Superoxide: A Critical Oxygen-Free Radical in Ischemic Bowel Injury By Stephen P. Dunn, Kirby R. Gross, Michael Dalsing, Richard Hon, and Jay L. Grosfeld Indianapolis, Indiana Q The radical apions of molecular oxygen reduc- tion, superoxide (0Z)_hydrogen peroxide (HzOz), and hydroxyl radical (OH), have been implicated in a number of disease processes, including ischemic bowel injury. This report evaluates the effect of superoxide dismutase (SOD), catalase (CAT), dimethyl sulfoxide (OMSO), selenium treatment, and selenium deficiency on bowel integrity and survival in experimental intestinal ischemia in rats. Ischemic bowel injury was produced in 204 male Sprague- Dawley rats (wt 90 to 100 g) by a one-minute occlusion of the superior mesenteric artery (SMA) with a microaneurysm clip. Experiment I treatment animals (n = 20) received 2.5 mg/kg SOD dissolved in Ringer's lactate, and control animals (n = 71) received Ringer's lactate alone. Experiment II treat- ment animals (n = 16J received 1 cc of 100% DMSO gavage, and control animals (n = 11) received no treatment. Experiment III treatment animals (n = 17} received 25 mg/kg CAT dissolved in phosphate buffered saline, and control animals (n = 11} received nothing. Experiment IV treatment animals (n = 14) received 300 /~g of sodium selenate by gavage dissolved in demonized water, and control animals (n = 15) receiving nothing. Experiment V treatment animals (n = 20} were raised from 35 to 50 g size on a selenium deficient diet, and control animals were raised (n = 20) on a normal rat chow diet, until they weighed 100 g when ischemia was induced. At seven days, survival, incidence of bowel perforation or necrosis, and length of survival were compared in each experiment between control and treatment groups using X = analysis. SOD increased survival (P < .05) and length of survival (P < .02) and decreased the perforation rate (P < ,025) in animals subject to ischemic bowel injury. CAT, DMSO, and selenium treatment were not beneficial. These data suggest a critical role for superoxide anion in the pathogenesis of intestinal ischemia. INDEX WORDS: Ischemic bowel; oxygen-free radi- cals; superoxide dismutase; selenium; catalase; dimethyl sulfoxide. From the Section of Pediatric Surgery, Indiana University School of Medicine, and the James Whitcomb Riley Hospi- tal for Children, Indianapolis, Indiana. Presented before the 15th Annual Meeting of the Ameri- can Pediatric Association, Marco Beach, Florida, May 9-12, 1984. Address reprint requests to Jay L. Grosfeld, MD, Sur- geon-in-Chief J. IV. Riley Hospital for Children (K21), 702 Barnhill Drive, Indianapolis, IN 46223. 1984 by Grune & Stratton, Inc. 00 22- 3468/84/1906~0 20/$03.00/0 R ECENT REPORTS suggest that free radi- cal anions may play a role in the pathogen- esis of diseases as diverse as arthritis, gastritis, and ischemic injury of the brain, myocardium, and bowel as well as in the aging process. 1~ A number of oxygen-free radicals, including su- peroxide (02), hydroxyl radical (OH), and hydrogen peroxide (H202), have been implicated in these conditions either individually or collec- tively. Studies by Parks et al and Dalsing et al present data that suggest the superoxide anion may contribute to the pathogenesis of ischemic bowel injuryY This report further evaluates this theory by comparing the effects of enzymes or scavengers specific for each oxygen-free radical in an animal model of intestinal ischemia. MATERIALS AND METHODS We studied 204 male Sprague Dawley rats (wt 100 to 125g) in five separate experiments. All animals not receiving a selenium-deficient diet were fed a Wayne lab bloc rodent diet and received water ad lib. Animals were maintained in separate stainless steel cages and were randomly subdivided into control and treatment groups at the start of each experiment. Experiment I treatment animals (n = 20) received superoxide dismutase (SOD) 2.5 mg/kg intrave- nously (IV) dissolved in Ringer's lactate, while control ani- mals (n = 73) received Ringer's lactate alone. In experiment II, treatment animals (n = 16) received 1 cc of 100% dimethyl sulfoxide (DMSO) by intragastric garage, and concurrent control animals (n = 11) received nothing. Exper- iment III treatment animals received catalase (CAT) 25 mg/kg IV dissolved in phosphate buffered saline, and control (n = 11) animals received nothing. Experiment IV treatment animals (n = 14) received 300/zg sodium selenite dissolved in demonized water, by gastric gavage, and control animals (n = 15) received nothing. Experiment V animals were raised from 35 to 50 g size and were fed a control (n = 20) or selenium (Se)-deficient diet (n = 20) (TEKLAD Diet #84092), until they weighed 100 g when ischemia was induced. All animals underwent midline laparotomy under light ether anesthetic using clean technique. The small bowel and cecum was eviscerated to the left, and a Heifitz neurovascu- lar clip was placed across the SMA at the base of the small bowel mesentery for exactly one minute. During SMA occlu- sion, the mid and distal small bowel and cecum lost pulsations and became quite blanched. After removal of the clip at one minute, the b|anched bowel became extremely hyperemic and pulsations in the small bowel arterial arcades were clearly visible. All abdominal incisions were closed with running 4-O silk suture. 740 Journal of Pediatric Surgery, Vol. 19, No. 6 (December),1984