Identifying the Role of Complement in Triggering Neuroinflammation after Traumatic Brain Injury

Abstract

The complement system is implicated in promoting acute secondary injury after traumatic brain injury (TBI), but its role in chronic post-traumatic neuropathology remains unclear. Using various injury-site targeted complement inhibitors that block different complement pathways and activation products, we investigated how complement is involved in neurodegeneration and chronic neuroinflammation after TBI in a clinically relevant setting of complement inhibition. The current paradigm is that complement propagates post-TBI neuropathology predominantly through the terminal membrane attack complex (MAC), but the focus has been on acute outcomes. Following controlled cortical impact in adult male mice, we demonstrate that although inhibition of the MAC (with CR2-CD59) reduces acute deficits, inhibition of C3 activation is required to prevent chronic inflammation and ongoing neuronal loss. Activation of C3 triggered a sustained degenerative mechanism of microglial and astrocyte activation, reduced dendritic and synaptic density, and inhibited neuroblast migration several weeks after TBI. Moreover, inhibiting all complement pathways (with CR2-Crry), or only the alternative complement pathway (with CR2-fH), provided similar and significant improvements in chronic histological, cognitive, and functional recovery, indicating a key role for the alternative pathway in propagating chronic post-TBI pathology. Although we confirm a role for the MAC in acute neuronal loss after TBI, this study shows that upstream products of complement activation generated predominantly via the alternative pathway propagate chronic neuroinflammation, thus challenging the current concept that the MAC represents a therapeutic target for treating TBI. A humanized version of CR2fH has been shown to be safe and non-immunogenic in clinical trials.SIGNIFICANCE STATEMENT Complement, and specifically the terminal membrane attack complex, has been implicated in secondary injury and neuronal loss after TBI. However, we demonstrate here that upstream complement activation products, generated predominantly via the alternative pathway, are responsible for propagating chronic inflammation and injury following CCI. Chronic inflammatory microgliosis is triggered by sustained complement activation after CCI, and is associated with chronic loss of neurons, dendrites and synapses, a process that continues to occur even 30 d after initial impact. Acute and injury-site targeted inhibition of the alternative pathway significantly improves chronic outcomes, and together these findings modify the conceptual paradigm for targeting the complement system to treat TBI.

Keywords: chronic injury; complement; inflammation; therapy.

Figure 1.

Acute recovery and complement deposition in the brain after CCI and targeted complement inhibition. a, Monitoring of weight loss acutely after CCI showing a significant reduction in weight loss over the 3 d period in animals treated with CR2Crry, CR2fH, or CR2CD59 compared with vehicle controls. ANOVA for overall effect with Bonferroni's test for multiple comparisons. **p < 0.01. n = 8/group. b, Acute assessment of forearm laterality on corner task showing that all three targeted inhibitors result in significant reduction in laterality on day 3 after CCI compared with vehicle controls. One-way ANOVA with Bonferroni's. *p < 0.05, **p < 0.01. c, Total distance moved by animals in open-field ambulation test showing that targeted complement inhibition does not influence the total distance moved by animals on day 3 compared with vehicle. One-way ANOVA with Bonferroni's. d, Kaplan–Meyer survival curve over 30 d period. Log rank (Mantel–Cox) test was used. No significant difference between groups on pair comparisons. e, IF staining for C5b-9 (MAC, green) and C3d (red) deposition in the perilesional brain 24 h after CCI. Scale bars, 100 μm.

Figure 2.

Motor recovery after CCI and treatment with complement inhibitors. a–c, Performance on corner task was assessed weekly after CCI showing that CR2fH and CR2Crry, but not CR2CD59, resulted in significantly faster recovery of forearm laterality compared with vehicle. One-way ANOVA with Bonferroni's multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. d, Motor scores on ladder task were assessed weekly after CCI showing that CR2fH and CR2Crry, but not CR2CD59, resulted in significantly high motor scores throughout 28 d of recovery compared with both vehicle controls and animals treated with CR2CD59. RM two-way ANOVA with Bonferroni's test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = mean ± SEM. e, Motor scores on day 28 after CCI showing significantly higher scores in CR2Crry and CR2fH-treated mice, but not CR2CD59-treated mice compared with vehicle controls. One-way ANOVA with Bonferroni's multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. f, g, Forelimb and hindlimb errors showing a significant reduction in the number of errors after CR2Crry or CR2fH therapy compared with both CR2CD59 and vehicle. RM two-way ANOVA with Bonferroni's test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = mean ± SEM.

Figure 3.

Effect of targeted complement inhibition on lesion volume and scarring after CCI. a, Representative Nissl stained brain section from the different treatment groups. b, 3D-reconstructed brains showing lesion (red) and scarring (orange) 30 d after TBI. Brains were reconstructed from Nissl quantification as described in Materials and Methods, Lesion volume reconstruction. Yellow, Cortex; blue, hippocampus; green, basal ganglia; gray, white matter. c, Similar to b but showing only scarring in the same brains shown in b. d, e, Quantification of lesion volume and scarring at 30 d after CCI showing significant reduction in lesion volume across the different treatment groups compared with vehicle controls. However, CR2fH and CR2Crry resulted in more effective reduction in overall lesion compared with CR2CD59. d, CR2Crry and CR2fH were effective at reduction in scarring 30 d after TBI, but not CR2CD59. One-way ANOVA with Bonferroni's multiple comparisons. n = 12–17/group. *p < 0.05, ***p < 0.001. Error bars = mean ± SEM.

Figure 4.

