Magdy Sourial

Influence of diet complexity on intestinal adaptation following massive small bowel resection in a preclinical model

Aims: To investigate the effect of dietary complexity on intestinal adaptation using a preclinical model.

GLP-2 administration results in increased proliferation but paradoxically an adverse outcome in a juvenile piglet model of short bowel syndrome.

Objective: The objective of the present study was to examine the effect of glucagon-like peptide-2 (GLP-2) administration in a piglet, juvenile model of short bowel syndrome. Materials and Methods: Four-week-old piglets underwent either a sham operation or 75% small bowel resection. Postoperatively, piglets received either polymeric infant formula diet or the diet and subcutaneous human recombinant GLP-2 (1600 μg/day for 7 days, 800 μg/day thereafter). Food intake was monitored throughout the experiment, and stool and serum samples obtained fortnightly. After the piglets were killed, tissues were obtained from the duodenum, jejunum, ileum, and terminal ileum, and used for morphological and functional analysis. Results: Treatment with GLP-2 resulted in significantly increased numbers of proliferating and apoptotic cells in the ileum of sham and small bowel resection piglets (P < 0.05). GLP-2 administration resulted in decreased weight gain, serum albumin, and disaccharidases in both sham and small bowel resection piglets (P < 0.001 compared with polymeric infant formula diet alone). Conclusions: This is the first study to our knowledge to examine the effect of GLP-2 administration in a juvenile short bowel syndrome model. Contrary to adult rodent studies, administration of GLP-2 resulted in adverse outcomes including reduced ability to gain weight; decreased serum albumin, tissue maltase, and sucrase; and villous atrophy. We anticipate this information will have important implications for future paediatric clinical trials.

Colostrum protein concentrate enhances intestinal adaptation after massive small bowel resection in juvenile pigs.

Objectives: Short bowel syndrome (SBS) usually results from the surgical removal of a large segment of small intestine. Patient outcome depends on the extent of intestinal resection and adaptation of the remaining intestine. We evaluated the impact of colostrum protein concentrate (CPC) on intestinal adaptation after massive small bowel resection in a porcine model of infant SBS. Methods: Four-week-old piglets underwent an approximate 75% small bowel resection (R, n = 23) or a control transection operation (C, n = 14). Postoperatively, animals from both groups received either pig chow (R = 6, C = 5), polymeric infant formula (R = 6, C = 3) or polymeric infant formula supplemented with CPC (R = 11, C = 6) for 8 weeks until sacrifice. Clinical outcome measures included weight gain and stool consistency. Morphologic measures were intestinal villus height and crypt depth. Functional outcome measure was mucosal disaccharidase activity. Results: Resected animals fed polymeric infant formula alone had reduced weight gain compared with controls fed the same diet (P < 0.005). Despite massive small bowel resection, animals fed pig chow or polymeric infant formula supplemented with CPC grew at an equivalent rate to controls fed polymeric infant formula alone. Resected animals supplemented with CPC had increased villus length and crypt depth in the jejunum (P < 0.001) and ileum (P < 0.001) compared with resected animals fed either pig chow or polymeric infant formula alone. Conclusion: In an animal model of SBS, CPC supplementation of polymeric infant formula resulted in normal weight gain and features of enhanced morphologic adaptation.

Plasma GLP-2 levels and intestinal markers in the juvenile pig during intestinal adaptation: effects of different diet regimens.

