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Mucosal permeability after subclinical intestinal ischemia-reperfusion injury: An exploration of possible mechanisms
Mucosal Permeability After Subclinical Intestinal Ischemia-Reperfusion Injury: An Exploration of Possible Mechanisms By Jacob C. Langer, Sarvjit S. Sohal, and Patricia Blennerhassett Hamilton, Ontario and St Louis, Missouri • Changes in mucosal permeability may be important in the etiology of necrotizing enterocoiitis. The authors have previously shown that subclinical ischemia-reperfusion injury results in increased permeability in the rat intestine, and have partially characterized this phenomenon. In the present study the authors attempt to determine the mechanism by which these changes occur. Six-week-old rats underwent 10-minute superior mesenteric artery occlusion (SMAO) or sham, and mucosal permeability to SlCrEDTA was measured after 30 minutes. Rats were pretreated with saline, inhibitors of oxygen free radicals (superoxide dismutase + catalase, vitamin E, allopurinol, ~-phenyI-N-tert butyl-nitrone}, inhibitors of eicosanoids (indomethacin, quinacrine, diethylcarbamazine, 13-azaprostanoic acid), the putative cytoprotective agent prostaglandin E2, or the inhibitor of neutrophil free radical production fructose 1-6 diphosphate. None of the agents significantly attenuated the increase in mucosal permeability caused by SMAO, although indomethacin and prostaglandin E2 significantly exacerbated the permeability changes. To further explore the role of neutrophils, tissue myeloperoxidase was measured 30 minutes after SMAO. There was no significant difference in myeloperoxidase levels between sham and SMAO animals. These data suggest that the early increase in mucosal permeability after subclinical ischemia-reperfusion injury is not mediated by oxygen free radicals, eicosanoids, or neutrophils. The deleterious effect of indomethacin and prostaglandin E2 suggests a possible protective role for the cyclooxygenase system, but further studies are necessary to elucidate this possibility. Copyright © 1995by W.B. Saunders Company INDEX WORDS: Necrotizing enterocolitis, mucosal permeability, ischemia-reperfusion injury, oxygen free radicals, eicosanoids, neutrophils, prostaglandin E2.
HE ETIOLOGY of neonatal necrotizing entero-
T colitis (NEC) is probably multifactorial, involving intestinal ischemia, intraluminal substrate, and bacterial infection to varying degrees in individual patients. 1,2 We have previously suggested that a subclinical insult to the gut may cause an increase in
From the Intestinal Disease Research Unit and Department of Surgery, McMaster University, Hamilton, Ontario, and the Division of Pediatric Surgery, St Louis Children's Hospital, St Louis, MO. Supported by Medical Research Council of Canada Grant No. MA-10878. Address reprint requests to Jacob C. Langer, MD, Division of Pediatric Surgery, Room 5W12, St Louis Children's Hospital, One Children's Place, St Louis, MO 63110. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3004-0014503.00/0
568
mucosal permeability to bacteria and toxic intraluminal proteins. The presence of these factors within the bowel wall may then result in the inflammation characteristic of NEC. As one example of such a subclinical insult, we have used a weanling rat model to show that ischemia-reperfusion injury (IRI) results in an early, reversible increase in mucosal permeability to 5aCr EDTA. 3 These changes are mediated by local tissue factors,4 and are not mediated by histamine s or ameliorated by prenatal or postnatal steroid administration.6 In a further study, we showed that mucosal permeability is altered in a similar fashion by a variety of insults to the immature rat intestine, 7 suggesting that mucosal permeability may be a common final pathway in the etiology of NEC. In the current study, we have further investigated the mechanism by which this increase in permeability occurs. Using our previous model of IRI in the weanling rat, we have tested the effect of various blockers in ameliorating the mucosal permeability changes after IRI and have examined the role of neutrophils in this process. MATERIALS AND METHODS
Animals Weanling male Sprague-Dawley rats aged 6 weeks (150 to 180 g) were housed under standard conditions, fed Purina (St Louis, MO) rat chow, and given only water for 12 hours before all experiments. All studies were approved by the McMaster University Committee on Animal Experimentation.
Superior Mesenteric Artery Occlusion Details of this technique have been previously described.3 Anesthesia was induced using intraperitoneal pentobarbital (45 mg/kg). A midline laparotomy was made, the small intestine was reflected to the left, and the superior mesenteric artery was exposed. The artery was occluded with a microaneurysm clip, taking care not to occlude the superior mesenteric vein. To ensure adequate occlusion, the bowel was examined for pallor and absence of pulsation in the mesenteric vessels. Upon reperfusion, the bowel was noted to become pink, and return of mesenteric pulsation was seen. Anesthesia was maintained by periodic intraperitoneal administration of pentobarbital (5 to 10 mg/kg). Sham operation involved the same technique and exposure, without clipping of the superior mesenteric artery.
