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Mast Cell Degranulation is Essential for Anastomotic Healing in Well Perfused and Poorly Perfused Rat Colon
Journal of Surgical Research 164, e73–e76 (2010) doi:10.1016/j.jss.2010.04.035
Mast Cell Degranulation is Essential for Anastomotic Healing in Well Perfused and Poorly Perfused Rat Colon John Coneely, M.D.,† Rory Kennelly, M.Ch.,*,1 D. Bouchier-Hayes, M.D.,† and Desmond C. Winter, M.D.* *Institute for Clinical Outcomes, Research, and Education (ICORE), St. Vincent’s University Hospital, Dublin, Ireland; and †Royal College of Surgeons, Ireland Submitted for publication February 12, 2010
INTRODUCTION Background. Mast cell degranulation is an important step in early wound healing in the skin however the role of the mast cell in anastomotic healing is less clear. The aim of this study was to investigate the importance of mast cell degranulation in anastomotic healing and to assess whether a promoter of mast cell degranulation could increase anastomotic healing in poorly perfused bowel. Methods. Fifty Wistar rats were divided into five groups: control, normally perfused bowel with mast cell stabilisation, normally perfused bowel with mast cell degranulation, hypoperfused bowel, and hypoperfused bowel with mast cell degranulation. A colo-colonic anastomosis was formed in each animal. Four d later, following sacrifice, the strength of the anastomosis was assessed in each animal. Results. Mast cell stabilisation reduced anastomotic healing in normally perfused bowel (P < 0.001). Hypoperfused bowel resulted in reduced anastomotic strength (P < 0.001) however the addition of a mast cell degranulating agent increased healing in hypoperfused bowel to levels comparable with control. Conclusions. Mast cell degranulation is essential for early anastomotic healing. Healing is reduced in hypoperfused bowel but the administration of a mast cell degranulation agent can compensate for the adverse effects of a poor blood supply on anastomotic healing. Ó 2010 Elsevier Inc. All rights reserved. Key Words: mast cell; wound healing; ischemia.
1 To whom correspondence and reprint requests should be addressed at Institute for Clinical Outcomes Research and Education, Department of Surgery, St. Vincent’s University Hospital, Dublin 00004, Ireland. E-mail: [email protected].
The management of colorectal cancer has changed considerably over the last 20 y. Total mesorectal excision coupled with low anterior resection has greatly improved local control [1], neo-adjuvant chemotherapy and radiation have contributed to increased survival, and advances in anesthetic practice have increased the number of patients who are considered fit to undergo colonic resection [2]. While all of these advances have improved oncologic outcomes, they have resulted in significant morbidity with the foremost cause being anastomotic leak [3]. Anastomotic leak rates are reported at 1%–30% [4]. While numerous contributing factors are known (irradiated bowel, atherosclerosis, diabetes mellitus [5]), the common underlying etiology is, arguably, impaired blood supply. The mast cell plays an integral role in skin wound healing through the secretion of histamine and cytokines and promotion of neovascularization through the release of vascular endothelial growth factor [6]. This is achieved by degranulation of the mast cell locally at the site of injury [7]. Mast cells are present in large numbers in the intestinal mucosa but their role in anastomotic healing is not known. Furthermore, it is unknown how ischemia affects mast cell function in the healing bowel. Ketotifen is a known mast cell stabilizing agent preventing degranulation [8] and compound 48/80 is a promoter of mast cell degranulation [9]. The purpose of this study is to examine the role of the mast cell in anastomotic wound healing in a rat model by inhibition and induction of mast cell degranulation. This is performed in normal colon and in a model of impaired colonic blood supply. MATERIALS AND METHODS Institutional ethical approval was granted for this study. Fifty male Wistar rats were divided into five groups of 10: control, mast cell
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0022-4804/$36.00 Ó 2010 Elsevier Inc. All rights reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 164, NO. 1, NOVEMBER 2010
inhibition, mast cell degranulation, induced ischemia, and induced ischemia with mast cell degranulation. All were maintained with standard 12 h light/dark cycles, were fed standard laboratory chow and allowed water ad libitum up to 6 h prior to initiation of the experimental protocol.
