Adaptation of the Gastric Mucosa to Stress Role of Prostaglandin and Epidermal Growth Factor S. J . KONTUREK, T. BRZOZOWSKI, J . MAJKA, D. DROZDOWICZ & J. STACHURA Institute of Physiology and Dept. of Cell Biology, University School of Medicine, Cracow, Poland
Konturek SJ, Brzozowski T, Majka J , Drozdowicz D, Stachura J. Adaptation of the gastric mucosa to stress. Role of prostaglandin and epidermal growth factor. Scand J Gastroenterol 1992;27 Suppl193:39-
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This study was designed to determine whether repeated exposures to stress lead to the adaptation of the gastric mucosa to stress ulcerogenesis. Wistar rats with intact or resected salivary glands were exposed to a standard period (3.5 h) of water-immersion and restraint stress every other day up to 8 days. The significant reduction in the severity of gastric lesions was first noticed after the second exposure to stress and was maximal after 6-day exposures to stress. This tolerance to stress ulcerogenesis disappeared after a 6-day rest during which animals were not exposed to stress. Histologically, the hemorrhages and edema seen after a single stress were less frequent during adaptation; instead the mucosa regenerated in spite of continuation of exposure to stress. During adaptation, the mucosal blood flow (MBF) and mucosal biosynthesis of PG were markedly increased. Administration of indomethacin ( 5 mg/kg i.p.) completely abolished gastric adaptation to stress and this was accompanied by about 85% reduction in mucosal generation of PG and signficant decrease in the MBF. Salivectomy, which significantly reduced the luminal contents of epidermal growth factor (EGF) in the stomach, delayed and reduced the adaptation. We conclude that the stomach has the ability to adapt to repeated exposures to stress and that this adaptation is mediated, at least in part, by endogenous PG and EGF. Key words: Adaptation; epidermal growth factor; gastric lesions; mucosal blood flow; prostaglandins Prof. D r . S. J . Konturek, Institute of Physiology, University School of Medicine, PL-31-531 Krakow, ul. Grzegorzecka 16, Poland
Gastric stress ulceration is a highly prevalent disease that arises as a complication following burns, sepsis, major surgery, trauma to the control nervous sywstem etc. (1). Among various stresses used in animals, the most reproducible results can be obtained by restraint plus water immersion or cold, which appear to act synergistically in the production of gastric ulcerations (1-3). The mechanism of stress ulcerations has not been fully clarified and remains controversial more than 60 years after their original description by Selye (4).The major factors implicated in the development of stress ulcers include the increase in gastric acid secretion, the decrease in mucosal protection due to the reduction in mucus-alkaline secretion, the attenuation of the mucosal blood flow and prostaglandin (PG) biosynthesis and the fall in DNA synthesis and cell proliferation in the mucosa (5-12). During the past few years it has become evident that the stomach can defend itself from irritating and ulcerogenic agents such as nonsteroidal anti-inflammatory drugs (NSAID) (13-16) or necrotizing substances (17). The ability of the gastric mucosa to withstand the damage by repeated application of gastric ulcerogens was called gastric adaptation (13-17) but the mechanism of this phenomenon has not been explained. No information is available whether repeated exposures to stress results in the development of any tolerance to stress ulcerogenesis and if so what could be the mechanism of the adaptation to stress.
In the present studies, we used as a model the formation of acute gastric erosions produced in fasted rats by the waterimmersion and restraint stress (18). We investigated whether repeated exposures to stress insults would result in adaptation or aggravation to the ulcerogenic effect of stress in rats with intact or resected salivary glands.
MATERIALS AND METHODS Male Wistar rats of an average body weight of 200g and fasted 18 h were used. The animals were divided into three major groups, group A for the studies on the development and duration of gastric adaptation, group B for the studies on the implication of endogenous PG in gastric adaptation and group C for the studies on the role of salivary glands and epidermal growth factor (EGF) in gastric adaptation. The animals of the latter group had sublingual-submandibular gland complexes removed under ether anesthesia. The salivectomized rats, were allowed 7-10 days of full recovery following the operation. In most experiments, 3.5 h of stress was applied by placing animals into stress cages causing immobilization and immersing in 23°C water to the rat’s xyphoid process as described previously (18). A period of 3.5 h of stress was chosen because in preliminary studies this stress period was sufficient to produce numerous gastric erosions and induced gastric
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Fig. 1. The number of gastric lesions induced by single exposure ('Once') to the water immersion and restraint stress for 3.5 or 7.0h or repeated every other day for up to 8 days. Means 5 SEM of 8-10 rats per group. Asterisk indicates significant decrease below the value obtained after stress applied once.
