Functional and morphologic changes of the ileal mucosa after ileoanal pouch procedure1

Functional and Morphologic Changes of the Ileal Mucosa after Ileoanal Pouch Procedure Akemi L Kawaguchi, MD, James CY Dunn, MD, PhD, Minn S Saing, BS, Galen Cortina, MD, Eric W Fonkalsrud, MD, FACS

Background: Restorative proctocolectomy is used widely for treatment of ulcerative colitis and familial polyposis coli. Limited information is available regarding the morphologic and functional adaptation of the mucosa in a functioning ileoanal pouch.

Conclusions: The AOP and maltase activities in mucosa from ileoanal pouches and colon were significantly lower than those in normal ileal mucosa. Ileoanal pouch mucosa from humans undergoes adaptive changes to resemble colonic mucosa both morphologically and functionally. (J Am Coll Surg 2000;190: 310–314. © 2000 by the American College of Surgeons)

Study Design: Ileal pouch specimens from patients who underwent pouch reconstruction (mean 7.5 years postcolectomy, nⴝ12) were compared with normal ileum (nⴝ15) and normal colon (nⴝ5). Aminooligopeptidase (AOP) and maltase activity were measured as parameters of normal ileal function. Histologic samples were examined for the presence of neutrophils and plasma cells, the villus to crypt height ratio, and the degree of crypt hyperplasia, villus blunting, and goblet cell mass. Data were analyzed by analysis of variance.

Restorative proctocolectomy has become the standard surgical treatment for ulcerative colitis and familial polyposis coli. This operative procedure allows removal of the diseased colon while preserving intestinal continuity and continence. The ileum used to create the pouch undergoes an adaptive process that affects the morphology and function of the ileal mucosa. Many studies have examined the morphologic changes of a functioning ileal pouch over time, several being in animal models. The majority of reports have evaluated samples with chronic or acute inflammatory changes.1,2 Many studies have also found that the ileal mucosa adapts to new environmental conditions with “colonic metaplasia,” which results in villus blunting or complete atrophy and crypt hyperplasia.3,4 Some investigators have also described the acute inflammatory changes of pouchitis that may correlate with clinical symptoms of increased frequency of bowel movements, urgency, diarrhea, fatigue, malaise, and fever.5 Sparse information is available regarding the exact functional changes of the ileal pouch mucosa in humans a few years after restorative proctocolectomy. The aim of this study is to investigate the longterm morphologic and functional changes of the ileal pouch mucosa.

Results: The AOP activity in the normal ileum was 73ⴞ32 units of enzymatic activity per gram of mucosal protein; the AOP activities of the pouch and colon were 21ⴞ22 and 16ⴞ10, respectively. The maltase activity of the normal ileum measured 254ⴞ116 units of enzymatic activity per gram of mucosal protein, and the maltase activities of the pouch and colon were 57ⴞ 71 units and 29ⴞ25 units, respectively. The ileal pouch mucosa demonstrated little acute inflammation and varying degrees of chronic inflammation. Morphologically, the ileal pouch mucosa demonstrated a range of adaptations, including villus blunting and crypt hyperplasia. Several specimens contained immature epithelial cells. No competing interests declared. Supported in part by the Hughes Employee Give Once Club, Los Angeles, CA. Received July 8, 1999; Revised October 19, 1999; Accepted October 21, 1999. From the Division of Pediatric Surgery (Kawaguchi, Dunn, Saing, Fonkalsrud) and Department of Pathology (Cortina), UCLA School of Medicine, Los Angeles, CA. Correspondence address: Eric W Fonkalsrud, MD, FACS, Division of Pediatric Surgery, UCLA School of Medicine, 10833 Le Conte Ave, 72-126 CHS, Los Angeles, CA 90095-1749. © 2000 by the American College of Surgeons Published by Elsevier Science Inc.

