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Histopathological effects of ischemia-reperfusion on intestinal anastomotic wound healing in dogs/Efectos histopatologicos de reperfusion isquemia en la anastomosis intestinal de heridas curadas en perros.

SUMMARY: The histopathological effects of three levels of ischemia-reperfusion (IR) on healing of intestinal anastomotic wound were investigated in dogs. Three groups of dogs in which superior mesenteric artery (SMA) and collaterals were isolated but not occluded (A-control), isolated and occluded immediately distal to the aorta with collateral interruption using an atraumatic arterial clip for fifteen minutes (B) and for forty-five minutes (C) prior to ileal resection and anastomosis after return of normal perfusion in occluded groups was done. Within each group, animals were anaesthesized on third and seventh post-operative day. Grossly, impaired abdominal wound healing, intra-abdominal adhesions and partial and complete anastomotic dehiscence occurred in groups B and C commensurate with intensity of ischemic injury. Histologically, light cellular infiltration with loose granulation tissue was observed in the control group with moderate neovascularization and epithelialization by the seventh day. With mild IR, moderate cellular infiltration was observed four days post operation, but heamorrhage and oedema persisted with minimal mucosal regeneration at seven days post operation. With profound IR, sloughing of the mucosa with cellular infiltration at four days with absence of mucosal regeneration and persistence of inflammatory cellular infiltrates minimal neovascularization and dense granulation tissue. Neutrophilia in the immediate post operative period appears significant to impaired healing due to ischemia-reperfusion.

KEY WORDS: Histopathology; Ischemia-reperfusion injury; Intestinal anastomoses; Wound healing.

RESUMEN: Se estudiaron, en perros, los efectos histopatologicos de tres niveles de reperfusion isquemia en la curacion de anastomosis intestinal. Fueron utilizados tres grupos de perros, en los cuales las colaterales de la arteria mesenterica superior fueron aisladas, pero no ocluidas (A-control), aisladas y ocluidas inmediatamente distal a la aorta con interrupcion colateral usando un clip arterial no traumatico, por 15 minutos (B) y por cuarenta y cinco minutos (C), previo a la reseccion ileal y la anastomosis despues de retornar a la perfusion normal en los grupos ocluidos. Dentro de cada grupo, los animales fueron anestesiados al tercer y septimo dias postcirugia. En la cicatrizacion del dano abdominal se observaron adhesiones y dehiscencias parcial y completa en los grupos B y C, correspondiendose con la intensidad del dano causado por la isquemia. Histologicamente, se observo en el grupo control, baja infiltracion celular con tejido de granulacion laxo, con moderada neovascularizacion y epitelizacion, desde el septimo dia. Con una suave IR, tambien se observo moderada infiltracion celular cuatro dias postcirugia, pero la hemorragia y el edema persistieron con una minima regeneracion mucosa hasta los sietes dias despues de la operacion. Con una profunda IR, la mucosa se desprendio con infiltracion celular hasta los cuatro dias con ausencia de regeneracion mucosa y persistencia de infiltrado celular inflamatorio con minima neovascularizacion y tejido denso de granulacion. Se observo una significativa neutrofilia en el postoperatorio inmediato lo que perjudico la cicatrizacion debido a la reperfusion-isquemia.

PALABRAS CLAVE: Histopatologia; Reperfusion isquemia; Anastomosis intestinal; Cicatrizacion de heridas.

INTRODUCTION

Healing of intestinal tissue wounds are affected by various factors, of which intrinsic vasculature is paramount as this determines the early movement of mediators of inflammatory process into the wound and commencement of wound cleansing to prevent infection which also affects healing significantly (Hunt et al., 1969).

Ischemia-reperfusion injury significantly impairs healing of intestinal anastomosis either directly or as a systemic phenomenon (Kuzu et al., 1998; Kuzu et al., 2000). It has been established that damage from an ischemic event is a product of two phases of injury with the most significant occurring after reperfusion, the severity being dependent on the intensity of the insult (Grace, 1994; Schiller et al., 1993; Olatunji-Akioye & Akinrinmade, 2006). When blood flow is restored to ischemic tissues, a local inflammatory response begins, which is then enhanced by the production of bioreactive oxygen radicals (Koksoy et al., 2000), which promote increased mitochondrial membrane permeability (Qian et al., 1997). Also, production and release of pro-inflammatory agents lead to polymorphonuclear leukocyte adherence and emigration causing microvascular leakage leading to cell death in remote organs by necrosis or apoptosis, which results in systemic immune response syndrome (MacLellan & Schneider, 1997; Austen et al., 1999).

