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Protection by Procaine Hydrochloride Against Reserpine-Induced Acute Gastric Mucosal Injury in the Rat: Implications for Stress-Induced Injury
Protection by Procaine Hydrochloride against Reserpine-Induced Acute Gastric Mucosal Injury in the Rat: Implications for Stress-lnduced Injury Aws S. SALIM Received July 27,1987, from the University Department of Surgery, Royal Infirmary, Glasgow G4 OSF, U.K. February 9, 1988. Present address: Department of Surgery, Royal Infirmary, ferth f H 1 INX, U.K. .
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C This study investigated whether oral procaine (1 mL of 5% solution) affords protection against reserpine-induced (5 mg/kg, ip) acute gastric mucosal injury in the rat. After 6 h, all rats in the reserpinealone group developed mucosal injury confined to the glandular stomach (40.1 5.2 mmZ, mean ? SEM), and neither atropine (5 mg/kg, ip) or cirnetidine (40 rnglkg ip) influenced this injury (38 _t 4 and 40.5 ? 4.6 mm2, respectively). Similarly, celiac ganglionectomy, to interrupt autonomic sympathetic delivery to the stomach, had no effect on the reserpine-induced injury (42 t- 6 versus 40.1 t 5.2 mm'). Dosedependent protection against the reserpine injury was produced by the alpha-adrenoceptor blocking drug phenoxybenzamine or phentolamine: a complete protection was noted with the 15-mg/kgdose. Vagotomy or procain completely protected the rat stomach against the reserpine injury. The data suggest that reserpine produces vagal adrenoceptor delivery to the rat stomach, resulting in mucosal injury, and that oral procaine blocks this delivery, thus achieving protection against injury development by a vagotomy action. The knowledge that the reserpine injury is a stress-induced injury indicates that oral procaine protects the rat stomach against stress-induced acute gastric mucosal injury. Abstract
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In experimental animals, parenteral administration of large doses of reserpine (2-10 mgikg) produces a vagal action on the stomach resulting in acute mucosal injury.'.z Recent rat studies2 demonstrated that this injury is produced by vagal adrenergic stimulation which causes severe gastric submucosal and mucosal vasoconstriction. This vasoconstriction injures the gastric mucosa, and H i or duodenal contents are not essential prerequisites for injury development.2 The reserpine injury represents stress-induced acute gastric mucosal injury in the Local anesthetics prevent transmission of impulses along nerve fibers and at nerve endings by inhibiting depolarization and ion exchange.6 The effects are reversible. Procaine hydrochloride, one of the least toxic local anesthetics with potent vasodilatation properties,6 was tested for protection against the reserpine-induced acute gastric mucosal injury.
Experimental Section Animals-Groups of 10 Sprague-Dawley rats of either sex, weighing between 210 and 240 g, were denied solid food for 24 h before the study. They were allowed free access to water and were housed in cages with wide mesh wire bottoms to prevent coprophagy. Source and Preparation of Drugs-A 1-mgimL solution of reserpine was prepared by dissolving 80 mg of crystalline powder (Sigma, St. Louis, MO) in 0.3 mL of glacial acetic acid (BDH Chemicals, Poole, England), and the volume was made up to 80 mL with double distilled water. Cimetidine (200-mg ampules; Smith Kline and French Labs. Ltd., Hertfordshire, England) was used to prepare an 8mg/mL solution with double distilled water. Atropine sulphate powder (Sigma, St. Louis, MO) was dissolved in double distilled water to prepare a 1-mgimL solution. A 5% solution of procaine hydrochloride (Sigma, St. Louis, MO) was prepared by dissolving 1g of the powder in 20 mL of double distilled water. Phenoxybenzamine 582 / Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 1988
Accepted for publication
hydrochloride (100-mg ampules; Dibenyline: Smith Kline and French Labs. Ltd., Hertfordshire, England) and phentolamine mesylate (50-mg ampules; Rogitine; CIBA Labs., Horsham, England) were diluted with double distilled water to prepare solutions of 1, 2, and 3 mg/mL for the 5-, lo-, and 15-mg/kg doses, respectively. Drugs were prepared in the mentioned concentrations so that a 200-g animal received 1mL. Drugs were prepared for each experimental day. Saline was given to control animals. Injections were administered intraperitoneally into the left iliac fossa using a 25 G needle, and gavage was undertaken under light ether anesthesia (diethyl ether) using a 6 FG Infant's Feeding Tube (Portex Ltd., Hythe, England). Surgery-Animals were anesthetized by inhalation of diethyl ether, and clean, but not sterilized, instruments were used to perform surgery through a 5-cm transverse abdominal incision situated midway between the xyphoid process and pubic symphysis. Vugotomy and Pyloroplasty-The gastrohepatic ligament was divided to expose the abdominal esophagus, then the anterior and