Experimental Endometriosis in the Rat Is Correlated With Colonic Motor Function Alterations but Not With Bacterial Load Caroline B. Appleyard, PhD, Myrella L. Cruz, BS, MT, Edelmarie Rivera, BS, Gerardo A. Hernández, MT, PhD, and Idhaliz Flores, PhD
Endometriosis commonly presents with symptoms that mimic chronic gastrointestinal disorders. The authors used the autotransplantion model of endometriosis in rats to investigate the possible underlying mechanisms. After the rats were killed, the presence of endometriotic vesicles, colonic inflammation, and white blood cell (WBC) numbers in the peritoneal fluid was determined. Sections of colon and of jejunum were collected for measurement of myeloperoxidase (MPO) activity and bacterial counts, and isometric recording in response to acetylcholine was measured in segments of longitudinal and circular smooth muscle. Experimental animals had significantly more colonic damage, MPO activity, and WBC numbers than controls did. There was no significant difference in the total bacterial load; however, experimental animals demonstrated an increased tension in the longitudinal muscle, which correlated with WBC numbers and colonic damage. In summary, this study presents evidence for a significant effect of peritoneal endometriosis on colonic function and integrity, which may help explain the gastrointestinal symptoms associated with this disease. KEY
WORDS:
Endometriosis, animal model, gastrointestinal symptoms, colonic inflammation, motility.
E
ndometriosis is defined as the presence of endometrial glands and stroma outside the uterus. In approximately 10% of all menstruating women, this tissue thrives and invades the ovaries, uterine ligaments,
From the Departments of Physiology and Pharmacology (CBA, MLC, ER, GAH) and Microbiology (IF), Ponce School of Medicine, Ponce, Puerto Rico. This study was supported by S06-GM08239 (to CBA and IF) from the National Institutes of Health (NIH) and by G12-RR03050 (to CBA) from the National Center of Research Resources (NCRR), a component of the NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or NIH. The authors wish to acknowledge the technical assistance of Olga I. Santiago and Lynnette A. Ruiz. We would also like to acknowledge statistical support for this article provided by the Epidemiology and Biostatistics Core Program of the Ponce School of Medicine, federally funded by NIH/NCRR/Research Centers in Minority Institutions (grant 2G12-RR03050). Address correspondence to: Caroline B. Appleyard, PhD, Department of Physiology and Pharmacology, Ponce School of Medicine, 395 Zona Ind Reparada 2, Ponce, Puerto Rico 00716-2347. E-mail:
[email protected]
Reproductive Sciences Vol. XX No. X Month XXXX xx-xx DOI. 10.1177/1933719107309722 © 2007 by the Society for Gynecologic Investigation
and pelvic peritoneum, but it can also involve the gastrointestinal (GI) tract.1 In fact, endometriosis occurs in the GI tract in up to 37% of all patients, affecting primarily the bowel segments located in the lower pelvis: the rectosigmoid colon, appendix, cecum, and distal ileum.2 Gastrointestinal symptoms associated with endometriosis include chronic abdominal pain, nausea, vomiting, early satiety, bloating, distention, hematochezia, and altered bowel habits (diarrhea and constipation).These symptoms may or may not be cyclic or temporally related to menstrual cycle phase.3 As a result, most intestinal endometriosis goes unrecognized for long periods of time, and coexistence of gynecological problems and GI symptoms often leads to misdiagnosis and inappropriate management of this important gynecological disease.1 Women with intestinal endometriosis present with symptoms that mimic GI disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).4-7 IBD is a chronic inflammatory condition of the intestinal tract encompassing Crohn disease and ulcerative colitis, whose main symptoms include abdominal
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pain, ulceration, bleeding, weight loss, and diarrhea.8 Like many other chronic inflammatory or autoimmune disorders, IBD seems to result from complex interactions between the immune system, the environment, and susceptibility genes, but its etiology and pathogenesis are still unknown.9,10 IBS, on the other hand, is a chronic GI disorder characterized by abdominal pain and alterations of bowel motility but no ulceration.11 Several contributory mechanisms for IBS have been suggested including psychosocial factors, altered motility, and abnormal visceral sensory perception, but the underlying pathophysiology remains unclear.12 Endometriosis can involve the intestinal tract extensively and may result in a spectrum of mucosal alterations6,13 and motility changes.14 