Experimental Protocol Twenty-four hours preoperatively, control animals received normal saline via tail-vein injection as vehicular control. The degranulation and inhibition groups received a dose of 1 mg/kg Compound 48/80 and 1 mg/kg ketotifen, respectively, via tail vein. General anesthesia was induced by inhalational halothane. All animals underwent laparotomy and the rodent colon was identified. The colon was transected and anastomosis performed, end to end using eight, full-thickness, interrupted 6/0 polypropylene sutures (Prolene; Ethicon Inc., Kirkton, Scotland) In the ischemia groups, identification and suture ligation of the mesenteric arcade supplying the proximal colon was performed. The hypoperfused segment was identified with intraoperative angiography and the watershed area of colon was transected and anastomosis performed. The anastomosed colon was returned to the peritoneal cavity and mass closure was performed using a continuous 3/0 nylon suture (Ethilon; Ethicon Inc.). The abdominal skin was closed using a continuous 3/0 nylon suture. All animals received 5 mL sodium chloride (0.9% NaCl) intraperitoneal fluid resuscitation at closure of laparotomy wounds and intramuscular injections of buprenorphine hydrochloride, 0.1 mg/kg (Temgesic) prior to termination of anesthesia. They were allowed access to water and chow ad libitum postoperatively.
Analysis Experimental animals were sacrificed on the fourth postoperative day by combination of anesthetise overdose and intra-peritoneal bolus injection of sodium pentobarbitone, 600 mg (Sagatal). Anastomotic healing was determined by measurement of anastomotic bursting pressure and tissue hydroxyproline content. Cytokine response to injury and ischemia was assessed by calculation of IL-6 and VEG-f serum levels.
FIG. 1. Mesenteric angiogram following ligation of mesenteric arcade. Reduced blood flow can be seen on left side of highlighted bowel loop.
Serum Specimens At time of sacrifice, 8 mL of whole blood was obtained by cardiac puncture using a 22 gauge needle. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum concentrations of Il-6 and VEG-f in all experimental groups. Standard, commercially available ELISA kits were used (Sigma-Aldrich, Ireland Ltd., Dublin, Ireland) Mean absorbance values were calculated for each duplicate set of standards and samples, less the average zero standard optical density. A standard curve was plotted and serum levels of each sample were calculated using this curve.
Statistical Analysis All values were calculated and expressed as mean 6 SD. The mean values of groups were compared using one way analysis of variance (ANOVA) or Kruskal-Wallis one way analysis of variance for nonparametric data. P < 0.05 was considered significant. Sigmastat was used for statistical calculations (Systat Software, San Jose, CA).
Tissue Specimen Retrieval Following sacrifice on d 4, the anastomosed segment was identified and excised, with a 2 cm segment of colon proximal and distal to the anastomotic site. Fresh specimens were immediately tested for anastomotic bursting pressure. Following bursting pressure determination, a standard sized segment of colon (5 g) incorporating equal lengths of proximal and distal colon was excised and retained for hydroxyproline estimation.
Anastomotic Bursting Pressure To determine anastomotic bursting pressure, the segment of bowel incorporating the anastomosis was clamped at one end and infused with a 10% betadine solution. Pressure change was measured in millimetres of mercury by a pressure transducer and an analog monitor (Hewlett Packard 78534C, Palo Alto, CA). Peak bursting pressure was determined as the peak value on the pressure trace immediately prior to the precipitous drop that indicated anastomotic bursting.
Hydroxyproline Estimation Hydroxyproline estimation was performed according to the protocol described by Reddy and Enwemeka [10]. Specimens of perianastomotic tissue (5 g) were dissected, flash-frozen in liquid nitrogen, and lyophilized. Absorbance for each sample was read at 550 nm using a Beckman spectrophotometer (Beckman Coulter Inc., Galway, Ireland).