posterior vagal trunks were divided just below the esophageal hiatus. Thereafter, a 0.5-cm longitudinal incision centered over the pyloric sphincter in the anterior wall of the pyloroduodenal segment was made dividing all coats. The wound was then closed in the transverse axis using continuous through and through sutures of 3/0 silk. Celiac Gunglionectomy-The abdominal viscera were retracted en mass to the right side of the peritoneal cavity. The celiac ganglion in the rat is situated on the ventral aspect of the aorta between the celiac and superior mesenteric arteries,7 which were clearly visible behind the peritoneum of the posterior abdominal wall. This peritoneum was stripped (by gauze) off the vessels and the celiac ganglion, lying within the loose areolar tissue on the aorta, was removed piecemeal, using nontoothed forceps, and placed in 4% buffered neutral formalin for histological confirmation. The ventral and lateral sides of the aorta, from 0.5 cm above the celiac artery to the origin of the superior mesenteric artery, were cleared of areolar tissue using a gauze pledget. Similarly, the first centimeter of the celiac artery was cleared of any areolar tissue. The abdomen was closed with continuous through and through sutures of 2/0 silk. Each celiac ganglion was fixed in formalin, embedded in paraffin, sectioned, stained with haematoxylin-eosin, and Holme's silver nitrate, and then examined microscopically to confirm complete removal as indicated by a layer of connective tissue, the epineurium, merging with the external capsule and surrounding the mass of ganglion cells. Mucosal Integrity Study-The experimental groups are shown in Table I. Vagotomy and pyloroplasty or a sham operation (exposing and identifying both vagal trunks and the pyloric sphincter), with celiac ganglionectomy or a sham operation (retracting the abdominal viscera and identifying the anatomical site of the ganglion behind the peritoneum), were performed. Animals were allowed to recover from anesthesia, and then 1mL of 5% procaine or saline was instilled by orogastric intubation into the stomach. Thereafter, rats were injected intraperitoneally with saline (5 mL/kg), atropine (5 mglkg), cimetidine (40 mg/kg), phenoxybenzamine (5, 10, or 15 mglkg), or phentolamine (5, 10, or 15 mg/kg). Finally, the rats were similarly injected with 5 mL/kg of saline (controls) or reserpine (5 mg/kg). Six hours later, animals were killed by ether overdose and the stomachs were removed and opened along the greater curvature. After wash-
0
0022-3549/88/0700-0582$01.00/0 1988, American Pharmaceutical Association
Table I-Reserpine-Induced Acute Gastric Yucosal Injury in the Rat % Incidence of Animals Showing Lesions
Lesion Area after 6 h (mean f SEM), mm'
Experimental Group
n
Control Reserpine (5 mg/kg, ip) Atropine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Cimetidine (40 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (10 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (15 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (10 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (15 mg/kg, ip) and resperine (5 mgikg, ip) 5% Procaine (1 mL, orally) and reserpine (5 mglkg, ip) Vagotomy and reserpine (5 mg/kg, ip) Celiac ganglionectomy and reserpine (5 mg/kg, ip)
10 10
100
40.1 t 5.2
10
100
38
10
100
40.5 ? 4.6
10
80
9.7 t 2a
10
40
3
10
0
0
10
80
11.5 t 1.6a
10
40
2.5 ? 0.7a
10
0
0
10
0
0
10
0
0
10
100
a
0
0
42
t4
t 0.5a
2
6
p < 0.001 by the Mann Whitney test comparing the reserpine-alone group with treatment group.
ing with a direct stream of cold water, the stomachs were pinned out and independently examined for the presence of mucosal injury. Each injury was measured in maximum length and width and the surface area (mm') was calculated. The total injury score was obtained for each animal and the mean injury score was calculated for each study group. Sections of injured and apparently uninjured gastric mucosa were cut off each stomach, fixed, embedded in paraffin, sectioned at right angles to the mucosa, stained (hematoxylin-eosin, Martius scarlet blue and periodic-acid-Schiff), and then examined microscopically. In order to minimize day-to-day variation in response to treatment, animals were allocated to the control and all of the treatment groups within the experiment on each experimental day. In preliminary studies, administration of intraperitoneal reserpine in a dose of 5 mglkg produced gastric mucosal injury in all rats, after 6 h, and was, therefore, considered a suitable dose for this study. The macroscopic and microscopic appearances of the r a t gastric mucosa were not affected after 6 h of intraperitoneal injection of phenoxybenzamine (5,10, or 15 mg/kg), phentolamine (5,10, or 15 mg/kg), or the vehicle solution of reserpine (5 mL/kg of 0.127 M glacial acetic acid). Statistical Analysis-Results are expressed as mean 5 SEM. The statistical significance (p 10.05) of observed differences between groups was determined using the Mann-Whitney U test for nonparametric data.
