Acute Complement Inhibition Potentiates Neurorehabilitation and Enhances tPA-Mediated Neuroprotection

Abstract

Because complement activation in the subacute or chronic phase after stroke was recently shown to stimulate neural plasticity, we investigated how complement activation and complement inhibition in the acute phase after murine stroke interacts with subsequent rehabilitation therapy to modulate neuroinflammation and neural remodeling. We additionally investigated how complement and complement inhibition interacts with tissue plasminogen activator (tPA), the other standard of care therapy for stroke, and a U.S. Food and Drug Administration preclinical requirement for translation of an experimental stroke therapy. CR2fH, an injury site-targeted inhibitor of the alternative complement pathway, significantly reduced infarct volume, hemorrhagic transformation, and mortality and significantly improved long-term motor and cognitive performance when administered 1.5 or 24 h after middle cerebral artery occlusion. CR2fH interrupted a poststroke inflammatory process and significantly reduced inflammatory cytokine release, microglial activation, and astrocytosis. Rehabilitation alone showed mild anti-inflammatory effects, including reduced complement activation, but only improved cognitive recovery. CR2fH combined with rehabilitation significantly potentiated cognitive and motor recovery compared with either intervention alone and was associated with higher growth factor release and enhanced rehabilitation-induced neuroblast migration and axonal remodeling. Similar outcomes were seen in adult, aged, and female mice. Using a microembolic model, CR2fH administered in combination with acute tPA therapy improved overall survival and enhanced the neuroprotective effects of tPA, extending the treatment window for tPA therapy. A human counterpart of CR2fH has been shown to be safe and nonimmunogenic in humans and we have demonstrated robust deposition of C3d, the CR2fH targeting epitope, in ischemic human brains after stroke.SIGNIFICANCE STATEMENT Complement inhibition is a potential therapeutic approach for stroke, but it is not known how complement inhibition would interact with current standards of care. We show that, after murine ischemic stroke, rehabilitation alone induced mild anti-inflammatory effects and improved cognitive, but not motor recovery. However, brain-targeted and specific inhibition of the alternative complement pathway, when combined with rehabilitation, significantly potentiated cognitive and motor recovery compared with either intervention alone via mechanisms involving neuroregeneration and enhanced brain remodeling. Further, inhibiting the alternative pathway of complement significantly enhanced the neuroprotective effects of thrombolytic therapy and markedly expanded the therapeutic window for thrombolytic therapy.

Keywords: complement; rehabilitation; thrombolysis.

Figure 1.

CR2fH treatment reduces intracranial hemorrhage and infarct acutely after stroke. a, Percentage of animals dying within 24 h of stroke that had intracranial hemorrhage between vehicle- and CR2fH-treated animals. Population proportion statistic, **p < 0.01. b, CR2fH treatment and fB deficiency significantly reduce hemoglobin content in the ipsilateral hemisphere 12 h after MCAO compared with vehicle. One-way ANOVA, Dunnett's multiple comparisons. n = 4/group, ***p < 0.001. c, Representative images of Nissl-stained brain sections showing peri-infarct intracerebral hemorrhage in vehicle controls but not in CR2fH-treated animals (90 min after MCAO administration). d, e, C3a and C5a ELISAs showing a significant reduction of MCAO-induced C3a and C5a generation by CR2fH treatment (90 min after MCAO administration) or fB deficiency 24 h after MCAO. One-way ANOVA, Tukey's multiple comparisons. n = 4/group, ****p < 0.0001, **p < 0.01. f, Correlation of hemoglobin content in the ipsilateral hemisphere with C3a levels (blue line, Pearson's correlation r² = 0.800, p < 0.001) and C5a levels (red line, Pearson's correlation r² = 0.5071, p < 0.001). g, Both CR2fH treatment and fB deficiency significantly reduced acute infarct volume quantified from Triphenyl tetrazolium chloride-stained 2-mm-thick brain slices 24 h after stroke. h, Mortality after MCAO and randomization to rehabilitation or regular housing. Kaplan–Meyer curve shows no significant difference between the groups. n = 21 vehicle, n = 16 rehabilitation, n = 12 CR2fH, and n = 12 CR2fH + rehabilitation.

