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Medullary thyrotropin-releasing hormone mediates vagal-dependent adaptive gastric protection induced by mild acid in rats
GASTROENTEROLOGY1995;109:861-865
Medullary Thyrotropin-Releasing Hormone Mediates VagaI-Dependent Adaptive Gastric Protection Induced by Mild Acid in Rats HIROSHI KANEKO, KIMITOSHI KATO, GORDON OHNING, and YVETTE TACHI~ CURE/Gastroenteric BiologyCenter, West Los AngelesVeteransAdministrationMedical Center, Departmentof Medicineand Brain Research Institute, UCLA, Los Angeles, California
Background & Aims: Adaptive gastric protection is dependent on vagal pathways in rats. It is hypothesized that medullary thyrotropin-releasing hormone (TRH), known to regulate vagal function, is part of the brain mechanisms mediating adaptive gastric protection. M e t h o d s : Urethane-anesthetized rats were pretreated with either acute bilateral subdiaphragmatic vagotomy, sham operation, or intracisternal injection of purified control, TRH, or peptide YY antibody. Gastric lesions were assessed 75 minutes after orogastric administration of I mL of either'vehicle or 0.35N HCI followed 15 minutes later by 0.6N or 1.ON HCI. Results: Injection of 0.6N and 1.ON HCl induced gastric lesions covering 23.1% +_ 2.7% and 37.8% +_ 3.3% of the corpus mucosa, respectively. Pretreatment with 0.35N HCI resulted in 67.3% and 50.5% reductions in gastric lesions induced by 0.6N and 1.ON HCI, respectively. Subdiaphragmatic vagotomy or intracisternal injection of TRH antibody increased gastric lesions induced by 0.6N HCI to 32.2% ___2.2% and 42.9% _ 5.6%, respectively, and completely abolished the protective effect of 0.35N HCI pretreatment. Control or peptide YY antibody injected intracisternally did not alter the gastric protection induced by mild acid. Conclusions: These results indicate that medullary TRH plays a role in the vagally mediated adaptive gastric protection induced by mild acid.
he vagus plays an important role not only in gastric lesion formation but also in gastric cytoprotection through release of gastric prostaglandins and nitric oxide. 1-5 Previous studies indicate that the vagus participates in adaptive protection whereby a mild irritant such as diluted alcohol protects against gastric injury induced by subsequent exposure to absolute alcohol) '4'5 However, little is known about the chemical coding within the brain that mediates the vagal-dependent adaptive cytoprotection. Medullary thyrotropin-releasing hormone (TRH) plays a physiological role in the vagal regulation of gas-
T
•
6
tric funcnon. In addition, it was recently shown that the stable T R H analogue, RX 77368, injected either into the cisterna magna or into the dorsal motor nucleus of the vagus (DMN) at a dose below the threshold necessary to increase gastric acid secretion prevented gastric mucosal damage induced by 60% ethanol via a vagalcholinergic prostaglandin, NO, and calcitonin gene-related peptide pathways in conscious and urethane-anesthetized rats. 7-9 These findings let us speculate that medullary T R H may play a role in the vagally mediated gastric adaptive cytoprotection induced by a mild irritant. The role of endogenous T R H in the brainstem was examined with the T R H antibody no. 8964 delivered into the cisterna magna. The T R H antibody was previously shown to be specific for prevention of the biological actions of exogenous and endogenous T R H in the medulla. 1()
Materials and Methods Animals Male Sprague-Dawley rats (260-330 g; Harlan Laboratories, San Diego, CA) were housed under standard conditions. Rats were deprived of food for 22 hours with free access to water up to 2 hours before the beginning of the study• All experiments were performed in rats anesthetized with urethane (1.5 g/kg intraperitoneally; Sigma Chemical Co., St, Louis, MO). Rectal temperature was kept at 36.5-37.5°C with a heating pad during the study.
Chemicals TRH (CURE no. 8964) and peptide YY (CURE no. 9153) rabbit polyclonal immunoglobulin (Ig) antibodies were produced and characterized as previously described 1°'11 (Antibody Core; CURE/Gastroenterology Biology Center, Los Angeles, CA). Control IgG antibody was obtained from nonimAbbreviations used in this paper: DMN, dorsal motor nucleus of the vagus; TRH, thyrotropin-releasinghormone. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/S3.00