Results The results are presented in Table I. Histological examination confirmed removal of the celiac ganglion in all members of the ganglionectomy group. All rats in the reserpine-alone group developed oval or round injury that was confined to the glandular stomach and which had no constant relationship to rugal crests (injury score 40.1 ? 5.2 mm'). Atropine or cimetidine did not influence the incidence, appearance, or score of this injury (38 4 and 40.5 ? 4.6 mm', respectively). Phenoxybenzamine or phentolamine, in doses of 5 mg/kg, significantly (p <0.001) protected rats against the reserpine-induced injury, with 80% of animals developing injury similar to that of the reserpine alone group (9.7 ? 2 and 11.5 t 1.6 mm', respectively versus 40.1 5.2 mm'). However, the protective action
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*
of the lO-mg/kg dose was significantly (p <0.01) more effective, with 40% of animals showing injury similar to that of the reserpine alone group (3 0.5 and 2.5 t 0.7 mm'). Celiac ganglionectomy had no effect on the incidence, pattern, or score of the reserpine injury (42 t 6 versus 40.1 ? 5.2 mm'). Vagotomy, 15 mg/kg of phenoxybenzamine or phentolamine, or 1 mL of 5%procaine protected all rats against the reserpine-induced acute gastric mucosal injury. Microscopically, the reserpine injury consisted of partial or full depth (including the muscularis mucosae) mucosal necrosis and/or loss with necrosis, hemorrhage, and polymorphonuclear leukocyte infiltration at the edges. The submucosal and mucosal blood vessels were severely constricted (Figure 1). The submucosa was infiltrated with polymorphonuclear leukocytes and was edematous, but the muscularis propria was completely intact. No pathological changes were detected in the forestomach or antrum. The reserpine injury developed in the mucosa and submucosa as foci of hemorrhage or necrosis. These foci were directly related to disrupted or constricted blood vessels and expanded to communicate with the lumen. Atropine, cimetidine, or celiac ganglionectomy had no effect on the microscopic appearances of the reserpine injury. Similarly, the injury developing with 5 or 10 mg/kg of phenoxybenzamine or phentolamine was microscopically similar to that of the reserpine alone group. Gastric sections from the groups having vagotomy, procaine (Figure 2), or 15 mglkg of phenoxybenzamine or phentolamine were microscopically similar to those of control animals.
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Discussion The results of the present study confirm the previous report2 that in the rat, 5 mglkg of reserpine produces vagal delivery of alpha-adrenoceptor stimulation to gastric submucosal and mucosal blood vessels causing vasoconstriction which, in turn, injures the mucosa. In the present study, 6 h Journal ofPharmaceutical Sciences / 583 Vol. 77,No. 7,July 1988
Figure 1-The glandular stomach after 6 h of intraperitoneal reserpine. Note the submucosal and mucosal vasoconstriction (periodic acidSchiff, x 1 16; original magnification x 29).
Flgure 2-The glandular stomach after 6 h of procaine and reserpine. Note the absence of injury and vasoconstriction(periodic acid-Schif, x 96; original magnification x 24).
