Effects of Different Mode Exercise on Depression-like Behaviors and Hippocampal Neurogenesis in Rats with Post-stroke Depression

Hui NIE, Qiang REN

Department of Rehabilitation Medicine, Taihe Hospital, Shiyan 442000, China

Abstract [Objectives] To explore the effects of high-intensity intermittent training (HIIT) on the degree of depression, neuronal morphology, and the expression of brain-derived neurotrophic factor (BDNF) in the hippocampal dentate gyrus (DG) of ischemic hemisphere in rats with post-stroke depression (PSD), and to investigate the potential mechanisms. [Methods] 40 male Wistar rats were randomly divided into control group, sham-operation group (Sham), PSD model group, moderate intensity aerobic training group (MIAT) and HIIT group (n=8 for each group). Except the control and Sham group, all other groups were given the middle cerebral artery occlusion (MCAO) using the intraluminal thread method, and chronic unpredictable mild stress (CUMS) was applied. After successful modeling, training programs of different intensity were performed in the MIAT and the HIIT group. Rats in the five groups were subjected to behavioral tests (open field test and tail suspension test) before and 28 d after training. Nissl staining was used to observe the morphological changes of neurons in the left hippocampal DG area. The expressions of BDNF protein were detected by western blotting and immunohistochemistry. [Results] After 28 d of training, compared with the other groups, the PSD group’s pause time of the central lattice was prolonged; the immobility time increased in tail suspension test; the expression of BDNF decreased in the left hippocampal DG area. The difference was statistically significant (P<0.01). Neurons of hippocampal DG neurons were loosely arranged; cells were missing. Nissl body was lightly stained and even dissolved; the ethological findings in MIAT and HIIT rats were reversed; the expression of BDNF was elevated. Nissl staining showed that the cells were arranged neatly; the layers were dense, and the Nissl body was deeply stained. The changes in HIIT group were more significant than that in MIAT group (P<0.05). [Conclusions] Compared with the traditional medium-intensity aerobic training, high-intensity interval training can improve the depression-like behavior in rats with post-stroke depression. The potential mechanism might be that HIIT increase the neuroplasticity and expression of BDNF in hippocampal DG area.

Key words Stroke, Depression, High-intensity interval training, Aerobic training

1 Introduction

Stroke has become the second leading cause of death in the world. After occurrence of stroke, many neuropsychiatric symptoms usually appear. The most common symptom is post-stroke depression (PSD)[1]. PSD seriously affects the prognosis of stroke patients, and the suicide rate and mortality are much higher than those of stroke patients without depressive symptoms[2]. In recent years, with the in-depth study of the pathogenesis of depression, the relationship between depression and synaptic plasticity has attracted more and more attention. In rodent experiments, neurons and glial cells in hippocampus and prefrontal cortex of experimental animals exposed to stress showed atrophy and loss in varying degrees, and synaptic plasticity was damaged[3]. The research on the pathogenesis of depression based on neuroplasticity has also become a hotspot, which is closely related to neurogenesis, development and the stability of neuronal structure and function. Quantitative evidence shows that brain-derived neurotrophic factor (BDNF) is related to the regulation of synaptic plasticity. The decrease of its expression will lead to the atrophy and loss of hippocampal neurons, while short-term BDNF treatment can induce the increase of hippocampal dendritic spine density[4].

High-intensity interval training (HIIT) is a training method characterized by short-term intermittent high-intensity activities combined with relatively low-intensity exercise or rest[5]. Clinically, HIIT has been found have effects of improving the symptoms of depression and anxiety in patients with a variety of chronic diseases[6-7]. The purpose of this study was to explore the effects of high-intensity interval training on the depressive behavioral performance, the number and density of hippocampal dentate gyrus (DG) neurons and the expression of BDNF protein in hippocampal DG area in rats with post-stroke depression, so as to explore the potential mechanism of high-intensity interval training to improve the symptoms of post-stroke depression.

2 Materials and methods

2.1 Animals and groupsClean grade male Wistar rats, weighing (250±20) g and aged 2-3 months, were provided by Jinan Pengyue Laboratory Animal Center. Animals were singly housed at room temperature of (21±2) ℃ and humidity of 50%-60% under 12-h light-dark cycle with food and water available. All rats were adapted to the laboratory environment for one week before the formal experiment. Forty rats were randomly divided into five groups: control group (control,n=8), sham operation group (sham,n=8), PSD model group (n=8), medium intensity aerobic training group (MIAT,n=8) and HIIT group (n=8).

