M1850 Bile Reflux Induces Higher COX-2 Expression Than Mixed Acid and Bile Reflux in a Rat Model of Esophagitis

bursting pressures of the gastrotomy wounds or in the peak force or total energy needed to disrupt the abdominal wounds when comparing the results of treated group to those of the control group on POD7 or 21. In regards to abdominal wound collagen content, no differences were noted between the control and treated groups at either time point. Importantly, there were no abdominal wall dehiscence or clinical gastrotomy leaks in CpG treated group. The histologic healing assessment scores were similar between groups. Conclusions: Perioperative administration of CpG doesn't appear to have a negative impact on abdominal wall or gastric wound healing in mice. Although it is not possible to extrapolate these results directly to the human setting, these findings lend support to the concept of a human Phase I perioperative CpG study.

M1850 Bile Reflux Induces Higher COX-2 Expression Than Mixed Acid and Bile Reflux in a Rat Model of Esophagitis Reginald V. N. Lord, Stefan Oberg, Jeffrey H. Peters, Steven R. DeMeester, Jeffrey A. Hagen, Tom R. DeMeester Background and Aims: A rat model of severe esophagitis is induced by performing either an esophagoduodenostomy (ED, acid and non-acid/bile reflux) or esophagoduodenostomy with total gastrectomy (ED + TG, non-acid reflux only). Esophagitis is not found in control animals in which total gastrectomy with Roux-en-Y reconstruction is performed (TG+RY, no reflux). All three operations result in formation of well-differentiated tumors at the esophageal anastomosis. Consequently, the nature of these tumors is controversial. COX-2 is overexpressed in a stepwise manner in esophagitis, Barrett's esophagus, and esophageal adenocarcinoma. The aim of this study was to (1) compare esophageal cyclo-oxygenase 2 (COX-2) expression in the three operation models, and (2) characterize the tumors. Methods: COX-2 enzyme expression was measured in the distal esophageal mucosa of 60 rats, 20 in each operation group, using a polyclonal rabbit antibody. Tumor and normal tissues were examined for DNA content and apoptotic index using flow cytometry with immunofluorescent propidium iodide and annexin V labeling, and for protein expression using immunohistochemistry. Results: COX-2 expression was higher than controls in severe esophagitis in both ED and ED+TG arms but was significantly higher in the non-acid bile reflux ED+TG arm. The tumors were diploid in 5 of the 6 examined and the remaining animal had aneuploidy in both tumor and normal tissues. None of 12 tumors examined showed immunoreactivity with p53, DAS-1, c-erbB-2, CEA, chromogranin A, or synaptophysin antibodies. Conclusions: Injury severity, as measured by COX-2 overexpression, was significantly higher in a nonacid reflux environment in this animal model of esophagitis. The esophageal tumors that developed in this surgical model without exogenous carcinogen exposure were dissimilar to human esophageal adenocarcinomas.