after intraperitoneal reserpine (5 mg/kg), gastric mucosal injury confined to the glandular stomach developed in all animals (Table I). This injury initiated intramurally as foci of necrosis or hemorrhage and was associated with submucosal and mucosal vasoconstriction. Pretreatment with potent antisecretory dosesHof the anticholinergic atropine or the H2-receptor antagonist cimetidine did not influence the appearances or severity of the reserpine-induced gastric mucosal injury (Table I). This observation suggests that H t is not the principal factor in the mechanism of this injury, but does not exclude the possibility that it might be an essential prerequisite. 584 !Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 1988
Phenoxybenzamine and phentolamine are alpha-adrenoceptor blocking drugs, the latter being more spe~ific.~ Dosedependent protection against the reserpine-induced acute gastric mucosal injury was produced by phenoxybenzamine or phentolamine; a complete protection against injury and its associated submucosal and mucosal vasoconstriction was noted with the 15-mg/kg dose (Table I). This finding and the microscopic observation that the reserpine injury initiates in relation to gastric submucosal and mucosal vasoconstriction suggest that ischemia is the principal factor behind the reserpine injury. Reserpine acts on the hypothalamus, producing central adrenergic discharge and stimulation of peripheral sympathetic pathways which release noradrenaline into the tissues.1o The results of the present study are consistent with the pharmacological action of reserpine in that they suggest that the reserpine-induced intragastric vasoconstriction is caused by alpha-adrenoceptor stimulation. Celiac ganglionectomy, to interrupt autonomic sympathetic delivery to the stomach,ll did not influence the reserpine-induced injury, but vagotomy afforded complete protection against this injury and its associated intragastric vasoconstriction (Table I). The similarity in action between vagotomy and large doses of alpha-adrenoceptor blocking drugs suggests that reserpine induces vagal alpha-adrenergic delivery to the rat stomach, causing submucosal and mucosal vasoconstriction which, in turn, injures the mucosa. Additional studies employing measurement of mucosal blood flow are needed to confirm these microscopic findings. F'rocaine, like vagotomy or large doses of alpha-adrenoceptor blocking drugs, protects against the reserpine-induced gastric submucosal and mucosal vasoconstriction and mucosal injury (Table I and Figure 2), demonstrating that it interrupts the vagal stimulation causing this vasoconstriction and injury, and thus providing a vagotomy action. It might be argued that vasodilation by procaine6 achieved protection against vasoconstriction. Since vagal stimulation produced this vasoconstriction, the conclusion that procaine has a vagotomy action still applies, regardless of whether this action is on blood vessels or neurotransmission. Reserpine-induced acute gastric mucosal injury is a stressinduced injury."-" Patients with major trauma, hemorrhage, burns, neurological injuries, or sepsis may survive their initial injury only to succumb to bleeding from stress-induced acute gastric mucosal injury.12This life-threatening injury13 may not respond to conservative treatment, making surgery the only option.14 The surgical procedure usually employed in such cases is that of vagotomy and drainage, however the predisposing factor to gastric bleeding increases the risk of the pera at ion.'^ The results of this study show that oral procaine protects against the development of stress-induced acute gastric mucosal injury. Detailed studies are needed to determine whether similar results can be obtained in humans. Procaine was chosen for this study because of its relatively low toxicity. Further studies are needed to determine the optimum dose of oral procaine for protection against the reserpine-induced injury and to examine its effect on gastric muscular tone, motility, and rate of emptying. Studies are also needed to determine whether relatively longer acting or more potent local anesthetics have actions similar to those of procaine observed in this study.
Conclusions The results of the present study demonstrate that in the rat, oral procaine prevents delivery of reserpine-induced vagal stimulation causing gastric submucosal and mucosal vasoconstriction and mucosal injury. Thus, procaine provides
a vagotomy action. Since the reserpine injury is a stressinduced injury, oral procaine protects the rat stomach against stress-induced acute gastric mucosal injury.
References and Notes 1. Blackman, J. G.;Campion, D. S.; Fastier, F. N. Br. J. Phurmacol. 1959,14,112-116. 2. Salim, A.S.J.Physiol. 1987,392,363-376. 3. Salim, A.S.J.Psychosom. Res. 1987,31,85-90. 4. Salim, A.S.J.Psychosom. Res. 1987,31,231-237. 5 . Salim, A. S. J. Psychiat. Res., in press. 6. Reynolds, J. E. F.; Prasad, A. B. In Martindale: The Ewtm Phurmacopoeia; Pharmaceutical Press: London, 1982;pp 877-879. 7. Greene, E. C. In The Rat in Laboratory Investigation; Farris, E. J.; Griffith, J. Q., Jr., Eds.; Hafner: New York, 1967;Chapter 3,pp 38-41.
8. Rainsford, K. D. Di est Dis. 1978,23,521-529. 9. Nickerson, M. In +he Pharmacological Basis of Therapeutics, Goodman, L. S.; Gilman, A., Eds.; Macmillan: London; 1970; Chapter 26,pp 550-570. 10. Paee. I. H. Phvsiol. Rev. 1958.38. 277-335. 11. Leinard, A.S;: Long, D.; French; L. A.; Peter, E. T.; Wangensteen, 0. H. Sur e 1964,56,109-120. 12. Butterfield, W. gurg. Ann. 1975,7 , 261-278. 13. Skillman, J.J.; Bushnell, L.S.:Goldman,. H.:. Silen, W. A m J. Surg. 1969,117,523-531. . 14. Lucas, C. E.; Sugawa, C.; Riddle, J.; Rector, F.; Rosenberg, B.; Walt, A. J. Arch. Surg. 1971,102,266-273.
8.