Adaptation of the residual small bowel following resection is dependent on luminal and humoral factors. We aimed to establish if circulating levels of glucagon-like peptide (GLP-2) change under different dietary regimens following resection and to determine if there is a relationship between plasma GLP-2 levels and markers of intestinal adaptation. Four-week-old piglets underwent a 75% proximal small bowel resection (n=31) or transection (n=14). Postoperatively they received either pig chow (n=14), nonpolymeric (elemental) infant formula (n=7), or polymeric infant formula alone (n=8) or supplemented either with fiber (n=6) or with bovine colostrum protein concentrate (CPC; n=10) for 8 weeks until sacrifice. Plasma GLP-2 levels were measured at weeks 0, 2, 4, and 8 postoperatively. In addition, end-stage parameters were studied at week 8 including weight gain, ileal villus height, crypt depth, and disaccharidase levels. Plasma GLP-2 levels were higher in resected animals compared to transected animals fed the same diet. Plasma GLP-2 levels were significantly increased in the colostrum protein isolate-supplemented animals following resection compared to all other diet groups. The increase in plasma GLP-2 (pM) was greatest in the first 2 weeks postresection (week 0, 15.5; week 2, 30.9), followed by a plateau at weeks 2 to 4 and a decrease in GLP-2 levels from week 4 to week 8. At week 8, no relationships were found between the plasma GLP-2 levels and the measurements of weight gain, villus height, lactase, sucrase, maltase, crypt depth, or villus/crypt ratio. Plasma GLP-2 levels increase in the first weeks following massive small intestinal resection. The increase in plasma GLP-2 levels was enhanced by supplementation of the diet with CPC. The changes in GLP-2 levels observed in this study may suggest that GLP-2 plays a role in the adaptive response in the intestine following resection in this preclinical model.

Colostrum Supplementation Restores Insulin-like Growth Factor -1 Levels and Alters Muscle Morphology Following Massive Small Bowel Resection

BACKGROUND Colostrum protein concentrate (CPC) contains a high level of insulin-like growth factor-1 (IGF-1). IGF-1 and IGF binding protein (IGFBPs) may play an important role during the postresection adaptation response. As smooth muscle is an important site for IGF-1 action in the intestine, this study aims to (1) investigate the effect of CPC supplementation on circulating levels and tissue expression of IGF-1, IGF-1 receptor, and IGFBPs following massive small bowel resection (MSBR), and (2) characterize the effect of CPC on the muscular adaptation response following MSBR. METHODS Four-week-old piglets underwent either a 75% MSBR or sham operation. Piglets received either a polymeric infant formula (PIF) diet or PIF supplemented with CPC for 8 weeks. Serum was analyzed by enzyme-linked immunosorbent assay, and ileal tissue assessed by molecular and histological analysis. RESULTS There was no difference in IGF-1 or IGFBPs mRNA among groups. CPC treatment resulted in significant increases in circulating levels of IGF-1 and IGFBPs and a concurrent increase in muscle width and the number of muscle cells, but did not alter muscle cell size. CONCLUSIONS Strategies aimed at increasing muscular adaptation may decrease Gl transit and allow greater mucosal contact time for absorption. We have shown that CPC supplementation following resection results in increased levels of circulating IGF-1, IGFBP-2, and IGFBP-3 and muscular hypertrophy. Our results suggest that IGF-1 and its mediators may play a role in the muscular adaptation response and warrant further exploration as a treatment option for short bowel syndrome.

Calcitonin gene–related peptide stimulates mitosis in the tip of the rat gubernaculum in vitro and provides the chemotactic signals to control gubernacular migration during testicular descent

BACKGROUND/AIMS We investigated whether calcitonin gene-related peptide (CGRP) released from sensory genitofemoral nerve branches could stimulate rodent gubernacular growth and provide chemotactic signals for directing inguinoscrotal gubernaculum migration in vitro. MATERIALS AND METHODS Neonatal rat gubernacula containing a developing cremaster sac (n = 60) were removed at days 0, 2, 4, 6, 8, and 10 (n = 10 per age; n = 5 per experimental group) and placed in organ culture for 24 hours with or without added CGRP (720 nmol/L). The gubernacula were stained for bromodeoxyuridine (BrdU) immunohistochemistry. Cells were counted (3 x 100 cells) in the mesenchymal tip of the gubernaculum to find the percentage of BrdU uptake. A further group of neonatal rat gubernacula (n = 21 per group) were placed in organ culture on an agar platform with 5 agarose beads soaked in either PBS or 10(-6) mol/L CGRP placed approximately 0.8 to 1 mm on each side of the tip of the cremaster sac. After 72 hours, the position of the gubernaculum was compared with its starting position and any deviation measured. RESULTS Exogenous CGRP caused a significant increase in BrdU uptake in the tip of the gubernaculum in 0-day-old rats compared with control cultures. Two-way analysis of variance in the cellular proliferation pattern between gubernacula cultured +/- CGRP between 0 and 10 days showed a significant difference (P < .001). The cultures containing CGRP-impregnated beads caused significant (P < .01) deviation of the tip of the gubernaculum toward the beads, whereas the controls demonstrated no net movement of the tip. CONCLUSIONS These studies demonstrate that mitosis in the tip of the rat gubernaculum is significantly increased in response to CGRP in vitro. Also, CGRP may provide chemotactic signals to control inguinoscrotal gubernacular migration in the rat.