Figure 2.

CR2fH treatment, but not rehabilitation (enriched environment and skilled handling), significantly reduced infarct size and scarring after MCAO. a, Representative T2-weighted MRI images at days 4 and 14 after MCAO showing large hyperintense lesions in vehicle- and rehabilitation-treated animals compared with CR2fH-treated animals with or without rehabilitation therapy. b, Quantification of a showing significant reduction of hyperintense lesion volume with CR2fH treatment compared with vehicle. Rehabilitation alone resulted in minimal reduction in lesion volume, which did not reach statistical significance. Kruskal–Wallis test with Dunn's multiple comparisons. n = 6 vehicle, n = 8 CR2fH, *p < 0.05. **p < 0.01. c, Representative Nissl-stained brains showing secondary injury and scarring in the ipsilateral hemisphere 15 d after MCAO that was inhibited by acute CR2fH therapy. c1, Nissl-stained brain slices. c2, 3D rendering of lesion (in red) on a brain showing the location of cortex (yellow), basal ganglia (green), white matter (gray), and hippocampus (blue). d, Quantification of lesion volume confirming histologically that CR2fH reduces secondary scarring 15 d after MCAO, with minimal contribution of rehabilitation therapy alone to reduced scarring. One-way ANVOA with Bonferroni's correction. n = 8/group, **p < 0.01, ***p < 0.001. e, Representative DTI and DKI images showing that CR2fH reduced MD and increased MK in the ipsilateral basal ganglia and hippocampus at day 4 after MCAO. f, Quantification of e using three ROIs drawn over the cortex, hippocampus, and basal ganglia both ipsilaterally and contralaterally. Shown in e are contralateral ROIs. Rations of ipsilateral to contralateral MDs and MKs are compared. Two-way ANOVA, Bonferroni's correction, n = 6 vehicle, n = 8 CR2fH, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Combination of CR2fH treatment and rehabilitation therapy resulted in faster and more pronounced motor recovery 15 d after MCAO. a, Overview of the experimental design. Survival curve for different groups is shown in Figure 1h. b, CR2fH administered 90 min after ischemia with or without rehabilitation resulted in significant and sustained improvement in neurological deficit over 15 d of recovery compared with vehicle or rehabilitation therapy alone. Compared with CR2fH alone, the CR2fH + rehabilitation group exhibited significantly faster recovery during the first week after MCAO. Two-way ANOVA, Bonferroni's correction, n = 11–21/group (individual n's are shown in Fig. 2e). *p < 0.05, ***p < 0.001. c, d, CR2fH also improved recovery of functional deficits when administered 6 h (c) or 24 h (d) after MCAO. e, Comparison of neurological deficits at day 15 after injury showing that CR2fH significantly improved long-term functional recovery compared with vehicle and that, when combined with rehabilitation, more pronounced recovery is observed. Comparisons were made against vehicle group. Kruskal–Wallis test with Dunn's multiple comparisons, not significant (ns), *p < 0.05, ***p < 0.001, ****p < 0.0001. f, Pairwise comparison of functional recovery between days 2 and 15 across the different groups showing that, despite significant acute improvement achieved with CR2fH alone compared with vehicle, combination of CR2fH with rehabilitation, but not CR2fH or rehabilitation alone, resulted in more pronounced recovery beyond the acute phase (between days 2 and 15). Two-way ANOVA with Bonferroni's correction, n = 11–21/group, *p < 0.05, ***p < 0.001. g, Among animals treated with rehabilitation, animals cotreated with CR2fH showed significantly more interaction with the enriched environment compared with vehicle-treated animals at day 3, but not day 10, after MCAO. Two-way ANOVA with Bonferroni's correction, n = 16 vehicle, n = 12 CR2fH 90 min, n = 7 CR2fH 6 and 24 h, *p < 0.05.