Acknowledgments The skillful technical assistance of Mr. Donald McMillan and secretarial work of Miss Lesley Cook are gratefully acknowledged.
Journal of Pharmaceutical Sciences / 585 Vol. 77, No. 7,July 1988
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C This study investigated whether oral procaine (1 mL of 5% solution) affords protection against reserpine-induced (5 mg/kg, ip) acute gastric mucosal injury in the rat. After 6 h, all rats in the reserpinealone group developed mucosal injury confined to the glandular stomach (40.1 5.2 mmZ, mean ? SEM), and neither atropine (5 mg/kg, ip) or cirnetidine (40 rnglkg ip) influenced this injury (38 _t 4 and 40.5 ? 4.6 mm2, respectively). Similarly, celiac ganglionectomy, to interrupt autonomic sympathetic delivery to the stomach, had no effect on the reserpine-induced injury (42 t- 6 versus 40.1 t 5.2 mm'). Dosedependent protection against the reserpine injury was produced by the alpha-adrenoceptor blocking drug phenoxybenzamine or phentolamine: a complete protection was noted with the 15-mg/kgdose. Vagotomy or procain completely protected the rat stomach against the reserpine injury. The data suggest that reserpine produces vagal adrenoceptor delivery to the rat stomach, resulting in mucosal injury, and that oral procaine blocks this delivery, thus achieving protection against injury development by a vagotomy action. The knowledge that the reserpine injury is a stress-induced injury indicates that oral procaine protects the rat stomach against stress-induced acute gastric mucosal injury. Abstract
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In experimental animals, parenteral administration of large doses of reserpine (2-10 mgikg) produces a vagal action on the stomach resulting in acute mucosal injury.'.z Recent rat studies2 demonstrated that this injury is produced by vagal adrenergic stimulation which causes severe gastric submucosal and mucosal vasoconstriction. This vasoconstriction injures the gastric mucosa, and H i or duodenal contents are not essential prerequisites for injury development.2 The reserpine injury represents stress-induced acute gastric mucosal injury in the Local anesthetics prevent transmission of impulses along nerve fibers and at nerve endings by inhibiting depolarization and ion exchange.6 The effects are reversible. Procaine hydrochloride, one of the least toxic local anesthetics with potent vasodilatation properties,6 was tested for protection against the reserpine-induced acute gastric mucosal injury.
Experimental Section Animals-Groups of 10 Sprague-Dawley rats of either sex, weighing between 210 and 240 g, were denied solid food for 24 h before the study. They were allowed free access to water and were housed in cages with wide mesh wire bottoms to prevent coprophagy. Source and Preparation of Drugs-A 1-mgimL solution of reserpine was prepared by dissolving 80 mg of crystalline powder (Sigma, St. Louis, MO) in 0.3 mL of glacial acetic acid (BDH Chemicals, Poole, England), and the volume was made up to 80 mL with double distilled water. Cimetidine (200-mg ampules; Smith Kline and French Labs. Ltd., Hertfordshire, England) was used to prepare an 8mg/mL solution with double distilled water. Atropine sulphate powder (Sigma, St. Louis, MO) was dissolved in double distilled water to prepare a 1-mgimL solution. A 5% solution of procaine hydrochloride (Sigma, St. Louis, MO) was prepared by dissolving 1g of the powder in 20 mL of double distilled water. Phenoxybenzamine 582 / Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 1988
Accepted for publication
hydrochloride (100-mg ampules; Dibenyline: Smith Kline and French Labs. Ltd., Hertfordshire, England) and phentolamine mesylate (50-mg ampules; Rogitine; CIBA Labs., Horsham, England) were diluted with double distilled water to prepare solutions of 1, 2, and 3 mg/mL for the 5-, lo-, and 15-mg/kg doses, respectively. Drugs were prepared in the mentioned concentrations so that a 200-g animal received 1mL. Drugs were prepared for each experimental day. Saline was given to control animals. Injections were administered intraperitoneally into the left iliac fossa using a 25 G needle, and gavage was undertaken under light ether anesthesia (diethyl ether) using a 6 FG Infant's Feeding Tube (Portex Ltd., Hythe, England). Surgery-Animals were anesthetized by inhalation of diethyl ether, and clean, but not sterilized, instruments were used to perform surgery through a 5-cm transverse abdominal incision situated midway between the xyphoid process and pubic symphysis. Vugotomy and Pyloroplasty-The gastrohepatic ligament was divided to expose the abdominal esophagus, then the anterior and posterior vagal trunks were divided just below the esophageal hiatus. Thereafter, a 0.5-cm longitudinal incision centered over the pyloric sphincter in the anterior wall of the pyloroduodenal segment was