Aerobic Exercise Promotes Hippocampal Neurogenesis and Ameliorates Cognitive Dysfunction Induced by Unilateral Labyrinthectomy methods
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Behavioral Analysis
Behavioral analysis was conducted by investigators blinded to the group assignments. All tests were conducted during the light phase. Prior to testing, mice were acclimated by handling for a minimum of 5 min, twice daily, for three consecutive days. No significant differences in body weight or whisker number were observed between groups.
Materials and Methods
Experimental procedures involving mice were approved by the Animal Welfare Committee of Huazhong University of Science and Technology (No. 3971). All experiments were conducted in strict accordance with the committee guidelines. C57BL/6J mice (8 weeks old) were obtained from the Experimental Animals Center of Tongji Medical College, Huazhong University of Science and Technology. To minimize variability associated with cyclical hormonal fluctuations in females, only male mice were used in this study. Animals were group-housed (≤ 5 per cage) under controlled environmental conditions (temperature: 22°C-24°C; humidity: 55%-80%) with a 12-h light/dark cycle and provided ad libitum access to food and water.
Unilateral Labyrinthectomy
Male mice (22-25 g) were randomly allocated to the UL or sham surgery groups. In the UL group, mice were anesthetized with 1% sodium pentobarbital (35 mg/kg, i.p.). A postauricular incision was made to expose the middle ear structures. The tympanic membrane was then punctured using fine forceps, and the malleus, incus, and stapes were carefully removed. The stapedial artery was then cauterized using an electrocautery device, and the vestibular window was widened with a probe to allow deep puncture of the labyrinth, ensuring complete destruction. Following this, a small amount of anhydrous ethanol was injected to further damage the labyrinth, and a gelatin sponge was placed in the vestibular window. The incision was then sutured closed. In the sham surgery group, the tympanic membrane was perforated, and the ossicles were removed, but the vestibular structures and lab...
Aerobic Exercise
During the first week, mice in the aerobic exercise groups performed adaptive running training in individual treadmill lanes (FT-200, Taimeng, China) at a speed of 5 m/min for 45 min per session. Over the following 3 weeks, the running protocol was adjusted to 5 m/min for 10 min, 8 m/min for 30 min, and 5 m/min for 10 min per session to minimize stress-induced inhibition of hippocampal neurogenesis [ ]. Mice in the static groups were placed on the stationary treadmill for the same duration.
BrdU Injection
Mice were administered 5′-Bromo-2′-Deoxyuridine (BrdU; B5002, Sigma) in saline via intraperitoneal injection at a dose of 100 mg/kg, once daily for five consecutive days. To avoid labeling the acute burst of progenitor proliferation induced by surgery or stimulation, we administered BrdU during the late phase of the intervention to more accurately capture the cell populations most relevant to exercise-responsive neurogenesis. Animals were sacrificed 24 h after the final BrdU injection.
Immunofluorescence Staining
Mice were anesthetized using 1% pentobarbital sodium (35 mg/kg, i.p.) and perfused transcardially with 0.1 M PBS, followed by 4% paraformaldehyde in 0.1 M PBS. The brains were then removed and fixed in 4% paraformaldehyde at 4°C overnight. After dehydration in 30% sucrose, brains were frozen in OCT medium (TissueTek, Sakura, Japan) and sliced into 30 µm coronal sections using a cryostat (HM550, Thermo Scientific). After three rinses with PBS, brain sections were rinsed in blocking buffer (PBS with 0.3% Triton X-100 containing 10% goat serum and 3% BSA) for 60 min at room temperature. Sections were incubated overnight at 4°C in blocking buffer containing rat anti-BrdU antibody (ab6326, Abcam; 1:300 dilution), rabbit anti-Ki67 antibody (ab15580, Abcam; 1:500 dilution), and rabbit anti-DCX antibody (13925-1-...
Inhibition of DG Neurogenesis in Mice at 28 Days After UL
In alignment with the observed cognitive deficits, UL mice exhibited a significant reduction in neural stem cell proliferation within the DG, as demonstrated by quantitative analysis of BrdU incorporation (Figure ), as well as Ki67- and DCX-positive cells (Figure ). To fully characterize the maturation process of neural stem cells in the DG, we used a retroviral labelling assay in which newborn neurons were specifically labeled with pROV-EF1a-EGFP by microinjection into the DG immediately after UL surgery in mice, and the maturation of neural stem cells was assessed 28 days later. Quantitative morphological analysis using the Sholl method revealed a significant reduction in total dendritic branching complexity compared to controls (Figure ). These findings indicate that UL leads to reduced proliferation and maturation of NSCs in the DG,...
Aerobic Exercise Improves Cognitive Function in Mice After UL
Cognitive dysfunction in mice after UL was improved by aerobic exercise (A) Schematic diagram of the experimental design of an exercise intervention in UL mice. (B, C) In the open field test, there were no significant differences in total movement distance and average speed among the four groups of mice: Sham + Static, Sham + UL, Run + Static, and Run + UL (two-way ANOVA: F (1,36) = 0.04837, p = 0.8272 and F (1,36) = 0.0009785, p = 0.9752). (D, E) There were no significant intergroup differences in terms of total immobility time and the duration spent in the central zone of the open field for the Sham + Static, Sham + UL, Run + Static, and Run + UL groups (two-way ANOVA: F (1,36) = 3.000, p = 0.0918 and F (1,36) = 0.2262, p = 0.6372, n = 10/group in OFT). (F, G) Aerobic exercise amelio...
