Multiple sclerosis is an autoimmune disease that affects the central nerve system, resulting in cumulative loss of motor function. Multiple sclerosis is induced through multiple mechanisms and is caused by inflammation and demyelination. This study aims to evaluate the neuroprotective effect of swimming exercise in experimental autoimmune encephalomyelitis (EAE) rats, an animal model of multiple sclerosis.
EAE was induced by an intradermal injection of 50-μg purified myelin oligodendrocyte glycoprotein 33–55 (MOG33-55) dissolved in 200-μL saline at the base of the tail. The rats in the swimming exercise group were made to swim for 30 minutes once pert a day for 26 consecutive days, starting 5 days after induction of EAE. To compare the effect of swimming exercise with interferon-β, a drug for multiple sclerosis, interferon-β was injected intraperitoneally into rats of the EAE-induced and interferon-β-treated group during the exercise period.
Injection of MOG33-55 caused weight loss, decreased clinical disability score, and increased level of pro-inflammatory cytokines and inflammatory mediators in the lumbar spinal cord. Loss of motor function and weakness increased demyelination score. Swimming exercise suppressed demyelination and expression of pro-inflammatory cytokines and inflammatory mediators. These changes promoted recovery of EAE symptoms such as body weight loss, motor dysfunction, and weakness. Swimming exercise caused the same level of improvement as interferon-β treatment.
The results of this experiment suggest the possibility of swimming exercise in urological diseases that are difficult to treat. Swimming exercises can be considered for relief of symptom in incurable multiple sclerosis.
- We investigated neuroprotective effect of swimming exercise in EAE rats, an animal model of multiple sclerosis.
- Injection of MOG33-55 caused EAE and showed weight loss, decreased clinical disability score, and increased level of pro-inflammatory cytokines and inflammatory mediators.
- Swimming exercise suppress inflammation and demyelination, resulting in improving motor function in EAE rats.
Multiple sclerosis is an immune-mediated neurological disease of the central nervous system (CNS). Multiple sclerosis causes inflammation, axonal demyelination, and neurodegeneration within the CNS [
The pathophysiological mechanisms of multiple sclerosis are complex, and inflammation and axonal demyelination are likely to play an important pathogenic role. According to previous studies, pro-inflammatory cytokines or inflammatory mediators affect neuronal function, alter synaptic transmission and plasticity [
Experimental autoimmune encephalomyelitis (EAE) is one of the most commonly used animal models to study multiple sclerosis [
Regular exercise can contribute to suppressing pathogenic infections by enhancing antigen-specific immune responses [
In this study, we evaluated whether swimming exercise affects functional recovery in MOG33-55-induced EAE rats. Clinical disability score was determined and rotarod test, vertical pole test, and grip strength test were performed. We conducted Luxol fast blue staining for the detecting of demyelination and enzyme-linked immunoassay (ELISA) for the measuring of TNF-α, IL-1β, IL-6 expression. Western blot analysis was performed to detect expression of MBP, PLP, nerve growth factor (NGF), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthases (iNOS).
Forty adult female Lewis rats weighing 210±5 g (9 weeks in age) were purchased commercially from breeder (Orient Bio Co., Seongnam, Korea). The rats were randomly divided into 4 groups (n=8 in each group): control group, EAE-induced group, EAE-induced and swimming exercise group, EAE-induced and interferon-β (IFN-β)-treated group. This experiment was approved by the Institutional Animal Care and Use Committee of Kyung Hee University [KHUASP(SE)-18-150]. All experimental procedures were performed according to the animal care guidelines from the National Institutes of Health and the Korean Institute of Medical Sciences.
The rats were anesthetized by inhalation of sevoflurane (4%) in oxygen (30%) and nitrogen (70%) delivered through a face mask. As described previously [
The body weight and clinical disability score were monitored, once every 2 days for 30 days (14 times, excluding one pretest before EAE induction). The clinical disability score was defined as follows: 0 with no clinical signs, 1 with tail paralysis (or loss of tail tone), 2 with tail paralysis and hind-limb weakness, 3 with hind-limb paralysis, and 4 with complete hindlimb paralysis and front-limb weakness [
The swimming exercise apparatus comprised a plastic tank (90-cm height, 30-cm diameter) filled with water to a depth of 50cm at 30°C. Starting 5 days after EAE induction, the rats in the swimming exercise group were made to swim for 30 minutes, one time a day for 26 consecutive days.
After 2 days of EAE induction, the rats in the IFN-β treatment group were injected intraperitoneally with 300,000 units of IFN-β (Bayer Schering Pharma, Berlin, Germany) once daily for 26 days (same schedule as the swimming exercise). This IFN-β concentration has been demonstrated to be effective as in previous study [
According to the previous described method [
To measure motor coordination and balance, the vertical pole test was performed according to the previous described method [
The forelimb strength was measured by the grip strength test using the grip strength meter 47200 (UgoBasile, Varese, Italy) according to the previous described method [
At 31 days after inducing of EAE, the rats were sacrificed after measuring grip strength. The rats were anesthetized with intraperitoneal injection of Zoletil 50 (10 mg/kg; Vibac Laboratories, Carros, France). Then, the rats were transcardially perfused with 50mM phosphate-buffered saline. The rats were fixed with the freshly prepared solution of 4% paraformaldehyde in 100mM phosphate buffer (pH, 7.4). The lumbar spinal cord was dissected, postfixed with the same fixative overnight, and transferred into 30% sucrose for cryoprotection. Using a freezing microtome (Leica, Nussloch, Germany), coronal sections of 20-μm thickness were cut. An average of 10 lumbar spinal cord sections were obtained from each rat.
