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Chronic mild restraint protects the rat gastric mucosa from injury by ethanol or cold restraint
GASTROENTEROLOGY
1983;85:370-5
Chronic Mild Restraint Protects the Rat Gastric Mucosa From Injury by Ethanol or Cold Restraint JOHN L. WALLACE, Department Canada
of Surgery,
NORMAN
University
of Toronto
The effects of chronic mild restraint on the susceptibility of the rat gastric mucosa to ethanol or coldrestraint injury were studied. Gastric mucosas of animals subjected to chronic mild restraint exhibited less damage when bathed with 40% ethanol than those of control animals. This reduced damage was observed with 2 days of mild restraint and was maximal (93% less damage; p < 0.0005) when the animal was mildly restrained for 10 days. Pretreatment with indomethacin abolished the protection afforded by chronic mild restraint, suggesting a mechanism involving endogenous prostaglandin synthesis. Chronic mild restraint significantly (p < 0.05) reduced the injury to the gastric mucosa caused by cold-restraint stress. This model of mild
restraint may prove useful in future studies on the mechanism of stress-induced and other gastric mucosal lesions. The gastric mucosa is very susceptible to different forms of stress, including those associated with critical illness, head injuries, burns, or surgical trauma. Selye (2) described the appearance of hemorrhagic erosions in the gastric mucosa of rats exposed to the severe stress of immobilization. The gastric mucosa of rats can also be damaged by the direct application of substances such as ethanol and aspi-
S. TRACK, and MAX M. COHEN and Mount
Sinai
Hospital,
0 1983 by the American Gastroenterological 0016.5085/83/$3.00
Association
Ontario,
rin (3). The damaging effects of these agents can be significantly reduced by their chronic administration. Ivey et al. (4) demonstrated an increased resistance of the rat gastric mucosa to 50% ethanol when it was administered daily for 4 wk. Similar findings have been reported with chronic administration of aspirin to rats (5) and dogs (6). All of these observations are based upon the direct exposure of the gastric mucosa to a damaging agent. The purpose of this study was to determine if a similar effect could be achieved by indirect methods, in an attempt to answer the question: will the gastric mucosa become more resistant to injury if the animal is subjected to chronic mild “stress” in the form of chronic mild restraint (CMR)? The present study evolved from an assessment of the effect of total parenteral nutrition (TPN) upon the susceptibility of the rat gastric mucosa to stress ulceration. In the control group, the mild restraint caused by the TPN catheter support system (without any intravenous infusion) reduced ethanol-induced injury to the gastric mucosa. In this report, the protective effects of CMR against both ethanol and cold-restraint gastric injury are investigated further. Materials Chronic
Received October 20, 1982. Accepted February 24, 1983. Address requests for reprints to: M.M. Cohen, Suite 440, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. This work was supported by grants from the Medical Research Council of Canada (MT 5316) and Mount Sinai Institute. This work was presented in part at the American Gastroenterological Association meeting in Chicago, Illinois, in May 1982, and published in abstract form (1). The authors thank Beth Chin, Susan Chiu, and Karen Gerry for their assistance in performing these studies; Dr. G. P. Morris for preparing the micrograph, and Mrs. B. M. Kruska for secretarial assistance.
Toronto,
and Methods
Mild Restraint
Technique
Male Wistar rats (300 g) were used in all experiments, and were housed, individually, in cages with a steel mesh bottom. Under pentobarbital anesthesia (60 mg/kg body wt i.p.), an area of about 9 cm* was shaved on the back of the neck of each animal. A z-cm incision was
made along the midline and a metal ring was inserted subcutaneously. The iricision the metal ring anchored to the skin with sutures. One end of a 24-cm metal spring this metal ring (see Figure 1). The other
(2 cm diameter) was closed and four additional was attached to
end was passed through a hole in the top of the animal’s cage. A bumper was attached to the free end of the spring on the outside of the cage to prevent the animal from pulling the spring into
August
MUCOSAL PROTECTION WITH CHRONIC STRESS
1983
stress 10 days after initiation or sham operation (n = 7). Gastric
Figure
1 Schematic diagram of the chronic mild restraint apparatus. A small metal ring is inserted subcutaneously in the back of the neck of the rat. A stainless steel spring is connected to the ring through a hole in the top of the cage. The animal can move to any part of its cage without stretching the spring.
Experimental
Design
The CMR and sham-operated rats were tested for susceptibility to direct mucosal injury by ethanol in a gastric chamber model. These experiments were performed on groups (n = 5-7) of animals after 2, 5, 10, or 12 days of CMR (or sham operation). Additional experiments were performed on chambered mucosas of IO-day CMR and sham-operated (n = 9) rats in which transmucosal potential difference and various ion fluxes were determined. The CMR and sham-operated rats were tested for susceptibility to indirect mucosal injury by cold-restraint
(n = 8)
Preparation
Each rat was deprived of food for 20 h, anesthetized, and a gastric chamber was prepared according to a modification (7) of the method of Mersereau and Hinchey (8). The animal was placed on a grooved Lucite platform. An electric heating pad placed under this platform was connected to a temperature controller (YSI Instruments, Yellow Springs, Ohio), which maintained the rectal temperature of the animal at 38°C. A midline laparotomy was performed and the pylorus and esophagus were ligated, with meticulous care taken to preserve the vasculature and the vagi. The stomach was opened by making an incision along the greater curvature and pulled gently through a small aperture in a Lucite plate placed over the animal. The stomach was pinned, mucosal side up, to the top of this plate, and a Lucite chamber (11 ml volume) was clamped to the mucosa and plate. Any hair or scales ingested during grooming were removed from the stomach by gently rinsing with 37”C, 0.3 M mannitol. Solutions were then added to the chamber by syringe and allowed to bathe the mucosa for periods of 10 min. A glass paddle turning at 200 rpm kept the contents of the chamber well mixed. Ethanol
the cage. The animal was free to feed ad libitum on standard food pellets and tap water (attached to the front of the cage) and to move to any part of the cage without stretching the spring. Routinely, sham-operated rats served as the controls. In one experiment, a second control group was included. These rats had the metal ring implanted, but a spring was not attached from the ring to the cage top. A group of CMR rats (n = 5) and a sham-operated group (n = 5) were weighed and placed in individual metabolic cages. Food and water consumption and the amount of feces and urine excreted were measured at 9:OO AM daily for 9 consecutive days. Each animal was given a daily ration of 100 ml of tap water and 35 g of standard pelleted rat diet. The food and water remaining on the following day were removed and measured. At the end of the 9 days, the animals were reweighed, killed by decapitation, and the adrenal glands were removed and weighed.