adaptation. The extension of stress to 7.0h resulted in greater number of erosions and more severe bleeding with poor recovery after each successive exposure to stress. Immediately after the stress, the animals of groups B were lightly anesthetized with ether, the abdomen was opened, the stomach exposed and the mucosal blood flow (MBF) was measured in the oxyntic gland area using H2 gas clearance technique described previously (19). Briefly, the double needle electrodes were inserted into the mucosa, one electrode was used for local generation of H2-gas and the other, for the measurement of tissue H2. With this method the H2 generated by water hydrolysis is carried away by the blood and the polarographic current detector gives the decreasing tissue H2 as the clearance curve which is used to calculate absolute flow rate (ml/min-100 g) in the tissue. After the measurement of MBF, the stomach was ligated at the cardia and the pylorus and 1 ml of cold saline was instilled into the stomach to wash out the gastric content for the determination of luminal content of epidermal growth factor (EGF). Then, the stomach was dissected out and opened along the greater curvature. The stomach was then examined with a 2x binocular magnifier for the presence of erosions by someone unaware of the treatment given. These lesions appeared as small round or linear hemorrhagic erosions occurring in the oxyntic gland area. The erosions were counted and the average number per stomach was calculated for each group. In animals of group A, the biopsy of the mocosa of the oxyntic gland area was obtained and used for the determination of tissue generation of PGE2 as described previously (12). In some experiments, the stomachs were fixed in 10% buffered formalin, and 3-pm
sections were stained with hematoxylin-eosin for histological evaluation. Several series of animals were used; in group A, one subgroup was sacrificed without stress and was used as a control for the measurement of EGF content in the gastric lumen and for the determination of mucosal PGE2 and MBF. Other subgroups (consisting of 8-10 rats each) were stressed for 3.5 or 7.0 h at 1100 h and used for the studies on gastric adaptation. One of these subgroups was stressed once (for 3.5 or 7.0 h) and after this single exposure to stress it was sacrificed. Another group was treated similarly, except that after stress they were removed from the stress cages, placed in a room temperature and refed until 1400 h next day. At that time, the animals were again fasted overnight and stress was repeated next morning. The animals were sacrificed after this second treatment with 3.5 or 7.0 h of water-immersion and restraint stress. Other groups underwent the same schedule of stress, refeeding and refasting for 4, 6 or 8 consecutive days. In all rats the MBF was determined and the stomachs were examined for gastric erosions. In studies on the duration of gastric adaptation after cessation of stress, rats were exposed to stress for 3.5 h every second day for 4 times in order to produce gastric adaptation. Following this 6 day treatment with stress, the animals were fed and remained without treatment for periods of 2, 4,6, 8, 10 or 12 days. At all these time intervals, they were fasted overnight and on the following day morning they were stressed by challenging 3.5 h period of water immersion and restraint. They were anaesthetized after the challenging treatment and examined for the MBF and the gastric erosions.
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Gastric Adaptation to Stress
In group B, the rats were subdivided into subgroups of 810 animals and stressed for 3.5 h at the same periods as in group A. In addition each animal received daily injection of either vehicle (saline i.p.) or indomethacin (5 mg/kg i.p.). After the stress, the rats were anaesthetized with ether, the abdomen was opened to measure MBF and then the number of gastric erosions was counted. Finally, the biopsy samples of the mucosa from the oxyntic gland area were taken for the determination of mucosal generation of PGEz as described before (12). In group C (with prior salivectomy), the animals were also subdivided in subgroups of 8-10 rats and stressed for 3.5 h at the same time periods as in group A they were sacrificed after first, second, third, fourth or fifth stress-treatment. The animals were anesthetized, the stomach exposed to measure the MBF, luminal contents were collected to measure the contents of EGF by radioimmunoassay as described previously (20). Briefly, the mucosal samples were weighed and homogenized in ice-cold 0.02 mM/l Tris-HCI buffer and centrifuged, the supernatant being collected and frozen at 20°C until EGF radioimmunoassay . The EGF antiserum raised in rabbits against human EGF was used in a final dilution of 1:210,000. Iodinated ([3-1251]iodotyrosyl)peptide and rat EGF were calibration standards (Amersham, England). The detection limit of the assay was 0.01 nM/I. The interassay and intra-assay precisions were about 12% and lo%, respectively. The results are reported as means SEM. Statistical significance was determined by analysis of variance and where appropriate by the unpaired Student’s t test, a value of less than 0.05 being considered significant.
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RESULTS Production of gastric erosions The water immersion and restraint stress applied once for 7.5 h produced numerous small erosions (about 29 m 2 per stomach) with severe bleeding. Similar exposure to stress for 3.5 h resulted in about 30% smaller number of erosions per stomach and less severe bleeding (Fig. 1). During the repeated exposures of stress for 7.5 h , the number of gastric lesions was similar but the reduction in the body weight of animals was observed and about 30% of rats died after 3rd or 4th stress exposure. In contrast, with repeated exposures to 3.5 h stress, a progressive decrease in the number of erosions was observed from 25% after first challenge to 53% after the fourth challenge. Thus, gastric adaptation is triggered by a single treatment with stress but becomes maximal after 6 days of repeated exposures to stress.
