Treadmill exercise rescues anxiety/depressive-like behaviors without the change of the HPA axis in mild stress rat model.
Kohei Koikea, Akiko Maruyamaa, Yuki Uenishia, Kazuto Ishidaa
a Department of Physical Therapy, Nagoya University Graduate School of Medicine, 1-1-20 Daikominami, Higashi-ku, Nagoya, Aichi 461-8673, Japan
Keywords
Depression; Treadmill exercise; Mild stress model; HPA axis
Abstract
Chronic stress is involved in the onset and development of depression. While exercise has stress-buffering effects, the therapeutic effect of exercise is not fully understood. The objectives of this study were, at first, confirm that chronic forced swim stress induces anxiety/depressive-like behaviors after stress exposure, and then investigate whether treadmill exercise alleviates the anxiety/depression-like behaviors induced by forced swim stress through the alteration of hypothalamic-pituary-adrenal (HPA) axis response. It has been found that forced swim stress exposure induced abnormal behaviors including forced swim 21 days after stress exposure using forced swim test, open field test and elevated open-platform test. These abnormalities were inhibited by repeated administration of the antidepressant imipramine, suggesting that these behaviors are anxiety/depressive behaviors. Moreover, treadmill exercise ameliorated depressive behaviors induced by chronic forced swim stress. Interestingly, there was no significant difference of the HPA axis response including serum corticosteron level among groups. These data suggest that chronic forced swim stress paradigm is mild stress model and exercise does not affect on the HPA axis. This is the first study that showed that treadmill exercise rescues anxiety/depression-like behaviors by not changing the HPA axis in mild stress model.
Abbreviations
Keywords
Depression, Treadmill exercise, Forced swim stress, Hypothalamic-pituary-adrenal axis
1. Introduction
Stress-related disorders such as anxiety, depression and post-traumatic stress disorder are serious problems in modern society. The majority of evidence has pointed to the hypothalamic-pituary-adrenal (HPA) axis abnormalities, which were reversed by antidepressant drugs in depressed patients [1][2]. However, antidepressant drugs use continues to be controversial due to their frequently reported negative side effects [3]. Consequently, there is obvious interest in developing better therapeutic treatments.
Many human studies suggest that physical exercise may play an antidepressant role in depression [4][5][6]. However, it is difficult to explore the mechanism of effect of exercise using human subjects. Therefore, further study using animal models to reveal the mechanism of treatment effect of exercise on psychiatry disorders still needs to be undertaken.
Some research using several exercise methods such as wheel running, swimming and treadmill exercise showed the therapeutic effect of exercise in stressed animal [4][5][6]. To date, whether exercise rescues the HPA abnormality as well as antidepressant drug is unclear. It was indicated that exercise has both positive (anti depressant) and negative (stressful) effects on the anxiety/depression-like behaviors and the HPA axis depending on the conditions of exercise including time and intensity [7][8][9][10][11][12]. It could not determined from these studies using while running and swimming exercise whether exercise truly revered the depression. Treadmill exercise is the appropriate method to design the rehabilitative exercise program for the depressed patient and to study the mechanism of the therapeutic effect of exercise. So far, very few attempts using treadmill exercise have been made in stressed animals [9][13]. Further study using treadmill exercise in different stress models needs to be tackled to understand the mechanism of the therapeutic effect of exercise.
Mizoguchi et al showed that chronic repeated forced swim stress induced anxiety/depression-like behaviors at the end of stress and these behaviors remained at least 14 days after stress [14]. This stress model might be useful in understanding the therapeutic effects of exercise.
The objectives of this study were, at first, confirm that forced swim stress induces anxiety/depressive-like behaviors after stress exposure, and then investigate whether treadmill exercise alleviates the anxiety/depressive-like behaviors induced by forced swim stress through the alteration of the HPA axis response. We, therefore, hypothesized that exercise rescues the stress-induced anxiety/depressive-like behaviors through the HPA axis response.
2. Materials and methods
2.1. Animals
Adult male Sprague-Dawley rats (250-300 g, 8 weeks of age, n=33) were used. The animals were housed under controlled temperature (25 °C) and a 12 h light/dark cycle with access to food and water ad libitum. All the experiments were conducted according to the animal care guidelines of the Nagoya University of Graduated School of Medicine (?).
