Short bowel syndrome (SBS) is a clinical condition resulting from massive enterotomy and is the most frequent cause of intestinal failure in children. In children with extensive intestinal resection, parenteral nutrition is required for survival, but this procedure is associated with frequent and severe complications (1). The length of the remnant intestine and the presence of the ileocecal valve predict the chance of acquiring intestinal sufficiency (1,2). After extensive small bowel resection, the remaining intestine undergoes compensatory changes to maintain its absorptive function (3–5) consisting of an increase in small intestinal mucosal thickness, villus length, and crypt depth (6,7). Intestinal crypt cells activate pathways of gene expression of adaptation and development resembling the developing immature intestinal tissues (8). These changes are interpreted as a homeostatic response to increase the remaining digestive-absorptive surface (9). A crucial factor of adaptation is the time of changes. When a large part of the intestine is suddenly removed, a prompt response is needed to ensure transepithelial ion fluxes, restore the barrier against bacterial translocation, and restart motility. Timely changes are essential for survival; however, intestinal adaptation, its mechanisms, and times are still largely unknown (10–12). In addition, the role and adaptive pattern of proximal to distal intestinal segments are also unknown.
Menge et al found that the total mucosal surface of ileal segments already was increased in the proximal but not in the distal remnants at the fourth day postresection (13). We tested the hypothesis that intestinal adaptation in SBS is segment and time specific and adaptive changes take place with a specific structural and temporal pattern in proximal to distal intestinal segments. To this end, we studied the qualitative and quantitative changes of proximal to distal intestinal epithelial architecture, including villus area and volume, at 3 time points after resection, in a model of extensive small bowel resection. Using the morphometric data obtained in the ileum and jejunum, we constructed a 4-dimensional (4D) geometric model of epithelial structure. The model provides an accurate experimental standard for investigations of postresection structural events and can be used to test the effects of nutrients and drugs on adaptation.
Experimental protocol: Wistar rats weighing between 210 and 270 g were used. The animals were housed in individual cages, under a 12-hour light-dark regimen and humidity and temperature control. They were divided into 3 groups: resected animals (n = 19), sham-resected control animals that underwent ileal transection with subsequent end-to-end anastomosis (n = 10), and nonoperated control animals (n = 7). The animals were killed by CO2 inhalation 2, 7, and 15 days after surgery because adaptive intestinal changes reach a plateau 15 days after intestinal resection in rats (14–16).
Surgery was performed on animals anesthetized with diazepam, ketamine, and medetomidine. The length of the small intestine from Treitz ligament to the ileocecal valve was measured in situ, still attached to the mesentery, under constant tension. Then, 75% of the mid-small bowel was resected, leaving the proximal 12.5% corresponding to the remaining jejunum, and the distal 12.5% corresponding to the remaining ileum (16,17). A primary end-to-end anastomosis was performed and the length of remaining small intestine ranged from 13 to 20 cm. Animals received 5% glucose solution after surgery, wetted food from the second day after surgery, and then were allowed free access to food. Postoperative analgesia and the antibiotic enrofloxacin were given and animals’ weight was recorded daily.
The animals were fed a normal standard diet with no restriction on food or water supply for 1 week before surgery. Before surgery, rats were fasted overnight and weighed. On the first postoperative day, rats had free access to water and rodent diet.
The experimental protocol was approved by the local ethics committee of the Ospedale Cardarelli (no. 1292/09/CB; February 3, 2009). The animals were treated and housed according to national and international regulations governing the use of animals in scientific research.
Tissue sampling: bowel samples were taken 2, 7, and 15 days postoperatively. Six proximal-to-distal intestinal segments were obtained from each animal as follows: antrum (1.5 cm proximal to the pylorus); duodenum (3 cm distal to the pylorus); proximal jejunum (3 cm proximal to the anastomosis in the resected group and 10 cm distal to the ligament of Treitz in the control group); distal ileum (3 cm distal to the anastomosis); right colon (10 cm distal to the cecum); left colon (proximal to the rectum). All of the intestinal segments were weighed, measured, and rinsed with ice-cold saline to remove any luminal contents. Tissue sections measuring 1 cm2 were cut from each segment along the longitudinal axis and used for histological and morphometric analysis.
