The role of apoptosis during intestinal adaptation after small bowel resection

BACKGROUND/PURPOSE: Adaptation after small bowel resection (SBR) is characterised by a new set point in the balance of enterocyte proliferation and apoptosis. Apoptosis is gene directed. The authors hypothesised that the adaptive response is influenced positively by antiapoptotic gene products (eg, bcl-2 gene-produced protein). The authors tested this hypothesis by studying the effect of bcl-2 overexpression on intestinal adaptation after SBR. METHODS: Male bcl-2 transgenic mice, overexpressing bcl-2 in the small intestinal epithelium, and wild type control mice underwent either a 75% mid-SBR, or a sham operation. The 4 experimental groups consisted of resection wild type (n = 8), transection wild type (n = 6), resection bcl-2 transgenic (n = 8), and transection bcl-2 transgenic (n = 8). Seven days postoperatively small bowel was harvested; total weight, mucosal weight, and mucosal protein, DNA, and RNA content in jejunal and ileal tissue were determined to quantitate the hyperplastic response. RESULTS: Compared with sham-operated animals, SBR resulted in increased total jejunal weight; mucosal weight; and mucosal protein, DNA, and RNA content. Furthermore, in the SBR groups, the jejunal mucosal weight and mucosal protein and DNA content were significantly higher in the bcl-2 transgenic mice compared with the wild-type mice. No differences were observed between any of these parameters in the transection wild-type and transgenic mice. In the ileum, similar changes were observed. The differences between resected and transected wild-type mice were less pronounced, and only total ileal weight and mucosal protein content reached statistical significance. In the transgenic animals, all ileal variables, with the exception of mucosal RNA content, were significantly higher in the SBR group than in the transected group. SBR in the transgenic mice resulted in higher ileal mucosal weight and mucosal protein, DNA, and RNA content compared with the wild-type mice. CONCLUSIONS: The results show that the murine SBR model is a true representation of the process of adaptation after SBR. Furthermore, major components of the adaptive response, both in the jejunum and in the ileum, are significantly more pronounced in the bcl-2 transgenic mice than in the wild-type control animals. Thus, it can be concluded that intestinal hyperplasia after SBR is significantly enhanced by overexpression of the anti-apoptotic bcl-2 gene product. This finding should prompt further research on the effects of antiapoptotic interventions on adaptation after SBR.     

Enterocyte expression of epidermal growth factor receptor is not required for intestinal adaptation in response to massive small bowel resection

PurposeIntestinal adaptation after massive small bowel resection (SBR) permits improved absorption of enteral nutrition despite significant loss of bowel length. Epidermal growth factor (EGF) and its receptor (EGFR) have previously been established to play major roles in the pathogenesis of adaptation. This study tested the hypothesis that EGFR signaling within the epithelial cell compartment (enterocytes) is required for intestinal adaptation.MethodsWe developed a tamoxifen-inducible Villin-Cre/LoxP recombinant system for enterocyte-directed EGFR deletion using EGFR-floxed mice. Epidermal growth factor receptor–null mice and wild-type littermates underwent either 50% proximal SBR or sham operation. Ileal tissue was harvested on postoperative day 7. To assess for adaptation, villus height and crypt depth as well as rates of crypt cell proliferation and apoptosis were measured.ResultsAdaptation after SBR occurred normally, as demonstrated by significant increases in villus height, crypt depth, and crypt proliferative and apoptotic index in both the wild-type and EGFR-null mice.ConclusionEnterocyte EGFR expression is not required for the adaptation response to massive SBR. This novel finding suggests that enterocyte proliferation during adaptation is regulated by EGFR signaling in cells other than enterocytes, perhaps within the mesenchymal cell compartment of the bowel wall via factor(s) that are presently unknown.     

