- Research article
- Open Access
Examining the safety of colon anastomosis on a rat model of ischemia-reperfusion injury
© Czeiger et al.; licensee BioMed Central Ltd. 2013
Received: 19 April 2013
Accepted: 27 June 2013
Published: 2 July 2013
Intestinal ischemia and reperfusion can impair anastomotic strength.
The purpose of this study was to evaluate the safety of delayed colon anastomosis following remote ischemia-reperfusion (IR) injury.
Rats divided into two groups underwent bilateral groin incisions, however only the study group had femoral artery clamping to inflict IR injury. Twenty-four hours following this insult, the animals underwent laparotomy, incision of the transverse colon and reanastomosis. End points included anastomotic leakage, strength and histopathological features.
Anastomotic leak among IR animals (22.2%) was not statistically different in comparison to the controls [10.5% (p = 0.40)]. Anastomotic mean burst pressures showed no statistically significant difference [150.6 ± 15.57 mmHg in the control group vs. 159.9 ± 9.88 mmHg in the IR group (p = 0.64)]. The acute inflammatory process in the IR group was similar to controls (p = 0.26), as was the chronic repair process (p = 0.88). There was no significant difference between the inflammation:repair ratios amongst the two groups (p = 0.67).
Primary colon repair is safe when performed 24 hours following systemic IR injury.
Ischemia-reperfusion (IR) injury represents a fundamental common pathway of tissue damage in a wide variety of disease and surgical processes such as major trauma, acute mesenteric ischemia, septic and hypovolemic shock, abdominal aortic aneurism surgery, and cardiopulmonary bypass [1, 2]. Interruption of blood supply results in ischemic injury to all body systems and especially to high metabolically active tissues; the intestine is a prominent example of a sensitive tissue to IR injury which is associated with high morbidity and mortality . Paradoxically, restoration of blood flow to the ischemic tissue augments cell injury by delivering toxic mediators induced in the ischemic tissue into the circulation thus affecting distant organs. This might lead to the development of systemic inflammatory response syndrome, which can progress to multiple organ failure and death . Among the toxic mediators produced in the IR injured tissue are acute-phase proteins, pro-inflammatory cytokines, oxygen free radicals, and components of the complement system . Emergency surgery and trauma situations may require abbreviated procedures during the initial phase of shock and organ ischemia. Definitive procedures including anastomosis to restore bowel continuity are undertaken 24 hours or more afterward. Two common examples of such situations are the strategy of damage control surgery in seriously injured patients, and acute mesenteric ischemia. In damage control laparotomy the goal in the emergency surgery is to stop bleeding and to control spillage from the intestine. In the second operation, which is done after the patient’s deranged physiology is corrected, bowel anastomosis may be created. In mesenteric ischemia gangrenous segments of the bowel are resected, while fluid resuscitation continues. Not infrequently, the patient condition does not allow performing primary anastomosis. This may be done during a second look operation 24 hours later. The spectrum of the effects of IR injury on the intestine is broad and ranges from a transient absorptive impair following mucosal damage to frank gangrene of the bowel . Previous reports have shown that ischemia and reperfusion of the intestinal wall can lead to impaired anastomotic strength [5–8]. However, there is not enough evidence in the literature to show the safety of delayed bowel anastomosis following systemic IR injury. We hypothesized that IR injury would adversely affect the safety of colonic anastomoses performed 24 hours following the injury. To evaluate this hypothesis we investigated the effects of IR injury on the healing of colon anastomoses in a rat model.
