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Effect of local and remote ischemia-reperfusion injury on healing of colonic anastomoses
Effect of local and remote ischemiareperfusion injury on healing of colonic anastomoses Murat Kologlu, MD, Kaya Yorganci, MD, Nurten Renda, MD, and Iskender Sayek, MD, Ankara, Turkey
Background. Although the effect of locally applied ischemia-reperfusion (I-R) injury on gastrointestinal anastomoses has been studied, to our knowledge there is no previous study that investigates the effect of remote I-R injury on gastrointestinal anastomotic healing. The aim of this study was to investigate and compare the effects of local I-R injury and remote I-R injury on the healing of colonic anastomoses. Methods. Anastomosis of the right colon was performed in 30 rats that were divided into 5 groups. Group 1 was the control group. In Group 2, I-R was applied to the colonic segment containing the anastomosis. Unilateral lower extremity I-R, unilateral renal I-R, and segmental small intestinal I-R was applied to the rats in Groups 3, 4, and 5, respectively, at the same time as colonic anastomosis. On the fourth postoperative day, animals were killed and bursting pressure and tissue hydroxyproline concentration of the anastomoses were analyzed and compared. Results. The mean bursting pressure values were: 143 mm Hg in Group 1, 40.8 mm Hg in Group 2, 82.8 mm Hg in Group 3, 46.1 mm Hg in Group 4, and 52.3 mm Hg in Group 5 (P < .0001; 1-way analysis of variance). Mean tissue hydroxyproline concentration values were: 5.3 µg/mg in Group 1, 1.6 µg/mg in Group 2, 2.2 µg/mg in Group 3, 1.3 µg/mg in Group 4, and 1.5 µg/mg in Group 5 (P < .0001, 1-way analysis of variance). Bursting pressure and tissue hydroxyproline concentration values had a good correlation r = 0.86, P < .001, Pearson correlation analysis). Conclusions. This study showed that I-R injury is a systemic phenomenon, and remote organ I-R can significantly delay anastomotic healing. This has to be kept in mind when constructing an intestinal anastomosis in the presence of local or remote I-R injury. (Surgery 2000;128:99-104) From the Departments of General Surgery and Biochemistry, Ankara, Turkey
THE EFFECT OF ISCHEMIA-REPERFUSION (I-R) injury on several organ systems is one of the most investigated subjects in experimental surgery. Clinically, I-R is important in transplantation, trauma surgery, vascular surgery, and in low-flow states.1 Today we know that reperfusion may be more harmful to tissues than the preceding ischemia, depending on the duration.2,3 In the 1990s, it became accepted that I-R injury has systemic effects and may induce a systemic inflammatory reaction.1,2,4-8 It has been concluded that the systemic effects of I-R injury are caused by activated neutrophils, the complement system, and proinflammatory and vasoactive mediators such as eicosanoids, nitric oxide, cytokines, and oxygen-free radicals.1,2,4-11 Although the effect of locally applied I-R injury on gastrointestinal anastomoses has been studAccepted for publication March 18, 2000. Reprint requests: Dr Murat Kologlu, Ahmet Mithat Efendi S 19/2, Çankaya, 06550, Ankara, Turkey. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.107414
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ied,12,13 to our knowledge there is no previous study that investigates the effect of remote I-R injury on gastrointestinal anastomotic healing. The aim of this study was therefore to investigate and compare the effects of local I-R injury and remote I-R injury on the healing of colonic anastomoses. For this purpose, we constructed anastomosis of the right colon in rats and compared the anastomotic healing in control group animals with the healing in animals having colonic (segment containing the anastomosis), lower extremity, renal, or small intestinal I-R injury. MATERIALS AND METHODS Thirty female Wistar albino rats weighing 150 to 200 g were used in the study. All animals were allowed standard rat chow and water ad libitum. Twelve hours before anesthesia, animals were deprived of food but had free access to water. After the intramuscular introduction of anesthesia with 5 mg/kg of xylazine (Rompun) and 30 mg/kg of ketamine hydrochloride (Ketalar), all rats underwent a midline laparotomy. The right colon was transected and an end-to-end anastomosis was perSURGERY 99
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formed by using one layer of interrupted 7/0 polypropylene sutures (Prolene, Ethicon, UK). After the operation, rats were fed with standard rodent chow and water. Rats were divided into 5 groups, each having 6 animals: Group 1, Control: These animals had anastomosis of the right colon and did not have any other procedure until the fourth postoperative day. Group 2, Colonic I-R (local I-R): At the same time as the anastomosis of the colon was performed, a microvascular clip was placed across the distal end of the superior mesenteric artery, occluding the ileocolic and right colic arteries. In addition, the collateral arcades in the mesentery of the terminal ileum and the right colon were occluded by microvascular clips. By this procedure, the terminal ileum, caecum, and right colon containing the