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Effects of scopolamine methylbromide on shock-induced gastric lesions in the unrestrained rat
Physiology and Behavior, Vol. 13, pp. 147-151. Brain Research Publications Inc., 1974. Printed in the U.S.A.
BRIEF COMMUNICATION Effects of Scopolamine Methylbromide on Shock-induced Gastric Lesions in the Unrestrained Rat R I C H A R D L. SEISER AND VINCENT P. HOUSER 1
Psychotropic Drug Laboratory, Veterans Administration Hospital, Perry Point, MD 21902
(Received 28 December 1973) SEISER, R. L. AND V. P. HOUSER. Effects of scopolamine methyibromide on shock-induced gastric lesions in the unrestrained rat. PHYSIOL. BEHAV. 13(1) 147-151, 1974. - Gastric lesions were produced in unrestrained rats subjected to a 6 hr shock stress session. A perch-contingent yoked design compared the degree of gastric pathology exhibited in animals subjected to an avoidance-avoidance conflict, with yoked animals receiving equivalent amounts of noncontingent shock. There were no differences in the amount of gastric lesion formation produced by these two procedures. Scopolamine methylbromide (0.50 and 1.0 mg/kg) significantly decreased ulcer development (i.e., percentage of animals exhibiting pathology, number of lesions per animal and severity of lesioning) in all groups tested. Gastric lesions
Unrestrained rat
Avoidance-avoidance conflict
Sc°polamine methylbromide
berg [ I I ] report that atropirac agents incompletely inhibit both vagal and gastrin provoked gastric secretions. Other reports [10] indicate that anticholinergics mimic a vagal resection, thereby reducing gastric secretion in volume and total acid content. Scopolamine methylbromide, a quaternary ammonium derivative o f scopolamine, possesses more potent parasympathetic blocking action than scopolamine and affects gastric secretion more selectively [ 15 ]. The actions of this synthetic agent are peripheral in contrast to the central actions of scopolamine [5 ] and its effects are more prolonged [10,15]. Previous experimentation [12] has indic a t e d t h a t s c o p o l a m i n e methylbromide significantly decreases gastric volume and total acid. The present work was undertaken in an a t t e m p t to determine whether the animal model described by Wald et aL [15,16] is able to produce significant gastric pathology in the unrestrained rat in a six-hour period. Secondly, an a t t e m p t was made to see if the introduction of a psychological variable (i.e., an avoidance-avoidance conflic4) would enhance the severity of the observed pathology. Finally, since scopolamine methylbromide is known to reduce acid output, it seemed advisable to explore whether this anticholinergic could reduce the severity of gastric ulceration in a dose related manner.
PREVIOUS reports in the literature indicate that psychological variables may intensify the physiological response to stress and may directly contribute to an increase in gastric pathology [13, 16, 17, 18, 19]. The present work is an a t t e m p t to evaluate the ulcerogenic properties of one such psychological variable (i.e., an avoidance-avoidance conflict). This investigation made use of an experimental paradigm developed by Wald and his colleagues [16,17] which administers equivalent amounts of electric shock to pairs o f animals in either a contingent or y o k e d procedure. The above design has been previously reported to rapidly produce gastric lesions (i.e., in 6 hr) in the unrestrained rat [16,17]. This technique thus provides an excellent functional model for the investigation of the ulcer inhibiting properties o f various pharmaceutical agents. The present report investigated the ulcer inhibiting effects of a peripheral acting anticholinergic, scopolamine methylbromide. The presence of hydrochloric acid is essential for gastric lesion development. Lesions may develop in stomachs secreting small quantities of acid, but not in stomachs totally absent of acid [6]. Since the primary phase of gastric secretion is under vaga] control, derivates o f the belladonna alkaloids, atropine and scopolamine, have been employed to block the effects of parasympathetic stimulation upon the stomach. Miiller-Wieland and Ossen-
J The authors wish to thank Mrs. Frances L. Houser for typing the manuscript, and Mr. Luther R. Gilliam, Chief, Medical Illustration Service, VA Hospital, Perry Point, Maryland who did the photographic work. 147
148
SEISER AND HOUSER EXPERIMENT 1 METHOD
Animals and Apparatus Animals for this study consisted of 50 mate albino rats of the Sprague-Dawley strain obtained from A.R.S. SpragueDawley Company,. Madison, Wisconsin. All animals were 48 hx food deprived at the start of the experimental session. They ranged in weight from 1 0 2 - 1 7 8 g just prior to the initiation of the stress sessions (i.e., after 48 hr of food deprivation). The animals were housed and tested under a reverse light-dark cycle (i.e., 12 hr illumination and 12 hr darkness) with the dark portion of this schedule occurring between 9:15 a.m. and 9:15 p.m. The experimental chambers consisted of 4 clear Plexiglas perch cages measuring 16-1/2 x 15-I/2 x 12 in. A perch platform 7 x 3 - I / 2 x 3 inches was elevated in the center of a 30-rod stainless steel grid floor which was wired for shock. The perch platform floor consisted o f 15 stainless steel rods. The grid rods of both the main