Intestinal adaptation in short bowel syndrome (SBS) is a compensatory physiological response that occurs following the loss of mucosal surface area, and is important in restoring the digestive and absorptive capacity of the intestine. Most models studying the adaptive responses to massive bowel resection use adult animals with fully developed intestinal tracts (1–6). Models in infant rats have focused on mucosal adaptation (7,8) or precocious intestinal maturation (9,10) after massive proximal small bowel resection. The surgery, as in most current SBS models, involves removal of only the proximal small bowel followed by jejunoileal anastomosis, and thus does not closely resemble the anatomy after bowel resection in humans with SBS (11). In human neonates, SBS most often occurs after surgical treatment for necrotizing enterocolitis (12–14), and usually involves removal of ileum, ileocecal valve (ICV), and sometimes some of or the entire colon. Thus, most currently used SBS models have limitations for studying human infantile SBS, and a more appropriate model in developing animal is needed.
In an adult rat model of SBS, with 60% jejunoileal resection (including removal of all of the ileum) and cecectomy, low survival was reported in the resection group (48% at 10 days after surgery), and only colonic adaptation occurred (15). In an adult mouse model, 50% ileocecal resection (ICR) along with removal of 1 cm of the ascending colon resulted in 91% survival, as well as marked adaptation in the residual jejunum at 7 days after ICR, but colonic adaptation was not reported (16). Neither of these models has been used in infant animals. In this study we adapted the 50% ICR mouse model (16) to weanling rats and investigated the intestinal adaptation in both residual jejunum and colon. In general, ileal resection is less well tolerated than jejunal resection, and the loss of ICV and/or colon has more adverse effects (11,12). To establish an acceptable survival rate, the ICR model was first validated in 20-day-old weanling rats.
MATERIALS AND METHODS
Animals and Surgical Procedures
The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee at Wake Forest University Health Sciences. Twenty-eight weanling male Sprague-Dawley rats were separated from their dams at 20 days of age (weight range 42–60 g). Five rats without surgery were used as controls and fed a liquid diet (Lieber-DeCarli Regular Control Rat Diet #710027, Dyets, Bethlehem, PA) for 7 days. Of the remaining rats 14 underwent ICR, and 9, sham surgery. These rats were allowed access to only water for 4 to 6 hours before surgery, and then provided the liquid diet for 7 days afterward. Water was provided only for the first 24 hours after surgery when intake of the liquid diet was minimal.
The ICR surgery followed a previously described procedure (16) with modifications. Surgery was performed aseptically, with 2% isoflurane and oxygen (1 L/min) given by a mask for anesthesia throughout the procedure. The abdomen was shaved and scrubbed first with povidone-iodine, then with 75% ethanol. All of the rats received 5 mg of piperacillin and tazobactam by intraperitoneal injection before surgery. The abdomen was incised along midline, and the small intestine, cecum, and proximal colon were spread out on gauze moistened with warm saline. The segment of intestine to be removed was located between the fifth to sixth mesenteric vessel arcade proximal to the ileocecal junction and 1 cm distal to the cecum in the ascending colon. The bowel in between was resected after ligation of the mesenteric vessels (Fig. 1A), resulting in the removal of an average of 23 to 33 cm (mean 29 cm) of small bowel. End-to-end anastomosis of the jejunum and proximal colon was performed (Fig. 1B) using interrupted 7-0 Vicryl ophthalmic suture (Ethicon Inc, Longhorn, PA). The sham surgery rats were subjected to bowel transection at the same proximal location as the ICR group, with subsequent anastomosis at the same site. Following the intestinal surgeries in all of the rats, 1 mL of warm saline was instilled into the abdomen and closure was accomplished using 2 layers of 5-0 Vicryl suture in a continuous pattern. The rats were held in an infant incubator at 35°C for at least 30 minutes after surgery, until fully recovered from anesthesia, and then returned to conventional individual housing.
Data and Tissue Collection
Body weight, food intake, and stool changes were recorded daily. At the end of the seventh postoperative day, the rats were humanely euthanized by cervical dislocation. The abdomen was opened and the intestinal tract, from the gastric pylorus to the rectum, was removed. Sections of intestine taken 1 cm from both sides of the anastomosis site were discarded in the surgery groups, and the adjacent 1 cm of jejunum was preserved in 10% neutral buffered formalin for at least 24 hours. Corresponding sections of distal jejunum were similarly processed for the control group, as well as 1-cm sections of proximal colon adjacent to the anastomosis sites in the ICR group and 1-cm sections of colon directly adjacent to the cecum in sham and control groups. These sections were trimmed, embedded in paraffin, processed routinely for histology, and stained with hematoxylin and eosin. The remaining intestine was thoroughly rinsed with ice-cold saline and gently blotted, and the weights and lengths of the small bowel and colon were recorded. Mucosal samples from 5-cm segments of the remaining distal jejunum, middle jejunum between the ligament of Treitz and anastomosis site, colon distal to the anastomosis site in the ICR rats, and corresponding segments in sham and control rats were collected. The segments were opened longitudinally, and the mucosa was gently scraped from the underlying intestinal wall using a glass slide, weighed, frozen in liquid nitrogen, and stored in −80°C.
