Resistance training restores skeletal muscle atrophy and satellite cell content in an animal model of Alzheimer’s disease

Animals and muscle tissue preparation

All experiments involving animals were performed following approved guidelines and ethical approval from Sport Sciences Research’s Institutional Animal Care and Use Committee (as registered under the code: IR.SSRI.REC.1401.1732) and according to the NIH Guidelines for the Care and Use of Laboratory Animals (NIH publication, 1996). Additionally, the present study was carried out in compliance with the ARRIVE guidelines. Two-month-old Wistar male rats (n = 48) were purchased from Lorestan University of Medical Sciences Laboratories and housed three-per-cage in an animal lab under standard conditions (12-h light/dark cycle in a room at a temperature of 20–25 °C) with access to food and water ad libitum. The animals were randomly assigned into four equal groups of 12: healthy-control (H-C), healthy-exercise (H-Ex), Alzheimer-control (A-C), and Alzheimer-exercise (A-Ex). At the end of the treatment periods, all rats were anesthetized with isoflurane inhalation. Gastrocnemius muscles from animals in all groups were dissected in optimal cutting temperature (OCT) medium, mounted on pieces of cork, secured with tragacanth gum, and frozen in liquid nitrogen-cooled isopentane and further stored at − 80 °C. Ten µm-thick cryosections were prepared and processed for immunostaining and used to test the program’s ability to recognize myofiber morphology and SCs content.

Alzheimer’s induction

To induce Alzheimer’s disease in the present study, beta-amyloid 1–42 (Aβ_1-42) was injected into the CA1 region of the dorsal hippocampus of Aβ rats using a stereotaxic device (RWD, China)¹². To prepare the injection solution, 1 mg/mL of Aβ_1-42 (Abcam, Cambridge, UK, Cat no. ab120301) was initially diluted with ice-cold 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and incubated at room temperature for at least 60 min with occasional vortexing. Next, the HFIP was removed using a SpeedVac, and the peptide film was stored at − 80 °C. Then for aggregation, the peptide was first resuspended in DMSO (5 mM), sterile phosphate buffer saline (PBS) was added to bring the peptide to a final concentration of 1 μg/μL, and the Aβ_1-42 peptide was incubated at 37 °C for 72 h. To perform stereotaxic surgery, the animals were anesthetized with isoflurane inhalation. Aβ_1-42 suspension (1 μl/site) was injected into the CA1 region of the dorsal hippocampus of A-C, and A-Ex groups (A-4.2, L ± 3.0, V-2.0 mm) based on the Paxinos and Watson atlas by a Hamilton syringe attached to an infusion pump¹². Moreover, isotonic saline solution was injected into the sham animals. To confirm Alzheimer’s induction, pathological slides were prepared from the hippocampus of three animals/per group 10 days after surgery using thioflavin immunostaining of beta-amyloid plaques.

Resistance training protocol

The rats in the H-Ex and A-Ex groups performed a 4-week training period. Resistance training was conducted using a 1-m ladder with 26 rungs and an inclined at 85° with a house chamber (20 × 20 × 20 cm) placed at the top. Initially, the rats were familiarized for one week with climbing ladders, and then the resistance training protocol was implemented for four weeks. Accordingly, in the familiarization period, the animals were taught to climb the ladder and perform four trials per day for four days. The rats were placed at the bottom of the climbing ladder with weights attached to their tails and were motivated to climb by touching the tail with tweezers to initiate the movement. Once the rats climbed to the house chamber they were allowed to rest inside the chamber for 120 s. The familiarization protocol was repeated until the animals voluntarily climbed the ladder three consecutive times without stimuli. Then, resistance training with progressively heavier loads was performed for four weeks at least with 48 h rest between each training session (12 sessions; on the same days each week: Saturday, Monday, and Wednesday). To determine the maximal carrying load (MCL), on the first day of every training week, each animal carried a load that was 75{530e870c018efe4faeaf1650f5521f73bbd950ecc9f753be09e2bfdaa8245e82} of their body mass, and 30 g were added for each additional climb repetition until the rat was unable to climb the entire length of the ladder. The next training sessions consisted of five ladder climbs with 65, 75, 85, 95, and 100{530e870c018efe4faeaf1650f5521f73bbd950ecc9f753be09e2bfdaa8245e82} of the rat’s previous MCL, as determined on the first day of every training week. Each resistance training session consisted of 5 sets / 4 reps with a 60-s break between reps and 3 min between sets. This type of resistance training protocol was adapted from the previous reports^13,14,15, and according to the needs of the current study.

