- Email: [email protected]
Protective Effect of Sucralfate Against Alcohol-Induced Gastric Mucosal Injury in the Rat
GASTROENTEROLOGY 1985;88:366-74
Protective Effect of Sucralfate Against Alcohol-Induced Gastric Mucosal Injury in the Rat Macroscopic, Histologic, Ultrastructural, and Functional Time Sequence Analysis DANIEL HOLLANDER, ANDRZEJ T ARNA WSKI, WILLIAM J. KRAUSE, and HELLA GERGELY Veterans Administration Medical Center. Long Beach California; University of California. Irvine; and University of Missouri. Columbia. Missouri
Histologic or ultrastructural evidence of the ability of sucralfate to protect the gastric mucosa against ethanol injury is lacking. Therefore we analyzed morphologic and functional changes in the mucosa of 120 rats receiving, intragastrically, 2 ml of either sucralfate 500 mg/kg body wt or a control solution and 1 h later 2 ml of 100% ethanol. At 15 min, 1,4,6, and 24 h after ethanol instillation, mucosal changes were assessed by macroscopic examination, quantitative histology, scanning electron microscopy, recordings of gastric potential difference, and measurements of volume, pH, and electrolytes in the gastric contents. Between 15 min and 24 h after ethanol instillation, macroscopic necrotic lesions in controls involved > 33% of mucosal area and in the sucralfate-treated group <4% (p < 0 .001 for each period). In controls, ethanol instillation produced surface epithelial cell disruption and deep (>0.2 mm) mucosal necrosis involving >55% ± 3% of the mucosal length. In sucralfate-pretreated animals, disruption of the surface epithelium was present at 15 min, 1 h, and 4 h after ethanol instillation, but deep necrotic lesions were virtually absent (0%-2%; p < 0.001 vs. controls) during the entire study period. The surface epithelium was mostly reestablished by 6 h after ethanol instillation in the sucralfate group but not in the controls. We concluded that sucralfate protects the gastric mucosa against ethanol-induced injury by preventing deep mucosal neReceived April 2, 1984. Accepted Jul y 16, 1984. Address requests for reprints to: Daniel Hollander, M.D ., C340. Medical Science I, Uni versity of California, Irvine, California 92717 . © 1985 by the American Gastroenterological Association 0016-5085/85/$3 .30
crosis and as a consequence the mucosal proliferative zone cells rapidly restitute mucosal integrity. Sucralfate, an aluminum salt of sucrose octasulfate, is effective in the therapy of acute peptic ulcer disease (1-5). In addition to its healing properties, sucralfate effectively reduces the recurrence of gastric ulcers in humans (6) and protects the gastric mucosa against several types of acute experimental injury (7-13) including damage produced by concentrated alcohol (12 ,13). Although the protective effect of sucralfate against ethanol-induced gastric mucosal injury in the rat has been documented (12,13), the histologic or ultrastructural assessment of the protection is lacking and some of the available functional data [e.g., potential difference (PD) changes] are contradictory (12 vs. 13) . Moreover, the mechanisms responsible for the protection against alcohol injury by sucralfate have not been delineated. Therefore , the present study was designed to explore the histologic and ultrastructural aspects of this protection of the gastric mucosa against alcohol injury, and to assess the degree and the time sequence of the protection and its relation to functional changes.
Materials and Methods Animals One hundred sixty male Sprague-Dawley rats weighing 225-250 g were maintained on regular feed (Wayne Lab-Blox, Allied Mills , Chicago, Ill .) and were fasted in individual wire-bottom cages for 24 h before starting the experiment. Rats received intragastric pretreatment with 2 ml of either solution A, sucralfate 500 mg/kg
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
367
Figure 1. A.Gross appearance of gastric mucosa. The stomach was opened along the greater curvature. The p hotograph shows one-half of a normal glandular mucosa. B. Light micrograph of normal gastric fundic mucosa (H & E, x 160) .
suspended in isotonic saline, or solution B, isotonic saline as a control. The pH and osmolarity of solution B were adjusted (pH of 4.6 and osmolality of 310 mosmol) to be identical to the sucralfate solution (A). One hour after pretreatment, 2 ml of 100% ethanol was instilled intragastrically and the rats were killed after 15 min, 1, 4 , 6, and 24 h.
Studies At the end of the experiment the rat was anesthetized with nembutal (50 mglkg intraperitoneally), the esophagus and pylorus were ligated, and the stomach was removed . The volume of intragastric contents was measured with a calibrated cylinder and contents were stored for pH and electrolyte determinations. The stomach was dissected along the greater curvature and rinsed with cold
saline. The mucosal surface was examined visually and photographed in a standardized fashion described previ· ously (14).
