Intestinal Perfusion in the Murine Embryonic Gastrointestinal Tract Precedes Neural Development

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS to prevent excessive BMP signal transduction through dephosphory lation and subsequent nuclear exclusion of Smad1/5/8. Our results not only gain insights into the important role of BMP signaling in hu man growth and development, but also are pertinent towards novel therapeutic approaches for the prevention and treatment of human diseases.

17.3. Murine Tissue-Engineered Small Intestine Demonstrates Intact Tight and Adherens Junctions. F. G. Sala, J. A. Matthews, A. L. Speer, T. C. Grikscheit; Childrens Hospital Los Angeles, Los Angeles, CA Objective: To evaluate the integrity of the intestinal barrier in tis sue engineered small intestine. Introduction: Increased perme ability of the intestinal barrier in premature infants has been associated with various diseases including necrotizing enterocolitis (NEC). NEC leads to short bowel syndrome and affected children suffer severe morbidity and mortality. Current therapies do not of

fer adequate palliation. In previous work, our laboratory demon strated that tissue engineered small intestine could be used to rescue the nutritional status of Lewis rats after massive small bowel resection. After transitioning to the mouse model, we demon strated that the tissue engineered small intestine successfully re capitulates the architecture of the native small intestine including a fully differentiated epithelium, features of stem cell niche and a muscularis. In this study, we investigated the presence of tight junctions and adherens junctions in the tissue engineered small intestine, a critical component for future human therapy. Methods: Small intestines from 1 week old mouse pups were har vested. Organoid units were isolated by a protocol previously estab lished and validated in our laboratory and loaded onto biodegradable polyglycolic acid/poly L lactic acid scaffold tubes. The constructs were then implanted into the omentum of adult NOD/SCID mice. Four weeks later, all implants were harvested and analyzed with histology and immunohistochemistry. Native murine small intestine was used as positive control. Results: Tis sue engineered small intestine stained identically to native small intestine, and was positive for markers of both tight junctions (Claudin5, Occludin and ZO 1) (Figures A F) and adherens junc tions (b catenin, E cadherin) at the apical and lateral sides of the epithelial cells (Figures G J). Both native and tissue engineered small intestine also contained Goblet cells and Paneth cells. Conclusion: Presence of key molecules composing both tight junctions and adherens junctions at the apical and lateral sides of the epithe lial cells in tissue engineered small intestine demonstrates the nor mal polarization of the epithelium. Moreover, identification in the tissue engineered small intestine of both the intrinsic barrier, which consists of the tight junctions and the adherens junctions, and the extrinsic barrier, which include the mucus layer generated by the Paneth cells and the Goblet cells, suggests proper integrity of the intestinal barrier necessary for normal intestinal function and prevention of bacterial translocation.

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17.4. Telomerase Null Mice Exhibit Attenuation of Alveolar Type 2 Cell Expansion Following Partial Pneumonectomy. S. Jackson, D. Warburton, B. Driscoll; Childrens Hospital Los Angeles, Los Angeles, CA Partial pneumonectomy (PNX) in the mouse is a valuable research tool for those investigating pulmonary development as well as lung injury and repair. While it is known that the injury model of PNX in mice results in compensatory lung growth, a truly mechanis tic elucidation of the biologic processes involved as well as the poten tial role of progenitor cell populations remains an area of active study. It has been demonstrated that telomerase, a ribonucleoprotein that stabalizes chromosomes in actively dividing and differentiating cells, is upregulated in alveolar type 2 cells (AEC2) following lung injury. AEC2 are epithelial progenitor cells critical for the maintai nance of epithelial integrity. We hypothesize that PNX in telome rase null mice, which exhibit acutely shortened telomeres, will demonstrate decreased proliferation of AEC2 and subsequent com promise of epithelial integrity leading to decreased overall survival. Following induction of anesthesia using inhalational isoflurane, ma ture C57BL (WT) and telomerase null (Terc / F3) mice did undergo resection of the left lung. 43% of terc / F3 mice survived surgery to 72 hrs, compared to 52% in the WT. While terc / F3 mice possess telomeres approximately one quarter the length of WT, F4 mice, with telomeres one fifth as long, did not survive the procedure, indi cating a potential correlation between telomere length and survival of PNX in the telomerase null model. At post surgery day (POD) 3, lungs from surviving mice were harvested and fixed for immunohis tochemistry identifying alveolar epithelial type 2 (AEC2) isolation. In WT mice, the number of AEC2 in the lung rose significantly at POD3 to 23.0% AEC2/total cells/microscopic field, versus 12.4% ob served in sham operated controls. This increase was significant (p 0.0067), a phenomenon not observed in terc / F3 lung, where an increase to 15.0% AEC2/total cells/microscopic field was not sig nificantly higher than the 10.6% observed in sham controls (p 0.3695). Thus, while WT mice show a greater ability to survive PNX, terc / F3 mice exhibit a significantly poorer response. The marked inability of terc / mice to respond to injury is evidenced by decreased animal survival, as well as the attenuated AEC2 re sponse to PNX. These data, along with the observation that terc / F3 lung possesses a markedly lower basal level of AEC2 than WT, suggest that AEC2 play an active role in lung repair and regenera tion following partial pneumonectomy.