Measurement of Mucosal Permeability A technique modified from Ramage was used. 3,8 The ileum was ligated 20 cm from the cecum and at the ileocecal junction, taking
Journal of Pediatric Surgery, Vo130, No 4 (April), 1995: pp 568-572
ISCHEMIA-REPERFUSION INJURY
care not to interfere with the blood supply to the isolated segment. At the time of intestinal reperfusion, 1.0 mL of 51Cr EDTA solution (0.065 mCi/mL in distilled water, McMaster Pharmacy Dept) was injected into the ileal segment. Blood was sampled (0.5 mL per sample) 30 minutes after reperfusion. Samples were counted using a 1282 Compugamma counter (LKB-Wallac Corporation, Turku, Finland). Results were expressed as the number of counts per sample/number of counts in a 10 I~L standard of 5aCr EDTA used in that day's experiment.
Blocker Experiments In all of the following experiments, rats underwent sham procedure or 10-minute superior mesenteric artery occlusion (SMAO), followed by assessment of mucosal permeability as described above. In the control group, 1 mL of normal saline was given intraperitoneally 1 hour before the experiment, and 1 mL was given intravenously at the time of reperfusion. All blockers were obtained from Sigma Chemical Corp (St Louis, MO). The dose and route of administration of each drug was based on previous reports of effectiveness in rodent models, 9-17and will be described below. Oxygenfree radicals. The role of oxygen free radicals was tested by pretreating the rats with one of several agents known to block the action of free radicals through a number of different mechanisms. The following agents were used: (1) superoxide dismutase (SOD) plus catalase, 5,000 units/kg of each before reperfusion, followed by an intravenous infusion of 18,000 units/kg/h during the following 30 minutes; (2) vitamin E (e~-tocopherol), 100 mg/kg by gavage for 7 days before the experiment, and 1 hour before the experiment; (3) allopurinol, 100 mg/kg by gavage for 2 days before the experiment, and 100 mg/kg intravenously before reperfusion; (4) e~-phenyl-N-tert-butyl-nitrone (PBN), 125 mg/kg intraperitoneally 1 hour before the experiment, and 125 mg/kg into the lumen of the isolated bowel loop at the time of reperfusion. Eicosanoids. The role of eicosanoids was tested by pretreating the rats with one of several agents known to block arachadonic acid metabolism, through a number of different mechanisms. The following agents were used: (1) indomethacin, a cyclooxygenase inhibitor, 5 mg/kg by gavage the day before the experiment, and intravenously at the time of reperfusion; (2) quinacrine, an inhibitor of phospholipase A2, 20 mg/kg intravenously at the time of reperfusion; (3) diethylcarbamazine, an inhibitor of lipoxygenase, 350 mg/kg intravenously at the time of reperfusion; (4) 13-azaprostanoic acid, a thromboxane receptor antagonist, 0.05 mg/kg intravenously at the time of repeffusion. 16,16-dimethylprostaglandin E2 (PGE2). The role of this putative cytoprotective agent was tested by pretreating the rats with PGE2, 300 mg/kg by gavage for 2 days before the experiment, and by injection into the lumen of the isolated bowel loop at the time of reperfusion. Fructose 1-6 diphosphate (FDP). This compound, which inhibits generation of oxygen free radicals by activated neutrophils, was tested by pretreating the rats with FDP, 350 mg/kg intraperitoneally for 2 days before the experiment, and intravenously at the time of reperfusion.
Measurement of Tissue Myeloperoxidase Myeloperoxidase is a neutrophil granule enzyme that acts as a marker for tissue neutrophil infiltration) 8 Tissue levels were assessed after either 10-minute SMAO or sham procedure. Thirty minutes after reperfusion, a segment of terminal ileum was excised, and the animal was killed. Myeloperoxidase levels in homogenized tissue samples were measured using the adsorbance assay originally described by Smith and modified by Bradley et al) 9 Myeloper-
569
oxidase was expressed as units of activity per gram of wet tissue, where i unit is defined as the quantity of myeloperoxidase required to convert 1 ixmol of hydrogen peroxide to water in 1 minute at 22°C. Control and experimental samples were examined in parallel, and values were assessed relative to a standard curve.
Data Analysis Data from each blocker group undergoing SMAO was compared with the saline group undergoing SMAO. To rule out an independent effect of blockers on mucosal permeability, each blocker group undergoing sham procedure was compared with the saline group undergoing sham procedure. All comparisons were done using an unpaired Student's t test, in which a P value of .05 was considered statistically significant.
RESULTS
Blocker Experiments The results of these experiments are summarized in Table 1. None of the oxygen free radical blockers significantly altered the degree of permeability after SMAO. Allopurinol appeared to have some effect in attenuating the increase in permeability, although this was not statistically significant. Diethylcarbamazine and 13-azaprosanoic acid had no effect on permeability, but both indomethacin and quinacrine exacerbated the increase in permeability (only indomethacin reached statistical significance). PGE2 significantly exacerbated the increase in permeability, and FDP had no effect.
Measurement of Tissue Myeloperoxidase There was no significant difference in myeloperoxidase levels between sham and SMAO animals (0.26 _+ .03 versus 0.28 _+ .07 units per gram) (five rats per group).