RESULTS
There was no intraoperative or perioperative mortality. On examination of the anastomosis post mortem, there was no evidence of anastomotic dehiscence in the 5 test groups. There was no observed difference in adhesion formation and no peri-anastomotic abscess formation was noted. Confirmation of Induction of Ischemia
Following ligation of the intestinal mesenteric arcade, intraoperative angiogram was performed on TABLE 1 Comparison of Serum Concentration of VEG-f and IL-6 in Control and Model of Intestinal Ischemia Test group Control Ischemia
IL-6 conc. (ng/mL)
VEG-f
54.8 (62.2) 76.6 (63.1) P < 0.05
63.9 (62.4) 94.6 (61.9) P < 0.001
e75
CONEELY ET AL.: MAST CELL FUNCTION EFFECTS WOUND HEALING
TABLE 2 Comparison of Anastomotic Bursting Pressure and Hydroxyproline Content in All Groups Test group
ABP (mmHg)
HP conc. (mg/mg)
Control Ketotifen (non-ischemic) Compound 48/80 (non-ischemic)) Ischemia Ischemia þ compound 48/80
25.9 (62.4) 12.3 (62.3) P < 0.001 25.2 (61.2) P ¼ 0.37 13.5 (61.20) P <0.001 25.4 (61.3) P ¼ 0.59
5.2 (60.2) 2.4 (60.4) P < 0.001 5.1 (60.2) P ¼ 0.41 3.0 (60.4) P <0.001 5.0 (60.3) P ¼ 0.13
each test animal. The successful induction of hypoperfusion was confirmed via reduced contrast flow (Fig. 1). Cytokine measurement showed a significant increase in serum concentration of VEG-f and IL-6 in animals in the ischemia group, consistent with appropriate systemic response to hypoperfusion (Table 1). Anastomotic Strength
Inhibition of mast cell degranulation by ketotifen caused a decrease in anastomotic bursting pressure indicating impaired anastomotic healing (Table 2). Similarly, hydroxyproline content was significantly reduced in animals treated with the mast cell stabilizing agent (Table 2). The administration of Compound 48/80, a promoter of mast cell degranulation did not affect anastomotic bursting pressure or anastomotic hydroxyproline content in normally perfused colon. Induction of ischemia significantly impaired anastomotic bursting pressure and hydroxyproline content compared with control (Table 2). Administration of Compound 48/80, in the presence of impaired blood supply, improved anastomotic healing allowing for bursting pressures and hydroxyproline content comparable to control (Table 2). DISCUSSION
The aim of this study was to examine the role of mast cell degranulation in anastomotic healing in the rat colon. This was performed in normally perfused colon and in colon with an induced impaired blood supply. Inhibition of mast cell degranulation resulted in reduced wound healing. Induction of ischemia reduced anastomotic healing, however, this event was reversed by the administration of a mast cell degranulating agent allowing for comparable anastomotic strength in both normal colon and hypoperfused colon. Major advances have occurred in the management of rectal cancer with more patients achieving cure than ever before. Neo-adjuvant chemo/radiotherapy downstages up to 60% of rectal cancers and can cause complete pathologic response in up to 20% of cases [11]. However, these results do not come without a cost. Patient morbidity following surgery on irradiated bowel is markedly increased. This is particularly relevant in the
elderly patient where anastomotic leak has a mortality of up to 50% [12]. When this is seen in the context of the median age of diagnosis of colorectal cancer (72 y) [13] the extent of the problem is realized. Indeed, such is the significance of operative complications in the elderly that in the Dutch rectal cancer trial, all benefits from total mesorectal excision and radiotherapy were negated by postoperative non-cancer-related mortality [14]. Current practice dictates that if an anastomosis is constructed in irradiated bowel, a defunctioning stoma should be fashioned primarily to reduce the sequelae of an anastomotic dehiscence [15]. While this technique appears to reduce the risk of life threatening complications, it