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Modulation of post-stroke degenerative and regenerative processes and subacute protection by site-targeted inhibition of the alternative pathway of complement.

Alawieh A, Elvington A, Zhu H, Yu J, Kindy MS, Atkinson C, Tomlinson S - J Neuroinflammation (2015)

Bottom Line: Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression.The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation.Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Children's Research Institute, Medical University of South Carolina, 173 Ashley Avenue BSB 201, Charleston, SC, 29425, USA. alawieh@musc.edu.

ABSTRACT

Background: Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion.

Methods: We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke.

Results: C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients.

Conclusions: Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke.

No MeSH data available.


Related in: MedlinePlus

CR2-fH increases neuroblasts migration to perilesional basal ganglia and hippocampus at 7 days post-MCAO. a Representative images of neuroblast migration to the basal ganglia. b Quantification of number of Dcx + cells in basal ganglia. Bar = Mean +/− SEM, n = 5. *p < 0.05. c Representative images of neuroblasts in the hippocampus. d Quantification of number of Dcx + cells in hippocampus. Bar = Mean +/− SEM, n = 5–6. *p < 0.05
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Fig5: CR2-fH increases neuroblasts migration to perilesional basal ganglia and hippocampus at 7 days post-MCAO. a Representative images of neuroblast migration to the basal ganglia. b Quantification of number of Dcx + cells in basal ganglia. Bar = Mean +/− SEM, n = 5. *p < 0.05. c Representative images of neuroblasts in the hippocampus. d Quantification of number of Dcx + cells in hippocampus. Bar = Mean +/− SEM, n = 5–6. *p < 0.05

Mentions: Since complement activation products have been implicated in ischemia-induced neurogenesis that contributes to recovery, we investigated the effect of complement inhibition on proliferation and neurogenesis in the brain after MCAO. Immunostaining for the proliferation marker Ki-67 was performed on sections from the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus, neurogenic niches of the adult brain previously studied in the context of ischemia-induced neurogenesis [22–24]. C3-deficient and CR2-Crry-treated mice, but not CR2-fH-treated mice, had a significant reduction in numbers of proliferating cells within the ipsilateral SVZ compared to wt controls, indicating that, unlike total complement blockade, alternative pathway blockade does not inhibit ischemia-induced neurogenesis (Fig. 4a). No differences were observed between any group in the ipsilateral SGZ (Fig. 4b), or in contralateral regions (data not shown). Immunostaining for doublecortin (Dcx), another marker of neurogenesis that is expressed by immature neurons (neuroblasts), similarly showed a higher number of Dcx-positive cells in the ipsilateral SVZ of CR2-fH-treated mice compared to CR2-Crry-treated mice or C3-deficient mice (Fig. 4c, d). No differences in numbers of Dcx-positive cells were observed between any group in the contralateral SVZ (data not shown). Since CR2-fH treatment did not significantly increase neurogenesis compared to wt mice at 7 days post-MCAO, we further investigated a potential effect of CR2-fH on neuronal migration by quantifying the number of Dcx-positive cells in the ipsilateral basal ganglia and hippocampus of mice post-MCAO; CR2-fH treatment significantly increased neuroblastic cell migration from the SVG to the basal ganglia (Fig. 5a, b) and hippocampus at 7 days after injury (Fig. 5c, d).Fig. 4


Modulation of post-stroke degenerative and regenerative processes and subacute protection by site-targeted inhibition of the alternative pathway of complement.

Alawieh A, Elvington A, Zhu H, Yu J, Kindy MS, Atkinson C, Tomlinson S - J Neuroinflammation (2015)

CR2-fH increases neuroblasts migration to perilesional basal ganglia and hippocampus at 7 days post-MCAO. a Representative images of neuroblast migration to the basal ganglia. b Quantification of number of Dcx + cells in basal ganglia. Bar = Mean +/− SEM, n = 5. *p < 0.05. c Representative images of neuroblasts in the hippocampus. d Quantification of number of Dcx + cells in hippocampus. Bar = Mean +/− SEM, n = 5–6. *p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696299&req=5

Fig5: CR2-fH increases neuroblasts migration to perilesional basal ganglia and hippocampus at 7 days post-MCAO. a Representative images of neuroblast migration to the basal ganglia. b Quantification of number of Dcx + cells in basal ganglia. Bar = Mean +/− SEM, n = 5. *p < 0.05. c Representative images of neuroblasts in the hippocampus. d Quantification of number of Dcx + cells in hippocampus. Bar = Mean +/− SEM, n = 5–6. *p < 0.05
Mentions: Since complement activation products have been implicated in ischemia-induced neurogenesis that contributes to recovery, we investigated the effect of complement inhibition on proliferation and neurogenesis in the brain after MCAO. Immunostaining for the proliferation marker Ki-67 was performed on sections from the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus, neurogenic niches of the adult brain previously studied in the context of ischemia-induced neurogenesis [22–24]. C3-deficient and CR2-Crry-treated mice, but not CR2-fH-treated mice, had a significant reduction in numbers of proliferating cells within the ipsilateral SVZ compared to wt controls, indicating that, unlike total complement blockade, alternative pathway blockade does not inhibit ischemia-induced neurogenesis (Fig. 4a). No differences were observed between any group in the ipsilateral SGZ (Fig. 4b), or in contralateral regions (data not shown). Immunostaining for doublecortin (Dcx), another marker of neurogenesis that is expressed by immature neurons (neuroblasts), similarly showed a higher number of Dcx-positive cells in the ipsilateral SVZ of CR2-fH-treated mice compared to CR2-Crry-treated mice or C3-deficient mice (Fig. 4c, d). No differences in numbers of Dcx-positive cells were observed between any group in the contralateral SVZ (data not shown). Since CR2-fH treatment did not significantly increase neurogenesis compared to wt mice at 7 days post-MCAO, we further investigated a potential effect of CR2-fH on neuronal migration by quantifying the number of Dcx-positive cells in the ipsilateral basal ganglia and hippocampus of mice post-MCAO; CR2-fH treatment significantly increased neuroblastic cell migration from the SVG to the basal ganglia (Fig. 5a, b) and hippocampus at 7 days after injury (Fig. 5c, d).Fig. 4

Bottom Line: Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression.The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation.Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Children's Research Institute, Medical University of South Carolina, 173 Ashley Avenue BSB 201, Charleston, SC, 29425, USA. alawieh@musc.edu.

ABSTRACT

Background: Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion.

Methods: We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke.

Results: C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients.

Conclusions: Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke.

No MeSH data available.


Related in: MedlinePlus