2.2. Experimental design
2.2.1 Experiment 1.
The rats were assigned into saline and imipramine groups. The forced swim stress was carried out as described previously with minor modifications [14]. All rats were subjected to daily forced swim test for 14 days. Rats were placed in a glass cylinder (35 cm high × 24 cm diameter) filled to a depth of 25 cm with water (25 ± 1 °C). A 6-min test was repeated each day. Rats were given vehicle (saline, i.p.) or imipramine (10 mg/kg, i.p.) after each forced swim test for 14 days. After the withdrawal of repeated forced swim stress, imipramine was no longer applied. At 3 times (before the stress (pre), at the end of stress (Day14) and 21 days after the withdrawal of stress), the rats were exposed to the behavior tests.
2.2.2 Experiment 2.
Stressed rats were assigned to non-exercise (stress) and exercise groups (stress + exercise). The animals in the exercise groups were forced to run on a motorized treadmill for 30 min a day for 3 weeks starting on the next day after stress exposure. Daily running exercise was performed on treadmill (50 cm (L) × 7.7 cm (W)). The exercise group ran at a speed of 5 m/min for the first 5 min, 8 m/min for the next 5 min, and 11 m/min for the remaining 20 min as previously described [15]. All rats underwent pre-training for 10 min a day for 3 days before experiment. Animals in the non exercise groups were left on the treadmill for the same period of time without running. At 0, 7, 14 and 21 days after the withdrawal of repeated forced swim stress, the rats were exposed to the behavior tests. After the last behavior test, corticosteron measurement and tissue were collected from rats. For comparison, we also prepared control samples that exposed to only behavior tests in order to examine the effects of stress on the HPA axis.
2.3. Behavioral tests
Behavioral paradigms were constructed so that each test had no impact on the other tests. All procedures were performed in a silent and dim room. The apparatus was cleaned with 70% alcohol before each animal was tested.
2.3.1 Forced swim test
The forced swim test (FST) was carried out as described previously with minor modifications [14]. Rats were placed in a glass cylinder (35 cm high × 24 cm diameter) filled to a depth of 25 cm with water (25 ± 1 °C). Immobility time (floating) was measured. A rat was judged to be immobile if it ceased struggling and remained floating motionless in water making only those movements necessary to keep its head above water.
2.3.2 Open field test (OFT)
To test the anxiety behaviors, the inactivity due to stress was measured using OFT as previously described [15] [16]. The apparatus consisted of an open wooden box (80 cm × 80 cm × 40 cm). The area was divided into 25 squares painted on the floor. Each rat was gently placed in the center of the arena and was allowed to explore freely for 5 min. Its behavior was recorded by a video camera mounted above the open field. The number of line crossings and standing was quantified as a measure of anxiety behavior.
2.3.3 Elevated open-platform test (EOPT)
EOPT was performed in accordance with previous report, with minor modifications [17]. A transparent acrylic cylinder (56.5 cm high × 14 cm diameter) was placed upside-down and rats were placed individually on the top (open-platform) for 6 min. When a rat slipped off the platform, it was immediately replaced on the open-platform and the measurement was continued. Head dips (exploratory movement of head/shoulders beyond the edge of the open-platform), a risk assessment behavior, were defined as behavioral activity in response to mild stress, and measured. The total number of times that an animal showed this behavior was recorded.
2.4.1. Serum corticosteron level
Following the completion of all behavior tests all animals were killed with a lethal dose of pentobarbital (?; ? mg/kg, i.p.). Using a heparinized syringe, blood samples were taken by transcardial puncture and then centrifuged at 3 000 g. for 15 min at 4°C. The resulting plasma was stored at −80°C until assayed. Serum corticosteron (CORT) levels were detected using the Rat Corticosteron Enzyme Immunoassay Kit (Yanaihara Inc., Shizuoka, Japan). The lower limit of detection was 0.21 ng/ml.
2.4.2. Tissue collection and Histology
Immediately following blood sampling, adrenal glands were removed. The rats were transcardially perfused with 0.9% saline followed by 0.1 M phosphate buffer (pH 7.5) containing 4% paraformaldehyde. The brains were removed and postfixed in the same fixative. Then, 40 μm thick coronal sections were taken every 440 μm from +2.0 to +2.2 using a cryostat after overnight cytoprotection in 30% sucrose solution. Eight sections were stained with H–E, and the volume of Hippocampus was calculated using the Image J version 1.42 (National institutes of Health, USA). The volume was analyzed in each section and averaged.