862 KANEKOET AL.
mune rabbits. All antibodies were purified using protein A affinity chromatography and reconstituted in phosphate buffer, pH 7.4, as previously described, m The mean effective doses for TRH and peptide YY that cause 50% inhibition of the initial bound-to-free ratio were 221 fmol/mL and 67 fmol/mL, respectively, at a final antibody dilution of 1:105.m']~
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Procedures In the first experiment, rats underwent either bilateral subdiaphragmatic vagotomy or sham operation. Two hours later, rats received by orogastric intubation (with stainless steel cannula) 1 mL of either distilled water (vehicle) or 0.35N HC1 (Fischer Scientific Co., Fair Lawn, NJ) followed 15 minutes later by 0.6N HC1. In a second experiment, rats received intracisternal injections (20 btL) of control antibody (260 [.tg), TRH antibody no. 8964 (260 ~tg), or peptide YY antibody no. 9153 (260 btg), followed 15 minutes later by orogastric intubation of 1 mL of either vehicle or 0.35N HC1. After an additional 15 minutes, 1 mL of 0.6N or 1.0N HCI was administered by orogastric intubation. Rats in both experiments were killed by cervical dislocation 1 hour after administration of the strong irritant. Stomachs were removed for examination of gastric mucosal lesions. In the last experiment, 1 mL of vehicle or 0.35N HCI was administered, and 15 minutes later 260 ltlg of either control or TRH antibody was injected intracisternally. Rats were killed 15 minutes later. The esophagogastric junction and pylorus were clamped by forceps, and gastric content was collected by gravity. After centrifugation (3000 rpm for 15 minutes), gastric volume was measured.
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Figure1. Effect of acute bilateral subdiaphragmatic vagotomy on mild acid-induced adaptive cytoprotection against strong acid in urethaneanesthetized rats. Two hours after the sham operation or subdiaphragmatic vagotomy, rats received intragastrically 1 mL of either vehicle or 0.35N HCl followed 15 minutes later by O.6N HCI. Gastric lesions were measured i hour after administration of the strong irritant. Each column represents the mean _+ SEM of the number of rats shown at the top. * * P < 0.01, #P < 0.05 compared with vehicle and shamoperation group; ÷+P < 0.01 compared with sham operation and 0.35N HCl pretreatment. D, Vehicle (1 mL intragastrically plus O.6N HCl (1 mL intragastrically); II, 0.35N HCl (1 mL intragastrically) plus O.6N HCI (1 mL intragastrically).
M e a s u r e m e n t of Gastric Lesions Gastric mucosal lesions were assessed by macroscopic criteria as previously described. 8 Each stomach was opened along the greater curvature, and the percentage of corpus mucosa containing lesions was determined using a computerized image analyzer device (MICRO/PDP-I 1; Digital Equipment Corp., Maynard, MA) equipped with imaging boards (Imaging Technology Inc., Woburn, MA).
Statistical Analysis Results are expressed as the mean -+- SEM. Multiplegroup comparisons were performed by analysis of variance followed by a Dunnet's contrast. A probability level of <0.05 was considered significant.
Results In sham-operated rats, orogastric administration of vehicle followed by 0.6N HCI induced macroscopic injury that involved 23.1% ___2.7% of the corpus mucosa (Figure 1). Lesions were characterized by long, dark red bands linearly oriented in a cranial-to-caudal axis confined to the corpus of the glandular stomach. Intragastric
administration of the mild irritant (0.35N HCI) inhibited 0.6N H C l - i n d u c e d gastric lesions by 67.3% (7.6% --- 1.1%; P < 0.01; Figure 1). Subdiaphragmatic vagotomy increased the 0.6N H C l - i n d u c e d gastric injury to 32.2% _+ 2.1% and abolished the protective effect of the pretreatment with 0.35N HCI (33.9% + 3.3%) (Figure 1). Intracisternal injection of purified T R H antibody (260 gg) increased the gastric mucosal injury induced by 0.6N HC1 to 42.9% --+ 5.6% compared with 30.2% + 4.9% in the control antibody-pretreated group (P < 0.01). T R H antibody injected intracisternally blocked the adaptive cytoprotective effect of 0.35N HCI pretreatment (36.5% + 5.1%), whereas control or peptide YY antibody (260 ILtg)pretreatment had no effect (Figure 2). The mild irritant significantly decreased the gastric mucosal injury induced by 0.6N HCI to 14.5% + 2.5% and 13.4% + 2.2% in control and peptide YY a n t i b o d y pretreated rats, respectively (Figure 2). In rats pretreated
MEDULLARY TRH AND ADAPTIVE CYTOPROTECTION
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Rgure 2. TRH antibody injected intracisternally prevents mild acidinduced adaptive cytoprotection against strong acid in urethane-anesthetized rats. Fifteen minutes after intracisternal injection of control, TRH, or peptide YY antibody, rats received orally either vehicle or 0.35N HCI followed by HCI (0.6N or 1.ON). Corpus mucosal lesions were determined 1 hour after administration of the strong irritant. Each column represents the mean _+ SEM of the number of rats shown at the top. * * P < 0.01 compared with respective vehicle group; ++P < 0.01 compared with control antibody and 0.35N HCI group; #P < 0.05 compared with control antibody and vehicle group. IC, intracisternal; IG, intragastric. 12, Vehicle (1 mL intragastrically); I , 0.35N HCI (1 mL intragastrically).
