Late exercise reduces neuroinflammation and cognitive dysfunction after traumatic brain injury

Abstract

Delayed secondary biochemical and cellular changes after traumatic brain injury continue for months to years, and are associated with chronic neuroinflammation and progressive neurodegeneration. Physical activity can reduce inflammation and facilitate recovery after brain injury. Here, we investigated the time-dependent effects, and underlying mechanisms of post-traumatic exercise initiation on outcome after moderate traumatic brain injury using a well-characterized mouse controlled cortical impact model. Late exercise initiation beginning at 5weeks after trauma, but not early initiation of exercise at 1week, significantly reduced working and retention memory impairment at 3months, and decreased lesion volume compared to non-exercise injury controls. Cognitive recovery was associated with attenuation of classical inflammatory pathways, activation of alternative inflammatory responses and enhancement of neurogenesis. In contrast, early initiation of exercise failed to alter behavioral recovery or lesion size, while increasing the neurotoxic pro-inflammatory responses. These data underscore the critical importance of timing of exercise initiation after trauma and its relation to neuroinflammation, and challenge the widely held view that effective neuroprotection requires early intervention.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1

Effect of exercise on TBI-induced neurological deficits in mice. (A) Experimental protocol. (B) TBI-induced spatial learning and working memory deficits were assessed using a standard Morris Water Maze (sMWM) test. Injured non-exercised mice (a; p<0.05 vs. naïve) and acute exercised mice (b; p<0.05 vs. naïve) showed significant impairment in working memory over time. Injured delayed exercised mice were not significantly different from naïve and showed significant improvements in working memory as compared to non-exercised animal (d; p<0.05 vs. TBI+noEX). (C) Reference memory was assessed using the sMWM probe test. There was a significant impairment in reference memory in the non-exercised injured animals (a; p<0.01 vs. naïve) and a significant improvement in reference memory in injured delayed exercised mice compared to non-exercised animals (d; p<0.05 vs. TBI+noEX). (D) Reversal spatial learning was assessed using a Reverse Morris Water Maze (rMWM) test immediately after sMWM. All injured mice showed significantly increased latency to find the sub-merged hidden platform (a-TBI+noEX, b-TBI+acEX, c-TBI+deEX; p<0.05 vs. naïve). Injured delayed exercise mice showed significant improvements in reversal spatial learning as compared to non-exercised (d; p<0.05 vs. TBI+noEX) or acute exercise animals (e; p<0.05 vs. TBI+acEX). (E) Reference memory in rMWM was assessed on the day after the fourth acquisition day. There was a significant impairment in reference memory in the non-exercised injured animals (a; p<0.01 vs. naïve) and a significant improvement in reference memory in injured delayed exercised mice compared to non-exercised animals (d; p<0.05 vs. TBI+noEX). Mice spent similar time with the two identical objects during the sample phase in each group. (F) Non-spatial memory was assessed using the novel object recognition (NOR) test. Significant discrimination with preference for the novel object was observed for each group (p<0.05; novel compared to familiar object exploration). Significantly shorter exploring time was observed in injured non-exercised (a; p<0.05 versus naïve) and acute exercised mice (b; p<0.05 versus naïve). The delayed exercised injured group exploring time did not significantly differ from naïve and was significantly longer than non-exercised injured (d; p<0.05 vs. TBI+noEX) and acute exercise injured animals (e; p<0.05 vs. TBI+acEX). Data included in this figure are expressed as mean ± SEM, n = 13-15/group, statistical analysis using repeated measures one-way ANOVA (Fig.1 B and D) or one-way ANOVA (Fig.1 C, E, F) followed by post hoc adjustments using Tukey’s test.