Chamber
of the mild restraint
371
Injury
to ethanol injury, For assessment of susceptibility the gastric chamber experiments consisted of four lo-min periods during which IO-ml aliquots of 37°C solutions were added to the chamber. The luminal solutions for the four periods were 0.3 M mannitol during first period, 40% ethanol (vol/vol) during the second period, and 0.05 M HCl made isosmotic with mannitol during the last two periods. At the end of the last period in each experiment, the mucosa was photographed with color slide film. These slides were projected onto 21.5 x 28.0-cm paper, traced, and labeled with a code number. The damaged area was measured from these tracings by planimetry with the single observer (J.L.W.) unaware to which group the photograph related. Damaged area was expressed as the percent of glandular mucosa showing gross damage. In previous studies (g), these macroscopic assessments of damage have been found to correlate well with light microscopic determinations of damage. The effect of indomethacin pretreatment upon ethanol injury was assessed in IO-day CMR and control animals in which indomethacin (Merck, Sharp and Dohme, Kirkland, Canada) was administered (1 mg/kg body wt i.p.) 75 min before the start of the gastric chamber experiments. The indomethacin was suspended in a 0.05% carboxymethylcellulose vehicle (#lOO sterile vehicle, Upjohn Co., Kalamazoo, Mich.). Electrophysiologic
Studies
Measurements of transmucosal potential difference (PD) and sodium, potassium, and hydrogen ion flux were
372
WALLACE ET AL.
GASTROENTEROLOGY Vol. 85, No. 2
assessed with the gastric chamber technique. The protocol was extended to seven IO-min periods. Mannitol (0.3 M) was added to the chamber in the first three periods. During the fourth period, 40% ethanol (vollvol) bathed the mucosa. This was followed by three periods in which 0.05 M HCl made isosmotic with mannitol was placed in the chamber. Sodium and potassium ion concentrations were measured
in the luminal
solutions
by flame
photometry
(Corning, Medfield, Mass.). Titratable acidity was measured by diluting a l-ml aliquot of the luminal solution with 20 ml deionized water and titrating to pH 7.0 with 0.01 M NaOH. In some cases, where the pH of the sample was >7.0, a back-titration with 0.01 N HCl was performed. An automatic titration system (Radiometer, Copenhagen, Denmark) was used for these analyses. The volume of the aliquot of luminal solution was determined gravimetrically before addition to the chamber and immediately after removal. The concentration of each ion (in milliequivalents per liter) was multiplied by the volume of the aliquot (in liters) of luminal solution, thereby yielding a value in units of milliequivalents. Gastric transmucosal PD was measured throughout each experiment using a system previously described in detail (9).
Cold-Restraint
Injury
Rats were deprived of food, but not water, for 16 h. Each animal was immobilized in a wire mesh and placed in a cold room (4°C) for 4 h. This procedure was always performed by the same person to ensure consistency in the degree of immobilization. The animals were then killed by decapitation, the abdomen was opened, and the stomach was removed. An incision was made along the greater curvature and the mucosa was pinned out on a wax platform. The mucosa was rinsed of any adherent blood or mucus with ice-cold phosphate (0.1 M; pH 7.4) buffer. The mucosa was then photographed and the damaged area was measured by planimetry (as described previously). The damage index was calculated as the number of square millimeters of damaged mucosa per thousand square millimeters of glandular mucosa.
Statistical
Analyses
Data were always expressed as mean ? SEM. Statistical comparisons between control and CMR groups were performed with Student’s two-tailed t-test for unpaired data. Probability values of ~5% were considered statistically significant.
Results Chronic Mild Restraint The implantation of a metal ring in the back of the rat’s neck and its attachment by a spring coil to the top of the cage allowed free movement to any part of the cage and ad libitum feeding. Despite this freedom, CMR still caused discomfort as evidenced by persistent scratching which gradually loosened
Table
I. Effect of 9 Days of Chronic Mild Restraint Metabolic Parameters Sham-operated
Number of animals Body wt gain (g/day) Food consumed (g/day) Water consumed (ml/day) Feces (g/day] Urine (ml/day]
5.0 25.3 33.4 12.4 11.9
5 + + 5 + +
0.4 0.8 1.1 0.6 0.5
on
CMR 2.9 25.4 36.3 12.7 13.5
5 2 k f f r
0.9” 0.6 1.1”
0.5 0.5b
o p < 0.05. b p < 0.01. All data represent
mean + SEM. Each parameter (except body weight] was measured daily for 9 consecutive days. CMR, chronic mild restraint.