with intracisternal injection of control antibody, gastric injury induced by orogastric administration of 1.0N HCI reached 37.8% + 3.3%, which was not different from the extent of gastric injury induced in TRH antibodypretreated rats receiving 0.6N HC1. However, pretreatment with 0.35N HCI decreased gastric lesions induced by 1.0N HCI to 18.7% + 2.5% in rats injected intracisternally with the control antibody (Figure 2). Intracisternal injection of TRH antibody did not influence the gastric fluid volume collected 15 minutes after orogastric administration of 1 mL of either vehicle (TRH antibody, 0.64 + 0.19 mL, n = 5; control antibody, 0.42 4- 0.10 mL, n = 6) or 0.35N HC1 (TRH antibody, 1.47 + 0.10 mL, n = 6; control antibody, 1.15 + 0.09 mL, n = 6). Mild acid treatment significantly increased (P < 0.01) the gastric fluid volume in both groups.
Discussion Oral administration of I mL of 0.6N or 1.0N HCI induced severe gastric damage located predominantly in the corpus of urethane-anesthetized rats. Similar findings have been reported in conscious rats. 12'1s Acute subdiaphragmatic vagotomy enhanced the percentage of gastric injury induced by 0.6N HC1 by 39.4%. Other stud-
863
ies also have shown that 70%-100% ethanol-induced gastric lesion formation was enhanced by acute subdiaphragmatic vagotomy. 1 ~,~4 ~6 These data indicate that vagal-dependent mechanisms enhance the resistance of the gastric mucosa to strong gastric irritants such as absolute ethanol or 0.6N HC1. In the present study, pretreatment with mild acid (0.35N HC1) reduced the macroscopic mucosal injury caused by subsequent exposure to strong acid (0.6N HCI) in urethane-anesthetized rats, similar to previous findings observed in conscious rats. 1~'1v Acute subdiaphragmatic vagotomy completely abolished the adaptive cytoprotection induced by 0.35N HCI pretreatment. These data indicate that adaptive cytoprotection induced by mild acid requires integrity of the vagus. Likewise, the adaptive gastric cytoprotection produced by intragastric administration of a low concentration of ethanol followed by absolute ethanol was no longer observed in vagotomized r a t s . 1'4'5'16'18 The brain transmitters mediating the vagal-dependent adaptive cytoprotection are unknown. Several lines of neuroanatomical and functional evidence support a role of medullary TRH in the vagally mediated regulation of gastric secretory and motor function and resistance of the gastric mucosa to injury. 6'v'19 The present study supports a role for medullary TRH in enhancement of gastric defense mechanisms in the presence of a gastric irritant such as acid. Intracisternal injection of the purified TRH antibody significantly increased gastric mucosal damage induced by 0.6N HCI and completely abolished the gastric cytoprotection induced by pretreatment with mild acid. This dose of TRH antibody has been shown to block the biological actions of TRH injected intracisternally or endogenously released in the dorsal vagal complex, io The TRH antibody effect was specific in that intracisternal injection of similar protein concentrations of control or peptide YY antibody did not influence the adaptive cytoprotective response to 0.35N HC1. The use of 1.0N HC1 resulted in a percentage of gastric injury (37.8% + 3.3%) in control antibody-pretreated rats similar to that found with 0.6N HC1 in TRH antibody-pretreated rats (42.9% + 5.6%). However, pretreatment with 0.35N HCI reduced gastric lesion formation caused by 1.0N HCI only in the control antibody-injected rats. These data indicate that the blockade of the protective effect of 0.35N HC1 by intracisternal injection of TRH antibody reflects the immunoneutralization of medullary TRH and not the inability of0,35N HCI administration to protect against the increased gastric damage induced by intracisternal injection of TRH antibody. Gastric prostaglandins play a role in the vagally medi-
864
KANEKO ET AL.