METHODS Specimens of ileoanal pouch, normal ileum, and normal colon were obtained with the approval of 310

ISSN 1072-7515/00/$21.00 PII S1072-7515(99)00276-8

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Table 1. Histologic Grading of Ileoanal Pouch Mucosa Score

Neutrophils

Plasma cells

0 1

Rare or none Intraepithelial or frequent

2

Exudate

No reactive changes Confluent plasma cells, reactive changes Expansion

3

Lymphoid hyperplasia

Crypt hyperplasia

Normal Reactive germinal center

Normal Mild increase in size and mitoses

Normal Mild

Normal ileum Mildly increased

Large mucosal ⫹ submucosal distant reactive follicles

Moderate increase in size and mitoses

Moderate

Moderately increased

Marked increase in size and mitoses

Mucosal Markedly ulceration increased, similar to colon

Submucosal chronic inflammation

the UCLA Human Subjects Protection Committee. Twelve specimens of ileoanal pouch were procured at the time of pouch revision for chronic dysfunction. The preoperative diagnosis, age, gender, and time period of the functioning pouch were determined from patient records. Normal ileal (n⫽15) and colonic (n⫽5) samples were obtained during ileostomy takedown, during ileoanal pouch construction, or from fresh cadaveric organ donors. For each sample, the mucosal tissue was removed with a sterile scalpel blade. A previously weighed portion of the harvested mucosa was placed in phosphate-buffered saline for the AOP assay and in a buffer (10mM NaH2P04 [monobasic, anhydrous], 5mM Tris-HCl, 1mM ethylenediaminetetraacetic acid [EDTA, disodium, anhydrous], 0.002% Triton-X-100) for the maltase assay. Each sample was homogenized at 15,000 rpm for 20 seconds using a Tissue Tearer (Biospec Products, Racine, WI). Maltase activity was determined by the method of Dahlqvist,6 and AOP activity was measured with the method of Wojnarowska and Gray.7 Protein content was measured by using the method of Lowry and coworkers.8 The specific activity of each enzyme was expressed as units of enzymatic activity per gram of mucosal protein (UE/g). Histologic specimens were preserved in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histologic specimens were examined under standard light microscopy by a pathologist unfamiliar with the origin of the individual specimens. The number of neutrophils, plasma cells, and the amount of lymphoid hyperplasia were measured to determine the type

Villus blunting

Goblet cell mass

and degree of inflammation. The morphologic characteristics of the histology samples were quantified by the villus to crypt height ratio, amount of crypt hyperplasia, degree of villus blunting, goblet cell mass, and neutrophil and plasma cell infiltration (Table 1). Grouped data were expressed as the mean and standard deviation. Differences among the groups were determined by analysis of variance. RESULTS Ten patients had ulcerative colitis and two had familial polyposis coli. There were four men and eight women. The reason for pouch reconstruction was the progressive enlargement of long lateral pouches resulting from elongated ileal spouts, which caused stasis in the absence of acute pouchitis. The mean age of the patients was 32⫾11 years. The mean time elapsed since construction of the pouch was 7.5⫾2.8 years. As an index of intestinal function, the enzymatic activities of maltase and AOP were determined. The mean maltase activity of the normal ileum (254⫾116UE/g) was significantly higher than that of the ileoanal pouch (57⫾71UE/g) and that of normal colon (29⫾25UE/g) (p⬍0.001, Fig. 1). The maltase activities of the ileoanal pouch and the colon samples were not significantly different (p⬎0.4). Similarly, the AOP activity of the normal ileum (73⫾32UE/g) was markedly higher than that in the ileoanal pouch (21⫾22UE/g) and that in the colon (16⫾10UE/g) (p⬍0.001, Fig. 2). The AOP activities of the ileoanal pouch and the

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Figure 1. Mean maltase enzymatic activity of the mucosa from ileum (n ⫽ 15), colon (n ⫽ 5), and ileoanal pouch (n ⫽ 12). UE, units of enzymatic activity.

colon were not statistically different (p⬎0.5). Analysis of variance of the pouch, ileal, and colon specimens for both the maltase and AOP groups was statistically significant (p⬍0.001). The morphology of the samples varied widely, but nearly all of the pouch samples showed little or no acute inflammatory changes, with a neutrophil index (defined in Table 1) of 0.6⫾0.7. Most of the specimens demonstrated mild chronic inflammation with a plasma cell index of 1.0⫾1.1. One sample showed both marked acute and chronic inflammatory changes. Several of the pouch samples