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Duration of gastric adaptation Fig. 2 shows the number of erosions per stomach after 6day exposures to stress in rats that were refed and were not stressed for varous time intervals (2-12 days). After each interval of rest, they were re-challenged with 3.5 h stress
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following an overnight fast. When the animals were rechallenged with stress at 2nd, 4th and 6th day, there was a gradual increase in the number of erosions per stomach reaching after 8 days the value not sitgnificantly different from that occurring after single treatment (‘Once’). Thus, the gastric adaptation to stress induced by a 6-day of repeated exposure to repeated stress persisted for up to 6 days after discontinuation of this stress. Histological evaluations A single exposure to 3.5 h stress produced deep and extending to the muscular is mucosae erosions in the oxyntic mucosa (Fig. 3). Portions of the mucosa were necrotic given rise to craters. The submucosa was edematous but without polymorphonuclear infiltration. After 6 days of exposures to stress every second day only few new erosions were observed. The mucosa appeared thicker and only occasional craters lined with a monolayer of newly formed surface epithelial cells were noticed (Fig. 4). In addition, mucosal scars of healed erosions were observed. The submucosa still showed the edema accompanied by minor inflammatory infiltrate in some rats. Gastric mucosal blood flow ( M B F ) The MBF in the mucosa of the oxyntic portion of the stomach of intact rats averaged about 51 2 6 ml/min-100 g. After a single exposure of group B to the 3.5 h stress, the MBF was reduced by about 43%. When the mucosa was adapted to repeated exposures to the stress, there was a gradual increase in the MBF reaching after the first challenge with stress about 80% of the value in the intact mucosa and attaining after the second challenge with stress the value not significantly different from that recorded in the intact mucosa. In rats injected daily with indomethacin (5 mg/kg i.p.), the adaptation of the gastric mucosa to stress completely disappeared and this was accompanied by a significant reduction in gastric MBF in these animals (Fig. 5). Effects of salivectomy and EGF on the adaptation of gastric mucosa to stress Following salivectomy, the number of gastric lesions in response to single exposure to stress showed a small but significant increase (by about 27%) (Fig. 6). After repeated exposure to stress every second day, the number of lesions per stomach was 33-50% higher than these in the corresponding group of rats with intact salivary glands. After 3rd and 4th consecutive exposure to stress in salivectomized rats, the number of lesions was significantly lower than that in rats after a single stress but it was still higher (by about 50%) than that in rats with intact salivary glands.
DISCUSSION The present results show that upon repeated exposures to stress, the rat gastric mucosa undergoes progressive adap-
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Fig. 2. Duration of gastric adaptation after cessation of exposure to stress. Animals were first exposed to repeated stress for 6 days and thereafter were without stress for various periods of time from 2-12 days after which were rechallenged with stress. Means 2 SEM of 8-10 rats. Asterisk indicates significant decrease below the value obtained after single exposure to stress (‘Once’).
Fig. 3. Acute stress erosion. Focal mucosal necrosis penetrating 3/4 of the mucosal thickness. In the ~ right bottom corner edematous submucosa. (H & E; magnification, 2 6 0 .)
tation to stress ulcerogenesis. This adaptation vanishes when the challenging stress is applied 6 days after the adaptation was induced by repeated exposures to stress. Previous studies (21) demonstrated that ‘mild’ stress such as 1 h immobilization is capable of preventing the formation of acute gastric lesions induced by 3 h of cold-restraint stress or by 100% ethanol. This adaptive gastroprotection by mild stress was found to disappear after the pretreatment with
indomethacin suggesting that endogenous PG may be implicated in this this phenomenon. Our present results indicate that the exposure to repeated stress insults greatly increases the tolerance of the gastric mucosa to stress-induced ulcerogenesis. This tolerance is triggered by a single exposure to stress and becomes maximal after 6 days. Histologically, the numerous necrotic hemorrhages and vasocongestion of submucosal edema
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Gastric Adaptation to Stress
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Fig. 4. Mucosal scar. Depression of the mucosa in the mucosa is reepithelized with surface epithelium (H & E; magnification, 260x .)
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Fig. 5 . The number of gastric lesions and the mucosal blood flow (MBF) in rats after single exposure to stress (‘Once’) and then after repeated exposures at 2nd, 4th. 6th and 8th day in rats with or without pretreatment with indomethacin ( 5 mg/kg i.p. daily). Asterisk indicates significant change as compared to the values obtained in rats exposed to single stress (‘Once’). Cross indicates significant change as compared to the values obtained in rats without administration of indomethacin.
observed after the single exposure to stress, appeared less frequently with the development of adaptation. The granulation tissue was seen in the areas that had become necrotic earlier and there was a proliferation of mucus cells lining as a monolayer at the sides of steep craters. These histological
data indicate that the process of active repair of earlier necrotic lesions accompanied by the active regeneration of gastric glands occur during the mucosal adaptation to stress. The mechanism of gastric adaptation to the stress is unknown but we tested several hypotheses to explain this
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S. J. Konturek et al.
DURATION (Day#) OF TREAT’MlCNT Fig. 6. The number of gastric lesions and luminal contents of EGF after single exposure to stress (‘Once’) and then after repeated exposures to stress at 2nd, 4th, 6th and 8th day in rats sham-operated with intact salivary glands and after salivectomy. Means ? SEM of 8-10 rats. Asterisk indicates significant decrease as compared to the value obtained after single stress. Cross indicates significant change as compared to the value obtained in rats with intact salivary glands.