The interplay of cells during inflammation and wound healing is macrophages and fibroblasts are predominant during early inflammation, whereas lymphocytes peak later (Witte & Barbul, 1997). Neutrophil infiltration is the primary mediator of reperfusion induced tissue damage through its effects on microvascular permeability and bacterial contamination when present (Megison et al., 1990).

Collagen production, which determines the gain in strength of anastomoses is by both fibroblasts and smooth muscle cells in the gastrointestinal tract (Graham et al., 1987) and influence of xanthine oxidase derived specie on collagen matrix is inhibitory (Arisawa et al., 1996).

MATERIAL AND METHOD

Ten dogs (six female and four male aged between nine months and a year) were used for this study. They were housed at the Department of Veterinary Surgery kennel. They were fed a meal of rice and fish pre-operatively and pap and milk peri- and post-operatively. They were brought to condition base-line blood samples were collected to establish normal parameters. Operation day was determined as day 0.

The dogs were pre-medicated using atropine sulphate(atropine injection B.P., Hamex Medica Ltd, UK) at a dose rate of 0.1mg/kgbwt intra-muscularly and xylazine hydrochloride (Xylazine 20 inj. Kepro, Holland) at a dose rate of 2mg/kgbwt intramuscularly. Anaesthesia was induced and maintained using Pentobarbitone sodium (6% solution, Kyron Laboratories, South Africa) at a dose rate of 25mg/kgbwt, intravenously.

Operative procedure. The ventral abdomen was shaved and prepared for aseptic surgery. A ventral mid-line incision was made on the skin from the xiphoid to the pubic region through the skin. The intestines were exteriorized and covered with sterile towel soaked in saline at 37[degrees]C in order to minimize fluid evaporation and heat loss. Maintenance of body temperature was achieved by using a heating lamp, and warm Lactated Ringer's solution is given intravenously during the operation, which also helps prevent dehydration of the animal.

The superior mesenteric artery (SMA) was located beneath the first lumbar vertebra close to the celiac artery within the root of the mesenteric lymph node just distal to the aorta.

In group A, the SMA and collateral vessels were not occluded; a 5cm length of the ileum was resected and anastomosed.

In group B, the SMA is clamped with an a-traumatic arterial clip along with the collateral supply for 15 minutes.

In group C, the SMA was similarly clamped along with the collateral blood supply for 45 minutes.

When declamped, restoration of vascular supply was assessed based on the color of the intestines, return of pulsation and peristalsis. After declamping, a 5cm length of the ileum about 5cm from the ileocecocolic junctionin bith groups B and C.

Following return of normal blood supply, anastomosis of the resected ends of the intestine was achieved by end-toend anastomotic technique. The anastomosis was tested for leakage by placing a syringe filled with 5mls normal saline, using fingers to occlude both ends of the intestine and delivering the normal saline therein.

The laparatomy incision was closed in 3 layers--the linea alba was closed using chromic cat-gut (size 2-0 Hospibrand), in a simple continuous suture pattern (Ahchong et al, 1996), the subcutis using a subcorticular suture pattern while the skin was closed using nylon (size 2-0 Hospibrand) in a horizontal mattress suture pattern.

Post-operative care. which was given routinely consisted of antibiotic therapy (penicillin and streptomycin in a 2:1 mixture, (Streptopen, Kepro, Holland) at a dose rate of 5ml/10kg bwt intramuscularly. The dogs were fed a diet of pap and milk and housed in individual kennels.

Sample collection. Re-operation of the animals was done on either day 4 or 7 postoperatively in all the groups in order to assess the intestinal anastomotic site after which the anastomotic site was removed and preserved in formalin. The samples were then embedded in paraffin for serial longitudinal sections to be cut and stained with Heamatoxylin and Eosin (H and E).

