Animals and experimental design
In this study, thirty- two adult male Wistar rats (eight-week-olds) were obtained from the Razi Vaccine and Serum Institute in Karaj, Iran. After two weeks of familiarization with the environment, the subjects were randomly divided into four groups (each group of eight rats) consisting of untrained + Placebo (UN + P), resistance training + Placebo (RT + P), untrained + Cr (UN + Cr) and resistance training + Cr (RT + Cr). All the rats were kept in standard cages made from polycarbonate and under controlled conditions with a 12:12-h light-dark cycle and room temperature maintained at 22℃. Furthermore, all rats were provided with water and food ad libitum. The rats had free access to food and water during the entire experimental period. Changes in body weight were measured weekly. The study design was approved by the animal research ethics committee of Kurdistan University of Medical Sciences (IR.MUK.REC.1397.5010).
In the present study, climbing a vertical ladder (110 cm high × 18 cm wide, 2 cm grid steps, 80 degrees of inclination) was considered the resistance training protocol. At the top of the ladder, the rats reached a resting chamber (L×W×H = 20 cm × 20 cm × 20 cm). Previous studies have shown that each rat is able to climb this device 8 to 12 times . Before the start of the main protocol, the rats were introduced to the ladder for 5 days. For this reason, each animal was placed in different parts of the ladder (base, middle, and upper stair) to practice climbing the ladder without extra load. Touching the rat's tail initiated the ladder climb without any electrical stimulation. After the adaptation period, the rats of the training groups performed maximal carrying load (MCL) test. A cylinder containing weights (metal balls) fixed to the base of each rat tail by a clip was used to create resistance. At the bottom of the climbing apparatus, the rats had to climb up with a load of 50% of their body weights. The overload (30 g) in each set was gradually increased over time. This process continued until the rats could not climb up the ladder after 3 consecutive trials. The last load to be carried up to the top of the ladder was recorded as MCL for that session.
The resistance training protocol was performed for 8 weeks, 5 days per week. The number of times that the rats climbed the ladders was 10 times per session and 120 seconds rest intervals was set between attempts. During training sessions, rats climbed a ladder with 50%, 75%, 90% and 100% of their individual MCL (each one twice), then 30 grams were added at each attempt to complete the 10 sets (if a rat was not able to climb the stairs, the climb was performed with the prior weight). The final highest load successfully carried at the end of the training was considered as the MCL for the next training session .
The doses of Cr supplementation was performed according to the recommendations of the International Society of Sports Nutrition . Cr supplementation began one week before the start of the main resistance training program (loading phase, 0.3 g/kg/day). Subjects then received 0.05 g/kg/day Cr (in powder form with a purity of 99.9%, Ultimate Sports Nutrition (USN), USA) at the maintenance phase for 8 weeks. In the present study, supplementation recipient groups (RT + Cr, UT + Cr) received Cr monohydrate diluted with 1.5 ml of dextrose solution (5%) using a syringe and by dripping the solution in the mouth. Placebo recipient groups (RT + P, UT + P) consumed the same amount of dextrose solution (1.5 ml) during this period. In the current study, an oral solution was used for supplementation which had a good taste (by adding dextrose). Therefore, without harming the rats, the prescribed dose was fully received. During the intervention period, the rats were weighed weekly to determine their supplemental dose.
48 hours after the last training session and following 12 hours of fasting, the rats were weighed and then anesthetized using ketamine (80 mg/kg of body mass) and xylazine (12 mg/kg of body mass). While waiting for the effects of anesthesia to appear, the skin that surrounds the lower right and left legs were shaved and removed carefully to show the leg muscles. The next step involved the Achilles tendon being cut to determine the flexor halluces longus (FHL) muscle (posterior and lateral surface of the body of the fibula). Then, the proximal and distal FHL muscle tendons were dissected and cut swiftly. Immediately after muscle separation, the weight of the FHL muscle was achieved with a precision analytical scale (Sartorius group (acculab) Germany, ATILON model, readability 0.0001 g). The ratio of FHL muscle weight to total body weight was also calculated. Finally, the left FHL muscle was fixed in 10% formalin solution to evaluate changes in muscle cross section, and the right FHL was frozen in liquid nitrogen and stored at -80℃ until western blotting analysis.