Macroscopic Mucosal Necrosis The areas of gross hemorrhagic necrosis were measured planimetrically and the results were expressed as a percentage of total glandular mucosal area (14) .
Mucosal Histology The gastric wall specimens were cut obliquely through the entire extent of glandular mucosa from the forestomach to the pylorus. They were fixed in 10% buffered formalin and stained with hematoxylin and eosin
Figure 2. Gross appearance of gastric mucosa after ethanol instillation. A. One. hour after ethanol in vehicle-pretreated rat. Hemorrhagic. necrotic, longitudinal lesions (red) are present in the fundic glandular mucosa. B. Six hours after ethanol in vehicle- pretreated group. Hemorrhagic necrosis became more extensive and severe. In sucralfatepretreated rats at 1 h (e) and 6 h (0) after ethanol, necrotic lesions are minimal. Sucralfate incrustations (white) are firmly adherrent to pyloric mucosa and focally to fundic mucosa (C).
w
N
:::.
OJ
"C
~
~
z
CD CD
~
<:
-<
C"l
ot"" o
:::c
tTl
--l
Z
E5 tTl
--l
> CIJ
C"l
r>
--l
tTl
;0
otTl
> Z
t"" t""
o
::r:
CD
'"
January 1985
SUCRALF ATE PROTECTION OF GASTRIC MUCOSA
Table 1. Macroscopic Necrosis After Alcohol (% of total glandular mucosal area) Time after ethanol instillation 15 min 1h 4h 6h 24 h
Vehicle (%)
33 33 37 37
46
± ± ± ± ±
4 2 4 2 5
Pretreatment Sucralfate (n) (%)
(n)
(10) (10) (8) (8) (8)
(10) (12) (12) (8) (8)
n = number of animals. Values are mean vehicle group.
5 ± 2° 4 ± 1° 3 ± 1° 4 ± 1° 3 ± 1° ±
SE. ° p < 0.01 vs.
as well as with alcian blue and periodic acid-Schiff. Coded mucosal specimens were evaluated qualitatively under light microscopy by two investigators unaware of the code. In addition, the extent of deep histologic necrosis (defined as necrotic lesions penetrating into the mucosa deeper than 0.2 mm) was quantitated morphometric ally with the aid of an ocular micrometer by measuring the length of mucosal strips and the total length of necrotic lesions per strip. Similarly, we quantitated disruption of the continuity of the surface epithelial layer by measuring the length of the mucosa devoid of the surface epithelium and expressing it as a percentage of total mucosal length of each studied strip.
Scanning Electron Microscopy Coded gastric mucosal specimens were fixed in 3.5% glutaraldehyde buffered in 0.1 M phosphate buffer. After glutaraldehyde fixation, the specimens were washed in phosphate buffer and then osmicated in 1.0% osmium tetroxide for 2 h. After a second wash in phosphate buffer and dehydration in a graded series of ethanol solutions, the specimens were dried at critical point by liquid carbon dioxide substitution, placed on spinner stubs, and coated with gold to a depth of 20 nm in a polaron SEM coating unit (15,16). Coded specimens were viewed in a JEOL-35 scanning electron microscope operated at 20 kV. Six rats were studied in each group.
Intragastric pH and Electrolytes The pH of gastric contents was measured with a microprobe (model MI-506; Microelectrodes, Inc., Londonderry, N.H.) connected to a Beckman 3560 digital pH meter (Beckman Instruments, Inc., Fullerton, Calif.). Sodium and potassium concentrations in the gastric contents were determined by means of a flame photometer (model 443; Instrumentation Laboratories, Inc., Lexington, Mass.). Chloride concentrations were measured with a BuchlerCotlove chloridometer (Buchler Instruments, Inc., Fort Lee, N.J.).
Gastric Mucosal Potential Difference Measurements Gastric mucosal PD was recorded by using saturated KCI in 5% agar electrodes; one exploring electrode was
369
introduced into the fundic mucosa and a reference electrode was introduced into the peritoneal cavity. Electrodes were connected to the standard calomel reference electrodes, immersed in saturated KCI, and connected to a self-balancing Keithley 610C electrometer (Keithley Instruments Inc., Cleveland, Ohio) (17). The Keithley electrometer was connected to a strip chart recorder (Fisher Recordall 5000; Houston Instrument Co., Austin, Tex.) (17). For this study, fasted rats (10 in each group) were anesthetized intraperitoneally with nembutal (50 mg/kg body wt), their abdomens were opened, and a PD electrode and a polyethylene catheter (for solution instillation and withdrawal) were introduced into the stomach through a small incision in the duodenum (17). The pylorus and lower esophagus were ligated. During the entire period, rats were anesthetized with repeated injections of nembutal. Potential difference measurements were first performed in groups of rats after instillation of 0.9% NaCI to obtain baseline values, then for 1 h after the instillation of pretreatment solutions, and finally for 3 h after the instillation of ethanol.
