Role of Leupeptin in Preventing Hind Limb Ischemic Tissue Injury

PURPOSE: Prolonged tourniquet ischemia leads to progressive muscle, nerve, and vascular injury. Currently, the only way to prevent injury to these tissues is by minimizing tourniquet time. Tissue ischemia leads to calpain activation and Wallerian-like degeneration. Calpain is expressed in the vascular wall and is implicated in several vascular inflammatory and degenerative disorders. Leupeptin inhibits the expression of calpain. We hypothesized that by inhibiting the expression of calpain with Leupeptin, we could diminish muscle, nerve, and vascular injury after prolonged tourniquet ischemia. We undertook a study to assess the role of Leupeptin in a rat model of prolonged hind limb ischemia. METHODS: Ten male Sprague-Dawley rats weighing 300–400 g were subjected to 2-hours of blood flow occlusion in the left hind limb by application of a neonatal blood pressure cuff set to 300 mm Hg.1 Half of the rats were then randomly selected to receive twice weekly intramuscular injections of Leupeptin at 12 mg/kg in saline starting right after tourniquet release, whereas the other half received injections of saline alone. Blood flow occlusion was confirmed by the loss of a pulse detectible by a pulse oximeter and cyanotic discoloration of the limb. All animals were monitored for gait quality using the sciatic functional index.2 Two weeks after the tourniquet applications, the animals were sacrificed. The sciatic nerves, gastrocnemius muscles, and saphenous veins and arteries were harvested from the left and right hind limbs, fixed in 10% formalin and imaged following Masson’s trichrome staining. RESULTS: The histologic images of the gastrocnemius muscle fascicle cross-sectional areas from both the hind limbs for the 2 groups—leupeptin and control—were imaged using a Nikon Eclipse E800 at 10× magnification and analyzed using an imaging software—ImageJ. The difference between the muscle cross-sectional areas of the left hind limbs (ischemic limbs) between these groups was found to be significant (leupeptin group: 774.57 µm2 versus 472.70 µm2 for the control; P = 0.043). These 2 values were significantly lower as compared to their respective right muscle fascicle areas (where no tourniquet was applied; 1,725.30 µm2 and 1,548.22 µm2 for the leupeptin and control groups, respectively). However, the differences in the sciatic functional index scores between these 2 groups were not found to be different (P = 0.785). CONCLUSIONS: The application of Leupeptin post-hind limb ischemia led to greater preservation of hind limb muscles. We postulate that by inhibiting calpain, Leupeptin inhibits the pathways that trigger cell death leading to greater tissue preservation. Studies focusing on the gross and histologic changes in the arteries, veins, and nerves between these groups are currently being performed. REFERENCES: 1. Kauvar DS, Baer DG, Walters TJ. Influence of systemic hypotension on skeletal muscle ischemia-reperfusion injury after 4-hour tourniquet application. J Surg Educ. 2007;64:273–277. 2. Wang GW, Yang H, Wu WF, et al. Design and optimization of a biodegradable porous vein conduit using microtubes as a guide for rat sciatic nerve defect repair. Biomaterials. 2017;131:145–159.