Materials and methods
The protocol employed in this study was approved by the Committee for the Ethical Care and Use of Laboratory Animals of the Ben-Gurion University of the Negev (approval code IL-41-7-2006). It included a provision that any rat exhibiting evidence of distress (such as restlessness or aggressive behavior) be immediately euthanized. Rats were acclimated to the laboratory for 2 weeks prior to the study and had free access to water and food at all times. A total of 40 male Sprague–Dawley rats (average weight 350 g) were used. The number of animals in each group was considered satisfactory based on a two-sided sample size determination (power analysis), assuming power of 0.80 and significance of 0.05. All rats were anesthetized with inhaled isoflurane 1% at a rate of 3–5 L/min. The study group (n = 20) underwent bilateral groin incision and clamping the femoral arteries for 30 minutes. The control group (n = 20) had a similar sham operation without inducing extremities ischemia. All wounds were then sutured with 4/0 silk. Twenty-four hours following this insult, all animals were anesthetized and underwent a midline laparotomy, full circumference incision of the transverse colon (including resection of 0.5 cm of mesentery on each side of the colon) and reanastomosis (end-to-end) using 4/0 polyglycolic acid sutures. The animals were then followed up and sacrificed one week later. The peritoneal cavity was subsequently explored for the presence of perforation, and local or generalized peritonitis.
Anastomotic healing was assessed by determining anastomotic burst pressures, as well as by formal histopathological examination. The transverse colon was dissected free of adhesions and resected. One end of this segment was ligated, and a catheter connected to a sphygmomanometer was secured to the other end. Air was then pumped into the segment of colon, which was submerged in water. Intraluminal pressure was monitored continuously while the air was injected. The intraluminal pressure at which air leakage from the anastomosis occurred was recorded as the burst pressure. More specifically, this parameter represents the mechanical strength of the anastomosis. The colon specimen was then processed and evaluated for three parameters of acute inflammation: (1) total number of polymorphonuclear cells, (2) total number of red blood cells and (3) the amount of fibrin, as well as for three parameters of chronic wound healing: (1) total number of mononuclear cells, (2) total number of fibroblasts and (3) total amount of collagen. Each parameter was graded from 0 to 4. The colon surgeon and the pathologist were each blinded with regard to the individual group allocation history of the animals. Statistical analysis was performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA. Parametric results are expressed as mean ± SEM and were compared using an unpaired t-test. Two-tailed p < 0.05 was considered as having a statistical significance.
The goal of this study was to examine the safety of colon anastomosis performed 24 hours after profound systemic ischemia-reperfusion injury. Our results show that resection and anastomosis of the colon is safe, when performed twenty-four hours following profound, remote IR injury. This is based on similar mortality and anastomotic leak ratios (although a non-significant trend towards a higher incidence of anastomotic leak among the IR animals was noted), comparable anastomotic mechanical strengths, and equivalent histological features of the anastomosis between the IR and the control groups.
Today, in 2013, anastomotic leak after colorectal resection still has lethality of 6-22% and morbidity leading to reoperation and permanent stoma in 56% . There is convincing evidence in the literature that primary repair or anastomosis is appropriate for the management of most colonic injuries and for other emergent surgical situations [10–17]. In contrast, there is little methodologically sound evidence outlining the outcome of a colon anastomosis in the setup of severe IR. Damage control surgery (DCS) is probably one of the most common situations where the surgeon faces the dilemma of creating colonic anastomosis in a delayed fashion after IR injury. Clinical retrospective series have revealed contradictory conclusions regarding the safety of this procedure. Miller et al.  concluded that delayed anastomoses in patients undergoing DCS is safe, whereas Weinberg and colleagues reported a significant colon related complication rate in patients who were treated by resection and anastomosis . A third group also identified a higher incidence of colonic anastomotic leakage among DCS patients who had resection followed by anastomosis; however they declared that resection and anastomosis is still considered safe . Ott pointed in a recently published manuscript that colon anastomosis is safe unless the abdomen remains open. He also regards the left colon as more vulnerable to leak under these conditions . It is obvious that limitations in these studies include heterogeneous patient populations, variance in patients’ clinical condition and surgeons’ preference, and even the very definition of DCS by different surgeons.