Histological Assessment. This was done by using a double blind method and it involves scoring done according to De Roy van Zuidewijn et al., 1992:

The examination of wound healing procedure was based on re-epithelialization of the mucosa and the repair of the muscularis propria and this was scored on a seven point scale while the repair of the muscularis propria was considered as positive or negative.

Necrosis, inflammatory exudates, granulation tissue and the level of granulocytes, macrophages and fibroblasts present in the granulation tissue was evaluated and scored on a fourpoint scale as 0-negative, 1+-low, 2+--moderate, 3+-high.

RESULTS AND DISCUSSION

In this study, it was observed that at four days postoperation, loose granulation tissue was present in the control group (Fig. A1), intact mucosa with foci of cellular infiltrates was present in the mild ischemia group (Fig. B1) whereas there was sloughing of canine intestinal mucosa with cellular infiltration, which was significantly more than the other groups (Fig. C1 and Table I). it is however important to note the increased inflammation and cellular infiltration, which is evident even in adipose tissue (Fig. C1b) and the presence of macrophages to the same degree, fibroplasia is least in the profound group (Table I) and this may be responsible for the impaired healing later observed (OlatunjiAkioye & Akinrinmade) as fibroblasts are responsible for collagen formation as well as smooth muscle cells in the intestines and this lays the framework for granulation tissue formation (Graham et al.). Also, the extent of fibroplasia is proportional to oxygen tension, which is dependent on vascularization.

Seven days postoperation, neovascularization and granulation tissue was observed in the control group (Fig. A2) which suggests that re-epithelialization is commencing as epithelial cells move and replicate most rapidly in hydrated and well oxygenated tissues. In the mild ischemia group, there's absence of mucosal regeneration with minimal neovascularization and cellular infiltrates (Fig. C2).

Wound healing is a complex process involving different biologic and immunologic systems and despite improvements in diagnostics and therapy, wound failures remain a critical problem (Witte & Barbul).

[FIGURE A1 OMITTED]

[FIGURE A2 OMITTED]

The histopathology of IR is based on hemostasis, which precedes inflammation where the fibrin clot serves as scaffolding for invading cells, such as neutrophils, monocytes fibroblasts, plasma cells and endothelial cells (Kurkinen et al., 1980). Chemotaxis of cells into wound is followed by functional activation, which is the phenotypic altering of cellular, biochemical and functional properties induced by local mediators. This induces new cell surface antigen expression, increased cytotoxicity, increased production and release of cytokines and other effects.

[FIGURE B1 OMITTED]

[FIGURE B2 OMITTED]

[FIGURE C1 OMITTED]

[FIGURE C1b OMITTED]

[FIGURE C2b OMITTED]

Fibroblasts in surrounding tissues need activation from their quiescent states and then migrate into the wound site. Granulocytes are responsible for a significant degree of collagenolytic activity and contamination and tissue necrosis increases their presence. They are however almost totally absent from ileal anastomoses by day seven (Hesp et al., 1985).

Received: 02-05-2007

Accepted: 12-09-2007

REFERENCES

Arisawa, S.; Arisawa, T.; Ohashi, M.; Nitta, Y.; Ikeya, T. & Asai, J. Effect of the hydroxyl radical on fibroblast mediated collagen remodeling in vitro. Clin. Exp. Pharmacol. Physiol., 23:222, 1996.

Austen, W. G.; Kyriakydes, C.; Faruzza, J.; Wang, Y.; Kobzik, L.; Moore, F. B. & Hechtman, H. B. Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex. Surgery, 126:343-8, 1999.

De Roy van Zuidewijn, D. B. W.; Schillings, P. H. M.; Wobbes, T. & de Boer, H. H. M. Histological evaluation of wound healing inexperimental intestinal anastomoses: Effects of antineoplastic agents. Int. J. Exp. Pathol., 73:465-84, 1992.

Graham, M. F.; Drucker, D. E.; Diegelmann, R. F. et al. Collagen synthesis by human intestinal smooth muscle cells in culture. Gastroenterology, 92:400, 1987. Grace, P. A. Ischemia-reperfusion injury. Br. J. Surg., 81:637-47, 1994.

Hesp, W. L.; Hendricks, T.; Schillings, P. H. et al. Histological features of wound repair. A comparison between experimental ileal and colonic anastomoses. Br. J. Exp. Pathol., 66:511, 1985.

