Volume 28, Issue 1 (3-2021)
RJMS 2021, 28(1): 12-22 |
Back to browse issues page
Research code: NO.19.33.2018
Ethics code: NO.19.33.2018
Clinical trials code: NO.19.33.2018
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Abdizadeh L, peeri M, Azarbayjani M A, Farzanegi P. The effect of aerobic exercise, mesenchymal stem cells (MSCs) and ozone therapy on anxiety and depression like behaviors caused by induction of osteoarthritis in rats. RJMS 2021; 28 (1) :12-22
URL: http://rjms.iums.ac.ir/article-1-6328-en.html
URL: http://rjms.iums.ac.ir/article-1-6328-en.html
Islamic Azad University, Tehran, Iran , m.peeri@iautcb.ac.ir
Abstract: (2743 Views)
Background & Aims: Osteoarthritis pain is a type of chronic pain. One of the reasons that make this pain one of the most problematic diseases of the present age is its coexistence with physical diseases and mental disorders. Studies show that anxiety and depression are commonly associated with chronic pain. Needless to say, due to its chronic, painful and debilitating nature, this disease has reduced the quality of life (1,2,3) and increase the prevalence of depression in these patients. Studies show that osteoarthritis pain, through its effect on fatigue and disability, causes a depressed mood. There is an extensive research literature today that shows that although behavioral and psychological factors may not play a significant role in the onset of pain, but these factors in the transformation of acute pain into chronic and its continuation and also in explaining the severity of pain and related problems, including Depression, disability and anxiety play a crucial role (4-7). Because depression is often undiagnosed in patients with chronic pain, this problem is often not treated, and on the other hand, depression is associated with reduced effectiveness of treatments methods (7,10). Studies have shown that osteoarthritis is a chronic inflammatory process with cellular and biochemical changes that justifies drug treatment (12). Accordingly, some studies support the efficacy and safety of ozone injection in the treatment of osteoarthritis (13,14), but there is no clear agreement on this, and ozone at high concentrations is likely to have different effects on osteoarthritis. Today, stem cells are used to treat many diseases, including osteoarthritis, and a recent study looked at their effect on depressive behaviors (15). In addition, exercise is a good way to achieve the desired mental state and improve states of depression, anxiety and self-confidence. Therefore, due to the few and sometimes contradictory studies in this regard and also due to the growing need for new therapies, we examined a new therapeutic approach for depressive disorders associated with osteoarthritis based on MSCs transplantation, ozone therapy and aerobic exercise in osteoarthritis rats.
Methods: In this experimental study, 40 two-month-old male Wistar rats were purchased from Pasteur Institute of Iran-Tehran, weighing about 230-250 g and transferred to the animal room. To prevent stress and change physiological conditions, they were kept under new conditions of temperature(22±2°C), humidity(%50±5) and light-dark cycle of 12:12 h for one week. Osteoarthritis was surgically treated (1). For this purpose, the animals were first anesthetized using a mixture of ketamine (50-50 mg/kg) and xylazine (3-5 mg/kg). Then, under sterile conditions, a vertical incision was made in the inner part of the knee of the right foot with a surgical blade and other surgical instruments on the skin that had been previously shaved. After removing the skin, the medial collateral ligament of the knee was removed to observe the internal meniscus. The osteoarthritis model was then induced by making an incision that incompletely resulted in rupture and injury to the meniscus, and the area was sutured again with a sterile method. All steps were performed in the least amount of pain to the animal according to the principles of working with laboratory animals. A three-week recovery period was considered after induction of the medial meniscus tear model. After induction of osteoarthritis, subjects were divided into eight groups of five, including 1) osteoarthritis patient control, 2) MSCs, 3) exercise, 4) ozone, 5) exercise+ozone, 6) Exercise+MSCs, 7) Ozone+MSCs, 8) Exercise+Ozone+MSCs were replaced. Also, to investigate the effects of osteoarthritis induction on the research variables, 5 rats were placed in a healthy control group. In order to get acquainted with the treadmill, were trained on the rodent treadmill for a week (3 times a week; for 10 minutes, without slope and at a speed of 5-8 m/min; 60%-70% VO2max). The main training program for 8 weeks in aerobic exercise groups, for three sessions per week and 30 