Effects of Aerobic Exercise in Alleviating Depressive Symptoms of Cerebral Ischemic Rats with Sedentary Behavior by Improving Inflammatory Microenvironment in Hippocampus

Qiang REN, Hui NIE

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

Abstract [Objectives] To explore the role and molecular mechanisms of aerobic exercise in post-stroke depression. [Methods] Wistar rats were randomly divided into sham, sedentary and aerobic exercise (Ex) groups. The rats in sedentary and Ex groups received middle cerebral artery occlusion (MCAO) surgery. Aerobic exercise was performed 24 h after MCAO for four weeks in Ex group. The sucrose preference test (SPT), open field test (OFT), tail suspension test (TST) was performed, respectively. The levels of IL-1β, IL-6, TNF-α were detected by ELISA. [Results] Aerobic exercise successfully prevented depression-like symptom induced by ischemic stroke with sedentary behavior as indicated by significant increase in sucrose preference (P<0.01) during SPT and decrease in immobility time (P<0.01) during FST and TST. Aerobic exercise significantly reduced the high levels of IL-1β, IL-6, TNF-α induced by ischemic stroke and sedentary behavior in the ischemic hippocampus (P<0.05). [Conclusions] Aerobic exercise after ischemic stroke can reverse depression and improve the inflammatory microenvironment in the hippocampus.

Key words Stroke, Depression, Exercise, Inflammation

1 Introduction

Stroke remains the leading cause of death second only to cancer and cardiac diseases[1], with high rate of mood and cognitive impairments. Around 31% stroke patients have been suffering from depression, namely post-stroke depression (PSD)[2]. The people with depression engage in low level of physical activity and high level of sedentary behavior[3]. The inpatient stroke survivors spent 76% of the daytime in bed or sitting, and only 23% of the time in standing or walking[3]. Extensive evidences demonstrate that aerobic exercise contributes to establishing recovery and prevent relapse of depression symptoms[4-5]. However, there is still no adequate evidence of the curative effectiveness of exercise, and research into its related molecular mechanisms in animal experiments. Previous studies have indicated that exercise training may alleviate spinal cord injury induced nerve injury by suppressing the expression of inflammatory mediators IL-1β, IL-6 and TNF-α via TLR4/NF-κB signaling[6]. However, whether exercise training can improve the symptoms of post-stroke depression by improving the inflammatory environment in the hippocampus and thus affecting the neuroplasticity of the hippocampus is unknown.

2 Materials and methods

2.1 AnimalsHealthy male Wistar rats (weighing 240-260 g, 8-week old) were obtained from Pengyue Experimental Animal Center. They were singly housed with a room temperature of (22±2) ℃ and humidity of 50%-60% in standard plastic cages with food and water availableadlibitum. All experiments and procedures were performed according to theNationalInstitutesofHealthGuidefortheCareandUseofLaboratoryAnimals. The rats were randomized by random number method into three groups before surgery: sham group (n=15), sedentary group (n=15), aerobic exercise (Ex) group (n=15). The timeline diagram illustrating experimental protocols is showed in Fig.1A.

2.2 Middle cerebral artery occlusion (MCAO) surgeryThe MCAO was performed with reference to the previously reported method[7]. Briefly, the rats were deeply anesthetized with 2% pentobarbital sodium (0.3 mL/100 g, i.p.) and then to expose the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The ECA was ligated, dissected and inserted gently with a round-tip nylon thread (Beijing Cinontech Co., Ltd., China) to block the blood flow for 90 min at the middle cerebral artery (MCA). The cerebral blood flow (CBF) was monitored by laser speckle flowmetry (RWD life science, China) (Fig.1B&C).

2.3 Exercise protocolsAerobic exercise training was performed 24 h after MCAO surgery on the 8-lane motor-driven treadmill (Zhenghua Biological Instrument Equipment Co. Ltd, China, Fig.1D). Sessions were composed of 4 min×5 min treadmill running at a speed of 15 m/min (40%-60% VO2max) interrupted by 30 s rest. The program was performed every day for 4 weeks. The sedentary rats in sham and sedentary groups were housed individually in a ventilated cage (cage size: 26 cm×19 cm×15 cm) with no access to the treadmill.

Note: A. Timeline diagram for experimental protocols. All rats were acclimatized for 1 week prior to MCAO, and 4-weeks sedentary behavior or exercise training. Behavioral tests, i.e., SPT, FST, TST, were performed after MCAO and treatment. B. MCAO model was established under cerebral blood flow monitored by laser speckle flowmetry. C. The ischemic cerebral blood flow dropped below 30% of the contralateral side during surgery. D. Aerobic exercise training was performed on the 8-lane motor-driven treadmill.

