Comparison of consequent small bowel anastomoses after transient ischemia: An experimental study in rats

Comparison By Billur

of Consequent Small Bowel Anastomoses After Ischemia: An Experimental Study in Rats Demirogullan,

Kaan Sijnmez, Zafer Nurten Ttirkijzkan,

Tijrkyllmaz, Gtilgen Ekingen, Ayse A. Can Baaaklar, and Nuri Kale

Ankara,

Background/Purpose: The role of ischemia/reperfusion (I/R) damage on intestinal anastomotic healing remains to be precisely determined. The objective of this study was to investigate healing of small bowel anastomoses performed at different times after transient ischemia. Methods:Thirty male Wistar-Albino rats were investigated in five groups (four study and one control). Under general anesthesia, the superior mesenteric artery (SMA) was occluded for 40 minutes in the study rats. Biopsy specimens, to document I/R histopathology, were obtained before small intestinal anastomoses at 20 minutes (group I), 90 minutes (group 21, 6 hours (group 31, and 24 hours (group 4) after reperfusion. In a control group, biopsy and intestinal anastomoses were performed after SMA disection without occlusion. The rats were relaparotomized on the fifth day to determine in situ bursting pressures and to obtain specimens for hydroxyproline content and histopathologic evaluation.

H

EALING of intestinal anastomoses is generally assessed by the principles of wound healing. Although a subject of extensive research, the effects of ischemia4reperfusion (I/R) on intestinal anastomotic healing have not been studied sufficiently. In the present study we aimed to compare the small bowel anastomoses performed at different times during the reperfusion period following transient ischemia, a condition that pediatric surgeons can face in the events of necrotizing enterocolitis, hypovolemia, inflammatory bowel diseases, pull-through operations, cardiac insufficiency, free pedicled bowel segment usage, and small bowel transplantation.1-3 MATERIALS

AND

METHODS

All groups were relaparotomized on the fifth postoperative day. Without adhesiolysis, the gut lumen was obliterated by 3-O silk 10 cm proximal and distal to the anastomosis. An 18G catheter was placed in the lumen, and infused with saline solution at a rate of 2 mL/min by an IVAC 770 infusion pump (IVAC Corporation, USA) while monitoring the pressure within the lumen continuously.” The pressure that caused anastomotic leakage was determined as the in situ bursting pressure. After the animal was killed, 0.5 cm of bowel segment with the anastomotic site was resected, divided longitudinally, and cleaned of debris and intestinal contents. Journalof

Pediatric

Surgery,Vol33,

No 1 (January),

1998:

pp 91-93

Dursun,

Transient Vakur

Bor,

Turkey

Results: Hydroxyproline content and bursting pressures were comparedstatistically with Mann-Whitney U test. Although there was no statistical difference between the control group and group 1, there were significant differences (P < ,051 between groups 2, 3, and 4, with both parameters decreasing as the duration after reperfusion increased.

Conclusion: Anastomosis formed sooner ratherthan event. J Pediatr Surg 33197-93. Company.

INDEX sis.

WORDS:

are less likely to leak when later after an ischemia/reperfusion Copyright

Ischemia/reperfusion,

o 7998 by W.6.

intestinal

per-

Saunders

anastomo-

Suture materials were removed. Half of each specimen was placed in 10% formaline for histopathologic examination, and the other half was wrapped in aluminum foil, frozen in liquid nitrogen, and kept at -30°C for biochemical studies. Paraffin blocks were cut in 5-p sections and stained using Hematoxylin-Eosin. The specimens were coded appropriately and studied blindly by one pathologist in random order. Intestinal tissue injury at the time of anastomosis was graded from 0 to 8: grade 0, normal mucosa; grade 1, development of a subepithelial space usually at the tips of the villi; grade 2, extension of the space with epithelial lifting at the villus tips; grade 3, epithelial elevation along the villus; grade 4, denuded villi; grade 5, loss of villus tissue; grade 6, crypt layer infarction; grade 7, necrosis of the entire intestinal mucosa; and grade 8, transmural infarction5 After the frozen tissues were dried using filter paper, they were divided into small pieces and weighted. From tlze Departments of Pediatric Surgery, Pathology and Biochemistry, Gazi University School of Medicine, Ankara, Turkey. Presented at the 44th Annual Internaiional Congress of the British Association of Paediatric Surgeons, Istanbul, Turkey, July 22-25, 1997. Address reprint requests to A. Can Bagaklar; MD, Sedat Simnvi Sokak 23B/6, 06550 Qankaya, Ankara, Turkey. Copyright o I998 by WB. Saunders Company

0022.3468/98/3301-0021$03.00/O 91

92

DEMiROGULLARl

ET AL

Biochemical studies defined hydroxyproline levels expressed as nanomoles. These absolute measures were proportioned to the tissue weights and the results were expressed as pmol/g tissue (wet weight), of hydroxy prolene (HP).6 Bursting pressures and HP content were statistically compared for controls and other groups using MannWhitney U test (P < .05 was considered significant). RESULTS Fig 2.