Table 1. Mucosal Permeability to 51CrEDTA 30 Minutes After 10-Minute SMAO or Sham Procedure Sham Group (n)
SMAO Group (n)
Saline SOD + catalase Vitamin E AIIopurinol PBN
Pretreatment
1.04 1.44 1.02 0.94 1.10
_+ 0.4 _+ 1,0 -+ 0.2 -+ 0.3 -+ 0.2
(10) (7) (5) (5) (5)
3.89 3.93 3.99 2.78 3.72
-+ 1.9 -+ 2.1 _+ 1.5 - 0.8 -+ 1.I
(9) (5) (5) (5) (5)
Indomethacin Quinacrine Diethylcarbamazine 13-Azaprostanoic acid PGE2 FDP
1.06 0.96 0.86 0.89 1.08 0.92
-+ 0.5 _+ 0.4 -+ 0.3 _+ 0.2 -+ 0.3 -+ 0.4
(5) (6) (7) (5) (5) (5)
7.00 6.61 4,06 3.72 10.99 3.54
+- 3.7 _+ 3.3 -+ 2.4 _+ 2.0 -+ 7.1 _+ 1.4
(5)* (6) (6) (5) (5)* (7)
NOTE. The increase in permeability was not attenuated by any of the pretreatments. There was significantly greater permeability in the rats that received PGE2 or indomethacin when compared with those that received saline. *P < .05.
570
LANGER, SOHAL, AND BLENNERHASSETT
DISCUSSION
The events that follow ischemia-reperfusion injury are complex and incompletely understood. 2° Mechanisms likely differ according to the length of ischemia, the specific tissue involved, and the species studied. In addition, the relative roles of ischemia and reperfusion in this phenomenon are still under debate. 21 In our model of short-term, subclinical ischemia-reperfusion injury, mucosal permeability is transiently increased, with a maximal effect noted 30 minutes after reperfusion. 3 We have previously determined that this phenomenon is mediated by release of local tissue factors. 4 Mediators that have been implicated in other systems of ischemia-reperfusion injury include histamine, oxygen free radicals, eicosanoids, and neutrophil factors, although it is likely that many events and mechanisms are involved simultaneously in most cases. We have previously shown that histamine does not mediate the permeability changes in our model. 5 In the present study we examined the roles of free radicals, eicosanoids, and neutrophils. Oxygen free radicals are thought to be generated during the reperfusion phase, with the addition of oxygen to previously ischemic tissues causing the formation of reactive oxygen species? 2 These radicals may be formed through the xanthine oxidase system, 23,24 neutrophil NADPH oxidase, 25 glutathione peroxidase, 26 or other mechanisms. 27 Oxygen free radicals cause epithelial damage largely through the peroxidation of lipid membranes, 28although they may also contribute to tissue damage by attracting neutrophils 29 and exaggerating eicosanoid release. 3° The premature neonate is thought to be more susceptible to free radical-induced injury because of increased levels of xanthine oxidase, 3~ decreased endogenous scavengers such as S O D , 32 and increased sensitivity of lipoproteins to oxidative stress, 33 all of which may contribute to the higher incidence of NEC in this population. We have tested the hypothesis that free radicals mediate the permeability changes in our model by attempting to block these changes at a number of different steps. SOD and catalase are free radical "scavengers," which together promote the conversion of reactive oxygen metabolites to oxygen and water. They have been successful in preventing mucosal injury in a number of intestinal ischemia-reperfusion injury models. 9,34,35 PBN is a "spin trapping" agent that was developed for analysis of free radicals in vitro, but that distributes widely to tissues when injected intraperitoneally, 36 and has been shown to decrease free radical-induced injury in rats. 12 Vitamin E has been shown to attenuate the peroxidation
of lipid membranes by free radicals, 1° and has been effective in ameliorating intestinal mucosal damage in a rabbit model of intestinal ischemia. 37 Allopurinol blocks xanthine oxidase, which generates free radicals through the conversion of hypoxanthine to xanthine in the presence of oxygen during reperfusion. Allopurinol has been shown in a number of models to attenuate free radical-induced intestinal injury, 23,38 although additional mechanisms other than xanthine oxidase inhibition have been proposed. 11,39 In our study the only agent in this group that had any effect on mucosal permeability was allopurinol, although the effect was not statistically significant. We conclude from these data that free radicals are not an important mediator of the permeability changes seen in our model. The products of arachidonic acid metabolism (eicosanoids) are generated either through the cyclooxygenase pathway (prostaglandins and thromboxanes) or the lipoxygenase pathway (leukotrienes). 