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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 localizes to the brain after MCAO and specifically targets the ipsilateral lesion site. Fluorescently labeled CR2-fH was administered as described for therapeutic protocol, and localization of CR2-fH was visualized by in vivo fluorescence tomography. a Representative images of head scans of a single mouse taken at indicated times. b Quantification of fluorescent signal. Mean +/− SEM, n = 5 (24–72 h), n = 4 (72 h), n = 3 (7 days). c, Ex vivo image of brain removed 7 days after MCAO and injection of labeled CR2-fH (lower) or PBS (upper)
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Fig11: CR2-fH localizes to the brain after MCAO and specifically targets the ipsilateral lesion site. Fluorescently labeled CR2-fH was administered as described for therapeutic protocol, and localization of CR2-fH was visualized by in vivo fluorescence tomography. a Representative images of head scans of a single mouse taken at indicated times. b Quantification of fluorescent signal. Mean +/− SEM, n = 5 (24–72 h), n = 4 (72 h), n = 3 (7 days). c, Ex vivo image of brain removed 7 days after MCAO and injection of labeled CR2-fH (lower) or PBS (upper)

Mentions: We have previously demonstrated C3d-specific binding of CR2-fH [15], and to investigate C3d deposition and the targeting and retention of CR2-fH in the post-ischemic brain, we performed in vivo fluorescence imaging. Fluorescently labeled CR2-fH was administered according to our therapeutic protocol, and shaved heads of mice (hair removed to prevent signal interference) were imaged over a 7-day period post-reperfusion. Targeting and localization of CR2-fH was clearly evident 6 h after reperfusion, with a gradual decline to near undetectable levels in live animals by 7 days (Fig. 11a, b). The calculated tissue half-life based on fluorescence signal was 48.5 h. No fluorescence was detected in the brains of sham-operated animals at any time point (not shown). Although fluorescent signal was barely detectable in live animals 7 days after MCAO, ex vivo imaging of the brain removed after sacrifice on day 7 demonstrated the continued presence of labeled CR2-fH, with localization to the ipsilateral parenchyma (Fig. 11c).Fig. 11


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 localizes to the brain after MCAO and specifically targets the ipsilateral lesion site. Fluorescently labeled CR2-fH was administered as described for therapeutic protocol, and localization of CR2-fH was visualized by in vivo fluorescence tomography. a Representative images of head scans of a single mouse taken at indicated times. b Quantification of fluorescent signal. Mean +/− SEM, n = 5 (24–72 h), n = 4 (72 h), n = 3 (7 days). c, Ex vivo image of brain removed 7 days after MCAO and injection of labeled CR2-fH (lower) or PBS (upper)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig11: CR2-fH localizes to the brain after MCAO and specifically targets the ipsilateral lesion site. Fluorescently labeled CR2-fH was administered as described for therapeutic protocol, and localization of CR2-fH was visualized by in vivo fluorescence tomography. a Representative images of head scans of a single mouse taken at indicated times. b Quantification of fluorescent signal. Mean +/− SEM, n = 5 (24–72 h), n = 4 (72 h), n = 3 (7 days). c, Ex vivo image of brain removed 7 days after MCAO and injection of labeled CR2-fH (lower) or PBS (upper)
Mentions: We have previously demonstrated C3d-specific binding of CR2-fH [15], and to investigate C3d deposition and the targeting and retention of CR2-fH in the post-ischemic brain, we performed in vivo fluorescence imaging. Fluorescently labeled CR2-fH was administered according to our therapeutic protocol, and shaved heads of mice (hair removed to prevent signal interference) were imaged over a 7-day period post-reperfusion. Targeting and localization of CR2-fH was clearly evident 6 h after reperfusion, with a gradual decline to near undetectable levels in live animals by 7 days (Fig. 11a, b). The calculated tissue half-life based on fluorescence signal was 48.5 h. No fluorescence was detected in the brains of sham-operated animals at any time point (not shown). Although fluorescent signal was barely detectable in live animals 7 days after MCAO, ex vivo imaging of the brain removed after sacrifice on day 7 demonstrated the continued presence of labeled CR2-fH, with localization to the ipsilateral parenchyma (Fig. 11c).Fig. 11

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