamate release from a presynaptic vesicle to the synaptic cleft coincides with postsynaptic membrane depolarization, i.e. when both pre - and postsynaptic neurons are activated (Figure 5). Post -synaptic depolarization is evoked by initial activation of glutamate-gated α-amino-3-hydroxyl -5-methyl-4-izoxazolepropionic receptors (AMPARs), an event that is required for expelling the Mg 2+ ion that blocks the NMDAR ion channel (Nicoll et al., 1988). Opened NMDAR allows for the influx of calcium, an intracellular second messenger that is bound by calcium-dependent kinases and induces a cascade of intracellular signaling. This activation initiates several pathways which lead to the induction of transcription factors and thus protein synthesis. The latter is the basis for lasting changes in properties of activated neurons (Kauer et al., 1988 ). These effector events might differ between different types of synapses but in the model hippocampal CA1-CA3 synapse, LTP causes potentiation of AMPAR conductance as well as recruitment of additional AMPARs from the peri-synaptic pool to the postsynaptic membrane (Malinow, 2003). As mentioned above, LTP has been described originally in the hippocampus but it is observed in many brain structures such as cerebral cortex, cerebellum and amygdala (Clugnet and LeDoux, 1990 ; Hess and Donoghue, 1994 ). Figure 5. L T P in hip p o c a m p a l cir c u i t r y ca u s e d by sy n c h r o n i z e d pr e - an d po s t s y n a p t i c ac t i v i t y . Single stimulus applied to Schaffer collateral synaptic input evokes normal excitatory postsynaptic potentials ( EPSPs ) in the postsynaptic CA1 neuron. However, if the CA1 neuronal membrane is briefly depolarized (by applying current pulses through the recording electrode) in conjunction with Schaffe r collateral stimulation, a persistent increase in the EPSP amplitude occurs (synapse undergoes LTP). Modified from Purves et al. (2008). Comple m ent in stroke and neural plasticity   26 Synapses can also undergo a long-lasting weakening termed long-term depression (LTD). Similar to LTP, this phenomenon seems to be dependent on the action of NMDARs although its mechanisms are far less understood. LTD was observed for the first time in the CA1 region of the hippocampus after a low-frequency stimulation protocol and is therefore believed to be evoked by synchronized weak neuronal activation (Mulkey and Malenka, 1992). Metaplasticity and homeostatic plasticity There are two additional forms of synaptic plasticity, namely metaplasticity and homeostatic plasticity. The term metaplasticity refers to the “ plasticity of synaptic plasticity” and assumes that previous history of activity of the synapse determines its current plasticity (Abraham and Bear, 1996). It consists of changes in induction thresholds for synaptic plasticity, which are caused by prior activity that itself did not change the synaptic efficacy. For example, if a given high frequency stimulation is not strong enough to evoke an increase in synaptic strength, it can inhibit subsequent induction of LTP (Huang et al., 1992 ). On the other hand, trains of low -frequency current pulses often do not produce LTD, however such stimulation protocol given after previously established LTP can result a depression of the responses, i.e. cause depotentiation (Bashir and Collingridge, 1994). When the general activity of the synaptic network is altered for a prolonged time, homeostatic mechanisms are activated and upregulate the synaptic strength if activity level was low, or downregulate it if activity was increased (Turrigiano and Nelson, 2000). This phenomenon is called homeostatic plasticity (also referred to as synaptic scaling) and is believed to occur in order to maintain functional stability of neuronal networks. Structural p lasticity Another aspect of plasticity has a structural character and involves changes in the number and complexity of dendrites, density of receptors, formation of new dendritic spines and the growth of new axon terminal, which lead to the increase in de nsity and size of synapses ., In some brain regions, structural plasticity involves also increase in number of neurons (as discussed further on). Consequently, existing pathways are Anna Stokowska   27 strengthened or new connections are developed, either within one neural network or between different neural networks. This may be a slow process taking place over weeks or months, since the growth of new connections takes time. These structural constituents of neural plasticity determine the complexity of neuronal networks and th eir activity, and can be demonstrated especially well during the process of motor learning. For example, rodents trained to traverse a demanding obstacle course had more synapses in the motor cortex and cerebellum than rats performing a simple walking task (Warraich and Kleim, 2010). Furthermore, animals trained to perform a skilled reaching task show dendritic growth, synaptogenesis, and enhanced synaptic responses which are connected to the expansion of wrist and digit movement representations within the forelimb motor cortex. Similar functional changes have been observed in primate and human motor cortex in connection with skill training (Pascual-Leone et al., 1995 ; Nudo et al., 1996). Forms of neural plasticity in recovery of function after ischemic stroke In cerebral ischemia, the initial pathological events associated with direct neuronal cell death are followed by the activation of regenerative processes for days, weeks or even months after stroke. After the ischemic attack, a majority of stroke patients e xhibit certain levels of motor weakness and sensory disturbances, however, over time, most will show a certain degree of functional recovery (Donnan et al., 2008), which may be explained by brain reorganization and plasticity . Recovery of function can be viewed as a relearning process, the basic principles of which do not differ from the ones in the intact CNS. Unlike the previously described mechanisms, which occur mainly on the level of single neurons and synapses, provision of a structural basis for new connections (including long projections) allows for more widespr ead changes in the form of functional brain remapping that is critical in the recovery of function after stroke and other CNS injuries. It allows the neurons in the spared regions to take over the sensory or motor functions that had previously been performed by the damaged areas. Ipsilateral axonal sprouting and synaptogenesis Following cerebral ischemia, extensive remodeling of dendritic spines occurs in the peri-infarct cortex. In rodent models of stroke, an increase in dendritic spine number and spine turnover rates is observed in somatosensory areas of the limbs during the first two Comple m ent in stroke and neural plasticity   26 Synapses can also undergo a long-lasting weakening termed long-term depression (LTD). Similar to LTP, this phenomenon seems to be dependent on the action of NMDARs although its mechanisms are far less understood. LTD was observed for the first time in the CA1 region of the hippocampus after a low-frequency stimulation protocol and is therefore believed to be evoked by synchronized weak neuronal activation (Mulkey and Malenka, 1992). Metaplasticity and homeostatic plasticity There are two additional forms of synaptic plasticity, namely metaplasticity and homeostatic plasticity. The term metaplasticity refers to the “ plasticity of synaptic plasticity” and assumes that previous history of activity of the synapse determines its current plasticity (Abraham and Bear, 1996). It consists of changes in induction thresholds for synaptic plasticity, which are caused by prior activity that itself did not change the synaptic efficacy. For example, if a given high frequency stimulation is not strong enough to evoke an increase in synaptic strength, it can inhibit subsequent induction of LTP (Huang et al., 1992 ). On the other hand, trains of low-frequency current pulses often do not produce LTD, howeve r such stimulation protocol given after previously established LTP can result a depression of the responses, i.e. cause depotentiation (Bashir and Collingridge, 1994). When the general activity of the synaptic network is altered for a prolonged time, homeostatic mechanisms are activated and upregulate the synaptic strength if activity level was low, or downregulate it if activity was increased (Turrigiano and Nelson, 2000). This phenomenon is called homeostatic plasticity (also referred to as synaptic scaling) and is believed to occur in order to maintain functional stability of neuronal networks. Structural p lasticity Another aspect of plasticity has a structural character and involves changes in the number and complexity of dendrites, density of receptors, formation of new dendritic spines and the growth of new axon terminal, which lead to the increase in de nsity and size of synapses ., In some brain regions, structural plasticity involves also increase in number of neurons (as discussed further on). Consequently, existing pathways are Anna Stokowska   27 strengthened or new connections are developed, either within one neural network or between different neural networks. This may be a slow process taking place over weeks or months, since the growth of new connections takes time. These structural constituents of neural plasticity determine the complexity of neuronal networks and th eir activity, and can be demonstrated especially well during the process of motor learning. For example, rodents trained to traverse a demanding obstacle course had more synapses in the motor cortex and cerebellum than rats performing a simple walking task (Warraich and Kleim, 2010). Furthermore, animals trained to perform a skilled reaching task show dendritic growth, synaptogenesis, and enhanced synaptic responses which are connected to the expansion of wrist and digit movement representations within the forelimb motor cortex. Similar functional changes have been observed in primate and human motor cortex in connection with skill training (Pascual-Leone et al., 1995 ; Nudo et al., 1996). Forms of neural plasticity in recovery of function after ischemic stroke In cerebral ischemia, the initial pathological events associated with direct neuronal cell death are followed by the activation of regenerative processes for days, weeks or even months after stroke. After the ischemic attack, a majority of stroke patients e xhibit certain levels of motor weakness and sensory disturbances, however, over time, most will show a certain degree of functional recovery (Donnan et al., 2008), which may be explained by brain reorganization and plasticity . Recovery of function can be viewed as a relearning process, the basic principles of which do not differ from the ones in the intact CNS. Unlike the previously described mechanisms, which occur mainly on the level of single neurons and synapses, provision of a structural basis for new connections (including long projections) allows for more widespread changes in the form of functional brain remapping that is critical in the recovery of function after stroke and other CNS injuries. It allows the neurons in the spared regions to take over the sensory or motor functions that had previously been performed by the damaged areas. Ipsilateral axonal sprouting and synaptogenesis Following cerebral ischemia, extensive remodeling of dendritic spines occurs in the peri-infarct cortex. In rodent models of stroke, an increase in dendritic spine number and spine turnover rates is observed in somatosensory areas of the limbs during the first two Comple m ent in stroke and neural plasticity   28 to three weeks (Brown et al., 2009). These changes represent synaptogenesis and circuit plasticity of the areas that exhibit remapping after stroke (Winship and Murphy, 2008). On the other arm of this structural plasticity is the axonal sprouting, as cortical dendritic spines are known to be postsynaptic partners for axonal boutons. Such a neuronal growth response after stroke has been demonstrated by anatomical tracing studies in rodents (Carmichael et al., 2001) and it is associated with increased expression of growth-promoting proteins and decrease of the growth-inhibitory ones (Carmichael et al., 2005). Massive axonal rearrangements are also seen in primates following brain injury (Dancause et al., 2005). For example, lesions to the primary motor area eliminate major inputs to premotor and somatosensory areas. Over time, new connections develop from the premotor area to the primary sensory cortex, replacing inputs from the primary motor area that had been lost. Tonic inhibition Some “global” changes can occur even before the new projections have sprouted, due to changes in the balance of excitatory and inhibitory connections between the whol e neural networks. This process depends on neurons and neuronal pathways having much larger region of anatomical connectivity than their usual territory of functional influence as well as a surprisingly high degree of overlap and redundancy of the connections across the cerebral cortex. Some zones may be kept relatively inactive by a phenomenon termed tonic inhibition. Tonic inhibition is mediated by a major inhibitory neurotran smitter of the brain, γ-aminobutyric acid (GABA) and its extrasynaptic GABA A receptor, the activation of which decreases neuronal excitability (Walker and Semyanov, 2008) . If this physiological inhibition is removed, the region of influence can be quickly increased or unmasked, which is associated with fundamental changes in cellular excitability including long-term potentiation (Jacobs and Donoghue, 1991 ; Hess et al., 1996 ). Increased tonic inhibition plays a role in the pathological context of ischemic stroke and it is considered to be a protective mechanism against possible overactivation of the cortical circuits after injury, which could be epileptogenic. It is caused for example by a decreased expression of astrocytic GABA transporters (GATs) in the peri -infarct cortex , resulting in the surplus of GABA in the neuronal surrounding that would normally be Anna Stokowska   29 rapidly cleaned up and recycled by astrocytes (Neumann-Haefelin et al., 1998; Frahm et al., 2004). The GABAergic mechanisms mediate changes in neuronal excitability that have a central role in functional recovery of peri-infarct cortex, however, increased and prolonged tonic inhibition after stroke may hamper recovery of function offered by cortical reorganization (Clarkson et al., 2010). Involvement of the contralesional hemisphere Mammalian brains are endowed with rich intracortical network that enables reciprocal communication among various sensory and motor areas. In contrast to the uninjured brain, the contralesional hemisphere can contribute to movement controlled from the ipsilesional cortex and after ischemic stroke, contralesional motor cortex increases its activity during the movement of the affected limb (Chollet et al., 1991; Enzinger et al., 2008 ). Moreover, animal studies show increased synaptogenesis in the non-lesioned motor and somatosensory cortical regions as well as transcallosal projections from the uninjured to the injured hemisphere following ischemic stroke (Stroemer et al., 1995; Carmichael and Chesselet, 2002; Takatsuru et al., 2009). Contralesional activation after stroke usually diminishes at the later stages of recovery. Persistent activation of contralateral cortical regions predicts slower and less complete recovery and is often associated with larger infarcts (Murphy and Corbett, 2009). Spontaneous behavioral recovery strongly correlates with remodeling of the contralateral corticospinal tracts (CST) at the level of cervical or lumbar spinal cord as well as with the accompanying reorganization of connections of pyramidal neurons in the cerebral cortex in rodents (Liu et al., 2009 ). CST consists of long projections descending from motor cortex that are organized in a topographic fashion and mediate voluntary movements of the limb musculature. Deafferentation due to stroke causes sprouting of collateral branches from both ipsi- and contralesional CST axons . These projections form new synapses on motor neurons that innervate the stroke-affected side of the body. Such a remodeling of CSTs has been linked to the recovery of fine motor function also in human subjects, so that the bilateral engagement of CST cor related with better recovery than the involvement of the ipsilesional tract alone (Schaechter et al., 2009). Comple m ent in stroke and neural plasticity   28 to three weeks (Brown et al., 2009). These changes represent synaptogenesis and circuit plasticity of the areas that exhibit remapping after stroke (Winship and Murphy, 2008 ). On the other arm of this structural plasticity is the axonal spr outing, as cortical dendritic spines are known to be postsynaptic partners for axonal boutons. Such a neuronal growth response after stroke has been demonstrated by anatomical tracing studies in rodents (Carmichael et al., 2001) and it is associated with increased expression of growth-promoting proteins and decrease of the growth-inhibitory ones (Carmichael et al., 2005). Massive axonal rearrangements are also seen in primates following brain injury (Dancause et al., 2005). For example, lesions to the primary motor area eliminate major inputs to premotor and somatosensory areas. Over time, new connections develop from the premotor area to the primary sensory cortex, replacing inputs from the primary motor area that had been lost. Tonic inhibition Some “global” changes can occur even before the new projections have sprouted, due to changes in the balance of excitatory and inhibitory connections between the whol e neural networks. This process depends on neurons and neuronal pathways having much larger region of anatomical connectivity than their usual territory of functional influence as well as a surprisingly high degree of overlap and redundancy of the connections across the cerebral cortex. Some zones may be kept relatively inactive by a phenomenon termed tonic inhibition. Tonic inhibition is mediated by a major inhibitory neurotran smitter of the brain, γ-aminobutyric acid (GABA) and its extrasynaptic GABA A receptor, the activation of which decreases neuronal excitability (Walker and Semyanov , 2008). If this physiological inhibition is removed, the region of influence can be quickly increased or unmasked, which is associated with fundamental changes in cellular excitability including long-term potentiation (Jacobs and Donoghue, 1991 ; Hess et al., 1996 ). Increased tonic inhibition plays a role in the pathological context of ischemic stroke and it is considered to be a protective mechanism against possible overactivation of the cortical circuits after injury, which could be epileptogenic. It is caused for example by a decreased expression of astrocytic GABA transporters (GATs) in the peri -infarct cortex , resulting in the surplus of GABA in the neuronal surrounding that would normally be Anna Stokowska   29 rapidly cleaned up and recycled by astrocytes (Neumann-Haefelin et al., 1998; Frahm et al., 2004). The GABAergic mechanisms mediate changes in neuronal excitability that have a central role in functional recovery of peri-infarct cortex, however, increased and prolonged tonic inhibition after stroke may hamper recovery of function offered by cortical reorganization (Clarkson et al., 2010). Involvement of the contralesional hemisphere Mammalian brains are endowed with rich intracortical network that enables reciprocal communication among various sensory and motor areas. In contrast to the uninjured brain, the contralesional hemisphere can contribute to movement controlled from the ipsilesional cortex and after ischemic stroke, contralesional motor cortex increases its activity during the movement of the affected limb (Chollet et al., 1991; Enzinger et al., 2008 ). Moreover, animal studies show increased synaptogenesis in the non-lesioned motor and somatosensory cortical regions as well as transcallosal projections from the uninjured to the injured hemisphere following ischemic stroke (Stroemer et al., 1995; Carmichael and Chesselet, 2002; Takatsuru et al., 2009). Contralesional activation after stroke usually diminishes at the later stages of recovery. Persistent activation of contralateral cortical regions predicts slower and less complete recovery and is often associated with larger infarcts (Murphy and Corbett, 2009). Spontaneous behavioral recovery strongly correlates with remodeling of the contralateral corticospinal tracts (CST) at the level of cervical or lumbar spinal cord as well as with the accompanying reorganization of connections of pyramidal neurons in the cerebral cortex in rodents (Liu et al., 2009 ). CST consists of long projections descending from motor cortex that are organized in a topographic fashion and mediate voluntary movements of the limb musculature. Deafferentation due to stroke causes sprouting of collateral branches from both ipsi- and contralesional CST axons. These projections form new synapses on motor neurons that innervate the stroke-affected side of the body. Such a remodeling of CSTs has been linked to the recovery of fine motor function also in human subjects, so that the bilateral engagement of CST correlated with better recovery than the involvement of the ipsilesional tract alone (Schaechter et al., 2009). Comple m ent in stroke and neural plasticity   30 Figure 6. P r o c e s s e s ac t i v a t e d fo l l o w i n g is c h e m i c st r o k e an d th e re l a t e d th e r a p e u t i c ap p r o a c h e s . The temporal sequence of events is shown along a schematic timeline. Darker shading highlights the maximum intensity of the specific mechanism. Processes that are detrimental for recovery are shown in pink. Regenerative responses in the form of cell genesis are shown in brown, whereas those that underlie adaptive plasticity are shown in green. Prospective therapies aimed at improving functional recovery are shown in gray. From Wieloch and Nikolich (2006). The temporal overview of plasticity-related changes occurring after stroke is presented in the Figure 6. Th e ro l e of ne u r o g e n e s i s in ne u r a l pla s t i c i t y In the adult rodent and human brains, neural stem cells continue producing new neurons, astrocytes and oligodendrocytes in two neurogenic niches (Figur e 7), namely the subgranular zone (SGZ) of hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles (Alvarez- Buylla and Lim, 2004) . In rodents, SVZ -derived neuronal progenitors migrate through the rostral migratory stream towards the olfactory Anna Stokowska   31 bulb, whereas SGZ -born cells move only short distances to the granular cell layer. The physiological role of adult mammalian neurogenesis has not been fully elucidated owing to the lack of highly specific and isolated ablation models. However, several line s of evidence indicate that neurogenesis is important for plasticity-dependent function of the target brain structure of the neurogenic niches. Correlative observations suggest that adult neurogenesis might contribute to the learning and memory functions subserved by the hippocampus, and to the perceptual and memory functions performed by the olfactory bulb. It is therefore plausible that newborn cells instruct changes in the networks that they join or, through a general alterable role, they simply permit c hanges to occur in those circuits. Figure 7. A d u l t ne u r o g e n e s i s in th e ro d e n t br a i n . Model of lineage relationships in the two neurogenic niches: SVZ and SGZ of the hippocamp al DG. Dashe d arrows represent infrequent events. DG - dentate gyrus, LV – lateral ventricle, RMS – rostral migratory stream, OB – olfactory bulb, GCL – granular cell layer. Adapted from Duan et al. (2008). Comple m ent in stroke and neural plasticity   30 Figure 6. P r o c e s s e s ac t i v a t e d fo l l o w i n g is c h e m i c st r o k e an d th e re l a t e d th e r a p e u t i c ap p r o a c h e s . The temporal sequence of events is shown along a schematic timeline. Darker shading highlights the maximum intensity of the specific mechanism. Processes that are detrimental for recovery are shown in pink. Regenerative responses in the form of cell genesis are shown in brown, whereas those that underlie adaptive plasticity are shown in green. Prospective therapies aimed at improving functional recovery are shown in gray. From Wieloch and Nikolich (2006) . The temporal overview of plasticity-related changes occurring after stroke is presented in the Figure 6. Th e ro l e of ne u r o g e n e s i s in ne u r a l pla s t i c i t y In the adult rodent and human brains, neural stem cells continue producing new neurons, astrocytes and oligodendrocytes in two neurogenic niches (Figur e 7), namely the subgranular zone (SGZ) of hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles (Alvarez -Buylla and Lim, 2004 ). In rodents, SVZ -derived neuronal progenitors migrate through the rostral migratory stream towards the olfactory Anna Stokowska   31 bulb, whereas SGZ -born cells move only short distances to the granular cell layer. The physiological role of adult mammalian neurogenesis has not been fully elucidated owing to the lack of highly specific and isolated ablation models. However, several line s of evidence indicate that neurogenesis is important for plasticity-dependent function of the target brain structure of the neurogenic niches. Correlative observations suggest that adult neurogenesis might contribute to the learning and memory functions subserved by the hippocampus, and to the perceptual and memory functions performed by the olfactory bulb. It is therefore plausible that newborn cells instruct changes in the networks that they join or, through a general alterable role, they simply permit changes to occur in those circuits. Figure 7. A d u l t ne u r o g e n e s i s in th e ro d e n t br a i n . Model of lineage relationships in the two neurogenic niches: SVZ and SGZ of the hippocamp al DG. Dashe d arrows represent infrequent events. DG - dentate gyrus, LV – lateral ventricle, RMS – rostral migratory stream, OB – olfactory bulb, GCL – granular cell layer. Adapted from Duan et al. (2008). Comple m ent in stroke and neural plasticity   32 At the systems level, physical activity and environmental enrichment increase both neurogenesis and performance on hippocampus-dependent learning tasks, whereas age and stress are associated with deficits in both phenomena. In addition, acquisition of spatial memory in the water maze correlates with the proliferation and survival of newborn dentate gyrus neuron, whereas in the olfactory bulb, olfactory enrichment links increased newborn cell survival with improvements in olfactory memory (Lledo et al., 2006). The mechanism by which neurogenesis is thought to be well suited to mediate formation of memory is that young granule cells in the adult dentate gyrus show a greater propensity for synaptic plasticity compared to older granule cells, whereas newborn granule and periglomerular cells in the olfactory bulb show markedly different active membrane properties compared to the existing neurons around them and show greater plasticity in response to sensory deprivation. In addition, for immature neurons, before glutamatergic synapses are developed, GABA is the major exc itatory neurotransmitter which further adds to a repertoire of distinct properties of newly-generated neurons that contribute to their increased plasticity (Ge et al., 2006 ). Ischemic stroke in rodents stimulates proliferation of stem cells in SVZ and a fract ion of NSPCs cells, which normally migrate through the rostral migratory stream towards olfactory bulb, divert form that route and migrate towards ischemic penumbra. There is evidence that neural stem cells may reside in the cortex or a subpopulation of as trocytes may acquire stem cells properties after injury (Magavi et al., 2000; Jiang et al., 2001; Shimada et al., 2010). Regardless of the origin, some of these cells will transiently persist in the vicinity of injury in an undifferentiated neuroblast state and a small fraction will turn into neurons and astrocytes. Manipulations that are directed at increasing neurogenesis have been shown to improve functional outcome, while the depletion of neuroblasts results in impaired performance after cerebral ischemia (Wang et al., 2004; Ohab et al., 2006 ; Leker et al., 2007 ; Wang et al., 2012). However the mechanism behind the positive effects of injury- induced neurogenesis seems unlikely to be mediated by direct neuronal replacement due to very few mature and functionally integrated neurons being generated. Instead, stem cells and progenitors, which are transiently present in penumbral tissue, may produce a variety of growth factors such as brain-derived neurotrophic factor (BDNF), neutrophin -3 (NT-3), nerve growth factor (NGF), glial - derived neurotrophic factor (GDNF), basic fibroblast growth factor bFGF, and vascular Anna Stokowska   33 endothelial growth factor (VEG F), all of which provide a supportive milieu for recovery - related plasticity (Chopp and L i, 2002; Lu et al., 2003 ). Remarkably, neurogenesis after stroke seems to be coupled to angiogenesis and revascularization of the ischemic brain tissue, possibly through production of VEG F by stem and pro genitor cells thus neurogenesis appears to contribute to tissue remodeling through enhanced perfusion as well as blood flow-independent mechanisms (H ermann and Chopp, 2012). Apart from plasticity-promoting trophic effects, newly-born, partially differentiated neural stem cells may protect the ischemic penumbra via direct cell-cell transfer of molecules (Jaderstad et al., 2010). T h e i m m u n e sy s t e m an d b r a i n p l a s t i c i t y As already mentioned, the limited access of peripheral immune cells to CNS due to the existence of BBB, made it necessary for the brain and s pinal cord cells to generate inflammatory mediators such as cytokines and complement proteins locally in order to maintain protection from invading microorganisms. However, in the recent years it became clear that the immune system is actively involved in brain plasticity and therefore its activation in CNS is important both for physiological functions and recovery after injury. Microglia One of the most prominent immune cell types that influence brain plasticity is microglia. Non-activated microglia in the healthy brain are highly active cells, extending and retracting their processes as they survey the microenvironment in CNS (Nimmerjahn et al., 2005). The most interesting mechanism by which this cell type influences neural plasticity under physiological conditions seems to be their interaction with synapses. Microglia have been recently identified as “the sculptors” of synaptic networks during development by performing elimination of weak synapses (Paolicelli et al., 2011). Moreover, microglial processes were observed to be in association with synapses during visual experience, including contacting axon terminals, dendritic spines, perisynaptic astrocytic processes and synaptic clefts in adult animals (Tremblay et al., 2010). Hence, developmental and experience -dependent plasticity may involve microglial interactions with synapses and physical remodeling of this component of neural circuits. Comple m ent in stroke and neural plasticity   32 At the systems level, physical activity and environmental enrichment increase both neurogenesis and performance on hippocampus-dependent learning tasks, whereas age and stress are associated with deficits in both phenomena. In addition, acquisition of spatial memory in the water maze correlates with the proliferation and survival of newborn dentate gyrus neuron, whereas in the olfactory bulb, olfactory enrichment links increased newborn cell survival with improvements in olfactory memory (Lledo et al., 2006). The mechanism by which neurogenesis is thought to be well suited to mediate formation of memory is that young granule cells in the adult dentate gyrus show a greater propensity for synaptic plasticity compared to older granule cells, whereas newborn granule and periglomerular cells in the olfactory bulb show markedly different active membrane properties compared to the existing neurons around them and show greater plasticity in response to sensory deprivation. In addition, for immature neurons, before glutamatergic synapses are developed, GABA is the major exc itatory neurotransmitter which further adds to a repertoire of distinct properties of newly-generated neurons that contribute to their increased plasticity (Ge et al., 2006 ). Ischemic stroke in rodents stimulates proliferation of stem cells in SVZ and a fract ion of NSPCs cells, which normally migrate through the rostral migratory stream towards olfactory bulb, divert form that route and migrate towards ischemic penumbra. There is evidence that neural stem cells may reside in the cortex or a subpopulation of as trocytes may acquire stem cells properties after injury (Magavi et al., 2000; Jiang et al., 2001 ; Shimada et al., 2010). Regardless of the origin, some of these cells will transiently persist in the vicinity of injury in an undifferentiated neuroblast state and a small fraction will turn into neurons and astrocytes. Manipulations that are directed at increasing neurogenesis have been shown to improve functional outcome, while the depletion of neuroblasts results in impaired performance after cerebral ischemia (Wang et al., 2004 ; Ohab et al., 2006 ; Leker et al., 2007 ; Wang et al., 2012 ). However the mechanism behind the positive effects of injury -induced neurogenesis seems unlikely to be mediated by direct neuronal replacement due to very few mature and functionally integrated neurons being generated. Instead, stem cells and progenitors, which are transiently present in penumbral tissue, may produce a variety of growth factors such as brain-derived neurotrophic factor (BDNF), neutrophin -3 (NT-3), nerve growth factor (NGF), glial - derived neurotrophic factor (GDNF), basi c fibroblast growth factor bFGF, and vascular Anna Stokowska   33 endothelial growth factor (VEG F), all of which provide a supportive milieu for recovery - related plasticity (Chopp and L i, 2002; Lu et al., 2003 ). Remarkably, neurogenesis after stroke seems to be coupled to angiogenesis and revascularization of the ischemic brain tissue, possibly through production of VEG F by stem and pro genitor cells thus neurogenesis appears to contribute to tissue remodeling through enhanced perfusion as well as blood flow-independent mechanisms (H ermann and Chopp, 2012). Apart from plasticity-promoting trophic effects, newly-born, partially differentiated neural stem cells may protect the ischemic penumbra via direct cell-cell transfer of molecules (Jaderstad et al., 2010). T h e i m m u n e sy s t e m an d b r a i n p l a s t i c i t y As already mentioned, the limited access of peripheral immune cells to CNS due to the existence of BBB, made it necessary for the brain and spinal cord cells to generate inflammatory mediators such as cytokines and complement proteins locally in order to maintain protection from invading microorganisms. However, in the recent years it became clear that the immune system is actively involved in brain plasticity and therefore its activation in CNS is important both for physiological functions and recovery after injury. Microglia One of the most prominent immune cell types that influence brain plasticity is microglia. Non-activated microglia in the healthy brain are highly active cells, extending and retracting their processes as they survey the microenvironment in CNS (Nimmerjahn et al., 2005). The most interesting mechanism by which this cell type influences neural plasticity under physiological conditions seems to be their interaction with synapses. Microglia have been recently identified as “the sculptors” of synaptic networks during development by performing elimination of weak synapses (Paolicelli et al., 2011). Moreover, microglial processes were observed to be in association with synapses during visual experience, including contacting axon terminals, dendritic spines, perisynaptic astrocytic processes and synaptic clefts in adult animals (Tremblay et al., 2010). Hence, developmental and experience -dependent plasticity may involve microglial interactions with synapses and physical remodeling of this component of neural circuits. Comple m ent in stroke and neural plasticity   34 Microglia also play an important physiological role in controlling adult neurogenesis. Mouse-derived microglia release soluble factors, which contribute to migration and differentiation of neural progenitors in vitro , and the number of activated microglia displaying ramified morphology correlates with increased neurogenesis and number of nestin-positive cells in SGZ of hippocampus (Aarum et al., 2003; Battista et al., 2006; Walton et al., 2006 ). Normal function of microglia has been also shown to be required for spatial learning and memory (Ziv et al., 2006 ). The effect of activated microglia in CNS seems to be dependent on the nature of the stimulating factor. Microglial activation phenotype has been deÞned as either the classical, M1 or the alternative, M2 state, adopted from descriptions used for peripheral macrophage activation (Ransohoff and Perry, 2009). For example, LPS and irradiation induce pro-inflammatory and cytotoxic