2.2 Preparation of PSD modelThe rats were briefly and deeply anesthetized with 2% pentobarbital sodium (0.3 mL/100 g, i.p.). The middle cerebral artery occlusion/reperfusion (MCAO) model was established by using the internationally recognized Longa external carotid artery thread occlusion method (embolization for 90 min). In sham group, only the common carotid artery, internal carotid artery and external carotid artery were isolated without wire insertion. MCAO model rats with a score of 1-3 were selected according to the Bederson motor function score standard 24 h after operation. They were isolated and given chronic unpredictable mild stimulation (CUMS) for 28 d to construct the PSD model. CUMS included: fasting for 24 h, water cutting for 24 h, tail clamping (1 cm away from the tip of the rat tail, 1 min), cage tilting at 45 degrees for 24 h, swimming in ice water for 5 min (4 ℃), and binding for 2 h (placing the rats in an empty bottle), day and night reversed for 24 h, wet environment (200 mL water per 100 g of padding). The same stimulation was not repeated within one week. The behavioral changes of rats before and after modeling (such as the prolongation of the central grid residence time in the open field test) were detected, and the difference was statistically significant (P<0.05). During the experiment, the rats with infection, death and unsuccessful modeling were excluded, and the model rats were supplemented to the original number. The rats in control and sham groups were only fed in a single cage without other stimuli.

2.3 Exercise training programAfter the PSD model was successfully prepared, the rats in HIIT and MIAT groups underwent treadmill training. Before formal training, the rats were trained for 3 d. The running speed was 5 m/min and the inclination angle were set at 10°. The rats were trained for 15 min every day.

According to the research results of Pin-Barre[8], the exercise plan is formulated: the running platform angle is 1°. The rats warmed up at the speed of 9 m/min for 5 min, and then increased by 3 m/min every 3 min until the rats were exhausted. At this time, the speed of the treadmill was recorded as the maximum exercise speed (Smax) of the rats. During running, the treadmill was paused for 10-20 s before each acceleration, and blood sampling (0.2 μL) was performed at the distal end of the rat tail vein. The blood lactate concentration (mmol/L) of rats was measured using a portable blood lactate meter (Lactate Scout+, EKF diagnostics®, Germany). When there was an obvious inflection point or 1 mmol/L difference in the blood lactate concentration measured for two consecutive times, the blood lactate concentration measured for the previous time was regarded as the lactate threshold (LT), and the corresponding treadmill speed was recorded as the lactate threshold speed (SLT) of rats.

HIIT: 4 min high-speed exercise (SLT+60%-70% (Smax-SLT) combined with 3 min low-speed exercise (SLT). Repeating 4 times without interruption for a total of 28 min. Training 5 d a week for 4 weeks.

MIAT: the speed was fixed at 80%-90% SLT. The exercise time of the rats in the MOD group was adjusted according to the energy consumption of the HIIT group by the following formula:

W (J/kg·m)=Mass (kg)×Speed (m/min)×Time (min)×Treadmill inclination (°)×9.8.

2.4 Behavioral testThe behavioral tests were carried out before and after PSD model preparation and 28 d after exercise training.

2.4.1OFT. During the test, put the rats to be tested into the open plastic box (28 cm×28 cm×28 cm). The free movement of rat was recorded for 10 min. The time of rat in the central area was analyzed by automatic video recording and analysis system. The longer the central grid stays, the more obvious the depression.

2.4.2TST. The rat tail 4 cm from the end was fixed on the horizontal wooden stick, and the rat was suspended for 6 min. The head was 10-15 cm away from the horizontal plane. The adjacent rat’s vision was separated by the wooden board, and the accumulative immobility time of the rat was recorded in the last 4 min by an independent observer blinded to the experiment.

