Participants and Study Design
This clinical trial study aimed to investigate the effects of 8-week home-based aerobic training and Vit D supplementation on BDNF and NGF in patients with multiple sclerosis. As detailed in Fig. 1, the study population consisted of 60 participants (aged 20–40 years) from the Kermanshah MS center with a disability scale of 3–5.
In this study, 40 women with multiple sclerosis (20–40 years) in Kermanshah were selected using purposive and available sampling method based on G. POWER 3.1 software with a statistical power of 99 %, the effect size of 95%, and significance level at 0.05.
Inclusion criteria included: at least 2 years MS history, disability scale of 3–5, lack of regular exercise, no history of other diseases, no smoking, and no immunosuppressive drug consumption. Exclusion criteria included a history of heart disease, hypertension, orthopedic disorders, and diabetes, consuming other drugs in addition to MS medications that might affect the individual's response to the intervention, lack of regular attendance in the exercise training and testing sessions muscle injuries, inability to perform the exercise, COVID-19 infection, and severe relapses during the study period.
The trial (IR.RAZI.REC.1400.002) was approved by the Ethics Committee of the Kermanshah Razi University and registered in the Iranian Clinical Trial Registration Center under the code IRCT IRCT20201129049525N; Written informed consent was obtained from all participants, including agreement of the patients to participate as volunteers and feasibility to leave the study. Forty subjects were randomly assigned into four equal (n = 10) groups; aerobic training plus vitamin D supplementation (AT + Vit D), aerobic training (AT), vitamin D supplementation (Vit D), and control (C) using the lottery method.
IRCT20201129049525N1
Intervention
The AT experimental groups exercised online three times per week for 2 months at home. All training sessions were supervised by an exercise physiologist. Vitamin D supplementation was performed based on the standard guidelines.
Aerobic Training
was executed online.
The subjects were instructed to do home-based walking aerobics three times per week, increasing the time and intensity as shown in Table 1 to 40 min and 70 % of HRmax [25] which was recommended by the Multiple Sclerosis Association of America (MSAA) [26] (Table 1). Every session included 10 min of warm-up and 10 min of cool-down.
The HRmax formula [HRmax = 220 − age] was used to determine the target heart rate [27]. The pulse palpation method was taught to the participants in an instructional session and they learned to monitor heart rate. Also, the 6–20 rating of perceived exertion (RPE) scale was considered to assure achieving and maintaining the desired exercise intensity during the walking aerobics phase, [28] (Table 1). The intervention continued for 8 weeks.
Table 1
Aerobic training protocol
Variables
|
Week
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
Intensity (HRmax)
|
50–55%
|
50–55%
|
55–60%
|
55–60%
|
60–65%
|
60–65%
|
65–70%
|
65–70%
|
Time (min)
|
20
|
25
|
25
|
30
|
30
|
35
|
35
|
40
|
Borg scale
|
10
|
10
|
10
|
11
|
11
|
11
|
12
|
12
|
Vitamin D supplementation and Diet
The participants filled the pre-participants physical activity readiness questionnaire (including demographic data, health status, and PA) before attending the training program, and a detailed semi-quantitative food frequency questionnaire to record and analyze 3-day food, recalls, adapted to the Iranian population (including common food items, serving sizes, and meals) before and at the end of the intervention. The Food Processor nutritionist 4 software (FPN4) was used to determine the amount of macronutrient consumption (protein, fat, and carbohydrates). Subjects were asked to consume the same food as the pre-test, the day before blood sampling in the post-test. In general, the subjects' diet consisted of 15% protein, 55% carbohydrates, and 30% fat.
In this study, 50000 IU of vitamin D (made by the Zahravi Pharmaceutical Company in Iran) were supplemented once per week for eight weeks in the Vit D groups [29, 30]. While, the C and AT received the placebo with the same shape, color, smell, and tastes as vitamin D supplement once per week (made by the Zahravi Pharmaceutical Company, Iran) for 8 weeks.
Body Composition and Anthropometric Measurements
Subjects were familiarized with the study procedure 3 days before the start of the intervention and primary measurements including anthropometric parameters and body composition were determined both at the start and end of the study. Waist circumference was measured to the nearest 0.5 cm with a non-elastic tape measure and Height to the nearest 0.5 cm using a stadiometer (DETECTO, Model 3PHTROD-WM, USA). Also, weight, fat mass, and BMI were determined by the INBODY test using bioelectric impedance analysis (Zeus 9.9 PLUS; Jawon Medical Co., Ltd., Kungsang Bukdo, South Korea). The body composition measurements were performed early in the morning (8–9 am) after at least 12 hours of fasting overnight. To minimize the effect of water consumption on the results, measurements were done after emptying the bladder, and subjects were asked to refrain from taking diuretic drugs and not to participate in intensive physical activity 48 hours before the test.
Outcome Variables and Biochemical Measurements
Subjects did not perform any exercise 48 hours before blood sampling and were on 12 hours of fasting before blood sampling. Then, 10 ml of blood samples were taken from the cubital vein, in the least possible time, at the beginning and the end of the study. For BDNF and NGF analyses Blood samples were collected in heparinized tubes, frozen, and stored at -80˚C. Then, BDNF and NGF were measured using a human BDNF and NGF kit (made in China under the license of Boster Biological Technology, America Company with a sensitivity of 2 and 2.48 pg/ml, respectively) by the ELISA method. Serum 25-hydroxyvitamin D level was measured by quantitative luminescence method (DiaSorin kit made in the USA).
Statistical analysis
To evaluate the normality of distribution the Shapiro–Wilk’s test was used. ANOVA and t-test were used to compare the mean of variables between and within groups, respectively. If significant differences were found Tukey's post hoc test was used. The SPSS statistical software (version 21; SPSS Inc., Chicago, IL, USA) was used to perform all statistical analyses at a significant level of P < 0.05.