Protective effects of immunosuppressants and steroids against ischemia-reperfusion injury in cremaster muscle flap at microcirculatory level

Abstract

The sequential events between leukocytes and endothelium have significant implications in surgical procedures, trauma, vascular injury, and wound healing. These sequential events are mediated by free oxygen radicals, leukocytes, red blood cells, and endothelial cells. In this study, we investigated the protective effects of steroids and immunosuppressants against ischemia-reperfusion injury in cremaster muscle flaps at the microcirculatory level. In this experimental study, 50 male Sprague-Dawley rats, weighing about 120-130 g, were used. The rats were divided into five groups of 10 rats each: the control group (group 1, n = 10), methyl prednisolone group (group 2, n = 10), dexamethasone group (group 3, n = 10), cyclosporin A group (group 4, n = 10), and azathioprine (group 5, n = 10). Surgical procedures were divided into two stages. In the first stage, a cremaster muscle end-organ tube flap was created by extracting the testes and spermatic cord. The flap was placed into a subcutaneous tunnel in the anteromedial aspect of the ipsilateral limb. In the experimental groups, ischemia was performed by clamping the femoral artery and vein above and below the cremaster pedicle for 4 h. Then the clamps were removed, and perfusion of the cremaster muscle was allowed for 24 h. In the second stage, a round flap was obtained from the cremaster muscle tube-flap to evaluate microcirculation after 24 h of reperfusion, and dissected along its front wall using cauterization. Vessel diameter, red blood cell velocities, leukocyte activation, perfused capillaries, and endothelial edema index were measured and evaluated statistically. There was a significant decrease in the number of rolling, sticking, and transmigrating neutrophils of groups 2 (cyclosporin A), 4 (methylprednisolone), and 3 (azathioprine) (P < 0.05), whereas those of group 5 (dexamethasone) were not decreased (P > 0.05). There was a significant increase in the number of perfused capillaries of groups 2 (cyclosporin A), 4 (methylprednisolone), and 3 (azathioprine) (P < 0.05), nearly 0.75-, 0.5-, and 0.75-fold, respectively. We conclude that cyclosporin and azathioprine showed a protective effect on muscle tissue against ischemia-reperfusion injury by inhibiting leukocyte infiltration, and that methylprednisolone had a beneficial effect against ischemia-reperfusion injury by reducing the synthesis of eicosanoids, edema formation, and leukocyte infiltration. However, we believe that dexamethasone might have a salutary effect due to reducing the synthesis of eicosanoids, edema formation, and release of free oxygen radicals, but not due to leukocyte infiltration.