Aaron P. Garrison

Early but not late administration of glucagon-like peptide-2 following ileo-cecal resection augments putative intestinal stem cell expansion

Expansion of intestinal progenitors and putative stem cells (pISC) occurs early and transiently following ileo-cecal resection (ICR). The mechanism controlling this process is not defined. We hypothesized that glucagon-like peptide-2 (GLP-2) would augment jejunal pISC expansion only when administered to mice immediately after ICR. Since recent reports demonstrated increases in intestinal insulin-like growth factor (IGF)-I following GLP-2 administration, we further hypothesized that increased intestinal IGF-I expression would correlate with pISC expansion following ICR. To assess this, GLP-2 or vehicle was administered to mice either immediately after resection (early) or before tissue harvest 6 wk following ICR (late). Histological analysis quantified proliferation and intestinal morphometrics. Serum levels of GLP-2 were measured by ELISA and jejunal IGF-I mRNA by qRT-PCR. Expansion of jejunal pISC was assessed by fluorescent-activated cell sorting of side population cells, immunohistochemistry for phosphorylated beta-catenin at serine 552 (a pISC marker), percent of crypt fission, and total numbers of crypts per jejunal circumference. We found that early but not late GLP-2 treatment after ICR significantly augmented pISC expansion. Increases in jejunal IGF-I mRNA correlated temporally with early pISC expansion and effects of GLP-2. Early GLP-2 increased crypt fission and accelerated adaptive increases in crypt number and intestinal caliber. GLP-2 increased proliferation and intestinal morphometrics in all groups. This study shows that, in mice, GLP-2 promotes jejunal pISC expansion only in the period immediately following ICR. This is associated with increased IGF-I and accelerated adaptive increases in mucosal mass. These data provide clinical rationale relevant to the optimal timing of GLP-2 in patients with intestinal failure.

Expansion of Intestinal Epithelial Stem Cells during Murine Development

Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

Intestinal Stem Cells

The epithelial cell lining of the gastrointestinal tract is the most rapidly proliferating tissue in the body. The constant state of renewal of differentiated epithelial cells is sustained by a continual supply of progeny from multipotent progenitors that originate from stem cells located within the intestinal crypts. In addition to supporting normal epithelial homeostasis, intestinal stem cells (ISC) are thought to play an important role in the rapid expansion of the gut during development, tissue regeneration following injury or surgical loss, and malignancy. Because of the lack of specific ISC markers required to isolate and characterize these cells, our current knowledge of the biology of ISC results largely from indirect measures of their behavior published during the past 40 years. The recent description of several potential ISC markers and the use of transgenic mice, both as a tool to lineage trace and to isolate specific cells expressing these markers, have provided a tremendous advancement to our current understanding of these cells. This brief review provides a general historical overview of our understanding of ISC and the tools available to study their behavior in the context of normal and pathological conditions, as well as potential future clinical applications that may result from this exciting area of research.

Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

BACKGROUND Bile acid (BA) reclamation following ileo-cecal resection (ICR) may prevent colonic mucosa from chronic injury. In this study, we hypothesized that in a murine model of ICR the remnant colon would upregulate the cellular machinery necessary for BA reclamation and would do so in an FXR- and bacteria-dependent manner. METHODS Conventional (WT), conventional FXR knockout (FXR null) and germ-free (GF) mice were randomized to undergo either ICR or sham operation. The ascending colon was harvested for histology and immunohistochemistry and changes in bile acid homeostatic gene expression determined by real-time polymerase chain reaction (RT-PCR) 7 days following surgery. RESULTS Following ICR WT mice showed significant increases in the expression of genes regulating bile acid transport including IBABP, Asbt, Ost beta and FGF 15. Increased expression of IBABP and Asbt was confirmed by immunohistochemistry. Induction of bile acid transport genes was absent or attenuated in FXR null and GF mice. CONCLUSION Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15.

Inflammation Enhances Resection-Induced Intestinal Adaptive Growth in IL-10 Null Mice

BACKGROUND Surgical resection of the ileum, cecum, and proximal right colon (ICR) is common in the management of Crohns disease, yet little is known about the effect of active inflammation on the adaptive response following intestinal loss. We recently developed a surgical model of ICR in germ-free (GF) IL-10 null mice that develop small intestinal inflammation only when mice undergo conventionalization with normal fecal microflora (CONV) before surgical intervention. In this study, we examined the effects of postsurgical small bowel inflammation on adaptive growth after ICR. METHODS GF 129SvEv IL-10 null mice, 8-10 wk old, were allocated to GF or CONV groups. Nonoperated GF and CONV mice provided baseline controls. Two wk later, GF and CONV mice were further allocated to ICR or sham operation. Small intestine and colon were harvested 7 d after surgery for histological analysis. RESULTS All mice within the gnotobiotic facility maintained GF status and did not develop small intestinal or colonic inflammation. CONV resulted in colitis in all groups, whereas small intestinal inflammation was only observed following ICR. Resection-induced small intestinal inflammation in CONV mice was associated with increases in proliferation, crypt depth, and villus height compared with GF mice after ICR. Resection-induced increases in crypt fission only occurred in CONV mice. CONCLUSION ICR-dependent small intestinal inflammation in CONV IL-10 null mice dramatically enhances early adaptive growth of the small intestine. Additional studies utilizing our model may provide clinical insight leading to optimal therapies in managing IBD patients after surgical resection.

Traumatic rupture of a choledochal cyst masking as a duodenal hematoma

Choledochal cysts are rare, congenital malformations of the intra and/or extrahepatic biliary tree. We describe a case in which a patient was transferred to our hospital with a reported duodenal hematoma. The patient ultimately required exploration when his condition deteriorated. Laparotomy resulted in the discovery and successful treatment of a ruptured type IV-A choledochal cyst.