floor and perch platform were placed at 1/2 in. intervals. The animals were fitted with tail electrodes which served to complete the electrical circuit. The tail electrodes were fabricated from a machine screw connected to a straight, heavily insulated, stranded copper wire and stainless steel plug. The head of the machine screw was held firmly against the base of the tail by Velcro strip adhesive and electrical tape. Standard electromechanical scheduling equipment was located in an adjacent hallway which automatically presented electric shock to the animals. Extraneous laboratory noises were masked by 90 dB o f white noise presented via a speaker mounted on the corner wall of the e x p e r i m e n t a l room. Current leakage through the cages was held to a minimum by insuring consistent temperature and humidity conditions in the experimental room and by thoroughly cleansing each cage after all sessions. The four perch cages were divided into two separate pairs (i.e., each pair consisting o f one experimental and one yoked cage) and each pair was wired in series to a separate Lafayette A-615A a.c. constant current stimulator. The main floor grid of the experimental cage was continuously electrified with 1.2 mA of current. The perch platform grid of the experimental cage was electrified intermittently for a duration of 2 sec (i.e., 1.2 mA) on a 60-see variable interval schedule. Thus, an experimental animal could escape the continuous main floor shock by climbing onto the perch, but was then subjected to 2 see of shock for every minute it remained on the perch. The y o k e d animal was wired in series with the experimental animal so that it received identical amounts o f shock, irrespective of its location within the yoked cage (i.e., either on the main grid floor or on the perch of the y o k e d cage). Procedure The animals were initially group housed with ad lib food and water immediately after they arrived from the supplier for a minimum o f 2 days. The animals were then subjected to a 48-hr food deprivation period prior to the stress session. Following deprivation, 50 animals were weighed and randomly assigned to 5 separate 10-animal groups: (1) experimental-saline; (2) y o k e d - s a l i n e ; (3) experim e n t a l - 0 . 1 2 5 mg/kg scopolamine methylbromide; (4) y o k e d - 0 . 1 2 5 mg/kg scopolamine methylbromide; and (5)
n o - s h o c k c o n t r o l - s a l i n e . Balancing procedures were employed in the assignment of taft electrodes and cages to compensate for possible inherent biases within the electrical apparatus. Electrode paste was applied between the electrode and the animal's tail to insure a good electrical contact. All drugs were dissolved in physiological saline (0.9%) and administered intraperitoneally in a volume of 1.0 ml/kg. The first injection occurred immediately preceding shaping procedures (8:00 a.m.), while the second injection occurred immediately following the termination of the stress session (2:30 p.m.). The sessions consisted of 6 hr of experimental, y o k e d or no-shock control procedures. Shaping procedures were performed one-half hr prior to the onset of the 6-hr stress session. Shaping consisted of physically placing the experimental animal onto the unshocked perch platform whenever he came into contact with the grid floor. Shaping (8:00 a.m.) was initially begun at 0.5 mA to prevent premature exhaustion of the animals. Following initial shaping to the complete safety of the perch (8:10 a.m.), the intermittent shock (0.5 mA) contingency was introduced to the perch. Shaping procedures were repeated to insure that the escape response (i.e., climbing onto the perch) would not be extinguished when shock was intermittently introduced to the perch. Current levels were then incremented: 8:20 a.m. - 0.75 mA; 8:30 a.m. - 1.0 mA; and 8:45 a.m. - 1.2 mA, where it remained for the rest of the 6 hr session. During shaping procedures, undesirable behaviors, such as taft electrode climbing, were extinguished by gently shaking the animal back to the perch. Following the termination of the stress session, the animals were again weighed and returned to their home cages. The animals were permitted free access to water for the 2-hr poststress delay period prior to being sacrificed. AU animals were sacrificed by exposure to chloroform. Their stomachs were then excised and inspected for ulceration. Extent of ulceration was measured by a scale of hundredths of an inch located within the eyepiece of a dissecting microscope. Ulceration was determined in measurements of percentage of rats exhibiting pathology, number of lesions, and ulcer score (i.e., length plus width of lesions in hundredths of an inch). The criterion for gastric lesioning was an obvious erosion or absence of mucosal tissue accompanied by hemorraging at the site of the lesion. Gastric lesions were characterized by a deterioration of epithelial cells extending through the mucosal lining. All evaluations concerning the extent of gastric lesions were confirmed through a blind procedure in which information as to which groups were shocked or drugged was not made available to the judge. RESULTS AND DISCUSSION Statistical analyses using the Mann-Whitney U test [14] indicated that none of the groups in Experiment l demonstrated significant amounts of stomach ulceration. This indicated the inability of the technique to consistently produce ulceration. Although no significant differences were noted, the data suggested an inverse relationship between body weight and occurrence of gastric lesions as has been reported earlier [ l ] . The few lesions which were observed in the shock groups principally appeared in the lightest weight animals. These findings suggest that body weight may have been an ulcerogenic factor operating in