Quantifications of Mucosal DNA, RNA, and Protein, and Assay for Maltase Activity
Mucosal DNA and RNA were extracted from frozen tissue using DNAzol and TRIzol reagents (Invitrogen, Carlsbad, CA), respectively, as suggested by the manufacturer, and expressed as micrograms per centimeter of bowel. Mucosal proteins were determined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol and expressed as milligrams per centimeter of bowel. Intestinal disaccharidase (maltase) activity was assayed using a previously described method (17). Glucose production was quantified using a glucose oxidase reagent (Pointe Scientific, Inc, Canton, MI), and maltase activity was expressed as micromoles of glucose per gram of protein per minute.
Histological examination of the distal jejunum and proximal colon was done by a board-certified veterinary pathologist. Length and width of small intestine villi, colonic mucosal thickness, and mitotic indices were determined qualitatively, and other lesions were noted.
All of the results are expressed as mean ± SEM. Mean values between control, sham, and ICR groups were compared using a 1-way ANOVA followed by Bonferroni posttests. The daily weight of each group was compared using a 2-way (age, group) ANOVA for repeated measures followed by Bonferroni posttests. A P value of <0.05 was considered statistically significant.
Survival Rate, Weight, Food Intake, and Stool Changes
Rats in both sham and ICR groups tolerated the surgery well. The survival rate for the ICR group was 85% (28/33) and 95% (19/20) for the sham group. Figure 2 shows the daily percentage of preoperative body weight in the control group during the 7-day feeding period and in sham and ICR groups for the same period after surgery. Preoperative body weights were similar in the 3 groups (49.55 ± 2.49 g in the control group, 51.22 ± 2.65 g in the sham group, and 50.42 ± 1.54 g in the ICR group). Postoperative body weights declined about 10% in the sham group and 15% in the ICR group 1 day after surgery, although the 5% additional weight loss in the ICR group was likely attributed to the weight of the segment of bowel removed. By the second and fourth postoperative days the body weights of rats in the sham and ICR groups, respectively, were back to baseline. Although both groups displayed linear growth afterward, rats in the ICR group weighed significantly less from the second postoperative day throughout the entire experimental period, during which the average of weight gain was 2.96 ± 0.22 g/day in the ICR group compared with 5.66 ± 0.33 g/day in the sham and 5.82 ± 0.20 g/day in the control groups (P < 0.05). At the end of the seventh postoperative day, the body weight of the ICR group was about 75% of that of the sham group (59.69 ± 1.81 g vs 79.84 ± 1.78 g, P < 0.05). The rats in the ICR group displayed hyperphagia beginning on the fourth postoperative day, with an average daily food intake of 50.91 ± 1.58 g/day from days 5 to 7, reflecting about 7 and 6 g/day more than that in the sham (43.99 ± 1.59 g/day) and control group (45.28 ± 1.64 g/day), respectively (P < 0.05). For each gram of increased weight the ICR rats consumed 18.26 ± 1.87 g of diet, which was about 2.5 times more than that consumed by the sham (7.63 ± 0.36 g) and the control rats (7.21 ± 0.11 g) (P < 0.05). Diarrhea with a foul odor was observed in the ICR rats as early as 36 hours after surgery and continued throughout the entire experimental period. Both sham and control rats had normal stool patterns.
Rats in the ICR group had significantly increased small intestinal mass, despite decreased total lengths and weights. Seven days after surgery, the residual small intestine of the ICR group was 79% and 56% in weight and length, respectively, of those of the sham group, whereas the weight per centimeter length of small intestine was 140% of that of the sham group (all P < 0.05). In contrast, the colon of the ICR group was similar in length but 132% of the weight, and 137% of the weight per centimeter length of the sham controls (all P < 0.05) (Table 1).
Table 2 shows the significant increases in mucosal mass and amounts of DNA, RNA, and protein in the middle, distal jejunum, and colon 7 days after surgery in the ICR group compared with that of the sham group. In addition, mucosal maltase activity in the distal jejunum segment was significantly higher in the ICR group (all P < 0.05). All of the measurements in sham rats were similar to those in the control rats (Tables 1 and 2).
