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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

Complement inhibition reduces microglia/macrophage activation and astrogliosis following MCAO and 7 days reperfusion. a Representative images of activated microglia/macrophages as assessed by Iba-1 immunohistochemical detection across the different groups. Scale bar = 200 μm. b Quantification of Iba-1 immunohistochemical staining. Mean +/− SEM (positive cells per square millimeter), n = 6–8 mice. *p < 0.05, **p < 0.01. c Quantification of reactive astrocytes as assessed by GFAP immunohistochemical detection. Mean +/− SEM (positive cells per square millimeter), n = 5–7 mice. *p < 0.05
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Fig2: Complement inhibition reduces microglia/macrophage activation and astrogliosis following MCAO and 7 days reperfusion. a Representative images of activated microglia/macrophages as assessed by Iba-1 immunohistochemical detection across the different groups. Scale bar = 200 μm. b Quantification of Iba-1 immunohistochemical staining. Mean +/− SEM (positive cells per square millimeter), n = 6–8 mice. *p < 0.05, **p < 0.01. c Quantification of reactive astrocytes as assessed by GFAP immunohistochemical detection. Mean +/− SEM (positive cells per square millimeter), n = 5–7 mice. *p < 0.05

Mentions: Microglia and astrocytes become activated in response to pathological changes, and microglia/macrophage activation and astrogliosis mark inflammation within the CNS, which is reduced at 24 h after MCAO by complement inhibition [4]. Microglia/macrophage activation and astrogliosis in the subacute phase at 7 days post-MCAO was assessed by immunohistochemical detection of Iba-1 and GFAP markers in the striatum, respectively. Compared to wt controls, there was a significant reduction in both activated microglia/macrophages and reactive astrocytes in CR2-Crry- and CR2-fH-treated mice (Fig. 2). In contrast, there was no significant difference in the detection of either marker in C3-deficient mice compared to wt. No differences were observed in the contralateral hemispheres between groups. These data indicate that C3 inhibition, but not C3 deficiency, reduce glial cell activation in the subacute phase after MCAO.Fig. 2


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)

Complement inhibition reduces microglia/macrophage activation and astrogliosis following MCAO and 7 days reperfusion. a Representative images of activated microglia/macrophages as assessed by Iba-1 immunohistochemical detection across the different groups. Scale bar = 200 μm. b Quantification of Iba-1 immunohistochemical staining. Mean +/− SEM (positive cells per square millimeter), n = 6–8 mice. *p < 0.05, **p < 0.01. c Quantification of reactive astrocytes as assessed by GFAP immunohistochemical detection. Mean +/− SEM (positive cells per square millimeter), n = 5–7 mice. *p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Complement inhibition reduces microglia/macrophage activation and astrogliosis following MCAO and 7 days reperfusion. a Representative images of activated microglia/macrophages as assessed by Iba-1 immunohistochemical detection across the different groups. Scale bar = 200 μm. b Quantification of Iba-1 immunohistochemical staining. Mean +/− SEM (positive cells per square millimeter), n = 6–8 mice. *p < 0.05, **p < 0.01. c Quantification of reactive astrocytes as assessed by GFAP immunohistochemical detection. Mean +/− SEM (positive cells per square millimeter), n = 5–7 mice. *p < 0.05
Mentions: Microglia and astrocytes become activated in response to pathological changes, and microglia/macrophage activation and astrogliosis mark inflammation within the CNS, which is reduced at 24 h after MCAO by complement inhibition [4]. Microglia/macrophage activation and astrogliosis in the subacute phase at 7 days post-MCAO was assessed by immunohistochemical detection of Iba-1 and GFAP markers in the striatum, respectively. Compared to wt controls, there was a significant reduction in both activated microglia/macrophages and reactive astrocytes in CR2-Crry- and CR2-fH-treated mice (Fig. 2). In contrast, there was no significant difference in the detection of either marker in C3-deficient mice compared to wt. No differences were observed in the contralateral hemispheres between groups. These data indicate that C3 inhibition, but not C3 deficiency, reduce glial cell activation in the subacute phase after MCAO.Fig. 2

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