To overcome these limitations inherent in clinical retrospective studies we created a rat model of IR injury followed by resection and reansatomosis of the transverse colon. IR injury has been intensively investigated since the 1970s. The IR phenomenon represents the common underlying pathophysiological process to a variety of medical conditions and surgical procedures. Tissue ischemia with inadequate oxygen supply followed by successful reperfusion initiates a wide and complex array of inflammatory responses that may both aggravate local injury, as well as induce impairment of remote organ function . Review of the literature reveals experimental studies evaluating the effect of transient preoperative IR on gut anastomotic strength [6, 8, 23–29]. The results of these studies were equivocal. This may partially be explained by the degree and duration of the inflicted ischemia . Also, the surgical procedure was done on small bowel in some of the studies , while in others large bowel anastomoses were tested . Furthermore, in the previous models the ischemia was done by clamping the blood supply of the resected segment of intestine, and/or performed the intestinal anastomosis immediately following the IR injury. Kuzu et al. attempted to demonstrate the systemic nature of IR by occluding the superior mesenteric artery and its collaterals and immediately thereafter they resected and reansatomose the left colon . Posma described the effect of a prolonged interval between IR and anastomotic construction on the anastomosis healing, but used a model of local mesenteric ischemia .
We believe that the present model, with severe systemic remote ischemia, performance of a colon anastomosis 24 hours later, and testing the anastomotic strength after one week, more closely resembles the true conditions of some emergent conditions that the surgical approach for them is still uncertain. Several mechanisms have been suggested to explain the blunting of the IR deleterious effect on bowel anastomoses when these are constructed late after the insult. One is subsidence of the harmful effects over the time elapsed from the insult to the creation of the anastomosis. Another explanation is the protective effect of ischemic preconditioning [30, 32]. Recently, studies have been published on prevention/alleviation the effect of IR injury by inhibiting compliment system activation , by applying antioxidants [34, 35], and trace elements . Another trend for attenuating effects of IR injury is ischemic postconditioning [37–39].
In our experiment we amplified the local ischemia at the site of anastomosis by resecting 0.5 cm of mesentery on each side of the divided transverse colon. Even under these stringent conditions we did not observe the expected IR harmful effects. On the other hand, our results showed no benefit to the ischemic group. This should question the protective effect of ischemic preconditioning in this setup.
In summary, this rat model augments the literature which support delayed primary repair after ischemia-reperfusion injury. However, more laboratory and clinical evidence is required before final conclusion can be drawn. More studies are also needed to understand the attenuation of the harmful effects of IR on intestinal anastomosis when performed 24 hours after the injury.
This work was not supported by any third party such as pharmaceutical or industrial company, or grants. No author has conflict of interest regarding the publication of this work. The study has not been presented, yet, at a scientific or medical conference.