ated resistance to necrotizing agents and adaptive gastric cytoprotection against ethanol or acid injury. 4'I3'~5'17 TRH, injected intracisternally or into the DMN at doses below the threshold for stimulation of gastric acid secretion, increased gastric prostaglandin E2 release via vagal cholinergic pathways and induced prostaglandin-dependent gastric protection against 60% ethanol in conscious or urethane-anesthetized r a t s . 8'9'2°'21 In contrast, recent studies show that mild irritants, such ethanol or acid, confer gastric adaptive cytoprotection through additional prostaglandin-independent mechanisms. 22-26 Factors such as luminal dilution of the strong irritant have been suggested as possible mechanisms in the adaptive cytoprotection because there is an increase in gastric fluid level after administration of 0 . 2 - 0 . 3 5 N HC1 and 1 mL of water immediately before 0.6N HC1 administration protects the gastric mucosa against lesions. 25'2v Likewise, in the present study intragastric administration of 0.3 5N HCI increased by 0.8 mL the gastric fluid volume collected 15 minutes after administration of mild acid in urethane-anesthetized rats, as previously reported in conscious rats. 25'27 However, intracisternal injection of TRH antibody did not modify the increase in fluid volume induced by the mild acid, although it counteracted the adaptive gastric cytoprotection. These data indicate that luminal dilution alone cannot account for the protection provided by intragastric administration of 0.35N HCI or be a mechanism through which TRH antibody blocks adaptive gastric protection against mild acid. In summary, the present data show that the vagus plays a role in increasing the resistance of the gastric mucosa against 0.6N HCI injury and adaptive protection by mild acid. In addition, medullary TRH is involved in mediating the vagal-dependent component of adaptive cytoprotection induced by mild acid.
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References 1. Henagan JM, Smith GS, Seidel ER, Miller TA. Influence of vagotomy on mucosal protection against alcohol-induced gastric damage in the rat. Gastroenterology 1984;87:903-908. 2. M6zsik G, Kir&ly A, Garamszegi M, J~vor T, Nagy L, SOt5 G, T6th G, Vincze A. Failure of prostacyclin, ~-carotene, atropine and cimetidine to produce gastric cyto- and general mucosal protection in surgically vagotomized rats. Life Sci 1991;49:13831389. 3. Cho CH, Chen BW, Hui WM, Lam SK, Ogle CW. The role of the vagus nerve in the protective action of acid inhibitors on ethanolinduced gastric mucosal damage in rats. J Gastroenterol Hepatol 1992;7:178-183. 4. Kato K, Matsuno Y, Matsuo Y, Shimamura M, Tanaka K, Murai I, Imai S. Role of mucosal prostaglandins in vagally-mediated adaptive cytoprotection in the rat. Gastroenterol Jpn 1992;27: 1-8. 5. Wallace JH, Morris GP, Cohen M. Gastric mucosal protection by
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chronic restraint: effects of vagotomy. Am J Physiol 1985; 248:G35-G39. Tach6 Y, Yang H, Yoneda M. Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex. Digestion 1993; 54:65-72. Tach6 Y, Yoneda M, Kato K, Kir~ly A, $0t6 G, Kaneko H. Intracisternal thyrotropin-releasing hormone-induced vagally mediated gastric protection against ethanol lesions: central and peripheral mechanisms. J Gastroenterol Hepatol 1994;9:$29-$35. Yoneda M, Tach6 Y. Central thyrotropin-releasing factor analog prevents ethanol-induced gastric damage through prostaglandins in rats. Gastroenterology 1992;102:1568-1574. Kato K, Yang Y, Tach6 Y. Low doses of analog act in the dorsal motor nucleus to induce gastric protection in rat. Am J Physiol (in press). Yang H, Ohning G, Tach6 Y. TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats. Am J Physiol 1993;265:G880-G886. Hines OJ, Whang EE, Liu CD, Bilchik AJ, Joffe H, Wong H, Zinner MJ, Ashley SW, McFadden DW. Effect of peptide YY and oxyntomodulin immuneutralizationfollowing intestinal resection (abstr). Gastroenterology 1994; 106:A815. Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCL, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 1979; 77:433-443. Takeuchi K, Nishiwaki H, Furukawa O, Okabe S. Cytoprotective action of histamine against O.6N HCl-induced gastric mucosal injury in rats; comparative study with adaptive cytoprotection induced by exogenous acid. Jpn J Pharmacol 1987;44:335344. M6zsik G, Kir~ly A, Garamszegi M, J~vor T, Nagy L, N~meth A, Silt6 G, Vincze A. Mechanisms of vagal nerve in gastric mucosal defense: unchanged gastric emptying and increased vascular permeability. J Clin Gastroenterol 1992;14(Suppl 1):$140S144. SiJt6 G, Kir~ly A, Vincze A, M6zsik G. Effect of acute surgical vagotomy on the mucosal content of 6-keto-PG~, PGE2and