The effects of blood flow restriction combined with low-intensity resistance exercise on the metabolic profiling of muscle-wasting rats.

1.1. Ethical statement All animal care and experimental procedures were completed strictly in accordance with China’s Regulations for the Administration of Laboratory Animals and the Guidelines on Welfare and Ethical Review for Laboratory Animals (GB/T 35892-2018).23 The whole experimental process, from sarcopenia induction through the blood flow restriction–enhanced low-intensity resistance training protocol, received approval from the Animal Ethics Committee of Chengdu Sport University (Approval No. Chengdu Sports University Ethics 2024-95). This manuscript is reported in accordance with the ARRIVE 2.0 guidelines for in vivo animal research.24 1.2. Study design 1.2.1. Animals A total of 60 female specific-pathogen-free (SPF) Sprague–Dawley rats, aged 8 months and weighing between 288 and 417 grams, were purchased from Beijing Speifu Biotechnology Co., Ltd. (License No. SCXK 2019-0010).1.2.2. Housing and husbandryAll animals were group-housed in standard cages and provided with ad libitum access to a national standard rodent diet and water. The environment was maintained at a relative humidity of 35.0 ± 5% and a temperature of 20 ± 3 °C, consistent with recommended ranges for laboratory rats (temperature: 20–24 °C; relative humidity: 30–70%). A computerized lighting system was used to simulate a 12-hour light/12-hour dark cycle, with lights on from 08:30 to 20:00, in line with standard photoperiod protocols . The facility was well‑ventilated to ensure optimal air exchange and maintain the micro-environment consistent with recognized housing guidelines.1.2.3. Modeling and randomization 1.2.3.1. Modeling A total of 54 eight-month-old female Sprague–Dawley rats underwent a 7-day acclimatization period. Rats were randomly assigned by using a random-number table allocated to the blank control group (NC; n = 8) or the sarcopenia model group (n =46). The sarcopenia model was established via bilateral ovariectomy (OVX) followed by dexamethasone (DXM) administration, as follows: ● Preoperative preparation: 2 hours of fasting (no food or water). ● Anesthesia: Intramuscular injection of Zoletil® 100 at a dose of 30 mg/kg. ● Surgical procedure: The rat was placed supine, and the abdominal area was shaved and disinfected with povidone-iodine, then covered with sterile gauze. A 1 cm incision was made — 1 cm dorsal to the third pair of nipples — from tail to head. Each ovary was ligated at both ends and excised. Then the incision was closed in layers. ● Postoperative care: Rats were kept warm and only returned to ad libitum feeding and drinking upon recovery. On the day of surgery and for the subsequent two days, each received an intramuscular injection of penicillin G (400,000 U) daily to prevent infection. ● The NC group underwent a sham surgery, where the ovaries were located but not removed, and the abdomen was sutured in layers. ● Seven days after OVX, rats in the model group received subcutaneous dexamethasone (5 mg/kg) once daily for 7 consecutive days. Rats in the NC group received an equal volume of 0.9% saline on the same schedule. ● Model validation: Body weight, grip strength, and rotarod performance were measured before and after model induction to confirm successful sarcopenia induction. During induction, four rats died, and one was excluded due to hind limb injury from improper blood flow restriction training. The final number of effective animals was 54, which were randomly re-assigned using a random‑number table into: ● NC group (n = 8)● Model control (DM) (n = 46) 1.2.3.2. Randomization and grouping Prior to model induction, rats were grouped into Blank Control (NC, n = 8) and Model Control (DM, n = 46). After confirming successful model induction, the 46 sarcopenia-model rats were further randomized (via random-number table) into different groups, the allocation results presented in (Table 1.). GroupGroup abbreviationnSarcopenia ControlDM8Low-Intensity Resistance TrainingDL10High-Intensity Resistance TrainingDH10Blood Flow Restriction onlyDR8Low-Intensity Resistance + BFRDLR10 1.2.4. Intervention 1.2.4.1. Training procedure Except for the NC, DM, and DR groups, the remaining two groups (DLR and DL) underwent progressive resistance training using a weighted ladder-climbing protocol. The resistance exercise was performed on a ladder measuring 1 meter in height, with 54 rungs spaced 0.5 cm apart. A resting platform was positioned at the top of the ladder to allow the rats to take a rest after each climb. During training, the ladder was inclined at an angle of 85°. The apparatus consisted of 10 parallel climbing lanes arranged bilaterally(S1). Prior to the formal training phase, rats assigned to the ladder-climbing groups received 5 days of adaptive learning sessions to familiarize them with this procedure. Following this acclimation, the maximal load capacity (one-repetition maximum, 1RM) of each rat was determined to individualize the training intensity. During the intervention, each rat was placed at the base of the ladder with a corresponding weight attached to its tail. The rats were required to complete a single climb within 10 seconds, and gentle tail stimulation was applied as needed to encourage climbing. A rest period of 1 minute was allowed between repetitions. If a rat failed to complete a climb, the training session was terminated(S2). Throughout the intervention period, body weight and muscular strength increased noticeably. To ensure that the training load remained within the target percentage of each animal’s 1RM and to maintain an appropriate intensity, a progressive loading protocol was implemented(S3). 