Voluntary running enhances glymphatic influx in awake behaving, young mice

Abstract

Vascular pathology and protein accumulation contribute to cognitive decline, whereas exercise can slow vascular degeneration and improve cognitive function. Recent investigations suggest that glymphatic clearance measured in aged mice while anesthetized is enhanced following exercise. We predicted that exercise would also stimulate glymphatic activity in awake, young mice with higher baseline glymphatic function. Therefore, we assessed glymphatic function in young female C57BL/6J mice following five weeks voluntary wheel running and in sedentary mice. The active mice ran a mean distance of 6km daily. We injected fluorescent tracers in cisterna magna of awake behaving mice and in ketamine/xylazine anesthetized mice, and later assessed tracer distribution in coronal brain sections. Voluntary exercise consistently increased CSF influx during wakefulness, primarily in the hypothalamus and ventral parts of the cortex, but also in the middle cerebral artery territory. While glymphatic activity was higher under ketamine/xylazine anesthesia, we saw a decrease in glymphatic function during running in awake mice after five weeks of wheel running. In summary, daily running increases CSF flux in widespread areas of the mouse brain, which may contribute to the pro-cognitive effects of exercise.

Keywords: Astrocyte; Cardiovascular changes; Convection; Exercise; Glymphatic system; Hypothalamus.

Conflict of interest statement

Competing interests statement

The authors have no competing interests to declare.

Copyright © 2017 Elsevier B.V. All rights reserved.

Figures

Figure 1. Exercise increases CSF tracer influx in awake, behaving mice

A. Female C57BL/6J mice are pair housed for five weeks before analysis of their glymphatic system and subsequent immunohistochemistry. The activity of the mice is measured as average daily running distance per cage. Mean distance per day is 6.7 km (n=8). The respiration and heart rate measured under isoflurane anesthesia (n=7–8) (Student’s t-test, P=0.7 and P=0.0061, respectively). B. Total CSF tracer concentration measured as percentage coverage of 7 coronal sections for a small (FITC-dextran, 3 kDa) and large (ovalbumin-AF647, 45 kDa) tracer (n=6–10). (Two-way ANOVA with Tukey’s correction, * = P<0.05). C. A schematic representation of the regional analysis. D. Regional analysis of the CSF tracers (3 and 45 kDa). The fluorescence intensities are measured as the percentage area coverage (Two-way ANOVA with Sidak correction, ** = P<0.01, *** = P<0.001). Regions without tracer fluorescence were omitted from the figure. E. Overlay of fluorescence influx in coronal sections −0.3 mm from bregma for the four groups (n=6–10). F. Retention of CSF tracers (3 and 45 kDa) assessed after 3 hours of circulation in awake mice (n=4) (Student’s t-test, P=0.21 and P=0.22).

Figure 2. Active running reduces CSF tracer influx

A. Schematic representing the dorsal macroscopic view of the whole brain after CM injection. B. Macroscopic image of the FITC (3 kDa) tracer distribution in the exercise awake and exercise running groups. C. Overlay of fluorescence influx in coronal sections (bregma −0.3 mm) for exercise awake and exercise running mice (n=3–4). D. The tracer intensity (FITC, 3 kDa and AF647, 45 kDa) as percentage coverage averaged over 7 coronal sections for the exercise running group (n=4) compared to awake exercised mice (n=3). (Student’s ttest, * = P<0.05). E. Relative difference in regional distribution in hypothalamus (HT) and ventral cortex (VC) for the running exercise group compared to the awake exercise group (normalized values, n=3–4).

Figure 3. Astrocytic expression of GFAP and AQP4 is unaltered

A. Confocal images of GFAP expression shown as maximum intensity projection of 10 µm z-stack, 40×. B. Glial fibrillary acidic protein (GFAP) expression in layer 1 of the lateral cortex (Lat Ctx), corpus callosum (CC), and CA3 of the hippocampus (HC) quantified as percentage area coverage (n=6) (Multiple t-testing with FDR, P=0.3–0.9). C. Representative confocal images of aquaporin-4 AQP4 expression (40×). D. AQP4 expression quantified as percentage area coverage in layer 1 of the lateral cortex (n=6) (Student’s t-test, P=0.8). E. AQP4 polarization calculated as low to high stringency as previously described [31]. Data are normalized. No significant difference (n=6) (Student’s t-test, P = 0.6). F. Our proposed model of glymphatic activity following five weeks of voluntary wheel running. When CSF tracers are injected into the cisterna magna of exercised awake animals, the glymphatic influx is increased compared to sedentary control mice.