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Figure 2. Mean amino-oligopeptidase enzymatic activity of the mucosa from ileum, colon, and ileoanal pouch. UE, units of enzymatic activity.

morphologically resembled colonic mucosa rather than the ileal mucosa from which they were constructed. The villi were mildly to severely blunted with an index of 1.4⫾1.0 (Fig. 3). The specimens with markedly blunted villi were noted to have flattened epithelial cells at the tip of the villi. These cells appeared poorly specialized and lacked the brush border of mature epithelial cells (Fig. 4). The average amount of crypt hyperplasia was graded as 1.1⫾1.0, with several samples showing a moderate

Figure 3. Morphologic changes of the ileal pouch mucosa (hematoxylin and eosin, 100⫻ magnification). A, normal ileum; B, moderately blunted villi and moderate crypt hyperplasia in ileoanal pouch mucosa; C, severely blunted villi and crypt hyperplasia in ileoanal pouch mucosa.

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Figure 4. Ileoanal pouch mucosa. Note the flattened, immature cells present on the tip of the villus (hematoxylin and eosin, 100⫻ magnification).

amount of hyperplasia. There was a wide variation of the villus to crypt ratio (2.7⫾2.0, ranging from less than 0.5 to 6, normal ileum being 6). The samples had predominantly normal or slightly elevated numbers of goblet cells, graded at 0.7⫾1.0. The samples with the lowest villus to crypt ratio tended to have the lowest disaccharidase enzymatic activity. Although some of the specimens had fairly well preserved villus architecture, these specimens still had a lower functional capacity than normal ileum. DISCUSSION After total proctocolectomy and construction of an ileoanal pouch, the ileal mucosa is exposed to new environmental stimuli and undergoes gradual adaptive changes. The present study demonstrates that the levels of maltase and AOP in the ileoanal pouch mucosa obtained more than 5 years after pouch construction are much lower than those in the normal ileal mucosa, but are similar to those in the normal colonic mucosa. Because these enzymes are normally present on the brush border of mature intestinal cells, the decreased expression of maltase and AOP suggests the loss of differentiation of enterocytes in the ileoanal pouch. These functional changes accompany the morphologic changes in the ileoanal pouch mucosa, demonstrating the loss

of normal villi and the hyperplasia of crypts. These observations are consistent with other reports in the literature that described the occurrence of colonic metaplasia in the ileoanal pouch several years after construction. None of the specimens showed evidence of dysplasia. In the present study, several interesting parallels can be drawn between the morphology of the ileal pouch specimens and decreased levels of maltase and AOP activity. In each specimen of ileoanal pouch, the villi of the ileal mucosa were either shortened or absent. So there were fewer epithelial cells available to produce maltase and AOP. In addition to having fewer epithelial cells, the cells that are present appear to be less functional. This may be partially explained by the lack of maturity of the enterocytes. The cells appeared morphologically immature. In addition, the crypt hyperplasia found in these specimens suggests a high turnover rate of the enterocytes. The decrease in enzymatic activity may also be from the excess of inflammatory cells or edema present in the mucosa. Lastly, it should be noted that the specimens were taken from enlarged malfunctioning ileoanal pouches with mild chronic inflammation that may not represent those that are experiencing normal function. A few previous studies have examined the functional capacity of ileoanal pouch mucosa. The ileo-

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anal pouch mucosa has been shown to express proteins such as sulphomucin that are normally produced by colonic cells.9,10 Stelzner and coworkers11 showed that the uptake of glucose, proline, taurocholate, and propionate were reduced from that in normal ileum to that measured in the colon in a canine model of ileoanal pouch. In another study, the transmucosal potential difference of the ileoanal pouch was found to be significantly different from normal ileum and approached the values of normal rectum.12 The ileoanal pouch mucosa has also been shown to have a significant reduction in glutamine oxidation.13 All of these observations are consistent with the findings of the present study, confirming the colonic transformation of the ileoanal pouch mucosa. Although the mechanisms and stimuli for the adaptation of the ileoanal pouch have not been elucidated, the fecal storage environment of the ileoanal pouch appears to stimulate the pouch mucosa to function more like the colon than the ileum. Although the normal ileum contains low levels of bacteria, the ileoanal pouch has extensive anaerobic and aerobic bacterial flora similar to that in the normal colon.14,15 The products of these bacteria may mediate the adaptive transformation of the ileal mucosa. For example, short-chain fatty acids are increased with the growth of bacterial flora. Within 1 year, the amount of short-chain fatty acids in the functioning ileoanal pouch approaches that in the normal colon.16 These short-chain fatty acids have been shown to alter the pattern of gene expression in enterocytes. Further studies are needed to more clearly elucidate the effects of a colonic environment on the metabolism of nutrients in the ileal pouch. The loss of AOP and maltase activity of the distal ileum used for pouch construction reflects the loss of normal ileal function. These studies support the idea that the longterm ileoanal pouch not only morphologically, but also functionally resembles the normal colon.17 By studying this adaptation process, future