the metal implant. The CMR animals appeared to be more active than the sham-operated animals during the daylight hours (normally the resting period). Chronic mild restraint could not be continued beyond 12 days as some animals eventually dislodged the implant. The CMR animals had significantly (p < 0.05) lower weight gain than sham-operated animals over the g-day period (Table 1). Food consumption and the amount of feces did not differ between the two groups. The CMR rats drank an average of 2.9 ml/day more water (p < 0.05) and excreted an average of 1.6 ml/day more urine (p < 0.01) than sham-operated rats. The adrenal glands in the CMR group were
significantly (p < 0.05) heavier (39.5 * 2.3 g) than those of the sham-operated animals (34.5 2 1.5 g). Mucosas of CMR animals maintained for periods of up to 12 days appeared normal when examined macroscopically. Ethanol Injury Exposure of the chambered gastric mucosa to ethanol (and subsequent acid) produced extensive hemorrhagic lesions in the sham-operated animals. This damage was usually confined to the fundic region of the stomach. In the 27 chamber experiments performed on sham-operated rats, the damaged area was 17.5 + 2.7% of the glandular mucosa. Light microscopic examination revealed that these lesions did not penetrate the muscularis mucosae (Figure 2). Consistently less damage was induced by the application of the ethanol to chambered mucosas of CMR animals (Figure 3). A slight reduction of mucosal damage was observed with only 2 days of CMR. Damage was significantly less in both the 5- (p < 0.001) and lo- (p < 0.0005) day CMR groups compared with their control groups. With 12 days of CMR, the damage was slightly higher than in the 5or IO-day CMR groups, and was not significantly different from the corresponding control group. The significant reduction in damage with 10 days
MUCOSAL
PROTECTION
WITH
CHRONIC
STRESS
373
Metal Implant
Figure
Figure
2. Light micrograph showing border between normal and ethanol-injured mucosa of sham-operated rat. Mucosa to right of arrow appeared normal upon macroscopic examination. Mucosa to left of arrow was hemorrhagic and was scored as damaged. Note disruption of surface epithelium and the vascular engorgement. The necrosis does not penetrate the muscularis mucosae (m). This section was taken from a sample of fundic mucosa fixed at the end of a gastric chamber experiment. (Toluidine blue, x40.)
of CMR was not observed in rats in which the metal ring was implanted (n = 5) without a spring coil connecting it to the cage top (Figure 4). The mucosas of lo-day control and CMR rats pretreated with indomethacin appeared normal when examined macroscopically. Application of ethanol to the chambered mucosas of these indomethacin-treated rats produced hemorrhagic lesions involving 23.6 -+ 6.6% (control group) and 33.5 k
4. Ethanol injury to chambered gastric mucosas of chronic mild restraint and sham-operated animals. Each column represents the mean 2 SEM percent of glandular mucosa damaged after bathing with 40% ethanol and acid. These experiments were performed 10 days after the sham operation (n = 5), initiation of chronic mild restraint (n = 6), or implantation of the metal ring (n = 5). *p < 0.001.
9.7% (CMR group) of the glandular mucosa. These areas of damage were not significantly different from each other or from the areas of damage observed in the sham-operated or metal-implant control groups which were not pretreated with indomethacin (Figure 4). Electrophysiologic
In the sham-operated animals in which PD and ion fluxes were measured, the ethanol injury involved 16.9 t 5.0% of the glandular mucosa. The damage to the mucosas of CMR animals was 5.3 * 1.8%. Despite this significant (p < 0.05) reduction in gross damage, the electrophysiologic parameters showed no marked differences. Potential difference values (Figure 5) for the two groups were not significantly different, although baseline PD values were higher in the control group. Both groups exhibited the same marked fall in PD in response to ethanol. Similarly, the ion flux data (Figure 6) did not differ greatly between the two groups. In both groups there was increased loss of titratable acidity and increased efflux of sodium and potassium ions in response to the application of ethanol. Cold-Restraint
Duration of CMR (days) Figure
of chronic mild restraint on ethanol 3. Effect of duration injury. Each column represents the mean ‘- SEM percent of glandular mucosa damaged after bathing with 40% ethanol and acid. Numbers in brackets represent the size of each group. * p < 0.001, ** p < 0.0005.
Studies
Injury
Exposure of immobilized sham-operated animals to cold (4°C) for 4 h resulted in the production of extensive hemorrhagic lesions in the fundic mucosa [Table 2). Ten-day CMR animals developed about half as many mucosal lesions. The lesion area of CMR animals was significantly (p < 0.05) less than that observed in the mucosa of sham-operated animals.
374
WALLACE
ET AL
GASTROENTEROLOGY
-6O-
F
c.
-5o-
8
s
-4o-
I/
-3o-
I!! E g
-2o-
?? -e
Sham
O-----O
CMR
85, No. 2
r
I
4.
/-
/ 00
k E n
1
I
_/” II
+40
Acid
Ethanol
Mannitol
Vol.
*P
n-I
1
pl’
-60 1
-60 L
a
\
1 60
10
20
30
40
50
60
70
60
Ii
Time (min) Figure
3 3 5
5. Transmucosal
potential difference across chambered gastric mucosas of lo-day chronic mild restraint and sham-operated rats. Each point represents the mean k SEM for a 5-min interval (n = 5-B). There are no significant differences during any 5-min interval. Solutions bathing the mucosa are indicated at the top of the figure.
f z zi =
40
20
Discussion It is well established that severe stress damages the gastric mucosa. The present study provides evidence that CMR protects the gastric mucosa from both direct (40% ethanol) and indirect (cold-restraint] forms of stress. The resistance to ethanol injury was maximal in lo-day CMR animals. The reduction in ethanol injury was less when CMR was extended to 12 days. It is possible that this diminished “protection” may be partially due to the looseness of the metal implant after 12 days. It is clear that all rat TPN models require catheter support. These support systems undoubtedly exert
Table
2.
Comparison
of Cold-Restraint
in IO-Day Chronic Operated Rats
Sham-operated Number of animals Lesions/stomach Damage index
Gastric
Mild Restraint
Damage
and ShamCMR
7
8
6.0 + 1.7
3.1 2 1.1
10.2
2 4.4
3.3 *
1.3”
” p < 0.05. All data represent mean + SEM. Animals were subjected to cold-restraint stress for 4 h after 10 days of chronic mild restraint (CMR) or sham operation. Damage index represents the number of square millimeters of damaged mucosa per thousand square millimeters of glandular mucosa.
