Thirty seven 7-week old male Sprague-Dawley rats (KIST, Daejun, Korea) were used for control (n=8) and experimental group (n=29). Throughout the experiment, all rats were housed in a controlled environment with 12 hr light/dark cycle and temperature of 22±2℃. After an acclimatization period of 1 week, 29 experimental rats were randomly divided into four groups in a time-course design and underwent an esophagoduodenostomy for inducing duodenal contents reflux for 10 (n=2), 20 (n=12), 30 (n=8) and 40 (n=7) weeks, respectively. Eight control rats underwent sham operation. At the end of the appropriate time period for each group, the control and experimental rats were killed with ether.

Solid food was withdrawn for 24 hr and water for 12 hr before surgery. Anesthesia was induced and maintained with isoflurane-air mixture. According to the Clark model, esophagoduodenal anastomosis was performed (Fig. 1) (20). In short, a midline laparotomy was performed, and the gastroesophageal junction was identified and mobilized while carefully preserving the vagus nerve. The gastroesophageal junction was ligated, and the distal esophagus was transected 2 mm above the ligature. A total of eight polypropylene 7-0 sutures were placed. Five mm transverse enterostomy was created on the antimesenteric border of duodenum 1 cm distal to pylorus. An end-to-side esophagoduodenstomy was performed. To compensate for blood loss, 1 mL of sterile 0.9% normal saline was instilled in the peritoneal cavity. The abdominal incision was closed in 2 layers and postoperatively the rats were allowed to drink water after 6 hr and were fed on the following day. Immediately after death, the entire esophagus, contiguous anastomotic site and 0.7 cm of duodenal mucosa were removed and the lumen was longitudinally opened (Fig. 2). After snap freezing, a representative sample of duodenum, and upper and lower esophagus was stored at -70℃ for reverse transcription-polymerase chain reaction (RT-PCR). The remaining esophagus including anastomotic site and 0.7 cm of duodenal mucosa was fixed in 10% neutral buffered formalin for 24 hr for histological study and immunohistochemistry.

For histologic evaluation, formalin-fixed tissues were embedded in paraffin, cut at 4 µm and stained with H&E. Two pathologists blinded to the experimental groups have assessed all tissues independently. Diagnosis of Barrett's esophagus was made by the presence of columnar metaplasia containing intestinal type goblet cells surrounded by squamous epithelium above the anastomotic site. In addition, Barrett's esophagus was divided into short and long one according to distance from anastomotic site; it was regarded as long one when the specialized intestinal type epithelium was present at a length of >0.5 cm above the anastomotic site. Atypical Barrett's esophagus was diagnosed when glands with atypical features were present both at the superficial and deep portions of the wall.

Serial sections of 4 µm thickness were made and spread on poly-L-lysine coated slides. Paraffin sections were immersed in three changes of xylene and hydrated using a graded series of alcohol. Antigen retrieval was performed routinely by immersing the sections in 0.01 M citrate buffer (pH 6.0) in a pressure cooker by autoclaving for 15 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min and then incubated with a primary antibody overnight in a humidified chamber at 4℃. Primary antibodies were polyclonal rabbit anti-iNOS (Transduction laboratories, Lexington, KY, U.S.A.) at a dilution of 1:300. Staining was achieved with a DAKO LSAB+kit (DAKO, Santa Barbara, CA, U.S.A.) and developed with diaminobenzidine tetrahydrochloride. Sections were counterstained for 5 min with Meyer's hematoxylin and then mounted. The hepatic tissue of rat in which lipopolysaccharide (Sigma Chemical Co., St. Louis, MO, U.S.A.) was intraperitoneally administered was used as a positive control. As a negative control, rabbit IgG (DAKO) was used instead of primary antibodies. An estimation of immunohistochemical expression of iNOS was evaluated according to both intensity and area of signal: 0, absent; 1, mild: 2, moderate; 3, severe.

Total RNA was extracted and purified from frozen tissues using GeneElute Mammalian Total RNA kit (Sigma Chemical Co.) acccording to manufacturer's instructions. The RNA was quantified by determining absorbance at 260 nm. Two µg of total RNA from each sample was reverse transcribed into cDNA using Superscript II reverse transcriptase (Life Technologies, Inc., Rockville, MD, U.S.A.) and random hexamer primers (Takara, Shiga, Japan). The PCR primers were as follows: glyceraldehydes 3-phosphate dehydrogenase (GAPDH) (311 bp), sense 5'-GAA CGG GAA GCT CAC TGG CAT GGC-3', antisense 5'-TGA GGT CCA CCA CCC TGT TGC TG-3'; iNOS (397 bp), sense 5'-CCA CAA TAG TAC AAT ACT ACT TGG-3', antisense 5'-ACG AGG TGT TCA GCG TGC TCC ACG-3'. After denaturation at 95℃ for 5 min, cDNA amplification was carried out by running 32 cycles of denaturation at 94℃ for 1 min, annealing at 58℃ for 45 sec and extension at 72℃ for 45 sec, followed by further incubation at 72℃ for 7 min. The PCR products were electrophoresed on 1.5% agarose gel containing ethidium bromide and then photographed under UV light.

The significance of differences between groups was evaluated by using chi-square test. Differences were considered to be statistically significant at p<0.05.