Both Interleukin-11 (IL-11), a multi-functional cytokine derived from bone marrow, and epidermal growth factor (EGF), a potent mitogen for epithelial and mesenchymal cells, have been shown to promote small intestinal crypt cell proliferation and differentiation and enhance bowel adaptation in animals with short bowel syndrome (1, 2, 3, 4, 5, 6, 7). IL-11 preserves small intestine crypt architecture and prolongs animal survival in mice subjected to cytoablative chemo irradiation, intestinal ischemia, and major burn insults (8-10). In addition, IL-11 stimulates megakaryocytopoiesis, erythropoiesis, and granulocytopoiesis and increases the number of immunoglobulin-secreting B-lymphocytes in vitro and in vivo (12). EGF a hormone produced in the Brunner glands of the duodenum and in salivary glands has a trophic effect on intestinal mucosa, increasing DNA synthesis, villus height, and protein content (13-17). EGF also increases brush border enteropeptidase activity and the uptake of glucose, electrolytes, and glutamine (18-21). The purpose of this study is to compare the effects of IL-11 and EGF on small intestine morphology after 85 % mid-small bowel resection in rats.
Materials and Methods
Animals
Male, Sprague Dawley rats weighing between 90 and 100g, (Harlan Laboratories, Indianapolis, IN) were kept in individual stainless steel cages and fed a standard rat chow and given water ad libitum. Surgical procedures were performed under anesthesia using aseptic technique and an intramuscular ketamine cocktail (ketamine, 100mg; promazine, 2.2 mg; and atropine 0,5 mg /mL, 1 mL/kg body weight) under guidelines approved by the Laboratory Animal Research Center, Indiana University.
Small bowel resection
In 40 rats, the abdomen was entered through a midline incision, and the bowel was eviscerated. An 85% mid-small bowel resection was performed by dividing the proximal bowel 4.0cm from the duodeno-jejunal junction and the distal bowel 4.0cm from the ileocecal valve. Bowel continuity was restored by a one-layer interrupted 6-0 silk end-to-end anastomosis. The abdominal wall was closed with a continuous 4-0 silk suture. Standard laboratory rat chow and water was offered ad libitum after operation.
Animals were divided randomly into four experimental groups of ten rats each. Animals in group II received IL-11, 125 mg/kg subcutaneously (SC) every 12 hours, whereas those in group III received EGF, 0.10 mg/g SC every 12 hours. Group IV animals received both IL-11 and EGF in the above noted doses every 12 hours, whereas group I animals received the same volume of 0.1% bovine serum albumin SC and served as controls. Recombinant IL-11 (provided by Genetics Institute, Cambridge, MA) was diluted in 0.1% BSA (Boehringer-Mannheim, Indianapolis, IN) and recombinant human EGF (Becton-Dickinson, Bedford, MA) was reconstituted in normal saline. Half of the rats in each group were killed on day 4 of therapy, and the rest were killed on day 8.
Animals were evaluated for weight gain, mucosal height, and bowel wall muscle thickness on postoperative day 4 and day 8 and expression of proliferating cell nuclear antigen (PCNA) in intestinal crypt and smooth muscle cells on day 8. After death, the remaining small bowel was harvested, and a portion of terminal ileum was fixed in 10% formaldehyde solution (2% to 3% methanol). Intestinal tissues were embedded in paraffin wax using standard techniques. Five-micrometer sections were cut and stained with hematoxylin and eosin and used for hystopathologic study. Mucosal length (villus height plus crypt depth) and muscle thickness were measured in a blinded fashion using an objective mounted micrometer at 200x magnification and an optical microscope at 10x100 magnification. Data collected from mucosal height and muscle thickness measurements consist of the average of eight separate measurements in each animal. PCNA analysis was performed on paraffin-embedded sections obtained from the same blocks used for the morphometric analysis and prepared as described by Orazi et al. (22). PCNA expression was measured at day 8 and is presented as a percentage of the number of cells staining positive over the total number of cells counted in 10 crypts (mucosa) or 10 high-power fields (muscle) at 40x magnification.
Statistical evaluation
Numerical results are expressed as the mean SD of the mean. The comparison of the different means between variables of body weight, small intestinal mucosal length, and PCNA analysis were performed by the Student's t-test. A P value of less than .05 was considered significant.