The manuscript is not under consideration for publication by any other journal.
- Mallick IH, Yang W, Winslet MC, Seifalian AM: Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig Dis Sci. 2004, 49 (9): 1359-1377.View ArticlePubMedGoogle Scholar
- Leng YF, Zhang Y, Zhang Y, Xue X, Wang T, Kang YQ: Ischemic post-conditioning attenuates the intestinal injury induced by limb ischemia/reperfusion in rats. Braz J Med Biol Res. 2011, 44 (5): 411-417.View ArticlePubMedGoogle Scholar
- Grootjans J, Lenaerts K, Derikx JP, Matthijsen RA, de Bruine AP, van Bijnen AA, van Dam RM, Dejong CH, Buurman WA: Human intestinal ischemia-reperfusion-induced inflammation characterized: experiences from a new translational model. Am J Pathol. 2010, 176 (5): 2283-2291. 10.2353/ajpath.2010.091069.PubMed CentralView ArticlePubMedGoogle Scholar
- Haglund U, Bulkley GB, Granger DN: On the pathophysiology of intestinal ischemic injury. Clinical review. Acta Chir Scand. 1987, 153 (5–6): 321-324.PubMedGoogle Scholar
- Posma LA, Bleichrodt RP, Lomme RM, de Man BM, van Goor H, Hendriks T: Early anastomotic repair in the rat intestine is affected by transient preoperative mesenteric ischemia. J Gastrointest Surg. 2009, 13 (6): 1099-1106. 10.1007/s11605-009-0827-5.View ArticlePubMedGoogle Scholar
- Kologlu M, Yorganci K, Renda N, Sayek I: Effect of local and remote ischemia-reperfusion injury on healing of colonic anastomoses. Surgery. 2000, 128 (1): 99-104. 10.1067/msy.2000.107414.View ArticlePubMedGoogle Scholar
- Kuzu MA, Tanik A, Kale IT, Aslar AK, Koksoy C, Terzi C: Effect of ischemia/reperfusion as a systemic phenomenon on anastomotic healing in the left colon. World J Surg. 2000, 24 (8): 990-994. 10.1007/s002680010170.View ArticlePubMedGoogle Scholar
- Posma LA, Bleichrodt RP, van Goor H, Hendriks T: Transient profound mesenteric ischemia strongly affects the strength of intestinal anastomoses in the rat. Dis Colon Rectum. 2007, 50 (7): 1070-1079. 10.1007/s10350-006-0822-9.View ArticlePubMedGoogle Scholar
- Daams F, Luyer M, Lange JF: Colorectal anastomotic leakage: aspects of prevention, detection and treatment. World J Gastroenterol. 2013, 19 (15): 2293-2297. 10.3748/wjg.v19.i15.2293.PubMed CentralView ArticlePubMedGoogle Scholar
- Demetriades D, Murray JA, Chan L, Ordonez C, Bowley D, Nagy KK, Cornwell EE, Velmahos GC, Munoz N, Hatzitheofilou C, Schwab CW, Rodriguez A, Cornejo C, Davis KA, Namias N, Wisner DH, Ivatury RR, Moore EE, Acosta JA, Maull KI, Thomason MH, Spain DA, Committee on Multicenter Clinical Trials: Penetrating colon injuries requiring resection: diversion or primary anastomosis? An AAST prospective multicenter study. J Trauma. 2001, 50 (5): 765-775. 10.1097/00005373-200105000-00001.View ArticlePubMedGoogle Scholar
- Gonzalez RP, Merlotti GJ, Holevar MR: Colostomy in penetrating colon injury: is it necessary?. J Trauma. 1996, 41 (2): 271-275. 10.1097/00005373-199608000-00012.View ArticlePubMedGoogle Scholar
- Sasaki LS, Allaben RD, Golwala R, Mittal VK: Primary repair of colon injuries: a prospective randomized study. J Trauma. 1995, 39 (5): 895-901. 10.1097/00005373-199511000-00013.View ArticlePubMedGoogle Scholar
- Stone HH, Fabian TC: Management of perforating colon trauma: randomization between primary closure and exteriorization. Ann Surg. 1979, 190 (4): 430-436. 10.1097/00000658-197910000-00002.PubMed CentralView ArticlePubMedGoogle Scholar
