Stasis Predisposes Ileal Pouch Inflammation in a Rat Model of Ileal Pouch-Anal Anastomosis1

Journal of Surgical Research 164, 75–83 (2010) doi:10.1016/j.jss.2009.03.049

Stasis Predisposes Ileal Pouch Inflammation in a Rat Model of Ileal Pouch-Anal Anastomosis1 Arthur F. Stucchi, Ph.D., Khaled O. Shebani, M.D., Karen L. Reed, Ph.D., Adam C. Gower, M.S., Michael F. Alapatt, M.D., Keith M. Crivello, M.D., James P. McClung, Ph.D., and James M. Becker, M.D.2 Department of Surgery Boston University School of Medicine, Boston, Massachusetts Submitted for publication December 23, 2008

Conclusions. These studies showed that ileal pouch stasis predisposes biochemical and histological evidence of ileal pouch mucosal inflammation. Studies such as this may provide the rationale for novel, adjunct therapies for the management of pouchitis in patients having undergone IPAA for CUC. Ó 2010 Elsevier

Background. While restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become the definitive surgical treatment for patients suffering from chronic ulcerative colitis (CUC), pouchitis still remains a major late complication. Fecal stasis has been implicated in the etiology of ileal inflammation; however, the mechanism(s) remain unclear, in part due to the lack of an animal model. Our goal was to surgically mimic the IPAA procedure in a rat to investigate the hypothesis that stasis leads to biochemical changes that predispose the ileal pouch to inflammation. Materials and Methods. Thirty-two Sprague-Dawley rats underwent total colectomy with either straight ileorectal (IRA) or IPAA, and 11 nonoperated rats served as controls (Controls). Twenty-one d postoperatively, 48 h serial barium radiographs and 12 h charcoal transit follow-through studies were performed. Following sacrifice, ileal tissue was harvested for the measurement of myeloperoxidase activity (MPO) activity, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) mRNA levels, and histology. Results. Serial barium radiographs showed stasis in the ileal pouch compared with IRA animals, and charcoal transit times that were two times longer (P £ 0.05) than that in the straight IRA rats. Ileal pouch MPO levels were significantly elevated in the IPAA rats compared with the straight IRA rats. ICAM-1 and VCAM-1 mRNA levels were not associated with neutrophil infiltration.

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Key Words: pouchitis; stasis; IPAA; restorative proctocolectomy; ileal pouch inflammation; adhesion molecules; rat models.

INTRODUCTION

Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become the definitive surgical treatment for patients suffering from chronic ulcerative colitis (CUC) and familial adenomatous polyposis (FAP) [1, 2]. Despite the significant improvements this surgical advance has made on the quality of life in these patients [3], inflammation of the surgically-created ileal pouch or pouchitis remains a major late postoperative complication that can often overshadow the benefits of this otherwise curative operation [4]. Pouchitis is a nonspecific inflammation of the ileal pouch, which manifests clinically with high stool frequency, diarrhea, urgency, abdominal cramping, and rectal bleeding [5, 6]. Some patients also experience symptoms consistent with active CUC, such as fever, pelvic discomfort, and extraintestinal manifestations [5, 7, 8]. The cumulative risk of experiencing at least one episode of pouchitis within 4 y of closure of the loop ileostomy can be as high as 50% [9]; however, the risk in patients experiencing extraintestinal manifestations, such as primary sclerosing cholangitis, can be considerably higher [10]. The typical IPAA procedure is a two stage operation in which a diverting loop ileostomy is created

1 Published, in part, in Gastroenterology 2000;118(Suppl 2):A1048. 2 To whom correspondence and reprint requests should be addressed at Department of Surgery, Boston University School of Medicine, 88 East Newton Street, C500 Boston, MA 02118. E-mail: James. [email protected].