4° Arachi, donic acid itself is generated from cell membrane phospholipids by the action of phospholipase A2.4° Eicosanoids are known to be mediators of intestinal ischemic injury, 41'42 and have been implicated in the pathogenesis of experimental 43 and clinical 44NEC. In our study, there was no attenuation of permeability changes by blockade of phospholipase A2, lipoxygenase, or thromboxane. However, we did see exacerbation of permeability changes by the cyclooxygenase inhibitor indomethacin. Although indomethacin has been shown to attenuate the hypotension seen in intestinal ischemia-reperfusion injury, 45 it is known to have an independent deleterious effect on intestinal mucosai permeability. 7,46 Despite being an eicosanoid, PGE2 has previously been shown to be a "cytoprotective" agent in several models of mucosal injury, 16'47'48 including injury induced by indomethacin. 49The profound exacerbation of mucosal permeability by PGE2 in our model was unexpected. The significant adverse effects of indomethacin and PGE2, and the milder adverse effect of quinacrine, suggest a possible role for eicosanoids, through the cyclooxygenase pathway, in protecting the mucosa after subclinical ischemia-reperfusion injury. The lack of effect of any of these agents on baseline permeability in sham-operated animals substantiates this theory by implying no direct toxicity to the normal intestinal mucosa. Further studies will be necessary to further elucidate this possibility. Neutrophils are also known to play a role in intestinal ischemia-reperfusion injury, both at the tissue level5°,5a and systemically.52 The role of neutrophils is complex. They are attracted by free radicals
ISCHEMIA-REPERFUSION INJURY
571
and eicosanoids, and also release these agents, t8 In our model, the lack of effect of FDP and lack of an increase in myeloperoxidase activity do not support a role for neutrophils in mediating the permeability
increase after subclinical ischemia-reperfusion injury. Despite this, however, it is likely that neutrophils do play an important role in subsequent events during the pathogenesis of NEC. 53
REFERENCES 1. Kliegman RM: Models of the pathogenesis of necrotizing enterocolitis. J Pediatr 117:$2-$5, 1990 (suppl) 2. Kosloske AM: A unifying hypothesis for pathogenesis and prevention of necrotizing enterocolitis. J Pediatr 117:$68-74, 1990 (suppl) 3. Langer JC, Sohal SS, Riddell RH: Mucosal permeability to 51Cr EDTA following subclinical intestinal ischemia-reperfusion injury in the weanling rat. J Pediatr Surg 28:601-605, 1993 4. Langer JC, Sohal SS: Increased mucosal permeability after ischemia-reperfusion injury is mediated by local tissue factors. J Pediatr Surg 27:329-332, 1992 5. Langer JC, Sohal SS, Marshall J: Histamine does not mediate mucosal permeability changes after subclinical intestinal ischemiareperfusion injury. J Pediatr Surg 28:1113-1116, 1993 6. Langer JC, Sohal SS: Corticosteroids do not alter mucosal permeability after subclinical intestinal ischemia-reperfusion injury in the rat. Pediatr Surg Int 9:66-69, 1994 7. Langer JC, Sohal SS, Mumford DA: Mucosal permeability in the immature rat intestine: Effects of ischemia-reperfusion, cold stress, hypoxia, and drugs. J Pediatr Surg 28:1380-1385, 1993 8. Ramage JK, St~nisz A, Scicchitano R, et al: Effect of immunological reactions on rat intestinal epithelium. Correlation of increased gut permeability to 51Cr-EDTA and ovalbumin during inflammation and anaphylaxis in the rat. Gastroenterology 94:1368-1375, 1988 9. Mittal VK, Dewan S, Amaria Y, et al: Role of oxygen free radical scavengers in preservation of intestinal villi in the presence of ischemia. Transplant Proc 20:1045-1047, 1988 10. Tappel AL, Dillard CJ: In vivo lipid peroxidation: Measurement via exhaled pentane and protection by vitamin E. Federation Proc 40:174-178, 1981 11. Garcia JG, Rollan CM, Enrinquez MAR, et al: Improved survival in intestinal ischemia by allopurinol not related to xanthineoxidase inhibition. J Surg Res 48:144-146, 1990 12. Janzen EG, Towner RA, Yamashiro S: The effect of phenyl tert-butyl nitrone (PBN) on CC14-induced rat liver injury detected by proton magnetic resonance imaging (MRI) in vivo and electron microscopy (EM). Free Radic Res Commun 9:325-335, 1990 13. Grosfeld JL, Kamman K, Gross K, et al: Comparative effects of indomethacin, prostaglandin El, and ibuprofen on bowel ischemia. J Pediatr Surg 18:738-741, 1983 14. Otamiri T, Lindahl M, Tagesson C: Phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia in rats. Gut 29:489-494, 1988 15. Hsueh W, Gonzalez-Crussi F, Arroyave JL: Plateletactivating factor-induced ischemic bowel necrosis: An investigation of secondary mediators in its pathogenesis. Am J Pathol 122:231239, 1986 16. Lancaster C, Robert A: Intestinal lesions produced by prednisolone: Prevention (cytoprotection) by 16,16-dimethyl prostaglandin E2. Am J Physio1235:E703-E708, 1978 17. Sun J, Farias LA, Markov AK: Fructose 1-6 diphosphate prevents intestinal ischemic reperfusion injury and death in rats. Gastroenterol 98:117-126, 1990 18. Anderson BO, Brown JM, Harken AH: Mechanisms of neutrophil-mediated tissue injury. J Surg Res 51:170-179, 1991