does necessitate a second operation for reversal of stoma, an undesirable situation in the elderly patient. For these reasons, techniques to enhance anastomotic healing are needed. Due to the important clinical implications of anastomotic failure, numerous methods have been attempted to improve healing with limited success. A moderate improvement was shown by the use of colloid resuscitation postoperatively in rats [16]. Platelet rich plasma showed no benefit in a pig model [17], and sutures impregnated with stem cells showed no benefit in a rat model [18]. No study to date has attempted to promote mast cell degranulation to aid healing. Mast cells are present in high concentration in mucosal tissue [19] and degranulation can occur due to a wide variety of stimuli [20]. The release of inflammatory mediators such as histamine and prostaglandin D2 are an integral part in the early stages of wound healing [21]. The role of the mast cell is well established in skin repair [22], however, the results presented here identify an important role for mast cell degranulation in anastomotic healing. The addition of a mast cell stabilizing agent reduced anastomotic strength showing that mast cell activation is necessary for normal anastomotic healing. Interestingly, the addition of a mast cell degranulating agent to the normally perfused anastomosis did not result in increased wound strength. This indicates that mast cell activation is optimized in adequately perfused bowel. The effect of ischemia on wound healing is no surprise, however, the addition of a degranulation agent (Compound 48/80) markedly improved anastomotic strength. While neither the specific mediator of mast
e76
JOURNAL OF SURGICAL RESEARCH: VOL. 164, NO. 1, NOVEMBER 2010
cell degranulation in this study nor the particular mast cell component have been elucidated, it is clear that an impaired blood supply reduces mast cell activation, resulting in reduced wound healing. While this study shows that mast cell degranulation improves healing in non-irradiated colon with a poor blood supply, to measure the role of the mast cell in healing irradiated bowel, further in vivo studies will need to be performed. Radiation reduces mast cell populations in the gut [23] and the role of a degranulation agent in this setting is unknown. The results presented in this paper help to elucidate the role of mast cells in the early healing of the colonic anastomosis. Secondly, these data suggest a potential therapeutic role for mast cell degranulation agents in the promotion of anastomotic healing in poorly vascularized tissue. REFERENCES 1. Heald RJ, Moran BJ, Ryall RD, et al. Rectal cancer: The Basingstoke experience of total mesorectal excision, 1978-1997. ArchSurg 1998;133:894. 2. Stipa F, Chessin DB, Shia J, et al. A pathologic complete response of rectal cancer to preoperative combined-modality therapy results in improved oncological outcome compared with those who achieve no downstaging on the basis of preoperative endorectal ultrasonography. Ann Surg Oncol 2006;3:1047. 3. Alves A, Panis Y, Pocard M, et al. Management of anastomotic leakage after nondiverted large bowel resection. J Am Coll Surg 1999;189:554. 4. Kingham TP, Pachter HL. Colonic anastomotic leak: Risk factors, diagnosis, and treatment. J Am Coll Surg 2009;208:269. 5. Matthiessen P, Hallbook O, Andersson M, et al. Risk factors for anastomotic leakage after anterior resection of the rectum. Colorectal Dis 2004;6:462. 6. Tonnesen MG, Feng X, Clark RA. Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5:40. 7. Norrby K. Mast cells and angiogenesis. APMIS 2002;110:355. 8. Serna H, Porras M, Vergara P. Mast cell stabilizer ketotifen [4-(1-methyl-4-piperidylidene)-4h-benzo[4,5]cyclohepta[1,2-b]t hiophen-10(9 H)-one fumarate] prevents mucosal mast cell hyperplasia and intestinal dysmotility in experimental T
9.
10.
11.
12.
13. 14.
15.
16.
17. 18.
19.
20. 21. 22. 23.