glutathion after intragastric 96% ethanol treatment in rats. Acta Physiol Hung 1992;80:205-211. Foschi D, Castoldi L, Del Soldato P, Musazzi M, Callioni F, Rovati V, Trabucchi E, Montorsi W. Effects of autonomic nervous system on gastric damage by ethanol in the rat. Dig Dis Sci 1989; 34:688-693. Robert A, Nezamis JE, Lancaster C, Davis JP, Field SO, Hanchar AJ. Mild irritants prevent gastric necrosis through "adaptive cytoprotection" mediated by prostaglandins. Am J Physiol 1983; 245:G113-G121. Tornwall MS, Smith SS, Barreto JC, Lopez RA, Henagan JM, Miller TA. Adverse effect of vagotomy on ethanol-induced gastric injury in the rat absence of a role for glutathione redox cycle. Dig Dis Sci 1993;38:2294-2298. Tach~ Y, Yoneda M. Central action of TRH to induce vagally mediated gastric cytoprotection and ulcer formation in rats. J Clin Gastroenterol 1993; 17(Suppl 1):S58-$63. Yoneda M, Tach~ Y. Vagal regulation of gastric prostaglandin E2 release by central TRH in rats. Am J Physiol 1993;264:G231G236. Kaneko H, Yang H, Ohning G, Tach~ Y. Medullary TRH is involved in gastric protection induced by low dose of kainic acid into the raphe pallidus. Am J Physiol 1995;268:G548-G552. MacNaughton WK, Williamson TE, Morris GP. Adaptive cytoprotection by 0.25 M HCl is truly "cytoprotective" and may not depend upon elevated levels of prostaglandin synthesis. Can J Physiol Pharmacol 1988;66:1075-1081.
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23. Hawkey CJ, Kemp RT, Walt RP, Bhaskar NK, Davies J, Filipowicz B. Evidence that adaptive cytoprotection in rats is not mediated by prostaglandins. Gastroenterology 1988;94:948-954. 24. Wallace JL. Increased resistance of the rat gastric mucosa to hemorrhagic damage after exposure to an irritant; role of the "mucoid cap" and prostaglandin synthesis. Gastroenterology 1988; 94:22 -32. 25. Hatakeyama Y, Matsuo M, Tomoi M, Mori J, Kohsaka M. Multiple mediators and mechanisms are involved in the adaptive cytoprotection provided by certain mild irritants. Jpn J Pharmacol 1993;63:251-256. 26. Cho CH, Ko JKS, Tang XL. The differential mechanisms of mild irritants on adaptive cytoprotection. J Gastroenterol Hepatol 1994; 9:$24-S28.
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27. Hatakeyama Y, Matsuo M, Tomoi M, Ohtsuka M, Shimomura K. Luminal dilution caused by certain mild irritants and capsaicin contributes to their gastric mucosal protection. Am J Physiol 1995; 268:G200-G206.
Received February 24, 1995. Accepted April 19, 1995. Address requests for reprints to: Yvette Tach6, Ph.D., CURE/Gastroenteric Biology Center, West Los Angeles Veterans Administration Medical Center, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, California 90073. Supported by National Institute of Mental Health grant MH-00663, National Institute of Arthritis Metabolism and Digestive Disease grant DK-30110, and Center grant DK 41301 (Antibody Cores). Dr. Kaneko is supported by a fellowship from Aichi Medical University.
Medullary Thyrotropin-Releasing Hormone Mediates VagaI-Dependent Adaptive Gastric Protection Induced by Mild Acid in Rats HIROSHI KANEKO, KIMITOSHI KATO, GORDON OHNING, and YVETTE TACHI~ CURE/Gastroenteric BiologyCenter, West Los AngelesVeteransAdministrationMedical Center, Departmentof Medicineand Brain Research Institute, UCLA, Los Angeles, California
Background & Aims: Adaptive gastric protection is dependent on vagal pathways in rats. It is hypothesized that medullary thyrotropin-releasing hormone (TRH), known to regulate vagal function, is part of the brain mechanisms mediating adaptive gastric protection. M e t h o d s : Urethane-anesthetized rats were pretreated with either acute bilateral subdiaphragmatic vagotomy, sham operation, or intracisternal injection of purified control, TRH, or peptide YY antibody. Gastric lesions were assessed 75 minutes after orogastric administration of I mL of either'vehicle or 0.35N HCI followed 15 minutes later by 0.6N or 1.ON HCI. Results: Injection of 0.6N and 1.ON HCl induced gastric lesions covering 23.1% +_ 2.7% and 37.8% +_ 3.3% of the corpus mucosa, respectively. Pretreatment with 0.35N HCI resulted in 67.3% and 50.5% reductions in gastric lesions induced by 0.6N and 1.ON HCI, respectively. Subdiaphragmatic vagotomy or intracisternal injection of TRH antibody increased gastric lesions induced by 0.6N HCI to 32.2% ___2.2% and 42.9% _ 5.6%, respectively, and completely abolished the protective effect of 0.35N HCI pretreatment. Control or peptide YY antibody injected intracisternally did not alter the gastric protection induced by mild acid. Conclusions: These results indicate that medullary TRH plays a role in the vagally mediated adaptive gastric protection induced by mild acid.