Results
All animals recovered from the operative procedure uneventfully. There were two deaths; both were 8-day controls that lost 30 g each of body weight by the time of death on day 7. By day 8 the average weight loss in the BSA control group was -18.8 ± 15.5% of initial weight, whereas the average weight loss in the IL-11, EGF, and combined IL-11/EGF groups was - 6.25 ± 8.7%, - 9.1 ± 8.3%, and -6.9 ± 3.2%, respectively when compared with baseline weights. Although there is a trend toward weight preservation in the experimental groups, this did not reach statistical significance, possibly because of the high standard deviations noted.
Mucosal thickness was significantly in both the IL-11 and the combined IL-11/EGF groups at both day 4 and day 8 when compared with either the EGF or BSA groups (Table 1). The increase in mucosal thickness appear to be secondary to the effects of IL-11 alone and are not augmented by the addition of EGF because mucosal thickness in the combined group was not statistically different from IL-11 alone at postoperative day 4 or 8 (Fig. 1). On the other hand, EGF appears trophic for the muscularis propria layer because muscle thickness was significantly increased in the EGF and the combined IL-11/EGF groups when compared with BSA and IL-11 alone (Table 2). By day 8, however the combined IL-11/EGF group demonstrates increased smooth muscle thickness compared with the EGF-only group, suggesting that IL-11 may enhance the trophic effect of EGF (Fig. 2).
Because mucosal thickness is dependent on proliferation and differentiation of intestinal postresectional stem and progenitor cells (23-24), the increased mucosal thickness is most likely the result of an increased postresectional intestinal crypt cell mitotic rate. This is supported by PCNA analysis which shows over 50% of the crypt cells staining positive in the IL-11 and EGF groups compared with 32% in the BSA control group (Table 3 ). Of interest is the observation that combined IL-11 and EGF treatment results in 60% of crypt cells staining positive, which is significantly increased when compared with all other groups (P<.01, Fig. 3).
PCNA staining in the muscularis propria cells after 85% bowel resection was minimal in the BSA (2%) and IL-11 (1.7%) groups. However, smooth muscle cells proliferate when treated with EGF (11.6% staining positive) alone or when EGF is combined with IL-11 (5.2%, Table 3 ). This increase in smooth muscle cell mitotic activity in the groups treated with EGF correlates directly with the increase in muscle thickness noted on morphological measurements.
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The loss of small intestinal mucosal surface area is a relatively common clinical situation seen in both the pediatric and adult population. The most frequent causes include mesenteric ischemia, trauma, inflammatory bowel disease (IBD), necrotizing enterocolitis and volvulus (3). The most common causes of short bowel syndrome in infants are necrotizing enterocolitis, intestinal atresia, and midgut volvulus, accounting for over two thirds of cases, whereas gastroschisis, total colonic Hirschsprung's disease and miscellaneous disorders account for the remaining third (25). Despite improvements in supportive care, including total parenteral nutrition (TPN) and defined enteral formulas, improved pediatric anesthesia, and surgical techniques over the past decade, survivorship has remained almost constant (range, 80 to 85 %) (31). After massive small bowel resection, intestinal adaptation is characterized by an increase in the bowel circumference, thickness of bowel wall, increase of villus height, increase in crypt depth, and an increased rate of cell proliferation and migration rate to the villus tip. The major stimulus for intestinal adaptation is enteral feeding (27). Children with ultra short bowel length (< 40.0 cm) continue to have problems with adapting enough to be free of long-term TPN support.
Common causes of death in patients with short bowel syndrome include infectious complications related to a compromised intestinal mucosal barrier and catheter sepsis from central venous lines needed to deliver the hyperalimentation. TPN-related liver cirrhosis and eventual liver failure are other common causes of patient death (25).
Intestinal transplantation has been used in patients with ultra short bowel that fail to adequately adapt. In a review from the University of Pittsburgh, 30 of 53 (56.6%) children with short bowel syndrome undergoing intestinal transplantation are alive with a follow-up ranging from 2 months to 6.3 years (31). The graft and patient survival rates at 5 years are 45% and 55%, respectively. However, the complication rate after bowel transplantation remains high. Complications include a 85-90% rejection rate, a 29 % incidence of cytomegalovirus infection, a 29% incidence of lymphoproliferative disease, as well as a 47% surgical complication rate. The mortality associated with lymphoproliferative disease is 75% (31).
Measures that accelerate small bowel adaptation with conversion to full enteral feeding may reduce the incidence of these complications, the need for intestinal transplantation, and may ultimately result in an improved outcome. Various growth factors including EGF, insulin-like growth factor (IGF-1), growth hormone and glutamine have been identified as agents that enhance intestinal adaptation (1-5, 6, 28, 29). Hepatocyte growth factor (HGF) has been shown to enhance intestinal epithelial cell function and mucosal mass beyond the normal adaptive response (30).
