A pilot study investigating the effects of voluntary exercise on capillary stalling and cerebral blood flow in the APP/PS1 mouse model of Alzheimer's disease

Abstract

Exercise exerts a beneficial effect on the major pathological and clinical symptoms associated with Alzheimer's disease in humans and mouse models of the disease. While numerous mechanisms for such benefits from exercise have been proposed, a clear understanding of the causal links remains elusive. Recent studies also suggest that cerebral blood flow in the brain of both Alzheimer's patients and mouse models of the disease is decreased and that the cognitive symptoms can be improved when blood flow is restored. We therefore hypothesized that the mitigating effect of exercise on the development and progression of Alzheimer's disease may be mediated through an increase in the otherwise reduced brain blood flow. To test this idea, we performed a pilot study to examine the impact of three months of voluntary wheel running in a small cohort of ~1-year-old APP/PS1 mice on short-term memory function, brain inflammation, amyloid deposition, and baseline cerebral blood flow. Our findings that exercise led to a trend toward improved spatial short-term memory, reduced brain inflammation, markedly increased neurogenesis in the dentate gyrus, and a reduction in hippocampal amyloid-beta deposits are consistent with other reports on the impact of exercise on the progression of Alzheimer's related symptoms in mouse models. Notably, we did not observe any impact of wheel running on overall baseline blood flow nor on the incidence of non-flowing capillaries, a mechanism we recently identified as one contributing factor to cerebral blood flow deficits in mouse models of Alzheimer's disease. Overall, our findings add to the emerging picture of differential effects of exercise on cognition and blood flow in Alzheimer's disease pathology by showing that capillary stalling is not decreased following exercise.

Fig 1. APP/PS1 mice reliably run when given unrestricted access to a running wheel in their home cage.

A. Timeline of the study. After three months of voluntary exercise on a wheel running (or standard housing for the sedentary control group), mice underwent behavioral testing. Next, a craniotomy was performed, after which mice were allowed three weeks to fully recover. The cortical vasculature was then imaged with in-vivo two photon excitation fluorescence microscopy. Finally, after four consecutive days of EdU-injections, mice were euthanized. Harvested brain tissue was then used for immunohistochemistry. B. Each dot is the distance covered in km per day over 45 days for the four APP/PS1 mice in the running group. Data show mean + SD. C. Daily distance in km per day for the four APP/PS1 mice in the running group across time.

Fig 2. Running improved performance in the object replacement test, but did not show strong effects in other short-term memory tests.

All behavioral testing results are shown for running (RUN) and sedentary (SED) APP/PS1 mice. A Preference score for OR (P = 0.01). B Correlation between OR preference score and total running distance (R² = 0.6; P = 0.03). C Spontaneous alternation score in Y-maze. D Correlation between spontaneous alternation and total running distance. E Preference score in NOR test. F Correlation between NOR preference score and total running distance. G Time spent in the arena center in the open field test. One value was excluded in the running group due to a software failure during the test. H Correlation between time spent in the arena center and total running distance. Animal numbers: RUN: n = 4 (OF: 3); SED: n = 4.

Fig 3. Running increased neural stem cell proliferation in the dentate gyrus, but did not decrease inflammation.

A Boxplot of the density of EdU positive cells in the dentate gyrus from running (RUN) and sedentary (SED) APP/PS1 mice (P = 0.03, Mann-Whitney test). B Representative confocal images of tissue sections from the hippocampus of sedentary (top) and running (bottom) APP/PS1 mice, with labeling of astrocytes (anti-GFAP, green), microglia (anti-Iba1, red), amyloid plaques (Methoxy-X04, blue; no amyloid plaques visible in these fields), cell nuclei (Hoechst, blue), and proliferating cells (EdU, yellow, indicated with arrows). C Representative confocal images of tissue sections from the cortex from running (top) and sedentary (bottom) mice. Labeling is the same as in panel B. Images to the right show individual channels. D Microglial density, represented as the fractional area that is Iba-1 positive; E Astrocyte density, represented as the fractional area that was GFAP positive; from the hippocampus and cortex of RUN and SED APP/PS1 mice. Animal numbers: RUN: n = 4; SED: n = 4. Scale bars indicates 50 μm.

Fig 4. Running decreased hippocampal amyloid burden.

A Representative confocal images of tissue sections from the hippocampus of sedentary (left) and running (right) APP/PS1 mice, with labeling of astrocytes (anti-GFAP, green), microglia (anti-Iba1, red), amyloid plaques (Methoxy-X04, blue), and cell nuclei (Hoechst, blue). The white arrows in the second column of images indicate Methoxy-X04 labeled amyloid plaques, which are distinguished from Hoechst-labeled cell nuclei by morphological differences. B Amyloid plaque density, represented as the fractional area that was Methoxy-X04 positive; from the hippocampus and cortex of RUN and SED APP/PS1 mice. Animal numbers: RUN: n = 4; SED: n = 4. Scale bars indicates 50 μm.

Fig 5. Running did not alter the levels of soluble and insoluble amyloid beta monomers in the brain.

Soluble (A and C) and insoluble (B and D) concentrations of Aβ1–42 (A and B) and Aβ1–40 (C and D) in brain lysates from running (RUN) and sedentary (SED) APP/PS1 mice, as measured using ELISA assays.

Fig 6. Running did not decrease the incidence of non-flowing capillaries, not increase capillary blood flow in the brain of APP/PS1 mice.

A Representative 2PEF image sequence showing both flowing and stalled capillary segments over 7 s. The blood plasma was labeled with Texas Red-dextran and the dark patches in the vessel lumen were formed by blood cells. The lack of motion of these dark patches in the lower capillary indicates stalled blood flow. (Stalled capillary is indicated with arrows). B Density of capillaries with stalled blood flow in running (RUN) and sedentary (SED) APP/PS1 mice. C Representative 2PEF image of a cortical capillary (left) and a space-time image from repetitive line scans along the centerline of the capillary segment (right). The diagonal streaks in the space-time image are formed by moving red blood cells, with a slope that is inversely proportional to the blood flow speed. D Capillary blood flow speed plotted as a function of vessel diameter for cortical capillaries from running (RUN) and sedentary (SED) APP/PS1 mice (points represent individual capillary measurements; 15 vessels were measured per mouse; nested two-way ANOVA).

Fig 7. Running did not alter capillary density or geometry.

A and B Representative 2PEF image stacks of fluorescently-labeled cortical vasculature (middle) and the segmentation of this image (right) from running (RUN) and sedentary (SED) APP/PS1 mice, respectively. C Density; D Diameter; E Length and; F Tortuosity of capillary segments from running (RUN; n = 5514 capillaries) and sedentary (SED; n = 7251 capillaries) APP/PS1 mice.