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approximately 40 cm proximal to the ileal pouch in the initial stage [1]. In the second stage, approximately 8 wk later, the diverting loop ileostomy is closed, signifying a major alteration in the primary function of the terminal ileum. Previously associated with nutrient absorption, the surgically-created ileal reservoir must now adapt to a new role as a functional neorectum. The storage of feces or stasis within the pouch is a major factor distinguishing the ileal pouch from a free flowing ileostomy [11]. Interestingly, ileal inflammation is nonexistent in unobstructed ileostomies [12]. In fact, ileal inflammation was not even described until about 30 y ago when Kock introduced the concept of the ileal reservoir or continent ileostomy (Kock pouch) as a surgical alternative for patients who had undergone proctocolectomy for CUC or FAP and wished to remain continent [13, 14]. It was at that time that ileal pouch inflammation was coined ‘‘pouchitis,’’ and it has since remained an enigmatic inflammatory syndrome. Interestingly, inflammation only occurs in a functioning ileal pouch and rarely, if ever, occurs prior to closure of the diverting ileostomy. Therefore, stasis must play at least some role in initiating pouch inflammation; however, it cannot be the sole underlying factor since patients who have undergone IPAA for FAP rarely experience pouchitis. Thus, some causal pathologic component inherent to ulcerative colitis must activate the manifestation of pouchitis in susceptible individuals [15, 16]. While there are numerous pathophysiologic mechanisms purportedly underlying the development of pouchitis, including altered mucosal immunoregulation, unsuspected Crohn’s disease, bacterial overgrowth and dysbiosis, epithelial nutrient deficiencies, ischemia, and stasis [4, 17, 18], attempts to resolve these idiopathic inflammatory episodes have been hindered significantly by our poor understanding of its etiology. This fact, coupled with the lack of a small animal model of IPAA, has significantly hampered our ability to study pathophysiologic mechanisms under closely controlled conditions. To date, there have been no mechanistic studies examining whether stasis is directly associated with inflammation in the ileal pouch. Hence, utilizing a rat model of IPAA developed in this laboratory [19], the primary goal of this study was to determine if, in fact, stasis does occur in the ileal pouch by comparing charcoal transit times and barium follow through in rats that have undergone IPAA with rats that have had a straight ileorectal anastomosis (IRA). By measuring mucosal myeloperoxidase (MPO) activity, a sensitive biochemical marker of neutrophil infiltration and inflammation, and intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in ileal pouch tissue from these two groups (IPAA versus IRA), we sought to determine whether stasis is associated with ileal pouch

inflammation, and investigate a plausible mechanism to explain why stasis initiates inflammation in ileal pouch mucosa. MATERIALS AND METHODS Materials All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.

Animals Male Sprague-Dawley (Charles River Labs, Wilmington, MA) rats weighing between 300 and 325 g were housed individually at a constant room temperature with 12-h light and dark cycles, and provided with standard rat chow (Purina no. 5001) and water ad libitum for 5 to 7 d prior to surgery. The Institutional Animal Care and Use Committee at the Boston University School of Medicine approved this study, and all procedures described were performed in accordance with recommendations outlined in the National Institutes of Health Guide for the Care and Use Laboratory Animals (NRC 1996).

Surgical Model Animals were fasted for 24 h prior to surgery, during which time they consumed water containing 5% dextrose in order to reduce the hypermetabolic effects associated with the prolonged postoperative fast required for anastomotic healing. Colectomy and proximal proctectomy was performed followed by the surgical construction of either a J pouch in the terminal ileum with anastomosis to the distal rectum (Fig. 1A), as we have previously described [19], or a straight ileorectal anastomosis (IRA) (Fig. 1B). Utilizing these surgical models comparatively provides a unique opportunity to assess the role of stasis in ileal pouch inflammation. We demonstrated earlier [19] that the inflammation occurring as a consequence of the ileal pouch surgery is localized to the suture line until healing occurs. Although the inflammation at the suture line subsided by 28 d postoperatively, a widespread inflammation of the ileal pouch persisted for up to 60 d postoperatively [19]. It is also important to note that the inflammation was localized strictly to the pouch, the proximal ileum was unaffected, and that no other chemical or dietary manipulations were utilized to initiate inflammation other than the resumption of solid food.

Experimental Design Colectomy with IPAA or IRA was performed on 32 Sprague-Dawley rats. Twenty-one d postoperatively, animals were administered charcoal via an orogastric tube to assess total gastrointestinal transit time. Barium follow-through studies were also performed to assess pouch evacuation times. Twenty-eight d postoperatively, animals were sacrificed by CO2 exposure, and the ileal pouch was removed for histology, mucosal myeloperoxidase (MPO) activity, and tissue ICAM-1 mRNA levels.