In previous animal studies, EGF was noted to enhance intestinal adaptation and preserve bowel morphology and function in animals with short bowel syndrome or a damaged intestinal mucosal barrier (1-5). Thompson et al. demonstra-ted that EGF and urogastrone stimulated neomucosal growth on serosal patches in rat and rabbit small bowel (32, 33). Read et al. showed an increased intestinal wet weight and mucosal sucrose activity after enteral administration of EGF after massive small bowel resection (34). Chaet et al. used a continuous intravenous infusion of EGF after 75% proximal small bowel resection in rats and noted enhanced mucosal hyperplasia in EGF treated animals. In addition, Zapata-Sirvent et al. reported preservation of intestinal structure and decreased bacterial translocation in mice receiving EGF after 32% scald burn (35). Thompson stated that the route, dose and timing of EGF administration are important factors with relevance to the potential clinical application of EGF therapy: 1. EGF should be given soon after resection; 2. early transient administration may lead to a substantial effect on adaptation; 3. both systemic and enteral therapy may be effective; 4. luminal nutrients are important but not essential in mediating EGF-stimulated adaptation; 5. combined therapy with other nutrients and other growth factors may have merit (5). In the present study, however, EGF-treated rats did not show increase in mucosal height as noted in other reports, but did demonstrate an increased proliferation of mucosal stem and progenitor cells that stained positive for PCNA. In our study EGF increased the thickness of the small bowel muscularis propria and enhanced myocyte proliferation. This observation has not been previously described in the small bowel and may in part explain the increase in intestinal weight reported by many investigators studying the morphological changes caused by EGF (6, 15, 17, 34). The functional significance of thickened smooth muscle wall in small bowel adaptation is yet unknown. EGF stimulates DNA synthesis in vascular smooth muscle cultured from adult rat aorta and mediates tyrosine kinase-dependent smooth muscle phasic contraction of the coronary vessels in various animal preparations, as well as in gastric muscle (36, 37). It is uncertain whether EGF plays a role in the regulation of small bowel motility.
IL-11, a multifunctional cytokine, is a 19 kD hemato-poietic growth factor cloned from an immortalized primate bone marrow stromal cell line (PU-34) (8). It stimulates bone marrow stem cell proliferation causing increased peripheral blood neutrophils and platelets, and stimulates erythroid elements, megakaryocytes, and B cell differentiation both in vivo and in vitro (12). IL-11 improves survival rates after bowel ischemia or cytoablative treatment in mice, and improves survival and decreases bacterial trans-location in an LD50 mouse burn model (9, 10, 11). IL-11 also is trophic to residual small bowel mucosa in rats with short bowel syndrome. This study corroborates the findings of Liu et al., which demonstrated significant increase in villus length and crypt cell mitotic rates in rats receiving IL-11 after a 90% small bowel resection (7). IL-11 is already considered as an adjuvant in the treatment of intestinal damage caused by Crohn's disease (38).
IL-11, like EGF is a potent stimulator of enterocyte proliferation. In this dosing regimen, rats receiving IL-11 had improved mucosal morphology compared with animals receiving EGF. This study does not address whether improved structure is associated with improved function. After extensive bowel resection in the clinical setting, the response of the residual small bowel to inadequate length (in term infants) is villus hyperplasia and subsequent hypertrophy of enterocytes as a means of increasing the absorptive area rather than increases in bowel length (25). Further, the ability of IL-11 to preserve the intestinal mucosal barrier and protect against insults such as cyto-ablative chemotherapy and radiation, burns, and ischemia indicate that IL-11 may play a role in decreasing bacterial translocation and infectious complications during the adaptive phase (22). Finally IL-11 reduces the high frequency of apoptosis of small intestinal mucosal cells after an insult from cytoablative therapy (22).
These data show that both IL -11 and EGF have a trophic effect on small bowel epithelium by increasing the crypt cell mitotic rate. IL-11 acts primarily on the enetrocytes, whereas EGF enhances proliferation of both enetrocytes and myocytes. A combination of the cytokine and growth factor doubles the mitotic index of small intestinal crypt cells compared with control animals undergoing an 85% small bowel resection. These preliminary results suggest that IL-11 with or without EGF may improve small bowel adaptation in instances of short bowel syndrome.
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