Morphological and functional changes in the colon after massive small bowel resection

PURPOSE Anecdotal evidence suggests that the colon plays an important role after small bowel resection (SBR). However, colonic changes have not previously been studied. The aim of this study was to characterize morphological and functional changes within the colon after SBR and elucidate the influence of diet complexity on adaptation. METHOD In study 1, 4-week-old piglets underwent a 75% SBR or sham operation and were studied at 2, 4, and 6 weeks postoperation to allow analysis of early and late adaptation responses. Piglets received a polymeric infant formula (PIF). In study 2, SBR piglets received an elemental diet and were studied at 6 weeks postoperation and compared with SBR + PIF piglets from study 1. For both studies, immunohistochemistry was used to quantitate intestinal cell types. Changes in functional proteins were measured by Western blot, enteroendocrine/peptide YY (PYY), enterocyte/liver fatty acid binding protein (L-FABP), and goblet cells/trefoil factor 3 (TFF3). RESULTS In study 1, early and late adaptation-related changes were observed after SBR. Early adaptation included increased numbers of enterocytes (P = .0001), whereas late adaptation included increased proliferative cell numbers (P = .02). Enteroendocrine, goblet, and apoptotic cells numbers were significantly elevated in the resected group at all time-points studied (P < .05). Functional changes included increased levels of L-FABP (P = .04) and PYY (P = .03). There was no change in TFF3 expression. In study 2, feeding with an elemental diet resulted in suboptimal adaptation as evidenced by reduced rate of weight gain and significant reductions in total cell numbers (P = .0001), proliferative (P = .0001) and apoptotic cells (P = .04), enteroendocrine cells (P = .001), and PYY expression (P .004). CONCLUSION These findings indicate that significant morphological and functional changes occur in the colon after massive SBR and that these occur as early and late adaptation responses. Elemental diet was associated with suboptimal adaptation suggesting an effect of diet complexity on colonic adaptation.

Proteomic analysis of the intestinal adaptation response reveals altered expression of fatty acid binding proteins following massive small bowel resection.

Intestinal adaptation in response to the loss of the small intestine is essential to restore enteral autonomy in patients who have undergone massive small bowel resection (MSBR). In a proportion of patients, intestinal function is not restored, resulting in chronic intestinal failure (IF). Early referral of such patients for transplant provides the best prognosis; however, the molecular mechanisms underlying intestinal adaptation remain elusive and there is currently no convenient marker to predict whether patients will develop IF. We have investigated the adaptation response in a well-characterized porcine model of intestinal adaptation. 2D DIGE analysis of ileal epithelium from piglets recovering from massive small bowel resection (MSBR) identified over 60 proteins that changed specifically in MSBR animals relative to nonoperational or sham-operated controls. Three fatty acid binding proteins (L-FABP, FABP-6, and I-FABP) showed changes in MSBR animals. The expression changes and localization of each FABP were validated by immunoblotting and immunohistochemical analysis. FABP expression changes in MSBR animals occurred concurrently with altered triglyceride and bile acid metabolism as well as weight gain. The observed FABP expression changes in the ileal epithelium occur as part of the intestinal adaptation response and could provide a clinically useful marker to evaluate adaptation following MSBR.