Mannitol
,
I 1
Figure
Ethanol
1
Acid
I
I
2 3 4 5 6 Time Period (10 min. each)
I
1 7
6. Net hydrogen sodium, and potassium flux across chambered gastric mucosas of to-day chronic mild restraint and sham-operated rats. Each column represents the mean 2 SEM flux for the lo-minute application period. Positive flux denotes a net gain in the luminal solution. *p < 0.05
some degree of stress. Birkhahn et al. (10) examined the effect of the restraint caused by a TPN harness system on food intake and body weight gain in rats. Their findings, similar to those reported here, were reduced weight gain with normal food intake. Sander et al. (11) used a similar TPN harness in their study of the effects of parenteral vs. oral feeding on susceptibility to cold-restraint-induced gastric injury, but did not examine the effects of the harness system itself. The CMR technique used in this study was derived from another TPN model (12). The consistent reduction in weight gain of CMR rats and the enlargement of adrenal glands in these animals
August
1983
demonstrates the ability of this technique to induce chronic stress. This stress, so mild that it did not upset the overall well being of the rat, reduced susceptibility to gastric mucosal injury. Although CMR significantly reduced the area of mucosa that became hemorrhagic when bathed with ethanol and acid, no significant differences in electrophysiologic parameters were observed. These findings are similar to a previous study using this gastric chamber model (9). In that study, topically applied prostaglandin E2 was found to significantly reduce the ethanol injury (gross damage) without reducing the alterations in ion fluxes or PD. It has also been observed that similar changes in ion fluxes and PD occur when the canine gastric mucosa is bathed by acetylsalicylic acid or salicylic acid, substances that have very different effects on prostaglandin synthesis and, presumably, on mucosal integrity (13). It is likely that some damage was present even in the most protected of CMR mucosas. This damage (destruction of surface epithelial cells] would cause significant alterations in electrophysiologic parameters (as observed) not necessarily distinguishable from the changes caused by extensive mucosal damage. The protective mechanism(s) of CMR is yet to be determined. The observation that indomethacin pretreatment abolished CMR protection suggests a role for endogenous prostaglandin synthesis. Exogenous prostaglandins have been shown in both animals (l4,15) and in humans (16)to be capable of preventing gastric mucosal damage induced by a variety of agents. This phenomenon has been termed “cytoprotection.” More recently, it has been shown that bathing the rat mucosa with a “mild irritant” protected it from subsequent injury by a necrotizing agent and, as this protection was abolished by indomethacin, it was deduced to be due to the stimulation of endogenous prostaglandin synthesis (17).A similar phenomenon has been observed in humans (18).These experiments, and those now reported, all support the concept that prostaglandin protection of the gastric mucosa is a physiologic phenomenon. The observation that the adrenal glands of CMR animals were enlarged and the known importance of the adrenal gland in the stress response (2) suggest that it plays a role in the protective mechanism of CMR. Also, the effect on the stomach of CMR is indirect (i.e., no manipulation of the gastric mucosa), suggesting the involvement of the central nervous system. The importance of the brain, in particular the hypothalamus, in the stress response has been recognized since Cushing described gastric damage in association with intracranial lesions (19). Recently, it was reported that central administration of bombesin (20) or neurotensin (21) protected the
MUCOSAL
PROTECTION
WITH
CHRONIC
STRESS
375
gastric mucosa from cold-restraint injury. Further studies are required to elucidate the roles of regulatory peptides and the adrenal gland in the CMR protective mechanism.
References 1. Wallace
JL, Cohen MM. Chronic minimal restraint in rats: a model for “adaptive cytoprotection”? (abstr). Gastroenterology 1982;82:1259. Book 2. Selye H. The stress of life. New York: McGraw-Hill Company Inc., 1956. 3. Brodie DA. Experimental peptic ulcer. Gastroenterology 1968; 55:125-34. 4. Ivey KJ, Tarnawski A, Stachura J, et al. The induction of mucosal tolerance to alcohol by chronic administration. J Lab Clin Med 1980;96:922-32. 5. St. John DJB, Yeomans ND, McDermott FT, et al. Adaptation of the gastric mucosa to repeated administration of aspirin in the rat. Am J Dig Dis 1973;18:881-6. 6. Bolton JP, Cohen MM. Effect of repeated aspirin administration on the gastric mucosal barrier and cell turnover. J Surg Res 1977;23:251-6. 7. Morris GP, Wallace JL. The roles of ethanol and of acid in the production of gastric mucosal erosions in rats. Virchows Arch (Cell Pathol) 1981;38:23-38. 8. Mersereau WA, Hinchey EJ. Effects of gastric acidity on gastric ulceration induced by haemorrhage in the rat, utilizing a gastric chamber technique. Gastroenterology 1973;64:11305. 9. Wallace JL, Morris GP, Krausse EJ, et al. Reduction of ethanolinduced gastric mucosal damage by cytoprotective agents: a correlated physiological and morphological study. Can J Physiol Pharmacol 1982;60:1686-99. 10. Birkhahn RH, Bellinger LL, Bernardis L, et al. The stress response in the rat from harnessing for chronic intravenous infusion. J Surg Res 1976;21:185-90. 11. Sander LD, Dudrick SJ, Johnson LR. Influence of method of feeding on stress ulcer development in the rat. Dig Dis Sci 1980;25:279-83. 12. Track NS. Gastrointestinal hormones during total parenteral nutrition. Front Horm Res 1980;7:258-64. 13. Ligumsky M, Grossman MI, Kauffman CL. Endogenous gastric mucosal prostaglandins: their role in mucosal integrity. Am J Physiol 1982;242:G337-41. 14. Cohen MM. Prostaglandin E, prevents gastric mucosal barrier damage. Gastroenterology 1975;68:876. 15. Robert A, Nezamis JE, Lancaster C, et al. Cytoprotection by prostaglandins. Gastroenterology 1979;77:433-43. 16. Cohen MM. Mucosal cytoprotection by prostaglandin EZ. Lancet 1978;2:1253-4. 17. Chaudhury TK, Robert A. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate. Dig Dis Sci 1980;25:830-6. 18. Cohen MM, Clark L. Protection against indomethacin damage to human gastric mucosa with diflunisal (abstr). Gastroenterology 1982;82:1035. 19. Cushing H. Peptic ulcers and the interbrain. Surg Gynecol Obstet 1932;55:1-33. 20. Tache Y. Simard P, Collu R. Prevention by bombesin of coldrestraint stress induced haemorrhagic lesions in rats. Life Sci 1979;24:1719-26. 21. Nemeroff CB, Hernandez DE, Orlando RC, et al. Cytoprotective effect of centrally administered neurotensin on stressinduced gastric ulcer. Am J Physiol 1982;242:G342-6.