Histopathologic Evaluation Average ischemic gradeswere 0 in control group, 3 in group 1,4 in group 2, and 0 in groups 3 and 4. The most severe insult was found in groups 1 and 2. Histopathologic study findings of the anastomotic sites showed nonspecific wound healing processin all groups. Bursting Pressures Average bursting pressuresof eachgroup are shownin Fig 1. The difference between bursting pressuresof the control and group 1 were not statistically significant. The differences between these two groups and the other groups were statistically significant (P < .05 for each comparison).Bursting pressuresof the groups2,3, and4 were also significant (P < .05 for eachcomparison). Hydroxyproline Contents Average HP content of each group is shown in Fig 2. The difference between hydroxyproline contents of the control and group 1 was not statistically significant. The differences between these two groups and the other groups were statistically significant (P < .05 for each comparison). HP content of the groups 2, 3, and 4 were also significant (P < .05 for each comparison). DISCUSSION

There arefew studiesof healing of intestinal anastomosesafter I/R and mostdeterminehealing of the anastomosis performed early in the reperfusion period.‘s7None study the effect of anastomosisperformed late after an I/R event. 250

204.8

Group

1

Group

Fig 1.

2

Mean

Group

bursting

3

Grow

pressures.

4

CO!ltiOl

Mean

hydroxyproline

values.

It has been shown that transient ischaemiacausedby SMA occlusion results in mucosal damage, which is apparent 10 minutes after reperfusion, is almost 80% healedby 6 hours, and almost completely resolved by 12 hours8 This study demonstratessimilar findings with the averagemucosaldamagegrade of 3 and 4 in group 1 and 2, respectively, and 0 in groups3 and 4. Histopathologic examination, mechanical burst pressures,and hydroxyproline estimation are acceptedmethods for assessingintestinal anastomosishealing.9These appear to demonstrateoptimal healing during the first week after repair.9-11 We therefore decided to evaluate the anastomosison the 5th postoperativeday. Although healing of an intestinal anastomosisdepends on the balancebetween collagen degradationand synthesis,in practice, HP content is regardedasa measureof net accumulationof collagen.l2Also, the anastomoticstrength depends on the type of collagen and the constituent crosslinks.Nevertheless,we found that bursting pressure did parallel HP content, although this is not a finding in other studies.13 The observation that there is no significant difference between the HP content of the control group and group 1 is consistentwith that in the literature.1,7The statistically significant decrease in the HP content and bursting pressure,asthe reperfusion period increased(Figs 1 and 2, P < .05 for all comparisonsother than controls and group l), indicates that anastomosesestablishedearly in the reperfusion period are stronger that those undertaken later. The reasonfor this is uncertain. It has been shown that free oxygen radicals directly cause collagenolysis, and it is recognised that these are increased in the intestine after transient ischaemia.r5Superoxide radicals derived from neutrophils at the anastomoticsite may also contribute to collagenolysis.14.15 These facts would suggest that oxidizing agents derived from neutrophis and other sourcesoverwhelm naturally occurring enzymatic antioxidans,and thesearethe causeof increasedcollagenolysis, which appearsto increasewith time after an I/R event. The clinical implication of this observation is that surgical reconstitution of the intestinal tract is better undertakensoonerrather than later after an I/R injury.

BOWEL

ANASTOMOSES

AFTER

TRANSIENT

93

ISCHEMIA

REFERENCES 1. Bergren CT, Bodzin JH, Cortez JA: Improved survival using oxygen free radical scavengers in the presence of ischemic bowel anastomosis. Am Surg 54:333-336, 1988 2. Park PO, Haglund U: Regeneration of small bowel mucosa after intestinal ischemia. Crit Care Med 20:135-139, 1992 3. Parks DA, Jacobson ED: Physiology of the splanchnic circulation. Arch Intern Med 145:1278-1281, 1985 4. Aszodi A, Ponsky JL: Effects of 5-Fluorourasil on the healing of bowel anastomosis in rats. Am Surg 51:671-674, 1985 5. Park PO, Haglund U, Bulkley GB, et al: The sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion. Surgery 107574.580,199O 6. Jamall IS, Finelli VN, Que Hee SS: A simple method to determine nanogram levels of 4-hidroxyproline in biological tissues. Anal Biothem 112:70-75, 1981 7. Ponsky J, McCollister DM, Aszodi A, et al: Effects of transient ischemia on the healing small bowel anastomosis. Am Surg 54:517-518, 1988 8. Udassin R, Vromen A, Haskel Y The time sequence of injury and recovery following transient reversible intestinal ischemia. J Surg Res 56:221-225, 1994

9. Hendriks T, Mastboom WJB: Healing of experimental intestinal anastomoses. Dis Colon Rectum 33:891-901, 1990 10. Asencio-Arana F, Martinez-Soriano F: Stimulation of the healing of experimental colon anastomoses in the rat. Exp Mol Path01 52:125127,1988 11. Puig LaCalle J, Greoles JM, Canal Pey G, et al: Rotated intestinal anastomoses. Surg Gynecol Obstet 154:662-666, 1982 12. Cronin K, Jackson DS, Dunphy JE: Specific activity of hidroxyproline-tritium in the healing colon. Surg Gynecol Obstet 126:10611065,1968 13. Jiborn H, Ahonon J, Zederfelt B: Healing of experimental colonic anastomoses. II. Breaking strength of the colon after left colon resection and anastomosis. Am J Surg 136:595-599, 1978 14. McGovan SE, Murray JJ: Direct effects of neutrophil oxidants on elastase-induced extracellular matrix proteolysis. Am Rev Respir Dis 135:1286-1293, 1987 15. Grisham MB, Hemandez LA, Granger DN: Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physio125 l:G567G574,1986 16. Reilly PM, Schiller HJ, Bulkley GB: Pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites. Am J Surg 161:488-503, 1991