2.5 Western blottingRats in each group were deeply anesthetized with 2% pentobarbital sodium (0.3 mL/100 g, i.p.), and transcardially perfused using a 0.9% saline solution. Then the brain was cut off. The brain tissue was taken at the hippocampal plane 4 mm after the optic chiasma and embedded in paraffin for sectioning. After the other rats were decapitated and killed quickly, the brain tissue was quickly removed, and the hippocampal tissue was separated on the ice surface of the super clean table and stored in the refrigerator at -80 ℃. After weighing, grinding, lysis, standing and centrifugation (4 ℃, 12 000 r/min, 10 min), take the supernatant and measure the protein concentration. Perform polyacrylamide gel electrophoresis for 1h and transfer to membrane for 1 h. After blocking with 5% skimmed milk powder for 1 h, the specific primary antibody BDNF (1∶2 000, Abcam) and β-actin (1∶3 000, Bioss) at 4 ℃ overnight. The membranes were washed three times for 10 min using TBS-T. Secondary antibody IgG (1∶5 000, Abcam) was incubated at room temperature for 2 h and washed three times for 10 min using TBS-T. The images were developed with the gel imaging system (UVP, USA), and the gray value was analyzed with Image J software.

2.6 Nissl stainingParaffin section with thickness of about 4 μm. Conventional dewaxing, tar violet staining and transparency were used to observe the staining of sections and the number of Nissl positive neurons in DG area of hippocampus under microscope.

2.7 ImmunohistochemistryParaffin section with thickness of about 4 μm. After dewaxing and dehydration, antigen repair and serum blocking, the specific primary antibody BDNF (1∶150, Abcam) was added and incubated at 37 ℃ for 2 h. The membranes were washed three times for 10 min using PBS. Secondary antibody (Goat anti rabbit IgG antibody, Abcam, 1∶300) was incubated at room temperature for 30 min. After immunohistochemical staining, Image J software was used for analysis.

2.8 Statistical analysisAll experimental data were analyzed by SPSS 23.0 statistical software, expressed as mean±standard deviation (SD). Multi group comparison was performed by one-way analysis of variance (ANOVA), and pairwise comparison between groups was performed by Tukey’s HSD test.P<0.05 was regarded as statistically significant.

3 Results and analysis

3.1 Changes of blood lactate concentration of rats in each groupAfter exercise training, the resting blood lactate concentration of rats in PSD group (3.5±0.6 mmol/L) was significantly higher than control group (2.1±0.3 mmol/L) and sham group (2.2±0.2 mmol/L), MIAT group (2.9±0.8 mmol/L) and HIIT group (2.5±0.7 mmol/L) (P<0.05).

3.2 Effects of different modes of exercise training on depression-like behaviors in each groupBefore exercise training, compared with the control and sham groups, the rats in the PSD, MIAT and HIIT groups had longer time in center in the open field test (P<0.01), and increased immobility time in the tail suspension test (P<0.01). After 28 d of exercise training, the behavioral performances of the rats in the HIIT and MOD groups were significantly reversed (P<0.01), and the changes in the HIIT group were more obvious than those in the MIAT group (P<0.01) (Fig.1).

Note: A. HIIT significantly increased the time in center during OPT; B. HIIT significantly increased immobility time during TST. All values are presented as means±SD. *P<0.05 and **P<0.01.Fig.1 Effects of different modes of exercise training on depression-like behaviors in each group

3.3 Expression of BDNF in the ischemic hippocampus of rats in each groupThe expression level of BDNF in the hippocampus of the ischemic hemisphere of the rats in the PSD group was significantly lower than that in the other groups (P<0.05). After 28 d of exercise training, the expression of BDNF in the hippocampus of the rats in the HIIT and MIAT groups was significantly increased (P<0.01). The expression level of BDNF was higher than that in the MIAT group, and the difference was statistically significant (P<0.01) (Fig.2).

Note: HIIT significantly increased the expression of BDNF in the ischemic hippocampus. All values are presented as means±SD. *P<0.05 and **P<0.01.Fig.2 Expression of BDNF in the ischemic hippocampus of rats in each group

3.4 Results of Nissl staining and BDNF expression in the DG area of the ischemic hippocampus in each groupCompared with other groups, the neurons in the hippocampal DG area of the PSD group were loosely arranged. Cells were missing. Nissl bodies were lightly stained or even dissolved. The layers are dense and the Nissl body is deeply stained in the HIIT group (Fig.3A). And compared with PSD and MIAT groups, HIIT significantly increased the number of granulosa cell in DG (P<0.01) (Fig.3C). Compared with other groups, the expression of BDNF in the ischemic hippocampal DG area of the rats in the PSD group was significantly decreased (P<0.05). After exercise training, the expression of BDNF in the hippocampal DG area of the HIIT and MIAT groups were significantly increased (P<0.01). The expression level of BDNF in the HIIT group was higher than that in the MIAT group, and the difference was statistically significant (P<0.01) (Fig.3B&D).