profile of microglia activation, which has detrimental consequences e.g. by inhibition of neurogenesis (Ekdahl et al., 2003 ; Monje et al., 2003). Therefore microglia inhibition has been shown to ameliorate these negative effects (Ekdahl et al., 2003 ; Hoehn et al., 2005 ). On the other hand, microglia stimulated by IFN γ or anti-inflammatory cytokine interleukin 4 (IL -4), are neuroprotective for cultured hippocampal slices and stimulate neurogenesis and oligodendrogenesis from adult neural stem cells (NSCs). The putative mechanism for these effects appears to be microglial secretion of insulin growth factor 1 (IGF -1), suppression of TNF α and upregulation of glutamate transporter 1 (GLT -1), which are important for clean-up of excitotoxic glutamate from the extracellular space (Butovsky et al., 2005; Shaked et al., 2005; Butovsky et al., 2006). Microglia can also support neuronal functions and promote neural plasticity by secretion of the main neurotrophic growth factors, BDNF and NGF (Elkabes et al., 1996 ; Madinier et al., 2009). Noteworthy, physical activity which increases both BDNF and NGF levels in the brain and stimulates neurogenesis, leads also to microglial proliferation in mouse cerebral cortex (Ehninger and Kempermann, 2003) . Macrophages Similar to microglia, blood-derived macrophages seem to play an important role in CNS plasticity. These cells are assumed to be one of the major contributors to successful Anna Stokowska   35 regeneration of damaged peripheral nerves as opposed to the limited direct regenerative properties of CNS pathways. Macrophages recruited to the damaged CNS are less effective in removal of growth-inhibiting myelin debris (Lazarov -Spiegler et al., 1996). However, increased numbers of macrophages at the injury sit e due to infiltration or transplantation, improve axonal regeneration of damaged optic nerve and spinal cord, effect of which is mediated by efficient myelin debris clean-up and secretion of factors such as oncomodulin and IL -10 (Lazarov -Spiegler et al., 1996; Rapalino et al., 1998; Yin et al., 2006; Shechter et al., 2009). Astrocytes Astrocytes are the important components of neurogenic niches and have been implicated in shaping their microenvironment by secreted factors as well as direct cell- cell contact and thus regulating neurogenesis (Song et al., 2002). They can also function as multipotent neural stem cells in both the developing and adult brain (Figure 7) . Furthermore astrocyte -derived factors regulate synaptogenesis and synapse maturation (Nagler et al., 2001; Christopherson et al., 2005; Eroglu et al., 2009 ). Moreover, astrocytes regulate and control the number of neurotransmitter molecules present in the synaptic cleft and the function of NMDARs, thus they influence synaptic transmission by being part of an entity termed the tri-partite synapse (Araque et al., 1998 ; Panatier et al., 2006; Jourdain et al., 2007 ). Upon CNS injury, astrocytes become active, which is hallmarked by cellular hypertrophy and upregulation of intermediate filaments proteins, collectively known as reactive astrogliosis. Reactive astrogliosis is neuroprotective in the acute stages after injury by restricting the secondary tissue damage though the formation of a glial scar (Li et al., 2008). However during the later stages of the recovery, this astrocyte barrier poses a serious obstacle for effective regeneration by secretion of powerful inhibitors of CNS regeneration (Pekny and Pekna, 2004). Astrocytes are also an essential component of the neurovascular unit and directly regulate the properties of BBB. Thus, they can also regulate neurogenesis and synaptogenesis indirectly, by determining the accessibility of blood-derived factors modulating neural plasticity (Barres, 2008). Comple m ent in stroke and neural plasticity   34 Microglia also play an important physiological role in controlling adult neurogenesis. Mouse-derived microglia release soluble factors, which contribute to migration and differentiation of neural progenitors in vitro , and the number of activated microglia displaying ramified morphology correlates with increased neurogenesis and number of nestin-positive cells in SGZ of hippocampus (Aarum et al., 2003; Battista et al., 2006; Walton et al., 2006 ). Normal function of microglia has been also shown to be required for spatial learning and memory (Ziv et al., 2006 ). The effect of activated microglia in CNS seems to be dependent on the nature of the stimulating factor. Microglial activation phenotype has been deÞned as either the classical, M1 or the alternative, M2 state, adopted from descriptions used for peripheral macrophage activation (Ransohoff and Perry, 2009). For example, LPS and irradiation induce pro-inflammatory and cytotoxic profile of microglia activation, which has detrimental consequences e.g. by inhibition of neurogenesis (Ekdahl et al., 2003 ; Monje et al., 2003). Therefore microglia inhibition has been shown to ameliorate these negative effects (Ekdahl et al., 2003 ; Hoehn et al., 2005 ). On the other hand, microglia stimulated by IFN γ or anti-inflammatory cytokine interleukin 4 (IL -4), are neuroprotective for cultured hippocampal slices and stimulate neurogenesis and oligodendrogenesis from adult neural stem cells (NSCs). The putative mechanism for these effects appears to be microglial secretion of insulin growth factor 1 (IGF -1), suppression of TNF α and upregulation of glutamate transporter 1 (GLT -1), which are important for clean-up of excitotoxic glutamate from the extracellular space (Butovsky et al., 2005; Shaked et al., 2005; Butovsky et al., 2006) Microglia can also support neuronal functions and promote neural plasticity by secretion of the main neurotrophic growth factors, BDNF and NGF (Elkabes et al., 1996 ; Madinier et al., 2009). Noteworthy, physical activity which increases both BDNF and NGF levels in the brain and stimulates neurogenesis, leads also to microglial proliferation in mouse cerebral cortex (Ehninger and Kempermann, 2003 ). Macrophages Similar to microglia, blood-derived macrophages seem to play an important role in CNS plasticity. These cells are assumed to be one of the major contributors to successful Anna Stokowska   35 regeneration of damaged peripheral nerves as opposed to the limited direct regenerative properties of CNS pathways. Macrophages recruited to the damaged CNS are less effective in removal of growth-inhibiting myelin debris (Lazarov -Spiegler et al., 1996). However, increased numbers of macrophages at the injury sit e due to infiltration or transplantation, improve axonal regeneration of damaged optic nerve and spinal cord, effect of which is mediated by efficient myelin debris clean-up and secretion of factors such as oncomodulin and IL -10 (Lazarov -Spiegler et al., 1996; Rapalino et al., 1998; Yin et al., 2006; Shechter et al., 2009). Astrocytes Astrocytes are the important components of neurogenic niches and have been implicated in shaping their microenvironment by secreted factors as well as direct cell- cell contact and thus regulating neurogenesis (Song et al., 2002). They can also function as multipotent neural stem cells in both the developing and adult brain (Figure 7) . Furthermore astrocyte- derived factors regulate synaptogenesis and synapse maturation (Nagler et al., 2001; Christopherson et al., 2005; Eroglu et al., 2009 ). Moreover, astrocytes regulate and control the number of neurotransmitter molecules present in the synaptic cleft and the function of NMDARs, thus they influence synaptic transmission by being part of an entity termed the tri-partite synapse (Araque et al., 1998; Panatier et al., 2006; Jourdain et al., 2007 ). Upon CNS injury, astrocytes become active, which is hallmarked by cellular hypertrophy and upregulation of intermediate filaments proteins, collectively known as reactive astrogliosis. Reactive astrogliosis is neuroprotective in the acute stages after injury by restricting the secondary tissue damage though the formation of a glial scar (Li et al., 2008). However during the later stages of the recovery, this astrocyte barrier poses a serious obstacle for effective regeneration by secretion of powerful inhibitors of CNS regeneration (Pekny and Pekna, 2004). Astrocytes are also an essential component of the neurovascular unit and directly regulate the properties of BBB. Thus, they can also regulate neurogenesis and synaptogenesis indirectly, by determining the accessibility of blood-derived factors modulating neural plasticity (Barres, 2008). Comple m ent in stroke and neural plasticity   36 T lym p hocytes A growing body of evidence suggests that the adaptive immune system and T lymphocytes in particular interact with CNS providing neuroprotection and regulating of neurogenesis. Especially T -cells directed against myelin binding protein (TMBP) reduce the spreading of damage following optic nerve and spinal cord injury (Moalem et al., 1999; Hauben et al., 2000 ). These autoreactive T-cells become activated and produce NGF and BDNF supporting neuronal growth and survival (Kerschensteiner et al., 1999; Moalem et al., 2000). T cells also play a role in the maintenance of the hippocampal neurogenic niche by the stimulation of progenitor cells proliferation. As a consequence, immunodeficiency leads to decreased neurogenesis, decreased BDNF expression in the brain and deficits in spatial learning in Morris Water M aze. Conversely, replenishing the CD4 + lymphocytes rescues precursor cell proliferation (Wolf et al., 2009 ). It seems that CNS-specific TMBP cells specific for the myelin basic protein are mainly responsible for these effects as transgenic TMBP mice show increased hippocampal neurogenesis and perform better in learning and memory tasks. T lymphocytes exert their beneficial properties partially through their interaction with microglia by production of M2-type cytokine, IL- 4 and without the need for infiltration of the brain parenchyma (Ziv et al., 2006 ; Derecki et al., 2010). Pro- inflammatory cytokines A number of immune proteins have been found to be associated with developing and mature synapses and some of them have been found to regulate synaptic transmission (Boulanger et al., 2001). A prominent example is TNF α released from microglia which promotes cell surface accumulation of AMPARs and concomitant decrease in GABA A receptors due to increased endocytosis in hippocampal neurons (Pascual et al., 2012). This way, TNF α increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal neurons in vitro (Beattie et al., 2002). TNF α is thought to be one of the important regulators of homeostatic plasticity by scaling-up the excitatory synapses as it may be released in response to drop in synaptic activity (Stellwagen and Malenka, 2006). Further, TNF α alters excitability throughout Anna Stokowska   37 CNS via its effects on glutamate transporters. It decreases the expression of the GLT -1 and decreases glutamate uptake by other glial transporters such as GLAST (Schafers and Sorkin, 2008). In vitro exposure of adult SVZ -derived NSCs to TNF α produces a dose- related increase in proliferation with no effect on differentiation (Widera et al., 2006 ).   Another pro-inflammatory cytokine, IL -1β, is thought to reduce neurotransmitter release, impair LTP in the hippocampus and modulate synaptic transmission in the neocortex (Schafers and Sorkin, 2008) by rapid inhibition of voltage–dependent calcium channels in CA1 and cortical neurons. On the other hand, IL -1β may have excitatory effect especially on nociceptive fibers as the IL -1 receptor is expressed in dorsal root ganglions, thus IL -1β can act directly on sensory neurons (Marchand et al., 2005). Evidence from several in vitro and in vivo experiments suggests the existence of a functional interaction between IL -1β and glutamate receptors: IL -1β reduces the frequency of AMPA dependent spontaneous excitatory postsynaptic currents (sEPSCs) and mEPSCs. Physiological levels of IL -1β in the hippocampus take part in memory consolidation and facilitation, while IL -1 receptor blockage impairs memory formation (Goshen et al., 2007 ). Interestingly, endogenous IL -1β may be required for peripheral nerve regeneration (Guenard et al., 1991 ). IL -6 has been shown to induce proliferation of brain microvessels in vitro , while IL -6-deficient mice show delayed tissue repair associated with increased neuronal death and slower revascularization in the injury site (Fee et al., 2000 ; Swartz et al., 2001 ). Furthermore, IL -6 levels are dramatically upregulated by physiological LTP in vivo , however, exogenous IL -6 inhibits LTP in hippocampal slices (Jankowsky et al., 2000 ; Balschun et al., 2004). It cannot be forgotten that an ample amount of studies show negative influence of CNS inflammation on learning, memory and neurogenesis in disease. However since the basal levels of cytokines in quiescent cond itions are required for all forms of neural plasticity, it means that cytokines effects are dose-, time- and context -dependent. Comple m ent in stroke and neural plasticity   36 T lym p hocytes A growing body of evidence suggests that the adaptive immune system and T lymphocytes in particular interact with CNS providing neuroprotection and regulating of neurogenesis. Especially T -cells directed against myelin binding protein (TMBP) reduce the spreading of damage following optic nerve and spinal cord injury (Moalem et al., 1999; Hauben et al., 2000 ). These autoreactive T-cells become activated and produce NGF and BDNF supporting neuronal growth and survival (Kerschensteiner et al., 1999 ; Moalem et al., 2000). T cells also play a role in the maintenance of the hippocampal neurogenic niche by the stimulation of progenitor cells proliferation. As a consequence, immunodeficiency leads to decreased neurogenesis, decreased BDNF expression in the brain and deficits in spatial learning in Morris Water M aze. Conversely, replenishing the CD4 + lymphocytes rescues precursor cell proliferation (Wolf et al., 2009 ). It seems that CNS-specific TMBP cells specific for the myelin basic protein are mainly responsible for these effects as transgenic TMBP mice show increased hippocampal neurogenesis and perform better in learning and memory tasks. T lymphocytes exert their beneficial properties partially through their interaction with microglia by production of M2-type cytokine, IL -4 and without the need for infiltration of the brain parenchyma (Ziv et al., 2006 ; Derecki et al., 2010). Pro- inflammatory cytokines A number of immune proteins have been found to be associated with developing and mature synapses and some of them have been found to regulate synaptic transmission (Boulanger et al., 2001). A prominent example is TNF α released from microglia which promotes cell surface accumulation of AMPARs and concomitant decrease in GABA A receptors due to increased endocytosis in hippocampal neurons (Pascual et al., 2012). This way, TNF α increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal neurons in vitro (Beattie et al., 2002). TNF α is thought to be one of the important regulators of homeostatic plasticity by scaling-up the excitatory synapses as it may be released in response to drop in synaptic activity (Stellwagen and Malenka, 2006). Further, TNF α alters excitability t hroughout Anna Stokowska   37 CNS via its effects on glutamate transporters. It decreases the expression of the GLT -1 and decreases glutamate uptake by other glial transporters such as GLAST (Schafers and Sorkin, 2008). In vitro exposure of adult SVZ -derived NSCs to TNF α produces a dose- related increase in proliferation with no effect on differentiation (Widera et al., 2006 ).   Another pro-inflammatory cytokine, IL -1β, is thought to reduce neurotransmitter release, impair LTP in the hippocampus and modulate synaptic transmission in the neocortex (Schafers and Sorkin, 2008) by rapid inhibition of voltage–dependent calcium channels in CA1 and cortical neurons. On the other hand, IL -1β may have excitatory effect especially on nociceptive fibers as the IL -1 receptor is expressed in dorsal root ganglions, thus IL -1β can act directly on sensory neurons (Marchand et al., 2005). Evidence from several in vitro and in vivo experiments suggests the existence of a functional interaction between IL -1β and glutamate receptors: IL -1β reduces the frequency of AMPA dependent spontaneous excitatory postsynaptic currents (sEPSCs) and mEPSCs. Physiological levels of IL -1β in the hippocampus take part in memory consolidation and facilitation, while IL -1 receptor blockage impairs memory formation (Goshen et al., 2007 ). Interestingly, endogenous IL -1β may be required for peripheral nerve regeneration (Guenard et al., 1991 ). IL -6 has been shown to induce proliferation of brain microvessels in vitro , while IL -6-deficient mice show delayed tissue repair associated with increased neuronal death and slower revascularization in the injury site (Fee et al., 2000 ; Swartz et al., 2001 ). Furthermore, IL -6 levels are dramatically upregulated by physiological LTP in vivo , however, exogenous IL -6 inhibits LTP in hippocampal slices (Jankowsky et al., 2000 ; Balschun et al., 2004). It cannot be forgotten that an ample amount of studies show negative influence of CNS inflammation on learning, memory and neurogenesis in disease. However since the basal levels of cytokines in quiescent cond itions are required for all forms of neural plasticity, it means that cytokines effects are dose-, time- and context -dependent. Comple m ent in stroke and neural plasticity   38 E merging roles of complement system in neural plasticity C3 seems to be the central complement-derived factor that regulates different forms of neural plasticity. As mentioned above, the discovery of anaphylatoxin receptors expression on the surface of NSPCs pointed to the role of complement in regulation of basal neurogenesis. While C3aR signaling was indeed found to stimulate b asal neurogenesis in olfactory bulb and dentate gyrus as well as migration and differentiation of NSPCs towards neurons in vitro , the role of C5aR in these processes has not been confirmed (Rahpeymai et al., 2006; Bogestål et al., 2007 ; Shinjyo et al., 2009) . Additionally, in a model of permanent cerebral ischemia, genetic deficiency of C3 resulted in greater brain tissue damage and decreased neurogenesis in the peri-infarct cortex (Rahpeymai et al., 2006). The latter finding suggests the importance of C3 and/or its activation fragments in neuroprotection or healing of ischemic brain tissue and extends the neurogenic role of the complement system to injury -related context. It is plausible that the positive complement-related effects are conveyed indirectly by other cells as in vitro studies revealed the stimulatory effect of C3a on the release of NGF by microglia and astrocytes (Heese et al., 1998 ; Jauneau et al., 2006 ). Both C3 and C1 have been found to mediate microglia-dependent elimination of synapses during early postnatal maturation of neuronal circuits (Stevens et al., 2007; Schafer et al., 2012). C1q, expressed by developing neurons, localizes to synapses that are thus tagged for elimination through the activation of the complement cascade and deposition of C3b fragments which are recognized by CR3 on microglia . Therefore, mice deficient either in C1q or C3 showed significant and sustained defects in synapse elimination in the thalamus. Additionally, C1q- deficient mice showed enhanced neocortical excitatory synaptic connectivity and epileptiform activity (Chu et al., 2010). Fi nally, significant upregulation of C3 mRNA in the growth-associated transcriptome of post-ischemic brain tissue points to the prominent role of the C3 protein or its derivatives in neural plasticity and recovery processes, although the mechanisms thereof are yet to be determined (Li et al., 2010 ). Anna Stokowska   39 A IM OF THE THESIS The overall aim of this thesis was to study the role of the complement system in brain plasticity and recovery after ischemic stroke as well as to test whether complement activation could be a predictive biomarker of functional outcome after stroke. Spe c i f i c aim s w e r e : ¥ To investigate the extent of complement activation in blood plasma of patients suffering from ischemic stroke of various etiologies and to test the hypothesis that these measurements can be helpful in predicting the functional outcome after ischemic stroke. (Paper I and II) ¥ To investigate whether genetic variation in the C3 gene is associated with ischemic stroke. (Paper III) ¥ To test the hypothesis that signaling through C3aR stimulates stroke induced neurogenesis and axonal sprouting/regeneration and leads to better functional outcome after experimental stroke. (Paper IV) ¥ To investigate the effect of C3 deficiency on hippocampal synaptic plasticity and cognitive performance. (Paper V) Comple m ent in stroke and neural plasticity   38 E merging roles of complement system in neural plasticity C3 seems to be the central complement-derived factor that regulates different forms of neural plasticity. As mentioned above, the discovery of anaphylatoxin receptors expression on the surface of NSPCs pointed to the role of complement in regulation of basal neurogenesis. While C3aR signaling was indeed found to stimulate b asal neurogenesis in olfactory bulb and dentate gyrus as well as migration and differentiation of NSPCs towards neurons in vitro , the role of C5aR in these processes has not been confirmed (Rahpeymai et al., 2006; Bogestål et al., 2007 ; Shinjyo et al., 2009 ). Additionally, in a model of permanent cerebral ischemia, genetic deficiency of C3 resulted in greater brain tissue damage and decreased neurogenesis in the peri-infarct cortex (Rahpeymai et al., 2006). The latter finding suggests the importance of C3 and/or its activation fragments in neuroprotection or healing of ischemic brain tissue and extends the neurogenic role of the complement system to injury -related context. It is plausible that the positive complement-related effects are conveyed indirectly by other cells as in vitro studies revealed the stimulatory effect of C3a on the release of NGF by microglia and astrocytes (Heese et al., 1998 ; Jauneau et al., 2006 ). Both C3 and C1 have been found to mediate microglia-dependent elimination of synapses during early postnatal maturation of neuronal circuits (Stevens et al., 2007; Schafer et al., 2012). C1q, expressed by developing neurons, localizes to synapses that are thus tagged for elimination through the activation of the complement cascade and deposition of C3b fragments which are recognized by CR3 on microglia . Therefore, mice deficient either in C1q or C3 showed significant and sustained defects in synapse elimination in the thalamus. Additionally, C1q -deficient mice showed enhanced neocortical excitatory synaptic connectivity and epileptiform activity (Chu et al., 2010). Fi nally, significant upregulation of C3 mRNA in the growth-associated transcriptome of post-ischemic brain tissue points to the prominent role of the C3 protein or its derivatives in neural plasticity and recovery processes, although the mechanisms thereof are yet to be determined (Li et al., 2010 ). Anna Stokowska   39 A IM OF THE THESIS The overall aim of this thesis was to study the role of the complement system in brain plasticity and recovery after ischemic stroke as well as to test whether complement activation could be a predictive biomarker of functional outcome after stroke. Spe c i f i c aim s w e r e : ¥ To investigate the extent of complement activation in blood plasma of patients suffering from ischemic stroke of various etiologies and to test the hypothesis that these measurements can be helpful in predicting the functional outcome after ischemic stroke. (Paper I and II) ¥ To investigate whether genetic variation in the C3 gene is associated with ischemic stroke. (Paper III) ¥ To test the hypothesis that signaling through C3aR stimulates stroke induced neurogenesis and axonal sprouting/regeneration and leads to better functional outcome after experimental stroke. (Paper IV) ¥ To investigate the effect of C3 deficiency on hippocampal synaptic plasticity and cognitive performance. (Paper V) Anna Stokowska   41 METHODS In this chapter, a summary of the methods used in the current studies will be given together with some methodological considerations. For further details of the experimental procedures, please refer to the papers. H u m a n su b j e c t s (I , II , II I ) The study population for the clinical component of the thesis consisted of a large case-control cohort, the Sahlgrenska Academy Study on Ischemic Stroke (SAHLS IS), which constitutes a well characterized sample of ischemic stroke patients and healthy controls form Western Sweden (Jood et al., 2005 ). Briefly, patients (n=8 44) younger than 70 years of age and presenting with first ever or recurrent stroke at four Stroke Units in Western Sweden have been prospectively recruited between 1998 and 2008. During the acute phase of stroke, all patients were examined by a physician trained in stroke medicine and all patients underwent electrocardiography and neuroimaging with computer tomography and/or magnetic resonance imaging. Additional diagnostic work - up was performed when clinically indicated. Based on clinical presentation and results of the diagnostics, cases were classified into etiological subtypes according to the TOAST criteria (Adams et al., 1993). Stroke severity at inclusion was scored using the Scandinavian Stroke Scale (SSS) (Scandinavian Stroke Study Group, 1985 ). At three- month follow-up, functional outcome was assessed with the modified Rankin Scale (mRS) (van Swieten et al., 1988) through examination by a physicia n trained in stroke medicine. At two-year follow-up, surviving patients were contacted by a research nurse trained in stroke medicine for a structured telephone interview. Healthy population controls (n=668), free fr om coronary or periphery artery disease, were randomly selected to match cases with regard to age, sex and geographical residence through the community-based health survey or from the Swedish Population Registry (Wilhelmsen et al., 1997 ). The control subjects were examined once by a research nurse trained in stroke medicine using the same questionnaire and protocol as for patients. All participants provided their written informed consent. Next -of-kin consented for those participants who were unable to communicate. The distribution of Anna Stokowska   41 METHODS In this chapter, a summary of the methods used in the current studies will be given together with some methodological considerations. For further details of the experimental procedures, please refer to the papers. H u m a n su b j e c t s (I , II , II I ) The study population for the clinical component of the thesis consisted of a large case-control cohort, the Sahlgrenska Academy Study on Ischemic Stroke (SAHLS IS), which constitutes a well characterized sample of ischemic stroke patients and healthy controls form Western Sweden (Jood et al., 2005 ). Briefly, patients (n=8 44) younger than 70 years of age and presenting with first ever or recurrent stroke at four Stroke Units in Western Sweden have been prospectively recruited between 1998 and 2008. During the acute phase of stroke, all patients were examined by a physician trained in stroke medicine and all patients underwent electrocardiography and neuroimaging with computer tomography and/or magnetic resonance imaging. Additional diagnostic work - up was performed when clinically indicated. Based on clinical presentation and results of the diagnostics, cases were classified into etiological subtypes according to the TOAST criteria (Adams et al., 1993). Stroke severity at inclusion was scored using the Scandinavian Stroke Scale (SSS) (Scandinavian Stroke Study Group, 1985). At three- month follow-up, functional outcome was assessed with the modified Rankin Scale (mRS) (van Swieten et al., 1988) through examination by a physician trained in stroke medicine. At two-year follow-up, surviving patients were contacted by a research nurse trained in stroke medicine for a structured telephone interview. Healthy population controls (n=668), free fro m coronary or periphery artery disease, were randomly selected to match cases with regard to age, sex and geographical residence through the community-based health survey or from the Swedish Population Registry (Wilhelmsen et al., 1997 ). The control subjects were examined once by a research nurse trained in stroke medicine using the same questionnaire and protocol as for patients. All participants provided their written informed consent. Next -of-kin consented for those participants who were unable to communicate. The distribution of Comple m ent in stroke and neural plasticity   42 etiological subtypes and baseline characteristics of the entire study population including vascular risk factors have been published elsewhere (Jood et al., 2005 ). In the studies addressing the associations between peripheral blood complement levels and outcome (papers I and II), a subset of individuals from the entire SAHLSIS population was selected in order to avoid sample size bias related to the uneven distribution of etiological subtypes among patients. To this end, all patients classified as having stroke due to LVD (n = 73) were included along with a similar number of patients with cryptogenic, SVD and CE stroke (n=79 for each ty pe). Because the cryptogenic and SVD groups have a relatively favorable functional outcome, to minimize the effect of uneven distribution of outcome categories in this subgroup and thus improve the statistical power in the outcome regression analysis, all patients form these two groups with mRS score >2 were selected. The remaining patients in these two groups were selected so that they represented an even distribution of the mRS scores 0, 1 and 2. Regarding the selection of control subjects, f or each of the four patient groups, half the number of controls was analyzed (selected so as to have the same mean age and sex distribution as the cases). In contrast, in paper III, data for all 844 patients and all 668 controls were analyzed as genetic association studies require large sample size to allow detection of relationships. Comment: SAHLSIS includes only patients younger than 70 years, which constitute the vast majority of all of the admissions to stroke units in the region. The upper age limit for inclusion was chosen as the SAHLSIS project was designed primarily to study genetic associations and genetic components show greater influence in younger individuals ( Flossmann, 200 6 ) . Generally, due to the younger age, unfavorable outcome including mortality was relatively rare. This fact together with the additional selection of patients that was required for reliable statistical analysis renders the presented results not necessarily representative of the general stroke population. Furthermore, as the SHALSIS project was not intended initially to study inflammatory markers, a detailed history of recent infections or inflammatory diseases was not obtained. This could have potentially influenced the levels of complement components in the blood. Anna Stokowska   43 M i c e (I V , V ) To study the role of the complement system in synapse elimination in the hippocampus (paper V), homozygous mice deficient in C3 (C3 KO) were used (Pekna et al. 1998). They were backcrossed onto the C57BL/6 background for 13 generations to obtain mice congenic with the control wild type (WT) strain (Jackson Laboratories, Bar Harbor, ME, USA). Male mice of various age s were used for these experiments. For electrophysiology, 17 to 30 days old mice were used to cover the postnatal circuit refinement period, whereas learning behavior and epileptiform brain activity were assessed in 2.5 to 3 months old mice to study post-developmental consequ ences of altered synaptic plasticity as well as for practical reasons. Microglial activation was assessed at 6 months of age, to assess the potential occurrence of delayed or chronic differences between the strains. To study the role of C3aR in experimenta l stroke (paper IV) , two sets of adult (7 to 9 months old) male mice were used to approximate the clinical situation, while avoiding age-related complications. In the first cohort, mice deficient in C3aR (C3aRKO ) and C3aRWT mice were used (Kildsgaard et al ., 2000). The second cohort consisted of transgenic mice expressing C3a under the control of glial fibrilary acidic protein (GFAP) promoter (C3a-GFAP) and their WT littermates which served as controls (Boos et al., 2004). All experiments on animals were pe rformed in accordance with the guidelines of the local ethical committee for animal research at the University of Gothenburg or the Malmö -Lund Ethical Committee for the use of laboratory animals , according to the location of the experiment was carried out. All experiments were conducted in accordance with European Union directives on animal rights. E L I S A (I , II ) The enzyme -linked immunosorbent assay (ELISA) is an antibody -based assay commonly used to quantitatively detect a specific antigen in plasma or se rum. In the current thesis, sandwich ELISAs were used. The principle of the assay consist in applying diluted plasma sample onto microtitter plate previously coated with capture Comple m ent in stroke and neural plasticity   42 etiological subtypes and baseline characteristics of the entire study population including vascular risk factors have been published elsewhere (Jood et al., 2005 ). In the studies addressing the associations between peripheral blood complement levels and outcome (papers I and II), a subset of individuals from the entire SAHLSIS population was selected in order to avoid sample size bias related to the uneven distribution of etiological subtypes among patients. To this end, all patients classified as having stroke due to LVD (n = 73) were included along with a similar number of patients with cryptogenic, SVD and CE stroke (n=79 for each ty pe). Because the cryptogenic and SVD groups have a relatively favorable functional outcome, to minimize the effect of uneven distribution of outcome categories in this subgroup and thus improve the statistical power in the outcome regression analysis, all patients form these two groups with mRS score >2 were selected . The remaining patients in these two groups were selected so that they represented an even distribution of the mRS scores 0, 1 and 2. Regarding the selection of control subjects, f or each of the four patient groups, half the number of controls was analyzed (selected so as to have the same mean age and sex distribution as the cases). In contrast, in paper III, data for all 844 patients and all 668 controls were analyzed as genetic association stu dies require large sample size to allow detection of relationships. Comment: SAHLSIS includes only patients younger than 70 years, which constitute the vast majority of all of the admissions to stroke units in the region. The upper age limit for inclusion was chosen as the SAHLSIS project was designed primarily to study genetic associations and genetic components show greater influence in younger individuals ( Flossmann, 200 6 ) . Generally, due to the younger age, unfavorable outcome including mortality was relatively rare. This fact together with the additional selection of patients that was required for reliable statistical analys is renders the presented results not necessarily representative of the general stroke population. Furthermore, as the SHALSIS project was not intended initially to study inflammatory markers, a detailed history of recent infections or inflammatory diseases was not obtained. This could have potentially influenced the levels of complement components in the blood. Anna Stokowska   43 M i c e (I V , V ) To study the role of the complement system in synapse elimination in the hippocampus (paper V), homozygous mice deficient in C3 (C3 KO) were used (Pekna et al. 1998). They were backcrossed onto the C57BL/6 background for 13 generations to obtain mice congenic with the control wild type (WT) strain (Jackson Laboratories, Bar Harbor, ME, USA). Male mice of various age s were used for these experiments. For electrophysiology, 17 to 30 days old mice were used to cover the postnatal circuit refinement period, whereas learning behavior and epileptiform brain activity were assessed in 2.5 to 3 months old mice to study post-developmental consequ ences of altered synaptic plasticity as well as for practical reasons. Microglial activation was assessed at 6 months of age, to assess the potential occurrence of delayed or chronic differences between the strains. To study the role of C3aR in experimenta l stroke (paper IV), two sets of adult (7 to 9 months old) male mice were used to approximate the clinical situation, while avoiding age-related complications. In the first cohort, mice deficient in C3aR (C3aRKO ) and C3aRWT mice were used (Kildsgaard et al ., 2000). The second cohort consisted of transgenic mice expressing C3a under the control of glial fibrilary acidic protein (GFAP) promoter (C3a-GFAP) and their WT littermates which served as controls (Boos et al., 2004). All experiments on animals were performed in accordance with the guidelines of the local ethical committee for animal research at the University of Gothenburg or the Malmö -Lund Ethical Committee for the use of laboratory