17.5. Intestinal Perfusion in the Murine Embryonic Gastrointestinal Tract Precedes Neural Development. J. L. Paredes, L. Li, K. A. Miller; Childrens Hospital Univesity of Pittsburgh, Pittsburgh, PA Introduction: Development of the enteric nervous system involves a complex migration of enteric neural crest cells (ENCCs) over the length of the gut in a predominant craniocaudal manner. Recent studies involving the avian intestinal neurovasculature development have identified endothelial cells prior to the arrival of migrating ENCCs and have shown that endothelial cells may promote migra tion of ENCCs.1 The spatiotemporal development of intestinal blood flow (perfusion) to the migration and patterning of the ENCCs is un clear and its impact is unknown. Methods: In order to study embry onal intestinal perfusion, we have developed an in vivo method of embryonic intracardiac microinjection of FITC labeled tomato lectin which labels perfused blood vessels of the gastrointestinal tract. Time induced pregnant mice carrying embryos from E11 E15 were anesthetized, and then underwent laparotomy with delivery of indi vidual gravid uterine horns into a customized petri dish filled with warm saline. Viable embryos were then evaluated by ultrasonogra phy and under ultrasound guidance, the embryonic heart was micro injected and FITC labeled tomato lectin was delivered. The

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS

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Levels of p65, p50, and IkB subunits of nuclear factor kappa light chain enhancer of activated B cells (NF kB), phosphorylation of c Jun N terminal kinases (JNK) and extracellular response kinases (ERK) were determined by Western blotting. Results: OmpA+ ES in duced higher levels of apoptosis compared to OmpA ES, as demon strated by TUNEL assay. OmpA ES induced greater NF kB and MAPK activation than OmpA+ ES. Conclusions: OmpA expression is required for efficient induction of enterocyte apoptosis by ES. OmpA may promote apoptosis by inhibiting anti apoptotic signaling through NF kB and MAPK.

microinjector was then removed and the uterus was returned to the mother’s abdomen for 15 minutes to let the tomato lectin circulate within the embryo. After this time, embryos were removed from the uterine horns and the gastrointestinal tract was dissected and kept intact to maintain vascular anatomy and avoid disruption. A five day staining protocol of embryonal intestinal whole mount was per formed for FITC labeled tomato lectin (TL) to labeled the perfused in testinal vessels, platelet endothelial cell adhesion molecule (PECAM) for the staining of all vascular structures (perfused or nonperfused), and early pan neuronal marker, protein gene product 9.5 (PGP 9.5), to evaluate enteric neuronal development. Samples were then evalu ated under confocal microscopy. Results: See Figure 1. Embryonic vasculogenesis is present throughout the gut length at all embryonic stages studied as evidenced by the significant network of PECAM staining throughout the bowel wall. Segmental embryonic intestinal perfusion stems from the mesenteric vessels into the bowel wall and increases with embryonic age. Furthermore, there is increased con nectivity of the perfused vasculature to the PECAM positive enteric vascular network. Distal intestinal vasculogenesis and perfusion are present prior to the presence of enteric neurons based on the lack of PGP 9.5 in distal bowel segments with concomitant staining of PECAM and TL. Conclusions: Intestinal vasculogenesis and per fusion in the murine embryonic gastrointestinal tract proceeds en teric neuronal development. In addition to possible mesenchymal signals expressed by the intestinal vasculature, substances carried to the developing gut via intestinal perfusion may also influence en teric neuronal development and patterning. 1. Nagy, N, Mwizerwa, O, et al., Endothelial cells promote migration and proliferation of en teric neural crest cells via b1 integrin signaling. Dev. Biol. 2009; 330:263 272.

