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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 cerebral injury and cell death following MCAO and 7 days reperfusion. a Infarct volumes after 60 min MCAO and either 24-h or 7 days reperfusion. Mean +/− SEM, n = 8–12 mice. **p < 0.01, ***p < 0.001, #p < 0.001. b Cell death in different brain regions as analyzed by TUNEL immunostaining at 7 days after MCAO. Mean +/− SEM (TUNEL-positive cells per square millimeter), n = 5–6 mice. *p < 0.05
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Fig1: Complement inhibition reduces cerebral injury and cell death following MCAO and 7 days reperfusion. a Infarct volumes after 60 min MCAO and either 24-h or 7 days reperfusion. Mean +/− SEM, n = 8–12 mice. **p < 0.01, ***p < 0.001, #p < 0.001. b Cell death in different brain regions as analyzed by TUNEL immunostaining at 7 days after MCAO. Mean +/− SEM (TUNEL-positive cells per square millimeter), n = 5–6 mice. *p < 0.05

Mentions: It has been shown previously that C3 deficiency [12, 13] or treatment of wt mice with CR2-Crry [16] or CR2-fH [15] significantly reduces infarct volume 24 h after MCAO and reperfusion. Here, we investigated the effect of targeted complement inhibition on subacute injury by assessing infarct volumes 7 days after MCAO, and compared subacute with acute (24 h) outcomes. Pharmacological inhibition in all studies reported here was achieved with a single dose of inhibitor (CR2-Crry or CR2-fH) administered 90 min after ischemia and 30 min after reperfusion. Compared to wt control mice, infarct volume was significantly decreased in C3-deficient mice and in wt mice treated with either CR2-Crry or CR2-fH at both 24 h and 7 days post-MCAO (Fig. 1a). At 24 h after MCAO, there was no significant difference in infarct volume between C3-deficient mice and CR2-fH- or CR2-Crry-treated mice. However, unlike CR2-fH-treated mice (and wt controls), C3-deficient and CR2-Crry-treated mice had a rebound increase in infarct volumes at 7 days compared to 24 h post-MCAO. CR2-fH-treated mice did not show any significant difference in infarct volume between the two time points post-reperfusion. We also assessed cell death in the ipsilateral cortex, basal ganglia, and hippocampus 7 days following MCAO using TUNEL staining. CR2-Crry- and CR2-fH-treated mice showed a significant reduction in cell death in the basal ganglia and cortex compared to C3−/− and PBS-treated mice, but only CR2-fH-treated mice had a significant reduction in hippocampal cell death compared to all other groups. Further, compared to CR2-Crry-treated mice, CR2-fH-treated mice had significantly less cell death in the cortex and hippocampus. There were no significant differences between C3−/− mice and control mice across the three brain regions (Fig. 1b). Thus, there is an evolution of secondary injury subacutely after MCAO in C3-deficient mice and CR2-Crry-treated mice, but not in CR2-fH-treated mice.Fig. 1


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 cerebral injury and cell death following MCAO and 7 days reperfusion. a Infarct volumes after 60 min MCAO and either 24-h or 7 days reperfusion. Mean +/− SEM, n = 8–12 mice. **p < 0.01, ***p < 0.001, #p < 0.001. b Cell death in different brain regions as analyzed by TUNEL immunostaining at 7 days after MCAO. Mean +/− SEM (TUNEL-positive cells per square millimeter), n = 5–6 mice. *p < 0.05
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696299&req=5

Fig1: Complement inhibition reduces cerebral injury and cell death following MCAO and 7 days reperfusion. a Infarct volumes after 60 min MCAO and either 24-h or 7 days reperfusion. Mean +/− SEM, n = 8–12 mice. **p < 0.01, ***p < 0.001, #p < 0.001. b Cell death in different brain regions as analyzed by TUNEL immunostaining at 7 days after MCAO. Mean +/− SEM (TUNEL-positive cells per square millimeter), n = 5–6 mice. *p < 0.05
Mentions: It has been shown previously that C3 deficiency [12, 13] or treatment of wt mice with CR2-Crry [16] or CR2-fH [15] significantly reduces infarct volume 24 h after MCAO and reperfusion. Here, we investigated the effect of targeted complement inhibition on subacute injury by assessing infarct volumes 7 days after MCAO, and compared subacute with acute (24 h) outcomes. Pharmacological inhibition in all studies reported here was achieved with a single dose of inhibitor (CR2-Crry or CR2-fH) administered 90 min after ischemia and 30 min after reperfusion. Compared to wt control mice, infarct volume was significantly decreased in C3-deficient mice and in wt mice treated with either CR2-Crry or CR2-fH at both 24 h and 7 days post-MCAO (Fig. 1a). At 24 h after MCAO, there was no significant difference in infarct volume between C3-deficient mice and CR2-fH- or CR2-Crry-treated mice. However, unlike CR2-fH-treated mice (and wt controls), C3-deficient and CR2-Crry-treated mice had a rebound increase in infarct volumes at 7 days compared to 24 h post-MCAO. CR2-fH-treated mice did not show any significant difference in infarct volume between the two time points post-reperfusion. We also assessed cell death in the ipsilateral cortex, basal ganglia, and hippocampus 7 days following MCAO using TUNEL staining. CR2-Crry- and CR2-fH-treated mice showed a significant reduction in cell death in the basal ganglia and cortex compared to C3−/− and PBS-treated mice, but only CR2-fH-treated mice had a significant reduction in hippocampal cell death compared to all other groups. Further, compared to CR2-Crry-treated mice, CR2-fH-treated mice had significantly less cell death in the cortex and hippocampus. There were no significant differences between C3−/− mice and control mice across the three brain regions (Fig. 1b). Thus, there is an evolution of secondary injury subacutely after MCAO in C3-deficient mice and CR2-Crry-treated mice, but not in CR2-fH-treated mice.Fig. 1

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