anastomosis were rendered ischemic. After 60 minutes of ischemia, the clips were removed and the reperfusion period began. It ended at the fourth postoperative day. Group 3, Lower extremity I-R: Simultaneously with the colonic anastomosis, unilateral lower extremity ischemia was induced by applying a rubber band tourniquet high around the left thigh. At the end of the 60-minute ischemia period, reperfusion was achieved by releasing the tourniquet. The reperfusion period ended at the fourth postoperative day. Group 4, Renal I-R: A microvascular clip was placed across the pedicle of the right kidney, at the same operation with the anastomosis of the colon. At the end of 60 minutes of renal ischemia, the clip was removed and the reperfusion period lasted until the fourth postoperative day. Group 5, Small intestinal I-R: At the same time as the anastomosis of the colon, a microvascular clip was placed across 2 or 3 ileal branches of the superior mesenteric artery; also, the collateral arcades in the mesentery of that ileal segment were occluded by microvascular clips. By this procedure, a segment of 8- to 10-cm ileum was rendered ischemic for 60 minutes. At the end of the ischemia period, the clips were removed and reperfusion began, which lasted until the fourth postoperative day. After the procedures, the laparotomy wounds were closed with continuous 4/0 silk sutures. Animals were fed with standard rat chow and water ad libitum postoperatively. At the fourth postoperative day, all animals were killed and a re-laparotomy was done. None of the organs that had I-R injury had macroscopic findings of irreversible ischemia and necrosis. The colonic anastomosis was dissected free of adhesions and a 4-cm segment
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of colon with the anastomosis in the middle was resected. One end of this segment was closed with a ligature, and a catheter was secured to the other end. Inside a glass jar filled with water, air was pumped into the segment of colon at a rate of 2 mL/min with an infusion pump. Intraluminal pressure was monitored while the air was pumped. The intraluminal pressure at which air leakage from the anastomosis occurred was recorded as the bursting pressure. This parameter showed the mechanical strength of the anastomoses. After the bursting pressure was measured, a 1cm segment of colon containing the anastomosis was resected to determine tissue hydroxyproline concentration, which represents the perianastomotic collagen concentration. Tissue hydroxyproline concentrations were determined by using the spectrophotometric method of Bergman and Loxley.14 Calculations were made to express the results as micrograms of hydroxyproline per milligram of tissue (µg/mg). The mean bursting pressures and tissue hydroxyproline concentrations of groups were calculated and expressed as mean ± SEM. The mean values of groups were compared by 1-way analysis of variance (ANOVA), and groups were compared separately by the Tukey-Kramer multiple comparisons test. Pearson correlation analysis was done to define the correlation of bursting pressure and hydroxyproline concentration values of the animals. In these tests, P < .05 was considered statistically significant. SPSS for Windows (6.0; SPSS Inc, Chicago, Ill) and Graphpad Instat (2.02; Graphpad Software Inc, San Diego, Calif) softwares were used for statistical analysis. RESULTS In this study, we used 2 parameters of wound healing to compare the healing in colonic anastomoses. These parameters were bursting pressure, which showed the mechanical strength of the anastomosis, and perianastomotic tissue hydroxyproline concentration, which represented the collagen accumulation around the anastomosis. When analyzing bursting pressures, all leaks occurred from the anastomosis. The mean bursting pressures of all the I-R groups were lower than the control groups. The mean bursting pressure of the lower extremity I-R group was higher than other I-R groups. These values in renal I-R and small intestinal I-R groups were very close to the mean value of the colonic I-R group (Table, Fig 1). The differences between the mean bursting pressures of the groups were statistically significant (P < .0001, 1-way ANOVA). When
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Fig 1. Bursting pressure values in the groups. Values of all animals are presented.
Table I. Mean bursting pressures and tissue hydroxyproline concentrations of the groups Group 1 Control
Group 2 Colonic (local) I-R
Mean ± SEM bursting pressure (mm Hg) 143 ± 2.9 Mean ± SEM tissue HP concentration (mg/mg) 5.3 ± 0.3
40.8 ± 4.1*† 1.6 ± 0.1‡
Group 3 Lower extremity I-R 82.8 ± 14.1* 2.2 ± 0.2‡
Group 4 Renal I-R 46.1 ± 7.7*† 1.3 ± 0.1‡
Group 5 Small intestinal I-R 52.3 ± 8.6* 1.5 ± 0.2‡
HP, Hydroxyproline. P < .0001 (1-way ANOVA) for the differences between mean bursting pressures of the groups. P < .0001 (1-way ANOVA) for the differences between mean tissue HP concentrations of the groups. *Compared with Group 1, difference is significant (P < .001, Tukey-Kramer test). †Compared with Group 3, difference is significant (P < .05, Tukey-Kramer test). ‡Compared with Group 1, difference is significant (P < .001, Tukey-Kramer test).