- Chappuis CW, Frey DJ, Dietzen CD, Panetta TP, Buechter KJ, Cohn I: Management of penetrating colon injuries. A prospective randomized trial. Ann Surg. 1991, 213 (5): 492-497. discussion 497–8.10.1097/00000658-199105000-00015.PubMed CentralView ArticlePubMedGoogle Scholar
- Singer MA, Nelson RL: Primary repair of penetrating colon injuries: a systematic review. Dis Colon Rectum. 2002, 45 (12): 1579-1587. 10.1007/s10350-004-7243-4.View ArticlePubMedGoogle Scholar
- Jimenez Fuertes M, Costa Navarro D: Resection and primary anastomosis without diverting ileostomy for left colon emergencies: is it a safe procedure?. World J Surg. 2012, 36 (5): 1148-1153. 10.1007/s00268-012-1513-4.View ArticlePubMedGoogle Scholar
- Constantinides VA, Tekkis PP, Athanasiou T, Aziz O, Purkayastha S, Remzi FH, Fazio VW, Aydin N, Darzi A, Senapati A: Primary resection with anastomosis vs. Hartmann’s procedure in nonelective surgery for acute colonic diverticulitis: a systematic review. Dis Colon Rectum. 2006, 49 (7): 966-981. 10.1007/s10350-006-0547-9.View ArticlePubMedGoogle Scholar
- Miller PR, Chang MC, Hoth JJ, Holmes JH, Meredith JW: Colonic resection in the setting of damage control laparotomy: is delayed anastomosis safe?. Am Surg. 2007, 73 (6): 606-609. discussion 609–10PubMedGoogle Scholar
- Weinberg JA, Griffin RL, Vandromme MJ, Melton SM, George RL, Reiff DA, Kerby JD, Rue LW: Management of colon wounds in the setting of damage control laparotomy: a cautionary tale. J Trauma. 2009, 67 (5): 929-935. 10.1097/TA.0b013e3181991ab0.View ArticlePubMedGoogle Scholar
- Kashuk JL, Cothren CC, Moore EE, Johnson JL, Biffl WL, Barnett CC: Primary repair of civilian colon injuries is safe in the damage control scenario. Surgery. 2009, 146 (4): 663-668.discussion 668–70. 10.1016/j.surg.2009.06.042.View ArticlePubMedGoogle Scholar
- Ott MM, Norris PR, Diaz JJ, Collier BR, Jenkins JM, Gunter OL, Morris JA: Colon anastomosis after damage control laparotomy: recommendations from 174 trauma colectomies. J Trauma. 2011, 70 (3): 595-602. 10.1097/TA.0b013e31820b5dbf.View ArticlePubMedGoogle Scholar
- Collard CD, Gelman S: Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury. Anesthesiology. 2001, 94 (6): 1133-1138. 10.1097/00000542-200106000-00030.View ArticlePubMedGoogle Scholar
- Aydin C, Teke Z, Aytekin F, Yenisey C, Kabay B, Simsek NG, Tekin K: Tempol prevents harmful effects of remote ischemia reperfusion injury on healing of experimental colonic anastomoses. Int J Colorectal Dis. 2007, 22 (3): 325-331. 10.1007/s00384-006-0149-y.View ArticlePubMedGoogle Scholar
- Colak T, Turkmenoglu O, Dag A, Polat A, Comelekoglu U, Bagdatoglu O, Polat G, Kanik A, Akca T, Aydin S: The effect of remote ischemic preconditioning on healing of colonic anastomoses. J Surg Res. 2007, 143 (2): 200-205. 10.1016/j.jss.2006.10.030.View ArticlePubMedGoogle Scholar
- Murthy S, Hui-Qi Q, Sakai T, Depace DE, Fondacaro JD: Ischemia/reperfusion injury in the rat colon. Inflammation. 1997, 21 (2): 173-190. 10.1023/A:1027318203971.View ArticlePubMedGoogle Scholar
- Posma LA, Bleichrodt RP, van Goor H, Hendriks T: A prolonged interval between deep intestinal ischemia and anastomotic construction does not impair wound strength in the rat. Int J Colorectal Dis. 2007, 22 (12): 1485-1491. 10.1007/s00384-007-0333-8.View ArticlePubMedGoogle Scholar