he vagus plays an important role not only in gastric lesion formation but also in gastric cytoprotection through release of gastric prostaglandins and nitric oxide. 1-5 Previous studies indicate that the vagus participates in adaptive protection whereby a mild irritant such as diluted alcohol protects against gastric injury induced by subsequent exposure to absolute alcohol) '4'5 However, little is known about the chemical coding within the brain that mediates the vagal-dependent adaptive cytoprotection. Medullary thyrotropin-releasing hormone (TRH) plays a physiological role in the vagal regulation of gas-
T
•
6
tric funcnon. In addition, it was recently shown that the stable T R H analogue, RX 77368, injected either into the cisterna magna or into the dorsal motor nucleus of the vagus (DMN) at a dose below the threshold necessary to increase gastric acid secretion prevented gastric mucosal damage induced by 60% ethanol via a vagalcholinergic prostaglandin, NO, and calcitonin gene-related peptide pathways in conscious and urethane-anesthetized rats. 7-9 These findings let us speculate that medullary T R H may play a role in the vagally mediated gastric adaptive cytoprotection induced by a mild irritant. The role of endogenous T R H in the brainstem was examined with the T R H antibody no. 8964 delivered into the cisterna magna. The T R H antibody was previously shown to be specific for prevention of the biological actions of exogenous and endogenous T R H in the medulla. 1()
Materials and Methods Animals Male Sprague-Dawley rats (260-330 g; Harlan Laboratories, San Diego, CA) were housed under standard conditions. Rats were deprived of food for 22 hours with free access to water up to 2 hours before the beginning of the study• All experiments were performed in rats anesthetized with urethane (1.5 g/kg intraperitoneally; Sigma Chemical Co., St, Louis, MO). Rectal temperature was kept at 36.5-37.5°C with a heating pad during the study.
Chemicals TRH (CURE no. 8964) and peptide YY (CURE no. 9153) rabbit polyclonal immunoglobulin (Ig) antibodies were produced and characterized as previously described 1°'11 (Antibody Core; CURE/Gastroenterology Biology Center, Los Angeles, CA). Control IgG antibody was obtained from nonimAbbreviations used in this paper: DMN, dorsal motor nucleus of the vagus; TRH, thyrotropin-releasinghormone. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/S3.00
862 KANEKOET AL.
mune rabbits. All antibodies were purified using protein A affinity chromatography and reconstituted in phosphate buffer, pH 7.4, as previously described, m The mean effective doses for TRH and peptide YY that cause 50% inhibition of the initial bound-to-free ratio were 221 fmol/mL and 67 fmol/mL, respectively, at a final antibody dilution of 1:105.m']~
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Procedures In the first experiment, rats underwent either bilateral subdiaphragmatic vagotomy or sham operation. Two hours later, rats received by orogastric intubation (with stainless steel cannula) 1 mL of either distilled water (vehicle) or 0.35N HC1 (Fischer Scientific Co., Fair Lawn, NJ) followed 15 minutes later by 0.6N HC1. In a second experiment, rats received intracisternal injections (20 btL) of control antibody (260 [.tg), TRH antibody no. 8964 (260 ~tg), or peptide YY antibody no. 9153 (260 btg), followed 15 minutes later by orogastric intubation of 1 mL of either vehicle or 0.35N HC1. After an additional 15 minutes, 1 mL of 0.6N or 1.0N HCI was administered by orogastric intubation. Rats in both experiments were killed by cervical dislocation 1 hour after administration of the strong irritant. Stomachs were removed for examination of gastric mucosal lesions. In the last experiment, 1 mL of vehicle or 0.35N HCI was administered, and 15 minutes later 260 ltlg of either control or TRH antibody was injected intracisternally. Rats were killed 15 minutes later. The esophagogastric junction and pylorus were clamped by forceps, and gastric content was collected by gravity. After centrifugation (3000 rpm for 15 minutes), gastric volume was measured.
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Figure1. Effect of acute bilateral subdiaphragmatic vagotomy on mild acid-induced adaptive cytoprotection against strong acid in urethaneanesthetized rats. Two hours after the sham operation or subdiaphragmatic vagotomy, rats received intragastrically 1 mL of either vehicle or 0.35N HCl followed 15 minutes later by O.6N HCI. Gastric lesions were measured i hour after administration of the strong irritant. Each column represents the mean _+ SEM of the number of rats shown at the top. * * P < 0.01, #P < 0.05 compared with vehicle and shamoperation group; ÷+P < 0.01 compared with sham operation and 0.35N HCl pretreatment. D, Vehicle (1 mL intragastrically plus O.6N HCl (1 mL intragastrically); II, 0.35N HCl (1 mL intragastrically) plus O.6N HCI (1 mL intragastrically).