Barium Follow-Through Studies Twenty-one d postoperatively, non-fasted IRA and IPAA animals (3 per group) were lightly anesthetized with ketamine/xylazine, and 4 mL barium sulfate/water (1:1; vol/vol) (E-Z Paque; E-Z-EM, Westbury, NY) was gavaged into the stomach with a 14 ga X 3 in. feeding needle [20] in order to assess pouch evacuation time. Animals were placed in a supine position, and anteroposterior abdominal radiographs were obtained immediately following gavage (time 0), then every 15 min for 3 h, then at 6, 12, 24, 32, and 48 h.

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FIG. 1. Surgical construction of ileal J pouch-anal anastomosis (A). After colectomy, the terminal ileum was oversewn, incised along its antimesenteric border, and the two limbs of the ileal loop were sutured together to form a pouch with the J configuration. Once both walls were completed, an anastomosis was performed between the distal end of the pouch and the proximal end of the rectal stump. The surgical construction of the straight ileorectal anastomosis (B) involved a colectomy with an anastomosis between the distal end of the ileum and the proximal end of the rectal stump. Adapted with permission from Shebani et al., Pouchitis in a rat model of ileal J pouch-anal anastomosis. Inflamm Bowel Dis 2002;8(1):23–34.

Charcoal Transit Time Studies To assess total gastrointestinal transit time, non-fasted IRA and IPAA animals (6 per group) were lightly anesthetized and administered 3 mL of activated charcoal (Liqui-Char; Jones Medical Industries, St. Louis, MO) via a 14 ga X 3 in. feeding needle, and then monitored every 30 min for the first passage of black stool [21]. This time point was recorded for each animal and represented the transit time.

Myeloperoxidase Activity MPO activity has been widely accepted as a sensitive biochemical marker to quantitate the degree of inflammation, and is indicative of the accumulation of neutrophils or polymorphonuclear cells in tissues [22, 23]. Distal ileal mucosa from nonoperated control and IRA animals, and pouch mucosa from animals with an IPAA (n ¼ 6 per group) were assayed for MPO activity in duplicate according to Barone et al. [24], with modifications as described by Shebani et al. [25]. One unit of MPO activity was defined as that degrading 1 mmol of hydrogen peroxide per minute.

in a reaction volume of 20 mL using bulk master mixes, except template, prepared for multiple reactions. Each PCR reaction consisted of 4 mL cDNA reaction added to 16 mL of master mix (13 PCR Buffer II, 2 mM MgCl2, 0.5 U AmpliTaq DNA polymerase, and 0.15 mM upstream and downstream primers). After the samples were heated for 5 min at 95 , PCR cycle, at 30 s 95 , 30 s 60 , and 30 s 72 , was conducted for 34 cycles. The ICAM-1 and VCAM-1 PCR primers, previously reported by Reed et al. [26] amplified a single PCR product of 388 and 570 bps, respectively. Following PCR amplification, PCR products (15 mL) were subjected to electrophoresis on 2% agarose gels containing 0.03 mg/ mL ethidium bromide. Quantitation of transcript level was carried out by scanning photographs of gels and analyzing the area under peaks, using Scion Imaging software available at http://www.scioncorp.com:8080/ Downloads/fr_login.htm [27]. Levels of mRNA expression were normalized by calculating as a percentage of glyceraldehydes-3phosphate dehydrogenase (GAPDH) mRNA expression levels, a constitutively expressed housekeeping gene that did not vary significantly between treatment groups. After values were normalized, mean expression values were determined and graphed as a percent of controls.