The effect of epidermal growth factor on differentiation of isolated enterocytes after small bowel resection

BACKGROUND/PURPOSE: In previous studies using mucosal scrapings or whole-bowel homogenates, epidermal growth factor (EGF) augments adaptation after massive small bowel resection (SBR). The purpose of this study was to determine directly the effect of adaptation and EGF on enterocyte differentiation using an explicit enterocyte cell population. METHODS: Male ICR mice underwent 50% proximal SBR or sham (bowel transection-reanastomosis) and were selected randomly to either orogastric saline or EGF (50 microg/kg/d). After 3 days, enterocytes were isolated from the remnant ileum by mechanical vibration and assayed for DNA and protein content as well as sucrase and alkaline phosphatase (AlkP) activity. RESULTS: Ileal wet weight, enterocyte protein, and DNA content were increased significantly after SBR and boosted even further with EGF. When normalized for protein, SBR caused an increase in AlkP and sucrase activity, and EGF treatment caused AlkP and sucrase activity to return to baseline. CONCLUSIONS: EGF enhances adaptation; however, when normalized for protein, the activity of two enterocyte-specific enzymes was not significantly altered by EGF. This analysis of an explicit enterocyte population supports the notion that the beneficial effects of EGF are more likely caused by increased numbers of enterocytes rather than an increase in the functional activity of each individual cell.     

Effects of interleukin-11 and epidermal growth factor on residual small intestine after experimental massive small bowel resection

Interleukin 11 (IL-11) is a multifunctional cytokine derived from bone marrow, which has a trophic effect on small bowel epithelium. This study compares the effects of IL-11 with epidermal growth factor (EGF), a growth factor known to enhance small bowel adaptation. Forty Sprague-Dawley rats (90-100g) underwent an 85% mid-small bowel resection with primary anastomosis on day 0. Rats were divided into four treatment groups: controls (group I) received bovine serum albumin (BSA), group II received IL-11, 125 microg/kg subcutaneously (SC) twice daily, group III received EGF, 0,10 microg/g SC bid, and group IV received EGF and IL-11 in the above doses. Half of the animals (five per group) were killed on day 4 of therapy, and the rest on day 8. Animals were evaluated for weight, mucosal length, and bowel wall muscle thickness on days 4 and 8, and expression of proliferating cell nuclear antigen (PCNA) in intestinal crypt and smooth muscle cells on day 8. Body weight was similar at day 4 and 8. Mucosal thickness in groups 11 (IL-11) and IV (IL-11 and EGF) was significantly increased at day 4 and 8 compared with controls (group I) and EGF (group III, P<.001). Muscle thickness was significantly increased in the EGF and combined group IV compared with the BSA controls and IL-11 groups (P < .001). Thirty-two percent of the mucosal crypt cells in group I stained positive for PCNA, whereas 51%, 53%, and 60% stained positive in groups II (IL-11), III (EGF), and IV (IL-11 and EGF), respectively. In groups I and II, 2% and 1.7% of the myocytes stained positive for PCNA, whereas 11.2% and 5.2% in group III and IV. These data suggest that IL-11 has a trophic effect on small intestinal enterocytes, causing cell proliferation and increased mucosal thickness. EGF has a more generalized effect causing proliferation of both enterocytes and myocytes. IL-11, with or without EGF may be a useful adjunct in treatment of short bowel syndrome.     

Serum from mice after small bowel resection enhances intestinal epithelial cell growth

BACKGROUND/PURPOSE: The adaptive response of the intestine to massive small bowel resection (SBR) is remarkably complex. An in vitro model of adaptation may facilitate the elucidation of signaling pathways involved in this process. In an effort to establish such a model, the effects of serum from resected mice on cultured intestinal epithelial cells were studied. METHODS: Serum was collected and pooled from male ICR mice 3 days after either 50% SBR or sham operation. Rat intestinal epithelial cells (RIEC-6) were plated at equal density and grown in the presence of 1% fetal bovine serum (FBS), 10% FBS, 1% FBS plus 9% sham serum, or 1% FBS plus 9% SBR serum. Cell number, proliferation, and caspase-3 activity were determined. RESULTS: RIEC-6 cell growth was reduced significantly in 1% FBS or sham serum. SBR serum markedly accelerated cell growth and proliferation when compared with all other groups and significantly suppressed caspase-3 activity. CONCLUSIONS: Massive intestinal resection in mice results in a serum factor that induces intestinal cell growth in vitro. This in vitro model of trophic signaling will permit further detailed investigations into the mechanisms of intestinal adaptation.     