19. Bradley PP, Priebat DA, Christensen RD, et al: Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206-209, 1982 20. Bulkley GB: Free radicals and other reactive oxygen metabolites: Clinical relevance and the therapeutic efficacy of antioxidant therapy. Surgery 113:479-483, 1993 21. Parks DA, Granger DN: Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 250:G749G753, 1986 22. McCord JM: Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med 312:159-163, 1985 23. Granger DN, McCord JM, Parks DA, et al: Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroentero190:80-84, 1986 24. Roldan EJA, Pinus CR, Turrens JF, et al: Chemiluminescence of ischaemic and reperfused intestine in vivo. Gut 30:184187, 1989 25. Grisham WB, Granger DN: Neutrophil-mediated mucosal injury: Role of reactive oxygen metabolites. Dig Dis Sci 33(suppl): 6S-15S, 1988 26. Schoenberg MH, Muhl E, Sellin D, et al: Posthypotensive generation of superoxide free radicals-possible role in the pathogenesis of the intestinal mucosal damage. Acta Chit Scand 150:301309, 1984 27. Schoenberg MH, Fredholm BB, Haglund U, et al: Studies on the oxygen radical mechanism involved in the small intestinal reperfusion damage. Acta Physiol Scand 124:581-589, 1985 28. Tien M, Svingen BA, Aust SD: Superoxide dependent lipid peroxidation. Federation Proc 40:179-182, 1981 29. Petrone WF, English DK, Wong K, et al: Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci 77:1159-1163, 1980 30. Miller MJS, McNeill H, Mullane KM, et al: SOD prevents damage and attenuates eicosanoid release in a rabbit model of necrotizing enterocolitis. Am J Physiol 255:G556-G565, 1988 31. Vettenranta K, Raivio KO: Xanthine oxidase during human fetal development. Pediatr Res 27:286-288, 1990 32. Kugo M, Sano K, Uetani Y, et al: Superoxide dismutase in polymorphonuclear leukocytes of term newborn infants and very low birth weight infants. Pediatr Res 26:227-231, 1989 33. Ogihara T, Kitagawa M, Miki M, et al: Susceptibility of neonatal lipoproteins to oxidative stress. Pediatr Res 29:39-45, 1991 34. Granger DN, Rutili G, McCord JM: Superoxide radicals in feline intestinal ischemia. Gastroenterology 81:22-29, 1981 35. Vohra K, Rosenfeld W, Singh I, et al: Ischemic injury to newborn rabbit ileum: Protective role of human superoxide dismutase. J Pediatr Surg 24:893-898, 1989 36. Chen GM, Bray TM, Janzen EG, et al: Excretion, metabolism and tissue distribution of a spin trapping agent, alpha-phenylN-tert-butyl-nitrone (PBN) in rats. Free Radic Res Commun 9:317-323, 1990 37. Telang VM, Stahl RE, Clark DA, et al: Ischemic injury to rabbit ileum: protective role of vitamin E (alpha-tocopherol: T). Pediatr Res 20:577A, 1986 (abstr)
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38. Megison SM, Horton JW, Chao H, et al: Prolonged survival and decreased mucosal injury after low-dose enteral allopurinol prophylaxis in mesenteric ischemia. J Pediatr Surg 25:917-921, 1990 39. Zimmerman BJ, Parks DA, Grisham MB, et al: Allopnrinol does not enhance antioxidant properties of extracellular fluid. Am J Physiol 255:H202-H206, 1988 40. Main IHM: Pharmacology of prostaglandins. Postgrad Med J 64:3-6, 1988 (suppl 1) 41. Hsueh W, Gonzalez-Crussi F, Arroyave JL: Release of lenkotriene C4 by isolated, perfused rat small intestine in response to platelet-activating factor. J Clin Invest 78:108-114, 1986 42. Mangino MJ, Anderson CB, Murphy MK, et al: Mucosal arachidonate metabolism and intestinal ischemia-reperfusion injury. Am J Physiol 257:G299-307, 1989 43. Miller MJS, Clark DA: Profile and sites of eicosanoid release in experimental necrotizing enterocolitis, in Samuelsson B, Wong PY-K, Sun FF (eds): Advances in Prostaglandin, Thromboxane, and Leukotriene Research. New York, Raven Press, 1989, pp 556-559 44. Hyman PE, Abrams CE, Zipser RD: Enhanced urinary immunoreactive thromboxane in neonatal necrotizing enterocolitis: a diagnostic indicator of thrombotic activity. Am J Dis Child 141:686-689, 1987 45. Mozes T, Wieszt E, Zahajszky T: Role of PGF2-alpha in the superior mesenteric artery-induced shock. J Surg Res 47:476-481, 1989