animals, according to the location of the experiment was carried out. All experiments were conducted in accordance with European Union directives on animal rights. E L I S A (I , II ) The enzyme- linked immunosorbent assay (ELISA) is an antibody -based assay commonly used to quantitatively detect a specific antigen in plasma or serum. In the current thesis, sandwich ELISAs were used. The principle of the assay consist in applying diluted plasma sample onto microtitter plate previously coated with capture Comple m ent in stroke and neural plasticity   44 antibodies directed against target protein and blocked to prevent unspecific binding of the analyte to the plate surface. Then the plate is washed to remove the unbound antigen followed by incubation with enzyme -linked detection antibody. After subsequent washing, the substrate is added, which is converted by the enzyme to the detectable form. Quantification is performed by absorbance measurements which are proportional to the antigen concentration in the sample and comparison against the standard values, i.e. values obtained from a serially diluted sample of known concentration of the target protein. Plasma C3 levels were measured by an in-house developed assay using polyclonal rabbit anti-C3c immunoglobulins (Dako, Danemark) as capture antibodies and horseradish peroxidase -conjugated sheep (Biogenesis, UK) or horseradish peroxidase - conjugated rabbit (Dako) anti-C3c antibodies for detection. As a color substrate, 1,2-phenylendiamine-dihydrochloride (Sigma, MO, USA) was used and reaction product measured at 490 nm and compared against standard which consisted of pooled human serum. Plasma C3a levels were measured by a commercial assay kit according to the manufacturer’s recommendations (Quidel Corporation, CA, USA). To minimize the occurrence of errors in both pre -analytical as well as analytical phase, venous blood samples were taken into EDTA -coated tubes, according to the standardized protocol (Jood et al., 2005 ). For patients, sampling was performed within 10 days after admission to the stroke unit (median 4.25 days) and at three months, while for control blood was collected once. Obtained EDTA -plasma samples were aliquoted and stored at -80°C until the analysis. During the analytical procedures, the quality control measures included accurate pipetting preceded by vortexing of samples and inclusion of samples with known antigen concentration. Comment: Plasma samples were chosen over serum as obtaining serum requires blood clotting, which can result in the activation of complement cascade. Conversely, ED TA used in plasma preparation prevents activation of coagulation - as well as complement cascade enzymes by chelating their essential co - factors Ca 2+ and Mg 2+ ions. Plasma C3 levels reflect the net effect of C3 synthesis in the liver and its c onsumption due to complement cascade activation. Anti - C3 antibodies used in our studies recognize the Anna Stokowska   45 internal fragment of C3 α chain but do not discriminate between intact C3 and its activation and degradation products (C3 b , iC3 b or C3c), thus they detect total C3 content. In contrast, the extent of activation of complement cascade is estimated by measurements of C3a levels. Of note, as C3a half - life in plasma is extremely short rendering it nearly impossible to correctly measure its content, the assay is designed to detect the more stable C3a desArg form. The latter peptide is devoid of the classical anaphylatoxic properties but its levels faithfully reflect the extent of C3 cleavage due to cascade activation. G e n o t y p i n g (I I I ) DNA for genotyping of the single nucleotide polymorphisms (SNPs) in the C 3 locus, was extracted from venous whole blood using commercially available kits. In the C3 gene, 16 tagSNPs (HapMap project) selected to cover a large amount of the variations in the gene using as few SNPs as possible, were analyzed, including rs344555, which has previously been shown to associate with C3 plasma levels. Genotyping was performed as a part of the analysis of a larger panel of SNPs using the Golden Gate assay (Illumina Inc., San Diego, CA, USA). The SNP assay for rs3745565 failed, so this SNP was genotyped using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA). Genotyping was performed blinded to case/control status by professional genotyping facilities at the Uppsala Univers ity and University of Gothenburg. Comment: The tagSNP strategy takes advantage of the phenomenon of linkage disequilibrium in the human genome and allows for informative analysis of putative associations for many SNPs without the need for genotyping all of them ( Barrett and Cardon, 200 6 ) . However, certain SNPs, which are indeed associated with the dise ase in question, may not be chosen as the tagSNP, although this information may be indirectly deduced. Additionally, high correlation of the remaining SNPs in the region with the tagSNP, allows for determination of haplotype associations, which might be mo re biologically informative than the association of a single marker. Comple m ent in stroke and neural plasticity   44 antibodies directed against target protein and blocked to prevent unspecific binding of the analyte to the plate surface. Then the plate is washed to remove the unbound antigen followed by incubation with enzyme -linked detection antibody. After subsequent washing, the substrate is added, which is converted by the enzyme to the detec table form. Quantification is performed by absorbance measurements which are proportional to the antigen concentration in the sample and comparison against the standard values, i.e. values obtained from a serially diluted sample of known concentration of the target protein. Plasma C3 levels were measured by an in-house developed assay using polyclonal rabbit anti-C3c immunoglobulins (Dako, Danemark) as capture antibodies and horseradish peroxidase -conjugated sheep (Biogenesis, UK) or horseradish peroxidase - conjugated rabbit (Dako) anti-C3c antibodies for detection. As a color substrate, 1,2-phenylendiamine-dihydrochloride (Sigma, MO, USA) was used and reaction product measured at 490 nm and compared against standard which consisted of pooled human serum. Plasma C3a levels were measured by a commercial assay kit according to the manufacturer’s recommendations (Quidel Corporation, CA, USA). To minimize the occurrence of errors in both pre -analytical as well as analytical phase, venous blood samples were taken into EDTA -coated tubes, according to the standardized protocol (Jood et al., 2005 ). For patients, sampling was performed within 10 days after admission to the stroke unit (median 4.25 days) and at three months, while for control blood was collected once. Obtained EDTA -plasma samples were aliquoted and stored at -80°C until the analysis. During the analytical procedures, the quality control measures included accurate pipetting preceded by vortexing of samples and inclusion of samples with known antigen concentration. Comment: Plasma samples were chosen over serum as obtaining serum requires blood clotting, which can result in the activation of complement cascade. Conversely, ED TA used in plasma preparation prevents activation of coagulation - as well as complement cascade enzymes by chelating their essential co - factors Ca 2+ and Mg 2+ ions. Plasma C3 levels reflect the net effect of C3 synthesis in the liver and its c onsumption due to complement cascade activation. Anti - C3 antibodies used in our studies recognize the Anna Stokowska   45 internal fragment of C3 α chain but do not discriminate between intact C3 and its activation and degradation products (C3 b , iC3 b or C3c), thus they detect total C3 content. In contrast, the extent of activation of complement cascade is estimated by measurements of C3a levels. Of note, as C3a half - life in plasma is extremely short rendering it nearly impossible to correctly measure its content, the assay is designed to detect the more stable C3a desArg form. The latter peptide is devoid of the classical anaphylatoxic properties but its levels faithfully reflect the extent of C3 cleavage due to cascade activation. G e n o t y p i n g (I I I ) DNA for genotyping of the single nucleotide polymorphisms (SNPs) in the C 3 locus, was extracted from venous whole blood using commercially available kits. In the C3 gene, 16 tagSNPs (HapMap project) selected to cover a large amount of the variations in the gene using as few SNPs as possible, were analyzed, including rs344555, which has previously been shown to associate with C3 plasma levels. Genotyping was performed as a part of the analysis of a larger panel of SNPs using the Golden Gate assay (Illumina Inc., San Diego, CA, USA). The SNP assay for rs3745565 failed, so this SNP was genotyped using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA). Genotyping was performed blinded to case/control status by professional genotyping facilities at the Uppsala University and University of Gothenburg. Comment: The tagSNP strategy takes advantage of the phenomenon of linkage disequilibrium in the human genome and allows for informative analysis of putative associations for many SNPs without the need for genotyping all of them ( Barrett and Cardon, 200 6 ) . However, certain SNPs, which are indeed associated with the disease in question, may not be chosen as the tagSNP, although this information may be indirectly deduced. Additionally, high correlation of the remaining SNPs in the region with the tagSNP, allows for determination of haplotype associations, which might be mo re biologically informative than the association of a single marker. Comple m ent in stroke and neural plasticity   46 E x p e r i m e n t a l st r o k e m o d e l (I V ) Cortical phothrombotic stroke was induced using the Rose Bengal method (Watson et al., 1985; Lee et al., 2007 ). The procedure requires anaesthetizing the animal with isoflurane and securing its head in a stereotaxic frame. Midline incision in the scalp is made to expose the scull surface. Photosensitive dye Rose Bengal is injected intraperitoneally (i.p.) and the target cortical area (in this study: left forelimb sensorimotor region) is illuminated through the intact skull for 12 min. As a consequence, photosensitive dye in the blood becomes focally activated by a cold light beam, which leads to production of singlet oxygen (a form of ROS). This leads to endothelial activation and formation of platelet-rich thrombi in the vessels within the illuminated area. Followin g illumination, scalp is sutured and animal is placed in a heated chamber until full recovery from anesthesia. Upon returning to their home cage, animals were provided with moist mashed food in a dish placed on the floor of the cage to encourage eating. Animals received 1 ml of sterile saline i.p. to replenish lost fluids 24 h after stroke if they lost more than 2 g of weight overnight. Comment: This stroke model is characterized by high reproducibility in the size and location of the infarct. Small cortical infarcts, producing deficits in the fine functions of the limbs represent a good model of clinical post- stroke rehabilitation. As opposed to ischemic stroke induced by classical middle cerebral artery occlusion (MCAo), the photothrombotic stroke model is minimally invasive and leads to an infarct with a clearly defined border. For these reasons, photothrombotic stroke model seems to be well suited for studies on post- stroke plasticity and regeneration without evoking an extensive inflammatory response, w hich may interfere with assessment of regenerative processes. However this form of cerebral injury does not allow significant collateral blood flow around the infarct and results in a relatively narrow penumbra region. Further, it does not permit recanalization, which occurs commonly in humans either spontaneously or as a result of tPA treatment. Anna Stokowska   47 B r d U ad m i n i s t r a t i o n (I V ) To be able to detect newly-born neurons, mice were injected with thymidine analogue bromo-deoxyuridine (BrdU). Systemic BrdU administrati on is a common way of labeling proliferating cells in vivo. The method relies on the incorporation of BrdU into DNA of dividing cells and its subsequent immunohistochemical detection in the nuclei of their daughter cells. Animals were injected with 50 mg/k g BrdU in saline i.p. twice a day, 8 h apart. First injection was given 24 h - and the last one on day 7 after stroke induction, as neurogenesis has been shown to peak during the first week after ischemic stroke in rodents. Comment: A potential problem wit h BrdU labeling is that this analogue can be taken- up by the apoptotic cells during the aberrant cell cycle re - entry to attempt the DNA repair. However, the extent of this type of labeling is very low in comparison with the amount of BrdU incorporated in t he process of DNA replication during normal cell division. Further, the labeled damaged cells do not last as long as the survival period for the animals used in our studies ( Kuan et al., 20 0 4 ) . Additionally, BrdU is available in the body for around 2 h after administration and high doses of this drug have been shown to be toxic to the neural stem cells ( Ross et al., 20 0 8 ) . There fore, two smaller doses were chosen instead of a single large dose to minimize the potential toxicity and maximize the effectiveness of BrdU uptake. I m m u n o h i s t o c h e m i s t r y an d flu o r e s c e n t - d y e ne u r o n lo a d i n g (I V , V ) Tissue preparation Animals were deeply anaesthetized with sodium pentobarbital and transcardially perfused with saline followed by ice-cold 4% paraformaldehyde solution in phosphate buffer saline (PBS) 21 days after stroke induction. Brains were removed and immersed in the same fixative at 4ºC for 16 to 24 h. In paper IV, brains were dehydrated in graded series of ethanol washes, cleared with xylene, embedded in paraffin and cut coronally in 6µm thick slices. In paper V, fixed brains were cryoprotected by infusion with 30% sucrose in 0.1 M phosphate buffer (PB) for 3 days at 4ºC, frozen and cut into 30 µ m thick floating sections. Comple m ent in stroke and neural plasticity   46 E x p e r i m e n t a l st r o k e m o d e l (I V ) Cortical phothrombotic stroke was induced using the Rose Bengal method (Watson et al., 1985; Lee et al., 2007 ). The procedure requires anaesthetizing the animal with isoflurane and securing its head in a stereotaxic frame. Midline incision in the scalp is made to expose the scull surface. Photosensitive dye Rose Bengal is injected intraperitoneally (i.p.) and the target cortical area (in this study: left forelimb sensorimotor region) is illuminated through the intact skull for 12 min. As a consequence, photosensitive dye in the blood becomes focally activated by a cold light beam, which leads to production of singlet oxygen (a form of ROS). This leads to endothelial activation and formation of platelet-rich thrombi in the vessels within the illuminated area. Followin g illumination, scalp is sutured and animal is placed in a heated chamber until full recovery from anesthesia. Upon returning to their home cage, animals were provided with moist mashed food in a dish placed on the floor of the cage to encourage eating. Animals received 1 ml of sterile saline i.p. to replenish lost fluids 24 h after stroke if they lost more than 2 g of weight overnight. Comment: This stroke model is characterized by high reproducibility in the size and location of the infarct. Small cortic al infarcts, producing deficits in the fine functions of the limbs represent a good model of clinical post - stroke rehabilitation. As opposed to ischemic stroke induced by classical middle cerebral artery occlusion (MCAo), the photothrombotic stroke model i s minimally invasive and leads to an infarct with a clearly defined border. For these reasons, photothrombotic stroke model seems to be well suited for studies on post - stroke plasticity and regeneration without evoking an extensive inflammatory response, w hich may interfere with assessment of regenerative processes. However this form of cerebral injury does not allow significant collateral blood flow around the infarct and results in a relatively narrow penumbra region. Further, it does not permit recanaliz ation, which occurs commonly in humans either spontaneously or as a result of tPA treatment. Anna Stokowska   47 B r d U ad m i n i s t r a t i o n (I V ) To be able to detect newly-born neurons, mice were injected with thymidine analogue bromo-deoxyuridine (BrdU). Systemic BrdU administration is a common way of labeling proliferating cells in vivo. The method relies on the incorporation of BrdU into DNA of dividing cells and its subsequent immunohistochemical detection in the nuclei of their daughter cells. Animals were injected with 50 mg/kg BrdU in saline i.p. twice a day, 8 h apart. First injection was given 24 h - and the last one on day 7 after stroke induction, as neurogenesis has been shown to peak during the first week after ischemic stroke in rodents. Comment: A potential problem wit h BrdU labeling is that this analogue can be taken- up by the apoptotic cells during the aberrant cell cycle re - entry to attempt the DNA repair. However, the extent of this type of labeling is very low in comparison with the amount of BrdU incorporated in the process of DNA replication during normal cell division. Further, the labeled damaged cells do not last as long as the survival period for the animals used in our studies ( Kuan et al., 20 0 4 ) . Additionally, BrdU is available in the body for around 2 h after administration and high doses of this drug have been shown to be toxic to the neural stem cells ( Ross et al., 20 0 8 ) . There fore, two smaller doses were chosen instead of a single large dose to minimize the potential toxicity and maximize the effectiveness of BrdU uptake. I m m u n o h i s t o c h e m i s t r y an d flu o r e s c e n t - d y e ne u r o n lo a d i n g (I V , V ) Tissue preparation Animals were deeply anaesthetized with sodium pentobarbital and transcardially perfused with saline followed by ice-cold 4% paraformaldehyde solution in phosphate buffer saline (PBS) 21 days after stroke induction. Brains were removed and immersed in the same fixative at 4ºC for 16 to 24 h. In paper IV, brains were dehydrated in graded series of ethanol washes, cleared with xylene, embedded in paraffin and cut coronally in 6µm thick slices. In paper V, fixed brains were cryoprotected by infusion with 30% sucrose in 0.1 M phosphate buffer (PB) for 3 days at 4ºC, frozen and cut into 30 µ m thick floating sections. Comple m ent in stroke and neural plasticity   48 Live hippocampal slices after patch-clamp recordings and concomitant neuron loading with neurobiotin (paper V) were fixed in 4% paraformaldehyde in PBS at 4°C overnight. Co mment: The advantage of using free - floating sections is that it allows deep penetration of antibody into the tissue and thus enables subsequent three - dimensional visualization of structures in thicker slices. On the other hand, paraffin embedding enables better preservation of the tissue morphology. This was important in the experimental stroke study as the fragile infarcted tissue is otherwise easily broken during sectioning or staining. Paraffin sections do not require special storage conditions and remain suitable for staining for many years. However, some antigens do not tolerate paraformaldehyde fixation and paraffin infiltration and may not be detectable on paraffin sections even after antigen retrieval. Im munofluorescent staining For the detection of post -stroke born neurons as well as synapses and axonal growth cones in the peri-infarct cortex (paper IV), brain sections were deparaffinized and underwent heat-induced antigen retrieval by intermittent gentle boiling in citrate buffer for total of 15 min. This step breaks the protein cross-links generated during fixation and is required for the unmasking of some antigens to be efficiently detected with antibodies. After cooling down and washing, unspecific protein binding was blocked by 30 min incubation at room temperature (RT) in PBS containing 0.05% Tween and 1 % bovine serum albumin (BSA) or 4% normal donkey serum (for BrdU/NeuN staining). For the quantification of microglia in the hippocampus (paper V), floating sections were washed and blocked with 5 % normal goat serum and 0.25 % Triton X -100 in PBS for 1 h at RT. Next, sections were incubated overnight at 4ºC with primary antibodies diluted in the respective blocking buffer. Following washing, sections were incubated with appropriate biotinylated or fluorescently-tagged secondary antibodies for 2 h at RT. Additional 1 h incubation with fluorescent dye-conjugated streptavidin was performed if the previous step involved biotin. Using the three -stage staining, less abundant antigens or antigens present in very small structures can be readily detected owing to the signal amplification Anna Stokowska   49 conveyed by each additional deposition step. After final washing, sections were mounted and coverslipped. For the dendritic spine quantification, fixed hippocampal slices were washed with PBS and permeabilized with 0.01% Triton -X in PBS for 1 h at RT. After washing in PBS, slices were incubated with dye-conjugate in PBS for 3 h, followed by additional washing, mounting and coverslipping. For detailed information about antib odies and conjugates used for staining, see Table 1.   Table 1. Antibodies and dye-conjugates used for tissue stainings Comment: Im munofluorescence is a sensitive method and a n invaluable tool in determination of cell morphology and the localizati on of cells or subcellular structures. It is based on specific binding of the primary antibody to the antigen in the tissue and its subsequent visualization with fluorescent dye - conjugated secondary or tertiary reagent. As op posed to bright - field immunohistochemistry, it allows for the detection of more than one type of antigen in the same tissue section using different fluorophores thus extending the possibilities of the characterization of cells and other structures in the t issues. It also allows for clear visualization of very fine structures even in thick sections due to Target structure Primary antibody (company) Dilution Secondary antibody (company) Dilution Fluorescent conjugate (company) Dilution rat anti-BrdU (AbD Serotec) 1:150 donk ey anti-rat- Alexa Fluor 488 (Invitrogen) 1:500 post-stroke - born mature neurons mouse anti- NeuN -biotin (Millipore) 1:100 streptavidin -Cy3 (Sigma) 1:100 pre-synaptic terminals goat anti - synapsin I a+b (Santa Cruz) 1:150 donkey anti -goat - biotin (Jackson Research Lab) 1:200 streptavid in-Cy3 (Sigma) 1:100 axonal growth cones mouse anti- GAP -43 (Millipore) 1:1000 rabbit anti- mouse-biotin (Dako) 1:200 streptavidin -Cy3 (Sigma) 1:100 microglia rabbit anti- Iba1 (Wako) 1:1000 goat anti -rabbit- Alexa Fluor 488 (Invitrogen) 1:200 dendritic spines on neurobiotin- filled neurons streptavidin - Alexa Fluor 488 (Invitrogen) 1:1000 Comple m ent in stroke and neural plasticity   48 Live hippocampal slices after patch-clamp recordings and concomitant neuron loading with neurobiotin (paper V) were fixed in 4% paraformaldehyde in PBS at 4°C overnight. Co mment: The advantage of using free - floating sections is that it allows deep penetration of antibody into the tissue and thus enables subsequent three - dimensional visualization of structures in thicker slices. On the other hand, paraffin embedding enables b etter preservation of the tissue morphology. This was important in the experimental stroke study as the fragile infarcted tissue is otherwise easily broken during sectioning or staining. Paraffin sections do not require special storage conditions and remai n suitable for staining for many years. However, some antigens do not tolerate paraformaldehyde fixation and paraffin infiltration and may not be detectable on paraffin sections even after antigen retrieval. Im munofluorescent staining For the detection of post-stroke born neurons as well as synapses and axonal growth cones in the peri-infarct cortex (paper IV), brain sections were deparaffinized and underwent heat-induced antigen retrieval by intermittent gentle boiling in citrate buffer for total of 15 min. This step breaks the protein cross-links generated during fixation and is required for the unmasking of some antigens to be efficiently detected with antibodies. After cooling down and washing, unspecific protein binding was blocked by 30 min incubation at room temperature (RT) in PBS containing 0.05% Tween and 1 % bovine serum albumin (BSA) or 4% normal donkey serum (for BrdU/NeuN staining). For the quantification of microglia in the hippocampus (paper V), floating sections were washed and blocked with 5 % normal goat serum and 0.25 % Triton X -100 in PBS for 1 h at RT. Next, sections were incubated overnight at 4ºC with primary antibodies diluted in the respective blocking buffer. Following washing, sections were incubated with appropriate biotinylated or fluorescently-tagged secondary antibodies for 2 h at RT. Additional 1 h incubation with fluorescent dye-conjugated streptavidin was performed if the previous step involved biotin. Using the three -stage staining, less abundant antigens or antigens present in very small structures can be readily detected owing to the signal amplification Anna Stokowska   49 conveyed by each additional deposition step. After final washing, sections were mounted and coverslipped. F or the dendritic spine quantification, fixed hippocampal slices were washed with PBS and permeabilized with 0.01% Triton -X in PBS for 1 h at RT. After washing in PBS, slices were incubated with dye-conjugate in PBS for 3 h, followed by additional washing, mounting and coverslipping. For detailed information about antibodies and conjugates used for staining, see Table 2.   Table 2. Antibodies and dye-conjugates us ed for tissue stainings Comment: Im munof luorescence is a sensitive method and an invaluable tool in determination of cell morphology and the localizatio n of cells or subcellular structures. It is based on specific binding of the primary antibody to the antigen in the tissue and its subsequent visualization with fluorescent dye - conjugated secondary or tertiary reagent. As opposed to bright - field immunohistochemistry, it allows for the detection of more than one type of antigen in the same tissue section using different fluorophores thus extending the possibilities of the characterization of cells and other structures in the t issues. It also allows for clear visualization of very fine structures even in thick sections due to Target structure Primary antibody (company) Dilution Secondary antibody (company) Dilution Fluorescent conjugate (company) Dilution rat anti-BrdU (AbD Serotec) 1:150 donk ey anti-rat- Alexa Fluor 488 (Invitrogen) 1:500 post-stroke - born mature neurons mouse anti- NeuN- biotin (Millipore) 1:100 streptavidin- Cy3 (Sigma) 1:100 pre-synaptic terminals goat anti- synapsin I a+b (Santa Cruz) 1:150 donkey anti-goat- biotin (Jackson Research Lab) 1:200 streptavid in-Cy3 (Sigma) 1:100 axonal growth cones mouse anti- GAP -43 (Millipore) 1:1000 rabbit anti- mouse-biotin (Dako) 1:200 streptavidin- Cy3 (Sigma) 1:100 microglia rabbit anti- Iba1 (Wako) 1:1000 goat anti- rabbit- Alexa Fluor 488 (Invitrogen) 1:200 dendritic spines on neurobiotin- filled neurons streptavidin- Alexa Fluor 488 (Invitrogen) 1:1000 Comple m ent in stroke and neural plasticity   50 adjustable high signal/background ratio. The disadvantage of using fluorescence as a detection system in comparison to light - stable chromogen, is the fact that it fades with time especially upon prolonged imaging. The quality of the staining depends on many factors and staining protocol needs to be optimized for each antigen and tissue. A negative control, most commonly consisting in the omission of the primary antibody, needs to be included in every assay to assess specificity. Eva l u a t i o n of th e in f a r c t vo l u m e (I V ) Infarct volume at 21 days post-stroke was evaluated on every 20th section containing the infarct (12-16 sections per mouse). After deparaffiniz ation, sections were rehydrated and incubated in hematoxylin (which stains basophilic cell nuclei violet/blue) followed by eosin solution (which stains cytoplasm and extracellular deposits) for 2 min each with washing in water in between (HE stain). Next, sections were dehydrated in ethanol and isopropanol, cleared in xylene and coverslipped. Infarct size was evaluated morphometrically on digital bright-field microscopic images (Eclipse 80i, Nikon, Japan) with ImageJ software (NIH, USA) by manual delineatio n of the infarct and hemisphere areas by an investigator blinded to the experimental group. Volume of the injury was derived by multiplying area of total tissue loss that includes shrinkage due to scarring [(contralateral hemisphere - ipsilateral hemisphere) + infarcted tissue] on each section by the total inter-section distance. Comment: HE staining is a simple and widely-used method of evaluating morphology on tissue sections. Clear infarct border created by a pronounced glial scar after photothrombotic stoke and negligible cytoplasmatic staining of the necrotic core enables straightforward and reliable delineation of the infarcted brain tissue. Measuring the area of the infarct on a few sections but within fixed coordinates with regard to bregma is genera lly used in experimental stroke studies (especially in MCAo models). However, in the present injury model with highly controlled location and clean border, analyzing sections spanning the entire infarct was considered a more accurate method. Anna Stokowska   51 Q u a n t i t a t i v e an a l y s i s of im m u n o s t a i n i n g s (V I , V ) All image acquisitions and quantifications were performed by an investigator blinded to the genetic background of the animals. Cell counting (IV , V ) BrdU + /NeuN + cells (paper IV) were counted on images obtained by a confo cal microscope (LSM TCS SP2, Leica Microsystems, Germany), using 40x objective, on 12 - 16 sections (separated by 120 µm intervals) per mouse. The results were expressed as cell density, obtained by dividing cell numbers by volume of tissue analyzed. The Iba1+ cell counting (paper V) was performed bilateral in 6 to 8 hippocampal sections per animal. Cells were counted in the dentate hilus and the granule cell layer (GCL) and the subgranular zone (SGZ), the latter defined as two cell body diameters below the G CL, using an epifluorescence microscope. Peri- infarct synap togenesis and axonal sprouting ( IV ) Highest signal intensity single plane images of sections stained for synapsin I and GAP -43 were obtained by laser scanning confocal microscope (LSM TCS SP2) with 63x objective. Images were taken from two adjacent but not overlapping optical fields (referred to as proximal and distal) in several regions of the peri -infarct cortex and dorsal striatum. Corresponding images were taken in the contralateral hemisphere. Three standard sections per animal spaced by 160 µm were analyzed. All sections were scanned with the same acquisition parameters and images were analyzed with MetaMorph® software (Molecular Devices, USA) to obtain average number, size and intensity of positive punctate structures per image. Comment: Because of the small size and high numbers of axonal terminals and synaptic puncta, automated high content image analysis (H C IM) was used for unbiased quantifications. Such an approach is highly reliable as opposed to meas uring the total intensity of pixels per image. Artefacts from staining and microfolds of the tissue usually have very high intensity, seriously confounding the whole - image measurements. Conversely, by assessing the average value of parameters of the indivi dual round - shaped Comple m ent in stroke and neural plasticity   50 adjustable high signal/background ratio. The disadvantage of using fluorescence as a detection system in comparison to light - stable chromogen, is the fact that it fades with time especially upon prolonged imaging. The quality of the staining depends on many factors and staining protocol needs to be optimized for each antigen and tissue. A negative control, most commonly consisting in the omission of the prim ary antibody, needs to be included in every assay to assess specificity. Eva l u a t i o n of th e in f a r c t vo l u m e (I V ) Infarct volume at 21 days post-stroke was evaluated on every 20th section containing the infarct (12-16 sections per mouse). After deparaffiniz ation, sections were rehydrated and incubated in hematoxylin (which stains basophilic cell nuclei violet/blue) followed by eosin solution (which stains cytoplasm and extracellular deposits) for 2 min each with washing in water in between (HE stain). Next, sections were dehydrated in ethanol and isopropanol, cleared in xylene and coverslipped. Infarct size was evaluated morphometrically on digital bright-field microscopic images (Eclipse 80i, Nikon, Japan) with ImageJ software (NIH, USA) by manual delineatio n of the infarct and hemisphere areas by an investigator blinded to the experimental group. Volume of the injury was derived by multiplying area of total tissue loss that includes shrinkage due to scarring [(contralateral hemisphere - ipsilateral hemisphere) + infarcted tissue] on each section by the total inter-section distance. Comment: HE staining is a simple and widely - used method of evaluating morphology on tissue sections. Clear infarct border created by a pronounced glial scar after photothrombotic s toke and negligible cytoplasmatic staining of the necrotic core enables straightforward and reliable delineation of the infarcted brain tissue. Measuring the area of the infarct on a few sections but within fixed coordinates with regard to bregma is genera lly used in experimental stroke studies (especially in MCAo models). However, in the present injury model with highly controlled location and clean border, analyzing sections spanning the entire infarct was considered a more accurate method. Anna Stokowska   51 Q u a n t i t a t i v e an a l y s i s of im m u n o s t a i n i n g s (V I , V ) All image acquisitions and quantifications were performed by an investigator blinded to the genetic background of the animals. Cell counting (IV , V ) BrdU + /NeuN + cells (paper IV) were counted on images obtained by a confo cal microscope (LSM TCS SP2, Leica Microsystems, Germany), using 40x objective, on 12 - 16 sections (separated by 120 µm intervals) per mouse. The results were expressed as cell density, obtained by dividing cell numbers by volume of tissue analyzed. The Iba1+ cell counting (paper V) was performed bilateral in 6 to 8 hippocampal sections per animal. Cells were counted in the dentate hilus and the granule cell layer (GCL) and the subgranular zone (SGZ), the latter defined as two cell body diameters below the G CL, using an epifluorescence microscope. Peri- infarct synaptogenesis and axonal sprouting (IV ) Highest signal intensity single plane images of sections stained for synapsin I and GAP -43 were obtained by laser scanning confocal microscope (LSM TCS SP2) with 63x objective. Images were taken from two adjacent but not overlapping optical fields (referred to as proximal and distal) in several regions of the peri -infarct cortex and dorsal striatum. Corresponding images were taken in the contralateral hemisphere. Three standard sections per animal spaced by 160 µm were analyzed. All sections were scanned with the same acquisition parameters and images were analyzed with MetaMorph® software (Molecular Devices, USA) to obtain average number, size and intensity of positive punctate structures per image. Comment: Because of the small size and high numbers of axonal terminals and synaptic puncta, automated high content image analysis (H C IM) was used for unbiased quantifications. Such an approach is highly reliable as opposed to measuring the total intensity of pixels per image. Artefacts from staining and microfolds of the tissue usually have very high intensity, seriously confounding the whole - image measurements. Conversely, by assessing the average value of parameters of the indivi dual round - shaped Comple m ent in stroke and neural plasticity   52 structures, this problem is largely eliminated due to these unwanted objects being automatically excluded as not fulfilling the set criteria. Moreover, such analysis is more informative as size of the puncta is related to number of pre - sy naptic vesicles in terminals or number of boutons in multiple - synapses. The key point o f this approach is to ensure homogenous conditions of staining and image acquisition, however even somewhat uneven intensity level of the staining would affect the other parameters only negligibly. Dendritc spines (V) Images of neurobiotin-filled neurons were obtained by a confocal microscope (LSM 700, Zeiss, Germany) at 63x magnification. Dendritic spines on apical secondary and tertiary dendrites of hippocampal CA1 pyramidal neurons were counted manually with the help of ImageJ software (NIH, USA). Spine densities were calculated as the number of spines divided by the length of the segment to approximate the density of excitatory synapses, which preferentially localize to the spines. E l e c t r o p h y s i o l o g y (V ) Acute slice preparation Mice were anesthetized with isoflurane and