METHYL SCOPOLAMINE AND ULCER FORMATION this technique. Furthermore, a previous report [4] has indicated that shock intensity significantly affects gastric ulceration in both frequency of occurrence and number of ulcers per animal. Thus, the higher the shock intensity, the greater the exhibited pathology. These results may suggest that body weight could interact with shock intensity to produce stomach ulceration. Finally, the results of Experiment 1 indicated that the two drug groups demonstrated a slight (nonsignificant) reduction in ulceration, suggesting that a higher dosage of this agent might be able to inhibit the development of stomach lesions. The second experiment was conducted to investigate the above speculations. EXPERIMENT
2
METHOD Animals and A pparatus
Animals for Experiment 2 consisted of 80 male SpragueDawley albino rats obtained from the previously mentioned supplier. They ranged in weight from 9 7 - 2 1 6 g at the beginning of the shock sessions. The data from the 10 noshock control animals of Experiment 1 were again used as the control data for Experiment 2. The experimental chambers and apparatus for this study were identical to those utilized in the first experiment. Procedure
The general procedures used throughout this experiment concerning treatment of animals, deprivation, and drug administration were identical to those outlined in Experiment 1. Following deprivation, the rats were weighed and assigned to eight I 0-animal weight matched groups. These 8 groups consisted of experimental and yoked saline groups and 6 drug groups. The drug groups were composed of an experimental and yoked group for each of 3 dosages (i.e., 0.125, 0.50 and 1.0 mg/kg) of scopolamine methylbromide. Since drug and saline injections were given immediately preceding and following the drug sessions, the effective dosages available to the various groups were actually 0.25, 1.0 and 2.0 mg/kg. T h e s h a p i n g procedures were identical to those employed in the first experiment except that shock intensity was adjusted proportionally to the body weights of the animals. Animals weighing 97 to 125 g received 1.2 mA; animals weighing 126 to 150 g received 1.4 mA; and animals weighing 151 to 216 g received 1.6 mA. Extreme care was exercised to insure that body weights were identical for all 9 groups of animals. All 9 groups had 4 animals in the lowest weight category, 2 in the middle category and 4 in the heaviest category. RESULTS The data for Experiment 2 consisted of the number of animals per group demonstrating pathology, number of ulcers per animal, and ulcer score per animal (i.e., length plus width of lesions in hundredths of an inch). As in Experiment I, all gastric lesions were observed in the glandular fundus of the stomach. All rumenal tissue was free of pathology. A Kruskal-Wallis one-way analysis of variance [14] was performed on the ulcer score and number of ulcers per animal data. It indicated that significant differences (p<0.001) existed between the various groups. Multiple statistical comparisons were then carried
149 out using the Mann-Whitney U test [14]. These comparisons indicated that there were no significant differences between any of the experimental and yoked groups in mean number of lesions and ulcer score (i.e., size of lesion). Since there were no experimental-yoked differences, these two groups were pooled within drug dosage and saline conditions. This procedure produced one 10-animal noshock control group and four 20-animal shock groups (i.e., saline, 0.125 mg/kg, 0.50 mg/kg, and 1.0 mg/kg scopolamine methylbromide). Further statistical analyses using the Mann-Whitney U test [I 4 ] were then performed on the number of ulcers and ulcer score data. The results of these analyses are summarized in Fig. I. The comparisons in Fig. 1 revealed a significant difference between the no-shock control and saline (shock) group (p<0.002) in mean number of ulcers per animal and mean ulcer score. A significant difference also occurred between the no-shock control and scopolamine methylbromide 0.125 mg/kg group (p
150
SEISER AND HOUSER
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CONTROL SALINE M SCOP M SCOP M StOP NO SHOCK .125 .5 1.0 SHOCK mg/kg mg/kg mg/kg I I SHOCK SHOCK SHOCK I pc.002 I I
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FIG. 1. Sumnutry of gastric pathology demomtrated by the various drug, saline or control groups. Degree of gastric pathology is shown in 3 separate mea~ares: % of aninuds showing pathology, mean number of ulcers per animal and mean ulcer score. Results of the MannWhitney U Tests are shown at the bottom of this figure. degree of physical exhaustion and severity of gastric pathology. Smaller animals would be expected to become exhausted sooner than heavier, more mature animals. Heavier animals, in turn, would require more intense shocks