RNA Isolation and Relative RT-PCR

Histology

Total RNA was isolated from 50–100 mg of ileal tissue using TRIzol reagent (Gibco BRL, Gaithersburg, MD), and contaminating DNA was removed using the S.N.A.P. total RNA Isolation Kit (Invitrogen, Carlsbad, CA). Reverse transcription of total RNA samples and PCR were accomplished using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Briefly, cDNA was synthesized by adding (final concentrations) 5 mM MgCl2 , 13 PCR Buffer II, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1 U RNase inhibitor, 2.5 U/mL MuLV reverse transcriptase, and 2.5 mM oligo d(T)16 to 0.5 mg total RNA. The cDNA reactions were incubated in a GeneAmp PCR System 2400 thermal cycler (Applied Biosystems, Foster City, CA) at 42 for 15 min, 99 for 5 min, followed by 5 for 5 min. The relative mRNA levels in all samples were then analyzed by amplification of the respective cDNA samples. PCR was carried out

Following the removal of the distal ileum from non-operated control animals and animals with either an IPAA or a straight IRA, two sections were fixed immediately in 10% neutral buffered formalin and embedded in paraffin for histological analysis. Full thickness sections were stained with hematoxylin and eosin and examined by a pathologist blinded to the groupings for evidence of neutrophil infiltration of the mucosa and submucosa, crypt microabscesses, blunting of intestinal villi, increased crypt depth, and ulcerations.

Statistics Group differences were determined by analysis of variance (ANOVA) followed by a post hoc Tukey’s multiple mean separation tests. Data were graphed and statistical analyses were performed

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using Origin (OriginLab, Northampton, MA) and SigmaStat (SPSS Inc., Chicago, IL), respectively. All data are expressed as mean 6 SEM.

RESULTS

Animals tolerated the IPAA and IRA procedures well with few postoperative complications. Although weight loss was noted through d 10 postoperatively in both the IRA and IPAA groups, nearly all animals gained weight steadily thereafter (5–8 g per d), eventually exceeding their preoperative weight prior to commencing any studies. Nonoperated controls gained, on average, an additional 7% body weight during the experimental period, while the IRA and IPAA animals gained approximately 9% and 10.3%, respectively. Stasis Occurs in the Ileal Pouch

Following an oral gavage of barium, anteroposterior abdominal radiographs of IPAA and IRA rats were taken at time 0, then every 15 min for 3 h, then at 6, 12, 24, 32, and 48 h. In the IRA rats, barium was not observed in the terminal ileum until the 6 h scan (Fig. 2,

FIG. 3. Charcoal transit times in straight ileorectal anastomosis (IRA) animals compared with animals having an ileal pouch-anal anastomosis (IPAA). The mean times noted represent the first appearance of black, charcoal-containing stool. Data represent the mean 6 SEM for six animals per group. *P  0.0001 compared with IRA.

top left panel); however, it was completely evacuated by 12 h in all animals (Fig. 2, top right panel). In the IPAA rats, trace barium was evident in the ileal pouch by as early as 12 h. However, in contrast to the IRA animals, barium still remained in the ileal pouch of IPAA animals at 32 h in all animals (Fig. 2, bottom left panel). Complete evacuation was not observed until 48 h (Fig. 2, bottom right panel). Transit Time Significantly Increases in IPAA Rats

In the IRA rats, the average total gastrointestinal charcoal transit time of 5.3 6 0.11 h approximated the time at which barium was evident in the terminal ileum of these animals. Conversely, in the IPAA rats, the average total gastrointestinal charcoal transit time of 13.2 6 0.60 h was significantly greater compared with the IRA animals (Fig. 3). This time also corresponded well with the barium follow through studies. Myeloperoxidase Levels Increase in the Ileal Pouch

Compared with nonoperated controls and IRA animals, ileal mucosal MPO levels were significantly increased (P < 0.05) in the IPAA animals, indicating inflammation associated with the increased infiltration of neutrophils (Fig. 4). ICAM-1 mRNA Levels Were Not Different FIG. 2. Top panels: representative barium follow-through in a straight ileorectal anastomosis animal. Barium is evident in the ileum at 6 h, and is completely evacuated by 12 h (n ¼ 3). Bottom panels: representative barium follow-through in an ileal pouch-anal anastomosis animal. Barium is still present in the ileal pouch at 32 h, but is completely evacuated by 48 h (n ¼ 3). Adapted with permission from Shebani et al., Pouchitis in a rat model of ileal J pouch-anal anastomosis. Inflamm Bowel Dis 2002;8(1):23–34. (Color version of figure is available online.)