the groups were compared one by one, differences between the control group and all other groups were statistically significant (P < .001 for Group 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group 4, and Group 1 vs Group 5; Tukey-Kramer multiple comparisons test). Differences between colonic I-R, renal I-R, and lower extremity I-R groups also were statistically significant (P < .05, for Group 2 vs Group 3, Group 4 vs Group 3; Tukey-Kramer multiple comparisons test). The mean tissue hydroxyproline concentrations showed the same trend; all I-R groups had mean values lower than the control group. Among I-R groups, the highest mean value was determined in the lower extremity I-R group. Colonic I-R, renal IR, and small intestinal I-R groups had very close mean values (Table, Fig 2). The differences between mean values of the groups were statistically significant (P < .0001, 1-way ANOVA). When the groups were compared one by one, the differences between the control group and all other groups were statistically significant (P < .001, for Group 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group
4, Group 1 vs Group 5; Tukey-Kramer multiple comparisons test). The correlation between bursting pressure and tissue hydroxyproline concentration values of all animals that were included in the study was determined by Pearson correlation analysis. These 2 parameters had a positive good correlation r = 0.86, P < .001, Pearson correlation analysis; Fig 3). The coefficient of determination (r2) was 0.74. DISCUSSION The effect of local I-R injury on intestinal anastomoses was investigated in a few studies, and it has been shown that anastomotic healing is delayed in intestinal segments that are subjected to I-R injury.12,13 Although systemic effects of I-R injury have gained more attention in recent years, the effect of remote organ I-R injury on gastrointestinal anastomotic healing remains unclear. Ischemia is mainly a local event but, after revascularization, the mediators from the ischemic tissue enter the systemic circulation and effect other organ systems, which may end up with multiorgan dysfunc-
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Fig 2. Tissue hydroxyproline concentration values in the groups. values of all animals are presented.
Fig 3. Bursting pressure values of animals are plotted against tissue hydroxyproline concentration values, and linear curve estimation is shown. r = 0.86, P < .001, Pearson correlation analysis.
tion; so I-R injury is a systemic phenomenon.* Neutrophils seem to play a central role in these systemic effects; after reperfusion, mediators like eicosanoids, complements, cytokines, and oxygenfree radicals derived from ischemic tissue activate the neutrophils and endothelium.1,2,4-11 The degree of neutrophil activation is related to the extent of I-R injury. Beginning with the first minutes of reperfusion, a systemic inflammatory reaction is induced.5,10,11 This is accompanied by a severe local microvascular dysfunction and a less severe microvascular dysfunction in the systemic circulation.4-6,10,15 Normally, oxygen-free radicals have a very short half-life and mainly have local effects. But xanthine oxidase can be released into the systemic circulation from ischemic tissues upon reperfusion, and *References 4, 5, 7, 8, 10, 11, 15-17
by reacting with circulating substrates it can produce intravascular oxygen-free radicals that may have effects on remote organ systems.17-20 In addition, circulating cytokines activate the endothelial cells in remote organ systems to increase the expression of adhesion molecules causing activated neutrophils to interact with endothelium and have injurious effects.1,4-7,11,15 The most significant and most investigated remote organ injury appears in lungs. Endothelial cell adenosine triphosphate depletion, increased vascular permeability, intraparenchymal hemorrhage, and neutrophil accumulation occurs in lungs after lower extremity or intestinal I-R.2,4-7,9,15,20 Other results of lower extremity I-R include: a decrease in the intestinal mucosal thickness and an increase in the intestinal mucosal permeability, in bacterial translocation, in systemic endotoxin concentration, and in hepatic and renal dysfunction.8,16 The administration of
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oxygen-free radical scavengers, xanthine oxidase inhibitor allopurinol, antineutrophil serum (or removal of neutrophils from reperfusate), antibodies directed against adhesion molecules and various cytokines, inhibitors of some arachidonic acid metabolites and complements can attenuate the local and the systemic effects of I-R injury.1-7,11 Blocking mediators can be more beneficial for the systemic effects of I-R, which are indirect and mediator dependent, compared with the local effects.5,6 In this study, we investigated and compared the effect of local and remote I-R injury on healing of colonic anastomoses. Both bursting pressures (mechanical parameter) and perianastomotic tissue hydroxyproline concentrations (biochemical parameter) of the anastomoses were determined as recommended in the literature.21-23 Healing was evaluated in the early postoperative period when the majority of anastomotic leaks take place. We showed that renal and small intestinal I-R injury can delay healing in colonic anastomoses as much as colonic (local) I-R injury. Lower extremity I-R injury also delayed anastomotic healing significantly but not as much as colonic (local) I-R injury did. Both parameters of anastomotic healing gave the same results, and they had a good correlation. Investigation of the reasons for these results and whether blocking various mediators in the presence of I-R injury