Note: A. Histological images of Nissl staining evaluated the neuronal survival in the hippocampal DG. B. Representative images of BDNF immunostaining showed the neurogenesis in hippocampal DG (Scale bar=200 μm). C. HIIT significantly increased the number of granulosa cell in DG. D. HIIT significantly increased the number of BDNF-positive cells in DG. All values are presented as means±SD. *P<0.05 and **P<0.01.Fig.3 Results of Nissl staining and BDNF expression in the DG region of the ischemic hippocampus in each group

4 Discussion

This study proved that exercise training can improve the depression like behavior of PSD model rats to a certain extent, and high-intensity interval training is more effective than the traditional moderate-intensity aerobic training. We measured the blood lactate threshold of PSD rats during exercise to quantify high-intensity exercise training (> 23 m/min) and low-intensity aerobic training (< 20 m/min). Unlike previous studies that quantify exercise intensity by measuring some physiological extreme values (VO2max, HRmax)[9-10], this study uses the sub physiological extreme value (LT) that can be achieved by most patients, which can predict better exercise stimulation intensity[10-11]. In this study, the energy consumption of rats in HIIT group and MIAT group was the same, but the total exercise duration was significantly lower than that in MIAT group. HIIT effectively improved the degree of depression compared with MIAT group, indicating that HIIT has higher time efficiency than MIAT[12].

Hippocampal dentate gyrus (DG) maintains the ability to generate new neurons throughout life, that is, neurogenesis. Dumanetal.[13]firstly proposed in 2000 that hippocampal neurogenesis is closely related to the pathogenesis of depression. They found that hippocampal volume decreased with atrophy and loss of nerve cells in the autopsy of brain tissue in patients with depression. Tadaetal.[14]and Santarellietal.[15]found that blocking hippocampal neurogenesis would eliminate the effect of antidepressants. Some studies have shown that sports training, such as running, can promote neurogenesis in DG area of hippocampus[13]. The Nissl staining results of this study showed that the arrangement of neurons in the DG area of the hippocampus of PSD rats was significantly sparse, there was the phenomenon of neuron loss, the cell gap widened. Nissl body in many cells became blurred or even disappeared, and the existing Nissl body was lightly colored. After 4 weeks of treadmill training, the Nissl staining of rats in HIIT and MIAT groups were significantly improved. The number and density of granule cells in DG area of hippocampus in HIIT group were higher than those in MIAT group, and the Nissl body staining was deeper. It suggests that HIIT may improve the depressive symptoms of PSD rats by promoting the neural plasticity in hippocampal DG.

BDNF regulates the growth, development, differentiation, repair and protection activities of neurons throughout life, and plays an important role in regulating the morphological structure and functional activities of neurons[4]. By intervening in the expression of BDNF in a single neuron, Pu Muming research group of Institute of Neuroscience clarified that BDNF secreted by adult hippocampal newborn neurons played a very important role in dendritic development. In addition, the phenomenon of increased dendritic growth of adult newborn neurons caused by autonomic movement such as running also depended on BDNF from autocrine source[16]. In addition, Freitasetal.[17]found that 6-week high-intensity interval training can significantly increase the expression of BDNF in the hippocampus of healthy adult rats. The results of this study showed that both HIIT and MIAT significantly up-regulated the expression of BDNF protein in the ischemic hippocampal DG region of PSD rats, and the effect of HIIT group was more obvious. It is suggested that HIIT may promote the neural plasticity of hippocampal DG region by inducing the expression of BDNF, so as to improve the depression-like behavior of PSD rats.

5 Conclusions

In conclusion, compared with the traditional moderate-intensity aerobic training, high-intensity interval training can significantly reduce the degree of depression in rats with post-stroke depression. Its mechanism may be related to the improvement of lactic acid accumulationinvivo, the increase of BDNF expression and neural plasticity in hippocampal DG area. The results can provide some experimental basis for related basic research and clinical rehabilitation treatment in the future.