made dividing all coats. The wound was then closed in the transverse axis using continuous through and through sutures of 3/0 silk. Celiac Gunglionectomy-The abdominal viscera were retracted en mass to the right side of the peritoneal cavity. The celiac ganglion in the rat is situated on the ventral aspect of the aorta between the celiac and superior mesenteric arteries,7 which were clearly visible behind the peritoneum of the posterior abdominal wall. This peritoneum was stripped (by gauze) off the vessels and the celiac ganglion, lying within the loose areolar tissue on the aorta, was removed piecemeal, using nontoothed forceps, and placed in 4% buffered neutral formalin for histological confirmation. The ventral and lateral sides of the aorta, from 0.5 cm above the celiac artery to the origin of the superior mesenteric artery, were cleared of areolar tissue using a gauze pledget. Similarly, the first centimeter of the celiac artery was cleared of any areolar tissue. The abdomen was closed with continuous through and through sutures of 2/0 silk. Each celiac ganglion was fixed in formalin, embedded in paraffin, sectioned, stained with haematoxylin-eosin, and Holme's silver nitrate, and then examined microscopically to confirm complete removal as indicated by a layer of connective tissue, the epineurium, merging with the external capsule and surrounding the mass of ganglion cells. Mucosal Integrity Study-The experimental groups are shown in Table I. Vagotomy and pyloroplasty or a sham operation (exposing and identifying both vagal trunks and the pyloric sphincter), with celiac ganglionectomy or a sham operation (retracting the abdominal viscera and identifying the anatomical site of the ganglion behind the peritoneum), were performed. Animals were allowed to recover from anesthesia, and then 1mL of 5% procaine or saline was instilled by orogastric intubation into the stomach. Thereafter, rats were injected intraperitoneally with saline (5 mL/kg), atropine (5 mglkg), cimetidine (40 mg/kg), phenoxybenzamine (5, 10, or 15 mglkg), or phentolamine (5, 10, or 15 mg/kg). Finally, the rats were similarly injected with 5 mL/kg of saline (controls) or reserpine (5 mg/kg). Six hours later, animals were killed by ether overdose and the stomachs were removed and opened along the greater curvature. After wash-
0
0022-3549/88/0700-0582$01.00/0 1988, American Pharmaceutical Association
Table I-Reserpine-Induced Acute Gastric Yucosal Injury in the Rat % Incidence of Animals Showing Lesions
Lesion Area after 6 h (mean f SEM), mm'
Experimental Group
n
Control Reserpine (5 mg/kg, ip) Atropine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Cimetidine (40 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (10 mg/kg, ip) and reserpine (5 mg/kg, ip) Phenoxybenzamine (15 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (5 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (10 mg/kg, ip) and reserpine (5 mg/kg, ip) Phentolamine (15 mg/kg, ip) and resperine (5 mgikg, ip) 5% Procaine (1 mL, orally) and reserpine (5 mglkg, ip) Vagotomy and reserpine (5 mg/kg, ip) Celiac ganglionectomy and reserpine (5 mg/kg, ip)
10 10
100
40.1 t 5.2
10
100
38
10
100
40.5 ? 4.6
10
80
9.7 t 2a
10
40
3
10
0
0
10
80
11.5 t 1.6a
10
40
2.5 ? 0.7a
10
0
0
10
0
0
10
0
0
10
100
a
0
0
42
t4
t 0.5a
2
6
p < 0.001 by the Mann Whitney test comparing the reserpine-alone group with treatment group.
ing with a direct stream of cold water, the stomachs were pinned out and independently examined for the presence of mucosal injury. Each injury was measured in maximum length and width and the surface area (mm') was calculated. The total injury score was obtained for each animal and the mean injury score was calculated for each study group. Sections of injured and apparently uninjured gastric mucosa were cut off each stomach, fixed, embedded in paraffin, sectioned at right angles to the mucosa, stained (hematoxylin-eosin, Martius scarlet blue and periodic-acid-Schiff), and then examined microscopically. In order to minimize day-to-day variation in response to treatment, animals were allocated to the control and all of the treatment groups within the experiment on each experimental day. In preliminary studies, administration of intraperitoneal reserpine in a dose of 5 mglkg produced gastric mucosal injury in all rats, after 6 h, and was, therefore, considered a suitable dose for this study. The macroscopic and microscopic appearances of the r a t gastric mucosa were not affected after 6 h of intraperitoneal injection of phenoxybenzamine (5,10, or 15 mg/kg), phentolamine (5,10, or 15 mg/kg), or the vehicle solution of reserpine (5 mL/kg of 0.127 M glacial acetic acid). Statistical Analysis-Results are expressed as mean 5 SEM. The statistical significance (p 10.05) of observed differences between groups was determined using the Mann-Whitney U test for nonparametric data.