1983;85:370-5
Chronic Mild Restraint Protects the Rat Gastric Mucosa From Injury by Ethanol or Cold Restraint JOHN L. WALLACE, Department Canada
of Surgery,
NORMAN
University
of Toronto
The effects of chronic mild restraint on the susceptibility of the rat gastric mucosa to ethanol or coldrestraint injury were studied. Gastric mucosas of animals subjected to chronic mild restraint exhibited less damage when bathed with 40% ethanol than those of control animals. This reduced damage was observed with 2 days of mild restraint and was maximal (93% less damage; p < 0.0005) when the animal was mildly restrained for 10 days. Pretreatment with indomethacin abolished the protection afforded by chronic mild restraint, suggesting a mechanism involving endogenous prostaglandin synthesis. Chronic mild restraint significantly (p < 0.05) reduced the injury to the gastric mucosa caused by cold-restraint stress. This model of mild
restraint may prove useful in future studies on the mechanism of stress-induced and other gastric mucosal lesions. The gastric mucosa is very susceptible to different forms of stress, including those associated with critical illness, head injuries, burns, or surgical trauma. Selye (2) described the appearance of hemorrhagic erosions in the gastric mucosa of rats exposed to the severe stress of immobilization. The gastric mucosa of rats can also be damaged by the direct application of substances such as ethanol and aspi-
S. TRACK, and MAX M. COHEN and Mount
Sinai
Hospital,
0 1983 by the American Gastroenterological 0016.5085/83/$3.00
Association
Ontario,
rin (3). The damaging effects of these agents can be significantly reduced by their chronic administration. Ivey et al. (4) demonstrated an increased resistance of the rat gastric mucosa to 50% ethanol when it was administered daily for 4 wk. Similar findings have been reported with chronic administration of aspirin to rats (5) and dogs (6). All of these observations are based upon the direct exposure of the gastric mucosa to a damaging agent. The purpose of this study was to determine if a similar effect could be achieved by indirect methods, in an attempt to answer the question: will the gastric mucosa become more resistant to injury if the animal is subjected to chronic mild “stress” in the form of chronic mild restraint (CMR)? The present study evolved from an assessment of the effect of total parenteral nutrition (TPN) upon the susceptibility of the rat gastric mucosa to stress ulceration. In the control group, the mild restraint caused by the TPN catheter support system (without any intravenous infusion) reduced ethanol-induced injury to the gastric mucosa. In this report, the protective effects of CMR against both ethanol and cold-restraint gastric injury are investigated further. Materials Chronic
Received October 20, 1982. Accepted February 24, 1983. Address requests for reprints to: M.M. Cohen, Suite 440, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. This work was supported by grants from the Medical Research Council of Canada (MT 5316) and Mount Sinai Institute. This work was presented in part at the American Gastroenterological Association meeting in Chicago, Illinois, in May 1982, and published in abstract form (1). The authors thank Beth Chin, Susan Chiu, and Karen Gerry for their assistance in performing these studies; Dr. G. P. Morris for preparing the micrograph, and Mrs. B. M. Kruska for secretarial assistance.
Toronto,
and Methods
Mild Restraint
Technique
Male Wistar rats (300 g) were used in all experiments, and were housed, individually, in cages with a steel mesh bottom. Under pentobarbital anesthesia (60 mg/kg body wt i.p.), an area of about 9 cm* was shaved on the back of the neck of each animal. A z-cm incision was
made along the midline and a metal ring was inserted subcutaneously. The iricision the metal ring anchored to the skin with sutures. One end of a 24-cm metal spring this metal ring (see Figure 1). The other
(2 cm diameter) was closed and four additional was attached to
end was passed through a hole in the top of the animal’s cage. A bumper was attached to the free end of the spring on the outside of the cage to prevent the animal from pulling the spring into
August
MUCOSAL PROTECTION WITH CHRONIC STRESS
1983
stress 10 days after initiation or sham operation (n = 7). Gastric
Figure
1 Schematic diagram of the chronic mild restraint apparatus. A small metal ring is inserted subcutaneously in the back of the neck of the rat. A stainless steel spring is connected to the ring through a hole in the top of the cage. The animal can move to any part of its cage without stretching the spring.
Experimental
Design
The CMR and sham-operated rats were tested for susceptibility to direct mucosal injury by ethanol in a gastric chamber model. These experiments were performed on groups (n = 5-7) of animals after 2, 5, 10, or 12 days of CMR (or sham operation). Additional experiments were performed on chambered mucosas of IO-day CMR and sham-operated (n = 9) rats in which transmucosal potential difference and various ion fluxes were determined. The CMR and sham-operated rats were tested for susceptibility to indirect mucosal injury by cold-restraint
(n = 8)
Preparation
Each rat was deprived of food for 20 h, anesthetized, and a gastric chamber was prepared according to a modification (7) of the method of Mersereau and Hinchey (8). The animal was placed on a grooved Lucite platform. An electric heating pad placed under this platform was connected to a temperature controller (YSI Instruments, Yellow Springs, Ohio), which maintained the rectal temperature of the animal at 38°C. A midline laparotomy was performed and the pylorus and esophagus were ligated, with meticulous care taken to preserve the vasculature and the vagi. The stomach was opened by making an incision along the greater curvature and pulled gently through a small aperture in a Lucite plate placed over the animal. The stomach was pinned, mucosal side up, to the top of this plate, and a Lucite chamber (11 ml volume) was clamped to the mucosa and plate. Any hair or scales ingested during grooming were removed from the stomach by gently rinsing with 37”C, 0.3 M mannitol. Solutions were then added to the chamber by syringe and allowed to bathe the mucosa for periods of 10 min. A glass paddle turning at 200 rpm kept the contents of the chamber well mixed. Ethanol
the cage. The animal was free to feed ad libitum on standard food pellets and tap water (attached to the front of the cage) and to move to any part of the cage without stretching the spring. Routinely, sham-operated rats served as the controls. In one experiment, a second control group was included. These rats had the metal ring implanted, but a spring was not attached from the ring to the cage top. A group of CMR rats (n = 5) and a sham-operated group (n = 5) were weighed and placed in individual metabolic cages. Food and water consumption and the amount of feces and urine excreted were measured at 9:OO AM daily for 9 consecutive days. Each animal was given a daily ration of 100 ml of tap water and 35 g of standard pelleted rat diet. The food and water remaining on the following day were removed and measured. At the end of the 9 days, the animals were reweighed, killed by decapitation, and the adrenal glands were removed and weighed.