decapitated. Brains were rapidly removed and placed in ice-cold preparation solution to lower the tissue metabolism and thus increase neuronal survival. Transverse hippocampal slices (400 µm thick) were cut with a vibratome in the same ice-cold solution. Slices were subsequently stored in artificial cerebrospinal fluid (ACSF) at 25 ºC for a minimum of 1 h. For the recordings, a single slice was transferred to a recording chamber where it was kept submerged in a constant flow of perfusion solution at 25-28 ºC. Calcium and magnesium chloride content in the perfusion solution varied between the different experimental paradigms depending on the purpose: in the single and paired pulse experiments, the solution contained physiological (2 mM) concentration of CaCl2 and MgCl2 each, while in the MK -801 experiments, the solution contained 3 mM CaCl 2 and no MgCl2 to enable NMDA receptor opening. In the whole-cell patch-clamp recordings and the burst experiments, the solution contained higher dicationic salt concentration, 4 mM each of CaCl2 and MgCl2, to suppress spontaneous network activity. Picrotoxin was always present in the perfusion Anna Stokowska   53 solution to block GABA receptor -mediated activity. All solutions were continuously bubbled with 95% O 2 and 5% CO 2 (pH 7.4). Comment: Hippocampal slices provide distinct experimental advantages over other in vitro and in vivo preparations of the hippocampus. The most significant advantages are that, except for the absence of afferent input, hippocampal slice preparations retain the cytoarchitecture and synaptic circuits of the intact hippocampus, yet are readily accessible for optical imaging or electrophysiological studies that can also include phar macological manipulation. Extracellular field recordings For field excitatory postsynaptic potential (fEPSP) recordings, electrical stimulation and recordings of synaptic responses were carried out in the CA1 region of the hippocampus with the stimulation and recording electrode positioned in the stratum radiatum . Biphasic constant current stimuli (at varying frequency dependent on the experiment type) were delivered via tungsten wires to the axons of the pyramidal cells of the CA3 area, called the Shaffer collaterals, while a glass pipette, placed in the layer of apical dendrites of the pyramidal CA1 neurons, was used for field recordings. The stimulation intensity was set such that it would not evoke firing in the postsynaptic neurons, as evidenced by the absence of a population spike distorting the fEPSP. Evoked responses were analyzed off -line in a blinded manner using custom-made IGOR -Pro software (WaveMetrics , Lake Oswego, OR, USA) Input/output measurements (synaptic efficacy) Synaptic efficacy evaluation in C3 KO and WT mice was performed by input/output measurements after pulse stimulation of 0.2 Hz. In this type of experiment, fEPSP, that is the response of the population of activated synapses, is preceded by a pre-synaptic fiber volley, the amplitude of which is proportional to the number of stimulated axons. Plotting the magnitude of fEPSP against size of the fiber volley at the different stimulation intensities allows for calculation of total synaptic efficacy per axon. Comple m ent in stroke and neural plasticity   52 structures, this problem is largely eliminated due to these unwanted objects being automatically excluded as not fulfilling the set criteria. Moreover, such analysis is more informative as size of the puncta is related to number of pre - sy naptic vesicles in terminals or number of boutons in multiple - synapses. The key point o f this approach is to ensure homogenous conditions of staining and image acquisition, however even somewhat uneven intensity level of the staining w ould affect the other parameters only negligibly. Dendritc spines ( V) Images of neurobiotin-filled neurons were obtained by a confocal microscope (LSM 700, Zeiss, Germany) at 63x magnification. Dendritic spines on apical secondary and tertiary dendrites of hippocampal CA1 pyramidal neurons were counted manually with the help of ImageJ software (NIH, USA). Spine densities were calculated as the number of spines divided by the length of the segment to approximate the density of excitatory synapses, which preferentially localize to the spines. E l e c t r o p h y s i o l o g y (V ) Acute slice preparation Mice were anesthetized with isoflurane and decapitated. Brains were rapidly removed and placed in ice-cold preparation solution to lower the tissue metabolism and thus increase neuronal survival. Transverse hippocampal slices (400 µm thick) were cut with a vibratome in the same ice-cold solution. Slices were subsequently stored in artificial cerebrospinal fluid (ACSF) at 25 ºC for a minimum of 1 h. For the recordings, a single slice was transferred to a recording chamber where it was kept submerged in a constant flow of perfusion solution at 25-28 ºC. Calcium and magnesium chloride content in the perfusion solution varied between the different experimental paradigms depending on the purpose: in the single and paired pulse experiments, the solution contained physiological (2 mM) concentration of CaCl2 and MgCl2 each, while in the MK -801 experiments, the solution contained 3 mM CaCl 2 and no MgCl2 to enable NMDA receptor opening. In the whole-cell patch-clamp recordings and the burst experiments, the solution contained higher dicationic salt concentration, 4 mM each of CaCl2 and MgCl2, to suppress spontaneous network activity. Picrotoxin was always present in the perfusion Anna Stokowska   53 solution to block GABA receptor -mediated activity. All solutions were continuously bubbled with 95% O 2 and 5% CO 2 (pH 7.4). Comment: Hippocampal slices provide distinct experimental advantages over other in vitro and in vivo preparations of the hippocampus. The most significant advantages are that, except for the absence of afferent input, hippocampal slice preparations retain the cytoarchitecture and synaptic circuits of the intact hippocampus, yet are readily accessible for optical imaging or electrophysiological studies that can also include phar macological manipulation. Extracellular field recordings For field excitatory postsynaptic potential (fEPSP) recordings, electrical stimulation and recordings of synaptic responses were carried out in the CA1 region of the hippocampus with the stimulation and recording electrode positioned in the stratum radiatum . Biphasic constant current stimuli (at varying frequency dependent on the experiment type) were delivered via tungsten wires to the axons of the pyramidal cells of the CA3 area, called the Shaffer collaterals, while a glass pipette, placed in the layer of apical dendrites of the pyramidal CA1 neurons, was used for field recordings. The stimulation intensity was set such that it would not evoke firing in the postsynaptic neurons, as evidenced by the absence of a population spike distorting the fEPSP. Evoked responses were analyzed off- line in a blinded manner using custom-made IGOR -Pro software (WaveMetrics , Lake Oswego, OR, USA). Input/output measurements (synaptic efficacy) Synaptic efficacy evaluation in C3 KO and WT mice was performed by input/output measurements after pulse stimulation of 0.2 Hz. In this type of experiment, fEPSP, that is the response of the population of activated synapses, is preceded by a pre-synaptic fiber volley, the amplitude of which is proportional to the number of stimulated axons. Plotting the magnitude of fEPSP against size of the fiber volley at the different stimulation intensities allows for calculation of total synaptic efficacy per axon. Comple m ent in stroke and neural plasticity   54 Release probability measurements The differences in release probability (p), were first assessed by measuring the response ratio to paired pulses (50 ms apart) to obtain a straightforward measure of facilitation or depression. Next, recordings with an irreversible NMDAR open channel blocker, MK -801, were performed (Tocris Cookson, UK). In these experiments, in response to a 0.5 Hz -stimulation, the release of the synaptic vesicle results in the opening of the opposing NMDARs. The resulting EPSP is recorded but due to the presence of MK -801, channels become immediately blocked, preventing further flow of ions i.e. they are being turned-off in a release-dependent way. The probability of the neurotransmitter release is estimated in a nearly direct manner by evaluating the propensity of EPSP magnitude to decay. In this experiment, AMPARs were blocked by the presence of NBQX compound (Tocris Cookson). To mimic the in vivo situation where the isolated stimuli, used in the experiments described above, happen very rarely, fEPSP were measured also as a response to a burst stimulation. In each recording, 30 trains of 10 high-frequency (20 Hz) impulses were delivered at 30 s intervals (total of 300 pulses). Comment: The extracellular signal from a single neuron is extremely small and thus hard to record in the brain. However, in the hippocampus, neurons are arranged in such a way that they all receive synaptic inputs in the same area. Because these neurons are in the same orientation, the extracellular signals from synaptic excitation do not cancel out, but add up to giving a robust signal that can easily be recorded with a field electrode. The advantages of extracellular recordings are experimental simplicity, unchanged intracellular environment and generation of stable recordings over long period of time. A common obstacle, however is that the fEPSP often becomes distorted by back propagating action potentials. To overcome this problem, the initial slope of the EPSP was measured instead of the amplitude of the burst response. Anna Stokowska   55 Intracellular recordings Whole cell patch -clamp recordings Patch-clamp technique is based on establishing a high resistance seal between the tip of glass pipette containing an electrode, and a cell, thus enabling control of the cell membrane voltage or current (Neher and Sakmann, 1976). In this thesis, by setting voltage over the membrane (”clamping the membrane potential”), miniature currents flowing over the cell membrane required to maintain that potential were measured and analysed in a blinded manner using Mini-analysis program (Synaptosoft, Decatur, GA, USA). Recordings were performed on visually identified neurons under the infrared differential-interference contrast microscope (Olympus BX51WI, Japan) . The recording pipette solution contained 0.5% of neurobiotin, which loaded the patched -clamped neuron (for subsequent visualization) during the r ecording. Comment: The major advantage of whole cell recordings is that they enable control the cellular environment and the assessment of the quantal (q) parameters of the neuron. However, patch - clamp recordings are technically demanding and more invasive (due to disruption of the intracellular milieu) than extracellular recordings, and can sometimes induce LTP ( Malinow and Tsien, 199 0 ) . In vivo ele c t r o e n c e p h a l o g r a p h y (V ) In order to detect possible spontaneous epileptiform activity, C3 KO and WT mice were unilateraly implanted with a recording electrode in the hippocampal CA3-CA1 region. EEG activity was recorded 10 days later, twice for 12 hours and evaluated for seizures and focal interictal spike activity by an investigator blind to the animal’s genotype. Beh a v i o r a l te s t i n g All tests were performed and scored by an investigator blinded to the genotype of the animals tested. Comple m ent in stroke and neural plasticity   54 Release probability measurements The differences in release probability (p), were first assessed by measuring the response ratio to paired pulses (50 ms apart) to obtain a straightforward measure of facilitation or depression. Next, recordings with an irreversible NMDAR open channel blocker, MK -801, were performed (Tocris Cookson, UK). In these experiments, in response to a 0.5 Hz -stimulation, the release of the synaptic vesicle results in the opening of the opposing NMDARs. The resulting EPSP is recorded but due to the presence of MK -801, channels become immediately blocked, preventing further flow of ions i.e. they are being turned-off in a release-dependent way. The probability of the neurotransmitter release is estimated in a nearly direct manner by evaluating the propensity of EPSP magnitude to decay. In this experiment, AMPARs were blocked by the presence of NBQX compound (Tocris Cookson). To mimic the in vivo situation where the isolated stimuli, used in the experiments described above, happen very rarely, fEPSP were measured also as a response to a burst stimulation. In each recording, 30 trains of 10 high-frequency (20 Hz) impulses were delivered at 30 s intervals (total of 300 pulses). Comment: The extracellular signal from a single neuron is extremely small and thus hard to record in the brain. However, in the hippocampus , neurons are arranged in such a way that they all receive synaptic inputs in the same area. Because these neur ons are in the same orientation, the extracellular signals from synaptic excitation do not cancel out, but add up to giving a robust signal that can easily be recorded with a field electrode. The advantages of extracellular recordings are experimental simp licity, unchanged intracellular environment and generation of stable recordings over long period of time. A common obstacle, however is that the fEPSP often becomes distorted by back propagating action potentials. To overcome this problem, the initial slop e of the EPSP was measured instead of the amplitude of the burst response. Anna Stokowska   55 Intracellular recordings Whole cell patch- clamp recordings Patch-clamp technique is based on establishing a high resistance seal between the tip of glass pipette containing an electrode, and a cell, thus enabling control of the cell membrane voltage or current (Neher and Sakmann, 1976). In this thesis, by setting voltage over the membrane (”clamping the membrane potential”), miniature currents flowing over the cell membrane required to maintain that potential were measured and analysed in a blinded manner using Mini-analysis program (Synaptosoft, Decatur, GA, USA). Recordings were performed on visually identified neurons under the infrared differential-interference contrast microscope (Olympus BX51WI, Japan). The recording pipette solution contained 0.5% of neurobiotin, which loaded the patched -clamped neuron (for subsequent visualization) during the r ecording. Comment: The major advantage of whole cell recordings is that they enable control the cellular environment and the assessment of the quantal (q) parameters of the neuron. However, patch - clamp recordings are technically demanding and more invasive (due to disruption of the intracellular milieu) than extracellular recordings, and can sometimes induce LTP ( Malinow and Tsien, 199 0 ) . In vivo ele c t r o e n c e p h a l o g r a p h y (V ) In order to detect possible spontaneous epileptiform activity, C3 KO and WT mice were unilateraly implanted with a recording electrode in the hippocampal CA3-CA1 region. EEG activity was recorded 10 days later, twice for 12 hours and evaluated for seizures and focal interictal spike activity by an investigator blind to the animal’s genotype. Beh a v i o r a l te s t i n g All tests were performed and scored by an investigator blinded to the genotype of the animals tested. Comple m ent in stroke and neural plasticity   56 E valuation of sensorimotor deficits (IV ) Spontaneous forelimb asymmetry task: Mouse was placed in a glass cylinder allowing it to stand on the base and small enough to encourage rearing. Mice were videotaped for 2 min with a mirror behind the cylinder to ensure good visibility from all directions. The number of times the right and the left forepaw were used to make first contact with the cylinder was recorded. Due to stroke-related deficits mice are expected to avoid using the right paw to support the body while rearing. Forelimb asymmetry index was calculated as the ratio of right (affected) first paw touches to total first paw touches [R/(R+L)]. Testing was performed at 48 h as well as at 7, 14 and 21 days after stroke. Adhesive removal: Circular adhesive stickers, cut in half, were used as bilateral tactile stimuli on the palmar surface of each forepaw of the mouse, and the time to remove each sticker was recorded. Mice were trained to readily remove both stickers within 15 seconds before stroke induction. Sensorimotor deficit due to stroke is expected to cause impaired perception and delayed removal of the sticker on the affected paw. Forelimb asymmetry was calculated as a ratio of time to remove the sticker from right (affected) paw to time to remove the sticker from the left paw [R/L]. Animals were tested 24 and 48 h as well as post-stroke day 7, 14 and 21. Comment: Behavioral tests are the invaluable tool for studying the effects of different factors on animals’ recovery from the experimental stroke. However, high variability of the individual performance within the group may easily pr eclude finding a between- group difference. Both of the tests used in this study depend on animal’s eagerness to explore and repeated testing may result in the decline in exploratory behavior. Additionally, stress related to the handling procedures (restraining prior adhesive removal test) may result in anxiety - like behavior, further increasing measurement variability. Evaluation of hippocampal - dependent cognitive performance (V) The IntelliCage® platform (New Behavior, Zurich, Switzerland) enabling unbias ed analysis of learning over time in a social context, in home cage environment was used to evaluate hippocampal function. First, mice were tested for baseline exploratory behavior Anna Stokowska   57 for 5 days. In the second experiment using different cohort of animals, mic e had an introduction period of 5 days in the IntelliCages, during which they were acclimatized to performing nose pokes to gain access to the water bottles. The introduction period was followed by a place learning period, for which each animal was randomized to one corner. This allocated corner was programmed as the correct corner while the other three corners were programmed as incorrect. The mice were only allowed to drink from the water bottles in the correct corner, where a nose poke would open a door and give them access to the water bottles. In the incorrect corners, the doors to the water bottles could not be opened by performing nose pokes. After 5 days, the animals were randomized to a new corner for the reversal learning. Food was provided ad libi tum during the experiments and mice were provided with shelters within the cage. Only the active (dark) period was analyzed. Comment: The great advantage of using the IntelliCage ® platform is that it eliminates stress resulting from handling and changing a nimals’ environment as e.g. in Morris water maze test. The system is fully automated, as mice are carrying a subcutaneous microtransponders to allow identification of individual animals and tracking of their behavior. However, drawing conclusions from the wealth of data generated by this system requires advanced statistical analysis. Besides, a number of considerations prior to the experiment as well as during data analysis are needed to avoid confounding the measures. For example, one should avoid testing too young animals to prevent two animals from being able to use the corner at the same time; exclude the long - lasting visits, which might indicate unintentional touching the sensor with the back while e.g. sleeping in the corner; or exclude animals with ve ry low frequency of visits and water licks which might indicate the animal’s failure to understand how the corners work or transponder registration failure. S t a t i s t i c s Different statistical approaches were employed throughout the studies depending on the nature of the data and the type of scientific question asked. Due to predominantly non-Gaussian distribution s of the clinical data (especially C3 and C3a measurement), almost exclusiv ely non-parametric statistics were used in the studies described in paper I Comple m ent in stroke and neural plasticity   56 E valuation of sensorimotor deficits (IV ) Spontaneous forelimb asymmetry task: Mouse was placed in a glass cylinder allowing it to stand on the base and small enough to encourage rearing. Mice were videotaped for 2 min with a mirror behind the cylinder to ensure good visibility from all directions. The number of times the right and the left forepaw were used to make first contact with the cylinder was recorded. Due to stroke-related deficits mice are expected to avoid using the right paw to support the body while rearing. Forelimb asymmetry index was calculated as the ratio of right (affected) first paw touches to total first paw touches [R/(R+L)]. Testing was performed at 48 h as well as at 7, 14 and 21 days after stroke. Adhesive removal: Circular adhesive stickers, cut in half, were used as bilateral tactile stimuli on the palmar surface of each forepaw of the mouse, and the time to remove each sticker was recorded. Mice were trained to readily remove both stickers within 15 seconds before stroke induction. Sensorimotor deficit due to stroke is expected to cause impaired perception and delayed removal of the sticker on the affected paw. Forelimb asymmetry was calculated as a ratio of time to remove the sticker from right (affected) paw to time to remove the sticker from the left paw [R/L]. Animals were tested 24 and 48 h as well as post-stroke day 7, 14 and 21. Comment: Behavioral tests are the invaluable tool for studying the effects of different factors on animals’ recovery from the experimental stroke. However, high variability of the individual performance within the group may easily pr eclude finding a between - group difference. Both of the tests used in this study depend on animal’s eagerness to explore and repeated testing may result in the decline in exploratory behavior. Additionally, stress related to the handling procedures (restrai ning prior adhesive removal test) may result in anxiety - like behavior, further increasing measurement variability. Evaluation of hippocampal - dependent cognitive performance (V) The IntelliCage® platform (New Behavior, Zurich, Switzerland) enabling unbias ed analysis of learning over time in a social context, in home cage environment was used to evaluate hippocampal function. First, mice were tested for baseline exploratory behavior Anna Stokowska   57 for 5 days. In the second experiment using different cohort of animals, mic e had an introduction period of 5 days in the IntelliCages, during which they were acclimatized to performing nose pokes to gain access to the water bottles. The introduction period was followed by a place learning period, for which each animal was randomized to one corner. This allocated corner was programmed as the correct corner while the other three corners were programmed as incorrect. The mice were only allowed to drink from the water bottles in the correct corner, where a nose poke would open a door and give them access to the water bottles. In the incorrect corners, the doors to the water bottles could not be opened by performing nose pokes. After 5 days, the animals were randomized to a new corner for the reversal learning. Food was provided ad libi tum during the experiments and mice were provided with shelters within the cage. Only the active (dark) period was analyzed. Comment: The great advantage of using the IntelliCage ® platform is that it eliminates stress resulting from handling and changing animals’ environment as e.g. in Morris water maze test. The system is fully automated, as mice are carrying a subcutaneous microtransponders to allow identification of individual animals and tracking of their behavior. However, drawing conclusions from the wealth of data generated by this system requires advanced statistical analysis. Besides, a number of considerations prior to the experiment as well as during data analysis are needed to avoid confounding the measures. For example, one should avoid testing too young animals to prevent two animals from being able to use the corner at the same time; exclude the long - lasting visits, which might indicate unintentional touching the sensor with the back while e.g. sleeping in the corner; or exclude animals with ve ry low frequency of visits and water licks which might indicate the animal’s failure to understand how the corners work or transponder registration failure. S t a t i s t i c s Different statistical approaches were employed throughout the studies depending on the nature of the data and the type of scientific question asked. Due to predominantly non-Gaussian distributions of the clinical data (especially C3 and C3a measurement), almost exclusiv ely non-parametric statistics were used in the studies described in paper I Comple m ent in stroke and neural plasticity   58 and II. Associations between complement levels and ischemic stroke or functional outcome after stroke (paper I and II) as well as between genetic variants of C3 SNPs and ischemic stroke or its subtypes (paper III) were analyzed by binary logistic regress ion (R statistical computing package v2.6, R DC Team, Vienna, Austria - http://www.R-­‐ project.org/   or SPSS v17.0, SPSS Inc. Chicago USA). To be able to practically determine the importance of the detected significant association, a measure of effect size is reported. To this end, odds ratios (ORs) and their confidence intervals (CIs) were estimated. All the association analyses were additionally adjusted for the effect of traditional vascular or outcome-related risk factors. Furthermore, the values of complement protein levels were divided into tertiles to obtain biologically informative unit of effect measures in the regression analyses. Associations between the genetic variants and C3 or C3a levels were estimated by linear regression with genotypes coded as ordinal categorical variables (R statistical computing package).. Values for complement measurements were first logarithmically transformed to obtain approximately normal distribution of the residuals, therefore back -calculated values of protein levels are reported instead of a difficult to interpret β coefficients of log-data. For the statistical analyses of all electrophysiology experiments (paper V), brain slices were treated as replicates, i.e., reported n equals number of slices analyzed. Input/output data were modeled by linear regression and the resulting functional coefficients were compared by Student’s t-test. Glutamate release probability in the MK - 801 experiment was modeled by non -linear regression (exponential decay function). Resulting best-fit curves for C3 KO and WT strains were com pared globally with the F - test, i.e., comparing the sum of squares of fits of two separate curves for each data set versus fitting one common curve for both strains. Additionally, the time constant of the decay was compared with t-test after adjusting for the correct number of degrees of freedom (GraphPad Prism, v5.0a, San Diego, CA, USA ). Behavioral stroke data (paper IV) were analyzed by two -way ANOVA (effect of time and strain) with repeated measurements, followed by Dunnett post-hoc tests (GraphPad Prism). For the analysis of different parameters measured in the IntelliCage (paper V), generalized estimating equations (GEE) were used to estimate the average response of the populations. These analyses were performed using SigmaStat 2.0 (SPSS Inc). Anna Stokowska   59 The remaining data were analyzed by unpaired t -tests between groups or paired t- tests within group (GraphPad Prism). For data sets with non -Gaussian distribution, non - parametric tests - Mann-Whitney U test and Wilcoxon signed rank test, respectively - were used. To avoid false positive results, Bonferroni correction was employed for testing with multiple pair-wise comparisons. Reported P- values are the corrected ones. In the genetic association study (paper III), correction for multiple testing was performed by multiplying the P-values by the number of haplotype blocks as Bonferroni correction would be an excessively conservative method due to genetic variants being dependent o n each other. Data are presented as mean ± SEM, or median ± interquartile ranges (IQRs), when data followed a non-Gaussian, unless stated otherwise. Comple m ent in stroke and neural plasticity   58 and II. Associations between complement levels and ischemic stroke or functional outcome after stroke (paper I and II) as well as between genetic variants of C3 SNPs and ischemic stroke or its subtypes (paper III) were analyzed by binary logistic regress ion (R statistical computing package v2.6, R DC Team, Vienna, Austria - http://www.R-­‐ project.org/   or SPSS v17.0, SPSS Inc. Chicago USA). To be able to practically determine the importance of the detected significant association, a measure of effect size is reported. To this end, odds ratios (ORs) and their confidence intervals (CIs) were estimated. All the association analyses were additionally adjusted for the effect of traditional vascular or outcome-related risk factors. Furthermore, the values of complement protein levels were divided into tertiles to obtain biologically informative unit of effect measures in the regression analyses. Associations between the genetic variants and C3 or C3a levels were estimated by linear regression with genotypes coded as ordinal categorical variables (R statistical computing package).. Values for complement measurements were first logarithmically transformed to obtain approximately normal distribution of the residuals, therefore back -calculated values of protein levels are reported instead of a difficult to interpret β coefficients of log-data. For the statistical analyses of all electrophysiology experiments (paper V), brain slices were treated as replicates, i.e. , reported n equals number of slices analyzed. Input/output data were modeled by linear regression an d the resulting functional coefficients were compared by Student’s t-test. Glutamate release probability in the MK - 801 experiment was modeled by non -linear regression (exponential decay function). Resulting best-fit curves for C3 KO and WT strains were com pared globally with the F - test, i.e., comparing the sum of squares of fits of two separate curves for each data set versus fitting one common curve for both strains. Additionally, the time constant of the decay was compared with t-test after adjusting for the correct number of degrees of freedom (GraphPad Prism, v5.0a , San Diego, CA, USA ). Behavioral stroke data (paper IV) were analyzed by two -way ANOVA (effect of time and strain) with repeated measurements, followed by Dunnett post-hoc tests (GraphPad Prism). For the analysis of different parameters measured in the IntelliCage (paper V), generalized estimating equations (GEE) were used to estimate the average response of the populations. These analyses were performed using SigmaStat 2.0 (SPSS Inc). Anna Stokowska   59 The remaining data were analyzed by unpaired t -tests between groups or paired t- tests within group (GraphPad Prism). For data sets with non- Gaussian distribution, non- parametric tests - Mann-Whitney U test and Wilcoxon signed rank test, respectively - were used. To avoid false positive results, Bonferroni correction was employed for testing with multiple pair-wise comparisons. Reported P- values are the corrected ones. In the genetic association study (paper III), correction for multiple testing was performed by multiplying the P-values by the number of haplotype blocks as Bonferroni correction would be an excessively conservative method due to genetic variants being dependent o n each other. Data are presented as mean ± SEM, or median ± interquartile ranges (IQRs), when data followed a non-Gaussian, unless stated otherwise. Comple m ent in stroke and neural plasticity   60 RESULTS AND DISCUSSION S y s t e m i c co m p l e m e n t re s p o n s e dif f e r s be t w e e n is c h e m i c st r o k e su b t y p e s (P a p e r s I an d II ). To date only a few and relatively small studies addressed the question of systemic complement activation during ischemic stroke while the reports on complement influence on functional outcome are even scarcer. Therefore, to evaluate the extent of complement response in ischemic stroke, we measured plasma C3 and C3a levels at the acute and delayed stage after stroke in patients classified to the four major ischemic stroke subtypes and compared them to the levels of the control subjects. We observed marked differences in the temporal pattern of plasma complement levels among patients suffering from ischemic stroke due to different etiologies. Plasma C3 levels were increased above the control level in all stroke groups in the acute phase while they remained elevated at three-month follow-up in LVD, SVD and cryptogenic but not CE stroke. Interestingly, only for the cryptogenic stroke patients the follow -up C3 levels did not decline as compared to acute stage, although this state was not associated with systemic elevation of C3a levels. In the remaining subtypes, C3a levels were still increased at three months after stroke. High (upper third) acute phase levels of both C3 and C3a were associated with CE and cryptogenic stroke independently of vascular risk factors, while high levels of C3 and C3a in both acute and convalescent phase showed independent association with the LVD and SVD stroke. Moreover, in the cryptogenic and CE stroke group, C3 levels at both time points were correlated with CRP levels. Levels of C3 -activation-derived C3a peptide were correlated with CRP levels at both time points only among the LVD patients. The finding that three-month levels (assumed to approximate the pre -stroke levels) of C3 and C3a were associated with patient status in LVD and SVD independently of traditional risk factors points to similarities between these subtypes, the chronic nature of the underlying pathology and the potential role of complement in this process. Indeed, atherosclerosis is often associated not only with LVD but also SVD stroke (Jung et al., 2012) and the complement system, and C3 in particular, is involved in the regulation of atherogenesis (Persson et al., 2004; Oksjoki et al ., 2007; Leung et al., 2009) . However, Anna Stokowska   61 with respect to the patterns of complement activation and the role of CRP, SVD and LVD subtypes appear to be two distinct entities as C3a and CRP levels correlate only in the LVD group. On the other hand, with regard to the correlation between systemic hsCRP and C3 levels, the cryptogenic stroke subtype appears to resemble CE subtype, and not the milder form of LVD as previously suggested (Bang et al., 2003). This may also indicate that a substantial fraction of cryptogenic strokes are indeed caused by a form of cardioembolism, at least in our study population. Such a possibility has been actually proposed based on gene expression profiling (Jickling et al., 2012 ). However, as that study did not distinguish between the two subcategories of undetermined etiology stroke, data presented there and our results cannot be directly compared. Pla s m a C 3 an d C 3 a le v e l s sh o w et i o l o g y - d e p e n d e n t as s o c i a t i o n s w it h fu n c t i o n a l ou t c o m e af t e r is c h e m i c st r o k e (P a p e r s I an d II ) . We also investigated the correlation of plasma C3 and C3a levels with the functional outcome after stroke. Three-month follow-up C3 levels in the LVD patients as well as C3 levels at both time points in the CE group showed a correlation with the extent of initial impairment measured by SSS at admission. Moreover, the high three-month C3 levels were associated with unfavorable outcome (mRS: 3-6) both at 3 months and 2 years after stroke in LVD and CE patients and this association was independent of age and sex. Such an association w as not found in the remaining groups. Interestingly, moderate levels (middle third) of C3a/C3 ratios at 3 -month follow-up in CE patients were associated with favorable outcome (mRS: 0-2) at 2 years post stroke. All the associations in the CE group withstoo d additional correction for SSS score and CRP levels. It cannot be ruled out that infarct size affects C3 levels in blood and the association with functional outcome among different subgroups. However, unlike high acute phase CRP levels, that are associated with long–term outcome and correlate with brain infarct volume (as shown here as well as in other studies), only C3 levels in the delayed phase after stroke showed significant and independent association with outcome in our study. The associations with outcome in CE stroke remained significant even after adjustment for CRP levels and SSS score, which is an approximated measure of the Comple m ent in stroke and neural plasticity   60 RESULTS AND DISCUSSION S y s t e m i c co m p l e m e n t re s p o n s e dif f e r s be t w e e n is c h e m i c st r o k e su b t y p e s (P a p e r s I an d II ) To date only a few and relatively small studies addressed the question of systemic complement activation during ischemic stroke while the reports on complement influence on functional outcome are even scarcer. Therefore, to evaluate the extent of complement response in ischemic stroke, we measured plasma C3 and C3a levels at the acute and delayed stage after stroke in