***p < 0.001. h, i, During the first week of recovery, CR2fH (90 min) alone or with rehabilitation significantly reduced forelimb asymmetry on the corner test (h) and increased open-field locomotor activity (i) compared with vehicle. Combination treatment has the largest effect on both tasks at any time point. j. No difference among groups was seen in the percentage time spent at center, thus controlling for potential difference in anxiety levels between the groups. Two-way ANOVA with Bonferroni's correction, n = 21 vehicle, n = 16 rehabilitation, n = 12 CR2fH ± rehabilitation, *p < 0.05, **p < 0.01, ***p < 0.001. k–m, On pasta-handling task, animals treated with rehabilitation, CR2fH, or CR2fH + rehabilitation showed signs of improvement in time to eat compared with vehicle (k), but the improvement in unilateral handling and number of adjustments was only significant in CR2fH ± rehabilitation animals compared with vehicle controls. Two-way ANOVA with Bonferroni's correction, n = 8/group, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Improvement in cognitive performance after MCAO with rehabilitation, CR2fH, and combination therapy. a–c, CR2fH treatment alone or in combination with rehabilitation significantly improved spatial learning (days 9–11 after MCAO) and retention of learned memory (day 15 after MCAO) compared with vehicle controls, as assessed by the total path length before reaching the target hole (a) or the number of error pokes (c). Cotreatment with CR2fH and rehabilitation also reduced path length and number of errors significantly compared with rehabilitation alone during learning. At the retention phase, rehabilitation alone showed significant improvement in both measures compared with vehicle, whereas CR2fH + rehabilitation was significantly better than CR2fH alone. Statistical results of retention day are displayed. Two-way ANOVA, Bonferroni's correction, n = 8/group, *p < 0.05, ***p < 0.001. Results of multiple comparisons: (1) day 8: no significant difference between groups; (2) day 9: vehicle versus CR2fH: p < 0.05, vehicle versus CR2fH + rehabilitation: p < 0.001, rehabilitation or CR2fH versus CR2fH + rehabilitation: p < 0.01; (3) day 10: vehicle versus CR2fH: p < 0.001, all groups compared with CR2fH + rehabilitation: p < 0.001; (4) day 11: vehicle versus CR2fH: p < 0.05, all groups compared with CR2fH + rehabilitation: p < 0.001; and (5) day 15: displayed on graph. d, Passive avoidance task revealed that animals treated with CR2fH with or without rehabilitation have better memory retention (longer time to enter the shock chamber) compared with vehicle and rehabilitation alone starting 7 d after MCAO. At both 7 and 14 d after injury, CR2fH was significantly better than rehabilitation alone. Rehabilitation alone was significantly better than vehicle, but combination therapy was significantly superior to all other groups. Two-way ANOVA, Bonferroni's correction, n = 8/group, *p < 0.05, **p < 0.01, ***p < 0.001. e, PCA of the performance on the different motor and cognitive tasks displayed in Figures 2 and 3 (see Materials and Methods) showed that three PCs (PC1–PC3) can explain 99.2% of the variance. Individual animal data including shams were plotted against the three PCs, showing that CR2fH combined with rehabilitation was most efficient at bringing the animals closer to sham compared with either single therapy. f, PCA analysis performed only on motor (right) or cognitive tasks (left) indicate that the effect of rehabilitation was more pronounced on cognitive rather than motor tasks. Two PCs explaining ∼90% of the variance were plotted.