Chamber
of the mild restraint
371
Injury
to ethanol injury, For assessment of susceptibility the gastric chamber experiments consisted of four lo-min periods during which IO-ml aliquots of 37°C solutions were added to the chamber. The luminal solutions for the four periods were 0.3 M mannitol during first period, 40% ethanol (vol/vol) during the second period, and 0.05 M HCl made isosmotic with mannitol during the last two periods. At the end of the last period in each experiment, the mucosa was photographed with color slide film. These slides were projected onto 21.5 x 28.0-cm paper, traced, and labeled with a code number. The damaged area was measured from these tracings by planimetry with the single observer (J.L.W.) unaware to which group the photograph related. Damaged area was expressed as the percent of glandular mucosa showing gross damage. In previous studies (g), these macroscopic assessments of damage have been found to correlate well with light microscopic determinations of damage. The effect of indomethacin pretreatment upon ethanol injury was assessed in IO-day CMR and control animals in which indomethacin (Merck, Sharp and Dohme, Kirkland, Canada) was administered (1 mg/kg body wt i.p.) 75 min before the start of the gastric chamber experiments. The indomethacin was suspended in a 0.05% carboxymethylcellulose vehicle (#lOO sterile vehicle, Upjohn Co., Kalamazoo, Mich.). Electrophysiologic
Studies
Measurements of transmucosal potential difference (PD) and sodium, potassium, and hydrogen ion flux were
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GASTROENTEROLOGY Vol. 85, No. 2
assessed with the gastric chamber technique. The protocol was extended to seven IO-min periods. Mannitol (0.3 M) was added to the chamber in the first three periods. During the fourth period, 40% ethanol (vollvol) bathed the mucosa. This was followed by three periods in which 0.05 M HCl made isosmotic with mannitol was placed in the chamber. Sodium and potassium ion concentrations were measured
in the luminal
solutions
by flame
photometry
(Corning, Medfield, Mass.). Titratable acidity was measured by diluting a l-ml aliquot of the luminal solution with 20 ml deionized water and titrating to pH 7.0 with 0.01 M NaOH. In some cases, where the pH of the sample was >7.0, a back-titration with 0.01 N HCl was performed. An automatic titration system (Radiometer, Copenhagen, Denmark) was used for these analyses. The volume of the aliquot of luminal solution was determined gravimetrically before addition to the chamber and immediately after removal. The concentration of each ion (in milliequivalents per liter) was multiplied by the volume of the aliquot (in liters) of luminal solution, thereby yielding a value in units of milliequivalents. Gastric transmucosal PD was measured throughout each experiment using a system previously described in detail (9).
Cold-Restraint
Injury
Rats were deprived of food, but not water, for 16 h. Each animal was immobilized in a wire mesh and placed in a cold room (4°C) for 4 h. This procedure was always performed by the same person to ensure consistency in the degree of immobilization. The animals were then killed by decapitation, the abdomen was opened, and the stomach was removed. An incision was made along the greater curvature and the mucosa was pinned out on a wax platform. The mucosa was rinsed of any adherent blood or mucus with ice-cold phosphate (0.1 M; pH 7.4) buffer. The mucosa was then photographed and the damaged area was measured by planimetry (as described previously). The damage index was calculated as the number of square millimeters of damaged mucosa per thousand square millimeters of glandular mucosa.
Statistical
Analyses
Data were always expressed as mean ? SEM. Statistical comparisons between control and CMR groups were performed with Student’s two-tailed t-test for unpaired data. Probability values of ~5% were considered statistically significant.
Results Chronic Mild Restraint The implantation of a metal ring in the back of the rat’s neck and its attachment by a spring coil to the top of the cage allowed free movement to any part of the cage and ad libitum feeding. Despite this freedom, CMR still caused discomfort as evidenced by persistent scratching which gradually loosened
Table
I. Effect of 9 Days of Chronic Mild Restraint Metabolic Parameters Sham-operated
Number of animals Body wt gain (g/day) Food consumed (g/day) Water consumed (ml/day) Feces (g/day] Urine (ml/day]
5.0 25.3 33.4 12.4 11.9
5 + + 5 + +
0.4 0.8 1.1 0.6 0.5
on
CMR 2.9 25.4 36.3 12.7 13.5
5 2 k f f r
0.9” 0.6 1.1”
0.5 0.5b
o p < 0.05. b p < 0.01. All data represent
mean + SEM. Each parameter (except body weight] was measured daily for 9 consecutive days. CMR, chronic mild restraint.