to produce equivalent amounts of physical exhaustion and, thus, similar degrees of stomach pathology. The data presented in Fig. 1. demonstrates a doseresponse relationship regarding the ulcer inhibiting qualities of scopolamine methylbromide. The two higher dosages (i.e., 0.50 and 1.0 mg/kg) significantly decreased gastric ulcer development while the lower dosage did not. These results suggest that this particular anticholinergic may inhibit the physiological mechanisms involved in gastric ulcer formation. Future research utilizing the present design should center upon investigating other cholinergic agents, in particular the cholinomimetics, to determine if enhanced cholinergic tone may increase the severity of gastric pathology. The above conclusions rest on the assumption that scopolamine methylbromide was able to exert its antiulcerogenic effects directly on the stomach by interfering with physiological processes (i.e., acid secretion) that are necessary to produce gastric ulceration. It is possible, however, that this agent was able to produce its effects indirectly by some other process. One possibility concerns the a b i l i t y o f anticholinergJcs to produce drug-induced analgesia. If the drug was able to reduce the severity of the stress (i.e., pain) perceived during the 6-hr session, it could explain why the drugged animals demonstrated fewer uJcers. Recent research from this laboratory [7,8], however, has indicated that both scopolamine hydrobromide and scopolamine methylbromide have no significant effects on the aversive threshold as measured in rats or monkeys. Therefore, it is unlikely that drug-induced analgesia can account for the present results. Another possibility concerns the ability of scopolamine methylbromide to affect avoidance acquisition on the perch. Since no measure of lime spent on the perch was available, it is possible that the drug was able to reduce stomach ulceration by enhancing the acquisition and/or performance of the avoidance response, thus reducing the total amount of shock received. Although this explanation is possible, it is unlikely, since this particular drug does not readily cross the blood brain barrier [5] and thus fails to affect a wide range of behaviors [ 2 ] including shock avoidance [8,9]. Furthermore, all drug and saline groups in both experiments showed similar rates of avoidance acquisition during shaping procedures. It would therefore seem more likely that this agent produced its antiulcer effect primarily through its peripheral actions (i.e., inhibiting vagally controlled acid secretion).
REFERENCES
1. Brodie, D. A. and A. M. Hanson. A study of factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38: 353-360, 1960. 2. Carlton, P. L. Brain acetylcholine and inhibition. In: Reinforcement and Behavior, edited by J. Tapp. New York: Academic Press, 1969, pp. 286-327. 3. Des/derato, O., J. R. MacKinnon and H. I-lissom.Development of gastric ulcers in rats following stress termination. J. comp. physioL Psychol. 1974, in press. 4. Gliner, J. A. Predictable vs unpredictable shock: preference behavior and stomach ulceration. Physiol. Behav. 9: 693-698, 1974. 5. Goodman, L. S. and A. Gilman. The Pharmacological Basis o f Therapeutics. New York: The Macmillan Company, 1965, pp. 83.
6. Grassman, M. I. No acid, no hemorrhage? Gastroenterology 57: 367, 1969. 7. Houser, V. P. and F. L. Houser. The alteration of aversive thresholds with choIinergic and adrenergic agents. Pharmac. ~'ochem. Behav. 1: 433-444, 1973. 8. Houser, V. P. and D. A. Van Hart. The effects of scopolamine and pilocarpine upon the aversive threshold of the rat. Pharmac. Biochem. Behav. 1: 427-431, 1973. 9. Houser, V. P. and F. L. Houser. The effects of agents that modify muscarinic tone upon behavior controlled by an avoidance schedule that employs signaled unavoidable shock. Psychopharmacologia (Bed) 32:133-150, 1973.
METHYL SCOPOLAMINE AND ULCER FORMATION 10. lnnes, I. R. and M. Nickerson. Drugs inhibiting the action of acetylcholine on structures innervated by postganglionic parasympathetic nerves. In: The Pharmacological Basis o f Therapeutics, edited by L. Goodman and A. Gilman. New York: The Macmillan Company, 1965, pp. 521-543. 11. Mfiller-Wieland, K. and F. W. Ossenberg. Medical management and treatment of duodenal ulcer. In: Advances in Psychosomatic Medicine, Vol. 6, edited by H. Weiner. New York: S. Karger, 1961, pp. 152-165. 12. Robert, A. and J . E . Nezamis. Effect of an anti-acetylcholine drug, methscopolamine bromide, on ulcer formation and gastric mucus. J. Pharm. Pharmac. 6: 690-695, 1964. 13. Sawrey, W. L., J. J. Conger and E. S. Turrell. An experimental investigation of the role of psychological factors in the production of gastric ulcers in rats. Y. comp. physiol. Psychol. 49: 457-461, 1956.
151 14. Siegel, S. Nonparametric Statistics. New York: McGraw-Hill Book Company, 1956, pp. 116-127. 15. Sollmann, T. A Manual o f Pharmacology. New York: W. B. Saunders Company, 1957, p. 400. 16. Wald, E. D. Endocrinological correlates of stress-induced ulcers. Unpublished Master's Thesis, Connecticut College, 1972. 17. Wald, E. D., J. R. MacKinnon and O. Desiderato. Production of gastric ulcers in the unrestrained rat. Physiol. Behav. 10: 825-827, 1973. 18. Weiss, J. M. Effects of coping responses on stress. J. comp. physiol. PsychoL 65: 251-260, 1968. 19. Weiss, J. M. Somatic effects of predictable and unpredictable shock. Psychosom. Med. 32: 397-404, 1970.