The lengths of the distal jejunal villi were not appreciably longer in any of the groups, but the villi were qualitatively thicker in the ICR group (Fig. 3). Both surgery groups had similar numbers of mitoses among the enterocytes, whereas those in the control group were qualitatively less abundant. The colonic mucosa from the ICR group was consistently thicker than that of the sham and control groups, and mitoses were more prominent, often extending more than halfway toward the surface (Fig. 4). Mitoses in the control group were consistently present at the base of the crypts and glands.
Because most investigators use adult rats (1–6,15) or mice (16,18) to model infantile SBS, there is little knowledge about the intestinal responses in younger animals. Data comparing intestinal adaptation between adult and infant rats with induced SBS are also limited. Only 1 study compared the mucosal adaptive responses, indicated by mucosal mass, DNA, RNA, and protein levels between 3- and 8-week-old rats, following massive proximal small bowel resection (7). The present study is a modification of a previously reported adult mouse ICR model (16), using weanling rats. In human infants, the intestine grows more rapidly during the last trimester of pregnancy, with a doubling in length by the 40th week or term gestational age (18). In contrast, the intestine in rats is immature at the time of birth, but grows rapidly starting from the time of weanling at 18 to 22 days until fully mature at 5 weeks of age (19), making the weanling rat model more reflective of the development of the human intestine. Following ICR, the weanling rats developed signs similar to those in humans with SBS, including diarrhea with a foul odor, hyperphagia, and poor weight gain, none of which except hyperphagia was reported in rats of similar age following massive proximal small bowel resection (7,8). These findings along with the significant intestinal adaptive responses makes ICR in weanling rats a more appropriate model to study infantile SBS than any of the currently used adult animal models.
The residual jejunum in the weanling rats underwent substantial adaptive response to ICR, which included increases in intestinal weight per centimeter, mucosal mass, DNA, RNA, and protein content, and maltase activity, all consistent with the anticipated proliferative response to compensate for the loss of mucosal surface area. These findings are comparable to the data previously reported (1–5,7,9,10). Morphologically, the apparent increased thickness of the villi in the ICR group likely contributed to the increased mucosal weight, although the height of the villi did not qualitatively differ from the sham group and mitoses were qualitatively more numerous than the control group. One study found increased villous height in both proximal small bowel resection and transaction groups compared with controls in 15-day-old nursing rats (9), but no difference in villous height between the resection and transection groups. However, a similar study in adult mice reported longer villi in the ICR group (16). It is possible that tissue reaction near the surgical sites may have induced similar changes in both resection and transaction groups of infant rats, making it necessary to examine sections more distant to the anastomosis site in the future studies. Also, the developing intestine of infants may be more susceptible to stimuli than that of adult intestine, possibly implying greater potential for adaptation or precocious maturation (9). Jejunal adaptation did not occur with 60% jejunoileal resection and cecectomy in adult rats fed a liquid diet (15), which may be diet related. The diet used in that study, as compared with that used in the present study, had lower fat (0.55% vs 3.96%) and higher carbohydrate (20.4% vs 11.5%). Diets high in fat are associated with accelerated structural bowel adaptation (20,21) and enhanced fat absorbability (22), whereas higher dietary carbohydrate concentrations can result in osmotic diarrhea (12), which may negatively impact bowel adaptation.
The colon also plays important roles in SBS. Colonic adaptation is crucial for small intestinal adaptation and function (23), and has been studied in adult rats (15,24,25) but not in infants. As far as the authors know, this is the first report of colonic adaptation to SBS in an infant model. In this study, the residual colon of ICR rats demonstrated significant increases in weight per centimeter, mucosal mass, and DNA, RNA, and protein content, similar to the findings in the residual jejunum. Histological findings were more dramatic in the colon than in the small intestine, which was unexpected. Mucosal hyperplasia with thickening of the mucosa up to twice of that in sham and control groups was typical, and mitotic figures were prominent at different levels of the mucosa in the ICR group, whereas mitoses were only occasionally present in the sham and control groups. The morphological adaptation of the colon may provide a structural foundation for its functional adaptation in many aspects of SBS, including absorption of fluid, electrolytes, and medium-chain triglycerides, production of short-chain fatty acids for malabsorbed energy salvage, hosting gut microbiota, and synthesis of vitamin K (24). This may explain why patients tolerate extensive small-bowel resections better if the colon is left in entirety and/or continuity (11).
In summary, weanling rats tolerated the ICR surgery well, with a survival rate of 85%, and developed signs similar to human patients with SBS afterward. Significant mucosal adaptation occurred 7 days after ICR in the residual middle and distal jejunum and colon. This model of SBS in weanling rats is appropriate for studying human infantile SBS, and suited particularly well to study enteral nutritional support strategies, and colonic adaptation after SBS induction.
The authors thank Dr April Ronca for generously providing the weanling rats, Ms Hermina Borgerink for preparation of the histological slides, and Dr Michael O'Shea for critical reading.
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