protects the anastomoses has to be the subject of another more detailed study. When an anastomosis is constructed in the gastrointestinal tract, an inflammation takes place as a response to traumatic injury and foreign material such as sutures.24,25 This inflammation is a normal constituent of wound healing. But if it is exaggerated, wound healing is delayed because of increased collagenolysis; it is why anastomotic healing is delayed in the presence of intraabdominal infection.23-27 Since remote organ I-R has a significant effect on healthy lungs, the effect of remote organ I-R on colonic anastomoses may be explained by similar mechanisms. The anastomotic area is already inflamed, and the endothelium in the perianastomotic area is already activated in the early phase of wound healing.24,25 As a result of proinflammatory and chemoattractant properties of the anastomosis, activated circulating neutrophils secondary to I-R injury in a remote organ system may accumulate easily in the perianastomotic area, increase the inflammatory reaction, and delay healing. Together with the proteolytic enzymes, oxygen-free radicals derived from activated neutrophils and circulating xanthine oxidase may increase collagenolysis in the perianastomotic area, which delays wound healing.12
On the other hand, I-R injury of an intraabdominal organ represents a form of acute intraabdominal trauma and may have humorally and/or neurally mediated local effects on splanchnic microcirculation leading to mesenteric vasoconstriction. It is shown that after intestinal I-R, splanchnic blood flow may be reduced by > 90% from baseline measurements.10 Defects of perfusion and oxygenation are known to delay healing in gastrointestinal anastomoses.23-25,28-30 This situation may explain why anastomotic healing is delayed more in renal and small intestinal I-R groups compared with the lower extremity I-R group. In light of our findings, we may draw some conclusions for clinical situations. Results of this study confirmed that I-R injury is a systemic phenomenon. The adverse effect of local I-R on anastomotic healing was also confirmed. Remote organ I-R was shown to delay anastomotic healing significantly. These have to be kept in mind when constructing an intestinal anastomosis in the presence of local or remote I-R injury. For instance, intestinal anastomoses constructed simultaneously with elective vascular reconstructions, emergency repairs of traumatic vascular lacerations, or in low-flow states may be under considerable risk because of reperfused ischemic tissues. When possible, avoiding intestinal anastomoses in these situations may be a reasonable approach.
REFERENCES 1. McMillen MA, Huribal M, Sumpio B. Common pathway of endothelial-leukocyte interaction in shock, ischemia, and reperfusion. Am J Surg 1993;166:557-62. 2. Hill J, Lindsay TF, Ortiz F, et al. Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemia-reperfusion in the rat. J Immunol 1992; 149:1723-8. 3. Sisley AC, Desai T, Harig JM, et al. Neutrophil depletion attenuates human intestinal reperfusion injury. J Surg Res 1994;57:192-6. 4. Seekamp A, Mulligan MS, Till GO, et al. Role of β2 integrins and ICAM-1 in lung injury following ischemia-reperfusion of rat hind limbs. Am J Pathol 1993;143:464-72. 5. Seekamp A, Warren JS, Remick DG, et al. Requirements for tumor necrosis factor-α and interleukin-1 in limb ischemia/ reperfusion injury and associated lung injury. Am J Pathol 1993;143:453-63. 6. Seekamp A, Till GO, Mulligan MS, et al. Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am J Pathol 1994;144:592-8. 7. Sorkine P, Setton A, Halpern P, et al. Soluble tumor necrosis factor receptors reduce bowel ischemia-induced lung permeability and neutrophil sequestration. Crit Care Med 1995;23: 1377-81. 8. Yassin MMI, Barros D’Sa AAB, Parks G, et al. Mortality following lower limb ischemia-reperfusion: a systemic inflammatory response? World J Surg 1996;20:961-7.
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9. Anner H, Kaufman RP, Valeri CR, et al. Reperfusion of ischemic lower limbs increases pulmonary microvascular permeability. J Trauma 1988;28:607-10. 10. LaNoue JL, Turnage RH, Kadesky KM, et al. The effect of intestinal reperfusion on renal function and perfusion. J Surg Res 1996;64:19-25. 11. Welbourn R, Goldman G, O’Riordan M, et al. Role for tumor necrosis factor as mediator of lung injury following lower torso ischemia. J Appl Physiol 1991; 70:2645-9. 12. Demirog˘ ullari B, Sönmez K, Türkyilmaz Z, et al. Comparison of consequent small bowel anastomoses after transient ischemia: an experimental study in rats. J Pediatr Surg 1998;33:91-3. 13. Kuzu MA, Köksoy C, Kale IT, et al. Reperfusion injury delays healing of intestinal anastomosis in a rat. Am J Surg 1998;176:348-51. 14. Bergman I, Loxley R. Two improved and simplified methods for the spectrophotometric determination of OH-proline. Anal Chem 1963;35:1961-5. 15. Gerkin TM, Oldham KT, Guice KS, et al. Intestinal ischemia-reperfusion injury causes pulmonary endothelial cell ATP depletion. Ann Surg 1993; 217: 48-56. 16. Willoughby RPN, Harris KA, Carson MW, et al. Intestinal mucosal permeability to 51Cr-ethylenediaminetetraacetic acid is increased after bilateral lower extremity ischemiareperfusion in the rat. Surgery 1996;120:547-53. 17. Yokoyama Y, Beckman JS, Beckman TK, et al. Circulating xanthine oxidase: potential mediator of ischemic injury. Am J Physiol 1990;258:G564-G570. 18. Friedl HP, Smith DJ, Till GO, et al. Ischemia-reperfusion in humans: appearance of xanthine oxidase activity. Am J Pathol 1990;136:491-5. 19. Tan S, Gelman S, Wheat JK, et al. Circulating xanthine oxidase in human ischemia reperfusion. South Med J 1995;88:479-82.