BRIEF COMMUNICATION Effects of Scopolamine Methylbromide on Shock-induced Gastric Lesions in the Unrestrained Rat R I C H A R D L. SEISER AND VINCENT P. HOUSER 1
Psychotropic Drug Laboratory, Veterans Administration Hospital, Perry Point, MD 21902
(Received 28 December 1973) SEISER, R. L. AND V. P. HOUSER. Effects of scopolamine methyibromide on shock-induced gastric lesions in the unrestrained rat. PHYSIOL. BEHAV. 13(1) 147-151, 1974. - Gastric lesions were produced in unrestrained rats subjected to a 6 hr shock stress session. A perch-contingent yoked design compared the degree of gastric pathology exhibited in animals subjected to an avoidance-avoidance conflict, with yoked animals receiving equivalent amounts of noncontingent shock. There were no differences in the amount of gastric lesion formation produced by these two procedures. Scopolamine methylbromide (0.50 and 1.0 mg/kg) significantly decreased ulcer development (i.e., percentage of animals exhibiting pathology, number of lesions per animal and severity of lesioning) in all groups tested. Gastric lesions
Unrestrained rat
Avoidance-avoidance conflict
Sc°polamine methylbromide
berg [ I I ] report that atropirac agents incompletely inhibit both vagal and gastrin provoked gastric secretions. Other reports [10] indicate that anticholinergics mimic a vagal resection, thereby reducing gastric secretion in volume and total acid content. Scopolamine methylbromide, a quaternary ammonium derivative o f scopolamine, possesses more potent parasympathetic blocking action than scopolamine and affects gastric secretion more selectively [ 15 ]. The actions of this synthetic agent are peripheral in contrast to the central actions of scopolamine [5 ] and its effects are more prolonged [10,15]. Previous experimentation [12] has indic a t e d t h a t s c o p o l a m i n e methylbromide significantly decreases gastric volume and total acid. The present work was undertaken in an a t t e m p t to determine whether the animal model described by Wald et aL [15,16] is able to produce significant gastric pathology in the unrestrained rat in a six-hour period. Secondly, an a t t e m p t was made to see if the introduction of a psychological variable (i.e., an avoidance-avoidance conflic4) would enhance the severity of the observed pathology. Finally, since scopolamine methylbromide is known to reduce acid output, it seemed advisable to explore whether this anticholinergic could reduce the severity of gastric ulceration in a dose related manner.
PREVIOUS reports in the literature indicate that psychological variables may intensify the physiological response to stress and may directly contribute to an increase in gastric pathology [13, 16, 17, 18, 19]. The present work is an a t t e m p t to evaluate the ulcerogenic properties of one such psychological variable (i.e., an avoidance-avoidance conflict). This investigation made use of an experimental paradigm developed by Wald and his colleagues [16,17] which administers equivalent amounts of electric shock to pairs o f animals in either a contingent or y o k e d procedure. The above design has been previously reported to rapidly produce gastric lesions (i.e., in 6 hr) in the unrestrained rat [16,17]. This technique thus provides an excellent functional model for the investigation of the ulcer inhibiting properties o f various pharmaceutical agents. The present report investigated the ulcer inhibiting effects of a peripheral acting anticholinergic, scopolamine methylbromide. The presence of hydrochloric acid is essential for gastric lesion development. Lesions may develop in stomachs secreting small quantities of acid, but not in stomachs totally absent of acid [6]. Since the primary phase of gastric secretion is under vaga] control, derivates o f the belladonna alkaloids, atropine and scopolamine, have been employed to block the effects of parasympathetic stimulation upon the stomach. Miiller-Wieland and Ossen-
J The authors wish to thank Mrs. Frances L. Houser for typing the manuscript, and Mr. Luther R. Gilliam, Chief, Medical Illustration Service, VA Hospital, Perry Point, Maryland who did the photographic work. 147
148
SEISER AND HOUSER EXPERIMENT 1 METHOD
Animals and Apparatus Animals for this study consisted of 50 mate albino rats of the Sprague-Dawley strain obtained from A.R.S. SpragueDawley Company,. Madison, Wisconsin. All animals were 48 hx food deprived at the start of the experimental session. They ranged in weight from 1 0 2 - 1 7 8 g just prior to the initiation of the stress sessions (i.e., after 48 hr of food deprivation). The animals were housed and tested under a reverse light-dark cycle (i.e., 12 hr illumination and 12 hr darkness) with the dark portion of this schedule occurring between 9:15 a.m. and 9:15 p.m. The experimental chambers consisted of 4 clear Plexiglas perch cages measuring 16-1/2 x 15-I/2 x 12 in. A perch platform 7 x 3 - I / 2 x 3 inches was elevated in the center of a 30-rod stainless steel grid floor which was wired for shock. The perch platform floor consisted o f 15 stainless steel rods. The grid rods of both the main floor and perch platform were placed at 1/2 in. intervals. The animals were fitted with tail electrodes which served to complete the electrical