phenomenon. Since the exposure to stress was reported to be accompanied by the decrease in the proliferation of mucosal cells and DNA synthesis (6-12) and the stressinduced lesions were aggravated in salivectomized rats (12), we carried out the studies with the gastric adaptation in salivectomized animals. Such studies revealed that the removal of salivary glands delayed the appearance of the gastric adaptation and reduced the degree of this adaptation. This impairment of gastric adaptation to stress ulcerogenesis in salivectomized rats was accompanied by a marked reduction in gastric luminal contents of E G F , suggesting that the deficiency of E G F could be responsible, at least in part, for the failure of the adaptation t o the stress in these animals. The possible involvement of EGF and its mucosal growth promoting action on the gastric adaptation is supported by the fact that salivectomy delayed this adaptation and it was followed by the decrease in luminal content of EGF. Furthermore, histology of the adapted mucosa showed the signs of active regeneration. Also luminal content of EGF that was almost doubled after exposure to the stress showed further significant increase in rats with preserved salivary glands but not in the salivectomized animals. Although the undisturbed mucosal proliferation and the presence of salivary glands appear t o be essential for the gastric adaptation to occur, other mechanisms may also be involved. Since adaptive cytoprotection to mild irritants, including mild stress, was abolished by the pretreatment with indomethacin (21,22), we tested whether endogenous
PG are also involved in gastric adaptation to stress. The pretreatment with indomethacin in a dose, that was shown previously (12) to reduce mucosal generation of PGE7 by about 80%, completely abolished the adaptation to stress. The number of lesions per rat stomach was 2-3 times higher in indomethacin-treated rats during repeated exposures to stress and no tendency of the mucosa to adapt was observed. The failure of the mucosa to adapt to stress ulcerogenesis was accompanied by a marked (by about 85%) decrease in the PG biosynthesis in the oxyntic mucosa. Furthermore, the usual increase in the mucosal blood flow observed during the development of adaptation was abolished by indomethacin. These results could be interpreted that mucosal PG play a crucial role in mucosal adaptation and that this involvement is mediated by the maintenance of the mucosal microcirculation impaired by the water immersion and restraint stress.
REFERENCES 1. Robert A, Kauffman GL. Stress ulcers. In: Sleisenger MH, Fordtran JS, editors. Gastrointestinal diseases, pathophysiology, diagnosis, management. Philadelphia: Saunders, 1983: 612-62.
2. Senay EC, Levine RJ. Synergism between cold and restraint for
rapid production of stress ulcers in rats. Proc SOC Exp Biol 1967;124:1221-3. 3. Brodie DA, Hanson HM. A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 1960;38:353-60.
Scand J Gastroenterol Downloaded from informahealthcare.com by Chulalongkorn University on 01/01/15 For personal use only.
Gastric Adaptation to Stress 4. Selye H. A syndrome produced by diverse nocuous agents. Nature 1936;l38:32. 5. Moody FG, Cheung LY, Simons MA, Zalewsky C. Stress and the acute gastric mucosal lesions. Dig Dis 1976;21:148-54. 6. Kim Y, Kerr RJ, Lipkin M. Cell proliferation during the development of stress erosions in the mouse stomach. Nature 1967:215:118(b1. 7. Lahtiharju A. Rytomaa T. DNA synthesis in fore and grandular stomach and in skin after nonspecific stress in mice. Exp Cell Res 1967;46:59>6. 8. Imondi AR, Balis ME, Lipkin M. Nucleic acid metabolism in the gastrointestinal tract of the mouse during fasting and restraintstress. Exp Mol Pathol 1968;9:339-48. 9.1 Ludwig WM, Lipkin M. Biochemical and cytological alterations in gastric mucosa of guinea pigs under restraint stress. Gastrocnterology 1060;56:895-902. 10. Takeuchi K, Johnson LR. Pentagastrin protects against stress ulcerations. Gastroenteroogy 1979;76:327-34. 1 1 . Kuwayama H, Eastwood GL. Effects of water immersion restraint stress and chronic indomethacin ingestion on gastric antral and fundic epithelial proliferation. Gastroenterology 198.5;XX:M2-5. 12. Konturek PR, Brzozowski T, Konturek SJ, Dembinski A. Role of epidermal growth factor, prostaglandins. and sulfhydryls in stress-induced gastric lesions. Gastroenterology 1990;99:60215. 13. St John DJB. Yeomans ND. McDermott FT.de Boer WGRM. Adaptation of the gastric mucosa to repeated administration of aspirin in the rat. Am J Dig Dis 1973;18:881-6. 14. Graham DY, Smith JL. Dobbs SM. Gastric adaptation occurs with aspirin administration in man. Dig Dis Sci 1983;28:1-6.
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15. Graham DY. Smith JL, Spjut HJ, Torres T. Gastric adaptation. Studies in humans during continuous aspirin administration. Gastroenterology 1988;85:327-33. 16. Robert A, Lancaster C, Olafsson AS, Gilbert-Beadling S, Zhang W. Gastric adaptation to the ulcerogenic effect of aspirin. Exp Clin Gastroenterol 1991:1:73-81. 17. Robert A , Lancaster C, Olafsson AS, Zhang W. Gastric adaptation to repeated administration of a necrotizing agent. In: Garner A, O'Brien PE, editors. Mechanisms of injury, protection and repair of the upper gastrointestinal tract. Wiley, 1991 i3.57-70. 18. Takeuchi K, Okabe S, Takagi K. A new model of stress ulcers in rats with pylorus ligation and its pathogenesis. Am J Dig Dis 1977;21:742-88. 19. Konturek SJ. Brzozowski T, Drozdeowicz D, et al. Nocloprost, a unique prostaglandin E, analog with local gastroprotective and ulcer-healing activity. Eur J Pharmacol 1991;195:347-57. 20. Konturek SJ. Brzozowski T. Dembinski A, Warzecha Z, Konturek PK, Yanaihara N. Interaction of growth hormone-releasing factor and somatostatin on ulcer healing and mucosal growth in rats: role of gastrin and epidermal growth factor. Digestion 1988;41:121-8. 21. Glavin GB, Lockhart LK, Rockman GE, Hall AM, Kiernan KM. Evidence of 'cross-stressor'-induced adaptive gastric cytoprotection. Life Sci 1987;41:2223-7. 22. Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection of prostaglandins in rats: prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCI and thermal injury. Gastroenterology 1979;77:433-43.