minutes of running on a treadmill without slope at a speed of 16 m/min was initiated in the first week, which gradually reached 50 minutes in the eighth week (19). Also, 5 minutes at a speed of 8 m/min at the beginning and end of exercise were performed for Warm-up and cool-down the animals. In this study, bone marrow mesenchymal stem cells were obtained from the National Center for Genetic Resources and then transferred to an incubator under sterile conditions. After 2 days, the supernatant was removed and after washing with PBS,DMEM/F12 warm and fresh medium was gently added to it. The cells were qualitatively examined for morphology and growth process. The cell environment was changed every 2 to 3 days for about 2 weeks until the cells in each group reached the appropriate density. Then the first passage was performed, so that the cells was removed from the bottom of the cultivation dishes using Trypsin/EDTA 0.2% made by the German company Gibco and cultured in a ratio of 3:1. About 10-15 days after each passage, the next passage was done. After filling the bottom of the culture dish and reaching a density of 90%, these cells were frozen in the desired passage to be used for injection purposes in the next stage of research. Then, after complete anesthesia of the animal in the cell therapy groups and during the recovery period, 1×106 cells/kg were injected as a single injection at the model induction site (20). It also injects oxygen into the ozone generator and produced ozone there using electricity. Then a 10 ml syringe was connected to the generator output and 10 ml of the produced gas was used to obtain ozone with a concentration of 20μg/ml. In the ozone treatment groups, 20 μg/ml was injected into the knee tibia-femoral articular line in 3 weeks after surgery, in 3 repetitions, at different times and for 3 consecutive weeks before the start of the training program (21). In the ozone+MSCs group, after intra-articular injection of ozone and in the fourth week, MSCs were injected as a single injection at a dose of 1×106 cells/kg. At the end of the treatment period, open field test was performed to evaluate the level of anxiety and depression in osteoarthritis rats (22). This test was performed for one day at light time. To perform the test, we held the rats from the base of the tail and placed them in one of the four corners of the device, allowing the animal to freely explore the environment of the device for 5 minutes. In this test, the parameters of total travel distance, time spend in periphery and time spend in center were evaluated to assess the level of anxiety and depression in rats. To analyze the results, Shapiro-Wilk statistical tests, independent t-test and 3-way ANOVA were used (p≥0.05).
Results: Induction of osteoarthritis led to anxiety and like depression behaviors (p≥0.05). However, exercise+MSCs significantly reduced circulation in the environment and significantly increased circulation in the center (p≥0.05); exercise, ozone, and MSCs each alone, exercise+ozone, and exercise+MSCs significantly increased the overall distance traveled (p≥0.05). Therefore, exercise, ozone, MScs, exercise+ozone interaction and exercise+MSCs had more favorable effects than the combination of MSCs+ozone and the interaction of all 3 interventions in increasing motor index and reducing depression.
Conclusion: Due to the discrepancy in the results of the reported studies, it is not possible to say with certainty, how chronic pain such as osteoarthritis is associated with the occurrence of behaviors related to depression and anxiety and the mechanism of effect of therapeutic interventions in the present study on osteoarthritis. However, exercise may reduce inflammatory factors, increase the activity of the serotonergic system, and reduce oxidative stress, which has anti-depressant effects, by increasing the expression and production of brain-derived neuronal growth factor (28). Also, injection of MSCs and ozone due to its anti-inflammatory properties and increase the oxygen content of the tissue improves the metabolism of cartilage tissue in the knee, which can reduce joint pain and stiffness by improving inflammatory factors, leading to improved function and quality of life in patients (32). On the other hand, ozone causes a feeling of well-being and happiness after ozone therapy by releasing endorphins and possibly increasing serotonin[33]. Overall, the results of this study showed that exercise, MSCs and MSCs+ozone have a protective effect in inducing the osteoarthritis model and modulate their anti-anxiety effects by increasing the circulation time in the center. Also, exercise, ozone, MSCs alone, and exercise+ozone and exercise+MSCs have more favorable effects in reducing depression due to osteoarthritis.