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Behavioral analysis was conducted by investigators blinded to the group assignments. All tests were conducted during the light phase. Prior to testing, mice were acclimated by handling for a minimum of 5 min, twice daily, for three consecutive days. No significant differences in body weight or whisker number were observed between groups.
Experimental procedures involving mice were approved by the Animal Welfare Committee of Huazhong University of Science and Technology (No. 3971). All experiments were conducted in strict accordance with the committee guidelines. C57BL/6J mice (8 weeks old) were obtained from the Experimental Animals Center of Tongji Medical College, Huazhong University of Science and Technology. To minimize variability associated with cyclical hormonal fluctuations in females, only male mice were used in this study. Animals were group-housed (≤ 5 per cage) under controlled environmental conditions (temperature: 22°C-24°C; humidity: 55%-80%) with a 12-h light/dark cycle and provided ad libitum access to food and water.
Male mice (22-25 g) were randomly allocated to the UL or sham surgery groups. In the UL group, mice were anesthetized with 1% sodium pentobarbital (35 mg/kg, i.p.). A postauricular incision was made to expose the middle ear structures. The tympanic membrane was then punctured using fine forceps, and the malleus, incus, and stapes were carefully removed. The stapedial artery was then cauterized using an electrocautery device, and the vestibular window was widened with a probe to allow deep puncture of the labyrinth, ensuring complete destruction. Following this, a small amount of anhydrous ethanol was injected to further damage the labyrinth, and a gelatin sponge was placed in the vestibular window. The incision was then sutured closed. In the sham surgery group, the tympanic membrane was perforated, and the ossicles were removed, but the vestibular structures and labyrinth remained intact.
During the first week, mice in the aerobic exercise groups performed adaptive running training in individual treadmill lanes (FT-200, Taimeng, China) at a speed of 5 m/min for 45 min per session. Over the following 3 weeks, the running protocol was adjusted to 5 m/min for 10 min, 8 m/min for 30 min, and 5 m/min for 10 min per session to minimize stress-induced inhibition of hippocampal neurogenesis [ ]. Mice in the static groups were placed on the stationary treadmill for the same duration.
Mice were administered 5′-Bromo-2′-Deoxyuridine (BrdU; B5002, Sigma) in saline via intraperitoneal injection at a dose of 100 mg/kg, once daily for five consecutive days. To avoid labeling the acute burst of progenitor proliferation induced by surgery or stimulation, we administered BrdU during the late phase of the intervention to more accurately capture the cell populations most relevant to exercise-responsive neurogenesis. Animals were sacrificed 24 h after the final BrdU injection.
Mice were anesthetized using 1% pentobarbital sodium (35 mg/kg, i.p.) and perfused transcardially with 0.1 M PBS, followed by 4% paraformaldehyde in 0.1 M PBS. The brains were then removed and fixed in 4% paraformaldehyde at 4°C overnight. After dehydration in 30% sucrose, brains were frozen in OCT medium (TissueTek, Sakura, Japan) and sliced into 30 µm coronal sections using a cryostat (HM550, Thermo Scientific). After three rinses with PBS, brain sections were rinsed in blocking buffer (PBS with 0.3% Triton X-100 containing 10% goat serum and 3% BSA) for 60 min at room temperature. Sections were incubated overnight at 4°C in blocking buffer containing rat anti-BrdU antibody (ab6326, Abcam; 1:300 dilution), rabbit anti-Ki67 antibody (ab15580, Abcam; 1:500 dilution), and rabbit anti-DCX antibody (13925-1-AP, Proteintech; 1:300 dilution). After three washes with PBST (PBS containing 0.05% Tween-20), sections were incubated with the appropriate fluorophore-conjugated secondary antibody (goat anti-rabbit IgG conjugated to Alexa Fluor 488, AS039, ABclonal; 1:300 dilution) in blocking b...
In alignment with the observed cognitive deficits, UL mice exhibited a significant reduction in neural stem cell proliferation within the DG, as demonstrated by quantitative analysis of BrdU incorporation (Figure ), as well as Ki67- and DCX-positive cells (Figure ). To fully characterize the maturation process of neural stem cells in the DG, we used a retroviral labelling assay in which newborn neurons were specifically labeled with pROV-EF1a-EGFP by microinjection into the DG immediately after UL surgery in mice, and the maturation of neural stem cells was assessed 28 days later. Quantitative morphological analysis using the Sholl method revealed a significant reduction in total dendritic branching complexity compared to controls (Figure ). These findings indicate that UL leads to reduced proliferation and maturation of NSCs in the DG, suggesting a potential causal relationship between impaired neurogenic processes and cognitive dysfunction.
Cognitive dysfunction in mice after UL was improved by aerobic exercise (A) Schematic diagram of the experimental design of an exercise intervention in UL mice. (B, C) In the open field test, there were no significant differences in total movement distance and average speed among the four groups of mice: Sham + Static, Sham + UL, Run + Static, and Run + UL (two-way ANOVA: F (1,36) = 0.04837, p = 0.8272 and F (1,36) = 0.0009785, p = 0.9752). (D, E) There were no significant intergroup differences in terms of total immobility time and the duration spent in the central zone of the open field for the Sham + Static, Sham + UL, Run + Static, and Run + UL groups (two-way ANOVA: F (1,36) = 3.000, p = 0.0918 and F (1,36) = 0.2262, p = 0.6372, n = 10/group in OFT). (F, G) Aerobic exercise ameliorated the UL-induced working memory impairment, manifested by the total entry to and time spent in the novel arm. Right arm (F) was set as the novel arm (two-way ANOVA: F (1,36) = 1.616, p = 0.2118, Tukey's multiple comparisons test: Static + Sham vs. Static + UL: p &#...
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