Materials and Methods

Animals

All experimental animals were obtained from the experimental center of Daping Hospital, Army Military Medical University (Third Military Medical University). A total of 48 10‐week‐old male Sprague–Dawley rats weighing 250 ± 20 g were used in this study. Before the experiment, the rats were housed in an environment with suitable temperature (22 ± 2°C) and relative humidity (50 ± 10%) for 1 week of acclimatization, with free access to food and water. This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (Approval No. SYXK (YU) 2022‐0018).

Construction of the ACS Model

The sample size of 48 rats was determined by a power analysis based on our pilot study (expected effect size = 0.8, α = 0.05, and power = 0.8), resulting in eight rats per group. The rats were randomly divided into six groups using a computer‐generated random number sequence: Sham, 0, 1, 2, 3, and 8 h. All behavioral and histological assessments were performed by an investigator blinded to group allocation. There were no exclusions. The rats were anesthetized with exhalation isoflurane (2%, 1.5 L/min) throughout the modeling procedure, and fixed in the supine position on the operating table. For the injury groups, a neonatal blood pressure cuff (Tempa‐Kuff, size #1; Shengmingfaxian Medical Products, Shenzhen, China) was placed and tightly wrapped around the hindlimb proximal to the rat, and a pressure of 300 mmHg was applied to compress the lower‐limb muscle for 3 h. Morphine (2 mg/kg) was administered intraperitoneally as an analgesic, followed by repeat doses every 3 h to maintain analgesia throughout the experiment. For the sham group, all surgical procedures were performed exactly as in the injury groups, except that the compression cuff was not applied and no balloon catheter was inserted into the fascial compartment. The sham animals were maintained under anesthesia for the same duration as the injury groups.

After the removal of the injury‐inducing device, an incision of approximately 2 mm was made in the Achilles tendon of the rat, and the posterior shallow fascial compartment was incised. The red connector of the pressure measuring device was connected to the pressure sensor (PT‐103N, Chengdu Coman Software Co. Ltd.), and the blue connector was connected to an injector containing 10 mL of saline. The injector was pulled back to create a vacuum between the balloon and tube. Immediately thereafter, the balloon catheter was inserted into the superficial fascial compartment. Appropriate saline solution (~0.3 mL) was slowly injected into the balloon to maintain a pressure of 8–10 mmHg, simulating normal ICP. The blue tube was then turned off to complete the ICP adjustment. The injured limb was immobilized with premade plaster to simulate the pathological process of ACS induced by improper immobilization of the lower limb after trauma. A data acquisition system (BL‐420N, Chengdu Coman Software Co. Ltd.) was used to collect and analyze data, including voltage measurements at 60 Hz. After completion of the above procedures, the injury groups were further maintained with plaster immobilization for different durations corresponding to the 0, 1, 2, 3, and 8 h groups, respectively. The 0 h group was euthanized immediately after plaster immobilization. The sham group was maintained under the same conditions and euthanized at 8 h after the sham procedure. After the operation, the rats were placed in a warm, quiet environment, and their activity, diet, and wound conditions were closely observed. No animals died during the experiment. Rats in each time point group (0, 1, 2, 3, and 8 h after injury) were euthanized at their respective designated time points. The overall experimental endpoint for the study was set at 8 h after injury, at which time the rats were euthanized by inhaling an excessive dose of isoflurane (5%, 3 L/min) until respiratory and cardiac arrest were confirmed, and muscle tissue was collected to assess irreversible injury.

To ensure animal welfare, at least one trained investigator remained in the laboratory to continuously observe the animals' general condition and to measure vital signs at irregular intervals throughout the experiment. These measurements were formally recorded every 15 min, but the animals were under near‑continuous visual and physiological monitoring between recordings.

Laser Speckle Imaging

Limb perfusion in both limbs of rats was continuously imaged using the Laser Speckle Contrast Imaging System RFLSI III (RWD, CA, USA). Briefly, the region of interest (ROI) of the limb vessels in both limbs was segmented, and the blood perfusion in the limb region was quantified by measuring the average intensity of the red pixels within the ROI using Image J.

Ischemic Score Analysis Laser Speckle Imaging

A modified ischemia scoring method was defined. Real‐time images of rat limbs after injury were captured by the Laser Speckle Contrast Imaging system, and ischemia scoring was accomplished by visual assessment of paw discoloration.

Histology Staining

The rat tibialis anterior muscle was taken at 0, 1, 2, 3, and 8 h after injury and stained with hematoxylin and eosin (H&E). The animals were euthanized, and the rat tibialis anterior muscles were taken and fixed in 4% paraformaldehyde solution for 24 h. The tissue was then dehydrated in ethanol of varying concentrations, cleared in xylene, and embedded in wax. The embedded tissues were cut into 4–5‐μm‐thick sections, deparaffinized in water, stained with H&E and observed under a microscope.

Inflammatory Factor Detection

Rat serum was collected via the abdominal aorta at 0, 1, 2, 3, and 8 h after injury. The levels of inflammatory factors Interleukin 1β (IL‐1β) and tumor necrosis factor α (TNF‐α) were determined according to the instructions of the enzyme‐linked immunosorbent assay (ELISA) kit. The optical density of each well was measured using an enzyme‐labeled instrument, and the levels of IL‐1β and TNF‐α in rat serum were quantified. Rat TNF‐α ELISA Kit (MM‐0180R1) and Rat IL‐1β ELISA Kit (MM‐0047R1) were purchased from Meimian Industrial Co. Ltd. (Jiangsu, China).

Biochemistry Indicator Measurements

The serum levels of lactose, malondialdehyde (MDA), creatine kinase (CK), and urea nitrogen (BUN) were measured according to the instructions of the biochemistry kits. Lactose, MDA, CK, and BUN kits were purchased from Aidisheng Biotechnology Co. Ltd. (Jiangsu, China). The optical density was measured in a spectrophotometer. Calculate the serum levels of LAC, MDA, CK, and BUN according to the formula provided with the kit.

Statistical Analysis

The data were analyzed using SPSS statistical software. First, the data were tested for normality using the Shapiro–Wilk test to determine whether the data were normally distributed. If the data were normally distributed, one‐way ANOVA was used to analyze the differences between the five groups of samples. If the ANOVA results showed significant differences, the Least Significant Difference or Tukey's test was further used to compare the two groups. If the data did not follow a normal distribution, the nonparametric Kruskal–Wallis rank‐sum test was chosen. Data are presented as mean ± SEM, p < 0.05 is considered statistically different.