Surgery July 2000 20. Terada LS, Dormish JJ, Shanley PF, et al. Circulating xanthine oxidase mediates lung neutrophil sequestration after intestinal ischemia-reperfusion. Am J Physiol 1992;63:L394L401. 21. Hendriks T, Mastboom WJB. Healing of experimental intestinal anastomoses: parameters for repair. Dis Colon Rectum 1990;33:891-901. 22. Koruda MJ, Rolandelli RH. Experimental studies on the healing of colonic anastomoses. J Surg Res 1990;48:504-15. 23. Kologlu M, Sayek I, Kologlu LB, et al. Effect of persistently elevated intraabdominal pressure on healing of colonic anastomoses. Am J Surg 1999;178:293-7. 24. Graham MF, Blomquist P, Zederfeldt B. The alimentary canal. In: Cohen IK, Diegelmann RF, Lindblad WJ, editors. Wound healing: biochemical and clinical aspects. First ed. Philadelphia: WB Saunders Co; 1992. p. 433-49. 25. Thornton FJ, Barbul A. Healing in the gastrointestinal tract. Surg Clin North Am 1997;77:549-73. 26. Ahrendt GM, Gardner K, Barbul A. Loss of colonic structural collagen impairs healing during intra-abdominal sepsis. Arch Surg 1994;129:1179-83. 27. Ahrendt GM, Tantry US, Barbul A. Intra-abdominal sepsis impairs colonic reparative collagen synthesis. Am J Surg 1996;171:102-7. 28. Kashiwagi H. The lower limit of tissue blood flow for safe colonic anastomosis: an experimental study using laser Doppler velocimetry. Surg Today 1993; 23: 430-38. 29. Senagore A, Milsom JW, Walshaw RK, et al. Intramural pH: quantitative measurement for predicting colorectal anastomotic healing. Dis Colon Rectum 1990;33:175-9. 30. Sheridan WG, Lowndes RH, Young HL. Tissue oxygen tension as a predictor of colonic anastomotic healing. Dis Colon Rectum 1987;30:867-71.
Background. Although the effect of locally applied ischemia-reperfusion (I-R) injury on gastrointestinal anastomoses has been studied, to our knowledge there is no previous study that investigates the effect of remote I-R injury on gastrointestinal anastomotic healing. The aim of this study was to investigate and compare the effects of local I-R injury and remote I-R injury on the healing of colonic anastomoses. Methods. Anastomosis of the right colon was performed in 30 rats that were divided into 5 groups. Group 1 was the control group. In Group 2, I-R was applied to the colonic segment containing the anastomosis. Unilateral lower extremity I-R, unilateral renal I-R, and segmental small intestinal I-R was applied to the rats in Groups 3, 4, and 5, respectively, at the same time as colonic anastomosis. On the fourth postoperative day, animals were killed and bursting pressure and tissue hydroxyproline concentration of the anastomoses were analyzed and compared. Results. The mean bursting pressure values were: 143 mm Hg in Group 1, 40.8 mm Hg in Group 2, 82.8 mm Hg in Group 3, 46.1 mm Hg in Group 4, and 52.3 mm Hg in Group 5 (P < .0001; 1-way analysis of variance). Mean tissue hydroxyproline concentration values were: 5.3 µg/mg in Group 1, 1.6 µg/mg in Group 2, 2.2 µg/mg in Group 3, 1.3 µg/mg in Group 4, and 1.5 µg/mg in Group 5 (P < .0001, 1-way analysis of variance). Bursting pressure and tissue hydroxyproline concentration values had a good correlation r = 0.86, P < .001, Pearson correlation analysis). Conclusions. This study showed that I-R injury is a systemic phenomenon, and remote organ I-R can significantly delay anastomotic healing. This has to be kept in mind when constructing an intestinal anastomosis in the presence of local or remote I-R injury. (Surgery 2000;128:99-104) From the Departments of General Surgery and Biochemistry, Ankara, Turkey
THE EFFECT OF ISCHEMIA-REPERFUSION (I-R) injury on several organ systems is one of the most investigated subjects in experimental surgery. Clinically, I-R is important in transplantation, trauma surgery, vascular surgery, and in low-flow states.1 Today we know that reperfusion may be more harmful to tissues than the preceding ischemia, depending on the duration.2,3 In the 1990s, it became accepted that I-R injury has systemic effects and may induce a systemic inflammatory reaction.1,2,4-8 It has been concluded that the systemic effects of I-R injury are caused by activated neutrophils, the complement system, and proinflammatory and vasoactive mediators such as eicosanoids, nitric oxide, cytokines, and oxygen-free radicals.1,2,4-11 Although the effect of locally applied I-R injury on gastrointestinal anastomoses has been studAccepted for publication March 18, 2000. Reprint requests: Dr Murat Kologlu, Ahmet Mithat Efendi S 19/2, Çankaya, 06550, Ankara, Turkey. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.107414