Figure 1

Effect of exercise on TBI-induced neurological deficits in mice. (A) Experimental protocol. (B) TBI-induced spatial learning and working memory deficits were assessed using a standard Morris Water Maze (sMWM) test. Injured non-exercised mice (a; p<0.05 vs. naïve) and acute exercised mice (b; p<0.05 vs. naïve) showed significant impairment in working memory over time. Injured delayed exercised mice were not significantly different from naïve and showed significant improvements in working memory as compared to non-exercised animal (d; p<0.05 vs. TBI+noEX). (C) Reference memory was assessed using the sMWM probe test. There was a significant impairment in reference memory in the non-exercised injured animals (a; p<0.01 vs. naïve) and a significant improvement in reference memory in injured delayed exercised mice compared to non-exercised animals (d; p<0.05 vs. TBI+noEX). (D) Reversal spatial learning was assessed using a Reverse Morris Water Maze (rMWM) test immediately after sMWM. All injured mice showed significantly increased latency to find the sub-merged hidden platform (a-TBI+noEX, b-TBI+acEX, c-TBI+deEX; p<0.05 vs. naïve). Injured delayed exercise mice showed significant improvements in reversal spatial learning as compared to non-exercised (d; p<0.05 vs. TBI+noEX) or acute exercise animals (e; p<0.05 vs. TBI+acEX). (E) Reference memory in rMWM was assessed on the day after the fourth acquisition day. There was a significant impairment in reference memory in the non-exercised injured animals (a; p<0.01 vs. naïve) and a significant improvement in reference memory in injured delayed exercised mice compared to non-exercised animals (d; p<0.05 vs. TBI+noEX). Mice spent similar time with the two identical objects during the sample phase in each group. (F) Non-spatial memory was assessed using the novel object recognition (NOR) test. Significant discrimination with preference for the novel object was observed for each group (p<0.05; novel compared to familiar object exploration). Significantly shorter exploring time was observed in injured non-exercised (a; p<0.05 versus naïve) and acute exercised mice (b; p<0.05 versus naïve). The delayed exercised injured group exploring time did not significantly differ from naïve and was significantly longer than non-exercised injured (d; p<0.05 vs. TBI+noEX) and acute exercise injured animals (e; p<0.05 vs. TBI+acEX). Data included in this figure are expressed as mean ± SEM, n = 13-15/group, statistical analysis using repeated measures one-way ANOVA (Fig.1 B and D) or one-way ANOVA (Fig.1 C, E, F) followed by post hoc adjustments using Tukey’s test.

Figure 2

Effect of exercise on TBI-induced brain lesion at three months post-injury. (A) Brain lesion was assessed with cresyl-violet stained brain sections in TBI mice. (B) Unbiased stereological assessment showed that lesion volume was significantly reduced in the delayed exercised TBI mice compared to both non-exercised (d; p<0.05 vs. TBI+noEX) and acute exercise TBI mice (e; p<0.05 vs. TBI+acEX). Data expressed as mean ± SEM, n=7-8/group, statistical analysis using one-way ANOVA followed by post hoc adjustments using Tukey’s test).

Figure 3

Effect of exercise on expression of IL-1β, IL-6, and IL-10 after TBI. Quantitative real-time PCR analysis was performed at 5 weeks or 9 weeks post-injury with/without exercise. (A) Acute exercise significantly up-regulated expression level of IL-1β at 5 weeks post-injury (**p<0.01, TBI+acEX vs. TBI+noEX 5w; n=4-6/group). No significant changes were observed in the expression of IL-6 and IL-10. (B) Delayed exercise significantly attenuated the TBI-induced IL-1β expression at 9 weeks post-injury. Moreover, delayed exercise resulted in a significant increase of both IL-6 and IL-10 expression (**p<0.01 TBI+deEX vs. TBI+noEX 9w; n=4-6/group). All data normalized to the expression level of the naïve group.

Figure 4

Effect of exercise on expression of microglial activation markers C1qB and Galectin-3 at three months post-injury. (A) Representative immunoblots for microglial activation markers (C1qB and galectin-3) and the loading control (β-actin). (B) Quantification of protein band intensity showed that expression of C1qB and galectin-3 was significantly increased in all TBI groups compared to naïve group (a-TBI+noEX, b-TBI+acEX, c-TBI+deEX; p<0.05 vs. naïve). The expression of C1qB and galectin-3 was significantly increased in the acute exercise injured animals compared to non-exercised TBI group (f, p<0.05 TBI+acEX vs. TBI+noEX) and attenuated by the delayed exercise compared to both non-exercise (d; p<0.05 TBI+deEX vs. TBI+noEX) and acute exercise (e, p<0.05 TBI+deEX vs. TBI+acEX) TBI groups. (C) Representative galectin-3 immunohistochemical images in the contused cortex at 3 months post-injury. (D) Galectin-3-positive cells (red) co-labeled with Iba1-positive microglia (green) and/or CD68-positive reactive microglia (magenta). Bar = 30 μm (C); 250 μm (D).