1.2.4.2. Blood restriction 1.2.4.2.1. Preparation Rats assigned to the modeling groups were first subjected to a preliminary assessment to ensure that their general condition and locomotor activity were adequate to tolerate anesthesia and subsequent measurements. Anesthesia was induced by intramuscular injection of Zoletil 100 at a dose of 30 mg/kg. The animals were positioned supine, and their limbs were secured to the examination table using medical adhesive tape to allow full exposure of the hindlimb musculature. Hair on both hind limbs was removed with an electric clipper. 1.2.4.2.2. Measurements A portable high-resolution ultrasound imaging system (Model M90 SCI, Mindray Medical) equipped with a linear-array transducer (L20-5s, frequency range 15–20 MHz) was used to assess baseline blood flow velocity and vessel diameter in the proximal thigh region without any occlusion. Adequate coupling gel was applied to ensure optimal acoustic contact between the transducer and the rat skin. Dental orthodontic elastic traction rings of various specifications were selected to apply blood flow restriction (BFR) to the femoral arteries of both hindlimbs. The model and quantity of elastic rings used were recorded (Table ). After securing the elastic bands, the target occlusion pressure was calculated, typically corresponding to 40–80% of each rat’s systolic blood pressure. The tension was gradually increased while monitoring changes in blood flow velocity and vessel diameter, and the degree of tension applied to the occlusion bands was documented. The occlusion bands were then slowly released, and the animals were observed for any abnormal respiratory patterns or heart rate fluctuations to prevent injury due to abrupt reperfusion. 1.2.4.3. Maximum voluntary load test Following 5 days of adaptive ladder-climbing training, the procedures were conducted as follows in S4: (1) Experimental Preparation:● A custom-built ladder was prepared for the resistance training protocol.● The body weight of each rat was measured with precision and recorded in detail.● Weighted loads corresponding to 10%, 20%, and 30% of each rat’s body weight (e.g., 20 g, 40 g, and 60 g) were prepared in advance.● A single adaptation climb was performed to allow each rat to familiarize itself with the testing environment and equipment. (2) Load Application and Determination of Maximal Voluntary Carrying Capacity:● Loads were attached to the base of the tail using secure fixation to prevent slippage. The attachment was tested prior to training to confirm stability.● Initial loading was set at 10% of body weight. After the rat successfully completed a single climb, the load was incrementally increased to 20%, and further increased stepwise. This process continued until the rat was unable to maintain grip on the ladder, dropped the load, or could no longer climb. The load at this point was defined as the maximal voluntary carrying capacity.● During ladder climbing, the rats’ performance was continuously observed. If a rat exhibited falling, excessive gasping, prolonged stalling, or failed to resume climbing after gentle tactile stimulation with a wooden rod, the training session was terminated. The load at the time of cessation was recorded accordingly. 1.3. Experimental procedures A total of 54 eight-month-old female Sprague Dawley rats were used in this study. After 8 days of adaptive feeding, the animals underwent bilateral ovariectomy to induce a sarcopenia model, except for the normal control group. Following a 16-day recovery period, dexamethasone was administered intraperitoneally to further promote muscle atrophy. After 5 days of adaptive ladder-climbing training, formal exercise interventions were initiated and continued for 8 weeks. During this period, behavioral assessments, body weight, grip strength, and muscle wet weight were recorded. At the end of the 8-week intervention, tissue samples were collected for histological examination (H&E staining), metabolomic analyses, and transcriptomic profiling. The main objective was to comprehensively investigate the regulatory mechanisms by which blood flow restriction combined with resistance exercise modulates muscle metabolism and gene expression networks in a sarcopenia rat model (Picture ). Timeline Summary (Table ): Days 1–8: Adaptive feeding Days 9–16: Bilateral ovariectomy D