M1848 Evaluation of the Cell Proliferation Expansion in the Columnar Mucosa of the Distal Esophagus in Patients with GERD: Immunohistochemical Analysis of Ki67 (MIB1) Antigen in Columnar Mucosa with and Without Intestinal Metaplasia Marcelo Binato, Renato B. Fagundes, Luise Meurer, Maria Isabel A. Edelweiss, Richard R. Gurski Introduction: Columnar mucosa without intestinal metaplasia in distal esophagus is considered to be the stage that precedes the metaplasia—dysplasia—adenocarcinoma sequence. In 20% of these cases, upper endoscopy and biopsy shall confirm diagnosis of intestinal metaplasia in subsequent tests. Histology alone cannot establish the percentage of patients that will develop intestinalization of columnar mucosa. Therefore, it is necessary to seek molecular markers capable of predicting progression to more severe stages of the disease. Objectives: Determine the proliferative activity of columnar mucosa in patients with gastroesophageal reflux subjected to digestive endoscopy and biopsy in the Unit of Digestive Endoscopy of Hospital Universitário de Santa Maria, between 2003 and 2006. Methods: The Ki67 antigen was assessed by immunohistochemical technique with use of monoclonal antibody Ki67 (MIB1) in the 1:400 dilution. Proliferative activity was determined by the Ki67 index in every proliferative compartment of the intestinal crypt (lower layer, middle layer and epithelial surface). The 62 patients who had columnar mucosa in the distal esophagus were divided into two groups: G1: 30 patients with columnar epithelium without intestinal metaplasia and G2: 32 patients with columnar epithelium with intestinal metaplasia. Results: In G1, proliferative activity of the Ki67 was limited to the lower layer of the gland in 83% of the patients, 3% showed positive reactivity up to the middle layer and 13% showed immunoreactivity up to the epithelial surface of the esophageal crypt. In G2, proliferative activity of the Ki67 was limited to the lower layer of the gland in 46.9% of the patients, 21.9% showed positive reactivity up to the middle layer and 31.2% showed immunoreactivity up to the epithelial surface. A significant increase in the prevalence of proliferative activity in the compartments above the lower layer of the gland in patients with metaplastic columnar mucosa was found (p<0.001). Conclusion: The expansion of cell proliferation was significantly associated to the process of intestinalization of the columnar mucosa in the distal esophagus. Therefore, the presence of proliferative activity above the lower layer of the intestinal crypt in patients with columnar mucosa may assist in the identification of patients more prone to malignant transformation of the gastric mucosa or intestinalization and, consequently, at a greater risk of disease progression (metaplasia— dysplasia—adenocarcinoma sequence). Keywords: Ki67 (MIB1). Barrett's esophagus. Columnar mucosa. Intestinal metaplasia.

M1851

BACKGROUND: The liver has an extremely effective regenerative capacity. There is some evidence, that a portosystemic shunt is beneficial for liver regeneration after extended resection. The aim of the study was to estimate, whether the regeneration is due to hypertrophy or hyperplasia of the remnant liver tissue and whether a portosystemic shunt is beneficial for liver regeneration. MATERIAL AND METHODS: An extended left hemihepatectomy (approximately 75% of liver volume) was performed in 6-8 weeks old domestic pigs (n=25, body weight 25-35 kg). In n=14 pigs a portosystemic H-shunt was inserted between the portal vein and the infrahepatic cava vein after extended resection. After surgery, animals were weaned from anesthesia and resumed oral feeding. Liver biopsies were histological examined before extended liver resection and weekly until the 3rd postoperative week. Liver regeneration was estimated by the liver volume, the size of the portal fields [mm2], the amount of hepatocytes per portal field and the amount of hepatocytes per mm2. RESULTS: Extended left resection was technically feasible in all animals with and without portosystemic shunt. Volume of the remnant right lateral segment reached 250% at the end of the first week after resection. The size of the portal fields increased significantly after resection (portal field [mm2]; animals with shunt before resection: 1.0±0.4, 1 week after resection: 1.8±0.9*; 3 weeks: 2.9±1.3*, *p<0.05; animals without shunt before resection: 1.1±0.3, 1 week after resection 1.8±0.7*; 3 weeks: 2.9±1.7*, *p<0.05). The number of hepatocytes in the portal fields increased significantly (hepatocytes/portal field, animals with shunt before resection: 3438±1281; 1 week after resection: 5831±3419*; 3 weeks: 8793±3690*; *p<0.05; animals without shunt before resection: 3053±1096, 1 week after resection: 4580±2007*; 3 weeks: 8014±4809*, *p<0.05). Interestingly there was no increase in hepatocytes/mm2 and there was no difference between animals with or without portosystemic shunt. CONCLUSION: After extended liver resection the restoration of the liver volume is accomplished by an extensive and significant hyperplasia of hepatocytes within the preexisting portal fields, indicated by increased portal fields and hepatocytes per portal field, but constant hepatocytes per mm2. However, there was no beneficial effect of a portosystemic shunt, regarding liver regeneration after extended liver resection.