Ileal ICAM-1 mRNA levels (Fig. 5, top) were not different between nonoperated controls (Control), straight IRA (IRA), and IPAA animals (IPAA). Ileal VCAM-1 mRNA levels (Fig. 5, bottom) were significantly lower in the IPAA (IPAA) animals compared with the straight IRA (IRA) animals (P < 0.05). Despite the lack of significant changes in ICAM-1 message levels in this study, we have shown in a previous study [28] that both

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FIG. 4. Myeloperoxidase levels in nonoperated control (Control), ileal pouch-anal anastomosis (IPAA), and straight ileorectal anastomosis (IRA) animals. Data represent the mean 6 SEM for six animals per group. *P  0.001 compared with control and IRA.

ICAM-1 AND VCAM-1 mRNA levels were significantly elevated in the colon following administration of dextran sodium sulfate to induce colitis in rats. Villous Blunting Occurs in the Ileal Pouch

Villi of control rats appeared histologically normal. While there was some villous shortening apparent in the distal ileum of IRA rats at 30 d postoperatively, the appearance of the ileum was otherwise normal. In contrast, mild to moderate villous atrophy with villous blunting was clearly apparent in the ileal pouch mucosa of IPAA animals in comparison with the nonoperated ileum, although there was no evidence of crypt microabscesses, ulcerations, or a significant inflammatory infiltrate (Fig. 6). These morphologic changes in the ileal pouch are found primarily in the setting of inflammation, and are quite similar to those we observed in ileal pouch mucosa in humans [29]. DISCUSSION

Dynamic defecographic studies have shown that even in the most efficiently functioning ileal pouch, residual volume remains following evacuation [30, 31]. Although the resultant stasis has been purported to play a role in the pathogenesis of ileal pouch inflammation or pouchitis [32], efforts to directly associate stasis as an initiating factor in the inflammatory response has been difficult. In the present study, we report new findings in a rat model of IPAA supporting a role for stasis in the pathophysiology of ileal pouch inflammation. We first demonstrated radiographically that significant stasis occurs in the ileal pouch of rats that have undergone IPAA compared with rats that underwent

FIG. 5. Intercellular adhesion molecule-1 (ICAM-1) mRNA levels (top panel) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels (bottom panel) in nonoperated controls (Control), straight ileorectal anastomosis (IRA), and ileal pouch-anal anastomosis (IPAA) animals. In each panel, respectively, a representative agarose gel of the PCR product is shown above the bar graph, representing the quantitation of transcript levels by scanning photographs of gels, and analyzing the area under peaks. After values were normalized to GAPDH mRNA expression levels, mean expression values were determined and graphed as a percent of nonoperated (nonop) control. Data represent the mean 6 SEM for six Control, four IRA, and five IPAA animals per group.

a straight IRA. We further showed that gastrointestinal charcoal transit times in rats with IPAA were over 2-fold longer compared with IRA rats, and that this extended transit time corresponded with significant elevations in MPO activity and inflammation in the ileal pouch. Several studies have shown that numerous factors contribute to pouch function [33], and that not all pouch configurations and sizes empty with the same efficiency [34]. However, there does not appear to be a direct relationship between incomplete pouch emptying and the incidence of pouchitis [35]. Nevertheless, while stasis, in and of itself, may not be implicated directly in the pathophysiology of pouchitis, it may initiate a cascade of synergistically pathogenic mechanisms that can

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FIG. 6. Representative cross-sectional views of hematoxylin and eosin stained ileum from a nonoperated control animal (Control), straight ileorectal anastomosis (IRA), and ileal pouch-anal anastomosis (IPAA) animals (magnification 340). Insets show further detail of histology (magnification 3100). (Color version of figure is available online.)

