YALE JOURNAL OF BIOLOGY AND MEDICINE 69 (1996), pp. 105-118. Copyright © 1997. All rights reserved. Epithelial Response of the Rat Gastric Mucosa to Chronic Superficial Injury Eric R. Lacya,b, Kathryn S. Cowartb, Jan S. Kingb, John DelVallec, and Adam J. Smolkad Departments ofbCell Biology and Anatomy, and dMedicine, Medical University of South Carolina, Charleston, South Carolina and cDepartment ofInternal Medicine, University ofMichigan Medical Center, Ann Arbor, Michigan (Received March 5, 1996; accepted July 15, 1996) Chronic injury to the healthy gastric mucosa with noxious agents such as aspirin or alcohol induces a progressive strengthening of the stomach wall against these insults. The present study examined the histologic response of the rat gastric mucosa to chronic destruction of the superficial mucosa for one month with hypertonic saline. The number, position and morphology of proliferating, pari- etal, G and D cells were followed during mucosal injury and one month of recovery. The results showed that chronic injury reduced parietal cell numbers by about 30 percent, particularly in the middle of the mucosal thickness where a clear zone was formed by hypertrophy of mucous neck-like cells. G cells were also reduced by about 50 percent, but there were no changes in D cells. Chronic injury induced a marked increase in the number of antral (+1 12 percent) and fundic (+250 percent) proliferating cells. Conclusion: The rat gastric mucosa responds to chronic superficial injury by down-regulation of acid secretory cells and gastrin secreting cells and an up-regulation of proliferating cells. The appearance of a prominent layer of mucous neck-like cells may indicate a new secretory function for these cells. INTRODUCTION Circumstantial evidence from patients ingesting noxious agents such as non-steroidal anti-inflammatory drugs and various mixtures of ethanol indicates that chronic insult to the gastric mucosa may be an important factor in the development of some gastropathies [1]. Nevertheless, most experimental studies of gastric damage and repair utilize a single, large dose of a strong necrotizing agent such as absolute ethanol to produce gross lesions in the stomach. These experimental protocols clearly have advantages for screening poten- tial gastro-protective drugs and to monitor the sequelae of healing events, but they proba- bly have little clinical relevance [2]. Relatively few experimental studies have monitored the progression of gastric mucos- al responses after successive doses of an orally administered damaging agent [3-12]. Surprisingly, repeated exposure to a strong necrotizing agent such as 80 percent ethanol induces a progressive mucosal resistance despite the agent's initial production of hemor- rhagic lesions. The mucosa also becomes progressively protected against the effects of strong necrotizing agents when it is chronically insulted with a mild damaging agent which destroys only the superficial epithelium. Very little is known about the mechanisms by which augmented mucosal protection is acquired under these conditions. A previous study showed that chronic superficial insult resulted in rapid re-establishment of the aTo whom all correspondence should be addressed: Eric R. Lacy, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. Tel: (803) 792-3549; Fax: (803) 792-0664; E-mail: [email protected]. eAbbreviations: PBS, phosphate buffered saline; BrdU, 5'-bromo-2'-deoxyuridine. 105 106 Lacy et al.: Chronic gastric injury mucosal surface epithelium and changes in the composition of antral mucus, but other his- tological responses to the repeated injury are unknown [12]. With this in mind, the present study utilized a clinically relevant model of chronically insulted gastric mucosa to exam- ine potential responses in the number, position and morphology of selected epithelial cells. Histochemical and immunohistochemical techniques were used to localize parietal cells, proliferating cells, and cells containing gastrin (G cells), and somatostatin (D cells) in the fundic and antral mucosa of rats subjected to chronic superficial mucosal injury for one month followed by one month of recovery. The results showed that chronic injury induced significant changes in the number and position of these cells, which may reflect the development of mucosal protection. MATERIALS AND METHODS Male Sprague-Dawley rats (150-250 g, Charles River Breeding Laboratories, Wilmington, DE) were fed standard laboratory chow and kept on a 12-hr light/dark cycle. Except for a 30-min dosing period, all rats were allowed water ad libitum. The experimental group of rats was placed on a four week regimen of chronic intra- gastric dosing with hypertonic saline followed by four weeks without dosing. Previous studies had shown that 2 M NaCl destroyed the superficial gastric epithelium without inducing hemorrhagic lesions [12]. Each rat received a single bolus (two to three ml) of 2 M NaCl orogastrically every 48 hours between 1:00 and 2:30 p.m. During this part of the animals' diurnal