M e a s u r e m e n t of Gastric Lesions Gastric mucosal lesions were assessed by macroscopic criteria as previously described. 8 Each stomach was opened along the greater curvature, and the percentage of corpus mucosa containing lesions was determined using a computerized image analyzer device (MICRO/PDP-I 1; Digital Equipment Corp., Maynard, MA) equipped with imaging boards (Imaging Technology Inc., Woburn, MA).
Statistical Analysis Results are expressed as the mean -+- SEM. Multiplegroup comparisons were performed by analysis of variance followed by a Dunnet's contrast. A probability level of <0.05 was considered significant.
Results In sham-operated rats, orogastric administration of vehicle followed by 0.6N HCI induced macroscopic injury that involved 23.1% ___2.7% of the corpus mucosa (Figure 1). Lesions were characterized by long, dark red bands linearly oriented in a cranial-to-caudal axis confined to the corpus of the glandular stomach. Intragastric
administration of the mild irritant (0.35N HCI) inhibited 0.6N H C l - i n d u c e d gastric lesions by 67.3% (7.6% --- 1.1%; P < 0.01; Figure 1). Subdiaphragmatic vagotomy increased the 0.6N H C l - i n d u c e d gastric injury to 32.2% _+ 2.1% and abolished the protective effect of the pretreatment with 0.35N HCI (33.9% + 3.3%) (Figure 1). Intracisternal injection of purified T R H antibody (260 gg) increased the gastric mucosal injury induced by 0.6N HC1 to 42.9% --+ 5.6% compared with 30.2% + 4.9% in the control antibody-pretreated group (P < 0.01). T R H antibody injected intracisternally blocked the adaptive cytoprotective effect of 0.35N HCI pretreatment (36.5% + 5.1%), whereas control or peptide YY antibody (260 ILtg)pretreatment had no effect (Figure 2). The mild irritant significantly decreased the gastric mucosal injury induced by 0.6N HCI to 14.5% + 2.5% and 13.4% + 2.2% in control and peptide YY a n t i b o d y pretreated rats, respectively (Figure 2). In rats pretreated
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TRH 0.6N
Control t,0N
Rgure 2. TRH antibody injected intracisternally prevents mild acidinduced adaptive cytoprotection against strong acid in urethane-anesthetized rats. Fifteen minutes after intracisternal injection of control, TRH, or peptide YY antibody, rats received orally either vehicle or 0.35N HCI followed by HCI (0.6N or 1.ON). Corpus mucosal lesions were determined 1 hour after administration of the strong irritant. Each column represents the mean _+ SEM of the number of rats shown at the top. * * P < 0.01 compared with respective vehicle group; ++P < 0.01 compared with control antibody and 0.35N HCI group; #P < 0.05 compared with control antibody and vehicle group. IC, intracisternal; IG, intragastric. 12, Vehicle (1 mL intragastrically); I , 0.35N HCI (1 mL intragastrically).
with intracisternal injection of control antibody, gastric injury induced by orogastric administration of 1.0N HCI reached 37.8% + 3.3%, which was not different from the extent of gastric injury induced in TRH antibodypretreated rats receiving 0.6N HC1. However, pretreatment with 0.35N HCI decreased gastric lesions induced by 1.0N HCI to 18.7% + 2.5% in rats injected intracisternally with the control antibody (Figure 2). Intracisternal injection of TRH antibody did not influence the gastric fluid volume collected 15 minutes after orogastric administration of 1 mL of either vehicle (TRH antibody, 0.64 + 0.19 mL, n = 5; control antibody, 0.42 4- 0.10 mL, n = 6) or 0.35N HC1 (TRH antibody, 1.47 + 0.10 mL, n = 6; control antibody, 1.15 + 0.09 mL, n = 6). Mild acid treatment significantly increased (P < 0.01) the gastric fluid volume in both groups.
