Enterocyte apoptosis is increased following small bowel resection

The intestinal mucosa is in a steady state of turnover as the rate of cellular proliferation is balanced by the rate of cell death. Although it is accepted that adaptation after small bowel resection (SBR) results in increased proliferation, its effect on apoptosis is not known. The purpose of this study was to determine the effect of adaptation following SBR on rates of enterocyte apoptosis. Male ICR mice underwent either 50% proximal SBR or sham operation (bowel transection/reanastomosis). After 12 and 24 hours, and 3 and 7 days, rates of proliferation were measured in the ileum as the percentage of crypt cells incorporating bromodeoxyuridine. Apoptosis was quantitated by end labeling of DNA strand breaks and propidium iodide staining of the number of apoptotic bodies per crypt and villus. Significant increases in enterocyte proliferation (30% to 40%) as well as apoptosis (57% to 87%) occurred at all time points following SBR when compared with sham-operated mice. Adaptation following SBR increases both the rate of enterocyte proliferation and the rate of apoptosis. Understanding the pathophysiology of intestinal adaptation and therapeutic interventions designed to augment this important response will require complete characterization of their effects on both proliferation and apoptosis. Presented at the Thirty-Eighth Annual Meeting of The Society for Surgery of the Alimentary Tract, May 11–14, 1997, Washington, D.C. Supported by a Trustee’s Grant from the Children’s Hospital Research Foundation awarded to Dr. Warner.     

Bax is required for increased enterocyte apoptosis after massive small bowel resection

BACKGROUND: Massive small bowel resection (SBR) increases rates of both enterocyte proliferation and apoptosis. Previous studies have demonstrated increased intestinal expression of proapoptotic bax mRNA and protein, as well as the appearance of an 18-kd bax cleavage product within 12 hours of SBR. This study tested the hypothesis that bax is required for postresection increases in enterocyte apoptosis. METHODS: Male bax-null and C57Bl/6 (control) mice underwent either a 50% proximal SBR or sham operation. After 3 days, the remnant ileum was harvested and weighed. Apoptotic indexes, proliferation indexes, villus heights, and crypt depths were determined. RESULTS: The usual adaptive increases in ileal wet weight, crypt depth, and rate of proliferation occurred in both the control and bax-null mice. Resection significantly increased the rate of apoptosis in the control mice; however, it failed to alter the apoptotic index in the bax-null mice. CONCLUSIONS: Bax is necessary for the increase in apoptosis that occurs after SBR, but its absence has no significant effect on short-term adaptation. These findings suggest that enterocyte proliferation and apoptosis are differentially regulated during intestinal adaptation.     

Bax deficiency rescues resection-induced enterocyte apoptosis in mice with perturbed EGF receptor function

BACKGROUND: Adaptation after massive smallbowel resection (SBR) is associated with increased cell turnover, increased rates of enterocyte proliferation, and apoptosis. Epidermal growth factor receptor (EGFR) inhibition attenuates adaptation and increases apoptosis. Intestinal levels of bax appear to correlate with EGFR signaling. This study tested the hypothesis that bax is required for the exaggerated postresection apoptosis induced by perturbed EGFR signaling. METHODS: Waved-2 mice with impaired EGFR signaling were crossbred with bax-null mice. Offspring were subjected to either 50% proximal SBR or sham operation (bowel transection and reanastomosis). After 7 days, parameters of adaptation (villus height, wet weight), proliferation (% Ki-67 immunostaining of crypt cells), and apoptosis (# apoptotic bodies per crypt) were recorded in the remnant ileum. RESULTS: Enterocyte apoptosis was increased in waved-2 mice and prevented in bax-null mice after SBR. The accelerated apoptosis in the waved-2 mice was rescued in the context of deficient bax expression. Other parameters of adaptation were restored in the bax-null/waved-2 mice. CONCLUSION: Bax is required for the induction of postresection enterocyte apoptosis. Defective EGFR signaling augments resection-induced enterocyte apoptosis via a mechanism that also requires bax expression. These data implicate a link between EGFR signaling and bax in the genesis of postresection apoptosis and 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.     

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.     

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 expression and activation of EGF and c-NEU receptors are increased in enterocytes during intestinal adaptation.