2.4 Behavioral testsBehavioral tests were performed one day after MCAO/r, 2 weeks after MCAO and 4 weeks after MCAO. The test sequence was as follows: (i) sucrose preference test (SPT); (ii) forced swimming test (FST); (iii) tail suspension test (TST).

2.4.1SPT. On the first day, rats were free to drink two bottles of 1% sucrose solution. On the next day, one of the sucrose was replaced by distilled water. On the third day, rats were deprived of water and food for 23 h. The rats were then given one bottle of 1% sucrose solution and one bottle of distilled water weighed in advanced. After 1 h, the volumes of consumed sucrose solution and distilled water were recorded to calculate the sucrose preference (%) by the following formula: Sucrose consumption/(Sucrose consumption+Qater consumption)×100%. A decrease of sucrose preference indicated anhedonia and depression-like behavior[8].

2.4.2FST. Briefly, rat was placed into an open transparent cylinder (37 cm diameter×60 cm height) containing 40 cm of water at (23±2) ℃. Rat was allowed to swim for 6 min. The immobile behavior during the last 5 min was measured manually by an experienced observer blind to the experiment design. Immobility was defined as the minimum movement and less struggle necessary to keep the head above water. An increase in immobility time indicated depression-like behavior[9].

2.4.3TST. The tail-suspension immobility time of rats was measured after the measurement of autonomic activity with reference to the method described previously. The 4 cm from the end of the rat tail 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[10].

2.5 Enzyme-linked immunosorbent assay (ELISA)After the behavioral tests, animals were sacrificed under deep anesthesia, hippocampus was collected and stored at -80 ℃ for further analysis. Protein content was determined by BCA protein assay reagents (Kangwei, Beijing, China). The levels of IL-1β, IL-6, and TNF-α were detected using the enzyme-linked immunosorbent assay (ELISA) kits (Fan Ke Wei, Shanghai, China) in accordance with the instructions of the manufacturer. The protein standards and samples were added to 96-well ELISA plates, and then added biotinylated antibodies of IL-1β, IL-6, and TNF-α. We used the microplate reader (Thermo Fisher Scientific, USA) to test the results. We calculated the concentrations using the linear equation derived from the standard curve.

2.6 Statistical analysisGraphPad Prism 9.0 software (CA, USA) and Image J 14.0 software were used for statistical analysis. The results of the experiments are presented as the means plus standard deviations (SD). The data were analyzed statistically by one-way analysis of variance (ANOVA) for multiple comparisons followed by Tukey’sposthoctest and the difference was significant (P<0.05).

3 Results and analysis

3.1 Effects of aerobic exercise on depression-like behaviors in the sedentary rats after ischemic strokeSedentary behavior after ischemic stroke manifested depression-like behavior, as demonstrated by SPT, FST and TST tests. In SPT study, rats after ischemic stroke and sedentary behavior for 4 weeks resulted in decreased sucrose preference (P<0.01). In FST and TST study, sedentary rats after ischemic stroke showed increased immobility time (P<0.01) compared with the sham group. Exercise training successfully prevented depression-like behaviors, as indicated by significant increase in sucrose preference (P<0.01) during SPT and decrease in immobility time (P<0.01) during FST and TST, suggesting that aerobic exercise had protective effects against ischemic stroke and sedentary behavior induced depression-like symptom (Fig.2).

Note: A. exercise significantly increased sucrose preference during SPT; B. exercise significantly increased immobility time during FST; C. exercise significantly increased immobility time during TST; all values are presented as means±SD. *P<0.05 and **P<0.01.

3.2 Effects of aerobic exercise on inflammatory cytokines in sedentary rats after ischemic strokeIn the present study, we observed that the levels of IL-1β, IL-6 and TNF-α in the ischemic hippocampus significantly increased (P<0.01) after ischemic stroke and sedentary behavior for 4 weeks. While aerobic exercise training significantly reduced the levels of IL-1β, IL-6, and TNF-α (P<0.01) in the ischemic hippocampus (Fig.3).

Note: A. aerobic exercise significantly increased the level of IL-1β in the ischemic hippocampus; B. aerobic exercise significantly increased the level of IL-6 in the ischemic hippocampus; C. aerobic exercise significantly increased the level of TNF-α in the ischemic hippocampus; all values are presented as means±SD. *P<0.05 and **P<0.01.