Results The results are presented in Table I. Histological examination confirmed removal of the celiac ganglion in all members of the ganglionectomy group. All rats in the reserpine-alone group developed oval or round injury that was confined to the glandular stomach and which had no constant relationship to rugal crests (injury score 40.1 ? 5.2 mm'). Atropine or cimetidine did not influence the incidence, appearance, or score of this injury (38 4 and 40.5 ? 4.6 mm', respectively). Phenoxybenzamine or phentolamine, in doses of 5 mg/kg, significantly (p <0.001) protected rats against the reserpine-induced injury, with 80% of animals developing injury similar to that of the reserpine alone group (9.7 ? 2 and 11.5 t 1.6 mm', respectively versus 40.1 5.2 mm'). However, the protective action
*
*
of the lO-mg/kg dose was significantly (p <0.01) more effective, with 40% of animals showing injury similar to that of the reserpine alone group (3 0.5 and 2.5 t 0.7 mm'). Celiac ganglionectomy had no effect on the incidence, pattern, or score of the reserpine injury (42 t 6 versus 40.1 ? 5.2 mm'). Vagotomy, 15 mg/kg of phenoxybenzamine or phentolamine, or 1 mL of 5%procaine protected all rats against the reserpine-induced acute gastric mucosal injury. Microscopically, the reserpine injury consisted of partial or full depth (including the muscularis mucosae) mucosal necrosis and/or loss with necrosis, hemorrhage, and polymorphonuclear leukocyte infiltration at the edges. The submucosal and mucosal blood vessels were severely constricted (Figure 1). The submucosa was infiltrated with polymorphonuclear leukocytes and was edematous, but the muscularis propria was completely intact. No pathological changes were detected in the forestomach or antrum. The reserpine injury developed in the mucosa and submucosa as foci of hemorrhage or necrosis. These foci were directly related to disrupted or constricted blood vessels and expanded to communicate with the lumen. Atropine, cimetidine, or celiac ganglionectomy had no effect on the microscopic appearances of the reserpine injury. Similarly, the injury developing with 5 or 10 mg/kg of phenoxybenzamine or phentolamine was microscopically similar to that of the reserpine alone group. Gastric sections from the groups having vagotomy, procaine (Figure 2), or 15 mglkg of phenoxybenzamine or phentolamine were microscopically similar to those of control animals.
*
Discussion The results of the present study confirm the previous report2 that in the rat, 5 mglkg of reserpine produces vagal delivery of alpha-adrenoceptor stimulation to gastric submucosal and mucosal blood vessels causing vasoconstriction which, in turn, injures the mucosa. In the present study, 6 h Journal ofPharmaceutical Sciences / 583 Vol. 77,No. 7,July 1988
Figure 1-The glandular stomach after 6 h of intraperitoneal reserpine. Note the submucosal and mucosal vasoconstriction (periodic acidSchiff, x 1 16; original magnification x 29).
Flgure 2-The glandular stomach after 6 h of procaine and reserpine. Note the absence of injury and vasoconstriction(periodic acid-Schif, x 96; original magnification x 24).