circuit. The tail electrodes were fabricated from a machine screw connected to a straight, heavily insulated, stranded copper wire and stainless steel plug. The head of the machine screw was held firmly against the base of the tail by Velcro strip adhesive and electrical tape. Standard electromechanical scheduling equipment was located in an adjacent hallway which automatically presented electric shock to the animals. Extraneous laboratory noises were masked by 90 dB o f white noise presented via a speaker mounted on the corner wall of the e x p e r i m e n t a l room. Current leakage through the cages was held to a minimum by insuring consistent temperature and humidity conditions in the experimental room and by thoroughly cleansing each cage after all sessions. The four perch cages were divided into two separate pairs (i.e., each pair consisting o f one experimental and one yoked cage) and each pair was wired in series to a separate Lafayette A-615A a.c. constant current stimulator. The main floor grid of the experimental cage was continuously electrified with 1.2 mA of current. The perch platform grid of the experimental cage was electrified intermittently for a duration of 2 sec (i.e., 1.2 mA) on a 60-see variable interval schedule. Thus, an experimental animal could escape the continuous main floor shock by climbing onto the perch, but was then subjected to 2 see of shock for every minute it remained on the perch. The y o k e d animal was wired in series with the experimental animal so that it received identical amounts o f shock, irrespective of its location within the yoked cage (i.e., either on the main grid floor or on the perch of the y o k e d cage). Procedure The animals were initially group housed with ad lib food and water immediately after they arrived from the supplier for a minimum o f 2 days. The animals were then subjected to a 48-hr food deprivation period prior to the stress session. Following deprivation, 50 animals were weighed and randomly assigned to 5 separate 10-animal groups: (1) experimental-saline; (2) y o k e d - s a l i n e ; (3) experim e n t a l - 0 . 1 2 5 mg/kg scopolamine methylbromide; (4) y o k e d - 0 . 1 2 5 mg/kg scopolamine methylbromide; and (5)
n o - s h o c k c o n t r o l - s a l i n e . Balancing procedures were employed in the assignment of taft electrodes and cages to compensate for possible inherent biases within the electrical apparatus. Electrode paste was applied between the electrode and the animal's tail to insure a good electrical contact. All drugs were dissolved in physiological saline (0.9%) and administered intraperitoneally in a volume of 1.0 ml/kg. The first injection occurred immediately preceding shaping procedures (8:00 a.m.), while the second injection occurred immediately following the termination of the stress session (2:30 p.m.). The sessions consisted of 6 hr of experimental, y o k e d or no-shock control procedures. Shaping procedures were performed one-half hr prior to the onset of the 6-hr stress session. Shaping consisted of physically placing the experimental animal onto the unshocked perch platform whenever he came into contact with the grid floor. Shaping (8:00 a.m.) was initially begun at 0.5 mA to prevent premature exhaustion of the animals. Following initial shaping to the complete safety of the perch (8:10 a.m.), the intermittent shock (0.5 mA) contingency was introduced to the perch. Shaping procedures were repeated to insure that the escape response (i.e., climbing onto the perch) would not be extinguished when shock was intermittently introduced to the perch. Current levels were then incremented: 8:20 a.m. - 0.75 mA; 8:30 a.m. - 1.0 mA; and 8:45 a.m. - 1.2 mA, where it remained for the rest of the 6 hr session. During shaping procedures, undesirable behaviors, such as taft electrode climbing, were extinguished by gently shaking the animal back to the perch. Following the termination of the stress session, the animals were again weighed and returned to their home cages. The animals were permitted free access to water for the 2-hr poststress delay period prior to being sacrificed. AU animals were sacrificed by exposure to chloroform. Their stomachs were then excised and inspected for ulceration. Extent of ulceration was measured by a scale of hundredths of an inch located within the eyepiece of a dissecting microscope. Ulceration was determined in measurements of percentage of rats exhibiting pathology, number of lesions, and ulcer score (i.e., length plus width of lesions in hundredths of an inch). The criterion for gastric lesioning was an obvious erosion or absence of mucosal tissue accompanied by hemorraging at the site of the lesion. Gastric lesions were characterized by a deterioration of epithelial cells extending through the mucosal lining. All evaluations concerning the extent of gastric lesions were confirmed through a blind procedure in which information as to which groups were shocked or drugged was not made available to the judge. RESULTS AND DISCUSSION Statistical analyses using the Mann-Whitney U test [14] indicated that none of the groups in Experiment l demonstrated significant amounts of stomach ulceration. This indicated the inability of the technique to consistently produce ulceration. Although no significant differences were noted, the data suggested an inverse relationship between body weight and occurrence of gastric lesions as has been reported earlier [ l ] . The few lesions which were observed in the shock groups principally appeared in the lightest weight animals. These findings suggest that body weight may have been an ulcerogenic factor operating in