Hunt, T. K.; Zederfeldt, B. & Goldstick, T. K. Oxygen and healing (review) Ann. J. Surg.,118:521, 1969.

Koksoy, C.; Kuzu, M.A.; Ergun, H.; Demiprence, E. & Zulfikaroglu, B. Intestinal ischemia-reperfusion impairs vasomotor functions of pulmonary vascular bed. Ann. Surg., 231:105-11, 2000.

Kurkinen, M.; Vaheri, A.; Roberts, P. J. et al. Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab. Invest., 43:47,1980.

Kuzu, M. A.; Koksoy, C.; Kale, I. T.; Tanik, A.; Terzi, C. & Elhan, A. Reperfusion injury delays healing of intestinal anastomosis in a rat. Am. J. Surg., 176:34851, 1998.

Kuzu, M. A.; Tanik, A.; Kale, I. T.; Aslar, A. K.; Koksoy, C. & Terzi, C. Ischemia-reperfusion as a systemic phenomenon impairs the anastomotic healing in left colon. World J. Surg., 24:990-4, 2000.

MacLellan, W. R. & Schneider, M. D. Death by design. Programmed cell death in cardiovascular biology and disease. Circ. Res., 81:137-44, 1997.

Megison, S. M.; Horton, J. W.; Chao, H. & Walker, P. P. A new model for intestinal ischemia in the rat. J. Surg. Res., 49:168-74, 1990.

Olatunji-Akioye, A. O. & Akinrinmade, J. F. Effect of Ischemia-Reperfusion on healing in anastomosis. Trop. Vet., 24(3):62-9,2006.

Qian, T.; Nieminen, A.L.; Herman, B. & Lemasters, J. J. Mitochondrial permeability transition in pHdependent reperfusion injury to rat hepatocytes Am. J. Physiol., 273:c1783-c92, 1997.

Schiller, H. J.; Reilly, P. M. & Bulkley, G. B. Anti oxidant therapy. Crit. Care Med., 20:92-102, 1993.

Witte, M. B. & Barbul, A. General Principles of Wound Healing 77(3) 509-29, 1997.

Correspondence to:

Dr. Adenike O. Olatunji-Akioye

Veterinary Teaching Hospital,

University of Ibadan

NIGERIA

Tel: 234-803-409-1407

Email: bonik2001@yahoo.com

* Akinrinmade, J. F. & ** Olatunji-Akioye, A. O.

* Department of Veterinary Surgery & Reproduction, Faculty of Veterinary Medicine, University of Ibadan, Nigeria.

** Veterinary Teaching Hospital, University of Ibadan, Nigeria.

AKINRINMADE, J. F. & OLATUNJI-AKIOYE, A. O. Histopathological effects of ischemia-reperfusion on intestinal anastomotic wound healing in dogs. Int. J. Morphol., 25(4):805-810, 2007.

AKINRINMADE, J. F. & OLATUNJI-AKIOYE, A. O. Efectos histopatologios de reperfusion-isquemia en la anastomosis intestinal de heridas curadas en perros. Int. J. Morphol., 25(4):805-810, 2007.
Table I. 4 days post-operation

Histopathological       Control      Mild     Profound
features                   A          B          C

Inflammatory               1+         1+         3+
Inflammatory cells:
Neutrophils                1+         3+         4+
Macrophages                2+         2+         2+
Lymphocytes                1+         1+         1+
Plasma cells               2+         0          0
Granulation tissue         3+         3+         3+
Fibroplasia                3+         2+         1+
Neovascularization         3+         3+         2+
Reepithelialization     Moderate   Moderate     Bad

Table II. 7 days post-operation.

Histopathological       Control      Mild     Profound
features                   A          B          C

Inflammatory exudates      1+         4+         1+
Inflammatory cells:
Neutrophils                1+         2+         1+
Macrophages                2+         2+         1+
Lymphocytes                1+         1+         3+
Plasma cells               2+         0          1+
Granulation tissue         3+         3+         1+
Fibroplasia                3+         2+         3+
Neovascularization         3+         2+         2+
Reepithelialization     Moderate     Bad        Bad
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Author:Akinrinmade, J.F.; Olatunji-Akioye, A.O.
Publication:International Journal of Morphology
Date:Dec 1, 2007
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