2.4.3. Western blotting
The brains were immediately removed after the last behavior test, and the Hippocampus was isolated. The tissues samples were homogenized in lysis buffer (50 mM Tri-HCl, 150 mM Nacl, 2 mM EDTA, 0.2% NP40) containing phosphatase and protease (Complete Phosphatase Inhibitor Cocktail Tablet, Diagnostics, Germany). The samples were centrifuged at 15,000 rpm at 4 °C for 30 min. The protein concentration in the tissue samples was determined using the Bradford method using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Next, 20 μg of protein was separated by SDS–PAGE (12% polyacrylamide gel) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% skim milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, followed by an overnight incubation at 4 °C with brain-derived neurotophic factor (BDNF)(1:100, Santa Cruz Biotechnology), TrkB (1:200, Santa Cruz Biotechnology) and β-actin (1:2000, MBL) primary antibodies diluted in TBS-T and 2.5% skim milk. The membranes were then washed and incubated with HRP secondary antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. After rinsing with buffer, the immune-complexes were visualized using standard chemical luminescence methods (Invitrogen). The film signals were digitally scanned and quantified using densitometric image software (CS Analyzer, ATTO Corporation, Tokyo, Japan) normalized to the β-actin level.
2.5. Statistical analysis
All statistical analyses were conducted using R (version 3.0.2). All data were expressed as the mean ± standard error of means (S.E.M.). Friedman test with Bonferroni’s test was performed between pre and each day of FST in experiment 1. The Man-Whitney U-test was used to assess the difference between group of OFT and EPOT in experiment 1. The difference between groups 21 days after stress was compared using the student’s t test in experiment 2. One-way ANOVA was used for analysis of the HPA axis response in experiment 2. The criteria for significance was set at p < 0.05.
3. Results
3.1. Effects of chronic forced swim stress (experiment 1)
Rats were exposed to the forced swim stress repeatedly. In FST, they exhibited longer immobility time compared with pre (day7, day14, p < 0.01 by Friedman test with Bonferroni), and high immobility remained even after 21 days (p < 0.01 by Friedman test with Bonferroni)(Fig. 1A). Furthermore, imipramine treatment significantly inhibited the increasing of immobility time due to forced swim stress at day7 (p < 0.01 by U-test).
We also examined anxiety-like behaviors, especially the activity inhibition induced by forced swim stress using the OFT and EOPT. All data was show in rates (the number of each day/the number of pre). Crossing activity in open field test was markedly decreased in vehicle-treated group, at day14 and after 21 days after stress, and this low activity remained even after 21 days (p < 0.01 by Friedman test with Bonferroni). Imipramine-treated groups showed the significant high activity compared with vehicle-treated groups 21 days after stress (p < 0.01 by U-test) (Fig. 1B). In addition, forced swim stress significantly decreased the standing activity of the OFT at day14 when compared with pre, and this low activity remained even after 21 days (p < 0.01 by Friedman test with Bonferroni). Imipramine-treated groups showed the significant high score compared with vehicle-treated groups at day14 and 21 days after stress (p < 0.05 by U-test) (Fig. 1C). The head dips activity in the EOPT was decreased in vehicle-treated group 21 days after stress. Imipramine-treated groups showed the significant high score when compared with vehicle-treated groups 21 days after stress (p < 0.01 by U-test) (Fig. 1D). These data suggest that abnormal behaviors in response to forced swim stress are equivalent to anxiety/depression-like behaviors, and these behaviors remain at least 21 days after stress exposure.
3.2. Effects of treadmill exercise on anxiety/depression-like behaviors (experiment 2)
Next, we examined the effect of treadmill exercise on the anxiety/depression-like behaviors in stress-exposed rats. Rats were subjected to forced swim stress for 14 days as well as experiment 1, and then exercise group was given treadmill exercise for 21 days. After 21 days after stress, exercise group showed the low immobility time in the FST (Fig. 2A, p < 0.05 by t test). This result indicates that exercise rescues stress-induced depressive-like behaviors.
We also examined the effect of exercise on the anxiety-like behaviors using the OFT and EOPT. The number of crossing and standing in exercise group was significantly increased compared to non exercise group in the OFT (Fig. 2B, p < 0.05 by t test; Fig. 2C, p < 0.01 by t test). Exercise significantly increased head dips activity when compared to non exercise group in the EOPT (Fig. 2D, p < 0.05 by t test). This result indicates that exercise also inhibits anxiety-like behaviors due to forced swim stress.