patients classified to the four major ischemic stroke subtypes and compared them to the levels of the control subjects. We observed marked differences in the temporal pattern of plasma complement levels among patients suffering from ischemic stroke due to different etiologies. Plasma C3 levels were increased above the control level in all stroke groups in the acute phase while they remained elevated at three-month follow-up in LVD, SVD and cryptogenic but not CE s troke. Interestingly, only for the cryptogenic stroke patients the follow-up C3 levels did not decline as compared to acute stage, although this state was not associated with systemic elevation of C3a levels. In the remaining subtypes, C3a levels were still increased at three months after stroke. High (upper third) acute phase levels of both C3 and C3a were associated with CE and cryptogenic stroke independently of vascular risk factors, while high levels of C3 and C3a in both acute and convalescent phase showed independent association with the LVD and SVD stroke. Moreover, in the cryptogenic and CE stroke group, C3 levels at both time points were correlated with CRP levels. Levels of C3 -activation-derived C3a peptide were correlated with CRP levels at both time points only among the LVD patients. The finding that three-month levels (assumed to approximate the pre -stroke levels) of C3 and C3a were associated with patient status in LVD and SVD independently of traditional risk factors points to similarities between these subtypes, the chronic nature of the underlying pathology and the potential role of complement in this process. Indeed, atherosclerosis is often associated not only with LVD but also SVD stroke (Jung et al., 2012) and the complement system, and C3 in particular, is involved in the regulation of atherogenesis (Persson et al., 2004; Oksjoki et al ., 2007; Leung et al., 2009 ). However, Anna Stokowska   61 with respect to the patterns of complement activation and the role of CRP, SVD and LVD subtypes appear to be two distinct entities as C3a and CRP levels correlate only in the LVD group. On the other hand, with regard to the correlation between systemic hsCRP and C3 levels, the cryptogenic stroke subtype appears to resemble CE subtype, and not the milder form of LVD as previously suggested (Bang et al., 2003). This may also indicate that a substantial fraction of cryptogenic strokes are indeed caused by a form of cardioembolism, at least in our study population. Such a possibility has been actually proposed based on gene expression profiling (Jickling et al., 2012 ). However, as that study did not distinguish between the two subcategories of undetermined etiology stroke, data presented there and our results cannot be directly compared. Pla s m a C 3 an d C 3 a le v e l s sh o w et i o l o g y - d e p e n d e n t as s o c i a t i o n s w it h fu n c t i o n a l ou t c o m e af t e r is c h e m i c st r o k e (P a p e r s I an d II ) . We also investigated the correlation of plasma C3 and C3a levels with the functional outcome after stroke. Three-month follow-up C3 levels in the LVD patients as well as C3 levels at both time points in the CE group showed a correlation with the extent of initial impairment measured by SSS at admission. Moreover, the high three-month C3 levels were associated with unfavorable outcome (mRS: 3-6) both at 3 months and 2 years after stroke in LVD and CE patients and this association was independent of age and sex. Such an association was not found in the remaining groups. Interestingly, moderate levels (middle third) of C3a/C3 ratios at 3 -month follow-up in CE patients were associated with favorable outcome (mRS: 0-2) at 2 years post stroke. All the associations in the CE group withstood additional correction for SSS score and CRP levels. It cannot be ruled out that infarct size affects C3 levels in blood and the association with functional outcome among different subgroups. However, unlike high acute phase CRP levels, that are associated with long–term outcome and correlate with brain infarct volume (as shown here as well as in other studies), only C3 levels in the delayed phase after stroke showed significant and independent association with outcome in our study. The associations with outcome in CE stroke remained significant even after adjustment for CRP levels and SSS score, which is an approximated measure of the Comple m ent in stroke and neural plasticity   62 extent of the injury, supporting the notion that the complement system plays a role in stroke and that for CE stroke patie nts, three-month plasma complement measurements may be better predictors of functional outcome than CRP. It is noteworthy, however, that low number of patients suffering from cryptogenic and SVD stroke displayed mRS score above 3 on either time point of outcome evaluation and therefore low statistical power might have precluded finding any significant associations in those groups. Additional factors, such as e.g. infection or subsequent more subtle thromboembolic events that can contribute to high systemic C3 levels and negative outcome in the CE and LVD patients could have also affected the results. The lack of a detailed record of recent infections or chronic inflammatory states in SAHLSIS is a clear limitation. The finding that moderate level of complement activation measured by C3a/C3 ratio (middle third) is associated with favorable outcome in the CE patients is intriguing and warrants the mechanistic studies on the beneficial properties of complement activation, such as the atheroprotective effects of C3adesArg (Lewis et al., 2011). Taken together, studies described in paper I and II show complex associations of plasma complement levels with ischemic stroke and patient’s functional outcome. Futu re larger studies controlling for infections and stratifying patients both according to stroke severity and etiology are warranted. G e n e t i c va r i a t i o n in co m p l e m e n t co m p o n e n t C 3 sh o w s as s o c i a t i o n w it h is c h e m i c st r o k e (P a p e r II I ) as w e l l as C 3 an d C 3 a le v e l s (u n p u b l i s h e d da t a ) . To evaluate the potential associations of genetic variation in the C 3 locus with ischemic stroke, we genotyped 16 tag SNPs within the C 3 region in entire SAHLIS population and analyzed the results with relation to clinical data. Two SNPs were found to be associated with ischemic stroke independently of the effect of age and sex. The minor allele (G) of rs2277984 was associated with increased risk of overall ischemic stroke, while the minor allele (C) of rs3745565 showed Anna Stokowska   63 association with decreased risk of stroke. These associations remained significant after additional adjustment for risk factors such as hypertension, diabetes, and smoking. Interestingly, when these two SNPs were analyzed in each of the main stroke subtypes individually, a positive association between the minor allele of rs2277984 and the cryptogenic stroke was observed. This association was also independent of all the mentioned risk factors in a multivariate model. Haplotype analysis did not add any further information. After correction for multiple testing, only the association of rs3745565 with overall ischemic stroke remained significant. Both rs2277984 and rs3745565 are intronic variations and are not linked to any known functional SNP(s). In contrast, no association was detected for rs2230199, a non- synonymous SNP in exon three resulting in a change in the electrophoretic properties of C3 protein, which is in line with other published studies (Kramer et al., 2000 ). The association of rs2277984 with cryptogenic stroke only may be caused by a possible subtype-specific effect, but also simply the lack of statistical power to detect associations in the remaining stroke subtypes. Indeed, our cryptogenic stroke population includes the largest number of participants out of the four subtypes, being around 1.5 to 2 fold greater than each of the remaining groups. We also investigated the possible influence of genetic variants of the C 3 gene on the plasma levels of C3 and C3a among the selected individuals (n=469, 310 patients and 159 controls, Table 3) from the SAHLSIS population (characterized in papers I and II). In this analysis, using pooled control and three-month patient measurements, two SNPs were found to be associated with complement protein levels in the univariate as well as in multiple-adjusted model. Inclusion of rs3745565 was found to significantly improve (P =0.0034) the basic prediction model of C3a levels that included age, sex and case/control status through the addition of 2.55% of the explanator y value of C3a variance, which then equaled 4.74% ( P =0.0007). Its rare allele (C), associated also with the control status in SAHLSIS population, was associated with the decrease of 12.04 ng/L (13 .32% of the geometric mean) in plasma C3a level (P=0.021) in heterozygotes. However, homozygous C allele carriers had on average 40.46 ng/L higher C3a levels (44.77%) than homozygous major allele carriers ( P =0.025), most probably due to predominance of patients in this genotype group. The other variant, the rs237554, Comple m ent in stroke and neural plasticity   62 extent of the injury, supporting the notion that the complement system plays a role in stroke and that for CE stroke patie nts, three-month plasma complement measurements may be better predictors of functional outcome than CRP. It is noteworthy, however, that low number of patients suffering from cryptogenic and SVD stroke displayed mRS score above 3 on either time point of outcome evaluation and therefore low statistical power might have precluded finding any significant associations in those groups. Additional factors, such as e.g. infection or subsequent more subtle thromboembolic events that can contribute to high systemic C3 levels and negative outcome in the CE and LVD patients could have also affected the results. The lack of a detailed record of recent infections or chronic inflammatory states in SAHLSIS is a clear limitation. The finding that moderate level of complement activation measured by C3a/C3 ratio (middle third) is associated with favorable outcome in the CE patients is intriguing and warrants the mechanistic studies on the beneficial properties of complement activation, such as the atheroprotective effects of C3adesArg (Lewis et al., 2011). Taken together, studies described in paper I and II show complex associations of plasma complement levels with ischemic stroke and patient’s functional outcome. Futu re larger studies controlling for infections and stratifying patients both according to stroke severity and etiology are warranted. G e n e t i c va r i a t i o n in co m p l e m e n t co m p o n e n t C 3 sh o w s as s o c i a t i o n w it h is c h e m i c st r o k e (P a p e r II I ) as w e l l as C 3 an d C 3 a le v e l s (u n p u b l i s h e d da t a ) . To evaluate the potential associations of genetic variation in the C 3 locus with ischemic stroke, we genotyped 16 tag SNPs within the C 3 region in entire SAHLIS population and analyzed t he results with relation to clinical data. Two SNPs were found to be associated with ischemic stroke independently of the effect of age and sex. The minor allele (G) of rs2277984 was associated with increased risk of overall ischemic stroke, while the minor allele (C) of rs3745565 showed Anna Stokowska   63 association with decreased risk of stroke. These associations remained significant after additional adjustment for risk factors such as hypertension, diabetes, and smoking. Interestingly, when these two SNPs were analyzed in each of the main stroke subtypes individually, a positive association between the minor allele of rs2277984 and the cryptogenic stroke was observed. This association was also independent of all the mentioned risk factors in a multivariate model. Haplotype analysis did not add any further information. After correction for multiple testing, only the association of rs3745565 with overall ischemic stroke remained significant. Both rs2277984 and rs3745565 are intronic variations and are not linked to any known functional SNP(s). In contrast, no association was detected for rs2230199, a non- synonymous SNP in exon three resulting in a change in the electrophoretic properties of C3 protein, which is in line with other published studies (Kramer et al., 2000 ). The association of rs2277984 with cryptogenic stroke only may be caused by a possible subtype-specific effect, but also simply the lack of statistical power to detect associations in the remaining stroke subtypes. Indeed, our cryptogenic stroke population includes the largest number of participants out of the four subtypes, being around 1.5 to 2 fold greater than each of the remaining groups. We also investigated the possible influence of genetic variants of the C 3 gene on the plasma levels of C3 and C3a among the selected individuals (n=469, 310 patients and 159 controls, Table 3) from the SAHLSIS population (characterized in papers I and II). In this analysis, using pooled control and three-month patient measurements, two SNPs were found to be associated with complement protein levels in the univariate as well as in multiple-adjusted model. Inclusion of rs3745565 was found to significantly improve (P =0.0034) the basic prediction model of C3a levels that included age, sex and case/control status through the addition of 2.55% of the explanator y value of C3a variance, which then equaled 4.74% ( P =0.0007). Its rare allele (C), associated also with the control status in SAHLSIS population, was associated with the decrease of 12.04 ng/L (13 .32% of the geometric mean) in plasma C3a level (P=0.021) in heterozygotes. However, homozygous C allele carriers had on average 40.46 ng/L higher C3a levels (44.77%) than homozygous major allele carriers ( P =0.025), most probably due to predominance of patients in this genotype group. The other variant, the rs237554, Comple m ent in stroke and neural plasticity   64 was found to significantly improve the basic model of plasma levels of C3 and C3a (P =0.0200 and P =0.0026, respectively), which then predicted 6.78% and 4.03% of the respective variances. Although the contribution of this variant to the determination of C3 and C3a level was only 1.66% and 1.84% respectively, homozygous minor allele (AA) carriers had 129.00 mg/L (31.22% of geometric mean) higher C3 levels than homozygotes for the dominant allele (GG) (P =0.014), while in heterozygotes (AG) the increase of C3a levels was on average 17.10 ng/L (18.92% of geometric mean, P =0.005). SNP Genotype Controls Ischemic stroke Total rs3745565 GG, n (%) 106 (66.7) 233 (76) 338 (72.8) CG, n (%) 50 (31.4) 64 (21) 114 (24.6) CC, n (%) 3 (1.9) 9 (3) 12 (2.6) rs237554 GG, n (%) 109 (6 8 . 6 ) 23 2 (75.6) 341 (73 . 2 ) AG, n (%) 4 7 (2 3 . 6 ) 70 (2 2 . 8 ) 117 (25.1) AA, n (%) 3 (1.9) 5 (1.6) 8 (1.7) Table 3. Genotype frequencies in controls and patients with the plasma complement levels data available Both rs237554 and 3745565 are deep-intronic SNPs in the central region of the C3 gene (intron 28 and 26, respectively), therefore it is difficult to speculate about any potential causal relation with plasma C3, and in particular C3a levels. It is unlikely that these SNPs represent any direct link with complement components level, although theoretically introns may contain sequences that could influence regulation of protein expression level (Cooper, 2010). Instead, these associations might be indicative for other, yet unidentified, causative variants remaining in the tight linkage disequilibrium with the analyzed SNPs, and localizing within the coding region of the C 3 gene or even outside its locus. Regardless of the actual type of relationship, the mechanism behind the effects of these variants seems to play only a subtle role in regulation of C3 production and activation. It is intriguing that the rare allele, which showed association with decreased risk of stroke, was also associated with lower levels of C3a independently of the case/control status. It is also possible that these associations are mediated by other factors. Anna Stokowska   65 Although potentially interesting, due to a very small sample size for the genetic association study and an extremely low number of homozygous uncommon allele carriers, these results should be treated with caution. The effects and strength of the association are rather small and some of them would not withstand correction for multiple testing. Therefore at least some of the above findings can be false-positive and future larger studies, with detailed fine mapping of these SNPs and controlling for factors such as levels of inflammatory markers, may clarify the issues raised by this study. R e c e p t o r fo r co m p l e m e n t pe p t i d e C 3 a st i m u l a t e s ne u r a l pla s t i c i t y af t e r ex p e r i m e n t a l br a i n is c h e m i a (P a p e r IV ) C3, C3a peptide and its receptor have been implicated in the neuroprotection, regulation of adult neurogenesis and synaptic reorganization during development in rodents. To determine the role of C3a-C3aR signaling in ischemia-induced neural plasticity and post-stroke recovery, we subjected C3aR -deficient (C3aRKO ) mice, transgenic mice with brain-specific and complement activation-independent C3a peptide expression (C3a -GFAP) and their respective wild type (WT) control mice to photothrombotic stroke. We found that the infarct size at 21 days after stroke did not differ between the groups but C3a overexpression incre ased, whereas C3aR deficiency decreased post- stroke neurogenesis as assessed by the number of newly-born neurons in the peri-infarct cortex. We found significantly increased number and size of synpasin I + pre-synaptic puncta in the proximal peri -infarct cortex as compared to the contralateral, uninjured cortical regions in all of the experimental groups. However, C3aR KO mice had markedly fewer synaptic puncta than C3aRWT mice in the contralateral hemisphere, whereas C3a- overexpression did not lead to furthe r increase in the number of putative synapses. Also in the striatum, the number and size of synaptic puncta was reduced in the uninjured hemisphere in C3aRKO as compared with C3aRWT animals. The difference between the strains was present also in the peri-infarct motoric cortex but only in regions located more than 240 µm away from the injury border. Comple m ent in stroke and neural plasticity   64 was found to significantly improve the basic model of plasma levels of C3 and C3a (P =0.0200 and P =0.0026, respectively), which then predicted 6.78% and 4.03% of the respective variances. Although the contribution of this variant to the determination of C3 and C3a level was only 1.66% and 1.84% respectively, homozygous minor allele (AA) carriers had 129.00 mg/L (31.22% of geometric mean) higher C3 levels than homozygotes for the dominant allele (GG) ( P =0.014 ), while in heterozygotes (AG) the increase of C3a levels was on average 17.10 ng/L (18.92% of geometric mean, P =0.005). SNP Genotype Controls Ischemic stroke Total rs3745565 GG, n (%) 106 (66.7) 233 (76) 338 (72.8) CG, n (%) 50 (31.4) 64 (21) 114 (24.6) CC, n (%) 3 (1.9) 9 (3) 12 (2.6) rs237554 GG, n (%) 109 (6 8 . 6 ) 23 2 (75.6) 341 (73 . 2 ) AG, n (%) 47 (2 3 . 6 ) 70 (2 2 . 8 ) 117 (25.1) AA, n (%) 3 (1.9) 5 (1.6) 8 (1.7) Table 3. Genotype frequencies in controls and patients with the plasma complement levels data available Both rs237554 and 3745565 are deep-intronic SNPs in the central region of the C3 gene (intron 28 and 26, respectively), therefore it is difficult to speculate about any potential causal relation with plasma C3, and in particular C3a levels. It is unlikely that these SNPs represent any direct link with complement components level, although theoretically introns may contain sequences that could influence regulation of protein expression level (Cooper, 2010). Instead, these associations might be indicative for other, yet unidentified, causative variants remaining in the tight linkage disequilibrium with the analyzed SNPs, and localizing within the coding region of the C 3 gene or even outside its locus. Regardless of the actual type of relationship, the mechanism behind the effects of these variants seems to play only a subtle role in regulation of C3 production and activation. It is intriguing that the rare allele, which showed association with decreased risk of stroke, was also associated with lower levels of C3a independently of the case/control status. It is also possible that these associations are mediated by other factors. Anna Stokowska   65 Although potentially interesting, due to a very small sample size for the genetic association study and an extremely low number of homozygous uncommon allele carriers, these results should be treated with caution. The effects and strength of the association are rather small and some of them would not withstand correction for multiple testing. Therefore at least some of the above findings can be false-positive and future larger studies, with detailed fine mapping of these SNPs and controlling for factors such as levels of inflammatory markers, may clarify the issues raised by this study. R e c e p t o r fo r co m p l e m e n t pe p t i d e C 3 a st i m u l a t e s ne u r a l pla s t i c i t y af t e r ex p e r i m e n t a l br a i n is c h e m i a (P a p e r IV ). C3, C3a peptide and its receptor have been implicated in the neuroprotection, regulation of adult neurogenesis and synaptic reorganization during development in rodents. To determine the role of C3a-C3aR signaling in ischemia-induced neural plasticity and post-stroke recovery, we subjected C3aR -deficient (C3aRKO ) mice, transgenic mice with brain-specific and complement activation-independent C3a peptide expression (C3a -GFAP) and their respective wild type (WT) control mice to photothrombotic stroke. We found that the infarct size at 21 days after stroke did not differ between the groups but C3a overexpression incre ased, whereas C3aR deficiency decreased post- stroke neurogenesis as assessed by the number of newly-born neurons in the peri-infarct cortex. We found significantly increased number and size of synpasin I + pre-synaptic puncta in the proximal peri -infarct cortex as compared to the contralateral, uninjured cortical regions in all of the experimental groups. However, C3aR KO mice had markedly fewer synaptic puncta than C3aRWT mice in the contralateral hemisphere, whereas C3a- overexpression did not lead to further increase in the number of putative synapses. Also in the striatum, the number and size of synaptic puncta was reduced in the uninjured hemisphere in C3aRKO as compared with C3aRWT animals. The difference between the strains was present also in the peri-infarct motoric cortex but only in regions located more than 240 µm away from the injury border. Comple m ent in stroke and neural plasticity   66 The quantification of GAP -43+ objects revealed a decreased number of activated axonal growth cones in the ipsilesional striatum and in both the ipsi - and contralesional somatosensory cortex of the C3aR KO mice. Interestingly, C3a-GFAP mice had more and larger GAP -43+ structures than WT animals, both in the injured and uninjured cortex as well as more growth cone-like objects in the striatum. Jointly, these findin gs suggest that C3aR signaling is important in several forms of structural plasticity after ischemic stroke. Two independent behavioral tests measuring fine sensorimotor performance indicated that mice with a deletion of C3aR have greater impairment of the stroke-affected forepaw function than their WT counterparts. However, the C3a -overexpressing mice did not show any difference in synaptogensesis and performance compared to their WT littermates. Although previous studies showed that C3 deficiency, that precludes production of the C3a peptide, is associated with increased infarct volume after MCAo, and C3a-GFAP mice are protected against neonatal ischemic brain injury, we did not observe any neuroprotection mediated by C3a-C3aR axis in the current stroke m odel. However , the photothrombotic stroke model is characterized by a limited penumbra region and therefore not likely to show any neuroprotective effects (Porritt et al., 2012). On the other hand, this study corroborates and extends the previous findings of the positive role of C3aR signaling in the regulation of neurogenesis. We showed that the overexpression of C3a in reactive astrocytes was associated with an increased number of newly born neurons in the peri-infarct region, implying that the endogenous neurogenic response to ischemia can be boosted by C3a. However, the comparable functional performance of C3a-GFAP mice and their WT littermates suggests that t he contribution of peri-infarct neurogenesis to sensorimotor function recovery in mice, at least during the first three weeks after stroke, is negligible. As the effect of C3aR deficiency on synaptic plasticity was observed primarily in the infarct-remote areas, it is conceivable that the potential positive effects of C3aR signaling on synaptic plasticity in the direct infarct vicinity are diminished by its other functions, such as glial cell activation or recruitment of blood-borne inflammatory cells in the infarct-proximal cortical region. Alternatively, as the contralateral synaptic plasticity in rats (Luke et al., 2004) has been associated with the engagement of the unaffected limb Anna Stokowska   67 and improvement of function, it is possible that employment of compensatory behaviors by the animals in our study contributed to the contralateral hemisphere response. Our observation that the expression of GAP -43 was reduced in the absence of C3aR, whereas it was increased when C3a was expressed in reactive astrocytes, together with the reports of C3 upregulation in sprouting neurons isolated from the rat cortex after ischemic stroke (Li et al., 2010) and the stimulatory effect of C3a on neurite outgrowth in vitro (Shinjyo et al., 2009 ), supports the conclusion that C3a signaling through C3aR plays a positive role in axonal sprouting after stroke. The unchanged functional performance of C3a-GFAP mice in spite of the marked increase in axonal growth marker expression might be due to the behavior tests used being not sensitive enough to detect subtle changes offered by C3a overexpression during the first three weeks post-stroke. It is also plausible that, although beneficial at a later stage, high C3a levels, resulting from reactive gliosis-driven C3a expression in the acute phase do not provide an optimal milieu for the induction of regeneration and instead hinder post-ischemic neural plasticity. In conclusion, these results indicate that C3aR signaling stimulates post-stroke cortical neurogenesis as well as synaptogenesis and axonal sprouting and should be considered as a target when designing therapeutic strategies to improve functional recovery. A l t e r e d co g n i t i v e pe r f o r m a n c e an d sy n a p t i c fu n c t i o n in th e hip p o c a m p u s of m ic e la c k i n g C 3 (P a p e r V ) As C3 was found to regulate both the number of synapses and hippocampal neurogenesis, we sought to determine the effects of constitutive genetic ablation of C3 on the functions of an unchallenged adult hippocampus by assessing place and reversal learning ability and synaptic function in C3 KO mice. Hippocam pus-dependent function test using the IntelliCage platform required mice to first learn where to access water by performing a nose poke (place learning, Comple m ent in stroke and neural plasticity   66 The quantification of GAP -43+ objects revealed a decreased number of activated axonal growth cones in the ipsilesional striatum and in both the ipsi - and contralesional somatosensory cortex of the C3aR KO mice. Interestingly, C3a-GFAP mice had more and larger GAP -43+ structures than WT animals, both in the injured and uninjured cortex as well as more growth cone-like objects in the striatum. Jointly, these findin gs suggest that C3aR signaling is important in several forms of structural plasticity after ischemic stroke. Two independent behavioral tests measuring fine sensorimotor performance indicated that mice with a deletion of C3aR have greater impairment of the stroke-affected forepaw function than their WT counterparts. However, the C3a -overexpressing mice did not show any difference in synaptogensesis and performance compared to their WT littermates. Although previous studies showed that C3 deficiency, that precludes production of the C3a peptide, is associated with increased infarct volume after MCAo, and C3a-GFAP mice are protected against neonatal ischemic brain injury, we did not observe any neuroprotection mediated by C3a-C3aR axis in the current stroke m odel. However , the photothrombotic stroke model is characterized by a limited penumbra region and therefore not likely to show any neuroprotective effects (Porritt et al., 2012). On the other hand, this study corroborates and extends the previous findings of the positive role of C3aR signaling in the regulation of neurogenesis. We showed that the overexpression of C3a in reactive astrocytes was associated with an increased number of newly born neurons in the peri-infarct region, implying that the endogenous neurogenic response to ischemia can be boosted by C3a. However, the comparable functional performance of C3a-GFAP mice and their WT littermates suggests that t he contribution of peri-infarct neurogenesis to sensorimotor function recovery in mice, at least during the first three weeks after stroke, is negligible. As the effect of C3aR deficiency on synaptic plasticity was observed primarily in the infarct-remote areas, it is conceivable that the potential positive effects of C3aR signaling on synaptic plasticity in the direct infarct vicinity are diminished by its other functions, such as glial cell activation or recruitment of blood-borne inflammatory cells in the infarct-proximal cortical region. Alternatively, as the contralateral synaptic plasticity in rats (Luke et al., 2004 ) has been associated with the engagement of the unaffected limb Anna Stokowska   67 and improvement of function, it is possible that employment of compensatory behaviors by the animals in our study contributed to the contralateral hemisphere response. Our observation that the expression of GAP -43 was reduced in the absence of C3aR, whereas it was increased when C3a was expressed in reactive astrocytes, together with the reports of C3 upregulation in sprouting neurons isolated from the rat cortex after ischemic stroke (Li et al., 2010) and the stimulatory effect of C3a on neurite outgrowth in vitro (Shinjyo et al., 2009 ), supports the conclusion that C3a signaling through C3aR plays a positive role in axonal sprouting after stroke. The unchanged functional performance of C3a-GFAP mice in spite of the marked increase in axonal growth marker expression might be due to the behavior tests used being not sensitive enough to detect subtle changes offered by C3a overexpression during the first three weeks post- stroke. It is also plausible that, although beneficial at a later stage, high C3a levels, resulting from reactive gliosis-driven C3a expression in the acute phase do not provide an optimal milieu for the induction of regeneration and instead hinder post-ischemic neural plasticity. In conclusion, these results indicate that C3aR signaling stimulates post-stroke cortical neurogenesis as well as synaptogenesis and axonal sprouting and should be considered as a target when designing therapeutic strategies to improve functional recovery. A l t e r e d co g n i t i v e pe r f o r m a n c e an d sy n a p t i c fu n c t i o n in th e hip p o c a m p u s of m ic e la c k i n g C 3 (P a p e r V ). As C3 was found to regulate both the number of synapses and hippocampal neurogenesis, we sought to determine the effects of constitutive genetic ablation of C3 on the functions of an unchallenged adult hippocampus by assessing place and reversal learning ability and synaptic function in C3 KO mice. Hippocampus -dependent function test using the IntelliCage platform required mice to first learn where to access water by performing a nose poke (place learning, Comple m ent in stroke and neural plasticity   68 corner 1) and then, in the subsequent phase (reversal learning, corner 2), to unlearn the previous behavior and learn to find the new allocated water corner. We observed a significant difference in both the number of incorrect visit ratio and absolute number of incorrect nose pokes ratio per day between C3 KO and WT mice over the five days of testing. For b oth of the measures, C3 KO mice performed better and displayed greater improvement per day in odds ratio in both place learning and reversal learning phase. Using extracellular field recordings in hippocampal slices, we found that the net efficacy of CA3-CA1 synapses was unchanged in C3 KO mice, whereas paired -pulse and MK -801 experiments revealed decreased glutamate release probability (p) in these mice. Since quantal parameters (q) of synapses of the single CA1 pyramidal neuron did not differ between the strains as judged by AMPA mESPC intracellular recordings, we concluded that C3 KO mice must have increased number of functional synapses in the hippocampus. Based on the electrophysiological data, the increase is estimated to be around 20% in C3 KO mice c lose to puberty. Despite the putative increase in number of glutamate release sites, we did not observe any spontaneous epileptiform activity in C3 KO mice, which is in contrast to studies investigating the neocortex of C1q KO mice (Chu et al., 2010). As both C1q and C3 are needed for synaptic circuit refinement and mice deficient in either of these components show the same phenotype, at least within the retinogeniculate system (Stevens et al., 2007), the decrease in release transmitter probability can be viewed as a homeostatic compensatory plasticity, the capacity of which might be brain region-dependent. Additional experiment revealed that in contrast to single pulse, high -frequency burst stimulation evokes significantly larger overall postsynaptic response in C3 KO than in WT mice. This finding points to a limited capacit y of these compensatory mechanisms in the hippocampal CA1 region of C3KO mice. Surprisingly, we did not find any significant difference between the strains in the number of dendritic spines, which are the primary excitatory synapse -bearing structures. The discrepancy between morphological and electrophysiological results is intriguing and merits further investigation. Possible explanation might be related to quantification inaccuracy due to limitations of conventional light microscopy and the type of labeling method, or predominant differences in the numbers of shaft, but not spiny synapses. Anna Stokowska   69 Although C3-deficincy has been shown to result in reduced hippocampal neurogenesis (Rahpeymai et al., 2006), enhanced place and reversal learning of C3 KO mice implies that neural plasticity at the level of synaptic function is a more important factor in determining hippocampus-dependent learning than the rate of generation of new neurons in the dentate gyrus, at least in the context of C3 deficiency. Therefore, the role of C3 in synapse elimination in the context of neurodegenerative pathology such as Alzheimer’s disease and the possible benefit for cognitive performance of sy napse sparing by C3 inhibition warrants further studies on the role complement in the aging or diseased hippocampus. Comple m ent in stroke and neural plasticity   68 corner 1) and then, in the subsequent phase (reversal learning, corner 2), to unlearn the previous behavior and learn to find the new allocated water corner. We observed a significant difference in both the number of incorrect visit ratio and absolute number of incorrect nose pokes ratio per day between C3 KO and WT mice over the five days of testing. For b oth of the measures, C3 KO mice performed better and displayed greater improvement per day in odds ratio in both place learning and reversal learning phase. Using extracellular field recordings in hippocampal slices, we found that the net efficacy of CA3-CA1 synapses was unchanged in C3 KO mice, whereas paired -pulse and MK -801 experiments revealed decreased glutamate release probability (p) in these mice. Since quantal parameters (q) of synapses of the single CA1 pyramidal neuron did not differ between the strains as judged by AMPA mESPC intracellular recordings, we concluded that C3 KO mice must have increased number of functional synapses in the hippocampus. Based on the electrophysiological data, the increase is estimated to be around 20% in C3 KO mice c lose to puberty. Despite the putative increase in number of glutamate release sites, we did not observe any spontaneous epileptiform activity in C3 KO mice, which is in contrast to studies investigating the neocortex of C1q KO mice (Chu et al., 2010). As both C1q and C3 are needed for synaptic circuit refinement and mice deficient in either of these components show the same phenotype, at least within the retinogeniculate system (Stevens et al., 2007), the decrease in release transmitter probability can be viewed as a homeostatic compensatory plasticity, the capacity of which might be brain region-dependent. Additional experiment revealed that in contrast to single pulse, high -frequency burst stimulation