Intestinal specimens were fixed in 10% buffered formalin for 24 hours, dehydrated and embedded in paraffin wax using standard techniques. Four-micrometer sections were cut perpendicular to the mucosa, placed on gelatin-coated glass, and stained with hematoxylin and eosin. At least 10 well-oriented sections from each intestinal segment were prepared and evaluated by linear quantitative methods using light microscopy. All of the morphometric and counting procedures were performed by 2 independent, blinded investigators. The following parameters were obtained for each segment: total wall thickness (micrometers), total mucosal thickness (micrometers), and inflammatory cells in the mucosa (number of cells/mm2 of mucosa and degree of inflammatory infiltrate). The glandular height in the antrum was also measured and is expressed in micrometers.
Villus height (in micrometers), number of villi (villi/mm2 of mucosal length), crypt depth (micrometers), and villus/crypt ratio were recorded in the duodenum, proximal jejunum, and distal ileum. In the small intestine, only villi and crypts cut throughout their length were measured. The distance from the tip to the base of the villus was taken as villus height. The distance from crypt base to villus-crypt junction was taken as crypt depth. Total mucosal thickness was measured in the proximal and distal jejunum by calculating the distance from the tip of the villus to the muscularis mucosae. Small bowel thickness was determined by calculating the distance from the villus tip to the serosal extremity of longitudinal muscle.
An adaptive response consisting of an increase in mucosal mass may occur through an increase in the number or in the size of intestinal villi or both. We tested the hypothesis that the adaptive response is segment-specific and that the specificity is associated with different mechanisms of adaptation. To address this issue, we applied the geometry of cylinders to villi and calculated epithelial surface expressed as cylinder surface lateral area. The formula to calculate the surface area is the following: surface area = 2(πr2) + (2πr) × h, where h is the height of the cylinder and r is half the measure of the base. Then we constructed a 4D geometric model using form-Z, version 6.0 software (Auto·Des·Sys Inc, Columbus, OH) by fitting the villi measures to obtain a spatial representation of adaptive changes in proximal to distal segments. This software is a 3D modeling program combining solids and surface modeling and is used for the first time to construct a biological model. The fourth dimension is time.
For the statistical analysis, results are mean ± standard deviation, with significance determined by analysis of variance test at the P < 0.05 level.
Two rats died 1 day after surgery. Three animals (1 in the control group and 2 in the 15-day resected group) were excluded from the study because a stricture developed at the anastomotic site. At postmortem analysis, both showed a dilated bowel proximal to the anastomosis. Mean body weight did not differ significantly before and after intestinal resection or between sham-resected and resected rats 2, 7, and 15 days after surgery. Data from nonoperated rats were also identical to the data obtained with the study groups.
The gut reacted to massive resection with a general adaptive response consisting in catch-up growth of the remaining small intestine. Fifteen days after surgery, the small intestine showed an increase in mass of 48.2% ± 7.6% over baseline. The adaptive changes had a well-defined segmental pattern. Figure 1A shows the major macroscopical findings 15 days postsurgery. Changes were clearly evident in the ileum: its weight/cm2 was almost 2-fold higher than in sham-resected animals. The stomach, the duodenum, and the jejunum underwent minor, not significant modifications, although there was a consistent trend toward an increase in all 3 segments, which could be interpreted as an expression of minimal adaptation. Finally, the proximal and the distal colonic segments were virtually unchanged after small bowel resection 2, 7, and 15 days after surgery compared with sham-resected and control rats. Total bowel thickness showed a similar segmental pattern with the ileum undergoing the most evident changes (Fig. 1B).