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ied,12,13 to our knowledge there is no previous study that investigates the effect of remote I-R injury on gastrointestinal anastomotic healing. The aim of this study was therefore to investigate and compare the effects of local I-R injury and remote I-R injury on the healing of colonic anastomoses. For this purpose, we constructed anastomosis of the right colon in rats and compared the anastomotic healing in control group animals with the healing in animals having colonic (segment containing the anastomosis), lower extremity, renal, or small intestinal I-R injury. MATERIALS AND METHODS Thirty female Wistar albino rats weighing 150 to 200 g were used in the study. All animals were allowed standard rat chow and water ad libitum. Twelve hours before anesthesia, animals were deprived of food but had free access to water. After the intramuscular introduction of anesthesia with 5 mg/kg of xylazine (Rompun) and 30 mg/kg of ketamine hydrochloride (Ketalar), all rats underwent a midline laparotomy. The right colon was transected and an end-to-end anastomosis was perSURGERY 99
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formed by using one layer of interrupted 7/0 polypropylene sutures (Prolene, Ethicon, UK). After the operation, rats were fed with standard rodent chow and water. Rats were divided into 5 groups, each having 6 animals: Group 1, Control: These animals had anastomosis of the right colon and did not have any other procedure until the fourth postoperative day. Group 2, Colonic I-R (local I-R): At the same time as the anastomosis of the colon was performed, a microvascular clip was placed across the distal end of the superior mesenteric artery, occluding the ileocolic and right colic arteries. In addition, the collateral arcades in the mesentery of the terminal ileum and the right colon were occluded by microvascular clips. By this procedure, the terminal ileum, caecum, and right colon containing the anastomosis were rendered ischemic. After 60 minutes of ischemia, the clips were removed and the reperfusion period began. It ended at the fourth postoperative day. Group 3, Lower extremity I-R: Simultaneously with the colonic anastomosis, unilateral lower extremity ischemia was induced by applying a rubber band tourniquet high around the left thigh. At the end of the 60-minute ischemia period, reperfusion was achieved by releasing the tourniquet. The reperfusion period ended at the fourth postoperative day. Group 4, Renal I-R: A microvascular clip was placed across the pedicle of the right kidney, at the same operation with the anastomosis of the colon. At the end of 60 minutes of renal ischemia, the clip was removed and the reperfusion period lasted until the fourth postoperative day. Group 5, Small intestinal I-R: At the same time as the anastomosis of the colon, a microvascular clip was placed across 2 or 3 ileal branches of the superior mesenteric artery; also, the collateral arcades in the mesentery of that ileal segment were occluded by microvascular clips. By this procedure, a segment of 8- to 10-cm ileum was rendered ischemic for 60 minutes. At the end of the ischemia period, the clips were removed and reperfusion began, which lasted until the fourth postoperative day. After the procedures, the laparotomy wounds were closed with continuous 4/0 silk sutures. Animals were fed with standard rat chow and water ad libitum postoperatively. At the fourth postoperative day, all animals were killed and a re-laparotomy was done. None of the organs that had I-R injury had macroscopic findings of irreversible ischemia and necrosis. The colonic anastomosis was dissected free of adhesions and a 4-cm segment
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of colon with the anastomosis in the middle was resected. One end of this segment was closed with a ligature, and a catheter was secured to the other end. Inside a glass jar filled with water, air was pumped into the segment of colon at a rate of 2 mL/min with an infusion pump. Intraluminal pressure was monitored while the air was pumped. The intraluminal pressure at which air leakage from the anastomosis occurred was recorded as the bursting pressure. This parameter showed the mechanical strength of the anastomoses. After the bursting pressure was measured, a 1cm segment of colon containing the anastomosis was resected to determine tissue hydroxyproline concentration, which represents the perianastomotic collagen concentration. Tissue hydroxyproline concentrations were determined by using the spectrophotometric method of Bergman and Loxley.14 Calculations were made to express the results as micrograms of hydroxyproline per milligram of tissue (µg/mg). The mean bursting pressures and tissue hydroxyproline