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. 2017 May 19;10:144.
doi: 10.3389/fnmol.2017.00144. eCollection 2017.

Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice

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Free PMC article

Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice

Xiao-Fei He et al. Front Mol Neurosci. .
Free PMC article

Abstract

Age is characterized by chronic inflammation, leading to synaptic dysfunction and dementia because the clearance of protein waste is reduced. The clearance of proteins depends partly on the permeation of the blood-brain barrier (BBB) or on the exchange of water and soluble contents between the cerebrospinal fluid (CSF) and the interstitial fluid (ISF). A wealth of evidence indicates that physical exercise improves memory and cognition in neurodegenerative diseases during aging, such as Alzheimer's disease (AD), but the influence of physical training on glymphatic clearance, BBB permeability and neuroinflammation remains unclear. In this study, glymphatic clearance and BBB permeability were evaluated in aged mice using in vivo two-photon imaging. The mice performed voluntary wheel running exercise and their water-maze cognition was assessed; the expression of the astrocytic water channel aquaporin 4 (AQP4), astrocyte and microglial activation, and the accumulation of amyloid beta (Aβ) were evaluated with immunofluorescence or an enzyme-linked immunosorbent assay (ELISA); synaptic function was investigated with Thy1-green fluorescent protein (GFP) transgenic mice and immunofluorescent staining. Voluntary wheel running significantly improved water-maze cognition in the aged mice, accelerated the efficiency of glymphatic clearance, but which did not affect BBB permeability. The numbers of activated astrocytes and microglia decreased, AQP4 expression increased, and the distribution of astrocytic AQP4 was rearranged. Aβ accumulation decreased, whereas dendrites, dendritic spines and postsynaptic density protein (PSD95) increased. Our study suggests that voluntary wheel running accelerated glymphatic clearance but not BBB permeation, improved astrocytic AQP4 expression and polarization, attenuated the accumulation of amyloid plaques and neuroinflammation, and ultimately protected mice against synaptic dysfunction and a decline in spatial cognition. These data suggest possible mechanisms for exercise-induced neuroprotection in the aging brain.

Keywords: aging; inflammation; interstitial fluid; paravascular space; spatial memory; wheel running.