initiate ileal pouch inflammation in susceptible individuals. For example, stasis can lead to the overgrowth of bacteria not typically indigenous to the terminal ileum [15, 16, 36]. Bacterial overgrowth disproportionately favors anaerobes, leading to bacterial dysbiosis, another purported cause of pouchitis [37]. Overgrowth or dysbiosis can lead to deficiencies in essential nutrients, such as butyrate [38] and glutamine [39], both purportedly required for optimal enterocyte function. In addition, the overgrowth of bacteria, especially anaerobes, can catalyze the excess production of secondary bile acids [40] not typically present in high concentrations in the ileum. Both of these mechanisms can compromise epithelial integrity and barrier function, thus predisposing the ileal pouch mucosa to a low-grade inflammatory response. Our earlier studies in this rat model showed that both anaerobic and aerobic bacteria overgrow the ileal pouch at 30 d postoperatively [19]. In addition, stasis has been associated with morphologic and histologic changes in ileal pouch mucosa that accompany the functional transition from ileum to colon known as colonic metaplasia [17, 32]. These histologic changes can include villous atrophy, crypt hyperplasia, and other more subtle, yet chronic, inflammatory changes in ileal pouch mucosa. Colonic metaplasia occurs to some degree in all functioning ileal pouches [29], although it is rarely evident in pouch mucosa, either prior to ileostomy closure [41] or in the terminal ileum, of patients with a unobstructed Brooke or end ileostomy, suggesting that stasis may predispose these morphologic changes. Although patients without a history of pouchitis have only a minimum degree of these inflammatory-induced histologic alterations

[29], when present, they are associated with inflammatory and reparative changes [29] that may predispose the pouch to subsequent inflammatory episodes. Although stasis is apparent, mild villous atrophy and villous blunting are evident histologically concurrent with elevated MPO levels in the ileal pouch mucosa of IPAA rats at 30 d, both typical findings in human ileal pouches [29, 42]. However, a significant inflammatory infiltrate was not apparent histologically, even at the higher magnification, which is not untypical in lowgrade inflammation, and the rationale for which MPO was used as a more sensitive marker of neutrophil infiltrate [23]. While there is no direct evidence implicating these morphologic changes in the pathophysiology of pouchitis, we have noted that they are associated with inflammatory mechanisms, such as the recruitment of neutrophils [29]. In the present study, we show biochemical evidence of neutrophil recruitment, elevated MPO levels, to the ileal pouch of IPAA animals as early as 30 d postoperatively. While these neutrophils may not be activated, their presence suggests a low level, chronic inflammation that may ‘‘prime’’ the ileal pouch to subsequent inflammatory insults that can precipitate acute, more severe episodes of ileal inflammation. We have also observed similar inflammatory changes, such as elevated MPO levels, in ileal pouch biopsies obtained from asymptomatic IPAA patients [42]. Neutrophil activation has been shown to cause oxidative stress and tissue injury in colonic mucosa of ulcerative colitis patients through the liberation of reactive oxygen metabolites, in concert with the consumption of tissue antioxidants [43].

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Neutrophil infiltration is one of the critical cellular components of the inflammatory response. One mechanism that may explain the increased numbers of neutrophils in the presence of stasis is the up-regulation of the endothelial adhesion molecules, ICAM-1 and VCAM-1, both of which facilitate leukocyte extravasation into tissues [44]. We chose to measure ICAM-1 because anti-ICAM-1 strategies have been shown to be efficacious in reducing intestinal inflammation in animal models [45], and are currently being developed as therapeutic tools in the management of refractory IBD, [46] chronic pouchitis [47], and other inflammatory disorders. Intestinal stasis has also been shown to increase the endothelial expression of ICAM-1 in adjacent capillary beds, which was subsequently reduced by antibiotic treatment, implicating a bacterial mechanism of action [48]. In the present study, although we showed that stasis did occur in the ileal pouch and was associated with both a neutrophil infiltrate and inflammation compared with animals that underwent the straight IRA, we were unable to demonstrate an increase in either ICAM-1 or VCAM-1 mRNA levels, suggesting that the increased infiltration of neutrophils was not mediated by these adhesion molecules. However, leukocyte infiltration is not completely dependent on the expression of adhesion molecules. Crockett et al. [49] recently showed that significant neutrophil infiltration can occur in P-selectin and ICAM-1 null mice, suggesting that alternative adhesion recruitment pathways independent of adhesion molecule expression do exist and can participate in neutrophil migration during an inflammatory response. Additional studies in mice have indicated that while adhesion molecules are important modulators of neutrophil infiltration, the mode of stimuli and duration of the injury can modulate neutrophil infiltration via the expression of chemotactic cytokines or CXC-chemokines, such as interleukin-8 (IL-8), that may play an equally central role in recruitment and migration [50]. Further studies supporting the importance of chemokines in leukocyte recruitment into inflamed ileal pouch tissue of CUC patients who have undergone IPAA demonstrated that the expression of monocyte chemotactic protein-1 and 3, as well as IL-8, were significantly elevated during episodes of acute ileal inflammation compared with the normal pouch ileum [51]. A study in humans by Thomas et al. [52], also showed that the expression of ICAM-1 and VCAM-1 in the ileal pouch was not different between FAP patients with no evidence of inflammation compared with UC patients with inflammation, suggesting that other recruitment pathways of leukocyte infiltration exist. Although the mechanism underlying the inflammatory response noted in the ileal pouches of IPAA animals was the result of bacterial overgrowth or dysbiosis, ischemia, or some other stimuli is unclear