M1849 The Pathogenesis of Barrett's Esophagus: the Combination of Acid and Bile Is Synergistic in the Induction of CDX2 and EGFR Activation in Esophageal Cells Nelly E. Avissar, Liana Toia, Yingchuan Hu, Alexi Matousek, Daniel Raymond, Carolyn E. Jones, Thomas J. Watson, Jeffrey H. Peters Clinical evidence strongly suggests that mixed reflux of duodenal and gastric content is associated with esophageal mucosal injury, particularly the development of Barrett's esophagus. Caudal homeobox 2 (CDX2), a transcription factor involved in normal intestinal development is thought to be the key factor responsible for the intestinal phenotype. We have previously shown that deoxycholic acid (DCA), at neutral pH induces CDX2 through transactivation of the epidermal growth factor receptor (EGFR). The aim of this study was to test the hypothesis that a combination of bile and acid pH is more potent in inducing CDX2 and activating the EGFR than either component alone. Methods: SEG-1 human esophageal adenocarcinoma cells were incubated with acid alone (pH 5), deoxycholic acid alone (100 or 300 µM), or their combination for up to 24 hours. CDX2 mRNA was determined by real-time PCR and EGFR site-specific tyrosine phosphorylation and receptor degradation were determined by Western blot analysis. Results: Neither acid (pH 5) nor the 100 µM DCA dose alone induced CDX2 mRNA. In contrast, there was a synergistic 55 fold increase in CDX2 mRNA expression with exposure to 100 µM DCA at pH5. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested, but in different time courses. Acid alone induced peak EGFR phosphorylation at 8h, DCA alone at 30 min and 8 hours and acid plus DCA at 1 hour. Interestingly, degradation of the EGF receptor occurred with the combination treatment but not with either acid or DCA alone, indicating differential transactivation pathways. The combination of pH 5 and 300 µM DCA resulted in significant cell death at all time points. Conclusion: The addition of acid markedly enhances bile salt induced activation of the transcription factor CDX2 and occurs coincident with activation of the epidermal growth factor receptor. CDX2 gene induction occurs at significantly lower concentrations of bile salt than in the absence of acid, and may be due to differential EGFR transactivation. This data provides further insight into the molecular pathogenesis of Barrett's columnar metaplasia and identifies molecular targets useful for diagnosis, risk assessment and therapeutic intervention.

M1852 Basics of Mouse Liver Anatomy from a Microsurgical Point of View Peter Studer, Daniel Sidler, Beat Gloor, Andre E. Dutly, Daniel Candinas, Daniel Inderbitzin Background: During the development of different mouse liver resection models for the study of hepatic regeneration and basic liver pathology a significant lack of relevant anatomical data of the mouse liver became obvious. It has been shown, however, that different mouse liver lobes exhibit different regenerative capacities (less for the caudate lobe). In order to create a mouse model with standardized resections allowing a meaningful study of adaptive and regenerative responses the following basic anatomical studies of the mouse liver were undertaken. Methods: In Balb/c mice (n=32, 18-26g) and Black 6 transgenic blue mice (n= 69, 19-27g) liver mass and individual liver lobe weights were determined. The detailed three-dimensional anatomy of the hepatic vascular system was studied with corrosion casts (n=8). For scanning electron microscopy (EM) the mouse liver was perfused with a Mercox solution. Casts were sputtered with gold (10 nm) and examined in a Philips XL 30 FEG scanning electron microscope. Results: When comparing liver weight (LW)/ bodyweight (BW) ratio significant strain specific differences were detected. In Balb/c mice liver weight (LW) was increasing up to a mouse body weight (BW) of 23.6g ± 1.6gr and decreasing thereafter (LW (g) = -0.0022*BW [g]2 + 0.1025*BW [g]).). In contrast the LW in the Black 6 transgenic mice showed a linear increase with increasing BW. In Balb/c mice vascular corrosion casts demonstrated a single vascular pedicle in the right superior, right inferior and caudate lobes. In contrast, a common pedicle was seen for medial and left lobes in 26%, explaining the necrosis of the left lobe after medial lobe resection in around 30% of

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SSAT Abstracts

SSAT Abstracts

Cellular Liver Regeneration After Extended Hepatic Resection in Pigs Ruth Ladurner, Martin Schenk, Frank Traub, Alfred Koenigsrainer, Jorg Glatzle