Targeted inhibition of C3-cleavage significantly improves cognitive recovery after CCI. a–c, Spatial learning and memory were assessed using the Barnes maze task. a, Path length traveled by the animal before reaching the escape hole and (b) the number of error pokes each animal made before reaching the scape hole. Treatment with CR2Crry and CR2fH but not CR2CD59 resulted in significant improvement compared with vehicle controls. RM two-way ANOVA with Bonferroni's multiple comparisons. n = 7–12/group. *p < 0.05, **p < 0.01, ***p < 0.001. d, Anxiety level was assessed using the percentage time spent at the periphery of an open field. Animals among the different groups did not show significant differences in anxiety levels. One-way ANOVA with Bonferroni's multiple comparisons. n = 7–12/group. Data shown as mean ± SEM.

Figure 5.

Targeted inhibition of C3 activation limits chronic neuroinflammation after CCI. a–c, Representative IHC staining of IgM deposition (a), C3d deposition (b), and Iba1+ cells (c) in the perilesional brain 30 d after CCI. Scale bars, 50 μm. d, Representative IF images of brain sections stained for C5b-9 (MAC) showing minimal MAC deposition 30 d after TBI. Red squares, High-magnification confocal microscopy of perilesional brain regions stained for C5b-9. Scale bars, 50 μm. e, f, Quantification of number and morphology of Iba1+ cells showing significantly lower number of Iba1+ cells and percentage of amoeboid Iba1+ cells in CR2Crry and CR2fH-treated animals. One-way ANOVA with Bonferroni's multiple comparisons. n = 5/group. *p < 0.05, ***p < 0.001. g, Quantification of C5b-9 deposition in the perilesional brain from the high fields shown in d demonstrating no significant difference in mean intensity of C5b-9 between vehicle-treated animals and the other treatment groups. One-way ANOVA with Bonferroni's multiple comparisons. n = 5/group. p = 0.573. h, i, Representative full-brain IF staining for Mac2 and GFAP 30 d after CCI. j, k, Quantification of f and g showing significant reduction in Mac2+ cells and GFAP+ cells in animals treated with CR2Crry or CR2fH but not CR2CD59. One-way ANOVA with Bonferroni's multiple comparisons. n = 7–9/group. *p < 0.05, **p < 0.01, ***p < 0.001. All error bars represent mean ± SEM.

Figure 6.

CR2Crry and CR2fH significantly increase neuroblast migration from the SVZ 30 d after CCI. a, IF staining for BrdU+/Dcx+ cells in and around the subventricular zone 30 d after CCI showing similar pattern of neurogenesis across the different groups. Scale bars, 50 μm. b–e, Representative Dcx-stained sections of the lesion area (b), perilesional cortex (c), basal ganglia (d), and hippocampus (e) showing significantly higher number of Dcx+ neuroblasts migrating to the lesion site, as well as to perilesional brain in animals treated with CR2Crry and CR2fH compared with both vehicle and CR2CD59. Scale bars, 50 μm. f–j, Quantification of a showing that CR2Crry and CR2fH do not affect SVZ neurogenesis but significantly increase neuronal migration after TBI. One-way ANOVA with Bonferroni's multiple comparisons. n = 5/group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = mean ± SEM.

Figure 7.

Interaction between chronic neuroinflammation and neurodegenerative processes after CCI. a, Representative Nissl stained brain section showing the location of field selection from the perilesional brain for immunostaining shown in this figure. b, Correlation between the number of NeuN+ neurons and the number of Mac2+ microglia in perilesional microscopic fields showing a significant negative correlation. Pearson correlation R² = 0.813, p < 0.0001. Blue dots, Vehicle controls; red dots, CR2Crry; green dots, CR2fH. c, Representative high resolution fields showing higher number of Mac2+ cells and lower numbers of NeuN+ neurons in the perilesional cortex in vehicle controls compared with CR2fH or CR2Crry-treated animals. Scale bars, 50 μm. d, Heat map of intensity of PSD95 staining in full brain sections from the different treatment groups. e, Super-resolution IF staining of dendritic arborization (MAP2, green) and complement deposition (C3d, red) in perilesional hippocampal sections showing significantly higher complement deposition on neurons and less elaborate dendritic arborization in vehicle controls compared with CR2Crry or CR2fH-treated animals. f, Higher-magnification fields of e stained for MAP2, NeuN, and C3d showing significantly higher deposition of C3d and loss of dendrites in vehicle compared with sham and to CR2Crry and CR2fH-treated mice. g, h, Quantification of staining in e and f. One-way ANOVA with Bonferroni's multiple comparisons. n = 5/group. *p < 0.05, **p < 0.01. Error bars = mean ± SEM.

Figure 8.

Targeted inhibition of C3 activation is also protective at 12 h after CCI. a, b, Quantification of lesion volume and scarring at 30 d after CCI showing significant reduction in lesion volume in animals treated with CR2Crry or CR2fH at 12 h after CCI compared with vehicle controls. One-way ANOVA with Bonferroni's multiple comparisons. **p < 0.01. c, Motor scores on day 28 after CCI showing significantly higher scores in CR2Crry and CR2fH-treated mice compared with vehicle controls. One-way ANOVA with Bonferroni's multiple comparisons. *p < 0.05. d, Barnes maze task: path length traveled by the animal before reaching the escape hole showing that treatment with CR2Crry and CR2fH resulted in significant reduction in path length during acquisition and retention of spatial memory compared with vehicle controls. Two-way ANOVA with Bonferroni's multiple comparisons. *p < 0.01.

Figure 9.

Interaction of different complement activation products with post-TBI pathology. Numbers in circles indicate the reference to figures that provide evidence on the associated step.