- Posma LA, Bleichrodt RP, van Goor H, Hendriks T: Ischemia and prolonged reperfusion before anastomotic construction do not reduce wound strength in the rat intestine. Surgery. 2006, 139 (5): 671-677. 10.1016/j.surg.2005.10.014.View ArticlePubMedGoogle Scholar
- Rolim MF, Riger CJ, Eleutherio EC, Colao Cda F, Pereira GC, Schanaider A: Colonic healing after portal ischemia and reperfusion: an experimental study with oxidative stress biomarkers. Redox Rep. 2007, 12 (6): 267-274. 10.1179/135100007X239261.View ArticlePubMedGoogle Scholar
- Teke Z, Aytekin FO, Kabay B, Yenisey C, Aydin C, Tekin K, Sacar M, Ozden A: Pyrrolidine dithiocarbamate prevents deleterious effects of remote ischemia/reperfusion injury on healing of colonic anastomoses in rats. World J Surg. 2007, 31 (9): 1835-1842. 10.1007/s00268-007-9106-3.View ArticlePubMedGoogle Scholar
- Holzner PA, Kulemann B, Kuesters S, Timme S, Hoeppner J, Hopt UT, Marjanovic G: Impact of remote ischemic preconditioning on wound healing in small bowel anastomoses. World J Gastroenterol. 2011, 17 (10): 1308-1316. 10.3748/wjg.v17.i10.1308.PubMed CentralView ArticlePubMedGoogle Scholar
- Kosmidis C, Efthimiadis C, Anthimidis G, Basdanis G, Apostolidis S, Hytiroglou P, Vasiliadou K, Prousalidis J, Fahantidis E: Myofibroblasts and colonic anastomosis healing in Wistar rats. BMC Surg. 2011, 11: 6-2482-11-6-View ArticleGoogle Scholar
- Moore-Olufemi SD, Kozar RA, Moore FA, Sato N, Hassoun HT, Cox CS, Kone BC: Ischemic preconditioning protects against gut dysfunction and mucosal injury after ischemia/reperfusion injury. Shock. 2005, 23 (3): 258-263.PubMedGoogle Scholar
- Diepenhorst GM, van Gulik TM, Hack CE: Complement-mediated ischemia-reperfusion injury: lessons learned from animal and clinical studies. Ann Surg. 2009, 249 (6): 889-899. 10.1097/SLA.0b013e3181a38f45.View ArticlePubMedGoogle Scholar
- Kabali B, Girgin S, Gedik E, Ozturk H, Kale E, Buyukbayram H: N-acetylcysteine prevents deleterious effects of ischemia/reperfusion injury on healing of colonic anastomosis in rats. Eur Surg Res. 2009, 43 (1): 8-12. 10.1159/000210673.View ArticlePubMedGoogle Scholar
- Teke Z, Bostanci EB, Yenisey C, Sacar M, Simsek NG, Akoglu M: Caffeic acid phenethyl ester alleviates mesenteric ischemia/reperfusion injury. J Invest Surg. 2012, 25 (6): 354-365. 10.3109/08941939.2012.677968.View ArticlePubMedGoogle Scholar
- Ersoy YE, Ayan F, Himmetoglu S: Trace element levels in ischemia-reperfusion injury after left colonic anastomosis in rats and effects of papaverine and pentoxiphylline on vascular endothelial growth factor in anastomosis healing. Acta Gastroenterol Belg. 2011, 74 (1): 22-27.PubMedGoogle Scholar
- Chu WW, Nie L, He XY, Yan AL, Zhou Y, Wu GL, Wang DH: Change of cytochrome c in postconditioning attenuating ischemia-reperfusion-induced mucosal apoptosis in rat intestine. Sheng Li Xue Bao. 2010, 62 (2): 143-148.PubMedGoogle Scholar
- Wen SH, Li Y, Li C, Xia ZQ, Liu WF, Zhang XY, Lei WL, Huang WQ, Liu KX: Ischemic postconditioning during reperfusion attenuates intestinal injury and mucosal cell apoptosis by inhibiting JAK/STAT signaling activation. Shock. 2012, 38 (4): 411-419. 10.1097/SHK.0b013e3182662266.View ArticlePubMedGoogle Scholar
- Wen SH, Ling YH, Li Y, Li C, Liu JX, Li YS, Yao X, Xia ZQ, Liu KX: Ischemic postconditioning during reperfusion attenuates oxidative stress and intestinal mucosal apoptosis induced by intestinal ischemia/reperfusion via aldose reductase. Surgery. 2013, 153 (4): 555-564. 10.1016/j.surg.2012.09.017.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.