Characterization of small bowel resection and intestinal adaptation in germ-free rats

BACKGROUND: After massive small bowel resection (SBR), the remnant bowel adapts by increasing enterocyte proliferation and apoptosis. The purpose of this study was to investigate the relevance of luminal bacteria on postresection intestinal cell turnover. METHODS: Male germ-free (GF) and normally colonized control rats underwent either a 75% mid-SBR or sham operation. In other experiments, normally colonized control rats were given antibiotics in the drinking water. After 7 days, the remnant ileum was harvested and adaptation verified by alterations in wet weight, crypt depth, and villus height. Proliferation and apoptosis were measured in crypts as the percent of crypt cells staining for Ki-67 or the number of apoptotic bodies per crypt. RESULTS: Both GF and control rats demonstrated significant increases in all adaptive parameters. Proliferation was increased after SBR in both groups, but significantly greater in the GF animals over control. This response could not be recapitulated after antibiotic treatment. Apoptosis increased equally after SBR in all groups. CONCLUSION: Resection-induced intestinal adaptation occurs normally in GF animals. Epithelial-microbial interactions are probably not involved in the activation of enterocyte apoptosis. The germ-free studies offer the possibility that luminal bacteria may attenuate the proliferative response of the enterocyte to massive small bowel resection.     

Intestinal adaptation after small bowel resection in human infants

Purpose

In animal models, the small intestine responds to massive small bowel resection (SBR) through a compensatory process termed adaptation, characterized by increases in both villus height and crypt depth. This study seeks to determine whether similar morphologic alterations occur in humans after SBR.

Methods

Clinical data and pathologic specimens of infants who had both an SBR for necrotizing enterocolitis and an ostomy takedown from 1999 to 2009 were reviewed. Small intestine mucosal morphology was compared in the same patients at the time of SBR and at the time of ostomy takedown.

Results

For all samples, there was greater villus height (453.6 ± 20.4 vs 341.2 ± 12.4 μm, P < .0001) and crypt depth (178.6 ± 7.2 vs 152.6 ± 6 μm, P < .01) in the ostomy specimens compared with the SBR specimens. In infants with paired specimens, there was an increase of 31.7% ± 8.3% and 22.1% ± 10.0% in villus height and crypt depth, respectively. There was a significant correlation between the amount of intestine resected and the percent change in villus height (r = 0.36, P < .05).

Conclusion

Mucosal adaptation after SBR in human infants is similar to what is observed in animal models. These findings validate the use of animal models of SBR used to understand the molecular mechanisms of this important response.     

Preventing enterocyte apoptosis after massive small bowel resection does not enhance adaptation of the intestinal mucosa

Background

After massive small bowel resection (SBR), increased rates of enterocyte apoptosis are observed in the remnant bowel via a mechanism requiring bax gene expression. This study tested the hypothesis that adaptive mucosal growth could be enhanced by the novel strategy of preventing postresection enterocyte apoptosis.

Methods

Male bax-null and corresponding wild-type (WT) mice underwent a 50% proximal SBR or sham operation (bowel transaction with reanastomosis alone). Mice were killed after a full adaptation interval of 1 month. Adaptation was measured in the remnant ileum as alterations in villus height, crypt depth, and wet weight. Rates of enterocyte proliferation were derived by immunostaining of crypt enterocytes for Ki-67 and apoptosis by the presence of apoptosis bodies.

Results

The expected increase in enterocyte apoptosis after SBR occurred in the WT mice but was unchanged in the bax-null mice. Despite the prevention of postresection apoptosis in the bax-null mice, all parameters of adaptation and proliferation increased equally after SBR in both groups of mice.

Conclusions

Bax deficiency prevents the increase in enterocyte apoptosis that occurs after massive SBR throughout the entire adaptation period. Attenuation of postresection enterocyte apoptosis does not augment mucosal adaptation to massive intestinal loss.     