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46. Bjarnason I, Williams P, Smethurst P, et al: The effect of nonsteroidal antiinflammatory drugs and prostaglandins on the permeability of the small intestine. Gut 27:1292-1297, 1986 47. Miller TA: Protective effects of prostaglandins against gastric mucosal damage: Current knowledge and proposed mechanisms. Am J Physio1245:G601-G623, 1983 48. Wallace JL, Whittle B JR, Boughton-Smith NK: Prostaglandin protection of rat colonic mucosa from damage induced by ethanol. Dig Dis Sci 30:866-876, 1985 49. Whittle B JR: Mechanisms underlying gastric mucosal damage induced by indomethacin and bile-salts, and the actions of prostaglandins. Br J Pharmacol 60:455-460, 1977 50. Otamiri T: Oxygen radicals, lipid peroxidation, and neutrophil infiltration after small-intestinal ischemia and reperfusion. Surgery 105:593-597, 1989 51. Brown MF, Ross AJ, Dasher J, et al: The role of leukocytes in mediating injury of intestinal ischemia/reperfusion. J Pediatr Surg 25:214-217, 1990 52. Schmeling DJ, Caty MG, Oldham KT, et al: Evidence for neutrophil-related acute lung injury after intestinal ischemiareperfusion. Surgery 106:195-202, 1989 53. Clark DA, Mullane K, Caravella S, et al: Eicosanoid and leukocyte participation in experimental necrotizing enterocolitis. Pediatr Res 21:407A, 1987 (abstr)
HE ETIOLOGY of neonatal necrotizing entero-
T colitis (NEC) is probably multifactorial, involving intestinal ischemia, intraluminal substrate, and bacterial infection to varying degrees in individual patients. 1,2 We have previously suggested that a subclinical insult to the gut may cause an increase in
From the Intestinal Disease Research Unit and Department of Surgery, McMaster University, Hamilton, Ontario, and the Division of Pediatric Surgery, St Louis Children's Hospital, St Louis, MO. Supported by Medical Research Council of Canada Grant No. MA-10878. Address reprint requests to Jacob C. Langer, MD, Division of Pediatric Surgery, Room 5W12, St Louis Children's Hospital, One Children's Place, St Louis, MO 63110. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3004-0014503.00/0
568
mucosal permeability to bacteria and toxic intraluminal proteins. The presence of these factors within the bowel wall may then result in the inflammation characteristic of NEC. As one example of such a subclinical insult, we have used a weanling rat model to show that ischemia-reperfusion injury (IRI) results in an early, reversible increase in mucosal permeability to 5aCr EDTA. 3 These changes are mediated by local tissue factors,4 and are not mediated by histamine s or ameliorated by prenatal or postnatal steroid administration.6 In a further study, we showed that mucosal permeability is altered in a similar fashion by a variety of insults to the immature rat intestine, 7 suggesting that mucosal permeability may be a common final pathway in the etiology of NEC. In the current study, we have further investigated the mechanism by which this increase in permeability occurs. Using our previous model of IRI in the weanling rat, we have tested the effect of various blockers in ameliorating the mucosal permeability changes after IRI and have examined the role of neutrophils in this process. MATERIALS AND METHODS
Animals Weanling male Sprague-Dawley rats aged 6 weeks (150 to 180 g) were housed under standard conditions, fed Purina (St Louis, MO) rat chow, and given only water for 12 hours before all experiments. All studies were approved by the McMaster University Committee on Animal Experimentation.
Superior Mesenteric Artery Occlusion Details of this technique have been previously described.3 Anesthesia was induced using intraperitoneal pentobarbital (45 mg/kg). A midline laparotomy was made, the small intestine was reflected to the left, and the superior mesenteric artery was exposed. The artery was occluded with a microaneurysm clip, taking care not to occlude the superior mesenteric vein. To ensure adequate occlusion, the bowel was examined for pallor and absence of pulsation in the mesenteric vessels. Upon reperfusion, the bowel was noted to become pink, and return of mesenteric pulsation was seen. Anesthesia was maintained by periodic intraperitoneal administration of pentobarbital (5 to 10 mg/kg). Sham operation involved the same technique and exposure, without clipping of the superior mesenteric artery.
Measurement of Mucosal Permeability A technique modified from Ramage was used. 3,8 The ileum was ligated 20 cm from the cecum and at the ileocecal junction, taking
Journal of Pediatric Surgery, Vo130, No 4 (April), 1995: pp 568-572
ISCHEMIA-REPERFUSION INJURY
care not to interfere with the blood supply to the isolated segment. At the time of intestinal reperfusion, 1.0 mL of 51Cr EDTA solution (0.065 mCi/mL in distilled water, McMaster Pharmacy Dept) was injected into the ileal segment. Blood was sampled (0.5 mL per sample) 30 minutes after reperfusion. Samples were counted using a 1282 Compugamma counter (LKB-Wallac Corporation, Turku, Finland). Results were expressed as the number of counts per sample/number of counts in a 10 I~L standard of 5aCr EDTA used in that day's experiment.