Konturek SJ, Brzozowski T, Majka J , Drozdowicz D, Stachura J. Adaptation of the gastric mucosa to stress. Role of prostaglandin and epidermal growth factor. Scand J Gastroenterol 1992;27 Suppl193:39-
Scand J Gastroenterol Downloaded from informahealthcare.com by Chulalongkorn University on 01/01/15 For personal use only.
45.
This study was designed to determine whether repeated exposures to stress lead to the adaptation of the gastric mucosa to stress ulcerogenesis. Wistar rats with intact or resected salivary glands were exposed to a standard period (3.5 h) of water-immersion and restraint stress every other day up to 8 days. The significant reduction in the severity of gastric lesions was first noticed after the second exposure to stress and was maximal after 6-day exposures to stress. This tolerance to stress ulcerogenesis disappeared after a 6-day rest during which animals were not exposed to stress. Histologically, the hemorrhages and edema seen after a single stress were less frequent during adaptation; instead the mucosa regenerated in spite of continuation of exposure to stress. During adaptation, the mucosal blood flow (MBF) and mucosal biosynthesis of PG were markedly increased. Administration of indomethacin ( 5 mg/kg i.p.) completely abolished gastric adaptation to stress and this was accompanied by about 85% reduction in mucosal generation of PG and signficant decrease in the MBF. Salivectomy, which significantly reduced the luminal contents of epidermal growth factor (EGF) in the stomach, delayed and reduced the adaptation. We conclude that the stomach has the ability to adapt to repeated exposures to stress and that this adaptation is mediated, at least in part, by endogenous PG and EGF. Key words: Adaptation; epidermal growth factor; gastric lesions; mucosal blood flow; prostaglandins Prof. D r . S. J . Konturek, Institute of Physiology, University School of Medicine, PL-31-531 Krakow, ul. Grzegorzecka 16, Poland
Gastric stress ulceration is a highly prevalent disease that arises as a complication following burns, sepsis, major surgery, trauma to the control nervous sywstem etc. (1). Among various stresses used in animals, the most reproducible results can be obtained by restraint plus water immersion or cold, which appear to act synergistically in the production of gastric ulcerations (1-3). The mechanism of stress ulcerations has not been fully clarified and remains controversial more than 60 years after their original description by Selye (4).The major factors implicated in the development of stress ulcers include the increase in gastric acid secretion, the decrease in mucosal protection due to the reduction in mucus-alkaline secretion, the attenuation of the mucosal blood flow and prostaglandin (PG) biosynthesis and the fall in DNA synthesis and cell proliferation in the mucosa (5-12). During the past few years it has become evident that the stomach can defend itself from irritating and ulcerogenic agents such as nonsteroidal anti-inflammatory drugs (NSAID) (13-16) or necrotizing substances (17). The ability of the gastric mucosa to withstand the damage by repeated application of gastric ulcerogens was called gastric adaptation (13-17) but the mechanism of this phenomenon has not been explained. No information is available whether repeated exposures to stress results in the development of any tolerance to stress ulcerogenesis and if so what could be the mechanism of the adaptation to stress.
In the present studies, we used as a model the formation of acute gastric erosions produced in fasted rats by the waterimmersion and restraint stress (18). We investigated whether repeated exposures to stress insults would result in adaptation or aggravation to the ulcerogenic effect of stress in rats with intact or resected salivary glands.
MATERIALS AND METHODS Male Wistar rats of an average body weight of 200g and fasted 18 h were used. The animals were divided into three major groups, group A for the studies on the development and duration of gastric adaptation, group B for the studies on the implication of endogenous PG in gastric adaptation and group C for the studies on the role of salivary glands and epidermal growth factor (EGF) in gastric adaptation. The animals of the latter group had sublingual-submandibular gland complexes removed under ether anesthesia. The salivectomized rats, were allowed 7-10 days of full recovery following the operation. In most experiments, 3.5 h of stress was applied by placing animals into stress cages causing immobilization and immersing in 23°C water to the rat’s xyphoid process as described previously (18). A period of 3.5 h of stress was chosen because in preliminary studies this stress period was sufficient to produce numerous gastric erosions and induced gastric
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Konturek el al.
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Fig. 1. The number of gastric lesions induced by single exposure ('Once') to the water immersion and restraint stress for 3.5 or 7.0h or repeated every other day for up to 8 days. Means 5 SEM of 8-10 rats per group. Asterisk indicates significant decrease below the value obtained after stress applied once.