A serum factor after intestinal resection stimulates epidermal growth factor receptor signaling and proliferation in intestinal epithelial cells

BACKGROUND: In vivo, intestinal adaptation after massive small bowel resection (SBR) requires a functional epidermal growth factor (EGF) receptor (EGFR). In vitro studies have shown that serum from mice after SBR induces rat intestinal epithelial cells to proliferate. This study tested the hypothesis that the proliferative response to SBR serum is mediated by EGFR signaling. METHODS: Serum was collected from male Sprague-Dawley rats 7 days after 75% SBR or sham operation. Rat intestinal epithelial cells were incubated in the presence of sham or SBR serum. Total EGFR expression and phosphorylation of several EGFR downstream pathways were determined by Western blotting. In other experiments, a specific EGFR inhibitor (ZD1839) was added and cell growth determined over 5 days. RESULTS: SBR serum significantly increased total EGFR expression (3-fold) over sham operation and consistently activated the phosphatidylinositol 3-kinase pathway. Furthermore, SBR serum markedly augmented rat intestinal epithelial cell growth, an effect that was abolished by EGFR inhibition. CONCLUSIONS: SBR serum contains a factor or factors that stimulates proliferation of intestinal epithelial cells by an EGFR and phosphatidylinositol 3-kinase signaling mechanism. These data recapitulate in vivo studies supporting the hypothesis that EGFR is a central mediator of postresection intestinal adaptation. This in vitro model may provide a novel means to gain insight into the pathophysiology of intestinal adaptation.     

Alteration of intestinal intraepithelial lymphocytes after massive small bowel resection

BACKGROUND: Intraepithelial lymphocytes (IEL) comprise the inner most layer of the gut immune system, and play a critical role in protecting the host from enteric organisms. Massive small bowel resection (MSBR) is one such clinical condition where patients are at particularly high risk for the development of such enteric infectious complications. Because of this, we hypothesized that the IEL may change significantly after the formation of a MSBR. To address this, a mouse model of MSBR was created and the acute phenotypic and functional characteristics of the IEL were studied. MATERIALS AND METHODS: Mice underwent a 70% mid-small bowel resection. After 7 days, IEL were isolated and analyzed for phenotypic changes by flow cytometry. IEL cytokine expression was performed with semiquantitative polymerase chain reaction techniques. To assess the functional significance of these changes, IEL proliferative response was assessed in vitro.Results. MSBR led to significant decreases in specific IEL subpopulations: CD 44+ (used as a marker of cell maturity); CD 8alphabeta+ (marker of thymic derivation), and CD 69+ (marker of T cell activation). Compared with controls, IEL TNF-alpha mRNA expression increased 84%, while IL-2 and IL-10 mRNA expression decreased by 69 and 72%, respectively. Spontaneous proliferation of IEL in the MSBR group was significantly higher than controls, however, proliferation failed to increase with T cell stimulation.Conclusion. These changes suggest a shift to a more immature and possibly less activated cell population. It is possible that such alterations may play an important role in the increase in enterically derived infections in patients with MSBR.     

Epidermal growth factor receptor signaling regulates goblet cell production after small bowel resection

Background/Purpose

Intestinal adaptation is a compensatory response to massive small bowel loss in which there are increased numbers of absorptive enterocytes. However, the generation of secretory epithelial cell subtypes in this process has not been investigated. The purpose of this study was to examine the adaptive changes of several small intestinal cell lineage changes in response to massive small bowel resection (SBR).

Methods

A 75% SBR or sham operation was performed on male Sprague-Dawley rats. On postoperative day 7, the remnant ileum was harvested and immunohistochemical staining for goblet, Paneth, and enteroendocrine cells was performed. Cell subtypes were evaluated as cells per micrometer of villus/crypt length and compared among operations.

Results

A significant increase in goblet cell density occurred after SBR. Intestinal resection did not alter the number of Paneth and enteroendocrine cells. In additional experiments, inhibition of epidermal growth factor receptor signaling was associated with a diminished goblet cell density.

Conclusions

The adaptive response of the intestine to massive bowel loss results in an expansion of the goblet cell population in addition to greater numbers of absorptive enterocytes. Although the mechanism and purpose for selective expansion of these stem cell-derived lineages are not presently known, epidermal growth factor receptor signaling appears to be a common pathway.     

Effect of sex and sex hormones on structural intestinal adaptation after massive small bowel resection in rats

Purpose

The gonadal steroids play a major role in the regulation of many functions. The purpose of the current study was to evaluate the effect of sex and sex hormones on intestinal adaptation in a rat model of short bowel syndrome (SBS).