concentrations of groups were calculated and expressed as mean ± SEM. The mean values of groups were compared by 1-way analysis of variance (ANOVA), and groups were compared separately by the Tukey-Kramer multiple comparisons test. Pearson correlation analysis was done to define the correlation of bursting pressure and hydroxyproline concentration values of the animals. In these tests, P < .05 was considered statistically significant. SPSS for Windows (6.0; SPSS Inc, Chicago, Ill) and Graphpad Instat (2.02; Graphpad Software Inc, San Diego, Calif) softwares were used for statistical analysis. RESULTS In this study, we used 2 parameters of wound healing to compare the healing in colonic anastomoses. These parameters were bursting pressure, which showed the mechanical strength of the anastomosis, and perianastomotic tissue hydroxyproline concentration, which represented the collagen accumulation around the anastomosis. When analyzing bursting pressures, all leaks occurred from the anastomosis. The mean bursting pressures of all the I-R groups were lower than the control groups. The mean bursting pressure of the lower extremity I-R group was higher than other I-R groups. These values in renal I-R and small intestinal I-R groups were very close to the mean value of the colonic I-R group (Table, Fig 1). The differences between the mean bursting pressures of the groups were statistically significant (P < .0001, 1-way ANOVA). When
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Fig 1. Bursting pressure values in the groups. Values of all animals are presented.
Table I. Mean bursting pressures and tissue hydroxyproline concentrations of the groups Group 1 Control
Group 2 Colonic (local) I-R
Mean ± SEM bursting pressure (mm Hg) 143 ± 2.9 Mean ± SEM tissue HP concentration (mg/mg) 5.3 ± 0.3
40.8 ± 4.1*† 1.6 ± 0.1‡
Group 3 Lower extremity I-R 82.8 ± 14.1* 2.2 ± 0.2‡
Group 4 Renal I-R 46.1 ± 7.7*† 1.3 ± 0.1‡
Group 5 Small intestinal I-R 52.3 ± 8.6* 1.5 ± 0.2‡
HP, Hydroxyproline. P < .0001 (1-way ANOVA) for the differences between mean bursting pressures of the groups. P < .0001 (1-way ANOVA) for the differences between mean tissue HP concentrations of the groups. *Compared with Group 1, difference is significant (P < .001, Tukey-Kramer test). †Compared with Group 3, difference is significant (P < .05, Tukey-Kramer test). ‡Compared with Group 1, difference is significant (P < .001, Tukey-Kramer test).
the groups were compared one by one, differences between the control group and all other groups were statistically significant (P < .001 for Group 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group 4, and Group 1 vs Group 5; Tukey-Kramer multiple comparisons test). Differences between colonic I-R, renal I-R, and lower extremity I-R groups also were statistically significant (P < .05, for Group 2 vs Group 3, Group 4 vs Group 3; Tukey-Kramer multiple comparisons test). The mean tissue hydroxyproline concentrations showed the same trend; all I-R groups had mean values lower than the control group. Among I-R groups, the highest mean value was determined in the lower extremity I-R group. Colonic I-R, renal IR, and small intestinal I-R groups had very close mean values (Table, Fig 2). The differences between mean values of the groups were statistically significant (P < .0001, 1-way ANOVA). When the groups were compared one by one, the differences between the control group and all other groups were statistically significant (P < .001, for Group 1 vs Group 2, Group 1 vs Group 3, Group 1 vs Group
4, Group 1 vs Group 5; Tukey-Kramer multiple comparisons test). The correlation between bursting pressure and tissue hydroxyproline concentration values of all animals that were included in the study was determined by Pearson correlation analysis. These 2 parameters had a positive good correlation r = 0.86, P < .001, Pearson correlation analysis; Fig 3). The coefficient of determination (r2) was 0.74. DISCUSSION The effect of local I-R injury on intestinal anastomoses was investigated in a few studies, and it has been shown that anastomotic healing is delayed in intestinal segments that are subjected to I-R injury.12,13 Although systemic effects of I-R injury have gained more attention in recent years, the effect of remote organ I-R injury on gastrointestinal anastomotic healing remains unclear. Ischemia is mainly a local event but, after revascularization, the mediators from the ischemic tissue enter the systemic circulation and effect other organ systems, which may end up with multiorgan dysfunc-
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Fig 2. Tissue hydroxyproline concentration values in the groups. values of all animals are presented.