Histological analysis of the FHL muscle
The FHL muscle was fixed in 10% formalin solution for 2 days, after which graded ethanol was used for dehydration and clearing. In the embedding phase, specimens were embedded in paraffin to produce paraffin blocks. These blocks were cut at 5 µm thickness using a microtome (Reichert-Jung, Model 2800 E Frigocut) and attached onto a slide glass. The slides were stained with hematoxylin and eosin (H&E) for analyzing the CSA of the FHL muscle. Photographs were taken using an optical microscope (Olympus IX81) equipped with a Ha- mamatsu EM-CCD (Model C9100). The CSA of the FHL muscle tissue was assessed by Image J software.
FHL muscle tissues (100 mg) were homogenized in ice-cold RIPA lysis buffer (50mM Tris, pH 8, 150mM sodium chloride, 1.0% NP-40, 1% sodium dodecyl sulfate (SDS), 1% ROCHE). Homogenized samples were centrifuged at 12000 × g for 15 minutes at 4°C. The supernatant was collected and the protein concentration measured (wavelength 595 nm) by a Bio-Rad protein-determining kit based on the Bradford method , and kept at a temperature of -20° C. The homogenized samples obtained were mixed in a ratio of 1:1 with a 2X loading buffer (50 mM tris - hydrogen chloride, pH = 6.8%, sodium dodecyl sulfate, 10% glycerol, 5% beta-mercaptoethanol and% 0.005 Bromophenyl blue). The samples were boiled for 5 minutes until all proteins were completely denatured. The proteins were isolated by SDS gel (10%) electrophoresis, then transferred to a nitrocellulose membrane, and blocked on the shaker for one hour with a combination of bovine serum albumin (BSA) 1%, tween 20 in phosphate-buffered saline (PBS) 0.1%. After washing in PBS-T solution, the membrane was incubated within a solution containing diluted anti-rabbit antibody in BSA-PBS-T solution. The antibodies used for immunoblotting were eIF2α (sc-517214; 1:500), p-eIF2α (sc-101670; 1:500), PERK (sc-377400; 1:500), p-PERK (sc-32577; 1:500), mTOR (sc-1550-R; 1:500), p-mTOR (sc-293133; 1:500), AKT (sc-135829; 1:500), p-AKT (sc-52940; 1:500) ATF4 (sc-390063; 1:500), and β-Actin (sc-130657) from the Santa Cruz Biotechnology company, and CHOP (ab-11419; 1:500) and BiP (ab-11419; 1:500) from Abcam company. The nitrocellulose membrane was removed from the solution and washed with PBST (three times each time for 5 minutes). Then, it was incubated for 1 hour with secondary antibody (anti rabbit/anti mouse depending on the primery antibody). The chemiluminescence chemical reaction and x-ray radiography film (Fujii) were used for visualization. Protein measurement densitometry analysis was carried out using Image J software (National Institutes of Health, Bethesda, Maryland, USA). The target genes band were normalized against β-Actin as loading control.
To assess normality of data distribution and equality of variances, the Shapiro–Wilk and Levene tests were used, respectively. Analysis of variance (ANOVA) with repeated measure [within-subject effects (time), between-subject effects (group), interaction between the two types of effects (group × time)] and Bonferroni post-hoc test were used to determine intra-group and inter-group changes in body weight and Maximal carrying load (MCL) variables. In relation to other research variables, one-way ANOVA and Tukey's post hoc tests were used to compare differences between groups (in the post test). SPSS software package (version 23) was used for data analysis and the statistically significant difference was set at p < 0.05.