17.6. Outer Membrane Protein A Expression Is Required for Enterobacter Sakazakii (ES)-Mediated Enterocyte Apoptosis. M. L. Williams, E. A. Adams, R. Mittal, M. Petrosyan, A. Franklin, C. L. Vendryes, A. Grishin, H. R. Ford, P. Nemani; Childrens Hospital Los Angeles, Los Angeles, CA Background: Previous studies from our lab have shown that ES, the opportunistic pathogen associated with hospital outbreaks of necro tizing enterocolitis (NEC), induces apoptosis in IEC 6 enterocytes. Be cause the outer membrane protein A (OmpA) of ES is necessary for the efficient induction of experimental NEC by this pathogen, we hypoth esize that OmpA may be required for ES induced apoptosis. Purpose: To determine the role of OmpA in ES induced apoptosis in IEC 6 cells. Methods: IEC 6 cells grown to 80% confluence were infected for 0 6 h with OmpA+ or OmpA ES. Apoptotic cells were identified using the terminal transferase deoxyuridine nick end labeling (TUNEL) assay.

17.7. Intestinal Subepithelial Myofibroblasts Demonstrate Cox-2 Expression in Early Stages of Epithelial Proliferation in Tissue Engineered Small Intestine. A. L. Speer, F. G. Sala, J. A. Matthews, T. C. Grikscheit; Childrens Hospital Los Angeles, Los Angeles, CA Introduction: Children with short bowel syndrome suffer severe morbidity and mortality. Tissue engineered small intestine (TESI) may serve as a viable replacement. In previous work, we have gen erated TESI in Lewis rats and Yorkshire swine, and TESI success fully rescued a model of short bowel syndrome in Lewis rats. We recently transitioned to a mouse model in order to investigate the mechanisms by which TESI forms. Intestinal subepithelial myofibro blasts (ISEMFs) are a specific population of cells involved in intesti nal epithelial proliferation, differentiation, inflammation, and neoplasia. Many factors such as cytokines (IL 1 and IL 17) and bac terial products (LPS) induce COX 2 expression and prostaglandin (PG) synthesis in isolated ISEMFs. Of the two known COX isoforms, COX 1 is constitutively expressed in most tissues, while COX 2 is expressed predominantly during inflammation and neoplasia. Fur thermore, synthesized PGE2 activates ISEMFs to promote prolifer ation and migration of intestinal epithelial cells and endothelial cells. We hypothesized that ISEMFs expressing COX 2 play a key role in signaling intestinal epithelial cells to proliferate and migrate during the generation of TESI. Methods: Small intestines from 1 week old CD1 mice were dissected. Organoid units, multicellular clusters containing both epithelium and mesenchyme, were isolated by a protocol previously established and validated in our laboratory and loaded onto biodegradable polyglycolic acd/poly L lactic acid scaffold tubes. The constructs were then implanted into the omen tum of adult NOD/SCID mice. Seven days later, all implants were harvested and analyzed with histology and immunohistochemistry. Adult native small intestine served as controls. Results: COX 2 pos itive cells are identified adjacent to developing epithelium in TESI at 7 days after implantation, but as in native small intestine are not identified in areas where the epithelium has already formed. The ep ithelium is marked by cytokeratin (data not shown). These COX 2 positive cells costain for alpha smooth muscle actin (alpha SMA) and are negative for desmin, which confirm their identity as ISEMFs. In the control, ISEMFs stain positive for alpha SMA, neg ative for desmin, and negative for COX 2 while the muscularis stains positive for alpha SMA and desmin. Conclusions: ISEMFs play important roles in intestinal epithelial proliferation, differenti ation, inflammation, and neoplasia by secreting cytokines, growth factors and PGs. Our data suggest that ISEMFs expressing COX 2