Fig 3. Bursting pressure values of animals are plotted against tissue hydroxyproline concentration values, and linear curve estimation is shown. r = 0.86, P < .001, Pearson correlation analysis.
tion; so I-R injury is a systemic phenomenon.* Neutrophils seem to play a central role in these systemic effects; after reperfusion, mediators like eicosanoids, complements, cytokines, and oxygenfree radicals derived from ischemic tissue activate the neutrophils and endothelium.1,2,4-11 The degree of neutrophil activation is related to the extent of I-R injury. Beginning with the first minutes of reperfusion, a systemic inflammatory reaction is induced.5,10,11 This is accompanied by a severe local microvascular dysfunction and a less severe microvascular dysfunction in the systemic circulation.4-6,10,15 Normally, oxygen-free radicals have a very short half-life and mainly have local effects. But xanthine oxidase can be released into the systemic circulation from ischemic tissues upon reperfusion, and *References 4, 5, 7, 8, 10, 11, 15-17
by reacting with circulating substrates it can produce intravascular oxygen-free radicals that may have effects on remote organ systems.17-20 In addition, circulating cytokines activate the endothelial cells in remote organ systems to increase the expression of adhesion molecules causing activated neutrophils to interact with endothelium and have injurious effects.1,4-7,11,15 The most significant and most investigated remote organ injury appears in lungs. Endothelial cell adenosine triphosphate depletion, increased vascular permeability, intraparenchymal hemorrhage, and neutrophil accumulation occurs in lungs after lower extremity or intestinal I-R.2,4-7,9,15,20 Other results of lower extremity I-R include: a decrease in the intestinal mucosal thickness and an increase in the intestinal mucosal permeability, in bacterial translocation, in systemic endotoxin concentration, and in hepatic and renal dysfunction.8,16 The administration of
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oxygen-free radical scavengers, xanthine oxidase inhibitor allopurinol, antineutrophil serum (or removal of neutrophils from reperfusate), antibodies directed against adhesion molecules and various cytokines, inhibitors of some arachidonic acid metabolites and complements can attenuate the local and the systemic effects of I-R injury.1-7,11 Blocking mediators can be more beneficial for the systemic effects of I-R, which are indirect and mediator dependent, compared with the local effects.5,6 In this study, we investigated and compared the effect of local and remote I-R injury on healing of colonic anastomoses. Both bursting pressures (mechanical parameter) and perianastomotic tissue hydroxyproline concentrations (biochemical parameter) of the anastomoses were determined as recommended in the literature.21-23 Healing was evaluated in the early postoperative period when the majority of anastomotic leaks take place. We showed that renal and small intestinal I-R injury can delay healing in colonic anastomoses as much as colonic (local) I-R injury. Lower extremity I-R injury also delayed anastomotic healing significantly but not as much as colonic (local) I-R injury did. Both parameters of anastomotic healing gave the same results, and they had a good correlation. Investigation of the reasons for these results and whether blocking various mediators in the presence of I-R injury protects the anastomoses has to be the subject of another more detailed study. When an anastomosis is constructed in the gastrointestinal tract, an inflammation takes place as a response to traumatic injury and foreign material such as sutures.24,25 This inflammation is a normal constituent of wound healing. But if it is exaggerated, wound healing is delayed because of increased collagenolysis; it is why anastomotic healing is delayed in the presence of intraabdominal infection.23-27 Since remote organ I-R has a significant effect on healthy lungs, the effect of remote organ I-R on colonic anastomoses may be explained by similar mechanisms. The anastomotic area is already inflamed, and the endothelium in the perianastomotic area is already activated in the early phase of wound healing.24,25 As a result of proinflammatory and chemoattractant properties of the anastomosis, activated circulating neutrophils secondary to I-R injury in a remote organ system may accumulate easily in the perianastomotic area, increase the inflammatory reaction, and delay healing. Together with the proteolytic enzymes, oxygen-free radicals derived from activated neutrophils and circulating xanthine oxidase may increase collagenolysis in the perianastomotic area, which delays wound healing.12
On the other hand, I-R injury of an intraabdominal organ represents a form of acute intraabdominal trauma and may have humorally and/or neurally mediated local effects on splanchnic microcirculation leading to mesenteric vasoconstriction. It is shown that after intestinal I-R, splanchnic blood flow may be reduced by > 90% from baseline measurements.10 Defects of perfusion and oxygenation are known to delay healing in gastrointestinal anastomoses.23-25,28-30 This situation may explain why anastomotic healing is delayed more in renal and small intestinal I-R groups compared with the lower extremity I-R group. In light of our findings, we may draw some conclusions for clinical situations. Results of this study confirmed that I-R injury is a systemic phenomenon. The adverse effect of local I-R on anastomotic healing was also confirmed. Remote organ I-R was shown to delay anastomotic healing significantly. These have to be kept in mind when constructing an intestinal anastomosis in the presence of local or remote I-R injury. For instance, intestinal anastomoses constructed simultaneously with elective vascular reconstructions, emergency repairs of traumatic vascular lacerations, or in low-flow states may be under considerable risk because of reperfused ischemic tissues. When possible, avoiding intestinal anastomoses in these situations may be a reasonable approach.
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