Figure 5.

CR2fH (administered 90 min after MCAO) is also protective in female mice and aged male mice (14 months old). a, CR2fH alone or in combination with rehabilitation significantly reduced neurological deficits in aged mice compared with vehicle; however, only CR2fH + rehabilitation was significantly better compared with rehabilitation only (p < 0.05). Two-way ANOVA, Bonferroni's correction, n = 7/group, *p < 0.05, **p < 0.01. b, Survival was significantly improved by CR2fH with or without rehabilitation in aged animals after MCAO. Experiments were terminated at day 10 due to loss of all vehicle controls. Log–rank (Mantel–Cox) test, n = 7/group, *p < 0.05. c, d, CR2fH improved neurological deficits and survival of adult female mice through 15 d after MCAO. c, Two-way ANOVA, Bonferroni's correction, n = 7/group, *p < 0.05, **p < 0.01. d, Log–rank (Mantel–Cox) test, n = 7/group. p = 0.057.

Figure 6.

CR2fH treatment blocks a robust inflammatory response that otherwise propagates to the chronic phase after MCAO. a, b, A sustained neuroinflammatory response manifested by C3d and IgM deposition 15 d after MCAO was inhibited by a single acute administration of CR2fH (90 min after MCAO). Shown in a are the lesion locations and in b the different treatment groups. Green, IgM; red, C3d; blue, DAPI. c, d, Quantification of IgM (c) and C3d (d) deposition showing that CR2fH significantly reduced deposition of both markers on the inflamed ipsilateral endothelium. Rehabilitation alone resulted in a modest but significant reduction in IgM and C3d deposition by 15 d after MCAO. ANOVA with Bonferroni's correction, n = 5 animals (4 fields per animal chosen from the peri-infarct lesion), *p < 0.05, ****p < 0.0001. e, GFAP immunofluorescence staining showing extensive astrogliosis in the peri-infarct area 15 d after MCAO that was significantly inhibited by CR2fH therapy alone, but not by rehabilitation alone. f, Mac2 immunofluorescence staining showing extensive gliosis and proliferation of M1-polarized (inflammatory type microglia/macrophages) in the perilesional area of vehicle- and rehabilitation-treated animals that was significantly inhibited by CR2fH therapy. g, Quantification of e and f showing that, although CR2fH more robustly reduced astrocytosis and microgliosis, rehabilitation nevertheless had a milder though significant effect on reducing inflammatory phenotype 15 d after stroke. ANOVA with Bonferroni's correction, n = 5 animals (3 fields per animal), *p < 0.05, ***p < 0.001.

Figure 7.

Gene expression analysis of the impact of CR2fH and rehabilitation on immune-related genes in the ipsilateral hemisphere 5 d after stroke. a, Clustergram of differentially expressed genes on Nanostring immunology gene expression assay with a p < 0.05 on at least one comparison. A detailed network of gene expression changes is provided in Figure 7-1, Figure 7-2, and Figure 7-3. b, PCA of differentially expressed genes showing PC1–PC3, which explain 69% of the variance. c, Enrichment analysis of GO biological processes enriched in genes that are significantly upregulated in MCAO versus sham animals. Enrichment statistics were performed through PANTHER (Mi et al., 2016). Processes with p < 0.0001 are displayed. d, Clustergram of genes significantly upregulated or significantly downregulated in rehabilitation compared with vehicle (p < 0.05). e, Pie charts showing the distribution of genes dysregulated by MCAO according to their changes by the different treatment categories. f, g, Gene expression panels of genes involved in microglial/macrophage activation (f) and complement activation (g). Two-way ANOVA, Bonferroni's correction, n = 5/group, *p < 0.05.

Figure 8.

CR2fH removes the inhibition on regenerative mechanisms promoting the outcome of rehabilitation therapy. a, Dcx immunostaining of perilesional hippocampi quantified in b showing a significant increase in the number of neuroblasts migrating to the ipsilateral hippocampus 15 d after injury with rehabilitation, CR2fH treatment, or combination therapy. However, combination of CR2fH and rehabilitation showed a more robust increase in Dcx+ cells compared with either single intervention. ANOVA. n = 6 animals (2 fields each), ***p < 0.001. c–f, Immunostaining for markers of regeneration and remodeling, including dendritic arborization (MAP2), synaptic density (PSD-95), and axonal growth (GAP-43) of full-brain slices showing that a combination of CR2fH and rehabilitation resulted in the most pronounced and significant increase in dendritic and axonal growth to the perilesional area with subsequent increase in synaptic density. Significant increase in regenerative markers was also seen in CR2fH-treated animals (all three markers) and in rehabilitation-only animals (MAP2 and PSD95). ANOVA, full hemispheres quantified, *p < 0.05, **p < 0.01, ***p < 0.001. Heat map shows mean luminescence. g, Costaining for GAP-43 and GFAP showing that robust astrogliosis inhibits the regrowth of regenerating (GAP-43+) axons into the perilesional area. CR2fH significantly reduced astrogliotic scarring, resulting in increased GAP-43 infiltration to perilesional brain, whereas CR2fH in combination with rehabilitation further increased the levels of GAP-43 in the perilesional areas. Rehabilitation-only animals showed limited GAP43 increase that was still blocked by astrogliosis. h, Quantification of GAP-43 density in the perilesional area showing that vehicle and rehabilitation animals showed a significant reduction in GAP43 density surrounding the areas of astrogliosis compared with CR2fH with or without rehabilitation. p < 0.01 comparing vehicle or rehabilitation with CR2fH with or without rehabilitation at locations −2 to 3. Two-way ANOVA, n = 3/group. i, Representative 3D-rendered fields from h at different positions relative to the astrogliotic scar. j, Notable reduction in GAP43 was observed near the areas of astrogliosis in vehicle-treated animals despite comparable neuronal density across the boundaries of the scar (NeuroTrace, cyan). k, Representative Western blots showing levels of neuronal growth factor (BDNF) and TNF-α across the different groups. l, Both CR2fH and rehabilitation alone resulted in a significant increase in the levels of BDNF in the ipsilateral hemisphere compared with vehicle. However, combination therapy resulted in a more robust and significant increase compared with either single intervention. ANOVA, n = 6/group, *p < 0.05, ***p < 0.001. m, Rehabilitation did not influence the levels of TNF-α. However, CR2fH with or without rehabilitation significantly reduced the levels of TNF-α compared with rehabilitation or vehicle groups. ANOVA, n = 5/group, **p < 0.01, ***p < 0.001.