adaptation. The extension of stress to 7.0h resulted in greater number of erosions and more severe bleeding with poor recovery after each successive exposure to stress. Immediately after the stress, the animals of groups B were lightly anesthetized with ether, the abdomen was opened, the stomach exposed and the mucosal blood flow (MBF) was measured in the oxyntic gland area using H2 gas clearance technique described previously (19). Briefly, the double needle electrodes were inserted into the mucosa, one electrode was used for local generation of H2-gas and the other, for the measurement of tissue H2. With this method the H2 generated by water hydrolysis is carried away by the blood and the polarographic current detector gives the decreasing tissue H2 as the clearance curve which is used to calculate absolute flow rate (ml/min-100 g) in the tissue. After the measurement of MBF, the stomach was ligated at the cardia and the pylorus and 1 ml of cold saline was instilled into the stomach to wash out the gastric content for the determination of luminal content of epidermal growth factor (EGF). Then, the stomach was dissected out and opened along the greater curvature. The stomach was then examined with a 2x binocular magnifier for the presence of erosions by someone unaware of the treatment given. These lesions appeared as small round or linear hemorrhagic erosions occurring in the oxyntic gland area. The erosions were counted and the average number per stomach was calculated for each group. In animals of group A, the biopsy of the mocosa of the oxyntic gland area was obtained and used for the determination of tissue generation of PGE2 as described previously (12). In some experiments, the stomachs were fixed in 10% buffered formalin, and 3-pm
sections were stained with hematoxylin-eosin for histological evaluation. Several series of animals were used; in group A, one subgroup was sacrificed without stress and was used as a control for the measurement of EGF content in the gastric lumen and for the determination of mucosal PGE2 and MBF. Other subgroups (consisting of 8-10 rats each) were stressed for 3.5 or 7.0 h at 1100 h and used for the studies on gastric adaptation. One of these subgroups was stressed once (for 3.5 or 7.0 h) and after this single exposure to stress it was sacrificed. Another group was treated similarly, except that after stress they were removed from the stress cages, placed in a room temperature and refed until 1400 h next day. At that time, the animals were again fasted overnight and stress was repeated next morning. The animals were sacrificed after this second treatment with 3.5 or 7.0 h of water-immersion and restraint stress. Other groups underwent the same schedule of stress, refeeding and refasting for 4, 6 or 8 consecutive days. In all rats the MBF was determined and the stomachs were examined for gastric erosions. In studies on the duration of gastric adaptation after cessation of stress, rats were exposed to stress for 3.5 h every second day for 4 times in order to produce gastric adaptation. Following this 6 day treatment with stress, the animals were fed and remained without treatment for periods of 2, 4,6, 8, 10 or 12 days. At all these time intervals, they were fasted overnight and on the following day morning they were stressed by challenging 3.5 h period of water immersion and restraint. They were anaesthetized after the challenging treatment and examined for the MBF and the gastric erosions.
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Gastric Adaptation to Stress
In group B, the rats were subdivided into subgroups of 810 animals and stressed for 3.5 h at the same periods as in group A. In addition each animal received daily injection of either vehicle (saline i.p.) or indomethacin (5 mg/kg i.p.). After the stress, the rats were anaesthetized with ether, the abdomen was opened to measure MBF and then the number of gastric erosions was counted. Finally, the biopsy samples of the mucosa from the oxyntic gland area were taken for the determination of mucosal generation of PGEz as described before (12). In group C (with prior salivectomy), the animals were also subdivided in subgroups of 8-10 rats and stressed for 3.5 h at the same time periods as in group A they were sacrificed after first, second, third, fourth or fifth stress-treatment. The animals were anesthetized, the stomach exposed to measure the MBF, luminal contents were collected to measure the contents of EGF by radioimmunoassay as described previously (20). Briefly, the mucosal samples were weighed and homogenized in ice-cold 0.02 mM/l Tris-HCI buffer and centrifuged, the supernatant being collected and frozen at 20°C until EGF radioimmunoassay . The EGF antiserum raised in rabbits against human EGF was used in a final dilution of 1:210,000. Iodinated ([3-1251]iodotyrosyl)peptide and rat EGF were calibration standards (Amersham, England). The detection limit of the assay was 0.01 nM/I. The interassay and intra-assay precisions were about 12% and lo%, respectively. The results are reported as means SEM. Statistical significance was determined by analysis of variance and where appropriate by the unpaired Student’s t test, a value of less than 0.05 being considered significant.
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RESULTS Production of gastric erosions The water immersion and restraint stress applied once for 7.5 h produced numerous small erosions (about 29 m 2 per stomach) with severe bleeding. Similar exposure to stress for 3.5 h resulted in about 30% smaller number of erosions per stomach and less severe bleeding (Fig. 1). During the repeated exposures of stress for 7.5 h , the number of gastric lesions was similar but the reduction in the body weight of animals was observed and about 30% of rats died after 3rd or 4th stress exposure. In contrast, with repeated exposures to 3.5 h stress, a progressive decrease in the number of erosions was observed from 25% after first challenge to 53% after the fourth challenge. Thus, gastric adaptation is triggered by a single treatment with stress but becomes maximal after 6 days of repeated exposures to stress.