richinella spiralis inflammation in the rat. J Pharmacol Exp Ther 2006;319:1104. McGowen AL, Hale LP, Shelburne CP, et al. The mast cell activator compound 48/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen. Vaccine 2009;27:3544. Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 1996; 29:225. Tulchinsky H, Rabau M, Shacham-Shemueli E, et al. Can rectal cancers with pathologic T0 after neoadjuvant chemoradiation (ypT0) be treated by transanal excision alone? Ann Surg Oncol 2006;13:347. Rutten H, den Dulk M, Lemmens V, et al. Survival of elderly rectal cancer patients not improved: Analysis of population based data on the impact of TME surgery. Eur J Cancer 2007; 43:2295. (http://seer.cancer.gov/csr/1975_2006/results_single/sect_01_ta ble.11_2pgs.pdf. Accessed January 10, 2010. Rutten HJ, den DM, Lemmens VE, van dV, et al. Controversies of total mesorectal excision for rectal cancer in elderly patients. Lancet Oncol 2008;9:494. Chude GG, Rayate NV, Patris V, et al. Defunctioning loop ileostomy with low anterior resection for distal rectal cancer: Should we make an ileostomy as a routine procedure? A prospective randomized study. Hepatogastroenterology 2008;55:1562. Marjanovic G, Juttner E, zur Hausen A, et al. Ischemic preconditioning improves stability of intestinal anastomoses in rats. Int J Colorectal Dis 2009;24:975. Fresno L, Fondevila D, Bambo O, et al. Effects of platelet-rich plasma on intestinal wound healing in pigs. Vet J 2009. Pascual I, de Miguel GF, Gomez-Pinedo UA, et al. Adipose-derived mesenchymal stem cells in biosutures do not improve healing of experimental colonic anastomoses. Br J Surg 2008; 95:1180. Moon TC, St Laurent CD, Morris KE, et al. Advances in mast cell biology: New understanding of heterogeneity and function. Mucosal Immunol 2010;3:111. Yu LC. The epithelial gatekeeper against food allergy. Pediatr Neonatol 2009;50:247. Noli C, Miolo A. The mast cell in wound healing. Vet Dermatol 2001;12:303. Weller K, Foitzik K, Paus R, et al. Mast cells are required for normal healing of skin wounds in mice. FASEB J 2006;20:2366. Sedgwick DM, Ferguson A. Dose-response studies of depletion and repopulation of rat intestinal mucosal mast cells after irradiation. Int J Radiat Biol 1994;65:483.
Mast Cell Degranulation is Essential for Anastomotic Healing in Well Perfused and Poorly Perfused Rat Colon John Coneely, M.D.,† Rory Kennelly, M.Ch.,*,1 D. Bouchier-Hayes, M.D.,† and Desmond C. Winter, M.D.* *Institute for Clinical Outcomes, Research, and Education (ICORE), St. Vincent’s University Hospital, Dublin, Ireland; and †Royal College of Surgeons, Ireland Submitted for publication February 12, 2010
INTRODUCTION Background. Mast cell degranulation is an important step in early wound healing in the skin however the role of the mast cell in anastomotic healing is less clear. The aim of this study was to investigate the importance of mast cell degranulation in anastomotic healing and to assess whether a promoter of mast cell degranulation could increase anastomotic healing in poorly perfused bowel. Methods. Fifty Wistar rats were divided into five groups: control, normally perfused bowel with mast cell stabilisation, normally perfused bowel with mast cell degranulation, hypoperfused bowel, and hypoperfused bowel with mast cell degranulation. A colo-colonic anastomosis was formed in each animal. Four d later, following sacrifice, the strength of the anastomosis was assessed in each animal. Results. Mast cell stabilisation reduced anastomotic healing in normally perfused bowel (P < 0.001). Hypoperfused bowel resulted in reduced anastomotic strength (P < 0.001) however the addition of a mast cell degranulating agent increased healing in hypoperfused bowel to levels comparable with control. Conclusions. Mast cell degranulation is essential for early anastomotic healing. Healing is reduced in hypoperfused bowel but the administration of a mast cell degranulation agent can compensate for the adverse effects of a poor blood supply on anastomotic healing. Ó 2010 Elsevier Inc. All rights reserved. Key Words: mast cell; wound healing; ischemia.
1 To whom correspondence and reprint requests should be addressed at Institute for Clinical Outcomes Research and Education, Department of Surgery, St. Vincent’s University Hospital, Dublin 00004, Ireland. E-mail: [email protected].
