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

RICHARD 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 lesion s per animal and severity of lesioning) in all groups tested.

Gastric lesions Unrestrained rat Avoidance-avoidance conflict Sc°polamine methylbromide

PREVIOUS reports in the l i terature indicate that psycho- logical variables may intensify the physiological response to stress and may directly contr ibute to an increase in gastric pathology [13, 16, 17, 18, 19]. The present work is an a t tempt to evaluate the ulcerogenic propert ies of one such psychological variable (i.e., an avoidance-avoidance con- flict). 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 of animals in either a contingent or yoked 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 func- tional model for the investigation of the ulcer inhibiting properties of various pharmaceutical agents.

The present report investigated the ulcer inhibiting effects of a peripheral acting anticholinergic, scopolamine methylbromide. The presence of hydrochloric acid is essen- tial for gastric lesion development. Lesions may develop in stomachs secreting small quantities of acid, but not in stomachs total ly absent of acid [6] . Since the primary phase of gastric secretion is under vaga] control, derivates of the belladonna alkaloids, atropine and scopolamine, have been employed to block the effects of parasympathet ic st imulation upon the stomach. Miiller-Wieland and Ossen-

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 para- sympathet ic 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 pro- longed [10,15] . Previous exper imentat ion [12] has indi- c a t e d t h a t s copo lamine methylbromide significantly decreases gastric volume and total acid. The present work was undertaken in an a t tempt 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 tempt was made to see if the introduct ion 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.

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. Sprague- Dawley Company,. Madison, Wisconsin. All animals were 48 hx food deprived at the start of the experimental session. They ranged in weight from 102-178 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 i l lumination and 12 hr darkness) with the dark port ion 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 of 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 fi t ted 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 automatical ly pre- sented electric shock to the animals. Extraneous laboratory noises were masked by 90 dB of white noise presented via a speaker mounted on the corner wall of the exper imenta l room. Current leakage through the cages was held to a minimum by insuring consistent temperature and humidi ty 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 of 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 intermit tent ly 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 yoked animal was wired in series with the experimental animal so that it received identical amounts of shock, irrespective of its location with- in the yoked cage (i.e., either on the main grid floor or on the perch of the yoked cage).

Procedure

The animals were initially group housed with ad lib food and water immediately after they arrived from the supplier for a minimum of 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) e x p e r i m e n t a l - s a l i n e ; (2) yoked-sa l ine ; (3) experi- men ta l -0 .125 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 elec- trode 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, yoked 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 un- shocked 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 intermit tent shock (0.5 mA) con- tingency 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 intermit tent ly 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 demon- strated 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 149

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 Experi- ment 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 experi- ment was conducted to investigate the above speculations.

EXPERIMENT 2

METHOD

Animals and A pparatus

Animals for Experiment 2 consisted of 80 male Sprague- Dawley 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 no- shock 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 Experi- ment 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.

The s h a p i n g procedures were identical to those employed in the first experiment except that shock inten- sity 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 iden- tical 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 signifi- cant differences (p<0.001) existed between the various groups. Multiple statistical comparisons were then carried

out using the Mann-Whitney U test [14]. These compari- sons 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 no- shock control group and four 20-animal shock groups (i.e., saline, 0.125 mg/kg, 0.50 mg/kg, and 1.0 mg/kg scopol- amine 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 signifi- cant difference also occurred between the no-shock con- trol and scopolamine methylbromide 0.125 mg/kg group (p<O.02). These results suggest that scopolamine methyl- bromide at this dosage lacks significant ulcer inhibiting properties, thus confh~ning the results of Experiment l. Figure 1 further indicates, however, that higher dosages of scopolamine methylbromide (i.e., 0.50 and 1.0 mg/kg) were able to significantly decrease (p< 0.002) ulcer formation by reducing the mean number and size of ulcers produced by the 6-hr stress session. In fact, the two higher dosages of the drug were so effective in reducing ulcer formation that these groups did not differ significantly from the no-shock control group with reference to mean number or size of ulcers.

DISCUSSION

The results of both experiments indicate that there was no difference between any of the experimental or yoked groups in terms of gastric pathology. These results, unlike those of Wald eta/. [ 16,17], indicate that the psychological variable (i.e., the avoidance-avoidance conflict) did not intensify the physiological response to stress as measured by degree of gastric pathology. It would appear, then, that the physical stressor (i.e., electric shock) was the major factor contributing to the amount of gastric pathology which occurred. Visual observation of the animals subjected to shock suggested that physical exhaustion may be highly correlated with the severity of gastric ulceration. Since degree of physical exhaustion should be a function of the amount of shock received, future research would do well to focus on investigating the possible correlation between shock duration and intensity and the severity of gastric pathology produced by this technique.

The enhanced amount of ulceration produced in Experi- ment 2 over that noted in the first experiment indicates that the ulcerogenic properties of this technique are con- siderably augmented when shock intensity is elevated in proportion to body weight. When the shock intensity was held constant (i.e., Experiment l), the ulcer incidence for animals under 125 g was 35%; for animals 125 -150 g it was 36%; and for animals over 150 g it was 0%. In contrast, the figures for comparable weight groups in Experiment 2 were 46%, 31% and 50% respectively. Thus, increasing shock intensity as a function of body weight was able to reduce the variability in ulcer results. Furthermore, the data of Experiment I confirm the previously reported [ 1 ] inverse relationship between body weight and ulcer susceptibility and supports the hypotheses, referred to above, concerning

150 SEISER AND HOUSER

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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 Mann-

Whitney U Tests are shown at the bottom of this figure.

degree of physical exhaustion and severity of gastric pathol- ogy. 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 dose- response 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 anti- ulcerogenic effects directly on the stomach by interfering with physiological processes (i.e., acid secretion) that are necessary to produce gastric ulceration. It is possible, how- ever, that this agent was able to produce its effects indi- rectly by some other process. One possibility concerns the a b i l i t y of 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], how- ever, 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 avoid- ance [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 con- trolled acid secretion).

R E F E R E N C E S

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M E T H Y L S C O P O L A M I N E A N D U L C E R F O R M A T I O N 151

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