Endometriosis lesions implant in the serosa, sometimes invading the muscularis propia and the mucosa. It has been proposed that the GI manifestations seen in patients with endometriosis are due to (1) growth of endometrial vesicles nearby or directly on local nerve endings,15 (2) sclerosing and fibrotic reactions in the affected bowel wall that leads to distension and stretching,16,17 and (3) the action of prostaglandins elaborated in response to local inflammation.18 We have shown that—similar to IBD—experimental endometriosis is characterized by the activation of proinflammatory mechanisms, mediated primarily by the overexpression of tumor necrosis factor α (TNF) and increased levels of soluble tumor necrosis factor receptor 2 (TNFRSF1B) in the peritoneal fluid and GI tissues of experimental rats as compared with controls.19 Moreover, the levels of TNF in the peritoneal fluid correlated with the colonic damage score of the experimental rats, which was significantly higher than that of controls.Whether local inflammatory mechanisms at play in endometriosis could be correlated with changes in intestinal motor function or with bacterial load is yet to be determined. The role of the microflora in endometriosis is unknown, but some reports suggest similarities between this condition and IBD in this respect. Recent studies have demonstrated that bacterial load is increased in animal models of acute colitis20 and is also increased in rhesus monkeys with endometriosis in comparison with controls.21 In addition, bacterial overgrowth has been observed in patients with IBD22 and also in patients with endometriosis.14 IBD is associated with changes in intestinal motor function dependent on the degree of disease activity,23 and a correlation with bacterial translocation has been suggested.24 No such information is yet available
Appleyard et al
for endometriosis. We hypothesized that increases in proinflammatory mediators such as TNF in the peritoneal environment in endometriosis may alter GI motility, which in turn could influence the profile of the intestinal microflora. The overall aim of this study was therefore to investigate whether ectopic growth of endometrial implants is associated with changes in intestinal motility and bacterial load.
MATERIALS AND METHODS The experiments reported herein were performed in accordance with the principles described in the Guide for the Care and Use of Laboratory Animals, Publication No. DHMS (NIH) 86-23.
Animal Model and Collection of Tissues Studies were performed with female Sprague-Dawley rats weighing 250 to 300 g (Southern Veterinary Service, Ponce School of Medicine, Puerto Rico). All animals were maintained in restricted-access rooms with a controlled temperature (23°C) and a 12-hour light-dark cycle. Standard laboratory chow and drinking water were provided ad libitum. All experimental procedures involving animals were approved by the Animal Care and Use Committee of Ponce School of Medicine. Intestinal endometriosis was surgically induced in mature female rats under pentobarbital anesthesia, based on the method by Vernon and Wilson.25 Briefly, the distal 1 cm of the right uterine horn was removed and immersed in a warm (37°C) sterile culture medium. The endometrium was exposed by opening the horn lengthwise with a pair of sterile scissors, and 4 pieces of uterine horn measuring 2 mm × 2 mm were cut. Four implants of uterine tissue were sutured next to the mesenteric vessels of the small intestine in the experimental group, with the endometrial side toward the mesentery. In the sham group, 4 silk sutures were attached to the mesentery of the intestine without implants, and the right uterine horn was massaged with fingertips for 2 minutes to minimize any effects on our results due simply to the mechanical handling of the uterine horn. All animals were allowed to recover for 60 days before being killed. This time was chosen because prior studies established that the size and development of vesicles appears to plateau after this point.25 A group of age-matched normal animals of comparable
Experimental Endometriosis
weight but undergoing no surgical procedures was also included.Vaginal cytologic smears were carried out for all rats for 5 to 7 days before and after surgical intervention to ensure that their reproductive cycles were occurring. A laparotomy was performed, and the peritoneal cavity was opened and systematically examined for the presence of vesicles and the original sutures.We classified all tissue growths at the implant site as vesicles, whether fluid filled or not. The classification of vesicles in terms of grade of growth was done based on size, following the criteria described by Ingelmo et al.26 Briefly, vesicles