Luxol fast blue staining was performed as described previously [
Demyelination of the lumbar spinal cord was assessed and we counted score as described previously [
ELISA was used to detect the concentration of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in lumbar spinal cord tissue. This assay was conducted according to the manufacturer’s protocol using an enzyme immunoassay kit (Abcam, Cambridge, UK) for TNF-α, IL-1β, and IL-6, as described previously [
According to a previous described method [
The western blot was initially incubated with following primary antibodies: rabbit MBP antibody, rabbit PLP antibody (1:2,000; Abcam, Cambridge, UK), mouse NGF antibody, mouse COX-2 antibody, mouse iNOS antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin antibody (1:1,000; Santa Cruz Biotechnology). Then, the membrane was treated with following secondary antibodies: horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories, Burlingame, CA, USA) for β-actin, NGF, COX-2, iNOS; and anti-rabbit antibodies (1:3,000; Vector Laboratories) for MBP and PLP.
The step-by-step experiment was conducted under normal laboratory conditions. All steps, except for membrane transfer, were performed at room temperature, which was done at 4°C using a cold pack and prechilled buffer. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection. In order to compare the relative protein expression of the detected bands, the bands were quantified using a Molecular Analyst version 1.4.1 (Bio-Rad). Control was set to 1.00 for relative quantification.
Statistical analysis was conducted using one-way analysis of variance and followed by Duncan
The body weight and clinical disability score of MOG33-35-induced EAE rats are presented in
Motor coordination and balance were assessed with the rotarod and vertical pole tests (
The concentration levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and inflammatory mediators (COX-2 and iNOS) in the lumbar spinal cord tissue are presented in
The expression of TNF-α, IL-1β, IL-6, COX-2, and iNOS of swimming rats was the same as IFN-β treated rats.
The histological characteristics and demyelination score in MOG33-55-induced EAE rats are presented in
MOG33-55 peptide is an important pathogenic component contributing to EAE induction. The EAE rat model of multiple sclerosis is a representative model of multiple sclerosis because this model markedly demonstrates hind-limb paralysis, motor dysfunction and nerve damage [
EAE induction enhances axonal damage through increase levels of pro-inflammatory cytokines and inflammatory exudates [
Regular physical exercise reduces neuronal degeneration and inflammation [
Increased MBP autocatalysis in the brain of multiple sclerosis may contribute to the production of immune dominant epitopes. A decrease in MBP indicates demyelinating status in multiple sclerosis [
Physical exercise has the effect of regulating immunity by balancing T helper cell-1 and T helper cell-2 [
Our results show that swimming exercise inhibits inflammation and demyelination and improves motor function and clinical impairment in MOG33-55-induced EAE rats. In addition, regular swimming caused the same level of improvement as IFN-β treatment. The results of this experiment suggest the possibility of improving the symptoms of swimming exercise in urological diseases that are difficult to treat. Thus, swimming exercises can be considered for symptom relief in incurable multiple sclerosis.
This study was approved by the Institutional Care and use Committee of Kyung Hee University (KHUASP[SE]-18-150).
No potential conflict of interest relevant to this article was reported.
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Experimental schedule. MOG, myelin oligodendrocyte glycoprotein; IFN, interferon.
Body weight and clinical disability score. (A) Body weight during the experiment. (B) Clinical disability score during the experiment. ●, control group; □, experimental autoimmune encephalomyelitis (EAE)-induced group; △, EAE-induced and swimming exercise group; ■, EAE-induced and interferon-β-treated group. *P<0.05 compared with the control group. #P<0.05 compared with the EAE-induced group.
Motor functions and grip strength. (A) Motor coordination in the rotarod test. ●, control group; □, experimental autoimmune encephalomyelitis (EAE)-induced group; △, EAE-induced and swimming exercise group; ■, EAE-induced and interferon (IFN)-β-treated group. (B) Balance in the vertical pole test. (C) Muscle strength in the grip strength test. CON, control group; EAE, EAE-induced group; EAE-swim, EAE-induced and swimming exercise group; EAE-IFN-β, EAE-induced and IFN-β-treated group. *P<0.05 compared with the control group. #P<0.05 compared with the EAE-induced group.
Expression of pro-inflammatory cytokines and inflammatory mediators. (A) Expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in the lumbar spinal cord tissue. (B) Expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in the lumbar spinal cord tissue. CON, control group; EAE, experimental autoimmune encephalomyelitis (EAE)-induced group; EAE-swim, EAE-induced and swimming exercise group; EAE-IFN-β, EAE-induced and IFN-β-treated group. IFN, interferon. *P<0.05 compared with the control group. #P<0.05 compared with the EAE-induced group.
Demyelination score and expression of nerve growth factor (NGF), myelin basic protein (MBP), and proteolipid protein (PLP). (A) Expression of demyelination in the lumbar spinal cord tissue. □ means demyelination evaluation site. The scale bar represents 50 μm (left) and 200 μm (right). (B) Demyelination score in each group. (C) Expression of NGF in the lumbar spinal cord tissue. (D) Expression of MBP in the lumbar spinal cord tissue. (E) Expression of PLP in the lumbar spinal cord tissue. CON, control group; EAE, experimental autoimmune encephalomyelitis (EAE)-induced group; EAE-swim, EAE-induced and swimming exercise group; EAE-IFN-β, EAE-induced and IFN-β-treated group. *P<0.05 compared with the control group. #P<0.05 compared with the EAE-induced group.