At microscopic evaluation, intestinal adaptation consisted of an increase in villus height, crypt depth, and mucosal thickness in the jejunum and ileum. The increase in villus height occurred 7 days after surgery in the ileum, whereas villus adaptation ceased 15 days after surgery in the jejunum (Fig. 1C). The crypt adaptive response was a distinct time-dependent process with a peak at day 15 in both ileum and jejunum (Fig. 1C). Two days after surgery, no changes were found in length and crypt depth, in the jejunum, or in the ileum. Overall, the ileum was the major site of intestinal adaptation; the major modifications were observed 15 days after surgery, and consisted of an increase in both villus height and crypt depth. Villus height and crypt depth were significantly increased also in the jejunum, albeit to a lesser extent than in the ileum. There were no substantial changes in the stomach, duodenum, or colon (data not shown).
To evaluate whether the different adaptive responses of intestinal segments were linked to segment-specific restructuring of the intestinal architecture, we analyzed the villus/crypt ratio in all of the intestinal segments. This ratio was conserved in all of the intestinal segments and there was a close overlapping of numerical values between resected and sham-resected animals at 15 days post-surgery (Fig. 2).
Light microscopy did not reveal evidence of inflammatory changes in the ileum or jejunum. This indicates that the observed changes were because of a true compensatory increase in epithelial cell mass, rather than to a change in inflammatory mucosal fluid content.
The different adaptive pattern in the jejunum and ileum supports the hypothesis that adaptation is the result of distinct mechanisms. To address this issue, we analyzed the segment-specific morphometric changes of the intestinal structure. The number of villi per linear millimeter of mucosa was calculated for each intestinal segment. The area and volume of villi were also measured in all of the segments. Finally, the overall increase of the intestinal surface was calculated for each segment. Minor quantitative and qualitative changes were observed 2 days after surgery, although differences were not significant. Adaptation became progressively more evident at subsequent observations. The number of villi in the ileum was significantly lower in resected animals than in sham-resected animals. In parallel, structural modifications were found in the ileal villus architecture, that is, there was an increase both in the diameter of the villus base and in villus height (Fig. 3). In contrast, the number of villi in the jejunum was similar to that observed in sham-resected animals; however, jejunal villi were longer, whereas the diameter of the villus base remained unchanged (Fig. 3).
We identified a time-related increase in morphometric parameters in the jejunum and ileum (Table 1), which indicates that the time course pattern and the type of architectural restructure were both segment specific. There were no modifications 2 days after surgery, whereas there was a significant increase in villus height and crypt depth at day 7 postsurgery in the ileum and at day 15 postsurgery in the ileum and jejunum segments. In both segments, the area and the volume of the intestinal surface increased, resulting in an increase of surface area, but the bulk of changes occurred in the ileum, which suggests that the latter possesses the highest adaptive plasticity (Fig. 4). Changes were also faster in the ileum than in the jejunum with an increase of >50% of surface area 7 days after surgery in the ileum versus 5% in the jejunum. The ileal segment located distally to the anastomosis was the major site of adaptive changes, which comprised mainly an increase of total bowel thickness, weight, and volume (Figs. 1 and 4).
Construction of a 4D Model of Segment-specific Intestinal Adaptation
The adaptive changes in jejunum and ileum were associated with a segment-specific restructuring of epithelial architecture. Based on the morphometrical data, a 4D geometric model of adaptation was constructed that provides a view of segment-specific changes (Fig. 5). Compared to sham-resected rats, the number of villi/mm2 of mucosa did not change in the jejunum, whereas it decreased in the ileum. This observation, together with the changes in bowel thickness, wet weight, height, and diameter of villi, strongly suggests that adaptation takes place in the jejunum and ileum with segment-specific events. In the former, the mucosal increase was associated with an increase in villus height, whereas their number and diameter did not change. In contrast, in the ileum, the mucosal increase was the result of a massive increase in villus height and diameter, whereas the villus number decreased (Fig. 5). Villus area and volume were much larger in the distal ileum than in the proximal jejunum; as a result, the nutrient absorptive surface area was strikingly larger in the distal ileum. In contrast, the stomach and colon were not involved in the adaption processes.