The management of colorectal cancer has changed considerably over the last 20 y. Total mesorectal excision coupled with low anterior resection has greatly improved local control [1], neo-adjuvant chemotherapy and radiation have contributed to increased survival, and advances in anesthetic practice have increased the number of patients who are considered fit to undergo colonic resection [2]. While all of these advances have improved oncologic outcomes, they have resulted in significant morbidity with the foremost cause being anastomotic leak [3]. Anastomotic leak rates are reported at 1%–30% [4]. While numerous contributing factors are known (irradiated bowel, atherosclerosis, diabetes mellitus [5]), the common underlying etiology is, arguably, impaired blood supply. The mast cell plays an integral role in skin wound healing through the secretion of histamine and cytokines and promotion of neovascularization through the release of vascular endothelial growth factor [6]. This is achieved by degranulation of the mast cell locally at the site of injury [7]. Mast cells are present in large numbers in the intestinal mucosa but their role in anastomotic healing is not known. Furthermore, it is unknown how ischemia affects mast cell function in the healing bowel. Ketotifen is a known mast cell stabilizing agent preventing degranulation [8] and compound 48/80 is a promoter of mast cell degranulation [9]. The purpose of this study is to examine the role of the mast cell in anastomotic wound healing in a rat model by inhibition and induction of mast cell degranulation. This is performed in normal colon and in a model of impaired colonic blood supply. MATERIALS AND METHODS Institutional ethical approval was granted for this study. Fifty male Wistar rats were divided into five groups of 10: control, mast cell
e73
0022-4804/$36.00 Ó 2010 Elsevier Inc. All rights reserved.
e74
JOURNAL OF SURGICAL RESEARCH: VOL. 164, NO. 1, NOVEMBER 2010
inhibition, mast cell degranulation, induced ischemia, and induced ischemia with mast cell degranulation. All were maintained with standard 12 h light/dark cycles, were fed standard laboratory chow and allowed water ad libitum up to 6 h prior to initiation of the experimental protocol.
Experimental Protocol Twenty-four hours preoperatively, control animals received normal saline via tail-vein injection as vehicular control. The degranulation and inhibition groups received a dose of 1 mg/kg Compound 48/80 and 1 mg/kg ketotifen, respectively, via tail vein. General anesthesia was induced by inhalational halothane. All animals underwent laparotomy and the rodent colon was identified. The colon was transected and anastomosis performed, end to end using eight, full-thickness, interrupted 6/0 polypropylene sutures (Prolene; Ethicon Inc., Kirkton, Scotland) In the ischemia groups, identification and suture ligation of the mesenteric arcade supplying the proximal colon was performed. The hypoperfused segment was identified with intraoperative angiography and the watershed area of colon was transected and anastomosis performed. The anastomosed colon was returned to the peritoneal cavity and mass closure was performed using a continuous 3/0 nylon suture (Ethilon; Ethicon Inc.). The abdominal skin was closed using a continuous 3/0 nylon suture. All animals received 5 mL sodium chloride (0.9% NaCl) intraperitoneal fluid resuscitation at closure of laparotomy wounds and intramuscular injections of buprenorphine hydrochloride, 0.1 mg/kg (Temgesic) prior to termination of anesthesia. They were allowed access to water and chow ad libitum postoperatively.
Analysis Experimental animals were sacrificed on the fourth postoperative day by combination of anesthetise overdose and intra-peritoneal bolus injection of sodium pentobarbitone, 600 mg (Sagatal). Anastomotic healing was determined by measurement of anastomotic bursting pressure and tissue hydroxyproline content. Cytokine response to injury and ischemia was assessed by calculation of IL-6 and VEG-f serum levels.
FIG. 1. Mesenteric angiogram following ligation of mesenteric arcade. Reduced blood flow can be seen on left side of highlighted bowel loop.
Serum Specimens At time of sacrifice, 8 mL of whole blood was obtained by cardiac puncture using a 22 gauge needle. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum concentrations of Il-6 and VEG-f in all experimental groups. Standard, commercially available ELISA kits were used (Sigma-Aldrich, Ireland Ltd., Dublin, Ireland) Mean absorbance values were calculated for each duplicate set of standards and samples, less the average zero standard optical density. A standard curve was plotted and serum levels of each sample were calculated using this curve.
Statistical Analysis All values were calculated and expressed as mean 6 SD. The mean values of groups were compared using one way analysis of variance (ANOVA) or Kruskal-Wallis one way analysis of variance for nonparametric data. P < 0.05 was considered significant. Sigmastat was used for statistical calculations (Systat Software, San Jose, CA).
Tissue Specimen Retrieval Following sacrifice on d 4, the anastomosed segment was identified and excised, with a 2 cm segment of colon proximal and distal to the anastomotic site. Fresh specimens were immediately tested for anastomotic bursting pressure. Following bursting pressure determination, a standard sized segment of colon (5 g) incorporating equal lengths of proximal and distal colon was excised and retained for hydroxyproline estimation.