BACKGROUND/PURPOSE: After massive small bowel resection (SBR), epidermal growth factor (EGF) and its intestinal receptor (EGF-R) play major roles during adaptation. The expression of a homologous enterocyte receptor termed c-neu (c-neu-R) is capable of forming heterodimers with EGF-R to facilitate cellular signaling. The purpose of this study was to determine the expression and activation of EGF-R and c-neu-R during the adaptive intestinal response to SBR. METHODS: Male ICR mice underwent either SBR or sham surgery. After 1, 3, and 7 days, enterocytes were isolated and protein immunoprecipitated with antibody to either EGF-R or c-neu-R. Receptor protein expression and activation status were determined. RESULTS: When compared with sham operation, the expression and activation status of both EGF-R (six- and twofold, respectively) and c-neu-R (nine- and twofold, respectively) were increased substantially in enterocytes from the adapting ileum after SBR by postoperative day 3. Minimal changes were appreciated for either EGF-R or c-neu-R expression or activation in the remnant bowel after enterocyte removal, liver, or kidney. CONCLUSIONS: Both the expression and activation status of EGF-R and c-neu-R are increased substantially in enterocytes from the adapting ileum by postoperative day 3 after massive SBR. These changes provide a unique mechanism for the enterocyte to enhance cellular signaling in response to EGF during intestinal adaptation.     

Combined pharmacotherapy that increases proliferation and decreases apoptosis optimally enhances intestinal adaptation

Background

Adaptation after massive small bowel resection (SBR) is associated with increased rates of enterocyte proliferation (P) and apoptosis (A). In the present study, we sought to determine the effect of dual therapy designed to increase P and simultaneously reduce A.

Methods

C57Bl/6 mice underwent a 50% small bowel resection (SBR) or sham operation, and then received an inhibitor of apoptosis (pan-caspase inhibitor), a stimulus for proliferation (epidermal growth factor; EGF), a combination, or vehicle control. After 3 days, adaptive morphology (villus height, crypt depth) and rates of enterocyte turnover (proliferation and apoptosis) were measured in the remnant ileum.

Results

Adaptation in controls and treated with the inhibitor was similar. EGF-treated mice demonstrated an even greater adaptive response. Combined therapy with the inhibitor and EGF resulted in maximal adaptation as gauged by the greatest increases in villus height and crypt depth and ratio of rates of P to A.

Conclusion

The capacity for adaptation following massive SBR is maintained via tight regulation of cell production and death. Pharmacologic intervention directed at increasing enterocyte proliferation while simultaneously decreasing apoptosis augments adaptation greater than either intervention alone and may provide a useful strategy to clinically amplify adaptation.     

Apoptosis and the pattern of DNase I expression following massive small bowel resection.

INTRODUCTION: Following massive small bowel resection (SBR), histologic evidence of increased enterocyte apoptosis has been demonstrated in several animal models. Deoxyribonuclease I (DNase I) is requisite for intranuclear cleavage of DNA during apoptosis; we therefore hypothesized that the expression of this gene would be increased following SBR. METHODS: Male ICR mice underwent either 50% proximal SBR or sham surgery (bowel transection/reanastomosis). After 12 h and 1, 3, and 7 days, rates of enterocyte proliferation and apoptosis were recorded as well as DNase I mRNA expression and activity. RESULTS: Adaptation after SBR was confirmed at each time point by increased proliferation. Enterocyte proliferation was increased by 12 h and apoptosis was increased by 24 h and remained elevated through Day 7. When compared with sham-operated mice, SBR resulted in a twofold increase in both DNase I expression and activity at 24 h postoperatively, which returned to baseline by Postoperative Day 3. CONCLUSIONS: DNase I expression and activity are increased early following massive SBR but return to baseline despite persistent increased rates of enterocyte apoptosis and proliferation. This enzyme may be important in the early induction of apoptosis following massive SBR, but not once a new set point has been established in the balance between the rate of enterocyte production and enterocyte loss.     

Effect of transforming growth factor-alpha on intestinal adaptation in a rat model of short bowel syndrome