Methods

In the first experiment, male and female Sprague-Dawley rats underwent bowel transection and re-anastomosis (sham group) or 75% small bowel resection and anastomosis (SBS group). Relative changes in parameters of intestinal adaptation (overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth) were measured on day 15 and were compared with respect to sex. In the second experiment, male rats were divided into 4 experimental groups: SBS rats, SBS castrated rats, SBS castrated rats treated with testosterone, and SBS castrated rats treated with estradiol. Parameters of intestinal adaptation were compared with respect to hormonal treatment. Statistical significance was determined by Student's t test and analysis of variance with P < .05 considered significant.

Results

Sex had minimal effects on intestinal adaptation. Both male and female rats showed a comparable increase in all parameters of intestinal adaptation. In the second experiment, castration led to significant decrease in bowel and mucosal weight, mucosal DNA and protein in both jejunum and ileum compared with SBS animals. Castrated rats also had lower jejunal villus height and crypt depth compared with SBS animals. Testosterone attenuated this negative effect of castration on bowel regrowth. Rats treated with testosterone showed a significant increase in bowel and mucosal weight, mucosal protein in both jejunum and ileum, mucosal DNA, villus height, and crypt depth in jejunum compared with castrated nontreated animals. Treatment with estradiol after resection and castration had minimal effect on bowel regrowth.

Conclusions

Bowel regrowth after massive small bowel resection is not sex-related. Depletion of androgens by castration inhibited intestinal adaptation. Testosterone has shown a strong stimulating effect on bowel regrowth.     

Neurotensin augments intestinal regeneration after small bowel resection in rats.

Massive small bowel resection (SBR) is characterized by increased proliferation of residual gut mucosa and pancreas. Neurotensin (NT), a gut tridecapeptide, stimulates growth of normal gut mucosa and pancreas. This study examined whether NT affected growth of the small intestine and the pancreas after either distal or proximal SBR. Male Fischer 344 rats were divided into four groups. Group 1 underwent ileal transection with reanastomosis (SHAM) and group 2 underwent 70% distal SBR. Group 3 underwent SHAM operation (jejunal transection), and group 4 underwent 70% proximal SBR. After operation, each group was further subdivided to receive either saline (control) or NT (300 micrograms/kg) subcutaneously in gelatin every 8 hours for 7 days. At death, the pancreas and proximal jejunum (from groups 1 and 2) or distal ileum (from groups 3 and 4) were removed, weighed, and analyzed for DNA, RNA, and protein content. Both proximal and distal SBR significantly increased mucosal growth in the remnant intestine; a more pronounced effect was noted with proximal SBR. Administration of NT significantly augmented the adaptive changes in both groups of rats by mechanisms involving increases in both cell size (hypertrophy) and cell number (hyperplasia). Pancreatic growth was stimulated by distal (but not proximal) SBR; NT did not augment this response. The authors conclude that NT augments intestinal growth after SBR by mechanisms involving an increase in overall mucosal cellularity. Administration of NT may be therapeutically useful to enhance mucosal regeneration during the early period of adaptive hyperplasia after SBR.     

The competence and bacteriologic effect of the telescoped intestinal valve after small bowel resection

In 20 dogs the distal one-third of the small intestine and the proximal 10 cm of the ascending colon were resected. Bowel continuity was established by the telescoping technique to create an intestinal valve in ten dogs (group A) and by conventional end-to-end anastomosis in ten dogs (group B). Diarrhea and weight loss were similar in both groups. The ileal bacterial growth was significantly higher than the jejunum in group A (P less than 0.001) but not in group B. The jejunal bacterial growth in group B was significantly higher than that in group A (P less than 0.01). Intestinal transit times were 212 +/- 16.0, 219 +/- 152.4, and 163 +/- 85.8 minutes for normal dogs, groups A and B respectively. The competence of the normal ileocecal valve, the telescoped valve, and conventional anastomosis were 56 +/- 1.7, 49.9 +/- 17.4 and 13.2 +/- 7.8 cm of barium, respectively. Artificial intestinal valve formed by the telescoping technique is safe, simple, and effective in reducing bacterial overgrowth in the jejunum and appears to prolong intestinal transit time.