cycle they were somnolent and their stomachs nearly void of food, which allowed direct access of saline to the gastric mucosal surface. After dosing, the rats were returned to their cages and deprived of food and water for 30 min followed by free access to both. During the four-week recovery period when rats were not dosed, they were given free access to both food and water and were handled in the same manner as the untreated control group. The two control groups of rats received either physiological saline (0.9 percent) in the same manner as the experimental rats or were left untreated. Rats were sacrificed at two and four weeks after the initiation of the dosing regimen and four weeks after its termination. At the termination of each segment of the experiment, rats were anesthetized with ether, the stomachs were exposed by laparotomy, and the gas- troesophageal and gastroduodenal junctions were ligated taking care not to occlude blood vessels and nerves. The nonglandular stomach was pierced with a 26-gauge needle, and three to four ml of either Carnoy's or Bouin's fixative was injected to gently inflate the gastric wall. Optimal immunohistochemistry for light microscopic identification of pro- liferating cells and parietal cells was achieved using Carnoy's fixative. Bouin's fixative was optimal for immunohistochemical localization of G and D cells. The same fixative as used for intragastric inflation also was poured over the external stomach surface and the preparation left in situ. After 20 min, the animals were killed and the ligated stomach was excised and submersed in fixative for an additional one to three hr at room temperature. The stomach was hemisected along the greater and lesser curvatures and full thickness pieces of the gastric wall were excised with a razor blade. Two strips of fundus (1.5-2.0 x 0.5 cm), each from the greater to lesser curvature adjacent to the limiting ridge, and one piece (0.5 x 0.5 cm) of antrum were excised. These tissues were processed for light microscopy as previously described [12] or for routine histochemistry and immunochem- istry as described below. All tissue samples were embedded in paraffin, sections cut five micrometers thick, placed on glass slides, deparaffinized and rehydrated using standard techniques. Some sections were stained with hematoxylin and eosin or Masson's Trichrome for his- tochemistry. Other sections were prepared for immunohistochemistry by pretreatment for five min in three percent H202 to eliminate endogenous peroxidase activity, then rinsed in phosphate buffered saline (PBS)e. Exposure of tissue sections to the four different antisera Lacy et al.: Chronic gastric injury 107 listed below was perforned at room temperature (except where specified) in a sealed, mois- turized chamber. Proliferating cells were labeled with antisera to 5'-bromo-2'-deoxyuridine (BrdU), which had been injected into rats for one hr according to previously published techniques [13]. Parietal cells were localized using a monoclonal antibody directed against H+/K+- ATPase [14]. This antibody was made by standard hybridoma techniques using as antigen pig gastric microsomal vesicles highly enriched in H+/K+-ATPase. Immunoblot analysis shows antibody specificity for the a subunit of H+/K+-ATPase, with an apparent molecu- lar mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 94 kDa (not shown). The antibody stains only parietal cells in pig, rabbit, rat, human, dog, guinea pig, and gerbil gastric mucosa. In these cells, tubulovesicular and secretory canalicular mem- branes are the site of antibody reactivity. Sections were preincubated in one percent bovine serum albumin (BSA) for 30 min at room temperature then incubated in H,K- ATPase monoclonal antibody at a 1:100 dilution in PBS overnight at 40C. The slides were rinsed in PBS then incubated in peroxidase-labeled goat anti-mouse IgG at a 1:100 dilu- tion in PBS for 20 minutes. Gastrin (G) cells were localized using specific antisera according to previously pub- lished techniques [15]. D cells were localized using a previously described sheep anti-somatostatin antiserum (8093) generated against somatostatin-14 [16]. The antiserum cross-reacts with both somatostatin-14 and somatostatin-28 and does not cross-react with gastrin, glucagon bombesin, pancreatic polypeptide, O-endorphin or neurotensin. Sections were preincubat- ed in a 1:100 dilution of blocking serum (normal rabbit serum) in PBS for 30 min then incubated overnight at 40C in a 1:1000 antiserum dilution in PBS. The sections were rinsed in PBS then incubated in a 1:50 dilution in PBS of rabbit anti-sheep IgG (secondary antibody) for 20 min, rinsed in PBS, then incubated in a 1:100 dilution of sheep peroxi- dase-antiperoxidase in PBS for 20 min. The final step for each of the four antiserum procedures described above was incuba- tion of the tissue sections in a 0.04 percent solution of 3.3 diaminobenzidine tetrahy- drochloride in Tris buffer, pH 7.3, to which 33 ml of 3.0 percent H202 was addedjust prior to use.