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at this point, the difference in MPO levels between the IPAA and IRA rats indicates that inflammatory cells, primarily neutrophils, are recruited to the ileal pouch in the presence of stasis. In either event, stasis appears to predispose the ileal pouch to a low grade chronic inflammation for which an exogenous inflammatory stimuli may trigger episodes of ileal pouch inflammation. We demonstrated this occurrence when a known intestinal inflammatory stimulus, dextran sulfate sodium, initiated pouchitis-like symptoms in IPAA rats within hours after oral consumption [19, 53]. Anecdotal evidence in humans also implicates bacterial overgrowth as underlying episodes of pouchitis, since these episodic flareups can often be ameliorated by a single course of the antibiotics metronidazole and ciprofloxacin. Interestingly, both these antibiotics have antioxidant properties and can potentially reduce the effects of oxidative stress by neutrophils. The longterm metabolic consequence of chronic, subclinical inflammation is the depletion of endogenous tissue antioxidants [54], thus shifting the delicate intracellular balance of oxidation and reduction (redox state) to favor oxidative stress [55]. The concurrent production of numerous proinflammatory cytokines and chemokines, driven in part by the up-regulation of redoxsensitive transcriptional factors such as nuclear factor-kB [56], facilitates a self-sustaining cycle of chronic, subclinical inflammation that appears to precede episodes of pouchitis. In further demonstration of these points in a similar rat model of ileal pouch-rectal anastomosis, antibiotic treatment with metronidazole and antioxidant treatment with allopurinol reduced both ileal pouch inflammation and histology score, again suggesting that stasis-induced inflammation may be of bacterial origin [57]. As mentioned earlier, patients undergoing the same IPAA procedure for FAP rarely experience pouchitis even though ileal pouch stasis occurs, suggesting that the mechanisms initiating ileal pouch inflammation extend beyond bacteria alone. Based on this line of reasoning, one might expect that an animal model of IPAA that lacks either a genetic predisposition to colonic inflammation, such as the HLA-B27 rat [58], or an inflammatory bowel disease, would not develop spontaneous ileal pouch inflammation. Interestingly, we have measured ileal pouch MPO levels in patients with FAP undergoing routine screening pouchoscopy and found elevated MPO levels compared with ileal biopsies taken proximal to the ileal pouch in the same patient (Stucchi, unpublished results). However, MPO levels were still lower than those we found in pouches from asymptomatic UC patients [42]. Thus, even though patients with FAP who underwent IPAA rarely experience pouchitis, they may in fact have low-grade asymptomatic ileal pouch inflammation presumably as a result of stasis.

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In conclusion, these studies showed that ileal pouch stasis was associated with biochemical evidence of ileal pouch mucosal inflammation. Studies such as this may provide the rationale for novel, adjunct therapies for the clinical management of pouchitis in patients having undergone IPAA for CUC. A better understanding of the pathophysiology of pouchitis can lead to the development of new interventions that may prevent or reduce severity and greatly improve the quality of life after IPAA surgery.

18. Stucchi AF, Becker JM. Pathogenesis of pouchitis. Problems Gen Surg 1999;16:139.

ACKNOWLEDGMENTS

23. Faith M, Sukumaran A, Pulimood AB, et al. How reliable an indicator of inflammation is myeloperoxidase activity? Clinica chimica acta. Int J Clin Chem 2008;396:23.

The authors wish to thank Dr. Timothy Heeren for his statistical expertise.

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