Morris Water Maze test

To study spatial learning and memory, we used Morris Water Maze (MWM) according to our previous studies^15,16. In summary, in the fourth week of resistance training, the MWM test was performed to test the animals’ cognition, including spatial learning and memory. MWM consisted of the following apparatus: (1) a black circular pool filled with water (22 °C ± 2 °C, 200 cm diameter, walls 76 cm depth, divided into four quadrants: N, S, W, and E), located in a room with visual signs on the walls equipped with a computerized tracking/image analyzer system, and (2) a platform with a diameter of 10 cm and a height of 35 cm, which was placed 2 cm below the water’s surface and in the middle of the S and E quartile during the spatial learning test. To test the animal spatial learning, the rats were randomly abandoned from the N, S, W, and E points in the water. In the spatial learning phase, (four trials/day for four consecutive days), rats were given up to 60 s per trial to find the hidden platform and were required to remain seated on the platform for 10 s, otherwise, the rats were guided by hand and allowed to remain on the platform for 10 s (in this case their escape latency was accepted as 60 s). Then, 24 h after the last spatial learning test, rats were tested for spatial memory (probe trial). To test the probe trial, after removing the platform, the time spent in the target quadrant was recorded for up to 60 s.

Biochemical assays for SOD, CAT, GPx, GSH, and MDA

All biochemical assays in the present study were carried out according to our previous study¹⁵. In summary, the activities of Superoxide dismutase (SOD), Catalase activity (CAT), and glutathione peroxidase (GSH) in the homogenate of the gastrocnemius muscle were measured by the specific ELISA kits developed for rats according to their manuals instruction (ZellBio GmbH, Germany).

Immunofluorescent staining

All immunohistochemical procedures in the present study were carried out according to our previous study⁷. To detect different myofibers, gastrocnemius muscle Sects. (10 µm-thick) were incubated with antibodies specific to myosin heavy chain (MyHC) types I, IIa, and IIb (BA-D5, SC-71, and BF-F3, respectively, University of Iowa Developmental Studies Hybridoma Bank, Iowa City, IA), supplemented with rabbit polyclonal anti-laminin antibody (L9393; Sigma-Aldrich, St. Louis, MO). Unstained myofibers were judged as MyHC IIx expression. Alexa Fluor 405, 488, and 546 secondary antibodies were used to detect MyHC types I, IIa, and IIb, respectively (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Further, laminin (L9393 Sigma-Aldrich, St. Louis, MO, USA) and Pax7 (Developmental Studies Hybridoma Bank, Iowa, IA, USA) were used to detect myofiber borders and satellite cells, respectively. Secondary antibodies coupled to anti-rabbit IgG Cy3 and Cy5-labeled (Jackson Immunoresearch Labs, West Grove, PA, USA) were used to detect laminin and Pax7.

Image acquisition and quantification

All images were captured at × 10 (for cross-sectional area (CSA) and fiber type measurements) and × 20 (for myonuclei and SCs measurements) magnification using a Carl Zeiss AxioImager fluorescent microscope (Carl Zeiss, Jena, Germany). To analyze whole muscle cross-section, consecutive fields from whole muscle sections were automatically acquired in multiple channels using the mosaic function in Image M1 Software (version 4.9.1.0, RRID: SCR_002677). For automatic quantification of mean CSA, myonuclei, fiber-type, and SCs images from various experimental conditions were analyzed using MyoView software as described⁷.

Imunofluorescence staining and morphometric analysis of isolated myofibers

In order to immunofluorescence staining of mono-myofibers, the gastrocnemius muscle blocks were fixed in 4{530e870c018efe4faeaf1650f5521f73bbd950ecc9f753be09e2bfdaa8245e82}PFA in PBS for 2H at RT. After several washes, 40 to 50 mono-myofibers were isolated per staining from each muscle. Then, isolated myofibers were mounted on glass slides in Vectashield mounting medium containing DAPI (Vector Laboratories, UK). To detect myonuclei, myofibers were mounted on slides using fluromount Aqueous mounting (Sigma, F4680-25 mL) and kept at 4° C. Isolated myofibers were analyzed using confocal laser scanning microscopy (Olympus Co. Ltd., Tokyo, Japan). CSA of isolated myofibers was calculated by the following formulas: CSA = π × (w/2) × (t/2) where w and t are the width and thickness, respectively. Additionally, the number of nuclei was counted and illustrated as the number of nuclei/100 µm fiber length or the number of nuclei/volume calculated for 100 µm fiber length. All of these analyses were determined on image stacks using the 3D manager plugin of Image J software¹⁷.

Statistical analyses

Statistical analysis was performed using the Graph-Pad Prism statistics software (Graph-Pad Software Inc., San Diego, La Jolla CA, USA free demo version 8.0.0, www.graphpad.com) and data was reported as mean ± S.E.M. Shapiro–Wilk and Levin’s tests were used to check normal data distribution and to determine the homogeneity of variances, respectively. Two-way ANOVA followed by Tukey’s post hoc test was performed for CSA, myofiber types, myonuclei and SC content changes with the resistance training program. Values of p < 0.05 were considered statistically significant.