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Duration of gastric adaptation Fig. 2 shows the number of erosions per stomach after 6day exposures to stress in rats that were refed and were not stressed for varous time intervals (2-12 days). After each interval of rest, they were re-challenged with 3.5 h stress
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following an overnight fast. When the animals were rechallenged with stress at 2nd, 4th and 6th day, there was a gradual increase in the number of erosions per stomach reaching after 8 days the value not sitgnificantly different from that occurring after single treatment (‘Once’). Thus, the gastric adaptation to stress induced by a 6-day of repeated exposure to repeated stress persisted for up to 6 days after discontinuation of this stress. Histological evaluations A single exposure to 3.5 h stress produced deep and extending to the muscular is mucosae erosions in the oxyntic mucosa (Fig. 3). Portions of the mucosa were necrotic given rise to craters. The submucosa was edematous but without polymorphonuclear infiltration. After 6 days of exposures to stress every second day only few new erosions were observed. The mucosa appeared thicker and only occasional craters lined with a monolayer of newly formed surface epithelial cells were noticed (Fig. 4). In addition, mucosal scars of healed erosions were observed. The submucosa still showed the edema accompanied by minor inflammatory infiltrate in some rats. Gastric mucosal blood flow ( M B F ) The MBF in the mucosa of the oxyntic portion of the stomach of intact rats averaged about 51 2 6 ml/min-100 g. After a single exposure of group B to the 3.5 h stress, the MBF was reduced by about 43%. When the mucosa was adapted to repeated exposures to the stress, there was a gradual increase in the MBF reaching after the first challenge with stress about 80% of the value in the intact mucosa and attaining after the second challenge with stress the value not significantly different from that recorded in the intact mucosa. In rats injected daily with indomethacin (5 mg/kg i.p.), the adaptation of the gastric mucosa to stress completely disappeared and this was accompanied by a significant reduction in gastric MBF in these animals (Fig. 5). Effects of salivectomy and EGF on the adaptation of gastric mucosa to stress Following salivectomy, the number of gastric lesions in response to single exposure to stress showed a small but significant increase (by about 27%) (Fig. 6). After repeated exposure to stress every second day, the number of lesions per stomach was 33-50% higher than these in the corresponding group of rats with intact salivary glands. After 3rd and 4th consecutive exposure to stress in salivectomized rats, the number of lesions was significantly lower than that in rats after a single stress but it was still higher (by about 50%) than that in rats with intact salivary glands.
DISCUSSION The present results show that upon repeated exposures to stress, the rat gastric mucosa undergoes progressive adap-
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S. J . Konturek el al.
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Fig. 2. Duration of gastric adaptation after cessation of exposure to stress. Animals were first exposed to repeated stress for 6 days and thereafter were without stress for various periods of time from 2-12 days after which were rechallenged with stress. Means 2 SEM of 8-10 rats. Asterisk indicates significant decrease below the value obtained after single exposure to stress (‘Once’).
Fig. 3. Acute stress erosion. Focal mucosal necrosis penetrating 3/4 of the mucosal thickness. In the ~ right bottom corner edematous submucosa. (H & E; magnification, 2 6 0 .)
tation to stress ulcerogenesis. This adaptation vanishes when the challenging stress is applied 6 days after the adaptation was induced by repeated exposures to stress. Previous studies (21) demonstrated that ‘mild’ stress such as 1 h immobilization is capable of preventing the formation of acute gastric lesions induced by 3 h of cold-restraint stress or by 100% ethanol. This adaptive gastroprotection by mild stress was found to disappear after the pretreatment with
indomethacin suggesting that endogenous PG may be implicated in this this phenomenon. Our present results indicate that the exposure to repeated stress insults greatly increases the tolerance of the gastric mucosa to stress-induced ulcerogenesis. This tolerance is triggered by a single exposure to stress and becomes maximal after 6 days. Histologically, the numerous necrotic hemorrhages and vasocongestion of submucosal edema
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Gastric Adaptation to Stress
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Fig. 4. Mucosal scar. Depression of the mucosa in the mucosa is reepithelized with surface epithelium (H & E; magnification, 260x .)
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Fig. 5 . The number of gastric lesions and the mucosal blood flow (MBF) in rats after single exposure to stress (‘Once’) and then after repeated exposures at 2nd, 4th. 6th and 8th day in rats with or without pretreatment with indomethacin ( 5 mg/kg i.p. daily). Asterisk indicates significant change as compared to the values obtained in rats exposed to single stress (‘Once’). Cross indicates significant change as compared to the values obtained in rats without administration of indomethacin.
observed after the single exposure to stress, appeared less frequently with the development of adaptation. The granulation tissue was seen in the areas that had become necrotic earlier and there was a proliferation of mucus cells lining as a monolayer at the sides of steep craters. These histological
data indicate that the process of active repair of earlier necrotic lesions accompanied by the active regeneration of gastric glands occur during the mucosal adaptation to stress. The mechanism of gastric adaptation to the stress is unknown but we tested several hypotheses to explain this
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S. J. Konturek et al.
DURATION (Day#) OF TREAT’MlCNT Fig. 6. The number of gastric lesions and luminal contents of EGF after single exposure to stress (‘Once’) and then after repeated exposures to stress at 2nd, 4th, 6th and 8th day in rats sham-operated with intact salivary glands and after salivectomy. Means ? SEM of 8-10 rats. Asterisk indicates significant decrease as compared to the value obtained after single stress. Cross indicates significant change as compared to the value obtained in rats with intact salivary glands.