OBJECTIVE: TGF-alpha has recently been shown to stimulate enterocyte proliferation. In the present study we investigated the effect of TGF-alpha on enterocyte proliferation and loss via apoptosis and its effects on intestinal adaptation in a rat following massive bowel resection. METHODS: Male Sprague-Dawley rats underwent bowel transection and reanastomosis (sham group) or 75% small bowel resection and anastomosis (SBS group) and were treated with intraperitoneal TGF-alpha (75 microg/kg) from the ninth postoperative day (SBS-TGF-alpha group). Parameters of intestinal adaptation (overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth), enterocyte proliferation, and apoptosis were determined on day 15. Statistical significance was determined by ANOVA with a P < 0.05 considered significant. RESULTS: SBS-TGF-alpha rats demonstrated a significant increase (vs SBS) in duodenal, jejunal, and ileal overall bowel and mucosal weights; ileal mucosal DNA and protein; and jejunal and ileal villus height. SBS-TGF-alpha rats also showed an increased cell proliferation index in jejunum (704 +/- 43 vs 499 +/- 63 BrdU-positive cells/10 crypts, P < 0.05) and ileum (715 +/- 84 vs 529 +/- 40 BrdU-positive cells/10 crypts, P < 0.05) and decreased apoptotic index in ileum (8.7 +/- 1.1 vs 21.8 +/- 3.2 apoptotic cells/1,000 villus cells, P < 0.05) compared to SBS animals. CONCLUSIONS: In a rat model of SBS, TGF-alpha enhances intestinal adaptation. Possible mechanisms may include increased cell proliferation and decreased enterocyte loss via apoptosis.     

EGF receptor signaling affects bcl-2 family gene expression and apoptosis after massive small bowel resection

Background

After massive small bowel resection (SBR), enterocyte apoptosis is elevated and inversely correlates with epidermal growth factor receptor (EGFR) signaling. The purpose of the current study was to determine whether EGFR manipulation affects the expression of specific bcl-2 family members.

Methods

A 50% proximal SBR or sham operation was performed in 3 groups of mice control, after exogenous EGF, or mutant mice with defective EGFR signaling (waved-2). Apoptotic index (no. of apoptotic bodies per crypt), and bax (pro-apoptosis) and bcl-w (anti-apoptosis) protein expression was measured in the remnant ileum after 12, 24, and 72 hours.

Results

Waved-2 mice with defective EGFR showed the greatest increase in apoptosis and altered the ratio of bax to bcl-w in favor of apoptosis after SBR. Conversely, EGF prevented the expected increase in apoptosis after SBR by shifting the ratio of bax to bcl-w in favor of cell survival.

Conclusions

After massive small bowel resection, inhibition of the EGFR accelerates the rate of apoptosis and modifies the expression of specific bcl-2 family members to favor apoptosis. These results further support a specific mechanistic pathway for the regulation of enterocyte apoptosis after SBR via EGFR signaling.     

Immune-enhancing enteral diet selectively augments ileal blood flow in the rat

BACKGROUND: Prior indirect studies have suggested that a functional epidermal growth factor receptor (EGFR) appears to be indispensable for the adaptive response of the remnant intestine to massive small bowel resection (SBR). The recent availability of a specific pharmacologic EGFR inhibitor enabled us to more directly test the hypothesis that EGFR signaling is required for postresection intestinal adaptation. METHODS: Mice (C57B1/6, n = 26) underwent a 50% SBR or sham operation and were then given orogastric EGFR inhibitor (ZD1839, 50 mg/kg/day) or vehicle. After 3 days, indices of adaptation (wet weight, crypt depth, and villus height) and apoptotic index (number of apoptotic bodies per crypt) were calculated in the ileum. The expression of proliferating cell nuclear antigen (PCNA) and activated EGFR was measured by Western blotting. RESULTS: ZD1839 prevented EGFR activation and the normal postresection increases in ileal wet weight, villus height, and crypt depth. Enterocyte proliferation was reduced twofold in the SBR group by ZD1839. Although not statistically significant, rates of enterocyte apoptosis were the highest in the inhibitor-treated mice. CONCLUSION: Following massive SBR, pharmacologic inhibition of the EGFR attenuates proliferation and the normal adaptive response of the intestine. These results more directly confirm the requirement of a functional EGFR as a mediator of the postresection adaptation response. This study demonstrates an in vivo application of a novel selective EGFR inhibitor and offers a unique experimental model to gain mechanistic insight into understanding postresection intestinal adaptation.     

Parenteral omega-3 fatty acids (Omegaven) modulate intestinal recovery after intestinal ischemia-reperfusion in a rat model

Background/purpose

Fatty acids from fish oil (omega-3 polyunsaturated fatty acids, 3PUFAs) are emerging as powerful yet safe disease-modifying nutrients and are protective in severe critical care conditions including ischemia-reperfusion (IR) injury. The purpose of the present study was to examine the effects of 3PUFAs on intestinal structural changes, enterocyte proliferation, and apoptosis after intestinal IR in a rat.

