Extremity Ischemic Reperfusion

Objective: Using the rats sustained hindlimbs ischemic reperfusion, this study wants, 1, to observe the level of NO in the blood and muscle and the morphology of the skeletal muscle; 2, to investigate the change of NO level in the blood and the morphology of muscle when administering L-Arg and L-NNA; 3, to study expression of NOS1 and NOS2 mRNA in the muscle; 4, to investigate NOS, SP and GRCP immunoreactivity in rat L₅ spinal cords. And 5, to investigate NO level in the blood of patients to whom pneumatic tourniquets were applied during orthopaedic operation.Materials and methods: 1. Adult male SD rats were divided into normal, sham operated controls, and experiment group, in which both hindlimbs were submitted to 6 hours of ischemia followed by 3, 12 or 24 hours of reprefusion. Griess method was used to assay NO₂^-/NO₃^- level in the blood and muscle. The muscle morphology were observed by HE, AKP, SDH and TEM. 2. In interventional experiment group, L-Arg (100mmol/L) and L-NNA (10mmol/L)was intraperitoneally administered at a volume of 1ml every 60 minutes, starting from 10 min before reperfusion. 3. Primers of NOS1 and NOS2 mRNA were synthesized, and then RT-PCR was used to detect the expression of NOS1 and NOS2 mRNA. 4. The rats were perfused via ascending aorta with 200 ml normal solution followed by 400 ml 4% paraformaldehyde in a 0.1 phosphate buffer. L_2.5 spinal cord segments were carefully removed and placed in 20% sucrose solution overnight. L₅ spinal cord were cut 30 mm thick transverse slices in a cryostat. The section were treated respectively with (a) mice anti-NOS1 (or SP or GRCP) overnight, (b) goat anti-mice IgG for 2-3 hours, (c)ABC complex for 2-3 hours, and finally were stained. The images of sections were analyzed with assistance of personal computer. 5. 8 patients (5 males and 3 females with mean age of 29.5 years) were studied, who underwent upper extremity operation. The tourniquet pressure was 0.045 mPa and duration 1 hour. The blood was obtained before and at 1h of the inflation and 3h after the deflation of the...