Methods: In this experimental study, 40 two-month-old male Wistar rats were purchased from Pasteur Institute of Iran-Tehran, weighing about 230-250 g and transferred to the animal room. To prevent stress and change physiological conditions, they were kept under new conditions of temperature(22±2°C), humidity(%50±5) and light-dark cycle of 12:12 h for one week. Osteoarthritis was surgically treated (1). For this purpose, the animals were first anesthetized using a mixture of ketamine (50-50 mg/kg) and xylazine (3-5 mg/kg). Then, under sterile conditions, a vertical incision was made in the inner part of the knee of the right foot with a surgical blade and other surgical instruments on the skin that had been previously shaved. After removing the skin, the medial collateral ligament of the knee was removed to observe the internal meniscus. The osteoarthritis model was then induced by making an incision that incompletely resulted in rupture and injury to the meniscus, and the area was sutured again with a sterile method. All steps were performed in the least amount of pain to the animal according to the principles of working with laboratory animals. A three-week recovery period was considered after induction of the medial meniscus tear model. After induction of osteoarthritis, subjects were divided into eight groups of five, including 1) osteoarthritis patient control, 2) MSCs, 3) exercise, 4) ozone, 5) exercise+ozone, 6) Exercise+MSCs, 7) Ozone+MSCs, 8) Exercise+Ozone+MSCs were replaced. Also, to investigate the effects of osteoarthritis induction on the research variables, 5 rats were placed in a healthy control group. In order to get acquainted with the treadmill, were trained on the rodent treadmill for a week (3 times a week; for 10 minutes, without slope and at a speed of 5-8 m/min; 60%-70% VO2max). The main training program for 8 weeks in aerobic exercise groups, for three sessions per week and 30 minutes of running on a treadmill without slope at a speed of 16 m/min was initiated in the first week, which gradually reached 50 minutes in the eighth week (19). Also, 5 minutes at a speed of 8 m/min at the beginning and end of exercise were performed for Warm-up and cool-down the animals. In this study, bone marrow mesenchymal stem cells were obtained from the National Center for Genetic Resources and then transferred to an incubator under sterile conditions. After 2 days, the supernatant was removed and after washing with PBS,DMEM/F12 warm and fresh medium was gently added to it. The cells were qualitatively examined for morphology and growth process. The cell environment was changed every 2 to 3 days for about 2 weeks until the cells in each group reached the appropriate density. Then the first passage was performed, so that the cells was removed from the bottom of the cultivation dishes using Trypsin/EDTA 0.2% made by the German company Gibco and cultured in a ratio of 3:1. About 10-15 days after each passage, the next passage was done. After filling the bottom of the culture dish and reaching a density of 90%, these cells were frozen in the desired passage to be used for injection purposes in the next stage of research. Then, after complete anesthesia of the animal in the cell therapy groups and during the recovery period, 1×106 cells/kg were injected as a single injection at the model induction site (20). It also injects oxygen into the ozone generator and produced ozone there using electricity. Then a 10 ml syringe was connected to the generator output and 10 ml of the produced gas was used to obtain ozone with a concentration of 20μg/ml. In the ozone treatment groups, 20 μg/ml was injected into the knee tibia-femoral articular line in 3 weeks after surgery, in 3 repetitions, at different times and for 3 consecutive weeks before the start of the training program (21). In the ozone+MSCs group, after intra-articular injection of ozone and in the fourth week, MSCs were injected as a single injection at a dose of 1×106 cells/kg. At the end of the treatment period, open field test was performed to evaluate the level of anxiety and depression in osteoarthritis rats (22). This test was performed for one day at light time. To perform the test, we held the rats from the base of the tail and placed them in one of the four corners of the device, allowing the animal to freely explore the environment of the device for 5 minutes. In this test, the parameters of total travel distance, time spend in periphery and time spend in center were evaluated to assess the level of anxiety and depression in rats. To analyze the results, Shapiro-Wilk statistical tests, independent t-test and 3-way ANOVA were used (p≥0.05).
Results: Induction of osteoarthritis led to anxiety and like depression behaviors (p≥0.05). However, exercise+MSCs significantly reduced circulation in the environment and significantly increased circulation in the center (p≥0.05); exercise, ozone, and MSCs each alone, exercise+ozone, and exercise+MSCs significantly increased the overall distance traveled (p≥0.05). Therefore, exercise, ozone, MScs, exercise+ozone interaction and exercise+MSCs had more favorable effects than the combination of MSCs+ozone and the interaction of all 3 interventions in increasing motor index and reducing depression.
Conclusion: Due to the discrepancy in the results of the reported studies, it is not possible to say with certainty, how chronic pain such as osteoarthritis is associated with the occurrence of behaviors related to depression and anxiety and the mechanism of effect of therapeutic interventions in the present study on osteoarthritis. However, exercise may reduce inflammatory factors, increase the activity of the serotonergic system, and reduce oxidative stress, which has anti-depressant effects, by increasing the expression and production of brain-derived neuronal growth factor (28). Also, injection of MSCs and ozone due to its anti-inflammatory properties and increase the oxygen content of the tissue improves the metabolism of cartilage tissue in the knee, which can reduce joint pain and stiffness by improving inflammatory factors, leading to improved function and quality of life in patients (32). On the other hand, ozone causes a feeling of well-being and happiness after ozone therapy by releasing endorphins and possibly increasing serotonin[33]. Overall, the results of this study showed that exercise, MSCs and MSCs+ozone have a protective effect in inducing the osteoarthritis model and modulate their anti-anxiety effects by increasing the circulation time in the center. Also, exercise, ozone, MSCs alone, and exercise+ozone and exercise+MSCs have more favorable effects in reducing depression due to osteoarthritis.
Type of Study: Research |
Subject:
Exercise Physiology
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
