Treadmill Exercise Exerts Neuroprotection and Regulates Microglial Polarization and Oxidative Stress in a Streptozotocin-Induced Rat Model of Sporadic Alzheimer's Disease

Abstract

Recent work has suggested that exercise may be beneficial in preventing or ameliorating symptoms of several neurological disorders, although the mechanism is not entirely understood. The current study was designed to examine the potential beneficial effect of treadmill exercise upon cognitive function in a streptozotocin (STZ)-induced rat model of Alzheimer's disease (AD). Animals underwent treadmill exercise (30 min/day, 5 days/week) for 4 weeks after bilateral STZ intracerebroventricular injection (2.4 mg/kg). We demonstrated that treadmill exercise significantly attenuated STZ-induced neurodegeneration in the rat hippocampal CA1 region and strongly preserved hippocampal-dependent cognitive functioning. Further mechanistic investigation displayed a marked suppression of STZ-induced amyloid-β accumulation and tau phosphorylation. Intriguingly, treadmill exercise remarkably inhibited reactive gliosis following STZ insult and effectively shifted activated microglia from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype, which was correlated with a significantly reduced expression of pro-inflammatory mediators and a corresponding enhancement of anti-inflammatory cytokine expression in the hippocampus. Furthermore, treadmill exercise caused a robust suppression of oxidative damage as evidenced by significantly reduced peroxynitrite production, lipid peroxidation, and oxidized DNA damage. Finally, treadmill exercise strongly attenuated STZ-induced mitochondrial dysfunction manifested by a dramatically elevated intra-mitochondrial cytochrome c oxidase activity and ATP synthesis, and markedly inhibited neuronal apoptosis in the hippocampus. These findings demonstrate that treadmill exercise has a multifactorial effect to attenuate many of the pathological processes that play a key role in AD, and provide further support for the beneficial role of exercise as a potential therapeutic option in AD treatment.

Keywords: Alzheimer’s disease; cognition; exercise; inflammation; microglia; oxidative stress; streptozotocin.

Fig. 1

Effect of treadmill exercise on STZ-induced spatial learning and memory deficits in rats. A) Representative tracking plots of Barnes Maze test for the indicated rats on the third trial day: (a) Control, (b) STZ, (c) STZ + Exe. Escape latencies and the average velocity were recorded and statistical data are shown in (d and e). B) Tracking plots and occupancy in target quadrant (green color) were recorded on probe day 4. (a)–(c) indicate the typical tracking plots for control, STZ, and STZ + Exe, respectively. Relative quadrant occupancy and errors made during Barnes Maze testing were recorded and statistically analyzed (d and e). C) The step-through latency (a) and numbers of rats that stayed in the bright chamber over 300 s (b) are shown in the passive avoidance test. D) Five-min novel object recognition tests were subsequently performed to compare the recognition memory. Representative traces are displayed for rat exploration of the familiar object (red) and a novel object (Cyan) (a: control, b: STZ, c: STZ + Exe). Diagram analysis of exploration time for different objects (d) and discrimination index (e) are indicated. n = 8 per group. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.

Fig. 2

Effect of treadmill exercise on STZ-induced hippocampal neuronal degeneration. A) Representative confocal pictures of rat hippocampal pyramidal CA1 neurons from control, STZ, STZ + Exe groups stained with the neuronal marker, NeuN and the neurodegenerative marker, F-Jade C, indicating robust neuron loss and neuronal degeneration following STZ insult, which was significantly attenuated by treadmill exercise. Scale bar = 20 μm. B) Quantification analysis was performed by counting surviving neurons and F–Jade C-positive neurons. The results indicate that neuronal loss and acute neuronal degeneration were induced by STZ administration at early stage of AD progression, whereas treadmill exercise exerted significant neuroprotection by increasing neuronal survival and decreasing neurodegeneration. n = 8 per group. Values are means ± SE. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.

Fig. 3

Effects of treadmill exercise on STZ-induced amyloidogenic processing of AβPP, Aβ accumulation, and tau phosphorylation in rat hippocampal CA1 region. A) Western blot analysis for C-terminal fragment of AβPP after α-secretase cleavage (C-83) and β-secretase cleavage (C-99), and the quantification analysis of C-99 to C-83 fragments ratios. B,C) The levels of Triton soluble Aβ_1–42 and Guanidine HCL soluble Aβ_1–42 were analyzed, and the data were expressed as fold changes versus control group. D) Representative Aβ_1–42 staining for control, STZ, and STZ + Exe groups in hippocampal CA1 region. Scale bar = 20 μm. E, F) Phosphorylation of tau protein (PHF) was examined by immunofluorescent staining and western blot analysis, respectively. Scale bar = 20 μm. n = 5 per group. Values are means ± SE. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.

Fig. 4

Treadmill exercise shifts microglial polarization from M1 to M2 phenotype and suppresses pro-inflammatory cytokines production. A) Confocal analysis of hippocampal CA1 neurons immunostained for IBA1, a marker of microglia. As shown above, STZ enhanced microglia activation and this effect was markedly suppressed by treadmill exercise. B, C) Western blot analysis for M1 and M2 markers of activated microglial revealed that treadmill exercise shifted activated microglia to M2 phenotype, while M1 phenotype was significantly inhibited. D) Levels of representative pro-inflammatory (a) & (b) and anti-inflammatory cytokines (c) & (d) were detected. Consistent with microglial polarization, treadmill exercise reversed STZ-induced elevation of pro-inflammatory cytokines and decrease of anti-inflammatory cytokines. Scale bar = 20 μm. n = 5 per group. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.

Fig. 5

Treadmill exercise ameliorates STZ-induced oxidative stress damage. A) Western blot analysis of reactive nitrogen species (RNS) generation, marked by 3-NT. STZ-induced RNS accumulation was markedly attenuated by treadmill exercise during AD progression. B) Confocal analysis for oxidative stress-induced damage to basic cellular components: lipid, histone and DNA, marked by 4-HNE (a–c), P-H2A.X (d–f), and 8-OHDG (g–i) respectively. Note that treadmill exercise significantly attenuated STZ-induced cellular oxidative damages. C) Relative immunoreactivity was analyzed in a diagram form following the confocal analysis above, with data expressed as fold changes versus normal control. Scale bar = 20 μm. n = 5 per group. Values are means ± SE. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.

Fig. 6

Treadmill exercise attenuates STZ-induced mitochondrial dysfunction and the consequent activation of caspase-9/caspase-3 apoptotic pathway. A, B) ELISA analysis of mitochondrial cytochrome c oxidase activity followed by the total ATP production were performed to examine mitochondrial oxidative phosphorylation level. C, D) The activities of pro-apoptotic proteins caspase 3/9 were subsequently analyzed, revealing that the enhanced activations of these pro-apoptotic proteins were inhibited by treadmill exercise. E) Representative confocal images of TUNEL staining revealed aggravated neuron apoptosis following dramatic mitochondrial dysfunction. F) TUNEL-positive cells per 250 μm in each confocal image were counted and expressed in graphic depiction. Values are means ± SE. Scale bar = 20 μm. n = 5 per group. *p < 0.05 versus normal control; ^#p < 0.05 versus STZ group.