Blocker Experiments In all of the following experiments, rats underwent sham procedure or 10-minute superior mesenteric artery occlusion (SMAO), followed by assessment of mucosal permeability as described above. In the control group, 1 mL of normal saline was given intraperitoneally 1 hour before the experiment, and 1 mL was given intravenously at the time of reperfusion. All blockers were obtained from Sigma Chemical Corp (St Louis, MO). The dose and route of administration of each drug was based on previous reports of effectiveness in rodent models, 9-17and will be described below. Oxygenfree radicals. The role of oxygen free radicals was tested by pretreating the rats with one of several agents known to block the action of free radicals through a number of different mechanisms. The following agents were used: (1) superoxide dismutase (SOD) plus catalase, 5,000 units/kg of each before reperfusion, followed by an intravenous infusion of 18,000 units/kg/h during the following 30 minutes; (2) vitamin E (e~-tocopherol), 100 mg/kg by gavage for 7 days before the experiment, and 1 hour before the experiment; (3) allopurinol, 100 mg/kg by gavage for 2 days before the experiment, and 100 mg/kg intravenously before reperfusion; (4) e~-phenyl-N-tert-butyl-nitrone (PBN), 125 mg/kg intraperitoneally 1 hour before the experiment, and 125 mg/kg into the lumen of the isolated bowel loop at the time of reperfusion. Eicosanoids. The role of eicosanoids was tested by pretreating the rats with one of several agents known to block arachadonic acid metabolism, through a number of different mechanisms. The following agents were used: (1) indomethacin, a cyclooxygenase inhibitor, 5 mg/kg by gavage the day before the experiment, and intravenously at the time of reperfusion; (2) quinacrine, an inhibitor of phospholipase A2, 20 mg/kg intravenously at the time of reperfusion; (3) diethylcarbamazine, an inhibitor of lipoxygenase, 350 mg/kg intravenously at the time of reperfusion; (4) 13-azaprostanoic acid, a thromboxane receptor antagonist, 0.05 mg/kg intravenously at the time of repeffusion. 16,16-dimethylprostaglandin E2 (PGE2). The role of this putative cytoprotective agent was tested by pretreating the rats with PGE2, 300 mg/kg by gavage for 2 days before the experiment, and by injection into the lumen of the isolated bowel loop at the time of reperfusion. Fructose 1-6 diphosphate (FDP). This compound, which inhibits generation of oxygen free radicals by activated neutrophils, was tested by pretreating the rats with FDP, 350 mg/kg intraperitoneally for 2 days before the experiment, and intravenously at the time of reperfusion.
Measurement of Tissue Myeloperoxidase Myeloperoxidase is a neutrophil granule enzyme that acts as a marker for tissue neutrophil infiltration) 8 Tissue levels were assessed after either 10-minute SMAO or sham procedure. Thirty minutes after reperfusion, a segment of terminal ileum was excised, and the animal was killed. Myeloperoxidase levels in homogenized tissue samples were measured using the adsorbance assay originally described by Smith and modified by Bradley et al) 9 Myeloper-
569
oxidase was expressed as units of activity per gram of wet tissue, where i unit is defined as the quantity of myeloperoxidase required to convert 1 ixmol of hydrogen peroxide to water in 1 minute at 22°C. Control and experimental samples were examined in parallel, and values were assessed relative to a standard curve.
Data Analysis Data from each blocker group undergoing SMAO was compared with the saline group undergoing SMAO. To rule out an independent effect of blockers on mucosal permeability, each blocker group undergoing sham procedure was compared with the saline group undergoing sham procedure. All comparisons were done using an unpaired Student's t test, in which a P value of .05 was considered statistically significant.
RESULTS
Blocker Experiments The results of these experiments are summarized in Table 1. None of the oxygen free radical blockers significantly altered the degree of permeability after SMAO. Allopurinol appeared to have some effect in attenuating the increase in permeability, although this was not statistically significant. Diethylcarbamazine and 13-azaprosanoic acid had no effect on permeability, but both indomethacin and quinacrine exacerbated the increase in permeability (only indomethacin reached statistical significance). PGE2 significantly exacerbated the increase in permeability, and FDP had no effect.
Measurement of Tissue Myeloperoxidase There was no significant difference in myeloperoxidase levels between sham and SMAO animals (0.26 _+ .03 versus 0.28 _+ .07 units per gram) (five rats per group).
Table 1. Mucosal Permeability to 51CrEDTA 30 Minutes After 10-Minute SMAO or Sham Procedure Sham Group (n)
SMAO Group (n)
Saline SOD + catalase Vitamin E AIIopurinol PBN
Pretreatment
1.04 1.44 1.02 0.94 1.10
_+ 0.4 _+ 1,0 -+ 0.2 -+ 0.3 -+ 0.2
(10) (7) (5) (5) (5)
3.89 3.93 3.99 2.78 3.72
-+ 1.9 -+ 2.1 _+ 1.5 - 0.8 -+ 1.I
(9) (5) (5) (5) (5)
Indomethacin Quinacrine Diethylcarbamazine 13-Azaprostanoic acid PGE2 FDP
1.06 0.96 0.86 0.89 1.08 0.92
-+ 0.5 _+ 0.4 -+ 0.3 _+ 0.2 -+ 0.3 -+ 0.4
(5) (6) (7) (5) (5) (5)
7.00 6.61 4,06 3.72 10.99 3.54
+- 3.7 _+ 3.3 -+ 2.4 _+ 2.0 -+ 7.1 _+ 1.4
(5)* (6) (6) (5) (5)* (7)
NOTE. The increase in permeability was not attenuated by any of the pretreatments. There was significantly greater permeability in the rats that received PGE2 or indomethacin when compared with those that received saline. *P < .05.