the metal implant. The CMR animals appeared to be more active than the sham-operated animals during the daylight hours (normally the resting period). Chronic mild restraint could not be continued beyond 12 days as some animals eventually dislodged the implant. The CMR animals had significantly (p < 0.05) lower weight gain than sham-operated animals over the g-day period (Table 1). Food consumption and the amount of feces did not differ between the two groups. The CMR rats drank an average of 2.9 ml/day more water (p < 0.05) and excreted an average of 1.6 ml/day more urine (p < 0.01) than sham-operated rats. The adrenal glands in the CMR group were
significantly (p < 0.05) heavier (39.5 * 2.3 g) than those of the sham-operated animals (34.5 2 1.5 g). Mucosas of CMR animals maintained for periods of up to 12 days appeared normal when examined macroscopically. Ethanol Injury Exposure of the chambered gastric mucosa to ethanol (and subsequent acid) produced extensive hemorrhagic lesions in the sham-operated animals. This damage was usually confined to the fundic region of the stomach. In the 27 chamber experiments performed on sham-operated rats, the damaged area was 17.5 + 2.7% of the glandular mucosa. Light microscopic examination revealed that these lesions did not penetrate the muscularis mucosae (Figure 2). Consistently less damage was induced by the application of the ethanol to chambered mucosas of CMR animals (Figure 3). A slight reduction of mucosal damage was observed with only 2 days of CMR. Damage was significantly less in both the 5- (p < 0.001) and lo- (p < 0.0005) day CMR groups compared with their control groups. With 12 days of CMR, the damage was slightly higher than in the 5or IO-day CMR groups, and was not significantly different from the corresponding control group. The significant reduction in damage with 10 days
MUCOSAL
PROTECTION
WITH
CHRONIC
STRESS
373
Metal Implant
Figure
Figure
2. Light micrograph showing border between normal and ethanol-injured mucosa of sham-operated rat. Mucosa to right of arrow appeared normal upon macroscopic examination. Mucosa to left of arrow was hemorrhagic and was scored as damaged. Note disruption of surface epithelium and the vascular engorgement. The necrosis does not penetrate the muscularis mucosae (m). This section was taken from a sample of fundic mucosa fixed at the end of a gastric chamber experiment. (Toluidine blue, x40.)
of CMR was not observed in rats in which the metal ring was implanted (n = 5) without a spring coil connecting it to the cage top (Figure 4). The mucosas of lo-day control and CMR rats pretreated with indomethacin appeared normal when examined macroscopically. Application of ethanol to the chambered mucosas of these indomethacin-treated rats produced hemorrhagic lesions involving 23.6 -+ 6.6% (control group) and 33.5 k
4. Ethanol injury to chambered gastric mucosas of chronic mild restraint and sham-operated animals. Each column represents the mean 2 SEM percent of glandular mucosa damaged after bathing with 40% ethanol and acid. These experiments were performed 10 days after the sham operation (n = 5), initiation of chronic mild restraint (n = 6), or implantation of the metal ring (n = 5). *p < 0.001.
9.7% (CMR group) of the glandular mucosa. These areas of damage were not significantly different from each other or from the areas of damage observed in the sham-operated or metal-implant control groups which were not pretreated with indomethacin (Figure 4). Electrophysiologic
In the sham-operated animals in which PD and ion fluxes were measured, the ethanol injury involved 16.9 t 5.0% of the glandular mucosa. The damage to the mucosas of CMR animals was 5.3 * 1.8%. Despite this significant (p < 0.05) reduction in gross damage, the electrophysiologic parameters showed no marked differences. Potential difference values (Figure 5) for the two groups were not significantly different, although baseline PD values were higher in the control group. Both groups exhibited the same marked fall in PD in response to ethanol. Similarly, the ion flux data (Figure 6) did not differ greatly between the two groups. In both groups there was increased loss of titratable acidity and increased efflux of sodium and potassium ions in response to the application of ethanol. Cold-Restraint
Duration of CMR (days) Figure
of chronic mild restraint on ethanol 3. Effect of duration injury. Each column represents the mean ‘- SEM percent of glandular mucosa damaged after bathing with 40% ethanol and acid. Numbers in brackets represent the size of each group. * p < 0.001, ** p < 0.0005.
Studies
Injury
Exposure of immobilized sham-operated animals to cold (4°C) for 4 h resulted in the production of extensive hemorrhagic lesions in the fundic mucosa [Table 2). Ten-day CMR animals developed about half as many mucosal lesions. The lesion area of CMR animals was significantly (p < 0.05) less than that observed in the mucosa of sham-operated animals.
374
WALLACE
ET AL
GASTROENTEROLOGY
-6O-
F
c.
-5o-
8
s
-4o-
I/
-3o-
I!! E g
-2o-
?? -e
Sham
O-----O
CMR
85, No. 2
r
I
4.
/-
/ 00
k E n
1
I
_/” II
+40
Acid
Ethanol
Mannitol
Vol.
*P
n-I
1
pl’
-60 1
-60 L
a
\
1 60
10
20
30
40
50
60
70
60
Ii
Time (min) Figure
3 3 5
5. Transmucosal
potential difference across chambered gastric mucosas of lo-day chronic mild restraint and sham-operated rats. Each point represents the mean k SEM for a 5-min interval (n = 5-B). There are no significant differences during any 5-min interval. Solutions bathing the mucosa are indicated at the top of the figure.
f z zi =
40
20
Discussion It is well established that severe stress damages the gastric mucosa. The present study provides evidence that CMR protects the gastric mucosa from both direct (40% ethanol) and indirect (cold-restraint] forms of stress. The resistance to ethanol injury was maximal in lo-day CMR animals. The reduction in ethanol injury was less when CMR was extended to 12 days. It is possible that this diminished “protection” may be partially due to the looseness of the metal implant after 12 days. It is clear that all rat TPN models require catheter support. These support systems undoubtedly exert
Table
2.
Comparison
of Cold-Restraint
in IO-Day Chronic Operated Rats
Sham-operated Number of animals Lesions/stomach Damage index
Gastric
Mild Restraint
Damage
and ShamCMR
7
8
6.0 + 1.7
3.1 2 1.1
10.2
2 4.4
3.3 *
1.3”
” p < 0.05. All data represent mean + SEM. Animals were subjected to cold-restraint stress for 4 h after 10 days of chronic mild restraint (CMR) or sham operation. Damage index represents the number of square millimeters of damaged mucosa per thousand square millimeters of glandular mucosa.