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studies may be able to better define the longterm function of ileoanal pouches and possibly the pathogenesis of pouchitis. References 1. Kojima Y, Sanada Y, Fonkalsrud E. Comparison of endorectal ileal pullthrough following colectomy with and without ileal reservoir. J Pediatr Surg 1982;17:653–659. 2. Shepard N, Jass J, Duval I, et al. Restorative proctocolectomy with ileal reservoir: a pathological and histochemical study of mucosal biopsy specimens. J Clin Pathol 1989;40:601–607. 3. Nasmyth D, Godwin P, Dixon M, et al. Ileal ecology after pouch-anal anastomosis or ileostomy. A study of mucosal morphology, fecal bacteriology, fecal volatile fatty acids and their interrelationship. Gastroenterology 1989;96:817–824. 4. Pronio A, Montesani C, Vecchione A, et al. Restorative proctocolectomy: histological assessment and cytometric DNA analysis of ileal pouch biopsies. Hepatogastroenterology 1997;44: 691–697. 5. Sandborn W. Pouchitis following ileal pouch-anal anastamosis: definition, pathogenesis, and treatment. Gastroenterology 1994;107:1856–1860. 6. Dahlqvist A. Method for assay of intestinal dissaccharides. Anal Biochem 1964;7:18–25. 7. Wojnarowska F, Gray G. Intestinal surface peptide hydrolases: identification and characterization of three enzymes from rat brush borders. Biochim Biophys Acta 1975;403:147–160. 8. Lowry O, Rosebrough N, Farr A, et al. Protein measurement in folin phenol reagent. J Biol Chem 1951;193:265–275. 9. De Silva S, Millard P, Soper N, et al. Effects of the faecal stream and stasis on the ileal pouch mucosa. Gut 1991;32:1166–1169. 10. De Silva S, Millard P, Ketlewell M, et al. Mucosal characteristics of pelvic ileal pouches. Gut 1991;32:61–65. 11. Stelzner M, Fonkalsrud E, Buddington R, et al. Adaptive changes in ileal mucosal nutrient transport following colectomy and endorectal ileal pull-through with ileal reservoir. Arch Surg 1990;125:586–590. 12. Garcia-Armengol J, Hinjosa J, Lledo S, et al. Prospective study of morphological and functional changes with time in the mucosa of the ileoanal pouch: functional appraisal using transmucosal potential differences. Dis Colon Rectum 1998;41:846– 853. 13. Chapman M, Hutton M, Grahn M, et al. Metabolic adaptation of terminal ileal mucosa after construction of an ileoanal pouch. Br J Surg 1993;80:1471–1478. 14. Gorbach S, Nahas L, Weinstein L, et al. Studies of intestinal microflora, IV: the microflora of ileostomy effluent, a unique microbial ecology. Gastroenterology 1967;22:965–968. 15. O’Connell R, Rankin D, Weiland L, et al. Enteric bacteriology, absorption, morphology, and emptying after the ileal pouch anal anastomosis. Br J Surg 1986;73:909–914. 16. Hove H, Mortensen P. Short-chain fatty acids in the nonadapted and adapted pelvic ileal pouch. Scand J Gastroenterol 1996;31:568–574. 17. Fonkalsrud E, Bustorff-Silva J. Reconstruction for chronic dysfunction of ileoanal pouches. Ann Surg 1999;229:197–204.