Anastomotic Bursting Pressure To determine anastomotic bursting pressure, the segment of bowel incorporating the anastomosis was clamped at one end and infused with a 10% betadine solution. Pressure change was measured in millimetres of mercury by a pressure transducer and an analog monitor (Hewlett Packard 78534C, Palo Alto, CA). Peak bursting pressure was determined as the peak value on the pressure trace immediately prior to the precipitous drop that indicated anastomotic bursting.
Hydroxyproline Estimation Hydroxyproline estimation was performed according to the protocol described by Reddy and Enwemeka [10]. Specimens of perianastomotic tissue (5 g) were dissected, flash-frozen in liquid nitrogen, and lyophilized. Absorbance for each sample was read at 550 nm using a Beckman spectrophotometer (Beckman Coulter Inc., Galway, Ireland).
RESULTS
There was no intraoperative or perioperative mortality. On examination of the anastomosis post mortem, there was no evidence of anastomotic dehiscence in the 5 test groups. There was no observed difference in adhesion formation and no peri-anastomotic abscess formation was noted. Confirmation of Induction of Ischemia
Following ligation of the intestinal mesenteric arcade, intraoperative angiogram was performed on TABLE 1 Comparison of Serum Concentration of VEG-f and IL-6 in Control and Model of Intestinal Ischemia Test group Control Ischemia
IL-6 conc. (ng/mL)
VEG-f
54.8 (62.2) 76.6 (63.1) P < 0.05
63.9 (62.4) 94.6 (61.9) P < 0.001
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CONEELY ET AL.: MAST CELL FUNCTION EFFECTS WOUND HEALING
TABLE 2 Comparison of Anastomotic Bursting Pressure and Hydroxyproline Content in All Groups Test group
ABP (mmHg)
HP conc. (mg/mg)
Control Ketotifen (non-ischemic) Compound 48/80 (non-ischemic)) Ischemia Ischemia þ compound 48/80
25.9 (62.4) 12.3 (62.3) P < 0.001 25.2 (61.2) P ¼ 0.37 13.5 (61.20) P <0.001 25.4 (61.3) P ¼ 0.59
5.2 (60.2) 2.4 (60.4) P < 0.001 5.1 (60.2) P ¼ 0.41 3.0 (60.4) P <0.001 5.0 (60.3) P ¼ 0.13
each test animal. The successful induction of hypoperfusion was confirmed via reduced contrast flow (Fig. 1). Cytokine measurement showed a significant increase in serum concentration of VEG-f and IL-6 in animals in the ischemia group, consistent with appropriate systemic response to hypoperfusion (Table 1). Anastomotic Strength
Inhibition of mast cell degranulation by ketotifen caused a decrease in anastomotic bursting pressure indicating impaired anastomotic healing (Table 2). Similarly, hydroxyproline content was significantly reduced in animals treated with the mast cell stabilizing agent (Table 2). The administration of Compound 48/80, a promoter of mast cell degranulation did not affect anastomotic bursting pressure or anastomotic hydroxyproline content in normally perfused colon. Induction of ischemia significantly impaired anastomotic bursting pressure and hydroxyproline content compared with control (Table 2). Administration of Compound 48/80, in the presence of impaired blood supply, improved anastomotic healing allowing for bursting pressures and hydroxyproline content comparable to control (Table 2). DISCUSSION
The aim of this study was to examine the role of mast cell degranulation in anastomotic healing in the rat colon. This was performed in normally perfused colon and in colon with an induced impaired blood supply. Inhibition of mast cell degranulation resulted in reduced wound healing. Induction of ischemia reduced anastomotic healing, however, this event was reversed by the administration of a mast cell degranulating agent allowing for comparable anastomotic strength in both normal colon and hypoperfused colon. Major advances have occurred in the management of rectal cancer with more patients achieving cure than ever before. Neo-adjuvant chemo/radiotherapy downstages up to 60% of rectal cancers and can cause complete pathologic response in up to 20% of cases [11]. However, these results do not come without a cost. Patient morbidity following surgery on irradiated bowel is markedly increased. This is particularly relevant in the