Discussion Oral administration of I mL of 0.6N or 1.0N HCI induced severe gastric damage located predominantly in the corpus of urethane-anesthetized rats. Similar findings have been reported in conscious rats. 12'1s Acute subdiaphragmatic vagotomy enhanced the percentage of gastric injury induced by 0.6N HC1 by 39.4%. Other stud-
863
ies also have shown that 70%-100% ethanol-induced gastric lesion formation was enhanced by acute subdiaphragmatic vagotomy. 1 ~,~4 ~6 These data indicate that vagal-dependent mechanisms enhance the resistance of the gastric mucosa to strong gastric irritants such as absolute ethanol or 0.6N HC1. In the present study, pretreatment with mild acid (0.35N HC1) reduced the macroscopic mucosal injury caused by subsequent exposure to strong acid (0.6N HCI) in urethane-anesthetized rats, similar to previous findings observed in conscious rats. 1~'1v Acute subdiaphragmatic vagotomy completely abolished the adaptive cytoprotection induced by 0.35N HCI pretreatment. These data indicate that adaptive cytoprotection induced by mild acid requires integrity of the vagus. Likewise, the adaptive gastric cytoprotection produced by intragastric administration of a low concentration of ethanol followed by absolute ethanol was no longer observed in vagotomized r a t s . 1'4'5'16'18 The brain transmitters mediating the vagal-dependent adaptive cytoprotection are unknown. Several lines of neuroanatomical and functional evidence support a role of medullary TRH in the vagally mediated regulation of gastric secretory and motor function and resistance of the gastric mucosa to injury. 6'v'19 The present study supports a role for medullary TRH in enhancement of gastric defense mechanisms in the presence of a gastric irritant such as acid. Intracisternal injection of the purified TRH antibody significantly increased gastric mucosal damage induced by 0.6N HCI and completely abolished the gastric cytoprotection induced by pretreatment with mild acid. This dose of TRH antibody has been shown to block the biological actions of TRH injected intracisternally or endogenously released in the dorsal vagal complex, io The TRH antibody effect was specific in that intracisternal injection of similar protein concentrations of control or peptide YY antibody did not influence the adaptive cytoprotective response to 0.35N HC1. The use of 1.0N HC1 resulted in a percentage of gastric injury (37.8% + 3.3%) in control antibody-pretreated rats similar to that found with 0.6N HC1 in TRH antibody-pretreated rats (42.9% + 5.6%). However, pretreatment with 0.35N HCI reduced gastric lesion formation caused by 1.0N HCI only in the control antibody-injected rats. These data indicate that the blockade of the protective effect of 0.35N HC1 by intracisternal injection of TRH antibody reflects the immunoneutralization of medullary TRH and not the inability of0,35N HCI administration to protect against the increased gastric damage induced by intracisternal injection of TRH antibody. Gastric prostaglandins play a role in the vagally medi-
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KANEKO ET AL.
ated resistance to necrotizing agents and adaptive gastric cytoprotection against ethanol or acid injury. 4'I3'~5'17 TRH, injected intracisternally or into the DMN at doses below the threshold for stimulation of gastric acid secretion, increased gastric prostaglandin E2 release via vagal cholinergic pathways and induced prostaglandin-dependent gastric protection against 60% ethanol in conscious or urethane-anesthetized r a t s . 8'9'2°'21 In contrast, recent studies show that mild irritants, such ethanol or acid, confer gastric adaptive cytoprotection through additional prostaglandin-independent mechanisms. 22-26 Factors such as luminal dilution of the strong irritant have been suggested as possible mechanisms in the adaptive cytoprotection because there is an increase in gastric fluid level after administration of 0 . 2 - 0 . 3 5 N HC1 and 1 mL of water immediately before 0.6N HC1 administration protects the gastric mucosa against lesions. 25'2v Likewise, in the present study intragastric administration of 0.3 5N HCI increased by 0.8 mL the gastric fluid volume collected 15 minutes after administration of mild acid in urethane-anesthetized rats, as previously reported in conscious rats. 25'27 However, intracisternal injection of TRH antibody did not modify the increase in fluid volume induced by the mild acid, although it counteracted the adaptive gastric cytoprotection. These data indicate that luminal dilution alone cannot account for the protection provided by intragastric administration of 0.35N HCI or be a mechanism through which TRH antibody blocks adaptive gastric protection against mild acid. In summary, the present data show that the vagus plays a role in increasing the resistance of the gastric mucosa against 0.6N HCI injury and adaptive protection by mild acid. In addition, medullary TRH is involved in mediating the vagal-dependent component of adaptive cytoprotection induced by mild acid.
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Received February 24, 1995. Accepted April 19, 1995. Address requests for reprints to: Yvette Tach6, Ph.D., CURE/Gastroenteric Biology Center, West Los Angeles Veterans Administration Medical Center, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, California 90073. Supported by National Institute of Mental Health grant MH-00663, National Institute of Arthritis Metabolism and Digestive Disease grant DK-30110, and Center grant DK 41301 (Antibody Cores). Dr. Kaneko is supported by a fellowship from Aichi Medical University.