phenomenon. Since the exposure to stress was reported to be accompanied by the decrease in the proliferation of mucosal cells and DNA synthesis (6-12) and the stressinduced lesions were aggravated in salivectomized rats (12), we carried out the studies with the gastric adaptation in salivectomized animals. Such studies revealed that the removal of salivary glands delayed the appearance of the gastric adaptation and reduced the degree of this adaptation. This impairment of gastric adaptation to stress ulcerogenesis in salivectomized rats was accompanied by a marked reduction in gastric luminal contents of E G F , suggesting that the deficiency of E G F could be responsible, at least in part, for the failure of the adaptation t o the stress in these animals. The possible involvement of EGF and its mucosal growth promoting action on the gastric adaptation is supported by the fact that salivectomy delayed this adaptation and it was followed by the decrease in luminal content of EGF. Furthermore, histology of the adapted mucosa showed the signs of active regeneration. Also luminal content of EGF that was almost doubled after exposure to the stress showed further significant increase in rats with preserved salivary glands but not in the salivectomized animals. Although the undisturbed mucosal proliferation and the presence of salivary glands appear t o be essential for the gastric adaptation to occur, other mechanisms may also be involved. Since adaptive cytoprotection to mild irritants, including mild stress, was abolished by the pretreatment with indomethacin (21,22), we tested whether endogenous
PG are also involved in gastric adaptation to stress. The pretreatment with indomethacin in a dose, that was shown previously (12) to reduce mucosal generation of PGE7 by about 80%, completely abolished the adaptation to stress. The number of lesions per rat stomach was 2-3 times higher in indomethacin-treated rats during repeated exposures to stress and no tendency of the mucosa to adapt was observed. The failure of the mucosa to adapt to stress ulcerogenesis was accompanied by a marked (by about 85%) decrease in the PG biosynthesis in the oxyntic mucosa. Furthermore, the usual increase in the mucosal blood flow observed during the development of adaptation was abolished by indomethacin. These results could be interpreted that mucosal PG play a crucial role in mucosal adaptation and that this involvement is mediated by the maintenance of the mucosal microcirculation impaired by the water immersion and restraint stress.
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2. Senay EC, Levine RJ. Synergism between cold and restraint for
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Gastric Adaptation to Stress 4. Selye H. A syndrome produced by diverse nocuous agents. Nature 1936;l38:32. 5. Moody FG, Cheung LY, Simons MA, Zalewsky C. Stress and the acute gastric mucosal lesions. Dig Dis 1976;21:148-54. 6. Kim Y, Kerr RJ, Lipkin M. Cell proliferation during the development of stress erosions in the mouse stomach. Nature 1967:215:118(b1. 7. Lahtiharju A. Rytomaa T. DNA synthesis in fore and grandular stomach and in skin after nonspecific stress in mice. Exp Cell Res 1967;46:59>6. 8. Imondi AR, Balis ME, Lipkin M. Nucleic acid metabolism in the gastrointestinal tract of the mouse during fasting and restraintstress. Exp Mol Pathol 1968;9:339-48. 9.1 Ludwig WM, Lipkin M. Biochemical and cytological alterations in gastric mucosa of guinea pigs under restraint stress. Gastrocnterology 1060;56:895-902. 10. Takeuchi K, Johnson LR. Pentagastrin protects against stress ulcerations. Gastroenteroogy 1979;76:327-34. 1 1 . Kuwayama H, Eastwood GL. Effects of water immersion restraint stress and chronic indomethacin ingestion on gastric antral and fundic epithelial proliferation. Gastroenterology 198.5;XX:M2-5. 12. Konturek PR, Brzozowski T, Konturek SJ, Dembinski A. Role of epidermal growth factor, prostaglandins. and sulfhydryls in stress-induced gastric lesions. Gastroenterology 1990;99:60215. 13. St John DJB. Yeomans ND. McDermott FT.de Boer WGRM. Adaptation of the gastric mucosa to repeated administration of aspirin in the rat. Am J Dig Dis 1973;18:881-6. 14. Graham DY, Smith JL. Dobbs SM. Gastric adaptation occurs with aspirin administration in man. Dig Dis Sci 1983;28:1-6.
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15. Graham DY. Smith JL, Spjut HJ, Torres T. Gastric adaptation. Studies in humans during continuous aspirin administration. Gastroenterology 1988;85:327-33. 16. Robert A, Lancaster C, Olafsson AS, Gilbert-Beadling S, Zhang W. Gastric adaptation to the ulcerogenic effect of aspirin. Exp Clin Gastroenterol 1991:1:73-81. 17. Robert A , Lancaster C, Olafsson AS, Zhang W. Gastric adaptation to repeated administration of a necrotizing agent. In: Garner A, O'Brien PE, editors. Mechanisms of injury, protection and repair of the upper gastrointestinal tract. Wiley, 1991 i3.57-70. 18. Takeuchi K, Okabe S, Takagi K. A new model of stress ulcers in rats with pylorus ligation and its pathogenesis. Am J Dig Dis 1977;21:742-88. 19. Konturek SJ. Brzozowski T, Drozdeowicz D, et al. Nocloprost, a unique prostaglandin E, analog with local gastroprotective and ulcer-healing activity. Eur J Pharmacol 1991;195:347-57. 20. Konturek SJ. Brzozowski T. Dembinski A, Warzecha Z, Konturek PK, Yanaihara N. Interaction of growth hormone-releasing factor and somatostatin on ulcer healing and mucosal growth in rats: role of gastrin and epidermal growth factor. Digestion 1988;41:121-8. 21. Glavin GB, Lockhart LK, Rockman GE, Hall AM, Kiernan KM. Evidence of 'cross-stressor'-induced adaptive gastric cytoprotection. Life Sci 1987;41:2223-7. 22. Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection of prostaglandins in rats: prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCI and thermal injury. Gastroenterology 1979;77:433-43.