elderly patient where anastomotic leak has a mortality of up to 50% [12]. When this is seen in the context of the median age of diagnosis of colorectal cancer (72 y) [13] the extent of the problem is realized. Indeed, such is the significance of operative complications in the elderly that in the Dutch rectal cancer trial, all benefits from total mesorectal excision and radiotherapy were negated by postoperative non-cancer-related mortality [14]. Current practice dictates that if an anastomosis is constructed in irradiated bowel, a defunctioning stoma should be fashioned primarily to reduce the sequelae of an anastomotic dehiscence [15]. While this technique appears to reduce the risk of life threatening complications, it does necessitate a second operation for reversal of stoma, an undesirable situation in the elderly patient. For these reasons, techniques to enhance anastomotic healing are needed. Due to the important clinical implications of anastomotic failure, numerous methods have been attempted to improve healing with limited success. A moderate improvement was shown by the use of colloid resuscitation postoperatively in rats [16]. Platelet rich plasma showed no benefit in a pig model [17], and sutures impregnated with stem cells showed no benefit in a rat model [18]. No study to date has attempted to promote mast cell degranulation to aid healing. Mast cells are present in high concentration in mucosal tissue [19] and degranulation can occur due to a wide variety of stimuli [20]. The release of inflammatory mediators such as histamine and prostaglandin D2 are an integral part in the early stages of wound healing [21]. The role of the mast cell is well established in skin repair [22], however, the results presented here identify an important role for mast cell degranulation in anastomotic healing. The addition of a mast cell stabilizing agent reduced anastomotic strength showing that mast cell activation is necessary for normal anastomotic healing. Interestingly, the addition of a mast cell degranulating agent to the normally perfused anastomosis did not result in increased wound strength. This indicates that mast cell activation is optimized in adequately perfused bowel. The effect of ischemia on wound healing is no surprise, however, the addition of a degranulation agent (Compound 48/80) markedly improved anastomotic strength. While neither the specific mediator of mast
e76
JOURNAL OF SURGICAL RESEARCH: VOL. 164, NO. 1, NOVEMBER 2010
cell degranulation in this study nor the particular mast cell component have been elucidated, it is clear that an impaired blood supply reduces mast cell activation, resulting in reduced wound healing. While this study shows that mast cell degranulation improves healing in non-irradiated colon with a poor blood supply, to measure the role of the mast cell in healing irradiated bowel, further in vivo studies will need to be performed. Radiation reduces mast cell populations in the gut [23] and the role of a degranulation agent in this setting is unknown. The results presented in this paper help to elucidate the role of mast cells in the early healing of the colonic anastomosis. Secondly, these data suggest a potential therapeutic role for mast cell degranulation agents in the promotion of anastomotic healing in poorly vascularized tissue. REFERENCES 1. Heald RJ, Moran BJ, Ryall RD, et al. Rectal cancer: The Basingstoke experience of total mesorectal excision, 1978-1997. ArchSurg 1998;133:894. 2. Stipa F, Chessin DB, Shia J, et al. A pathologic complete response of rectal cancer to preoperative combined-modality therapy results in improved oncological outcome compared with those who achieve no downstaging on the basis of preoperative endorectal ultrasonography. Ann Surg Oncol 2006;3:1047. 3. Alves A, Panis Y, Pocard M, et al. Management of anastomotic leakage after nondiverted large bowel resection. J Am Coll Surg 1999;189:554. 4. Kingham TP, Pachter HL. Colonic anastomotic leak: Risk factors, diagnosis, and treatment. J Am Coll Surg 2009;208:269. 5. Matthiessen P, Hallbook O, Andersson M, et al. Risk factors for anastomotic leakage after anterior resection of the rectum. Colorectal Dis 2004;6:462. 6. Tonnesen MG, Feng X, Clark RA. Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5:40. 7. Norrby K. Mast cells and angiogenesis. APMIS 2002;110:355. 8. Serna H, Porras M, Vergara P. Mast cell stabilizer ketotifen [4-(1-methyl-4-piperidylidene)-4h-benzo[4,5]cyclohepta[1,2-b]t hiophen-10(9 H)-one fumarate] prevents mucosal mast cell hyperplasia and intestinal dysmotility in experimental T
9.
10.
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