Figures

Figure 1
Figure 1
Schematic diagram of the timeline of this study and the effects of voluntarily running on spatial memory in a water-maze task. (A) Timeline of the behavioral tests and biochemical parameters used in this study. (B) Time to reach the platform in the control and running groups during Morris water-maze training. (C) Representative swim paths of mice in the control and running groups during the probe trial. (D) Mean velocities of mice in the control and running groups during the probe trial. (E) Number of times the target area (former platform) was crossed in the control and running groups during the probe trial. (F,G) Time (F) and % time (G) spent in the target quadrant (formerly contained the platform) in the control and running groups during the probe trial. Datasets are expressed as means ± SD, n = 12. *P ≤ 0.05; ***P ≤ 0.001.
Figure 2
Figure 2
Analysis of two-photon microscopy data on glymphatic clearance, including the influx through paravascular space (PVS)–interstitial fluid (ISF) exchange and the efflux through ISF drainage. (A) Schema showing the infusion of the fluorescein isothiocyanate (FITC)-dextran tracer into the cisterna magna for in vivo two-photon imaging (250×, scale bar = 200 μm). (B) Three-dimensional (3D) images of the brain vasculature and the distribution of the cerebrospinal fluid (CSF) tracer at different time points in the control and running groups (250×, scale bar = 200 μm). (C) Quantitative analysis of the mean pixel intensity of the tracer in the 3D image stacks in (B), which shows that the influx and clearance of the CSF tracer were markedly accelerated in the running group compared with the control group (n = 6 per group). (D) Representative image of the CSF tracer along the perivascular spaces penetrating into the brain parenchyma, 100 μm below the cortical surface (250×, scale bar = 200 μm). (E) Quantitative analysis of the fluorescence intensity of the CSF tracer in the PVS shown in (D) (n = 6 per group). (F) Schema showing the dissipation of the FITC-dextran tracer in the brain parenchyma during in vivo two-photon imaging, which indicates the efflux of the glymphatic system (100×, scale bar = 200 μm). (G) Representative 3D images of dye alignment at different time points in the control and running groups (250×, scale bar = 200 μm). (H) Comparison of the average fluorescence intensity in the parenchyma of the control and running groups at different time points (n = 6 per group). (I) Representative image of the dye alignment along the PVS, 100 μm below the cortical surface (250×, scale bar = 200 μm). (J) Representative image of the ISF drainage into the deep cervical lymph nodes in the control and running groups at 1 h after FITC-dextran was injected into the brain parenchyma (50×, scale bar = 1 mm). (K) Comparison of the average fluorescence intensity in the deep cervical lymph nodes of the control and running groups (n = 6 per group). Datasets are expressed as means ± SD, n = 6. **P ≤ 0.01.
Figure 3
Figure 3
Effects of voluntarily running on aquaporin 4 (AQP4) expression and AQP4 polarity. (A) Immunofluorescent staining of AQP4 in the cortex and hippocampus in the control and running groups (250×, scale bar = 100 μm). (B) Histograms comparing the average fluorescence intensity of AQP4 in the cortex and hippocampus (n = 6 per group). (C) Representative images of AQP4 polarity in the control and running groups (250×, zoomed in three-fold, scale bar = 50 μm). (D) Schematic diagram of the calculation of AQP4 polarity. (E) Histograms comparing AQP4 polarity in the cortex and hippocampus (n = 6 per group). Datasets are expressed as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Figure 4
Figure 4
Effects of voluntarily running on the permeability of the blood–brain barrier (BBB). (A) 3D reconstructed images of the brain vessels at different time points after rhodamine B was injected into the mouse tail vein. (B) xyz stacks of the brain vessels at different time points, indicating dye permeation. (C) Line diagram of the pixel intensity in the extravascular compartment. Datasets are expressed as means ± SD, n = 6.
Figure 5
Figure 5
Effects of voluntarily running on the activation of astrocytes and microglia. (A) Immunofluorescent staining of glial fibrillary acidic protein (GFAP)-positive astrocytes in the control and running groups (200×, scale bar = 100 μm). (B) Histogram comparing the numbers of GFAP-positive astrocytes in the cortex and hippocampus (n = 6 per group). (C) Immunofluorescent staining of ionized calcium-binding adapter molecule 1 (IBA1)-positive microglia in the control and running groups (200×, scale bar = 100 μm). (D) Histogram comparing the numbers of IBA1-positive microglia in the cortex and hippocampus (n = 6 per group). Datasets are expressed as means ± SD, n = 6. *P ≤ 0.05; ***P ≤ 0.001.
Figure 6
Figure 6
Effect of voluntarily running on amyloid β accumulation. (A) Immunofluorescent staining of Aβ1–16 in the control and running groups (200×, scale bar = 100 μm). (B) Histograms comparing the average Aβ1–16 fluorescence intensity in the cortex and hippocampus in the control and running groups (n = 6 per group). (C) Immunofluorescent staining of Aβ1–40 and Aβ1–42 in the control and running groups (250×, zoomed in three-fold, scale bar = 50 μm). (D) Histograms comparing the average Aβ1–40 and Aβ1–42 fluorescence intensity in the cortex and hippocampus in the control and running groups (n = 6 per group). (E) Comparison of the Aβ1–40 and Aβ1–42 levels detected with enzyme-linked immunosorbent assays (ELISAs). Datasets are expressed as means ± SD, n = 6. **P ≤ 0.01; ***P ≤ 0.001.
Figure 7
Figure 7
Effects of voluntarily running on synaptic function detected in Thy1–green fluorescent protein (GFP) transgenic mice and on immunofluorescent staining for postsynaptic density protein (PSD95). (A) Immunofluorescent staining of IBA1-positive cells in the cortices and hippocampi of Thy1–GFP mice in the control and running groups (250×, scale bar = 200 μm). (B) Histogram comparing the GFP intensity in the cortices and hippocampi of the control and running groups. (C) Representative images of dendrites and dendritic spines in the cortices and hippocampus of the control and running groups (630×, zoomed in three-fold, scale bar = 20 μm). To show the spines more clearly, the images were coded with different colors. (D) Histogram comparing the number of spines in the cortices and hippocampi of the control and running groups (n = 6 per group). (E) Immunofluorescent staining of PSD95 in the cortices and hippocampi of the control and running groups (630×, scale bar = 50 μm). (F) Histogram comparing the PSD95-positive particles in the cortices and hippocampi of the control and running groups (n = 6 per group). Datasets are expressed as means ± SD, n = 6. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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