570
LANGER, SOHAL, AND BLENNERHASSETT
DISCUSSION
The events that follow ischemia-reperfusion injury are complex and incompletely understood. 2° Mechanisms likely differ according to the length of ischemia, the specific tissue involved, and the species studied. In addition, the relative roles of ischemia and reperfusion in this phenomenon are still under debate. 21 In our model of short-term, subclinical ischemia-reperfusion injury, mucosal permeability is transiently increased, with a maximal effect noted 30 minutes after reperfusion. 3 We have previously determined that this phenomenon is mediated by release of local tissue factors. 4 Mediators that have been implicated in other systems of ischemia-reperfusion injury include histamine, oxygen free radicals, eicosanoids, and neutrophil factors, although it is likely that many events and mechanisms are involved simultaneously in most cases. We have previously shown that histamine does not mediate the permeability changes in our model. 5 In the present study we examined the roles of free radicals, eicosanoids, and neutrophils. Oxygen free radicals are thought to be generated during the reperfusion phase, with the addition of oxygen to previously ischemic tissues causing the formation of reactive oxygen species? 2 These radicals may be formed through the xanthine oxidase system, 23,24 neutrophil NADPH oxidase, 25 glutathione peroxidase, 26 or other mechanisms. 27 Oxygen free radicals cause epithelial damage largely through the peroxidation of lipid membranes, 28although they may also contribute to tissue damage by attracting neutrophils 29 and exaggerating eicosanoid release. 3° The premature neonate is thought to be more susceptible to free radical-induced injury because of increased levels of xanthine oxidase, 3~ decreased endogenous scavengers such as S O D , 32 and increased sensitivity of lipoproteins to oxidative stress, 33 all of which may contribute to the higher incidence of NEC in this population. We have tested the hypothesis that free radicals mediate the permeability changes in our model by attempting to block these changes at a number of different steps. SOD and catalase are free radical "scavengers," which together promote the conversion of reactive oxygen metabolites to oxygen and water. They have been successful in preventing mucosal injury in a number of intestinal ischemia-reperfusion injury models. 9,34,35 PBN is a "spin trapping" agent that was developed for analysis of free radicals in vitro, but that distributes widely to tissues when injected intraperitoneally, 36 and has been shown to decrease free radical-induced injury in rats. 12 Vitamin E has been shown to attenuate the peroxidation
of lipid membranes by free radicals, 1° and has been effective in ameliorating intestinal mucosal damage in a rabbit model of intestinal ischemia. 37 Allopurinol blocks xanthine oxidase, which generates free radicals through the conversion of hypoxanthine to xanthine in the presence of oxygen during reperfusion. Allopurinol has been shown in a number of models to attenuate free radical-induced intestinal injury, 23,38 although additional mechanisms other than xanthine oxidase inhibition have been proposed. 11,39 In our study the only agent in this group that had any effect on mucosal permeability was allopurinol, although the effect was not statistically significant. We conclude from these data that free radicals are not an important mediator of the permeability changes seen in our model. The products of arachidonic acid metabolism (eicosanoids) are generated either through the cyclooxygenase pathway (prostaglandins and thromboxanes) or the lipoxygenase pathway (leukotrienes). 4° Arachi, donic acid itself is generated from cell membrane phospholipids by the action of phospholipase A2.4° Eicosanoids are known to be mediators of intestinal ischemic injury, 41'42 and have been implicated in the pathogenesis of experimental 43 and clinical 44NEC. In our study, there was no attenuation of permeability changes by blockade of phospholipase A2, lipoxygenase, or thromboxane. However, we did see exacerbation of permeability changes by the cyclooxygenase inhibitor indomethacin. Although indomethacin has been shown to attenuate the hypotension seen in intestinal ischemia-reperfusion injury, 45 it is known to have an independent deleterious effect on intestinal mucosai permeability. 7,46 Despite being an eicosanoid, PGE2 has previously been shown to be a "cytoprotective" agent in several models of mucosal injury, 16'47'48 including injury induced by indomethacin. 49The profound exacerbation of mucosal permeability by PGE2 in our model was unexpected. The significant adverse effects of indomethacin and PGE2, and the milder adverse effect of quinacrine, suggest a possible role for eicosanoids, through the cyclooxygenase pathway, in protecting the mucosa after subclinical ischemia-reperfusion injury. The lack of effect of any of these agents on baseline permeability in sham-operated animals substantiates this theory by implying no direct toxicity to the normal intestinal mucosa. Further studies will be necessary to further elucidate this possibility. Neutrophils are also known to play a role in intestinal ischemia-reperfusion injury, both at the tissue level5°,5a and systemically.52 The role of neutrophils is complex. They are attracted by free radicals
ISCHEMIA-REPERFUSION INJURY
571
and eicosanoids, and also release these agents, t8 In our model, the lack of effect of FDP and lack of an increase in myeloperoxidase activity do not support a role for neutrophils in mediating the permeability
increase after subclinical ischemia-reperfusion injury. Despite this, however, it is likely that neutrophils do play an important role in subsequent events during the pathogenesis of NEC. 53
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