Results

Modeling and Study Design

The present study was conducted to develop an animal model of ACS and investigate the physiological changes in rats following injury. The experimental design is shown in Figure 1. First, the neonatal blood pressure cuff was attached to the rat's hindlimb proximal to the rat, inflated to about 300 mmHg, and squeezed continuously for 3 h, thereby inducing ACS. After the injury, a 2‐mm incision was made in the Achilles tendon of rats to open the skin and fascia. Subsequently, the adjusted balloon catheter was inserted into the rat's fascia chamber for continuous ICP monitoring. According to the results of the previous pretest, the fluctuation of blood flow and pressure in the hindlimb proximal of rats tends to stabilize after 2 h, so the ICP was recorded continuously for 2 h in this study. At the same time, the rats' hindlimbs were scanned with an imaging device to obtain blood perfusion images.

Figure 1: Experimental flow pattern diagram.

Pressure Changes During ACS Formation

Significant swelling of the hindlimb of rats was observed in the ACS group after injury (Figure 2A). And the ICP data showed the dynamic evolution of ICP over time (Figure 2B). As time progressed, the hindlimb ICP increased continuously (0–80 min) and then entered a plateau phase (80–120 min). During this process, ICP increased from baseline to 30 mmHg in about 35 min and to 50 mmHg in about 75 min, meeting the diagnostic criteria for ACS [22, 23]. Notably, the ICP rose sharply, peaking at 60–80 min, consistent with the exponential growth of the pressure–volume curve. When intrafascial swelling reaches a certain level, a small increase in content or muscle swelling may result in a large increase in ICP. These suggested that the model could effectively reproduce the evolution of pressure during ACS development.

Figure 2: The measurement process and results of the intracompartmental pressure (ICP). (A) Operation and processing of the ACS animal model construction. From left to right: neonatal blood pressure cuff, injury process in rats, swollen hindlimb proximal after injury and immobilized with plaster, balloon manometry catheter, and attaching balloon catheter to hindlimb. (B) Line graph of ICP over time during ACS modeling. The horizontal coordinate is time (min), and the vertical coordinate is ICP (mmHg). ACS, acute compartment syndrome.

Circulatory Hemodynamic Changes in the Hindlimbs

Visual images of blood flow distribution in the hindlimbs were obtained by the imaging system. Blood perfusion in the ACS group showed characteristic three‐step changes (Figure 3A). At the beginning of the injury, the blood perfusion of the ACS group was at about 50% of the basal value (Figure 3B). This was followed by a period of compensatory recovery to the level of the Sham group at about 27 min. However, starting at about 30 min, a progressive decrease in hindlimb perfusion was observed in both the Sham and ACS groups, accompanied by a gradual increase in ICP, which plateaued at 80 ± 3 min. Blood flow‐pressure correlation analysis showed a negative correlation between perfusion and pressure at ICP > 30 mmHg. Notably, this experiment included 24 rats, of which 20 provided valid data. Among these, 16 successfully reached an ICP of 30 mmHg, yielding an 80% success rate, and the remaining four were excluded from the analysis due to data acquisition anomalies. These results suggested that changes in ICP were a key factor affecting hindlimb blood flow, and that increased pressure could decrease hindlimb perfusion.

Figure 3: Circulatory perfusion monitoring of the hindlimbs. (A) Representative graph of the thermographic distribution of blood flow in the hindlimbs of rats during 120 min. Left hindlimb, ACS injury; right hindlimb, Sham. The redder colors indicate higher perfusion, and the bluer colors indicate lower perfusion. (B) Line graph comparing the changes in perfusion over time in the ACS and Sham groups during 120 min. The horizontal coordinate is time (min), and the vertical coordinate is perfusion volume (PU). ACS, acute compartment syndrome.

Pathological Process of Lower Limb Injury in Rats

The muscle tissues at different time points after injury (0, 1, 2, 3, and 8 h) were observed through H&E staining, and typical time‐dependent pathological changes were observed (Figure 4). The muscle fibers of the Sham group were arranged regularly, with clear transverse boundaries, and no abnormalities in the mesenchyme. At 0 h after injury, the tissue structure was normal, and no obvious difference was found in the morphology and arrangement of muscle fibers. After 1 h, some of the muscle fibers began to show mild swelling, while the overall structure remained relatively orderly. The changes in muscle fibers in the 2 h postinjury group were more obvious than those in the 1 h group, with more significant swelling. The abnormal behavior of the muscle fibers in the 3‐h postinjury group was further worsened, with more prominent swelling and disorganization, and inflammatory cell infiltration began to appear. When the injury progressed to 8 h, the muscle fiber structure suffered more serious damage, with phenomena such as muscle fiber rupture and dissolution observed, and the intermuscular space between the muscle fibers was enlarged, and the inflammatory reaction was more intense, indicating that the muscle tissue had been seriously damaged after a long period.

Figure 4: Representative images of hematoxylin and eosin staining of rat hindlimb muscle tissue at different time points after injury (200× and 100×). From left to right: Sham, 0 h after injury, 1 h after injury, 2 h after injury, 3 h after injury, and 8 h after injury.

Changes of Injury on Serum Indices in Rats

To further evaluate the persistent damage caused by blood pressure cuff‐induced injury in rats, we examined the biochemical changes related to ACS formation at 0, 1, 2, 3, and 8 h postinjury. The results showed that the proinflammatory cytokines IL‐1β and TNF‐α remained at low levels in the Sham group, similar to 0 h after the injury group (Figure 5A,B). As time passed, they increased and reached their highest value at 8 h, indicating that the inflammatory response was progressively enhanced after injury. The content of CK, an indicator of muscle injury, was relatively low in the Sham group (Figure 5C). It gradually increased from 0 h and was significantly higher at 8 h than at the other time points, suggesting that the degree of muscle damage aggravated over time. The expressions of renal function indicators BUN, the anaerobic metabolic product LAC, and the lipid peroxidation product MDA also continuously increase over time, indicating that the degree of injury is gradually worsening (Figure 5D–F).

Figure 5: Changes of biochemical indexes in the serum of rats at different time points after injury (0, 1, 2, 3, and 8 h). (A, B) Inflammatory factors IL‐1β and TNF‐α. (C) Muscle injury index CK. (D) Kidney function index BUN. (E) Anaerobic metabolism index LAC. (F) Lipid peroxidation index MDA. Black scattered dots are specific data points, and letters on the bars indicate statistically significant differences (p < 0.05). BUN, urea nitrogen; CK, creatine kinase; IL‐1β, interleukin 1β; LAC, lactate; MDA, malondialdehyde; TNF‐α, tumor necrosis factor α.