The total intestinal absorptive surface area is approximately 250 m2 in adult humans; however, there is a functional and structural segmental pattern of the intestine, and distinct intestinal segments have different mechanisms for nutrient absorption and for the transepithelial flux of electrolytes. In SBS, extensive intestinal resection suddenly leads to a dramatic reduction in intestinal surface producing an imbalance of hydroelectrolyte transport and hampered nutrient absorption. Most patients require parenteral nutrition for survival (18,19). In recent years, the anatomic definition of irreversible SBS has changed and, in parallel, the outcome of patients has improved. The longer survival of children with severe SBS provided the opportunity to observe the ability of the remaining intestine to adapt over time (20). Adaptation is affected by the time of refeeding and other clinical variables such as age and nutritional state (21); however, in our rat model, there was a clear segment-specific pattern of adaptation. A major difference in the adaptive responses of jejunum and ileum was the time-related pattern of adaptation. Two days after resection, the mucosal morphology of the rat jejunum and ileum did not differ from that of controls. Adaptation was completed in the ileum within 2 to 7 days, whereas adaptive changes continued in the jejunum at 15 days postsurgery. We do not know whether jejunal changes were completed at 15 days; however, the adaptive response was found to reach a plateau after 2 weeks in similar animal models of short gut (22,23).
The pattern of intestinal adaptation that we observed resembles the intrinsic development of intestine during embryonic development. In human infants, the intestine grows more rapidly during the last trimester of pregnancy, doubling its length by the 40th week or term gestational age (24,25). In contrast, the intestine in rats is immature at birth, but grows rapidly starting from the time of weanling at 18 to 22 days until reaching a plateau at 5 weeks of age (26). In our model, ileal architecture underwent major modifications. A major increase in surface area and volume was associated with a reduction in villi number. This led to an overall increase in villi surface. From the functional point of view, these changes enhance the adaptive response because the ileum is the main site of absorption of liquids, thereby allowing prompt restoration of transepithelial ion fluxes and of nutrient absorption, which obviously is an advantage in terms of postresection structural/functional intestinal modifications.
In an attempt to restore the intestinal functions, the adaptive response is fully functional in terms of the restructuring because there is an increase in absorptive surface area in a relatively short period.
Both drugs and nutrients have been used to stimulate cell growth and proliferation in SBS, namely, insulin (27), growth hormone and glucagon-like peptide-2 (28,29), glutamine, arginine, zinc, and, more recently, lactoferrin (30–32). The 3D model provides a tool to investigate the type and amount of fuel necessary to optimize absorptive changes and promote intestinal sufficiency. The 3D model was approached by other authors. Liao et al (33) constructed a 3D surface model of different gastrointestinal tracts, from the stomach to the colon. Their macroscopic model was developed to study the geometry and the morphology of the visceral organs to evaluate the visceral distention and curvature during stress events. The 3D modeling approach is a quantitative method that could be used as a useful and analytical tool to study the biochemical properties of intestinal mucosa in different physiological and pathophysiological states.
In conclusion, extensive intestinal resection results in a differential adaptive response in the remaining proximal and distal small intestinal segments. The bulk of changes is observed in the ileum distal to the anastomosis, whereas the adaptive response is less evident in the jejunum and does not involve the colon. The responses follow a different time- and segment-related pattern. Our findings show that the ileum plays a major role in postresection adaptation. This is reflected in the better outcome observed in patients with SBS with preserved rather than removed ileocecal valve (34,35). Sparing even small segments of ileum could result in adaptive changes that may be eventually associated with restoration of full intestinal digestive absorptive functions in children undergoing extensive intestinal resection.
The authors thank Mario Nasti and Dr Marilena Oliva for expert technical assistance. This manuscript was edited for English language, grammar, punctuation, spelling, and overall style by Jean Ann Gilder.
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