Voluntary running prevents progressive memory decline and increases adult hippocampal neurogenesis and growth factor expression after whole-brain irradiation

Abstract

Whole-brain irradiation (WBI) therapy produces progressive learning and memory deficits in patients with primary or secondary brain tumors. Exercise enhances memory and adult hippocampal neurogenesis in the intact brain, so we hypothesized that exercise may be an effective treatment to alleviate consequences of WBI. Previous studies using animal models to address this issue have yielded mixed results and have not examined potential molecular mechanisms. We investigated the short- and long-term effects of WBI on spatial learning and memory retention and determined whether voluntary running after WBI aids recovery of brain and cognitive function. Forty adult female C57Bl/6 mice given a single dose of 5 Gy or sham WBI were trained 2.5 weeks and up to 4 months after WBI in a Barnes maze. Half of the mice received daily voluntary wheel access starting 1 month after sham or WBI. Daily running following WBI prevented the marked decline in spatial memory retention observed months after irradiation. Bromodeoxyuridine (BrdUrd) immunolabeling and enzyme-linked immunosorbent assay indicated that this behavioral rescue was accompanied by a partial restoration of newborn BrdUrd+/NeuN+ neurons in the dentate gyrus and increased hippocampal expression of brain-derived vascular endothelial growth factor and insulin-like growth factor-1, and occurred despite irradiation-induced elevations in hippocampal proinflammatory cytokines. WBI in adult mice produced a progressive memory decline consistent with what has been reported in cancer patients receiving WBI therapy. Our findings show that running can abrogate this memory decline and aid recovery of adult hippocampal plasticity, thus highlighting exercise as a potential therapeutic intervention.

Figure 1

Experimental design and timeline of procedures. Adult female mice were given sham-(Sham) or whole-brain irradiation (WBI; IRR) and allowed to recover. Mice were then tested in the Barnes maze (BM 1). Approximately 1 month after sham- or WBI, mice either remained socially housed (Sham or IRR) or were given daily access to an individual running wheel for 8–12 h per day during the dark phase of their light-dark cycle (Sham-Run or IRR-Run) throughout the duration of the experiment. At 90 days after sham- or WBI, mice were retested on the Barnes maze (BM 2). Mice were then given 5 daily injections of BrdU, and tested once more on the Barnes maze (BM 3) prior to being sacrificed (21 days after the last BrdU injection) at ~5 mo after sham- or WBI.

Figure 2

Slower spatial learning was evident shortly after WBI in IRR mice during BM1 testing, but spatial memory remained intact (Sham, n = 20; IRR, n = 20). All data represent group means. Error bars indicate SEM. A, spatial learning performance was analyzed using mean escape latencies for each day of training (across 3 trials/day) for each mouse. * significantly different from Sham mice at p < 0.05. B, probe trial performance at 1 hr and 4 d after BM1 training (during which the escape box was removed) was analyzed by dividing the maze into four quadrants and recording the percentage of time spent in each quadrant. A target quadrant bias was evident if the percent time spent in the target quadrant was significantly greater than all other quadrants. The dashed line represents chance performance. T, target; AR, adjacent right; OP, opposite; AL, adjacent left. * significantly different from all other quadrants at p < 0.05. C, both Sham and IRR mice showed a significant increase in mean escape latency from the last training trial where spatial cues were visible (cues present) to the trial where spatial cues were occluded by a curtain (cues removed). * significantly different from “cues present” at p < 0.05.

Figure 3

Daily running prevented decline in spatial memory observed months after WBI during BM2 testing (Sham, n = 10; Sham-Run, n = 10; IRR, n = 10; IRR-Run, n = 10). All data represent group means. Error bars indicate SEM. A, Sham-Run and IRR-Run mice increased total distance traveled (km) per week from weeks 1 to 2, which subsequently plateaued. Over weeks 2 to 6, IRR-Run mice ran significantly less (242.34 ± 19.66 total km) than Sham-Run mice (300.47 ± 18.35 total km), F_{1, 18} = 4.72, p < 0.05. B, body weight at the time of perfusion did not significantly differ between groups (F < 1). C, spatial learning performance was analyzed using mean escape latencies for each day of training (across 3 trials/day) for each mouse. D, probe trial performance for each group at 1 hr, 4 d, and 18 d after BM2 training. A target quadrant bias was evident if the percent time spent in the target quadrant was significantly greater than all other quadrants. The dashed line represents chance performance. T, target; AR, adjacent right; OP, opposite; AL, adjacent left. * significantly different from all other quadrants at p < 0.05.

Figure 4

Daily running partially rescued adult hippocampal neurogenesis months after WBI (Sham, n = 10; Sham-Run, n = 10; IRR, n = 10; IRR-Run, n = 9). All data represent group means. Error bars indicate SEM. A, number of BrdU+ cells in the dentate gyrus at 3 weeks after 5 daily injections of BrdU prior to BM3 training. * p < 0.05; ** main effect of WBI at p < 0.05. B, dentate gyrus volume estimated using the optical fractionator and according to Cavalleri’s principle. C, newly divided cells (peroxidase-stained with anti-BrdU) in the dentate gyrus of Sham, Sham-Run, IRR, and IRR-Run mice. Scale bars indicate 50 μm. Photomicrographs taken at a 10x. D, confocal images of BrdU+ cells in the dentate gyrus that co-expressed NeuN (yellow arrow), GFAP (yellow arrowhead), or neither (white arrow head). Scale bars indicate 25 μm. Confocal images taken at 40x. GCL, granule cell layer. SGZ, subgranular zone.

Figure 5

Elevated cytokine expression in the adult hippocampus at ~5 months after WBI (Sham, n = 10; Sham-Run, n = 10; IRR, n = 10; IRR-Run, n = 9). All data are expressed as pg of cytokine protein per μl of total protein, and represent group means. Error bars indicate SEM. A, hippocampal TNF-α expression at ~5 mo after sham- or WBI. * significantly different from Sham and Sham-Run groups at p < 0.001. B, hippocampal INF-γ expression at ~5 mo after sham- or WBI. IFN-γ protein levels in Sham and Sham-Run mice were < 1 pg/μl. * significantly different from Sham and Sham-Run groups at p < 0.001. C, hippocampal IL-6 expression at ~5 mo after sham- or WBI. * significantly different from Sham and Sham-Run groups at p < 0.001.

Figure 6

WBI and running alter hippocampal neurotrophic/growth factor expression at ~5 mo after treatment (Sham, n = 10; Sham-Run, n = 10; IRR, n = 10; IRR-Run, n = 9). All data expressed as percent of control (Sham) levels and represent group means. Error bars indicate SEM. A, hippocampal BDNF expression at ~5 mo after sham- or WBI. ** main effect of WBI at p < 0.05. B, hippocampal VEGF expression at ~5 mo after sham- or WBI. * p < 0.05; ** main effect of WBI at p < 0.05. C, hippocampal IGF-1 expression at ~5 mo after sham- or WBI. * p < 0.05; ** main effect of WBI at p < 0.05; # main effect of running at p < 0.05.