METHYL SCOPOLAMINE AND ULCER FORMATION this technique. Furthermore, a previous report [4] has indicated that shock intensity significantly affects gastric ulceration in both frequency of occurrence and number of ulcers per animal. Thus, the higher the shock intensity, the greater the exhibited pathology. These results may suggest that body weight could interact with shock intensity to produce stomach ulceration. Finally, the results of Experiment 1 indicated that the two drug groups demonstrated a slight (nonsignificant) reduction in ulceration, suggesting that a higher dosage of this agent might be able to inhibit the development of stomach lesions. The second experiment was conducted to investigate the above speculations. EXPERIMENT
2
METHOD Animals and A pparatus
Animals for Experiment 2 consisted of 80 male SpragueDawley albino rats obtained from the previously mentioned supplier. They ranged in weight from 9 7 - 2 1 6 g at the beginning of the shock sessions. The data from the 10 noshock control animals of Experiment 1 were again used as the control data for Experiment 2. The experimental chambers and apparatus for this study were identical to those utilized in the first experiment. Procedure
The general procedures used throughout this experiment concerning treatment of animals, deprivation, and drug administration were identical to those outlined in Experiment 1. Following deprivation, the rats were weighed and assigned to eight I 0-animal weight matched groups. These 8 groups consisted of experimental and yoked saline groups and 6 drug groups. The drug groups were composed of an experimental and yoked group for each of 3 dosages (i.e., 0.125, 0.50 and 1.0 mg/kg) of scopolamine methylbromide. Since drug and saline injections were given immediately preceding and following the drug sessions, the effective dosages available to the various groups were actually 0.25, 1.0 and 2.0 mg/kg. T h e s h a p i n g procedures were identical to those employed in the first experiment except that shock intensity was adjusted proportionally to the body weights of the animals. Animals weighing 97 to 125 g received 1.2 mA; animals weighing 126 to 150 g received 1.4 mA; and animals weighing 151 to 216 g received 1.6 mA. Extreme care was exercised to insure that body weights were identical for all 9 groups of animals. All 9 groups had 4 animals in the lowest weight category, 2 in the middle category and 4 in the heaviest category. RESULTS The data for Experiment 2 consisted of the number of animals per group demonstrating pathology, number of ulcers per animal, and ulcer score per animal (i.e., length plus width of lesions in hundredths of an inch). As in Experiment I, all gastric lesions were observed in the glandular fundus of the stomach. All rumenal tissue was free of pathology. A Kruskal-Wallis one-way analysis of variance [14] was performed on the ulcer score and number of ulcers per animal data. It indicated that significant differences (p<0.001) existed between the various groups. Multiple statistical comparisons were then carried
149 out using the Mann-Whitney U test [14]. These comparisons indicated that there were no significant differences between any of the experimental and yoked groups in mean number of lesions and ulcer score (i.e., size of lesion). Since there were no experimental-yoked differences, these two groups were pooled within drug dosage and saline conditions. This procedure produced one 10-animal noshock control group and four 20-animal shock groups (i.e., saline, 0.125 mg/kg, 0.50 mg/kg, and 1.0 mg/kg scopolamine methylbromide). Further statistical analyses using the Mann-Whitney U test [I 4 ] were then performed on the number of ulcers and ulcer score data. The results of these analyses are summarized in Fig. I. The comparisons in Fig. 1 revealed a significant difference between the no-shock control and saline (shock) group (p<0.002) in mean number of ulcers per animal and mean ulcer score. A significant difference also occurred between the no-shock control and scopolamine methylbromide 0.125 mg/kg group (p
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FIG. 1. Sumnutry of gastric pathology demomtrated by the various drug, saline or control groups. Degree of gastric pathology is shown in 3 separate mea~ares: % of aninuds showing pathology, mean number of ulcers per animal and mean ulcer score. Results of the MannWhitney U Tests are shown at the bottom of this figure. degree of physical exhaustion and severity of gastric pathology. Smaller animals would be expected to become exhausted sooner than heavier, more mature animals. Heavier animals, in turn, would require more intense shocks