evokes significantly larger overall postsynaptic response in C3 KO than in WT mice. This finding points to a limited capacit y of these compensatory mechanisms in the hippocampal CA1 region of C3KO mice. Surprisingly, we did not find any significant difference between the strains in the number of dendritic spines, which are the primary excitatory synapse -bearing structures. The discrepancy between morphological and electrophysiological results is intriguing and merits further investigation. Possible explanation might be related to quantification inaccuracy due to limitations of conventional light microscopy and the type of labeling method, or predominant differences in the numbers of shaft, but not spiny synapses. Anna Stokowska   69 Although C3-deficincy has been shown to result in reduced hippocampal neurogenesis (Rahpeymai et al., 2006), enhanced place and reversal learning of C3 KO mice implies that neural plasticity at the level of synaptic function is a more important factor in determining hippocampus-dependent learning than the rate of generation of new neurons in the dentate gyrus, at least in the context of C3 deficiency. Therefore, the role of C3 in synapse elimination in the context of neurodegenerative pathology such as Alzheimer’s disease and the possible benefit for cognitive performance of sy napse sparing by C3 inhibition warrants further studies on the role complement in the aging or diseased hippocampus. Anna Stokowska   71 MA IN CONCLUSSIONS The results of this thesis support the view that complement system is involved in the pathophysiology of ischemic stroke. Further, this work demonstrates that complement system regulates different forms of basal as well as stroke-induced synaptic plasticity. Spe c i f i c a l l y w e fo u n d th a t : ¥ Plasma C3 and C3a levels are elevated in ischemic stroke as long as 3 months after stroke but the dynamics of this increase as well as the potential prognostic value of these markers are strongly influenced by ischemic stroke etiology. Importantly, for CE stroke patients, C3 levels measured at follow -up may be a better predictor of outcome than CRP. ¥ In the C 3 gene, rare allele of rs3745565 SNP is associated with decreased risk of overall ischemic stroke as well as lower C3a levels, while rs2277984 shows association with cryptogenic stroke subtype . Additionally, rs237554 is associated with increased level of both C3 and C3a. ¥ C3aR signaling plays a positive role in neural plasticity after experimental ischemic stroke and deletion of C3aR leads to disturbance of these processes and greater functional impairment. Brain-targeted overexpression of C3a stimulates post-stroke cortical neurogenesis and axonal sprouting, which may potentially contribute to functional recovery. ¥ C3 deficiency alters synaptic function and most probably the synapse number in the maturing hippocampus. These changes are associated with improved spatial learning. Anna Stokowska   71 MA IN CONCLUSSIONS The results of this thesis support the view that complement system is involved in the pathophysiology of ischemic stroke. Further, this work demonstrates that complement system regulates different forms of basal as well as stroke-induced synaptic plasticity. Spe c i f i c a l l y w e fo u n d th a t : ¥ Plasma C3 and C3a levels are elevated in ischemic stroke as long as 3 months after stroke but the dynamics of this increase as well as the potential prognostic value of these markers are strongly influenced by ischemic stroke etiology. Importantly, for CE stroke patients, C3 levels measured at follow -up may be a better predictor of outcome than CRP. ¥ In the C 3 gene, rare allele of rs3745565 SNP is associated with decreased risk of overall ischemic stroke as well as lower C3a levels, while rs2277984 shows association with cryptogenic stroke subtype. Additionally, rs237554 is associated with increased level of both C3 and C3a. ¥ C3aR signaling plays a positive role in neural plasticity after experimental ischemic stroke and deletion of C3aR leads to disturbance of these processes and greater functional impairment. Brain-targeted overexpression of C3a stimulates post-stroke cortical neurogenesis and axonal sprouting, which may potentially contribute to functional recovery. ¥ C3 deficiency alters synaptic function and most probably the synapse number in the maturing hippocampus. These changes are associated with improved spatial learning. Comple m ent in stroke and neural plasticity   72 CONCLUDNG REMARKS AND FUTURE DIRECTIONS It needs to be noted that the results from human subjects and animal models cannot be directly compared due to profoundly different aspects of the role of complement in stroke investigated in these studies. Nevertheless, they both are relevant for the development of a holistic approach to treat ischemic stroke. Deciphering inflammatory mechanisms in ischemic stroke, including those involving the complement system, is of a great importance for better understanding of stroke pathophysiology. This in turn is indispensible for designing successful and safe therapies that could help limit the extent of brain damage resulting in smaller initial deficit. Equally or even more important is the investigation of the mechanisms underlying post-stroke plasticity and repair processes and possibilities to boost them in order to promote recovery of lost functions. Apart from the limitations due the lack of data on possible underlying inflammatory conditions in SAHLSIS, the inclusion criterion of age restricts the generalizability of our results from this relatively young cohort of patients as ischemic stroke incidence increases with age. Therefore, a replication study including older patients would be necessary to confirm our findings. Furthermore, it would be useful to measure plasma levels of complement proteins at several strictly defined time points to obtain a better temporal profile of the complement response. Such information could help in defining a suitable time frame for eventual acute phase therapies aimed at inhibiting complement activation. Our current data do not allow us to establish the mechanism by which C3a and its receptor regulate synaptogenesis and axonal sprouti ng. As most or all brain cells ex press C3aR, it remains to be determined whether the observed effects are neuron-autonomous or are mediated by the responses of glia or other cell type. These issues could be resolved by using WT, C3 and C3aR -deficient primary cultures of for example astrocytes and microglia and their co-cultures with neurons under normal as well as ischemic conditions. We are currently addressing the possibility that C3aR -deficient mice have altered synaptic density in the cortex in the abse nce of ischemic injury. Such a finding would indicate yet another aspect of the complement-dependent regulation of synapse number during development. Anna Stokowska   73 Our findings of the beneficial effects of brain specific C3a expression on post -stroke neurogenesis and axonal plasticity together with a recent report showing that C3a overexpression confers a robust neuroprotection against neonatal hypoxic -ischemic injury and intraventricular administration of C3a peptide ameliorates hypoxia -ischemia induced cognitive deficits (Järlestedt et al., 2013 ), are very encouraging. Despite the differences in ischemic injury model and the maturation stage, the latter results prove the feasibility and effectiveness of exogenous administration of C3a peptide to the brain. However, determining the precise timing of such a therapy for ischemic stroke would be crucial as it might be suboptimal or even deleterious to administer C3a immediately after stroke. Another challenging aspect of designing such a complement-based therapy for modulation of plasticity response after ischemic stroke would be the development of an efficient, safe and non-invasive administration route for the drug to avoid systemic effects and problems with crossing BBB. Although previous studies from our laboratory showed that mouse NSPCs express receptors for both C3a and C5a, neither C5aR-deficient nor C5a-overexpressing animals showed an altered basal neurogenesis (Bogestål et al., 2007 ). However, it is possible that the effects of C5a may not observable in unchallenged animals. Therefore, we are in the process of evaluating the role of C5a-C5aR signaling using the methods described in paper IV. Taking into consideration that the half -life of C3a and C5a is extremely short, it is possible that at least some of the effects of C3a and C5a in CNS are mediated by their more stable desarginated forms. Therefore, due to its affinity for both C5adesArg and C3adesArg, and a possible modulatory effect on C5aR signaling, it would be interesting to evaluate the role of C5L2 receptor in different forms of neural plasticity including basal neurogenesis. Despite clear results obtained by electrophysiological methods, we could not confirm at a structural level the conclusion of higher number of synapses in the hippocampus of C3KO mice. Therefore, an alternative labeling techniqu e allowing for simultaneous visualization of pre - and post-synaptic components together with a super-resolution confocal microscopy could help clarify this issue. As C3 was shown to mediate synapse elimination from spinal cord motor neurons after peripheral nerve axotomy (Berg et al., Comple m ent in stroke and neural plasticity   72 CONCLUDNG REMARKS AND FUTURE DIRECTIONS It needs to be noted that the results from human subjects and animal models cannot be directly compared due to profoundly different aspects of the role of complement in stroke investigated in these studies. Nevertheless, they both are relevant for the development of a holistic approach to treat ischemic stroke. Deciphering inflammatory mechanisms in ischemic stroke, including those involving the complement system, is of a great importance for better understanding of stroke pathophysiology. This in turn is indispensible for designing successful and safe therapies that could help limit the extent of brain damage resulting in smaller initial deficit. Equally or even m ore important is the investigation of the mechanisms underlying post-stroke plasticity and repair processes and possibilities to boost them in order to promote recovery of lost functions. Apart from the limitations due the lack of data on possible underlying inflammatory conditions in SAHLSIS, the inclusion criterion of age restricts the generalizability of our results from this relatively young cohort of patients as ischemic stroke incidence increases with age. Therefore, a replication study including older patients would be necessary to confirm our findings. Furthermore, it would be useful to measure plasma levels of complement proteins at several strictly defined time points to obtain a better temporal profile of the complement response. Such information could help in defining a suitable time frame for eventual acute phase therapies aimed at inhibiting complement activation. Our current data do not allow us to establish the mechanism by which C3a and its receptor regulate synaptogenesis and axonal sprouti ng. As most or all brain cells ex press C3aR, it remains to be determined whether the observed effects are neuron-autonomous or are mediated by the responses of glia or other cell type. These issues could be resolved by using WT, C3 and C3aR -deficient primary cultures of for example astrocytes and microglia and their co-cultures with neurons under normal as well as ischemic conditions. We are currently addressing the possibility that C3aR -deficient mice have altered synaptic density in the cortex in the abse nce of ischemic injury. Such a finding would indicate yet another aspect of the complement-dependent regulation of synapse number during development. Anna Stokowska   73 Our findings of the beneficial effects of brain specific C3a expression on post -stroke neurogenesis and axonal plasticity together with a recent report showing that C3a overexpression confers a robust neuroprotection against neonatal hypoxic -ischemic injury and intraventricular administration of C3a peptide ameliorates hypoxia -ischemia induced cognitive deficits (Järlestedt et al., 2013 ), are very encouraging. Despite the differences in ischemic injury model and the maturation stage, the latter results prove the feasibility and effectiveness of exogenous administration of C3a peptide to the brain. However, determining the precise timing of such a therapy for ischemic stroke would be crucial as it might be suboptimal or even deleterious to administer C3a immediately after stroke. Another challenging aspect of designing such a complement-based therapy for modulation of plasticity response after ischemic stroke would be the development of an efficient, safe and non-invasive administration route for the drug to avoid systemic effects and problems with crossing BBB. Although previous studies from our laboratory showed that mouse NSPCs express receptors for both C3a and C5a, neither C5aR-deficient nor C5a-overexpressing animals showed an altered basal neurogenesis (Bogestål et al., 2007 ). However, it is possible that the effects of C5a may not observable in unchallenged animals. Therefore, we are in the process of evaluating the role of C5a-C5aR signaling using the methods described in paper IV. Taking into consideration that the half -life of C3a and C5a is extremely short, it is possible that at least some of the effects of C3a and C5a in CNS are mediated by their more stable desarginated forms. Therefore, due to its affinity for both C5adesArg and C3adesArg, and a possible modulatory effect on C5aR signaling, it would be interesting to evaluate the role of C5L2 receptor in different forms of neural plasticity including basal neurogenesis. Despite clear results obtained by electrophysiological methods, we could not confirm at a structural level the conclusion of higher number of synapses in the hippocampus of C3KO mice. Therefore, an alternative labeling techniqu e allowing for simultaneous visualization of pre- and post-synaptic components together with a super-resolution confocal microscopy could help clarify this issue. As C3 was shown to mediate synapse elimination from spinal cord motor neurons after peripheral nerve axotomy ( Berg et al., Comple m ent in stroke and neural plasticity   74 2012), it remains to be tested whether C3 plays any role in microglia-mediated in synapse elimination after stroke (Wake et al., 2009 ). Another important question is the role of the complement system and in particular synapse elimination and altered synaptic plasticity in cognitive decline during aging. The first observations in this regard point, perhaps not surprisingly, to an even larger complexity of this problem. As Stephan and colleagues recently showed, C1q KO mice are protected from age-related cognitive impairment independently of synapse elimination. This study further showed that the absence of C3 and C1q had opposing effects on activity-dependent synaptic potentiation of perforant path to dentate gyrus synapses (Stephan et al., 2013). These results show that C1q contributes to age- related decline of hippocampus-dependent cognitive functions independently of complement activation and without affecting the synapse numbers in the hippocampal dentate gyrus. Better understanding of the complement activation dependent as well as independent functions of the complement proteins, in particular C1q and C3 may therefore pave the way to tackle one of the greatest health threats of old age. In summary, this thesis contributes to the growing body of evidence for the role of complement in ischemic stroke and stroke recovery. The roles of individual complement proteins and their activation products in stroke may, however, be profoundly different depending on both the temporal and spatial context. The currently established roles of the complement system range from the more classically immune functions such as initiation of inflammation, opsonization, regulation of antibody responses and cell lysis through the elimination of dead cells and cell debris, atherogenesis and stem cell homing to stem cell differentiation, neuroprotection and synapse elimination in CNS. The next decade will undoubtedly unravel even less expected functions for a system that was once regarded as “an antibody helper”. Anna Stokowska   75 A CKNOWLEDGEMENTS I would like to express my sincere gratitude to all the people that have contributed to the completion of this work and who made my “tough life” of a PhD student not only easier but also turned it into a fantastic experience. Therefore I would like to especially acknowledge the following people: First and foremost, I would like to thank my supervisor, Mar c e l a P e k n a for giving me the opportunity to take part in the extremely interesting scientific projects and for an excellent guidance through it combined with a “humane” approach. I am grateful for your always comprehensive feedback, inspiration and reminding me to see behind the difficulties. I have learned a lot from you! C h r i s t i n a Je r n - my co-supervisor and the clinical and genetics expert, for sharing your expertise, invaluable comments and a thoughtful approach to a non -clinician such as myself. C h r i s t i a n B lo m s t r a n d an d K a t a r i n a Jo o d - for your expert feedback that helped improve my work as well as your kind and friendly attitude. M i l o s P e k n y for your contagious enthusiasm, spreading the passion for science, encouragement and nice chats about science and life in general. M a r c e l a ’ s gr o u p : No r i k o Sh i n j y o , my first friend and flat mate in Göteborg – for always being so kind and helpful and many pleasant moments during hard work in the lab and in the outside world; Alis o n A t k i n s - for co-running some of the laborious parts of the experimental stroke project, including 12 -hour cycle in EBM on weekends and the help with proofreading ; M a r t a Pe r e z - A l c a z a r , N a n c y B e y e r and Mic h a e l a P a s c o e - for creating friendly and positive atmosphere, interesting discussions as well as many good laughs  ; R o s a n n a O ls e n - for all the efforts and help in the experimental stroke proj ect in its final stage and excellent proofreading of my thesis. Our “symbiotic lab” - M i l o s ’ gr o u p : especially Ulr i k a W il h e l m s s o n - for good advice and making sure lots of things actually work in the labs; D a n i e l A n d e r s s o n , Y o l a n d a de Pa b l o , T il l P u s c h m a n n , Is a b e l l L e b k u e c h n e r , E li n M ö l l e s t r ö m , M e n g C h e n , X ia o g u a n g Y a n g , A n d e r s St å h l b e r g , Å s a W i d e s t r a n d , P e t e Sm i t h , M a r y a m F a i z and t h e su m m e r an d pr o j e c t st u d e n t s – for all the jokes and laughs, interesting literature seminars, borrowing of some lab materials and being helpful, friendly and cool people . The C B R P I s ( G e o r g K u h n , K la s B lo m g r e n , M ic h a e l N il s s o n an d M a u r i c e C u r t i s ) and a l l th e pr e s e n t an d fo r m e r co l l e a g u e s , te c h n i c i a n s an d st u d e n t s from the 4 th flo o r - for creating and excellent and cozy atmosp here for scientific work, friendship, all sorts of help and great “get -together” events; special thanks to Mic h e l l e P o r r i t t - for enabling me to kick-start with the photothrombotic stroke project by teaching me the basics of animal surgery and setting up the behavioral testing as well as lots of fun times and chats. All the coll a b o r a t o r s and co - a u t h o r s for a fruitful co-operation – especially San d r a O ls s o n and T a r a St a n n e - for a friendly attitude, tips and giving me the possibility to Comple m ent in stroke and neural plasticity   74 2012), it remains to be tested whether C3 plays any role in microglia-mediated in synapse elimination after stroke (Wake et al., 2009 ). Another important question is the role of the complement system and in particular synapse elimination and altered synaptic plasticity in cognitive decline during aging. The first observations in this regard point, perhaps not surprisingly, to an even larger complexity of this problem. As Stephan and colleagues recently showed, C1q KO mice are protected from age-related cognitive impairment independently of synapse elimination. This study further showed that the absence of C3 and C1q had opposing effects on activity-dependent synaptic potentiation of perforant path to dentate gyrus synapses (Stephan et al., 2013). These results show that C1q contributes to age -related decline of hippocampus-dependent cognitive functions independently of complement activation and without affecting the synapse numbers in the hippocampal dentate gyrus. Better understanding of the complement activation dependent as well as independent functions of the complement proteins, in particular C1q and C3 may therefore pave the way to tackle one of the greatest health threats of old age. In summary, this thesis contributes to the growing body of evidence for the role of complement in ischemic stroke and stroke recovery. The roles of individual complement proteins and their activation products in stroke may, however, be profoundly different depending on both the temporal and spatial context. The currently established roles of the complement system range from the more classically immune functions such as initiation of inflammation, opsonization, regulation of antibody responses and cell lysis through the elimination of dead cells and cell debris, atherogenesis and stem cell homing to stem cell differentiation, neuroprotection and synapse elimination in CNS. The next decade will undoubtedly unravel even less expected functions for a system that was once rega rded as “an antibody helper”. Anna Stokowska   75 A CKNOWLEDGEMENTS I would like to express my sincere gratitude to all the people that have contributed to the completion of this work and who made my “tough life” of a PhD student not only easier but also turned it into a fantastic experience. Therefore I would like to especially acknowledge the following people: First and foremost, I would like to thank my supervisor, Mar c e l a P e k n a for giving me the opportunity to take part in the extremely interesting scientific projects and for an excellent guidance through it combined with a “humane” approach. I am grateful for your always comprehensive feedback, inspiration and reminding me to see behind the difficulties. I have learned a lot from you! C h r i s t i n a Je r n - my co-supervisor and the clinical and genetics expert, for sharing your expertise, invaluable comments and a thoughtful approach to a non -clinician such as myself. C h r i s t i a n B lo m s t r a n d an d K a t a r i n a Jo o d - for your expert feedback that helped improve my work as well as your kind and friendly attitude. M i l o s P e k n y for your contagious enthusiasm, spreading the passion for science, encouragement and nice chats about science and life in general. M a r c e l a ’ s gr o u p : No r i k o Sh i n j y o , my first friend and flat mate in Göteborg – for always being so kind and helpful and many pleasant moments during hard work in the lab and in the outside world; Alis o n A t k i n s - for co-running some of the laborious parts of the experimental stroke project, including 12 -hour cycle in EBM on weekends and the help with proofreading ; M a r t a Pe r e z - A l c a z a r , N a n c y B e y e r and Mic h a e l a P a s c o e - for creating friendly and positive atmosphere, interesting discussions as well as many good laughs  ; R o s a n n a O ls e n - for all the efforts and help in the experimental stroke project in its final stage and excellent proofreading of my thesis. Our “symbiotic lab” - M i l o s ’ gr o u p : especially Ulr i k a W il h e l m s s o n - for good advice and making sure lots of things actually work in the labs; D a n i e l A n d e r s s o n , Y o l a n d a de Pa b l o , T il l P u s c h m a n n , Is a b e l l L e b k u e c h n e r , E li n M ö l l e s t r ö m , M e n g C h e n , X ia o g u a n g Y a n g , A n d e r s St å h l b e r g , Å s a W i d e s t r a n d , P e t e Sm i t h , M a r y a m F a i z and t h e su m m e r an d pr o j e c t st u d e n t s – for all the jokes and laughs, interesting literature seminars, borrowing of some lab materials and being helpful, friendly and cool people . The C B R P I s ( G e o r g K u h n , K la s B lo m g r e n , M ic h a e l N il s s o n an d M a u r i c e C u r t i s ) and a l l th e pr e s e n t an d fo r m e r co l l e a g u e s , te c h n i c i a n s an d st u d e n t s from the 4 th flo o r - for creating and excellent and cozy atmosp here for scientific work, friendship, all sorts of help and great “get- together” events; special thanks to Mic h e l l e P o r r i t t - for enabling me to kick-start with the photothrombotic stroke project by teaching me the basics of animal surgery and setting up the behavioral testing as well as lots of fun times and chats. All the coll a b o r a t o r s and co - a u t h o r s for a fruitful co-operation – especially San d r a O ls s o n and T a r a St a n n e - for a friendly attitude, tips and giving me the possibility to Comple m ent in stroke and neural plasticity   76 train my Swedish, as well as Eri c H a n s e - for sharing your expertise in electrophysiology and having the patience to explain the details many times. C o l l e a g u e s form the nei g h b o r i n g la b s : the groups of Sve n E n e r b ä c k , Sar a L in d é n, M a d e l e i n e Z e t t e r b e r g / J a n - O l o f K a r l s s o n , C a r i n a M a l l a r d and S u z a n n e D ic k s o n – for friendliness and nice chats in the corridor or in the “fikaroom”. Ann a B lo m , M a r c i n O k r ó j an d A r e k P i e r z c h a l s k i - thanks to whom my adventure in the world of complement and in Scandinavia began in the first place. Krz y s z t o f B a r t o s z e k - for coaching in statistical programming, proofreadings, friendship and support. And last but not least - special thanks to my de a r f a m i l y , especially my parents Kry s t y n a and Ada m and my brother R a d e k as well as my friends both in Sweden and Poland – for your care, constant support and believing in me. Thank you all!  The work presented in this thesis was supported by Swedish Research Council, Swedish governmental grants for researchers working in health care (ALF Gothenb urg and ALF Lund), The Swedish Brain Foundation (Hjärnfonden), The Swedish Heart and Lung Foundation (Hjärt - Lungfonden) , The Swedish Childhood Cancer Foundation (Barncancerfonden), The Swedish Stroke Foundation, Swedish Society of Medicine, The Gothenburg Medical Society, Goteborg as well as Y. Land’s, J. and B. Wennerström’s Foundations for Neurological Research , STENA Foundation, Alzheimer’s Foundation, The King Gustav V Jubilee Clinic Cancer Research Foundation (JK - fonden), The Frimurare Barnhus Foundati on, The, the New Zealand National Research Centre for Growth and Development, and the New Zealand Health Research Council, AFA Insurance, Sahlgrenska Foundation, S. A. Olsson Foundation for Research and Culture , as well as W. and M. Lundgren’s, R., T. Söd erberg’s, E. Jacobson’s, R. and U. Amlöv’s, T. Nilsson’s, Emelle’s, O.E . and E. Johanssons and L. Hierta Memorial Foundations. Anna Stokowska   77 REFERENCES Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003) Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A 100:15983-15988. Abraham WC, Bear MF (1996) Metaplasticity: the plasticity of synaptic plasticity. Trends Neurosci 19:126-130. Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, G ordon DL, Marsh EE, 3rd (1993) Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 24:35-41. Alper CA, Johnson AM, Birtch AG, Moore FD (1969) H uman C'3: evidence for the liver as the primary site of synthesis. Science 163:286-288. Alvarez -Buylla A, Lim DA (2004) For the long run: maintaining germinal niches in the adult brain. Neuron 41:683-686. Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Ac ker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber -Lang M (2010) Molecular intercommunication between the complement and coagulation systems. J Immunol 185:5628 -5636. Araque A, Parpura V, Sanzgiri RP, Haydon PG (1998) Glutamate -dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur J Neurosci 10:2129 -2142. Arboix A, Marti -Vilalta JL (2004) New concepts in lacunar stroke etiology: the constellation of small-vessel arterial disease. Cerebrovasc Dis 17 Suppl 1:58-62. Ballabio E, Bersano A, Bresolin N, Candelise L (2007) Monogenic vessel diseases related to ischemic stroke: a clinical approach. J Cereb Blood Flow Metab 27:1649-1662. Balschun D, Wetzel W, Del Rey A, Pitossi F, Schneider H, Zuschratter W, Besedov sky HO (2004) Interleukin -6: a cytokine to forget. FASEB J 18:1788 -1790. Bamford J, Sandercock P, Jones L, Warlow C (1987) The natural history of lacunar infarction: the Oxfordshire Community Stroke Project. Stroke 18:545 -551. Bang OY, Lee PH, Joo SY, Lee JS, Joo IS, Huh K (2003) Frequency and mechanisms of stroke recurrence after cryptogenic stroke. Ann Neurol 54:227-234. Barnum SR (1995) Complement biosynthesis in the central nervous system. Crit Rev Oral Biol Med 6:132 -146. Barres BA (2008) The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60:430-440. Barrett JC, Cardon LR (2006) Evaluating coverage of genome -wide association studies. Nat Genet 38:659 -662. Bashir ZI, Collingridge GL (1994) An investigation of depote ntiation of long-term potentiation in the CA1 region of the hippocampus. Exp Brain Res 100:437 -443. Battista D, Ferrari CC, Gage FH, Pitossi FJ (2006) Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus. Eur J Neurosci 23:83 -93. Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC (2002) Control of synaptic strength by glial TNFalpha. Science 295:2282-2285. Benard M, Raoult E, Vaudr y D, Leprince J, Falluel -Morel A, Gonzalez BJ, Galas L, Vaudry H, Fontaine M (2008) Role of complement anaphylatoxin receptors Comple m ent in stroke and neural plasticity   76 train my Swedish, as well as Eri c H a n s e - for sharing your expertise in electrophysiology and having the patience to explain the details many times. C o l l e a g u e s form the nei g h b o r i n g la b s : the groups of Sve n E n e r b ä c k , Sar a L in d é n , M a d e l e i n e Z e t t e r b e r g / J a n - O l o f K a r l s s o n , C a r i n a M a l l a r d and S u z a n n e D ic k s o n – for friendliness and nice chats in the corridor or in the “fikaroom”. Ann a B lo m , M a r c i n O k r ó j an d A r e k P i e r z c h a l s k i - thanks to whom my adventure in the world of complement and in Scandinavia began in the first place. Krz y s z t o f B a r t o s z e k - for coaching in statistical programming, proofreadings, friendship and support. And last but not least - special thanks to my de a r f a m i l y , especially my parents Kry s t y n a and Ada m and my brother R a d e k as well as my friends both in Sweden and Poland – for your care, constant support and believing in me. Thank you all!  The work presented in this thesis was supported by Swedish Research Council, Swedish governmental grants for researchers working in health care (ALF Gothenb urg and ALF Lund), The Swedish Brain Foundation (Hjärnfonden), The Swedish Heart and Lung Foundation (Hjärt - Lungfonden) , The Swedish Childhood Cancer Foundation (Barncancerfonden), The Swedish Stroke Foundation, Swedish Society of Medicine , The Gothenburg Medical Society, Goteborg as well as Y. Land’s, J. and B. Wennerström’s Foundations for Neurological Research , STENA Foundation, Alzheimer’s Foundation, The King Gustav V Jubilee Clinic Cancer Research Foundation (JK - fonden), The Frimurare Barnhus Foundati on, The, the New Zealand National Research Centre for Growth and Development, and the New Zealand Health Research Council, AFA Insurance, Sahlgrenska Foundation, S. A. Olsson Foundation for Research and Culture , as well as W. and M. Lundgren’s, R., T. Söd erberg’s, E. Jacobson’s, R. and U. Amlöv’s, T. Nilsson’s, Emelle’s, O.E . and E. Johanssons and L. Hierta Memorial Foundations. Anna Stokowska   77 REFERENCES Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003) Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A 100:15983-15988. Abraham WC, Bear MF (1996) Metaplasticity: the plasticity of synaptic plasticity. Trends Neurosci 19:126-130. Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, G ordon DL, Marsh EE, 3rd (1993) Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 24:35-41. Alper CA, Johnson AM, Birtch AG, Moore FD (1969) Human C'3: evidence for the liver as the primary site of synthesis. Science 163:286-288. Alvarez- Buylla A, Lim DA (2004) For the long run: maintaining germinal niches in the adult brain. Neuron 41:683-686. Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Ac ker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber -Lang M (2010) Molecular intercommunication between the complement and coagulation systems. J Immunol 185:5628 -5636. Araque A, Parpura V, Sanzgiri RP, Haydon PG (1998) Glutamate- dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur J Neurosci 10:2129 -2142. Arboix A, Marti -Vilalta JL (2004) New concepts in lacunar stroke etiology: the constellation of small-vessel arterial disease. Cerebrovasc Dis 17 Suppl 1:58-62. Ballabio E, Bersano A, Bresolin N, Candelise L (2007) Monogenic vessel diseases related to ischemic stroke: a clinical approach. J Cereb Blood Flow Metab 27:1649-1662. Balschun D, Wetzel W, Del Rey A, Pitossi F, Schneider H, Zuschratter W, Besedov sky HO (2004) Interleukin- 6: a cytokine to forget. FASEB J 18:1788 -1790. Bamford J, Sandercock P, Jones L, Warlow C (1987) The natural history of lacunar infarction: the Oxfordshire Community Stroke Project. Stroke 18:545 -551. Bang OY, Lee PH, Joo SY, Lee JS, Joo IS, Huh K (2003) Frequency and mechanisms of stroke recurrence after cryptogenic stroke. Ann Neurol 54:227-234. Barnum SR (1995) Complement biosynthesis in the central nervous system. Crit Rev Oral Biol Med 6:132 -146. Barres BA (2008) The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60:430-440. Barrett JC, Cardon LR (2006) Evaluating coverage of genome- wide association studies. Nat Genet 38:659 -662. Bashir ZI, Collingridge GL (1994) An investigation of depotentiation of long -term potentiation in the CA1 region of the hippocampus. Exp Brain Res 100:437 -443. Battista D, Ferrari CC, Gage FH, Pitossi FJ (2006) Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus. Eur J Neurosci 23:83 -93. Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC (2002) Control of synaptic strength by glial TNFalpha. Science 295:2282-2285. Benard M, Raoult E, Vaudr y D, Leprince J, Falluel- Morel A, Gonzalez BJ, Galas L, Vaudry H, Fontaine M (2008) Role of complement anaphylatoxin receptors Comple m ent in stroke and neural plasticity   78 (C3aR, C5aR) in the development of the rat cerebellum. Mol Immunol 45:3767- 3774. Berg A, Zelano J, Stephan A, Thams S, Barres BA, Pekny M, Pekna M, Cullheim S (2012) Reduced removal of synaptic terminals from axotomized spinal motoneurons in the absence of complement C3. Exp Neurol 237:8 -17. Bliss TV, Lomo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331 -356. Bogestål YR, Barnum SR, Smith PL, Mattisson V, Pekny M, Pekna M (2007) Signaling through C5aR is not involved in basal neurogenesis. J Neurosci Res 85:2892 - 2897. Bolliger MF, Martinelli DC, Sudhof TC (2011) The cell- adhesion G protein -coupled receptor BAI3 is a high-affinity receptor for C1q -like proteins. Proc Natl Acad Sci U S A 108:2534 -2539. Boos L, Szalai AJ, Barnum SR (2005) C3a expressed in the central nervous system protects against LPS -induced shock. Neurosci Lett 387:68- 71. Boulanger LM, Huh GS, Shatz CJ (2001) Neuronal plasticity and cellular immunity: shared molecular mechanisms. Curr Opin Neurobiol 11:568 -578. Brown CE, Aminoltejari K, Erb H, Winship IR, Murphy TH (2009) In vivo voltage- sensitive dye imaging in adult mice reveals that somatosensory maps lost to stroke are replaced over weeks by new structural and functional circuits with prolonged modes of activation within both the peri-infarct zon e and distant sites. J Neurosci 29:1719 -1734. Butovsky O, Talpalar AE, Ben -Yaakov K, Schwartz M (2005) Activation of microglia by aggregated beta-amyloid or lipopolysaccharide impairs MHC -II expression and renders them cytotoxic whereas IFN -gamma and IL -4 render them protective. Mol Cell Neurosci 29:381-393. Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino S, Martino G, Schwartz M (2006) Microglia activated by IL -4 or IFN -gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. Mol Cell Neurosci 31:149-160. Cain SA, Monk PN (2002) The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). J Biol Chem 277:7165- 7169. Carmichael ST, Chesselet MF (2002) Synchron ous neuronal activity is a signal for axonal sprouting after cortical lesions in the adult. J Neurosci 22:6062 -6070. Carmichael ST, Wei L, Rovainen CM, Woolsey TA (2001) New patterns of intracortical projections after focal cortical stroke. Neurobiol Dis 8 :910-922. Carmichael ST, Archibeque I, Luke L, Nolan T, Momiy J, Li S (2005) Growth -associated gene expression after stroke: evidence for a growth -promoting region in peri- infarct cortex. Exp Neurol 193:291- 311. Carroll MC (2004) The complement system in regulation of adaptive immunity. Nat Immunol 5:981-986. Chollet F, DiPiero V, Wise RJ, Brooks DJ, Dolan RJ, Frackowiak RS (1991) The functional anatomy of motor recovery after stroke in humans: a study with positron emission tomography. Ann Neurol 29:63-71. Chopp M, Li Y (2002) Treatment of neural injury with marrow stromal cells. Lancet Neurol 1:92-100. Anna Stokowska   79 Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA (2005) Thrombospondins are astrocyte - secreted proteins that promote CNS synaptogenesis. Cell 120:421-433. Chu Y, Jin X, Parada I, Pesic A, Stevens B, Barres B, Prince DA (2010) Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proc Natl Acad Sci U S A 107:7975-7980. Cianflone KM, Sniderman AD, Walsh MJ, Vu HT, Gagnon J, Rodriguez MA (1989) Purification and characterization of acylation stimulating protein. J Biol Chem 264:426-430. Clarkson AN, Huang BS, Macisaac SE, Mody I, Carmichael ST (2010) Reducing excessive GABA -mediated tonic inhibition promotes functional recovery after stroke. Nature 468:305-309. Clugnet MC, LeDoux JE (1990) Synaptic plasticity in fear conditioning circuits: induction of LTP in the lateral nucleus of the amygdala by stimulation of the medial geniculate body. J Neurosci 10:2818 -2824. Cole FS, Matthews WJ, Jr., Rossing TH, Gash DJ, Lichtenberg NA, Pennington JE (1983) Complement biosynthesis by human bronchoalveolar macrophages. Clin Immunol Immunopathol 27:153-159. Cooper DN (2010) Functional intronic polymo rphisms: Buried treasure awaiting discovery within our genes. Human genomics 4:284 -288. Costa C, Zhao L, Shen Y, Su X, Hao L, Colgan SP, Stahl GL, Zhou T, Wang Y (2006) Role of complement component C5 in cerebral ischemia/reperfusion injury. Brain Res 1100:142-151. Coyle JT, Puttfarcken P (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689-695. Cronin CA (2010) Intravenous tissue plasminogen activator for stroke: a review of the ECASS III results in relation to prior clinica l trials. J Emerg Med 38:99 -105. Cui W, Simaan M, Laporte S, Lodge R, Cianflone K (2009) C5a - and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I -C5L2 mutation. Mol Immunol 46:3086-3098. Cuzzocrea S, Riley DP, Caputi AP, Salvemini D (2001) Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacol Rev 53:135 -159. Dahl MR, Thiel S, Matsushita M, Fujita T, Willis AC, Christensen T, Vorup -Jensen T, Jensenius JC (2001) MASP -3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15:127-135. Dancause N, Barbay S, Frost SB, Plautz EJ, Chen D, Zoubina EV, Stowe AM, Nudo RJ (2005) Extensive cortical rewiring after brain inj ury. J Neurosci 25:10167 - 10179. Daveau M, Benard M, Scotte M, Schouft MT, Hiron M, Francois A, Salier JP, Fontaine M (2004) Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat. J Immunol 173:3418-3424. Davoust N, Nataf S, Holers VM, Barnum SR (1999) Expression of the murine complement regulatory protein crry by glial cells and neurons. Glia 27:162 -170. de Jong G, Kessels F, Lodder J (2002) Two types of lacunar infarcts: further arguments from a study on prognosis. Stroke; a journal of cerebral circulation 33:2072 - 2076. Comple m ent in stroke and neural plasticity   78 (C3aR, C5aR) in the development of the rat cerebellum. Mol Immunol 45:3767- 3774. Berg A, Zelano J, Stephan A, Thams S, Barres BA, Pekny M, Pekna M, Cullheim S (2012) Reduced removal of synaptic terminals from axotomized spinal motoneurons in the absence of complement C3. Exp Neurol 237:8 -17. Bliss TV, Lomo T (1973) Long -lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331 -356. Bogestål YR, Barnum SR, Smith PL, Mattisson V, Pekny M, Pekna M (2007) Signaling through C5aR is not involved in basal neurogenesis. J Neurosci Res 85:2892 - 2897. Bolliger MF, Martinelli DC, Sudhof TC (2011) The cell -adhesion G protein -coupled receptor BAI3 is a high-affinity receptor for C1q -like proteins. Proc Natl Acad Sci U S A 108:2534 -2539. Boos L, Szalai AJ, Barnum SR (2005) C3a expressed in the central nervous system protects against LPS -induced shock. Neurosci Lett 387:68 -71. Boulanger LM, Huh GS, Shatz CJ (2001) Neuronal plasticity and cellular immunity: shared molecular mechanisms. Curr Opin Neurobiol 11:568 -578. Brown CE, Aminoltejari K, Erb H, Winsh ip IR, Murphy TH (2009) In vivo voltage - sensitive dye imaging in adult mice reveals that somatosensory maps lost to stroke are replaced over weeks by new structural and functional circuits with prolonged modes of activation within both the peri-infarct zon e and distant sites. J Neurosci 29:1719 -1734. Butovsky O, Talpalar AE, Ben -Yaakov K, Schwartz M (2005) Activation of microglia by aggregated beta-amyloid or lipopolysaccharide impairs MHC -II expression and renders them cytotoxic whereas IFN -gamma and IL -4 render them protective. Mol Cell Neurosci 29:381-393. Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino S, Martino G, Schwartz M (2006) Microglia activated by IL -4 or IFN -gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. Mol Cell Neurosci 31:149-160. Cain SA, Monk PN (2002) The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). J Biol Chem 277:7165 -7169. Carmichael ST, Chesselet MF (2002) Synchron ous neuronal activity is a signal for axonal sprouting after cortical lesions in the adult. J Neurosci 22:6062 -6070. Carmichael ST, Wei L, Rovainen CM, Woolsey TA (2001) New patterns of intracortical projections after focal cortical stroke. Neurobiol Dis 8 :910-922. Carmichael ST, Archibeque I, Luke L, Nolan T, Momiy J, Li S (2005) Growth -associated gene expression after stroke: evidence for a growth -promoting region in peri- infarct cortex. Exp Neurol 193:291 -311. Carroll MC (2004) The complement system in regulation of adaptive immunity. Nat Immunol 5:981-986. Chollet F, DiPiero V, Wise RJ, Brooks DJ, Dolan RJ, Frackowiak RS (1991) The functional anatomy of motor recovery after stroke in humans: a study with positron emission tomography. Ann Neurol 29:63-71. Chopp M, Li Y (2002) Treatment of neural injury with marrow stromal cells. Lancet Neurol 1:92-100. Anna Stokowska   79 Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA (2005) Thrombospondins are astrocyte - secreted proteins that promote CNS synaptogenesis. Cell 120:421-433. Chu Y, Jin X, Parada I, Pesic A, Stevens B, Barres B, Prince DA (2010) Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proc Natl Acad Sci U S A 107:7975-7980. Cianflone KM, Sniderman AD, Walsh MJ, Vu HT, Gagnon J, Rodriguez MA (1989) Purification and characterization of acylation stimulating protein. J Biol Chem 264:426-430. Clarkson AN, Huang BS, Macisaac SE, Mody I, Carmichael ST (2010) Reducing excessive GABA -mediated tonic inhibition promotes functional recovery after stroke. Nature 468:305-309. Clugnet MC, LeDoux JE (1990) Synaptic plasticity in fear conditioning circuits: induction of LTP in the lateral nucleus of the amygdala by stimulation of the medial geniculate body. J Neurosci 10:2818 -2824. Cole FS, Matthews WJ, Jr., Rossing TH, Gash DJ, Lichtenberg NA, Pennington JE (1983) Complement biosynthesis by human bronchoalveolar macrophages. Clin Immunol Immunopathol 27:153-159. Cooper DN (2010) Functional intronic polymorphisms: Buried treasure awaiting discovery within our genes. Human genomics 4:284 -288. Costa C, Zhao L, Shen Y, Su X, Hao L, Colgan SP, Stahl GL, Zhou T, Wang Y (2006) Role of complement component C5 in cerebral ischemia/reperfusion injury. Brain Res 1100:142-151. Coyle JT, Puttfarcken P (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689-695. Cronin CA (2010) Intravenous tissue plasminogen activator for stroke: a review of the ECASS III results in relation to prior clinical trials. J Emerg Med 38:99 -105. Cui W, Simaan M, Laporte S, Lodge R, Cianflone K (2009) C5a - and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I- C5L2 mutation. Mol Immunol 46:3086-3098. Cuzzocrea S, Riley DP, Caputi AP, Salvemini D (2001) Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacol Rev 53:135- 159. Dahl MR, Thiel S, Matsushita M, Fujita T, Willis AC, Christensen T, Vorup -Jensen T, Jensenius JC (2001) MASP -3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15:127-135. Dancause N, Barbay S, Frost SB, Plautz EJ, Chen D, Zoubina EV, Stowe AM, Nudo RJ (2005) Extensive cortical rewiring after brain injury. J Neurosci 25:10167 - 10179. Daveau M, Benard M, Scotte M, Schouft MT, Hiron M, Francois A, Salier JP, Fontaine M (2004) Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat. J Immunol 173:3418-3424. Davoust N, Nataf S, Holers VM, Barnum SR (1999) Expression of the murine complement regulatory protein crry by glial cells and neurons. Glia 27:162 -170. de Jong G, Kessels F, Lodder J (2002) Two types of lacunar infarcts: further arguments from a study on prognosis. Stroke; a journal of cerebral circulation 33:2072 - 2076. Comple m ent in stroke and neural plasticity   80 De Simoni MG, Storini C, Barba M, Catapano L, Arabia AM, Rossi E, Bergamaschini L (2003) Neuroprotection by complement (C1) inhibitor in mouse transient brain ischemia. J Cereb Blood Flow Metab 23:232 -239. Del Rio-Tsonis K, Tsonis PA, Zarkadis IK, Tsagas AG, Lambris JD (1998) Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles. J Immunol 161:6819 -6824. Derecki NC, Cardani AN, Yang CH, Quinnies KM, Crihfield A, Lynch KR, Kipnis J (2010) Regulation of learning and memory by meningeal immunity: a key role for IL -4. J Exp Med 207:1067- 1080. Donnan GA, Fisher M, Macleod M, Davis SM (2008) Stroke. Lancet 371:1612 -1623. Duan X, Kang E, Liu CY, Ming GL, Song H (2008) Development of neural stem cell in the adult brain. Curr Opin Neurobiol 18:108 -115. Ehninger D, Kempermann G (2003) Regional effects of wheel running and environmental enrichment on cell genesis and microglia proliferation in the adult murine neocortex. Cereb Cortex 13:845 -851. Einstein LP, Hansen PJ, Ballow M, Davis AE, 3rd, Davis JSt, Alper CA, Rosen FS, Colten HR (1977) Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3 -deficient humans. J Clin Invest 60:963-969. Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003) Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci U S A 100:13632-13637. Elkabes S, DiCicco -Bloom EM, Black IB (1996) Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci 16:2508-2521. Enzinger C, Johansen- Berg H, Dawes H, Bogdanovic M, Collett J, Guy C, Ropele S, Kischka U, Wade D, Fazekas F, Matthews PM (2008) Functional MRI correlates of lower limb function in stroke victims with gait impairment. Stroke 39:1507-1513. Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Ozkan E, Chakraborty C, Mulinyawe SB, Annis DS, Huberman AD, Green EM, Lawler J, Dolmetsch R, Garcia KC, Smith SJ, Luo ZD, Rosenthal A, Mosher DF, Barres BA (2009) Gabapentin receptor alpha2delta- 1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139:380 -392. Fee D, Grzybicki D, Dobbs M, Ihyer S, Clotfelter J, Macvilay S, Hart MN, Sandor M, Fabry Z (2000) Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells. Cytokine 12:655-665. Ferro JM (2003) Brain embolism - Answers to practical questions. J Neurol 250:139- 147. Ferro JM, Massaro AR, Mas JL (2010) Aetiological diagnosis of ischaemic stroke in young adults. Lancet Neurol 9:1085 -1096. Fischer WH, Jagels MA, Hugli TE (1999) Regulation of IL -6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). J Immunol 162:453-459. Flossmann E (2006) Genetics of ischaemic stroke; single gene disorders. Int J Stroke 1:131-139. Frahm C, Haupt C, Weinandy F, Siegel G, Bruehl C, Witte OW (2004) Regulation of GABA transporter mRNA and protein after photothrombotic infarct in rat brain. J Comp Neurol 478:176 -188. Anna Stokowska   81 Freeman WD, Aguilar MI (2011) Prevention of cardioembolic stroke. Neurotherapeutics 8:488-502. Fujita T, Matsushita M, Endo Y (2004) The lectin -complement pathway--its role in innate immunity and evolution. Immunol Rev 198:185-202. Gavrilyuk V, Kalinin S, Hilbush BS, Middlecamp A, McGuire S, Pelligrino D, Weinberg G, Feinstein DL (2005) Identification of complement 5a -like receptor (C5L2) from astrocytes: characterization of anti -inflammatory properties. J Neurochem 92:1140-1149. Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H (2006) GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 439:589-593. Gendrel M, Rapti G, Richmond JE, Bessereau JL (2009) A secreted complement-control- related protein ensures acetylcholine receptor clustering. Nature 461:992-996. Goshen I, Kreisel T, Ounallah -Saad H, Renbaum P, Zalzstein Y, Ben -Hur T, Levy -Lahad E, Yirmiya R (2007) A dual role for interleukin -1 in hippocampal-dependent memory processes. Psychoneuroendocrinology 32:1106-1115. Guenard V, Dinarello CA, Weston PJ, Aebischer P (1991) Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel. J Neurosci Res 29:396-400. Guercini F, Acciarresi M, Agnelli G, Paciaroni M (2008) Cryptogenic stroke: time to determine aetiology. J Thromb Haemost 6:549 -554. Hankey GJ (2006) Potential new risk factors for ischemic stroke: what is their potential? Stroke 37:2181-2188. Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, Mor F, Leibowitz - Amit R, Pevsner E, Akselrod S, Neeman M, Cohen IR, Schwartz M (2000) Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J Neurosci 20:6421-6430. Hebb D (1949) The organization of behaviour. New York: Wiley. Heese K, Hock C, Otten U (1998) Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem 70:699 -707. Hermann DM, Chopp M (2012) Promoting brain remodelling and plasticity for stroke recovery: therapeutic promise and potential pitfalls of clinical translation. Lancet Neurol 11:369-380. Hess G, Donoghue JP (1994) Long -term potentiation of horizontal connections provides a mechanism to reorganize cortical motor maps. J Neurophysiol 71:2543 -2547. Hess G, Aizenman CD, Donoghue JP (1996) Conditions for the induction of long -term potentiation in layer II/III horizontal connections of the rat motor cortex. J Neurophysiol 75:1765-1778. Hoehn BD, Palm er TD, Steinberg GK (2005) Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin. Stroke 36:2718-2724. Hossmann KA (1994) Viability thresholds and the penumbra of focal ischemia. Ann Neurol 36:557-565. Huang J, Kim LJ, Mealey R, Ma rsh HC, Jr., Zhang Y, Tenner AJ, Connolly ES, Jr., Pinsky DJ (1999) Neuronal protection in stroke by an sLex -glycosylated complement inhibitory protein. Science 285:595-599. Huang YY, Colino A, Selig DK, Malenka RC (1992) The influence of prior synaptic activity on the induction of long-term potentiation. Science 255:730-733. Huber -Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Comple m ent in stroke and neural plasticity   80 De Simoni MG, Storini C, Barba M, Catapano L, Arabia AM, Rossi E, Bergamaschini L (2003) Neuroprotection by complement (C1) inhibitor in mouse transient brain ischemia. J Cereb Blood Flow Metab 23:232 -239. Del Rio-Tsonis K, Tsonis PA, Zarkadis IK, Tsagas AG, Lambris JD (1998) Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles. J Immunol 161:6819 -6824. Derecki NC, Cardani AN, Yang CH, Quinnies KM, Crihfield A, Lynch KR, Kipnis J (2010) Regulation of learning and memory by meningeal immunity: a key role for IL -4. J Exp Med 207:1067 -1080. Donnan GA, Fisher M, Macleod M, Davis SM (2008) Stroke. Lancet 371:1612 -1623. Duan X, Kang E, Liu CY, Ming GL, Song H (2008) Development of neural stem cell in the adult brain. Curr Opin Neurobiol 18:108 -115. Ehninger D, Kempermann G (2003) Regional effects of wheel running and environmental enrichment on cell genesis and microglia proliferation in the adult murine neocortex. Cereb Cortex 13:845 -851. Einstein LP, Hansen PJ, Ballow M, Davis AE, 3rd, Davis JSt, Alper CA, Rosen FS, Colten HR (1977) Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3 -deficient humans. J Clin Invest 60:963-969. Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003) Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci U S A 100:13632-13637. Elkabes S, DiCicco -Bloom EM, Bl ack IB (1996) Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci 16:2508-2521. Enzinger C, Johansen -Berg H, Dawes H, Bogdanovic M, Collett J, Guy C, Ropele S, Kischka U, Wade D, Faz ekas F, Matthews PM (2008) Functional MRI correlates of lower limb function in stroke victims with gait impairment. Stroke 39:1507-1513. Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Ozkan E, Chakraborty C, Mulinyawe SB, Annis DS, Huberman AD, Green EM, Lawler J, Dolmetsch R, Garcia KC, Smith SJ, Luo ZD, Rosenthal A, Mosher DF, Barres BA (2009) Gabapentin receptor alpha2delta -1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139:380 -392. Fee D, Grzybicki D, D obbs M, Ihyer S, Clotfelter J, Macvilay S, Hart MN, Sandor M, Fabry Z (2000) Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells. Cytokine 12:655-665. Ferro JM (2003) Brain embolism - Answers to practical questions. J Neurol 250:139-147. Ferro JM, Massaro AR, Mas JL (2010) Aetiological diagnosis of ischaemic stroke in young adults. Lancet Neurol 9:1085 -1096. Fischer WH, Jagels MA, Hugli TE (1999) Regulation of IL -6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). J Immunol 162:453-459. Flossmann E (2006) Genetics of ischaemic stroke; single gene disorders. Int J Stroke 1:131-139. Frahm C, Haupt C, Weinandy F, Siegel G, Bruehl C, Witte OW (2004) Regulation of GABA transporter mRNA and protein after photothrombotic infarct in rat brain. J Comp Neurol 478:176 -188. Anna Stokowska   81 Freeman WD, Aguilar MI (2011) Prevention of cardioembolic stroke. Neurotherapeutics 8:488-502. Fujita T, Matsushita M, Endo Y (2004) The lectin -complement pathway--its role in innate immunity and evolution. Immunol Rev 198:185-202. Gavrilyuk V, Kalinin S, Hilbush BS, Middlecamp A, McGuire S, Pelligrino D, Weinberg G, Feinstein DL (2005) Identification of complement 5a -like receptor (C5L2) from astrocytes: characterization of anti -inflammatory properties. J Neurochem 92:1140-1149. Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H (2006) GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 439:589-593. Gendrel M, Rapti G, Richmond JE, Bessereau JL (2009) A secreted complement -control- related protein ensures acetylcholine receptor clustering. Nature 461:992-996. Goshen I, Kreisel T, Ounallah -Saad H, Renbaum P, Zalzstein Y, Ben- Hur T, Levy- Lahad E, Yirmiya R (2007) A dual role for interleukin -1 in hippocampal-dependent memory processes. Psychoneuroendocrinology 32:1106-1115. Guenard V, Dinarello CA, Weston PJ, Aebischer P (1991) Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel. J Neurosci Res 29:396 -400. Guercini F, Acciarresi M, Agnelli G, Paciaroni M (2008) Cryptogenic stroke: time to determine aetiology. J Thromb Haemost 6:549 -554. Hankey GJ (2006) Potential new risk factors for ischemic stroke: what is their potential? Stroke 37:2181-2188. Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, Mor F, Leibowitz - Amit R, Pevsner E, Akselrod S, Neeman M, Cohen IR, Schwartz M (2000) Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J Neurosci 20:6421 -6430. Hebb D (1949) The organization of behaviour. New York: Wiley. Heese K, Hock C, Otten U (1998) Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem 70:699 -707. Hermann DM, Chopp M (2012) Promoting brain remodelling and plasticity for stroke recovery: therapeutic promise and potential pitfalls of clinical translation. Lancet Neurol 11:369-380. Hess G, Donoghue JP (1994) Long- term potentiation of horizontal connections provides a mechanism to reorganize cortical motor maps. J Neurophysiol 71:2543 -2547. Hess G, Aizenman CD, Donoghue JP (1996) Conditions for the induction of long -term potentiation in layer II/III horizontal connections of the rat motor cortex. J Neurophysiol 75:1765-1778. Hoehn BD, Palm er TD, Steinberg GK (2005) Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin. Stroke 36:2718-2724. Hossmann KA (1994) Viability thresholds and the penumbra of focal ischemia. Ann Neurol 36:557-565. Huang J, Kim LJ, Mealey R, Ma rsh HC, Jr., Zhang Y, Tenner AJ, Connolly ES, Jr., Pinsky DJ (1999) Neuronal protection in stroke by an sLex -glycosylated complement inhibitory protein. Science 285:595-599. Huang YY, Colino A, Selig DK, Malenka RC (1992) The influence of prior synaptic activity on the induction of long-term potentiation. Science 255:730-733. Huber -Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Comple m ent in stroke and neural plasticity   82 Wetsel RA, Ward PA (2006) Generatio n of C5a in the absence of C3: a new complement activation pathway. Nat Med 12:682-687. Ignatius A, Schoengraf P, Kreja L, Liedert A, Recknagel S, Kandert S, Brenner RE, Schneider M, Lambris JD, Huber- Lang M (2011a) Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL -1beta. J Cell Biochem 112:2594 -2605. Ignatius A, Ehrnthaller C, Brenner RE, Kreja L, Schoengraf P, Lisson P, Blakytny R, Recknagel S, Claes L, Gebhard F, Lambris JD, Huber -Lang M (2011b) The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro. J Trauma 71:952 -960. Jacobs KM, Donoghue JP (1991) Reshaping the cortical motor map by unmasking latent intracortical connections. Science 251:944-947. Jaderstad J, Jaderstad LM, Li J, Chintawar S, Salto C, Pandolfo M, Ourednik V, Teng YD, Sidman RL, Arenas E, Snyder EY, Herlenius E (2010) Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host. Proc Natl Acad Sci U S A 107:5184 - 5189. Janeway C, Travers P, Walport M, Shlomchik M (2005) Immunobiology : the immune system in health and disease, Chapter: The complement system and innate immunity, 6th Edition. London: Garland Science Publishing. Jankowsky JL, Derrick BE, Patterson PH (2000) Cytokine responses to LTP induction in the rat hippocampus: a comparison of in vitro and in vivo techniques. Learn Mem 7:400-412. Janssen BJ, Christodoulidou A, McCarthy A, Lamb ris JD, Gros P (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444:213-216. Järlestedt K, Rousset CI, Ståhlberg A, Sourkova H, Atkins AL, Thornton C, Barnum SR, Wetsel RA, Dragunow M, Pekny M, Mallard C, Hag berg H, Pekna M (2013) Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic - ischemic brain injury. FASEB J 27:3797-3804. Jauneau AC, Ischenko A, Chatagner A, Benard M, Chan P, Schouft MT, Patte C, Vaudry H, Fontaine M (2006) Interleukin- 1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes. J Neuroinflammation 3:8. Jiang W, Gu W, Brannstrom T, Rosqvist R, Wester P (2001) Cortical neurogenesis in adult rats after transient middle cerebral artery occlusion. Stroke 32:1201-1207. Jickling GC, Stamova B, Ander BP, Zhan X, Liu D, Sison SM, Verro P, Sharp FR (2012) Prediction of cardioembolic, arterial, and lacunar causes of cryptogenic stroke by gene expression and infarct location. Stroke 43:2036 -2041. Jood K, Ladenvall C, Rosengren A, Blomstrand C, Jern C (2005) Family history in ischemic stroke before 70 years of age: the Sahlgrenska Academy Study on Ischemic Stroke. Stroke 36:1383-1387. Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, Matute C, Tonello F, Gundersen V, Volterra A (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci 10:331-339. Jung KW, Shon YM, Yang DW, Kim BS, Cho AH (2012) Coexisting carotid atherosclerosis in patients with intracranial small- or large-vessel disease. J Clin Neurol 8:104-108. Anna Stokowska   83 Kalant D, Cain SA, Maslowska M, Sniderman AD, Cianflone K, Monk PN (2003) The chemoattractant receptor-like protein C5L2 binds the C3a des -Arg77/acylation - stimulating protein. J Biol Chem 278:11123 -11129. Kau er JA, Malenka RC, Nicoll RA (1988) A persistent postsynaptic modification mediates long-term potentiation in the hippocampus. Neuron 1:911-917. Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T, Klinkert WE, Kolbeck R, Hoppe E, Oropeza -Wekerle RL, Bartke I, Stadelmann C, Lassmann H, Wekerle H, Hohlfeld R (1999) Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 18 9:865-870. Kildsgaard J, Hollmann TJ, Matthews KW, Bian K, Murad F, Wetsel RA (2000) Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin -shock. J Immunol 165:5406-5409. Kim CH, Wu W, Wysoczynski M, Abdel -Latif A, Sunkara M, Morris A, Kucia M, Ratajczak J, Ratajczak MZ (2011) Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b- C9 as homing factors. Leukemia 26:106 -116. Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA, Welch KM (1995) Expression of monocyte chemoattractant protein -1 and macrophage inflammatory protein-1 after focal cerebral ischemia in the rat. J Neuroimmunol 56:127-134. Kimura Y, Madhavan M, Call MK, Santiago W, Tsonis PA, Lambris JD, Del Rio -Tsonis K (2003) Expression of complement 3 and complement 5 in newt limb and lens regeneration. J Immunol 170:2331 -2339. Kirshner HS (2 009) Differentiating ischemic stroke subtypes: risk factors and secondary prevention. J Neurol Sci 279:1 -8. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Kohl J (2009) The role of the anaphylatoxins in health and disease. Mol Immunol 46:2753 -2766. Koku bo Y (2012) Traditional risk factor management for stroke: a never-ending challenge for health behaviors of diet and physical activity. Curr Opin Neurol 25:11-17. Konorski J (1948) Conditioned Reflexes and Neuron Organization. Cambridge, MA.: Cambridge Uni versity Press. Korn H, Faber DS (1991) Quantal analysis and synaptic efficacy in the CNS. Trends Neurosci 14:439-445. Kramer J, Harcos P, Prohaszka Z, Horvath L, Karadi I, Singh M, Csaszar A, Romics L, Fust G (2000) Frequencies of certain complement protei n alleles and serum levels of anti-heat-shock protein antibodies in cerebrovascular diseases. Stroke 31:2648-2652. Kuan CY, Schloemer AJ, Lu A, Burns KA, Weng WL, Williams MT, Strauss KI, Vorhees CV, Flavell RA, Davis RJ, Sharp FR, Rakic P (2004) Hypoxia - ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci 24:10763 -10772. Lakhan SE, Kirchgessner A, Hofer M (2009) Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 7:97. Comple m ent in stroke and neural plasticity   82 Wetsel RA, Ward PA (2006) Generatio n of C5a in the absence of C3: a new complement activation pathway. Nat Med 12:682-687. Ignatius A, Schoengraf P, Kreja L, Liedert A, Recknagel S, Kandert S, Brenner RE, Schneider M, Lambris JD, Huber -Lang M (2011a) Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL -1beta. J Cell Biochem 112:2594 -2605. Ignatius A, Ehrnthaller C, Brenner RE, Kreja L, Schoengraf P, Lisson P, Blakytny R, Recknagel S, Claes L, Gebhard F, Lambris JD, Huber -Lang M (2 011b) The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro. J Trauma 71:952 -960. Jacobs KM, Donoghue JP (1991) Reshaping the cortical motor map by unmasking latent intracortical connections. Science 251:944-947. Jaderstad J, Jaderstad LM, Li J, Chintawar S, Salto C, Pandolfo M, Ourednik V, Teng YD, Sidman RL, Arenas E, Snyder EY, Herlenius E (2010) Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host. Proc Natl Acad Sci U S A 107:5184 - 5189. Janeway C, Travers P, Walport M, Shlomchik M (2005) Immunobiology : the immune system in health and disease, Chapter: The complement system and innate immunity, 6th Edition. Lon don: Garland Science Publishing. Jankowsky JL, Derrick BE, Patterson PH (2000) Cytokine responses to LTP induction in the rat hippocampus: a comparison of in vitro and in vivo techniques. Learn Mem 7:400-412. Janssen BJ, Christodoulidou A, McCarthy A, Lamb ris JD, Gros P (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444:213-216. Järlestedt K, Rousset CI, Ståhlberg A, Sourkova H, Atkins AL, Thornton C, Barnum SR, Wetsel RA, Dragunow M, Pekny M, Mallard C, Hag berg H, Pekna M (2013) Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic - ischemic brain injury. FASEB J. Jauneau AC, Ischenko A, Chatagner A, Benard M, Chan P, Schouft MT, Patte C, Vaudry H, Fontaine M (2006) Interleukin -1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes. J Neuroinflammation 3:8. Jiang W, Gu W, Brannstrom T, Rosqvist R, Wester P (2001) Cortical neurogenesis in adult rats after transient middle cerebral artery occlusion. Stroke 32:1201-1207. Jickling GC, Stamova B, Ander BP, Zhan X, Liu D, Sison SM, Verro P, Sharp FR (2012) Prediction of cardioembolic, arterial, and lacunar causes of cryptogenic stroke by gene expression and infarct location. Stroke 43:2036 -2041. Jood K, Ladenvall C, Rosengren A, Blomstrand C, Jern C (2005) Family history in ischemic stroke before 70 years of age: the Sahlgrenska Academy Study on Ischemic Stroke. Stroke 36:1383-1387. Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, Matute C, Tonello F, Gundersen V, Volterra A (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci 10:331-339. Jung KW, Shon YM, Yang DW, Kim BS, Cho AH (2012) Coexisting carotid atherosclerosis in patients with intracranial small- or large-vessel disease. J Clin Neurol 8:104-108. Anna Stokowska   83 Kalant D, Cain SA, Maslowska M, Sniderman AD, Cianflone K, Monk PN (2003) The chemoattractant receptor-like protein C5L2 binds the C3a des -Arg77/acylation - stimulating protein. J Biol Chem 278:11123 -11129. Kau er JA, Malenka RC, Nicoll RA (1988) A persistent postsynaptic modification mediates long-term potentiation in the hippocampus. Neuron 1:911-917. Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T, Klinkert WE, Kolbeck R, Hoppe E, Oropeza- Wekerle RL, Bartke I, Stadelmann C, Lassmann H, Wekerle H, Hohlfeld R (1999) Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 18 9:865-870. Kildsgaard J, Hollmann TJ, Matthews KW, Bian K, Murad F, Wetsel RA (2000) Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin- shock. J Immunol 165:5406-5409. Kim CH, Wu W, Wysoczynski M, Abdel -Latif A, Sunkara M, Morris A, Kucia M, Ratajczak J, Ratajczak MZ (2011) Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b- C9 as homing factors. Leukemia 26:106 -116. Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA, Welch KM (1995) Expression of monocyte chemoattractant protein -1 and macrophage inflammatory protein-1 after focal cerebral ischemia in the rat. J Neuroimmunol 56:127-134. Kimura Y, Madhavan M, Call MK, Santiago W, Tsonis PA, Lambris JD, Del Rio -Tsonis K (2003) Expression of complement 3 and complement 5 in newt limb and lens regeneration. J Immunol 170:2331- 2339. Kirshner HS (2 009) Differentiating ischemic stroke subtypes: risk factors and secondary prevention. J Neurol Sci 279:1 -8. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Kohl J (2009) The role of the anaphylatoxins in health and disease. Mol Immunol 46:2753 -2766. Kokubo Y (2012) Traditional risk factor management for stroke: a never -ending challenge for health behaviors of diet and physical activity. Curr Opin Neurol 25:11-17. Konorski J (1948) Conditioned Reflexes and Neuron Organization. Cambridge, MA.: Cambridge Uni versity Press. Korn H, Faber DS (1991) Quantal analysis and synaptic efficacy in the CNS. Trends Neurosci 14:439-445. Kramer J, Harcos P, Prohaszka Z, Horvath L, Karadi I, Singh M, Csaszar A, Romics L, Fust G (2000) Frequencies of certain complement protei n alleles and serum levels of anti-heat-shock protein antibodies in cerebrovascular diseases. Stroke 31:2648-2652. Kuan CY, Schloemer AJ, Lu A, Burns KA, Weng WL, Williams MT, Strauss KI, Vorhees CV, Flavell RA, Davis RJ, Sharp FR, Rakic P (2004) Hypoxia- ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci 24:10763 -10772. Lakhan SE, Kirchgessner A, Hofer M (2009) Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 7:97. Comple m ent in stroke and neural plasticity   84 Lam bertsen KL, Gregersen R, Meldgaard M, Clausen BH, Heibol EK, Ladeby R, Knudsen J, Frandsen A, Owens T, Finsen B (2004) A role for interferon -gamma in focal cerebral ischemia in mice. J Neuropathol Exp Neurol 63:942- 955. Lazarov -Spiegler O, Solomon AS, Zeev- Brann AB, Hirschberg DL, Lavie V, Schwartz M (1996) Transplantation of activated macrophages overcomes central nervous system regrowth failure. FASEB J 10:1296 -1302. Lee HM, Wu W, Wysoczynski M, Liu R, Zuba -Surma EK, Kucia M, Ratajczak J, Ratajczak MZ (20 09) Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes. Leukemia 23:2052 -2062. Lee JK, Park MS, Kim YS, Moon KS, Joo SP, Kim TS, Kim JH, Kim SH (2007) Photochemically induced cerebral ischemia in a mouse model. Surg Neurol 67:620-625; discussion 625. Leker RR, Soldner F, Velasco I, Gavin DK, Androutsellis- Theotokis A, McKay RD (2007) Long -lasting regeneration after ischemia in the cerebral cortex. Stroke 38:153-161. Leung VW, Yun S, Botto M, Mason JC, Malik TH, Song W, Paixao -Cavalcante D, Pickering MC, Boyle JJ, Haskard DO (2009) Decay -accelerating factor suppresses complement C3 activation and retards atherosclerosis in low-density lipoprotein receptor-deficient mice. Am J Pathol 175:1757 -1767. Levine SR (2005) Hypercoagulable states and stroke: a selective review. CNS spectrums 10:567-578. Lewis RD, Perry MJ, Guschina IA, Jackson CL, Morgan BP, Hughes TR (2011) CD55 deficiency protects against atherosclerosis in ApoE -deficient mice via C3a modulation of lipid metabolism. Am J Pathol 179:1601 -1607. Li L et al. (2008) Protective role of reactive astrocytes in brain ischemia. J Cereb Blood Flow Metab 28:468- 481. Li R, Coulthard LG, Wu MC, Taylor SM, Woodruff TM (2013) C5L2: a controversial receptor of complement anaphylatoxin, C5a. FASEB J 27:855- 864. Li S, Overman JJ, Katsman D, Kozlov SV, Donnelly CJ, Twiss JL, Giger RJ, Coppola G, Geschwind DH, Carmichae l ST (2010) An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke. Nat Neurosci 13:1496-1504. Lienenklaus S, Ames RS, Tornetta MA, Sarau HM, Foley JJ, Crass T, Sohns B, Raffetseder U, Grove M, Holzer A, Klos A, Kohl J, Bautsch W (1998) Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor. J Immunol 161:2089 -2093. Liszewski MK, Leung M, Cui W, Subramanian VB, Parkinson J, Barlow PN, Manchester M, Atkinson JP (2000) Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46). J Biol Chem 275:37692 - 37701. Liu Z, Zhang RL, Li Y, Cui Y, Chopp M (2009) Remodeling of the corticospinal innervation and spontaneous behavioral recovery after ischemic stroke in adult mice. Stroke 40:2546-2551. Lledo PM, Alonso M, Grubb MS (2006) Adult neurogenesis and functional plasticity in neuronal circuits. Nat Rev Neurosci 7:179-193. Anna Stokowska   85 Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem cells constitut ively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 181:115 -129. Luke LM, Allred RP, Jones TA (2004) Unilateral ischemic sensorimotor cortical damage induces contralesional synaptogenesis and enhances skilled reaching with the ipsilateral forelimb in adult male rats. Synapse 54:187-199. Madinier A, Bertrand N, Mossiat C, Prigent-Tessier A, Beley A, Marie C, Garnier P (2009) Microglial involvement in neuroplastic changes following focal brain ischemia in rats. PLoS One 4:e8101. Magavi SS, Leavitt BR, Macklis JD (2000) Induction of neurogenesis in the neocortex of adult mice. Nature 405:951-955. Malinow R (2003) AMPA receptor trafficking and long-term potentiation. Philos Trans R Soc Lond B Biol Sci 358:707-714. Malinow R, Tsien RW (1990) Presynaptic enhancement shown by whole -cell recordings of long-term potentiation in hippocampal slices. Nature 346:177-180. Mamane Y, Chung Chan C, Lavallee G, Morin N, Xu LJ, Huang J, Gordon R, Thomas W, Lamb J, Sch adt EE, Kennedy BP, Mancini JA (2009) The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation. Diabetes 58:2006-2017. Manev H, Favaron M, Guidotti A, Costa E ( 1989) Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death. Mol Pharmacol 36:106-112. Marchand F, Perretti M, McMahon SB (2005) Role of the immune system in chronic pain. Nat Rev Neurosci 6:521-532. Markiewski MM, Mastellos D, Tudoran R, DeAngelis RA, Strey CW, Franchini S, Wetsel RA, Erdei A, Lambris JD (2004) C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury. J Immunol 173:747 -754. Martin RL, Lloyd H G, Cowan AI (1994) The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Trends Neurosci 17:251 -257. Mastellos D, Papadimitriou JC, Franchini S, Tsonis PA, Lambris JD (2001) A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration. J Immunol 166:2479 -2486. Moalem G, Leibowitz -Amit R, Yoles E, Mor F, Cohen IR, Schwartz M (1999) Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 5:49 -55. Moalem G, Gdalyahu A, Shani Y, Otten U, Lazarovici P, Cohen IR, Schwartz M (2000) Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun 15:331 -345. Mocco J, Wilson DA , Komotar RJ, Sughrue ME, Coates K, Sacco RL, Elkind MS, Connolly ES, Jr. (2006a) Alterations in plasma complement levels after human ischemic stroke. Neurosurgery 59:28-33; discussion 28 -33. Mocco J, Mack WJ, Ducruet AF, Sosunov SA, Sughrue ME, Hassid BG, Nair MN, Laufer I, Komotar RJ, Claire M, Holland H, Pinsky DJ, Connolly ES, Jr. (2006b) Complement component C3 mediates inflammatory injury following focal cerebral ischemia. Circ Res 99:209-217. Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade r estores adult hippocampal neurogenesis. Science 302:1760-1765. Morgan BP, Gasque P (1996) Expression of complement in the brain: role in health and disease. Immunol Today 17:461-466. Comple m ent in stroke and neural plasticity   84 Lam bertsen KL, Gregersen R, Meldgaard M, Clausen BH, Heibol EK, Ladeby R, Knudsen J, Frandsen A, Owens T, Finsen B (2004) A role for interferon -gamma in focal cerebral ischemia in mice. J Neuropathol Exp Neurol 63:942 -955. Lazarov -Spiegler O, Solomon AS, Zeev -Brann AB, Hirschberg DL, Lavie V, Schwartz M (1996) Transplantation of activated macrophages overcomes central nervous system regrowth failure. FASEB J 10:1296 -1302. Lee HM, Wu W, Wysoczynski M, Liu R, Zuba -Surma EK, Kucia M, Ratajczak J, Ratajczak MZ (20 09) Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes. Leukemia 23:2052 -2062. Lee JK, Park MS, Kim YS, Moon KS, Joo SP, Kim TS, Kim JH, Kim SH (2007) Photochemically induced cerebral ischemia in a mouse model. Surg Neurol 67:620-625; discussion 625. Leker RR, Soldner F, Velasco I, Gavin DK, Androutsellis -Theotokis A, McKay RD (2007) Long -lasting regeneration after ischemia in the cerebral cortex. Stroke 38:153-161. Leung VW, Yun S, Botto M, Mason JC, Malik TH, Song W, Paixao -Cavalcante D, Pickering MC, Boyle JJ, Haskard DO (2009) Decay -accelerating factor suppresses complement C3 activation and retards atherosclerosis in low-density lipoprotein receptor-deficient mice. Am J Pathol 175:1757 -1767. Levine SR (2005) Hypercoagulable states and stroke: a selective review. CNS spectrums 10:567-578. Lewis RD, Perry MJ, Guschina IA, Jackson CL, Morgan BP, Hughes TR (2011) CD55 deficiency protects against atherosclerosis in ApoE -deficient mice via C3a modulation of lipid metabolism. Am J Pathol 179:1601 -1607. Li L et al. (2008) Protective role of reactive astrocytes in brain ischemia. J Cereb Blood Flow Metab 28:46 8-481. Li R, Coulthard LG, Wu MC, Taylor SM, Woodruff TM (2013) C5L2: a controversial receptor of complement anaphylatoxin, C5a. FASEB J 27:855 -864. Li S, Overman JJ, Katsman D, Kozlov SV, Donnelly CJ, Twiss JL, Giger RJ, Coppola G, Geschwind DH, Carmichae l ST (2010) An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke. Nat Neurosci 13:1496-1504. Lienenklaus S, Ames RS, Tornetta MA, Sarau HM, Foley JJ, Crass T, Sohns B, Raffetseder U, Grove M, Holzer A, Klos A, Kohl J, Bautsch W (1998) Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor. J Immunol 161:2089 -2093. Liszewski MK, Leung M, Cui W, Subramanian VB, Parkinson J, Barlow PN, Manchester M, Atkinson JP (2000) Dissectin g sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46). J Biol Chem 275:37692 - 37701. Liu Z, Zhang RL, Li Y, Cui Y, Chopp M (2009) Remodeling of the corticospinal innervation and spontaneous behavioral recovery after ischemic stroke in adult mice. Stroke 40:2546-2551. Lledo PM, Alonso M, Grubb MS (2006) Adult neurogenesis and functional plasticity in neuronal circuits. Nat Rev Neurosci 7:179-193. Anna Stokowska   85 Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 181:115 -129. Luke LM, Allred RP, Jones TA (2004) Unilateral ischemic sensorimotor cortical damage induces contralesional synaptogenesis and enhances skilled reaching with the ipsilateral forelimb in adult male rats. Synapse 54:187-199. Madinier A, Bertrand N, Mossiat C, Prigent-Tessier A, Beley A, Marie C, Garnier P (2009) Microglial involvement in neuroplastic changes following focal brain ischemia in rats. PLoS One 4:e8101. Magavi SS, Leavitt BR, Macklis JD (2000) Induction of neurogenesis in the neocortex of adult mice. Nature 405:951-955. Malinow R (2003) AMPA receptor trafficking and long-term potentiation. Philos Trans R Soc Lond B Biol Sci 358:707 -714. Malinow R, Tsien RW (1990) Presynaptic enhancement shown by whole -cell recordings of long-term potentiation in hippocampal slices. Nature 346:177-180. Mamane Y, Chung Chan C, Lavallee G, Morin N, Xu LJ, Huang J, Gordon R, Thomas W, Lamb J, Sch adt EE, Kennedy BP, Mancini JA (2009) The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation. Diabetes 58:2006-2017. Manev H, Favaron M, Guidotti A, Costa E (1989) Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death. Mol Pharmacol 36:106-112. Marchand F, Perretti M, McMahon SB (2005) Role of the immune system in chronic pain. Nat Rev Neurosci 6:521-532. Markiewski MM, Mastellos D, Tudoran R, DeAngelis RA, Strey CW, Franchini S, Wetsel RA, Erdei A, Lambris JD (2004) C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury. J Immunol 173:747 -754. Martin RL, Lloyd HG, Cowan AI (1994) The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Trends Neurosci 17:251 -257. Mastellos D, Papadimitriou JC, Franchini S, Tsonis PA, Lambris JD (2001) A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration. J Immunol 166:2479 -2486. Moalem G, Leibowitz -Amit R, Yoles E, Mor F, Cohen IR, Schwartz M (1999) Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 5:49 -55. Moalem G, Gdalyahu A, Shani Y, Otten U, Lazarovici P, Cohen IR, Schwartz M (2000) Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun 15:331 -345. Mocco J, Wilson DA, Komotar RJ, Sughrue ME, Coates K, Sacco RL, Elkind MS, Connolly ES, Jr. (2006a) Alterations in plasma complement levels after human ischemic stroke. Neurosurgery 59:28-33; discussion 28 -33. Mocco J, Mack WJ, Ducruet AF, Sosunov SA, Sughrue ME, Hassid BG, Nair MN, Laufer I, Komotar RJ, Claire M, Holland H, Pinsky DJ, Connolly ES, Jr. (2006b) Complement component C3 mediates inflammatory injury following focal cerebral ischemia. Circ Res 99:209-217. Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade r estores adult hippocampal neurogenesis. Science 302:1760-1765. Morgan BP, Gasque P (1996) Expression of complement in the brain: role in health and disease. Immunol Today 17:461-466. Comple m ent in stroke and neural plasticity   86 Mukherjee P, Pasinetti GM (2001) Complement anaphylatoxin C5a neuroprotec ts through mitogen-activated protein kinase-dependent inhibition of caspase 3. J Neurochem 77:43-49. Mukherjee P, Thomas S, Pasinetti GM (2008) Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo. J Neuroinflammation 5:5. Mulkey RM, Malenka RC (1992) Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975. Murphy TH, Corbett D (2009) Plasticity during stroke recovery: from synapse to behaviour. Nat Rev Neurosci 10:861-872. Nagler K, Mauch DH, Pfrieger FW (2001) Glia -derived signals induce synapse formation in neurones of the rat central nervous system. J Physiol 533:665 -679. Nataf S, Davoust N, Barnum SR (1998) Kinetics of anaphylatoxi n C5a receptor expression during experimental allergic encephalomyelitis. J Neuroimmunol 91:147-155. Nauta AJ, Raaschou -Jensen N, Roos A, Daha MR, Madsen HO, Borrias -Essers MC, Ryder LP, Koch C, Garred P (2003) Mannose -binding lectin engagement with late apoptotic and necrotic cells. Eur J Immunol 33:2853 -2863. Nayak A, Pednekar L, Reid KB, Kishore U (2012) Complement and non -complement activating functions of C1q: a prototypical innate immune molecule. Innate Immun 18:350-363. Neher E, Sakmann B (1976) Sin gle-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799-802. Neumann-Haefelin T, Staiger JF, Redecker C, Zilles K, Fritschy JM, Mohler H, Witte OW (1998) Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience 87:871-879. Nicoll RA, Kauer JA, Malenka RC (1988) The current excitement in long -term potentiation. Neuron 1:97-103. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314-1318. Nudo RJ, Milliken GW, Jenkins WM, Merzenich MM (1996) Use -dependent alterations of movement representations in primary motor cortex of adult squirrel monke ys. J Neurosci 16:785 -807. O'Barr SA, Caguioa J, Gruol D, Perkins G, Ember JA, Hugli T, Cooper NR (2001) Neuronal expression of a functional receptor for the C5a complement activation fragment. J Immunol 166:4154 -4162. Ohab JJ, Fleming S, Blesch A, Carmich ael ST (2006) A neurovascular niche for neurogenesis after stroke. J Neurosci 26:13007 -13016. Oksjoki R, Kovanen PT, Meri S, Pentikainen MO (2007) Function and regulation of the complement system in cardiovascular diseases. Front Biosci 12:4696 -4708. Panatier A, Theodosis DT, Mothet JP, Touquet B, Pollegioni L, Poulain DA, Oliet SH (2006) Glia -derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125:775-784. Pangburn MK, Schreiber RD, Muller -Eberhard HJ (1981) Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b -like activities by spontaneous hydrolysis of the putative thioester in native C3. J Exp Med 154:856-867. Anna Stokowska   87 Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, Gross CT (2011) Synaptic pruning by microglia is necessary for normal brain development. Science 333:1456-1458. Pascual-Leone A, Nguyet D, Cohen LG, Brasil -Neto JP, Cammarota A, Hallett M (1995) Modulation of muscle responses evoked by transcranial magnetic stimulation during the acquisition of new fine motor skills. J Neurophysiol 74:1037 -1045. Pascual O, Ben Achour S, Rostaing P, Triller A, Bessis A (2012) Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission. Proc Natl Acad Sci U S A 109:E197 -205. Pedersen ED, Waje -Andreassen U, Vedeler CA, Aamodt G, Mollnes TE (2004) Systemic complement activation following human acute ischaemic stroke. Clin Exp Immunol 137:117-122. Pedersen ED, Loberg EM, Vege E, Daha MR, Maehlen J, Mollnes TE (2009) In situ deposition of complement in human acute brain ischaemia. Scand J Immunol 69:555-562. Pekna M, Pekny M, Nilsson M (2012) Modulation of neural plasticity as a basis for stroke rehabilitation. Stroke 43:2819-2828. Pekny M, Pekna M (2004) Astrocyte intermediate filaments in CNS pathologies and regeneration. J Pathol 204:428 -437. Persson L, Boren J, Robertson AK, Wallenius V, Hansson GK, Pekna M (2004) Lack of complement factor C3, but not factor B, increases hyperlipidemia and atherosclerosis in apolipoprotein E -/ - low-density lipoprotein receptor-/ - mice. Arterioscler Thromb Vasc Biol 24:1062 -1067. Petersen SV, Thiel S, Jensen L, Vorup -Jensen T, Koch C, Jensenius JC (2000) Control of the classical and the MBL pathway of complement activation. Mol Immunol 37:803-811. Peterson KL, Zhang W, Lu PD, Keilbaugh SA, Peerschke EI, Ghebrehiwet B (1997) The C1q -binding cell membrane proteins cC1q -R and gC1q -R are released from activated cells: subcellular distribution and immunochemical characterization. Clin Immunol Immunopathol 84:17-26. Porritt MJ, Andersson HC, Hou L, Nilsson A, Pekna M, Pekny M, Nilsson M (2012) Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane. PLoS One 7:e41090. Purves D, Augustine GJ, Fitzpatrick D, C. HW, LaMantia A -S, McNamara JO, White LE (2008) Neuroscience, 4th Edition. Sunderland, MA: Sinauer Associates, Inc. Rahpeymai Y, Hietala MA, Wilhelmsson U, Fothe ringham A, Davies I, Nilsson AK, Zwirner J, Wetsel RA, Gerard C, Pekny M, Pekna M (2006) Complement: a novel factor in basal and ischemia-induced neurogenesis. EMBO J 25:1364 - 1374. Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli, specia lized responses. Annu Rev Immunol 27:119-145. Rapalino O, Lazarov -Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solomon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M (1998) Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med 4:814-821. Ratajczak MZ, Wysoczynski M, Reca R, Wan W, Zuba -Surma EK, Kucia M, Ratajczak J (2008) A pivotal role of activation of complement cascade (CC) in mobilization of hematopoietic stem/progenitor cells (HSPC). Adv Exp Med Biol 632:47 -60. Comple m ent in stroke and neural plasticity   86 Mukherjee P, Pasinetti GM (2001) Complement anaphylatoxin C5a neuroprotec ts through mitogen-activated protein kinase-dependent inhibition of caspase 3. J Neurochem 77:43-49. Mukherjee P, Thomas S, Pasinetti GM (2008) Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo. J Neuroinflammation 5:5. Mulkey RM, Malenka RC (1992) Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975. Murphy TH, Corbett D (2009) Plasticity during stroke recovery: from synapse to behaviour. Nat Rev Neurosci 10:861-872. Nagler K, Mauch DH, Pfrieger FW (2001) Glia -derived signals induce synapse formation in neurones of the rat central nervous system. J Physiol 533:665 -679. Nataf S, Davoust N, Barnum SR (1998) Kinetics of anaphylatoxi n C5a receptor expression during experimental allergic encephalomyelitis. J Neuroimmunol 91:147-155. Nauta AJ, Raaschou -Jensen N, Roos A, Daha MR, Madsen HO, Borrias -Essers MC, Ryder LP, Koch C, Garred P (2003) Mannose -binding lectin engagement with late apoptotic and necrotic cells. Eur J Immunol 33:2853 -2863. Nayak A, Pednekar L, Reid KB, Kishore U (2012) Complement and non -complement activating functions of C1q: a prototypical innate immune molecule. Innate Immun 18:350-363. Neher E, Sakmann B (1976) Sin gle-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799-802. Neumann-Haefelin T, Staiger JF, Redecker C, Zilles K, Fritschy JM, Mohler H, Witte OW (1998) Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience 87:871-879. Nicoll RA, Kauer JA, Malenka RC (1988) The current excitement in long -term potentiation. Neuron 1:97-103. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314-1318. Nudo RJ, Milliken GW, Jenkins WM, Merzenich MM (1996) Use -dependent alterations of movement representations in primary motor cortex of adult squirrel monke ys. J Neurosci 16:785 -807. O'Barr SA, Caguioa J, Gruol D, Perkins G, Ember JA, Hugli T, Cooper NR (2001) Neuronal expression of a functional receptor for the C5a complement activation fragment. J Immunol 166:4154 -4162. Ohab JJ, Fleming S, Blesch A, Carmich ael ST (2006) A neurovascular niche for neurogenesis after stroke. J Neurosci 26:13007 -13016. Oksjoki R, Kovanen PT, Meri S, Pentikainen MO (2007) Function and regulation of the complement system in cardiovascular diseases. Front Biosci 12:4696 -4708. Panatier A, Theodosis DT, Mothet JP, Touquet B, Pollegioni L, Poulain DA, Oliet SH (2006) Glia -derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125:775-784. Pangburn MK, Schreiber RD, Muller -Eberhard HJ (1981) Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b -like activities by spontaneous hydrolysis of the putative thioester in native C3. J Exp Med 154:856-867. Anna Stokowska   87 Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, Gross CT (2011) Synaptic pruning by microglia is necessary for normal brain development. Science 333:1456-1458. Pascual-Leone A, Nguyet D, Cohen LG, Brasil- Neto JP, Cammarota A, Hallett M (1995) Modulation of muscle responses evoked by transcranial magnetic stimulation during the acquisition of new fine motor skills. J Neurophysiol 74:1037 -1045. Pascual O, Ben Achour S, Rostaing P, Triller A, Bessis A (2012) Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission. Proc Natl Acad Sci U S A 109:E197 -205. Pedersen ED, Waje -Andreassen U, Vedeler CA, Aamodt G, Mollnes TE (2004) Systemic complement activation following human acute ischaemic stroke. Clin Exp Immunol 137:117-122. Pedersen ED, Loberg EM, Vege E, Daha MR, Maehlen J, Mollnes TE (2009) In situ deposition of complement in human acute brain ischaemia. Scand J Immunol 69:555-562. Pekna M, Pekny M, Nilsson M (2012) Modulation of neural plasticity as a basis for stroke rehabilitation. Stroke 43:2819-2828. Pekny M, Pekna M (2004) Astrocyte intermediate filaments in CNS pathologies and regeneration. J Pathol 204:428 -437. Persson L, Boren J, Robertson AK, Wallenius V, Hansson GK, Pekna M (2004) Lack of complement factor C3, but not factor B, increases hyperlipidemia and atherosclerosis in apolipoprotein E -/- low-density lipoprotein receptor-/- mice. Arterioscler Thromb Vasc Biol 24:1062- 1067. Petersen SV, Thiel S, Jensen L, Vorup -Jensen T, Koch C, Jensenius JC (2000) Control of the classical and the MBL pathway of complement activation. Mol Immunol 37:803-811. Peterson KL, Zhang W, Lu PD, Keilbaugh SA, Peerschke EI, Ghebrehiwet B (1997) The C1q- binding cell membrane proteins cC1q -R and gC1q -R are released from activated cells: subcellular distribution and immunochemical characterization. Clin Immunol Immunopathol 84:17-26. Porritt MJ, Andersson HC, Hou L, Nilsson A, Pekna M, Pekny M, Nilsson M (2012) Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane. PLoS One 7:e41090. Purves D, Augustine GJ, Fitzpatrick D, C. HW, LaMantia A -S, McNamara JO, White LE (2008) Neuroscience, 4th Edition. Sunderland, MA: Sinauer Associates, Inc. Rahpeymai Y, Hietala MA, Wilhelmsson U, Fothe ringham A, Davies I, Nilsson AK, Zwirner J, Wetsel RA, Gerard C, Pekny M, Pekna M (2006) Complement: a novel factor in basal and ischemia-induced neurogenesis. EMBO J 25:1364 - 1374. Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli, specia lized responses. Annu Rev Immunol 27:119-145. Rapalino O, Lazarov -Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solomon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M (1998) Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med 4:814-821. Ratajczak MZ, Wysoczynski M, Reca R, Wan W, Zuba-Surma EK, Kucia M, Ratajczak J (2008) A pivotal role of activation of complement cascade (CC) in mobilization of hematopoietic stem/progenitor cells (HSPC). Adv Exp Med Biol 632:47- 60. Comple m ent in stroke and neural plasticity   88 Reca R, Mastellos D, Majka M, Marquez L, Ratajczak J, Franchini S, Glodek A, Honcza renko M, Spruce LA, Janowska -Wieczorek A, Lambris JD, Ratajczak MZ (2003) Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing -related responses to SDF -1. Blood 101:3784-3793. Ringelstein EB, Biniek R, Weiller C, Ammeling B, Nolte PN, Thron A (1992) Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization. Neurology 42:289 -298. Ross HH, Levkoff LH, Marshall GP, 2nd, Caldeira M, Steindler DA, Reynolds BA, Laywell ED (2008) Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells 26:3218-3227. Rovira A, Grive E, Rovira A, Alvarez -Sabin J (2005) Distribution territories and causative mechanisms of ischemic stroke. Eur Radiol 15:416 -426. Sahu A, Lambris JD (2001) Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol Rev 180:35 - 48. Scandinavian Stroke Study Group (1985) Multicenter trial of hemodilution in ischemic stroke--background and study protocol. Stroke 16:885-890. Schaechter JD, Fricker ZP, Perdue KL, Helmer KG, Va ngel MG, Greve DN, Makris N (2009) Microstructural status of ipsilesional and contralesional corticospinal tract correlates with motor skill in chronic stroke patients. Hum Brain Mapp 30:3461-3474. Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR , Yamasaki R, Ransohoff RM, Greenberg ME, Barres BA, Stevens B (2012) Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74:691-705. Schafers M, Sorkin L (2008) Effect of cytokines on neuronal excitability. Ne urosci Lett 437:188-193. Schraufstatter IU, Discipio RG, Zhao M, Khaldoyanidi SK (2009) C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol 182:3827 -3836. Shaked I, Tchoresh D, Gersn er R, Meiri G, Mordechai S, Xiao X, Hart RP, Schwartz M (2005) Protective autoimmunity: interferon-gamma enables microglia to remove glutamate without evoking inflammatory mediators. J Neurochem 92:997 -1009. Shechter R, London A, Varol C, Raposo C, Cusiman o M, Yovel G, Rolls A, Mack M, Pluchino S, Martino G, Jung S, Schwartz M (2009) Infiltrating blood -derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med 6:e1000113. Shimada IS, Peterson BM, Spees JL (2010) Isolation of locally derived stem/progenitor cells from the peri-infarct area that do not migrate from the lateral ventricle after cortical stroke. Stroke 41:e552-560. Shinjyo N, Stå hlberg A, Dragunow M, Pekny M, Pekna M (2009) Complement-derived anaphylatoxin C3a regulates in vitro differentiation and migration of neural progenitor cells. Stem Cells 27:2824-2832. Sniderman AD, Maslowska M, Cianflone K (2000) Of mice and men (and women) and the acylation-stimulating protein pathway. Curr Opin Lipidol 11:291 -296. Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39-44. Anna Stokowska   89 Stahel PF, Morganti -Kossmann MC, Kossmann T (1998) The role of the complement system in traumatic brain injury. Brain Res Brain Res Rev 27:243-256. Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF -alpha. Nature 440:1054-1059. Stephan AH, Madison DV, Mateos JM, Fraser DA, Lovelett EA, Coutellier L, Kim L, Tsai HH, Huang EJ, Rowitch DH, Berns DS, Tenn er AJ, Shamloo M, Barres BA (2013) A Dramatic Increase of C1q Protein in the CNS during Normal Aging. J Neurosci 33:13460 -13474. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A , Litke AM, Lambris JD, Smith SJ, John SW, Barres BA (2007) The classical complement cascade mediates CNS synapse elimination. Cell 131:1164-1178. Stevens SL, Bao J, Hollis J, Lessov NS, Clark WM, Stenzel -Poore MP (2002) The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice. Brain Res 932:110-119. Streit WJ, Walter SA, Pennell NA (1999) Reactive microgliosis. Prog Neurobiol 57:563 - 581. Strey CW, Markiewski M, Mastellos D, Tudoran R, Spruce LA, Greenbaum LE, Lambris JD (2003) The proinflammatory mediators C3a and C5a are essential for liver regeneration. J Exp Med 198:913 -923. Stroemer RP, Kent TA, Hulsebosch CE (1995) Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke 26:2135-2144. Swanson RA, Ying W, Kauppinen TM (2004) Astrocyte influences on ischemic neuronal death. Curr Mol Med 4:193-205. Swartz KR, Liu F, Sewell D, Schochet T, Campbell I, Sandor M, Fabry Z (2001) Interleukin-6 promotes post-traumatic healing in the central nervous system. Brain Res 896:86-95. Szeplaki G, Szegedi R, Hirschberg K, Gombos T, Varga L, Karadi I, Entz L, Szeplaki Z, Garred P, Prohaszka Z, Fust G (2009) Strong complement activation after acute ischemic stroke is associated with unfavorable outcomes. Atherosclerosis 204:315-320. Takabayashi T, Vannier E, Burke JF, Tompkins RG, Gelfand JA, Clark BD (1998) Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuclear cells. J Infect Dis 177:1622 -1628. Takatsuru Y, Fukumoto D, Yoshitomo M, Nemoto T, Tsukada H, Nabekura J (2009) Neuronal circuit remodeling in the contralateral cortical hemisphere during functional recovery from cerebral infarction. J Neurosci 29:10081 -10086. Tamam Y, Iltumur K, Apak I (2005) Assessment of acute phase proteins in acute ischemic stroke. Tohoku J Exp Med 206:91 -98. Thomas A, Gasque P, Vaudry D, Gonzalez B, Fontaine M (2000) Expression of a complete and functional complement system by human neuronal cells in vitro. Int Immunol 12:1015-1023. Tremblay ME, Lowery RL, Majewska AK (2010) Microglial interactions with synapses are modulated by visual experience. PLoS Biol 8:e1000527. Turnberg D, Botto M (2003) The regulation of the complement system: insights from genetically-engineered mice. Mol Immunol 40:145-153. Turrigiano GG, Nelson SB (2000) Hebb and homeostasis in neuronal plasticity. Curr Opin Neurobiol 10:358 -364. Comple m ent in stroke and neural plasticity   88 Reca R, Mastellos D, Majka M, Marquez L, Ratajczak J, Franchini S, Glodek A, Honczarenko M, Spruce LA, Janowska -Wieczorek A, Lambris JD, Ratajczak MZ (2003) Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing -related responses to SDF -1. Blood 101:3784-3793. Ringelstein EB, Biniek R, Weiller C, Ammeling B, Nolte PN, Thron A (1992) Type and extent of hemispheric brain infarctions and clinical outcome i n early and delayed middle cerebral artery recanalization. Neurology 42:289 -298. Ross HH, Levkoff LH, Marshall GP, 2nd, Caldeira M, Steindler DA, Reynolds BA, Laywell ED (2008) Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells 26:3218-3227. Rovira A, Grive E, Rovira A, Alvarez -Sabin J (2005) Distribution territories and causative mechanisms of ischemic stroke. Eur Radiol 15:416 -426. Sahu A, Lambris JD (2001) Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol Rev 180:35 - 48. Scandinavian Stroke Study Group (1985) Multicenter trial of hemodilution in ischemic stroke--background and study protocol. Stroke 16:885-890. Schaechter JD, Fricker ZP, Perdue KL, Helmer KG, Va ngel MG, Greve DN, Makris N (2009) Microstructural status of ipsilesional and contralesional corticospinal tract correlates with motor skill in chronic stroke patients. Hum Brain Mapp 30:3461-3474. Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR , Yamasaki R, Ransohoff RM, Greenberg ME, Barres BA, Stevens B (2012) Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74:691-705. Schafers M, Sorkin L (2008) Effect of cytokines on neuronal excitability. Ne urosci Lett 437:188-193. Schraufstatter IU, Discipio RG, Zhao M, Khaldoyanidi SK (2009) C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol 182:3827 -3836. Shaked I, Tchoresh D, Gersn er R, Meiri G, Mordechai S, Xiao X, Hart RP, Schwartz M (2005) Protective autoimmunity: interferon-gamma enables microglia to remove glutamate without evoking inflammatory mediators. J Neurochem 92:997 -1009. Shechter R, London A, Varol C, Raposo C, Cusiman o M, Yovel G, Rolls A, Mack M, Pluchino S, Martino G, Jung S, Schwartz M (2009) Infiltrating blood -derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med 6:e1000113. Shimada IS, Peterson BM, Spees JL (2010) Isolation of locally derived stem/progenitor cells from the peri-infarct area that do not migrate from the lateral ventricle after cortical stroke. Stroke 41:e552-560. Shinjyo N, Stahlberg A, Dragunow M, Pekny M, Pekna M (2009) Compleme nt-derived anaphylatoxin C3a regulates in vitro differentiation and migration of neural progenitor cells. Stem Cells 27:2824-2832. Sniderman AD, Maslowska M, Cianflone K (2000) Of mice and men (and women) and the acylation-stimulating protein pathway. Curr Opin Lipidol 11:291 -296. Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39-44. Anna Stokowska   89 Stahel PF, Morganti- Kossmann MC, Kossmann T (1998) The role of the complement system in traumatic brain injury. Brain Res Brain Res Rev 27:243-256. Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF -alpha. Nature 440:1054-1059. Stephan AH, Madison DV, Mateos JM, Fraser DA, Lovelett EA, Coutellier L, Kim L, Tsai HH, Huang EJ, Rowitch DH, Berns DS, Tenner AJ, Shamloo M, Barres BA (2013) A Dramatic Increase of C1q Protein in the CNS during Normal Aging. J Neurosci 33:13460- 13474. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A , Litke AM, Lambris JD, Smith SJ, John SW, Barres BA (2007) The classical complement cascade mediates CNS synapse elimination. Cell 131:1164-1178. Stevens SL, Bao J, Hollis J, Lessov NS, Clark WM, Stenzel -Poore MP (2002) The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice. Brain Res 932:110-119. Streit WJ, Walter SA, Pennell NA (1999) Reactive microgliosis. Prog Neurobiol 57:563 - 581. Strey CW, Markiewski M, Mastellos D, Tudoran R, Spruce LA, Greenbaum LE, Lambris JD (2003) The proinflammatory mediators C3a and C5a are essential for liver regeneration. J Exp Med 198:913- 923. Stroemer RP, Kent TA, Hulsebosch CE (1995) Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke 26:2135-2144. Swanson RA, Ying W, Kauppinen TM (2004) Astrocyte influences on ischemic neuronal death. Curr Mol Med 4:193-205. Swartz KR, Liu F, Sewell D, Schochet T, Campbell I, Sandor M, Fabry Z (2001) Interleukin-6 promotes post-traumatic healing in the central nervous system. Brain Res 896:86-95. Szeplaki G, Szegedi R, Hirschberg K, Gombos T, Varga L, Karadi I, Entz L, Szeplaki Z, Garred P, Prohaszka Z, Fust G (2009) Strong complement activation after acute ischemic stroke is associated with unfavorable outcomes. Atherosclerosis 204:315-320. Takabayashi T, Vannier E, Burke JF, Tompkins RG, Gelfand JA, Clark BD (1998) Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuclear cells. J Infect Dis 177:1622 -1628. Takatsuru Y, Fukumoto D, Yoshitomo M, Nemoto T, Tsukada H, Nabekura J (2009) Neuronal circuit remodeling in the contralateral cortical hemisphere during functional recovery from cerebral infarction. J Neurosci 29:10081 -10086. Tamam Y, Iltumur K, Apak I (2005) Assessment of acute phase proteins in acute ischemic stroke. Tohoku J Exp Med 206:91 -98. Thomas A, Gasque P, Vaudry D, Gonzalez B, Fontaine M (2000) Expression of a complete and functional complement system by human neuronal cells in vitro. Int Immunol 12:1015-1023. Tremblay ME, Lowery RL, Majewska AK (2010) Microglial interactions with synapses are modulated by visual experience. PLoS Biol 8:e1000527. Turnberg D, Botto M (2003) The regulation of the complement system: insights from genetically-engineered mice. Mol Immunol 40:145-153. Turrigiano GG, Nelson SB (2000) Hebb and homeostasis in neuronal plasticity. Curr Opin Neurobiol 10:358 -364. Comple m ent in stroke and neural plasticity   90 Van Beek J, Bernaudin M, Petit E, Gasque P, Nouvelot A, MacKenzie ET, Fontaine M (2000) Expression of receptors for complement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Exp Neurol 161:373 - 382. van Beek J, Nicole O, Ali C, Ischenko A, MacKenzie ET, Buisson A, Fontaine M (2001) Complement anaphylatoxin C3a is selectively protective against NMDA - induced neuronal cell death. Neuroreport 12:289-293. van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J (1988) Interobserver agreement for the assessment of handicap in stroke patients. Stroke 19:604-607. Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci 29:3974 -3980. Walker MC, Semyanov A (2008) Regulation of excitability by extrasynaptic GABA(A) receptors. Results Probl Cell Differ 44:29-48. Walton NM, Sutter BM, Laywell ED, Levkoff LH, Kearns SM, Marshall GP, 2nd, Scheffler B, Steindler DA (2006) Microglia instruct subventricular zone neurogenesis. Glia 54:815-825. Wamba PC, Mi J, Zhao XY, Zhang MX, Wen Y, Cheng H, Hou DQ, Cianflone K (2008) Acylation stimulating protein but not complement C3 associates with metabolic syndrome components in Chinese children and adolescents. Eur J Endocrinol 159:781-790. W ang L, Zhang Z, Zhang R, Hafner MS, Wong HK, Jiao Z, Chopp M (2004) Erythropoietin up -regulates SOCS2 in neuronal progenitor cells derived from SVZ of adult rat. Neuroreport 15:1225 -1229. Wang X, Feuerstein GZ (1995) Induced expression of adhesion molecule s following focal brain ischemia. J Neurotrauma 12:825 -832. Wang X, Mao X, Xie L, Sun F, Greenberg DA, Jin K (2012) Conditional depletion of neurogenesis inhibits long-term recovery after experimental stroke in mice. PLoS One 7:e38932. Warraich Z, Kleim JA (2010) Neural plasticity: the biological substrate for neurorehabilitation. PM & R : the journal of injury, function, and rehabilitation 2:S208-219. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD (1985) Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 17:497-504. Weber F, Meinl E, Aloisi F, Nevinny -Stickel C, Albert E, Wekerle H, Hohlfeld R (1994) Human astrocytes are only partially competent antigen presenting cells. Possible implications for lesion development in multiple sclerosis. Brain 117 ( Pt 1):59- 69. WH O (2010) Global burden of the disease, http://www.who.int/ Widera D, Mikenberg I, Elvers M, Kaltschmidt C, Kaltschmidt B (2006) Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF- kappaB signaling. BMC Neurosci 7:64. Wieloch T, Nikolich K (2006) Mechanisms of neural plasticity following brain injury. Curr Opin Neurobiol 16:258- 264. Wigstrom H, Gustafsson B (1986) Postsynaptic control of hippocampal long -term potentiation. J Physiol (Paris) 81:228 -236. Anna Stokowska   91 Wilhelmsen L, Johansson S, Rosengren A, Wallin I, Dotevall A, Lappas G (1997) Risk factors for cardiovascular disease during the period 1985-1995 in Goteborg, Sweden. The GOT -MONICA Project. J Intern Med 242:199 -211. Winship IR, Murphy TH (2008) In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke. The Journal of neuroscience : the official journal of the Society for Neuroscience 28:65 92-6606. Wolf SA, Steiner B, Akpinarli A, Kammertoens T, Nassenstein C, Braun A, Blankenstein T, Kempermann G (2009) CD4 -positive T lymphocytes provide a neuroimmunological link in the control of adult hippocampal neurogenesis. J Immunol 182:3979-3984. Woo druff TM, Costantini KJ, Crane JW, Atkin JD, Monk PN, Taylor SM, Noakes PG (2008) The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis. J Immunol 181:8727 -8734. Wu MC, Brennan FH, Lynch JP, Mantovani S, Phipps S, Wetsel RA, Ruitenberg MJ, Taylor SM, Woodruff TM (2013) The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization. Proc Natl Acad Sci U S A 110:9439 -9444. Xu Y, Nara yana SV, Volanakis JE (2001) Structural biology of the alternative pathway convertase. Immunol Rev 180:123-135. Yin Y, Henzl MT, Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R, Benowitz LI (2006) Oncomodulin is a macrophage -derived signal for axon rege neration in retinal ganglion cells. Nat Neurosci 9:843-852. Yuzaki M (2008) Cbln and C1q family proteins: new transneuronal cytokines. Cell Mol Life Sci 65:1698 -1705. Yuzaki M (2010) Synapse formation and maintenance by C1q family proteins: a new class of secreted synapse organizers. Eur J Neurosci 32:191 -197. Zimmet P, Boyko EJ, Collier GR, de Courten M (1999) Etiology of the metabolic syndrome: potential role of insulin resistance, leptin resistance, and other players. Ann N Y Acad Sci 892:25-44. Ziv Y, R on N, Butovsky O, Landa G, Sudai E, Greenberg N, Cohen H, Kipnis J, Schwartz M (2006) Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood. Nat Neurosci 9:268-275. Zucker RS, Regehr WG (2002) Short -term synaptic plasticity. Annu Rev Physiol 64:355- 405. Comple m ent in stroke and neural plasticity   90 Van Beek J, Bernaudin M, Petit E, Gasque P, Nouvelot A, MacKenzie ET, Fontaine M (2000) Expression of receptors for complement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Exp Neurol 161:373 - 382. van Beek J, Nicole O, Ali C, Ischenko A, MacKenzie ET, Buisson A, Fontaine M (2001) Complement anaphylatoxin C3a is selectively protective against NMDA - induced neuronal cell death. Neuroreport 12:289-293. van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J (1988) Interobserver agreement for the assessment of handicap in stroke patients. Stroke 19:604-607. Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci 29:3974 -3980. Walker MC, Semyanov A (2008) Regulatio n of excitability by extrasynaptic GABA(A) receptors. Results Probl Cell Differ 44:29-48. Walton NM, Sutter BM, Laywell ED, Levkoff LH, Kearns SM, Marshall GP, 2nd, Scheffler B, Steindler DA (2006) Microglia instruct subventricular zone neurogenesis. Glia 54:815-825. Wamba PC, Mi J, Zhao XY, Zhang MX, Wen Y, Cheng H, Hou DQ, Cianflone K (2008) Acylation stimulating protein but not complement C3 associates with metabolic syndrome components in Chinese children and adolescents. Eur J Endocrinol 159:781-790. W ang L, Zhang Z, Zhang R, Hafner MS, Wong HK, Jiao Z, Chopp M (2004) Erythropoietin up -regulates SOCS2 in neuronal progenitor cells derived from SVZ of adult rat. Neuroreport 15:1225 -1229. Wang X, Feuerstein GZ (1995) Induced expression of adhesion molecule s following focal brain ischemia. J Neurotrauma 12:825 -832. Wang X, Mao X, Xie L, Sun F, Greenberg DA, Jin K (2012) Conditional depletion of neurogenesis inhibits long-term recovery after experimental stroke in mice. PLoS One 7:e38932. Warraich Z, Kleim JA (2010) Neural plasticity: the biological substrate for neurorehabilitation. PM & R : the journal of injury, function, and rehabilitation 2:S208-219. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD (1985) Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 17:497-504. Weber F, Meinl E, Aloisi F, Nevinny -Stickel C, Albert E, Wekerle H, Hohlfeld R (1994) Human astrocytes are only partially competent antigen presenting cells. Possible implications for lesion development in multiple sclerosis. Brain 117 ( Pt 1):59- 69. WH O (2010) Global burden of the disease, http://www.who.int/ Widera D, Mikenberg I, Elvers M, Kaltschmidt C, Kaltschmidt B (2006) Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF -kappaB signaling. BMC Neurosci 7:64. Wieloch T, Nikolich K (2006) Mechanisms of neural plasticity following brain injury. Curr Opin Neurobiol 16:258 -264. Wigstrom H, Gustafsson B (1986) Postsynapt ic control of hippocampal long-term potentiation. J Physiol (Paris) 81:228 -236. Anna Stokowska   91 Wilhelmsen L, Johansson S, Rosengren A, Wallin I, Dotevall A, Lappas G (1997) Risk factors for cardiovascular disease during the period 1985-1995 in Goteborg, Sweden. The GOT-M ONICA Project. J Intern Med 242:199 -211. Winship IR, Murphy TH (2008) In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke. The Journal of neuroscience : the official journal of the Society for Neuroscience 28:65 92-6606. Wolf SA, Steiner B, Akpinarli A, Kammertoens T, Nassenstein C, Braun A, Blankenstein T, Kempermann G (2009) CD4 -positive T lymphocytes provide a neuroimmunological link in the control of adult hippocampal neurogenesis. J Immunol 182:3979-3984. Woodruff TM, Costantini KJ, Crane JW, Atkin JD, Monk PN, Taylor SM, Noakes PG (2008) The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis. J Immunol 181:8727 -8734. Wu MC, Brennan FH, Lynch JP, Mantovani S, Phipps S, Wetsel RA, Ruitenberg MJ, Taylor SM, Woodruff TM (2013) The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization. Proc Natl Acad Sci U S A 110:9439 -9444. Xu Y, Nara yana SV, Volanakis JE (2001) Structural biology of the alternative pathway convertase. Immunol Rev 180:123-135. Yin Y, Henzl MT, Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R, Benowitz LI (2006) Oncomodulin is a macrophage- derived signal for axon regeneration in retinal ganglion cells. Nat Neurosci 9:843-852. Yuzaki M (2008) Cbln and C1q family proteins: new transneuronal cytokines. Cell Mol Life Sci 65:1698 -1705. Yuzaki M (2010) Synapse formation and maintenance by C1q family proteins: a new class of secreted synapse organizers. Eur J Neurosci 32:191 -197. Zimmet P, Boyko EJ, Collier GR, de Courten M (1999) Etiology of the metabolic syndrome: potential role of insulin resistance, leptin resistance, and other players. Ann N Y Acad Sci 892:25-44. Ziv Y, R on N, Butovsky O, Landa G, Sudai E, Greenberg N, Cohen H, Kipnis J, Schwartz M (2006) Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood. Nat Neurosci 9:268-275. Zucker RS, Regehr WG (2002) Short -term synaptic plasticity. Annu Rev Physiol 64:355- 405.