3.3. Effects of treadmill exercise on the HPA axis (experiment 2)
Finally, we examined the mechanism of exercise treatment against anxiety/depressive-like behaviors. We focused on the HPA axis response, and compared the plasma corticosteron level (Fig. 3A), the volume of the hippocampus (Fig. 3B), BDNF (Fig. 3C) and Trkß mRNA level in the hippocampus (Fig. 3D) among control group, non exercise (only stress) group and exercise (stress + exercise) group. There were no differences observed in all data between non exercise group and exercise group. These data suggest that the mechanism of exercise treatment is not involved in the HPA axis. Moreover, there was no significant difference between control group and non exercise group (only stress) in all analysis, suggesting that the alteration of HPA axis does not happen 21 days after stress.
4. Discussion
In the present study, we found that forced swim stress induced anxiety/depression-like behaviors at least 21 days after stress exposure. Treadmill exercise ameliorated anxiety/depression-like behaviors induced by chronic forced swim stress. Interestingly, there was no significant difference of the HPA axis response among control, non exercise and exercise groups. These data suggest that chronic forced swim stress paradigm is mild stress model and exercise does not affect on the HPA axis. This is the first study that showed that treadmill exercise rescues anxiety/depression-like behaviors by not changing the HPA axis in mild stress model.
It is well known that chronic stress plays an important role in development of depression [18]. Many animal studies showed abnormal behaviors, but only some stress models have shown that chronic stress can induce long-term behavior disturbances [19][20]. The forced swim test is used to assess the effects of antidepressants. The previous study confirmed that repeated forced swim stress induced increasing the immobility time in FST and inactivity in OPFT [14], but the effects of chronic forced stress on behaviors have not been established and it was not understood whether this stress model is useful to examine the effect of exercise on anxiety/depression state after stress. Firstly, we confirmed that forced swim stress induces anxiety/depressive-like behaviors after stress exposure. In this study, chronic forced swim stress increased the immobility time in FST, decreased crossing and standing activity in OFT and heap dip activity in EOPT. These behaviors remained for at least 21 days after stress exposure. Also, these abnormal behaviors induced by chronic forced swim stress were inhibited by antidepressant treatment, which suggest that these abnormal behaviors are anxiety/depression-like behaviors. These results using behavior tests indicate that chronic forced swim stress induces long term disturbances, and is good stress model to examine the therapeutic effect of exercise.
Interestingly, we observed no significant difference between control and stress groups immediately (data was not shown) and 21 days after stress. It is known that only 50% of depression patients show cortisol abnormality [21], and corticosteron level represents the stressful condition [22]. Thus this forced swim chronic stress model is mild/moderate stress model, and could be useful to examine the mechanism of depression without the HPA axis abnormality.
Many clinical studies have suggested a therapeutic role of exercise [4][5][6]. We also showed that treadmill exercise for 21 days decreased the immobility time in FST, and increased crossing and standing activity in OFT and heap dip activity in EOPT. To our knowledge, this is the first study to show that treadmill exercise has antidepressant effect on mild stress model. The effectiveness of exercise may be independent on the severity of disease.
How does exercise ameliorate the anxiety/depression-like behaviors? The principle physiological responses to stress stimuli are mediated by the HPA axis. It is unclear whether the therapeutic effect of exercise is thorough the change of the HPA axis, while exercise has both positive and negative effects on the HPA axis [9][10][11][12]. We showed that there was no significant difference of the HPA axis response including serum corticosteron, the Hippocampus volume, BDNF and Trkß level in the Hippocampus between non exercise group and exercise groups. In addition, the previous study also showed that treadmill exercise ameliorates chronic stress induced-anxiety/depression-like behaviors, but slightly decrease high corticosteron level [9]. Considering this result together with our studies above, treatment effect of exercise may be not through the alteration of the HPA axis. These results also suggest that the HPA axis changes may be not as responsive as behavioral measures. Further research is needed to explore the mechanism of chronic forced swim stress.
In summary, forced swim stress induces anxiety/depression-like behaviors without the change of the HPA axis. Moreover, treadmill exercise ameliorates anxiety/depression-like behaviors induced by chronic forced swim stress not affecting on the HPA axis respons. These data suggest that treadmill exercise may rescue anxiety/depression-like behaviors without the HPA axis in mild stress rat model.