Methods

Male rats were divided into three experimental groups: sham rats underwent laparotomy, IR rats underwent occlusion of both superior mesenteric artery and portal vein for 30 minutes followed by 48 hours of reperfusion, and 3PUFA-treated IR (IR-3PUFA) rats underwent IR and were treated with Omegaven (Fresenius Kabi, Bad Homburg, Germany) given intraperitoneally at a dose of 1 mL twice a day. Intestinal structural changes (Park injury score, overall bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, cell proliferation, and apoptosis) were determined 48 hours after IR. Real-time polymerase chain reaction (PCR) was used to determine the level of bax and bcl-2 messenger RNA.

Results

A significant decrease in bowel and mucosal weight was observed in the ileum of untreated IR rats compared with sham animals. Forty-eight hours after IR, cell apoptosis remained increased in the jejunum and ileum, which coincided with increased bax/bcl-2 ratio. Cell proliferation was increased 48 hours after IR, suggesting tissue repair. Treatment with Omegaven resulted in a significant increase in bowel and mucosal weight in the jejunum and ileum, villus height in the jejunum and ileum, and crypt depth in the jejunum compared with untreated IR animals. IR-3PUFA rats also demonstrated a significantly lower Park injury score in the jejunum and ileum as well as a lower apoptotic index in the ileum compared with untreated IR animals.

Conclusions

Parenteral Omegaven administration decreases the intestinal mucosal injury and inhibits enterocyte apoptosis after intestinal IR in a rat.     

Extent of small bowel resection does not influence the magnitude of intestinal adaptation in the mouse

Purpose

The magnitude of intestinal adaptation is considered to correlate with the extent of small bowel resection (SBR). However, this association has never been tested in mice. We sought to test the hypothesis that a greater SBR will induce a greater adaptation response.

Methods

C57/B6 mice underwent 50% SBR, 75% SBR, or sham operation and were killed on postoperative day 7. The magnitude of adaptation was compared between 50% SBR and 75% SBR as changes in villus height, crypt depth, as well as rates of apoptosis and proliferation.

Results

Seventy-five percent SBR led to decreased survival and increased weight loss compared with 50% SBR. The remnant ileum of both 50% SBR and 75% SBR displayed similar crypt expansion, enhanced villi, and increased apoptotic indices. Proliferation rates increased after 50% and 75% SBR equally.

Conclusion

Models of resection greater than 50% in mice result in greater morbidity and mortality and do not magnify the adaptation response to massive SBR. The use of more extreme resection models does not appear to provide added benefit for investigating mechanisms of intestinal adaptation.     

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.     

Pretreatment With the Tumor Nerosis Factor-α Blocker Etanercept Attenuated Ischemia-Reperfusion Renal Injury

Introduction

Tumor necrosis factor (TNF)-α mediates inflammation and apoptosis in ischemia-reperfusion (IR) injury of the kidneys. Etanercept, a soluble TNF-α receptor, has shown anti-inflammatory and anti-apoptotic effects in several animal models of renal injury, including chronic insufficiency and unilateral ureteral obstruction. We evaluated the protective effect of etanercept against experimental renal IR injury.

Methods

Male Sprague-Dawley (SD) rats were divided into 4 groups: saline-treated sham rats, etanercept-treated sham rats, saline-treated IR rats, and etanercept-treated IR rats. Renal messenger RNA (mRNA) levels of TNF-α and monocyte chemotactic protein-1 (MCP-1) were measured by real-time polymerase chain reaction (PCR) at 24 hours after IR injury. The protein levels of renal Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl), extracellular signal-regulated kinase (ERK), and caspase-3 activation were evaluated using Western blot analysis. The degree of apoptosis of renal tubular cells was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.

Results

At 24 hours after IR injury, the serum levels of blood urea nitrogen (BUN) and creatinine were significantly lower among etanercept-treated than saline-treated IR rats. Renal mRNA levels of TNF-α and MCP-1 in saline-treated IR rats were significantly higher than the levels in saline-treated sham rats, and TNF-α and MCP-1 mRNA levels in etanercept-treated IR rats were significantly lower than those in saline-treated IR rats. Etanercept pretreatment of IR-injured rats significantly increased EKR phosphorylation and reduced the renal Bcl-2/Bax ratio, the renal caspase-3 activation, and the number of TUNEL-positive apoptotic cells.

Conclusion

Etanercept improved resistance to renal injury during IR by enhancing the activation of ERK and increasing the Bcl-2/Bax ratio.     

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.