Cremaster Island Flap Model

Male Lewis rats (4 to 6 weeks old) weighing 200 to 250 g were anesthetized with urethane intraperitoneally based on a dosing regimen of 1000 mg/kg, and if required, incremental doses of 50 mg until hind limb pain reflexes were undetectable. The cremaster muscle and external fascia were exposed with a surgical incision from the anterior iliac spine to the tip of the scrotum. The cremaster muscle was dissected out by blunt dissection. Fascia superficial to the cremaster muscle was cleared under a stereomicroscope. The cremaster muscle was then opened along its least vascular sector (ventral side). The testicle and its appendages were separated from their dorsal connections while preserving the testicular artery and vein, and then reduced into the abdomen. The cremaster muscle was dissected out as an island flap attached to the body through a vascular pedicle that included the pudic-epigastric artery and vein. Finally, it was laid out upon a slide mounted on an in-house designed acrylic stage and fully spread out by means of five 4-0 silk sutures. The exposed tissue was superfused with normal saline through a temperature controller (Warner Instruments, USA) to prevent dessication throughout the study.

After establishing the island flap model, a 30-minute stabilization period was allowed. During this period, 4 potential postcapillary venules in the central sector of the flap were located and mapped out. Each venule was then recorded for 3 minutes to provide baseline data. Ischemia-reperfusion injury was induced by clamping of the pedicle with a microvascular clamp for 4 hours, followed by 2 hours of reperfusion. After reperfusion, the 4 venules were observed and the venule with the best return of flow was recorded.

A total of 15 rats were used (n = 5, 3 groups). The control group was subjected to 4 hours of ischemia and 2 hours of reperfusion without treatment. The ANV-6L15 treatment group received 1 dose of ANV-6L15 (200 μg/kg) through the penile vein immediately after starting reperfusion. For the ANV group, 1 dose of ANV (200 μg/kg) was given through the penile vein immediately after starting reperfusion.