Figure 5

Effect of exercise on expression of NADPH oxidase at three months post-injury. (A) Representative immunoblots for the NADPH oxidase subunits gp91phox and p22phox and the loading control (β-actin). (B) Quantification of protein band intensity showed that in all injured groups expression of gp91phox and p22phox was significantly increased compared to naïve group (a-TBI+noEX, b-TBI+acEX, c-TBI+deEX; p<0.05 vs. naïve). The expression of gp91phox and p22phox was significantly attenuated by delayed exercise compared to either non-exercised TBI (d; p<0.05 TBI+deEX vs. TBI+noEX) or acute exercise TBI (e; p<0.05 TBI+deEX vs. TBI+acEX) groups. (C) Gp91phox-positive cells (green) co-localized with CD68-positive reactive microglia (red) that displayed a hypertrophic morphology in the injured cortex of non-exercised TBI sections at three months post-injury. Delayed exercise has reduced gp91phox-positive reactive microglia at this time point. Bar = 75 μm.

Figure 6

Effect of exercise on activated microglial phenotypes in the hippocampus at three months post-injury. (A) Representative Iba-1 immunohistochemical images displayed resting (ramified morphology, upper left) or activated (hypertrophic morphology, lower left) microglial phenotypes and the corresponding Neurolucida reconstructions (right). (B) Unbiased stereological quantitative assessment of microglial phenotypes in the hippocampus of each group. There was no significant difference in the number of ramified microglia in the CA2/3 and DG regions across groups. Injured non-exercised (a; p<0.05 TBI+noEX vs. naïve) or acute exercise tissue (b; p<0.05 TBI+acEX vs. naïve) showed significantly increased activated microglia in CA2/3 and DG regions compared to naïve. Delayed exercise tissue showed no significant differences in activated microglia when compared to naïve and demonstrated significantly reduced numbers of activated microglia in CA2/3 and DG compared to acute exercise (e; p<0.05 TBI+deEX vs. TBI+acEX; n=4-6/group).

Figure 7

Effect of exercise on the expression of BDNF, CREB, IGF-1, and neurogenesis in post-traumatic brain. (A) BDNF, CREB and IGF-1 genes expression were compared across naïve, non-exercised TBI (1w, 5w, and 9w) and acute/delayed exercised TBI animals using quantitative real-time PCR. There were no differences in the expression of BDNF and CREB genes in non-exercised TBI mice compared to naïve mice. Delayed exercise significantly increased the expression of BDNF and CREB genes compared to either uninjured naïve (**p<0.01, versus naive) or injured non-exercised controls (**p<0.01, versus TBI+noEX 1w, 5w, 9w). Notably, there was significant TBI-induced IGF-1 up-regulation at 1 week (**p<0.01, TBI+noEX 1w vs. naïve) post-injury, which gradually returned to uninjured naïve levels at 9 weeks post-injury. Delayed exercise significantly increased IGF-1 (**p<0.01, TBI+deEX vs. TBI+noEX 9w or naïve) expression compared to uninjured controls or non-exercised TBI groups at 9 weeks post-injury. No significant changes in any of these genes were observed in acute exercise TBI group compared to uninjured or injured controls at 5 weeks post-injury. (B and C) Representative confocal images showed NeuN-positive (green) neurons and newly generated BrdU-positive (red) cells in ipsilateral DG with exercised or non-exercised TBI mice. Arrowheads indicated double positive NeuN+/BrdU+ cells, representing newly generated neurons that had reached maturity. (D) Semiquantitative analysis was performed for assessment of BrdU+/NeuN+- cells in the ipsilateral DG in each section at 3 months post-injury. The injured delayed exercise animals had significantly more NeuN+/BrdU+ neurons compared to non-exercised (9w) group (d; p<0.05, TBI+deEX vs. TBI+noEX (9w)). (E) Unbiased stereological assessment of surviving neurons in the DG. All injured mice showed significantly lower number of neurons compared to naïve (a-TBI+noEX, b-TBI+acEX, c-TBI+deEX; p<0.05 vs. naïve). Injured delayed exercise mice showed significantly improved DG neuronal survival after TBI compared to both injured non-exercised (d; p<0.05 TBI+deEX vs. TBI+noEX) and acute exercise (e; p<0.05 TBI+deEX vs. TBI+acEX) groups. Data represents mean ± SEM, statistical analysis using one-way ANOVA followed by post hoc adjustments using Tukey’s test, n=5-6/group. Bar = 75 μm.