Figure 9.

CR2fH acts cooperatively with tPA to reduce mortality and improve outcomes after embolic stroke. a, Kaplan–Meyer curve of poststroke survival showing a significant reduction in mortality in animals treated with CR2fH or cotreated with CR2fH and tPA compared with vehicle treatment. Log–rank (Mantel–Cox) test, n = 10–14/group, *p < 0.05. b, Infarct volume was assessed using Nissl staining of serially cut brain sections 200 μm apart. tPA, CR2fH, and the combination significantly reduced infarct volume compared with vehicle. Combination of CR2fH and tPA resulted in the most profound effect in reducing infarct volume compared with single therapies. One-way ANOVA with Bonferroni's multiple comparisons. n = 6/group, *p < 0.05, **p < 0.01, ***p < 0.001. c, Neurological deficit assessed daily starting 6 h after high-dose embolic stroke showed a significant additive effect of CR2fH and tPA therapy on overall deficit after stroke. Repeated-measures two-way ANOVA with Bonferroni's multiple comparisons, n = 6–8/group, *p < 0.05, ***p < 0.001. Representative TTC-stained sections are also shown, with arrows indicating infarcted area. d, Corner task was used to assess forearm laterality compared with baseline laterality before stroke. Similar to neurological deficits, combination therapy with CR2fH and tPA showed the most robust and sustainable effect in reducing deficits over 14 d of recovery compared with vehicle treatment. Two-way ANOVA with Bonferroni's multiple comparisons, n = 6–8/group, *p < 0.05, **p < 0.01. e, Passive avoidance task was used to assess cognitive recovery after high-dose embolic stroke. On day 3 after emboli administration, animals treated with tPA, CR2fH, or a combination showed a significant improvement in memory retention (longer latency to enter the dark chamber) compared with vehicle controls. This improvement was only maintained at days 7 and 14 after stroke in animals treated with CR2fH and combination therapy, but not tPA alone. Two-way ANOVA with Bonferroni's multiple comparisons, n = 6–8/group, *p < 0.05, **p < 0.01.

Figure 10.

Complement and IgM deposition in the ischemic hemisphere of human patients who died from acute stroke. a, Immunofluorescence microscopy showing IgM (green) and C3d (red) deposition in the ischemic but not contralateral hemisphere of patients who died from acute stroke. HB1, 24 h poststroke; HB2, 24–48 h poststroke; HB3, 48 h poststroke; blue, DAPI. b, High-resolution 3D rendering of IgM and C3d deposition showing colocalization of C3d and IgM in the ischemic penumbra. c, Pairwise comparisons of C3d and IgM deposition in the ipsilateral compared with the contralateral hemisphere showing significantly higher levels in the ipsilateral hemisphere of acute stroke patients. Pairwise two-way ANOVA, Bonferroni's correction, n = 3, *p < 0.05, **p < 0.01.

Figure 11.

Illustration of the main hypothesis on how complement inhibition and rehabilitation interact to affect recovery after stroke.