Calcitonin gene-related peptide is a survival factor, inhibiting apoptosis in neonatal rat gubernaculum in vitro

BACKGROUND Testicular descent is proposed to occur in 2 stages. During the second stage, calcitonin gene-related peptide (CGRP) released from the genitofemoral nerve (GFN) causes maximal mitosis in the gubernacular bulb. As normal development requires a balance between cell proliferation and apoptosis, this study explored the effect of CGRP on apoptosis in the rat gubernacular bulb. METHODS Gubernacula were collected from male Sprague-Dawley rats at birth (D0) or 2 days post birth (D2), and placed in organ culture for 24 hours with or without CGRP (0.001 mol/L). The D2 rats were pretreated with capsaicin (sensory nerve toxin) or flutamide (antiandrogen) or untreated. D0 rats were untreated (n = 64). Sections of the bulb were stained using the TUNEL method to identify apoptotic cells. Apoptosis was calculated as the percentage of positive cells per hundred cells. RESULTS Normal Sprague-Dawley rat gubernacula showed reduced apoptosis when cultured with CGRP, in D0 (7.0% vs 4.8%, P < .05) and D2 (4.9% vs 2.3%, P < .001). Greater apoptosis occurred at D0 compared to D2, without CGRP added (7.0% vs 4.9%, P < .05) and with CGRP (4.8% vs 2.3%, P < .001). For D2 gubernacula, capsaicin treatment increased apoptosis compared to controls, without CGRP added (4.9% vs 7.3%, P < .05) and with CGRP (2.3% vs 6.7%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.3% vs 6.7%, nonsignificant) after capsaicin treatment. Flutamide treatment increased apoptosis compared to controls, but only with CGRP (2.3% vs 7.3%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.1% vs 7.3%, nonsignificant) after flutamide. CONCLUSIONS Calcitonin gene-related peptide (CGRP) acts as a survival factor in the rat gubernaculum, possibly to steer cells away from a defined apoptotic pathway. Greater apoptosis occurs earlier in development. However, in vivo CGRP released from the genitofemoral nerve may be required to prevent apoptosis, as shown by pretreatment with the sensory nerve toxin capsaicin. Androgen is also involved in the pathway controlling apoptosis, as androgen blockade with flutamide inhibited the action of CGRP.

An individualized approach to sustained inflation duration at birth improves outcomes in newborn preterm lambs

A sustained first inflation (SI) at birth may aid lung liquid clearance and aeration, but the impact of SI duration relative to the volume-response of the lung is poorly understood. We compared three SI strategies: 1) variable duration defined by attaining volume equilibrium using real-time electrical impedance tomography (EIT; SIplat); 2) 30 s beyond equilibrium (SIlong); 3) short 30-s SI (SI30); and 4) positive pressure ventilation without SI (no-SI) on spatiotemporal aeration and ventilation (EIT), gas exchange, lung mechanics, and regional early markers of injury in preterm lambs. Fifty-nine fetal-instrumented lambs were ventilated for 60 min after applying the allocated first inflation strategy. At study completion molecular and histological markers of lung injury were analyzed. The time to SI volume equilibrium, and resultant volume, were highly variable; mean (SD) 55 (34) s, coefficient of variability 59%. SIplat and SIlong resulted in better lung mechanics, gas exchange and lower ventilator settings than both no-SI and SI30. At 60 min, alveolar-arterial difference in oxygen was a mean (95% confidence interval) 130 (13, 249) higher in SI30 vs. SIlong group (two-way ANOVA). These differences were due to better spatiotemporal aeration and tidal ventilation, although all groups showed redistribution of aeration towards the nondependent lung by 60 min. Histological lung injury scores mirrored spatiotemporal change in aeration and were greatest in SI30 group (P < 0.01, Kruskal-Wallis test). An individualized volume-response approach to SI was effective in optimizing aeration, homogeneous tidal ventilation, and respiratory outcomes, while an inadequate SI duration had no benefit over positive pressure ventilation alone.