Mannitol
,
I 1
Figure
Ethanol
1
Acid
I
I
2 3 4 5 6 Time Period (10 min. each)
I
1 7
6. Net hydrogen sodium, and potassium flux across chambered gastric mucosas of to-day chronic mild restraint and sham-operated rats. Each column represents the mean 2 SEM flux for the lo-minute application period. Positive flux denotes a net gain in the luminal solution. *p < 0.05
some degree of stress. Birkhahn et al. (10) examined the effect of the restraint caused by a TPN harness system on food intake and body weight gain in rats. Their findings, similar to those reported here, were reduced weight gain with normal food intake. Sander et al. (11) used a similar TPN harness in their study of the effects of parenteral vs. oral feeding on susceptibility to cold-restraint-induced gastric injury, but did not examine the effects of the harness system itself. The CMR technique used in this study was derived from another TPN model (12). The consistent reduction in weight gain of CMR rats and the enlargement of adrenal glands in these animals
August
1983
demonstrates the ability of this technique to induce chronic stress. This stress, so mild that it did not upset the overall well being of the rat, reduced susceptibility to gastric mucosal injury. Although CMR significantly reduced the area of mucosa that became hemorrhagic when bathed with ethanol and acid, no significant differences in electrophysiologic parameters were observed. These findings are similar to a previous study using this gastric chamber model (9). In that study, topically applied prostaglandin E2 was found to significantly reduce the ethanol injury (gross damage) without reducing the alterations in ion fluxes or PD. It has also been observed that similar changes in ion fluxes and PD occur when the canine gastric mucosa is bathed by acetylsalicylic acid or salicylic acid, substances that have very different effects on prostaglandin synthesis and, presumably, on mucosal integrity (13). It is likely that some damage was present even in the most protected of CMR mucosas. This damage (destruction of surface epithelial cells] would cause significant alterations in electrophysiologic parameters (as observed) not necessarily distinguishable from the changes caused by extensive mucosal damage. The protective mechanism(s) of CMR is yet to be determined. The observation that indomethacin pretreatment abolished CMR protection suggests a role for endogenous prostaglandin synthesis. Exogenous prostaglandins have been shown in both animals (l4,15) and in humans (16)to be capable of preventing gastric mucosal damage induced by a variety of agents. This phenomenon has been termed “cytoprotection.” More recently, it has been shown that bathing the rat mucosa with a “mild irritant” protected it from subsequent injury by a necrotizing agent and, as this protection was abolished by indomethacin, it was deduced to be due to the stimulation of endogenous prostaglandin synthesis (17).A similar phenomenon has been observed in humans (18).These experiments, and those now reported, all support the concept that prostaglandin protection of the gastric mucosa is a physiologic phenomenon. The observation that the adrenal glands of CMR animals were enlarged and the known importance of the adrenal gland in the stress response (2) suggest that it plays a role in the protective mechanism of CMR. Also, the effect on the stomach of CMR is indirect (i.e., no manipulation of the gastric mucosa), suggesting the involvement of the central nervous system. The importance of the brain, in particular the hypothalamus, in the stress response has been recognized since Cushing described gastric damage in association with intracranial lesions (19). Recently, it was reported that central administration of bombesin (20) or neurotensin (21) protected the
MUCOSAL
PROTECTION
WITH
CHRONIC
STRESS
375
gastric mucosa from cold-restraint injury. Further studies are required to elucidate the roles of regulatory peptides and the adrenal gland in the CMR protective mechanism.
References 1. Wallace
JL, Cohen MM. Chronic minimal restraint in rats: a model for “adaptive cytoprotection”? (abstr). Gastroenterology 1982;82:1259. Book 2. Selye H. The stress of life. New York: McGraw-Hill Company Inc., 1956. 3. Brodie DA. Experimental peptic ulcer. Gastroenterology 1968; 55:125-34. 4. Ivey KJ, Tarnawski A, Stachura J, et al. The induction of mucosal tolerance to alcohol by chronic administration. J Lab Clin Med 1980;96:922-32. 5. St. John DJB, Yeomans ND, McDermott FT, et al. Adaptation of the gastric mucosa to repeated administration of aspirin in the rat. Am J Dig Dis 1973;18:881-6. 6. Bolton JP, Cohen MM. Effect of repeated aspirin administration on the gastric mucosal barrier and cell turnover. J Surg Res 1977;23:251-6. 7. Morris GP, Wallace JL. The roles of ethanol and of acid in the production of gastric mucosal erosions in rats. Virchows Arch (Cell Pathol) 1981;38:23-38. 8. Mersereau WA, Hinchey EJ. Effects of gastric acidity on gastric ulceration induced by haemorrhage in the rat, utilizing a gastric chamber technique. Gastroenterology 1973;64:11305. 9. Wallace JL, Morris GP, Krausse EJ, et al. Reduction of ethanolinduced gastric mucosal damage by cytoprotective agents: a correlated physiological and morphological study. Can J Physiol Pharmacol 1982;60:1686-99. 10. Birkhahn RH, Bellinger LL, Bernardis L, et al. The stress response in the rat from harnessing for chronic intravenous infusion. J Surg Res 1976;21:185-90. 11. Sander LD, Dudrick SJ, Johnson LR. Influence of method of feeding on stress ulcer development in the rat. Dig Dis Sci 1980;25:279-83. 12. Track NS. Gastrointestinal hormones during total parenteral nutrition. Front Horm Res 1980;7:258-64. 13. Ligumsky M, Grossman MI, Kauffman CL. Endogenous gastric mucosal prostaglandins: their role in mucosal integrity. Am J Physiol 1982;242:G337-41. 14. Cohen MM. Prostaglandin E, prevents gastric mucosal barrier damage. Gastroenterology 1975;68:876. 15. Robert A, Nezamis JE, Lancaster C, et al. Cytoprotection by prostaglandins. Gastroenterology 1979;77:433-43. 16. Cohen MM. Mucosal cytoprotection by prostaglandin EZ. Lancet 1978;2:1253-4. 17. Chaudhury TK, Robert A. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate. Dig Dis Sci 1980;25:830-6. 18. Cohen MM, Clark L. Protection against indomethacin damage to human gastric mucosa with diflunisal (abstr). Gastroenterology 1982;82:1035. 19. Cushing H. Peptic ulcers and the interbrain. Surg Gynecol Obstet 1932;55:1-33. 20. Tache Y. Simard P, Collu R. Prevention by bombesin of coldrestraint stress induced haemorrhagic lesions in rats. Life Sci 1979;24:1719-26. 21. Nemeroff CB, Hernandez DE, Orlando RC, et al. Cytoprotective effect of centrally administered neurotensin on stressinduced gastric ulcer. Am J Physiol 1982;242:G342-6.