after intraperitoneal reserpine (5 mg/kg), gastric mucosal injury confined to the glandular stomach developed in all animals (Table I). This injury initiated intramurally as foci of necrosis or hemorrhage and was associated with submucosal and mucosal vasoconstriction. Pretreatment with potent antisecretory dosesHof the anticholinergic atropine or the H2-receptor antagonist cimetidine did not influence the appearances or severity of the reserpine-induced gastric mucosal injury (Table I). This observation suggests that H t is not the principal factor in the mechanism of this injury, but does not exclude the possibility that it might be an essential prerequisite. 584 !Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 1988
Phenoxybenzamine and phentolamine are alpha-adrenoceptor blocking drugs, the latter being more spe~ific.~ Dosedependent protection against the reserpine-induced acute gastric mucosal injury was produced by phenoxybenzamine or phentolamine; a complete protection against injury and its associated submucosal and mucosal vasoconstriction was noted with the 15-mg/kg dose (Table I). This finding and the microscopic observation that the reserpine injury initiates in relation to gastric submucosal and mucosal vasoconstriction suggest that ischemia is the principal factor behind the reserpine injury. Reserpine acts on the hypothalamus, producing central adrenergic discharge and stimulation of peripheral sympathetic pathways which release noradrenaline into the tissues.1o The results of the present study are consistent with the pharmacological action of reserpine in that they suggest that the reserpine-induced intragastric vasoconstriction is caused by alpha-adrenoceptor stimulation. Celiac ganglionectomy, to interrupt autonomic sympathetic delivery to the stomach,ll did not influence the reserpine-induced injury, but vagotomy afforded complete protection against this injury and its associated intragastric vasoconstriction (Table I). The similarity in action between vagotomy and large doses of alpha-adrenoceptor blocking drugs suggests that reserpine induces vagal alpha-adrenergic delivery to the rat stomach, causing submucosal and mucosal vasoconstriction which, in turn, injures the mucosa. Additional studies employing measurement of mucosal blood flow are needed to confirm these microscopic findings. F'rocaine, like vagotomy or large doses of alpha-adrenoceptor blocking drugs, protects against the reserpine-induced gastric submucosal and mucosal vasoconstriction and mucosal injury (Table I and Figure 2), demonstrating that it interrupts the vagal stimulation causing this vasoconstriction and injury, and thus providing a vagotomy action. It might be argued that vasodilation by procaine6 achieved protection against vasoconstriction. Since vagal stimulation produced this vasoconstriction, the conclusion that procaine has a vagotomy action still applies, regardless of whether this action is on blood vessels or neurotransmission. Reserpine-induced acute gastric mucosal injury is a stressinduced injury."-" Patients with major trauma, hemorrhage, burns, neurological injuries, or sepsis may survive their initial injury only to succumb to bleeding from stress-induced acute gastric mucosal injury.12This life-threatening injury13 may not respond to conservative treatment, making surgery the only option.14 The surgical procedure usually employed in such cases is that of vagotomy and drainage, however the predisposing factor to gastric bleeding increases the risk of the pera at ion.'^ The results of this study show that oral procaine protects against the development of stress-induced acute gastric mucosal injury. Detailed studies are needed to determine whether similar results can be obtained in humans. Procaine was chosen for this study because of its relatively low toxicity. Further studies are needed to determine the optimum dose of oral procaine for protection against the reserpine-induced injury and to examine its effect on gastric muscular tone, motility, and rate of emptying. Studies are also needed to determine whether relatively longer acting or more potent local anesthetics have actions similar to those of procaine observed in this study.
Conclusions The results of the present study demonstrate that in the rat, oral procaine prevents delivery of reserpine-induced vagal stimulation causing gastric submucosal and mucosal vasoconstriction and mucosal injury. Thus, procaine provides
a vagotomy action. Since the reserpine injury is a stressinduced injury, oral procaine protects the rat stomach against stress-induced acute gastric mucosal injury.
References and Notes 1. Blackman, J. G.;Campion, D. S.; Fastier, F. N. Br. J. Phurmacol. 1959,14,112-116. 2. Salim, A.S.J.Physiol. 1987,392,363-376. 3. Salim, A.S.J.Psychosom. Res. 1987,31,85-90. 4. Salim, A.S.J.Psychosom. Res. 1987,31,231-237. 5 . Salim, A. S. J. Psychiat. Res., in press. 6. Reynolds, J. E. F.; Prasad, A. B. In Martindale: The Ewtm Phurmacopoeia; Pharmaceutical Press: London, 1982;pp 877-879. 7. Greene, E. C. In The Rat in Laboratory Investigation; Farris, E. J.; Griffith, J. Q., Jr., Eds.; Hafner: New York, 1967;Chapter 3,pp 38-41.
8. Rainsford, K. D. Di est Dis. 1978,23,521-529. 9. Nickerson, M. In +he Pharmacological Basis of Therapeutics, Goodman, L. S.; Gilman, A., Eds.; Macmillan: London; 1970; Chapter 26,pp 550-570. 10. Paee. I. H. Phvsiol. Rev. 1958.38. 277-335. 11. Leinard, A.S;: Long, D.; French; L. A.; Peter, E. T.; Wangensteen, 0. H. Sur e 1964,56,109-120. 12. Butterfield, W. gurg. Ann. 1975,7 , 261-278. 13. Skillman, J.J.; Bushnell, L.S.:Goldman,. H.:. Silen, W. A m J. Surg. 1969,117,523-531. . 14. Lucas, C. E.; Sugawa, C.; Riddle, J.; Rector, F.; Rosenberg, B.; Walt, A. J. Arch. Surg. 1971,102,266-273.
8.
Acknowledgments The skillful technical assistance of Mr. Donald McMillan and secretarial work of Miss Lesley Cook are gratefully acknowledged.
Journal of Pharmaceutical Sciences / 585 Vol. 77, No. 7,July 1988