to produce equivalent amounts of physical exhaustion and, thus, similar degrees of stomach pathology. The data presented in Fig. 1. demonstrates a doseresponse relationship regarding the ulcer inhibiting qualities of scopolamine methylbromide. The two higher dosages (i.e., 0.50 and 1.0 mg/kg) significantly decreased gastric ulcer development while the lower dosage did not. These results suggest that this particular anticholinergic may inhibit the physiological mechanisms involved in gastric ulcer formation. Future research utilizing the present design should center upon investigating other cholinergic agents, in particular the cholinomimetics, to determine if enhanced cholinergic tone may increase the severity of gastric pathology. The above conclusions rest on the assumption that scopolamine methylbromide was able to exert its antiulcerogenic effects directly on the stomach by interfering with physiological processes (i.e., acid secretion) that are necessary to produce gastric ulceration. It is possible, however, that this agent was able to produce its effects indirectly by some other process. One possibility concerns the a b i l i t y o f anticholinergJcs to produce drug-induced analgesia. If the drug was able to reduce the severity of the stress (i.e., pain) perceived during the 6-hr session, it could explain why the drugged animals demonstrated fewer uJcers. Recent research from this laboratory [7,8], however, has indicated that both scopolamine hydrobromide and scopolamine methylbromide have no significant effects on the aversive threshold as measured in rats or monkeys. Therefore, it is unlikely that drug-induced analgesia can account for the present results. Another possibility concerns the ability of scopolamine methylbromide to affect avoidance acquisition on the perch. Since no measure of lime spent on the perch was available, it is possible that the drug was able to reduce stomach ulceration by enhancing the acquisition and/or performance of the avoidance response, thus reducing the total amount of shock received. Although this explanation is possible, it is unlikely, since this particular drug does not readily cross the blood brain barrier [5] and thus fails to affect a wide range of behaviors [ 2 ] including shock avoidance [8,9]. Furthermore, all drug and saline groups in both experiments showed similar rates of avoidance acquisition during shaping procedures. It would therefore seem more likely that this agent produced its antiulcer effect primarily through its peripheral actions (i.e., inhibiting vagally controlled acid secretion).
REFERENCES
1. Brodie, D. A. and A. M. Hanson. A study of factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38: 353-360, 1960. 2. Carlton, P. L. Brain acetylcholine and inhibition. In: Reinforcement and Behavior, edited by J. Tapp. New York: Academic Press, 1969, pp. 286-327. 3. Des/derato, O., J. R. MacKinnon and H. I-lissom.Development of gastric ulcers in rats following stress termination. J. comp. physioL Psychol. 1974, in press. 4. Gliner, J. A. Predictable vs unpredictable shock: preference behavior and stomach ulceration. Physiol. Behav. 9: 693-698, 1974. 5. Goodman, L. S. and A. Gilman. The Pharmacological Basis o f Therapeutics. New York: The Macmillan Company, 1965, pp. 83.
6. Grassman, M. I. No acid, no hemorrhage? Gastroenterology 57: 367, 1969. 7. Houser, V. P. and F. L. Houser. The alteration of aversive thresholds with choIinergic and adrenergic agents. Pharmac. ~'ochem. Behav. 1: 433-444, 1973. 8. Houser, V. P. and D. A. Van Hart. The effects of scopolamine and pilocarpine upon the aversive threshold of the rat. Pharmac. Biochem. Behav. 1: 427-431, 1973. 9. Houser, V. P. and F. L. Houser. The effects of agents that modify muscarinic tone upon behavior controlled by an avoidance schedule that employs signaled unavoidable shock. Psychopharmacologia (Bed) 32:133-150, 1973.
METHYL SCOPOLAMINE AND ULCER FORMATION 10. lnnes, I. R. and M. Nickerson. Drugs inhibiting the action of acetylcholine on structures innervated by postganglionic parasympathetic nerves. In: The Pharmacological Basis o f Therapeutics, edited by L. Goodman and A. Gilman. New York: The Macmillan Company, 1965, pp. 521-543. 11. Mfiller-Wieland, K. and F. W. Ossenberg. Medical management and treatment of duodenal ulcer. In: Advances in Psychosomatic Medicine, Vol. 6, edited by H. Weiner. New York: S. Karger, 1961, pp. 152-165. 12. Robert, A. and J . E . Nezamis. Effect of an anti-acetylcholine drug, methscopolamine bromide, on ulcer formation and gastric mucus. J. Pharm. Pharmac. 6: 690-695, 1964. 13. Sawrey, W. L., J. J. Conger and E. S. Turrell. An experimental investigation of the role of psychological factors in the production of gastric ulcers in rats. Y. comp. physiol. Psychol. 49: 457-461, 1956.
151 14. Siegel, S. Nonparametric Statistics. New York: McGraw-Hill Book Company, 1956, pp. 116-127. 15. Sollmann, T. A Manual o f Pharmacology. New York: W. B. Saunders Company, 1957, p. 400. 16. Wald, E. D. Endocrinological correlates of stress-induced ulcers. Unpublished Master's Thesis, Connecticut College, 1972. 17. Wald, E. D., J. R. MacKinnon and O. Desiderato. Production of gastric ulcers in the unrestrained rat. Physiol. Behav. 10: 825-827, 1973. 18. Weiss, J. M. Effects of coping responses on stress. J. comp. physiol. PsychoL 65: 251-260, 1968. 19. Weiss, J. M. Somatic effects of predictable and unpredictable shock. Psychosom. Med. 32: 397-404, 1970.