4 Discussion

In the present study, we investigated both the depression-like behavioral and molecular mechanisms of exercise training in a cerebral ischemic rodent model with sedentary behavior. We demonstrated that chronic treatment with aerobic exercise training after ischemic stroke reversed depression-like behaviors and improved the inflammatory microenvironment in the hippocampus.

The stroke is a severe life-threatening disease with high fatality and disability rate[11]. Currently, depression, with a significant and lasting low mood as the main clinical symptom, is one of the most frequently appearing psychiatric disorders after a stroke. It is estimated that post-stroke depression has a high morbidity of 31% in stroke patients, which also might lead to reduced quality of life and increased suicidal deaths in stroke patients[2,12]. In recent years, the diagnostic and nursing techniques for depression have greatly improved; however, traditional antidepressants usually have limited satisfactory cure due to the weak curative effects, a series of side effects and the high recurrent rate. There are one-third patients with depression remaining treatment-resistant and less than one-third achieving remission after initial treatment with the first-line antidepressants, such as the selective serotonin reuptake inhibitors (SSRIs)[13]. Therefore, it is urgently necessary to develop new alternative strategies for treating depression. Depressed patients have low level of physical activity and long-lasting sedentary behavior[6]. In agreement with previous reports, our study also demonstrated that sedentary behavior after ischemic stroke manifested depression-like behavior. The rat after ischemic stroke and sedentary behavior for 4 weeks resulted in decreased sucrose preference, and increased immobility time in open field test and forced tail suspension. Aerobic exercise training had protective effects against ischemic stroke and sedentary behavior induced depression-like symptom, as indicated by significant increase in sucrose preference during SPT and decrease in immobility time during FST and TST. Moreover, a study examined the relationship between intensity of exercise and depression-like symptom, which found that moderate-intensity and high-intensity exercise contributing to improving depression, while very-low intensity exercise was not as beneficial as the other intensities[14]. Studies that included a measure of both increased physical activity frequency and intensity demonstrated stronger associations with depressive symptoms than those that used measures of intensity alone[15].

The positive effects of exercise on mental health may well be due to the ability of exercise to reduce inflammation. Previous studies have demonstrated that the anti-inflammatory effects of exercise may be attributed to 4 main mechanisms, (i) change in cytokine release[16], (ii) reduction of visceral fat mass[17], (iii) down regulation of toll-like receptors[18]and (iv) increase in vagal tone[19]. As inflammation plays a key role in the pathogenesis of depression and anxiety for a subset of individuals, it is clear there is a pertinent purpose for studying the link between inflammation and mental health. There are two key questions, the first is whether successful treatment of depression is associated with a reduction in inflammation, and the other is whether anti-inflammatory therapy can have an effective antidepressant effect, especially in depressed patients with an increased inflammatory response. At present, there is a lot of evidence to give a positive answer to the above two questions. For example, a recent cumulative meta-analysis reported that IL-6 and C-reactive protein (CRP) were most closely associated with depression among inflammatory markers[20], and were highly associated with pleasure deficiency and mental retardation symptoms. A recent meta-analysis of 82 studies including 3 212 major depression disorder (MDD) patients and 2 798 healthy controls showed that MDD patients had higher levels of IL-6, TNF, IL-10, sIL-2, CCL-2, IL-13, IL-18, IL-12, IL-1RA, sTNFR-2, and low level of IFN-γ[21]. Although there are currently no approved treatments for depression aimed at modulating the immune response, there is evidence that traditional antidepressants have anti-inflammatory effects and that this response may depend in part on the immune phenotype. In the present study, we also observed that the levels of IL-1β, IL-6 and TNF-α in the ischemic hippocampus were markedly increased after ischemic stroke and sedentary behavior for 4 weeks. While aerobic exercise significantly reduced the levels of IL-1β, IL-6, and TNF-α in the ischemic hippocampus.

5 Conclusions

In conclusion, we intended to provided evidence and mechanism for the effectiveness of aerobic exercise in the treatment of post-stroke depression, from the aspects of anti-inflammatory. Neuroinflammation plays an important role in the development of depression. Long-term exercise training reversed depression-like behaviors, possibly by improving the inflammatory microenvironment in the hippocampus. Future research will focus on exploring whether the dosage of exercise has different effects on inflammatory response, so as to have different or even opposite effects on improving post-stroke depression or other disease-related depressive symptoms.