Prophylactic versus therapeutic CORM-3 compared with Ginkgo biloba extract, N-acetylcysteine and pentoxifylline in rat hind-limb ischemia–reperfusion
Description
Complete data underlying every analysis reported in the related article. Fifty-six female Sprague-Dawley rats were allocated to seven groups of eight. Group 1 was a non-ischemic control. Groups 2-7 underwent 4 h of right hind-limb tourniquet ischemia followed by 4 h of reperfusion: Group 2 untreated; Groups 3-6 received one intraperitoneal dose of CORM-3 (10 mg/kg), Ginkgo biloba extract EGb-761 (100 mg/kg), N-acetylcysteine (150 mg/kg) or pentoxifylline (40 mg/kg) immediately before reperfusion; Group 7 received CORM-3 10 mg/kg 1 h before ischemia. Endpoints: body weight, gastrocnemius wet mass, serum creatine kinase, lactate dehydrogenase and myoglobin, indocyanine green (SPY) perfusion at three angiosomes of both hind limbs, and blinded semi-quantitative scoring (0-3) of nine histopathological criteria. One row per animal.
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Animals. Fifty-six adult female Sprague-Dawley rats (300-350 g), housed four per cage at 21 +/- 2 C, 40-60% humidity, 12-h light/dark cycle, standard chow and water ad libitum, were allocated to seven groups of eight by drawing lots. Protocol. Anesthesia was induced with sevoflurane in a closed chamber and maintained with intramuscular ketamine 25 mg/kg and xylazine 10 mg/kg; animals stayed anesthetized throughout the 8-h protocol and were euthanized in a carbon dioxide chamber without recovery. Group 1 was a non-ischemic control. In Groups 2-7 the right hind limb was exsanguinated with an Esmarch bandage and occluded for 4 h with a locked cable tie (2.5 x 150 mm) at the greater trochanter, occlusion verified by pediatric pulse oximetry, followed by 4 h of normothermic reperfusion. Group 2 received no drug. Groups 3-6 received one intraperitoneal dose of CORM-3 10 mg/kg, Ginkgo biloba extract (EGb-761) 100 mg/kg, N-acetylcysteine 150 mg/kg or pentoxifylline 40 mg/kg immediately before tourniquet release; Group 7 received CORM-3 10 mg/kg 1 h before ischemia. Measurements. Intracardiac blood sampling at 4 h of reperfusion; creatine kinase and lactate dehydrogenase on a Beckman Coulter AU5800, myoglobin by rat-specific ELISA; hemolyzed samples excluded. Indocyanine green angiography (0.3 mg/kg; SPY system, Stryker) at proximal, middle and distal landmarks of both limbs, one frame per animal, tail as 100% reference. Right gastrocnemius weighed, formalin-fixed, paraffin-embedded, sectioned at 5 micrometres, stained with hematoxylin and eosin and graded 0-3 on nine criteria by two blinded pathologists. Analysis. Kruskal-Wallis for global comparisons; Dunn's procedure with tie correction and Bonferroni adjustment for all 21 pairs; Wilcoxon signed-rank for paired limbs; two-tailed, experimental unit the animal. Reproducible from Dataset.csv with any package offering these tests. Data corrections are listed in Data_Reconciliation_Record.docx.