Statistical Analysis Statistical comparisons of the degree of macroscopic and histologic mucosal necrosis were performed with the Kruskall-Wallis and Wilcoxon tests (18). The statistical significance of other data was evaluated by Student's ttest.
Results Macroscopic Necrosis of the Gastric Mucosa When compared with the normal gastric appearance (Figure 1), intragastric instillation of 2 ml of 100% ethanol to controls produced extensive visible hemorrhagic necrosis occupying >30% of the mucosal area (Figures 2A and 2B, Table 1) seen from 15 min to 24 h after ethanol instillation. In contrast sucralfate pretreatment reduced the extent of deep mucosal necrosis after ethanol instillation to a great extent (Figures 2C and 2D, Table 1).
Histologic Changes Mucosal histology examined between 15 min and 4 h after ethanol instillation revealed three major characteristic changes in the control groups (groups Band C) that included (a) disruption and desquamation of the surface epithelium, (b) extensive necrotic lesions penetrating deeply (>0.2 mm) into the mucosa, and (c) prominent edema of the submucosa as well as leukocyte infiltration (Figures 3A and 3B). In the control group deep necrotic lesions and submucosal edema persisted for 24 h after ethanol instillation, whereas the surface epithelium was restored only in the mucosal areas outside deep necrotic lesions.
Figure 3. Light micrographs of gastric mucosa after ethanol instillation (H & E. x 80). A. One hour after ethanol in vehicle-pretreated rat, disruption of the surface epithelium and necrotic lesions penetrate deeply into the mucosa and extensive edema of submucosa and leukocyte infiltration are present. B. Six hours after ethanol in vehiclepretreated rat. Deep necrotic lesions are better delineated; surface epithelium outside necrotic lesion is partially restored. Submucosal edema persists. C. One hour after ethanol in sucralfate-pretreated rat, disruption of the surface epithelium and extensive edema of the submucosa are present but deep mucosal necrosis is absent. D. Six hours after ethanol in sucralfate-pretreated rat. The surface epithelium is continuous and fully restored; deep mucosal necrosis is absent. The submucosal edema persists.
w
N
~
'1:l
~
~
z
5'"
OJ
<: ~
-<
C'l
o r o
:;<:l
ttl
--l
Z
ottl
:;<:l
--l
(fJ
>-
C'l
r>-
--l
ttl
:;<:l
ttl
(3
>-
r
5r
"o
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
Table 2. Disruption of the Surface Epithelium After Alcohol Instillation a Time after ethanol instillation 15 min 1h 4h 6h 24 h a
Pretreatment Vehicle
(n)
± ± ± ± ±
(7) (10) (8) (8) (8)
96 96 72 65 63
1.3 1.6 5 1.8 5.4
Sucralfate
(n)
± ± ± ± ±
(6) (10) (8) (6) (8)
92 87 50 22 5
Percent of total mucosal length. Mean ± SE. vehicle group. C p < 0.001 vs. vehicle group.
b
1.3 3.4 4.6 b 1.8 e 0.8 e
P < 0.01 vs.
In sucralfate-pretreated rats disruption of the surface epithelium and edema of the submucosa with leukocyte infiltration were found between 15 min and 1 h after ethanol injury (Figure 3C, Table 2). These changes were similar in extent to the changes seen in control rats (Figure 3, Table 2). However, deep necrotic lesions were absent or drastically reduced in sucralfate-pretreated animals through the entire study period (Figures 3C and 3D, Table 3). The surface epithelium was mostly restored (Figure 3, Table 2) in sucralfate-pretreated rats by 6 h after ethanol instillation. Scanning Electron Microscopy Instillation of ethanol produced prominent disruption of the surface epithelium in both sucralfate-pretreated and control rats at 15 min to 1 h after ethanol instillation (Figures 4B and 4C). Unlike controls (Figure 4DJ, the surface epithelium was mostly restored by 6 h in sucralfate-pretreated rats (Figure 4E). Volume, pH, and Electrolytes in Gastric Contents
After ethanol instillation, the gastric volume and pH's were significantly lower in sucralfatepretreated animals at 1-24 h, whereas sodium concentration was lower at 6 and 24 h (Table 4). Gastric Mucosal Potential Difference Instillation of ethanol produced a rapid drop in gastric PD of 47 ± 2 mV in controls and 36 ± 3 mV in sucralfate-pretreated rats (Figure 5). The rapid drop in PD coincided with disruption of the surface epithelium in both groups. Potential difference remained less reduced, however, in the sucralfatepretreated group than in the control group (Figure 5).
Discussion The present study confirmed previous reports indicating that sucralfate protects the gastric mucosa
371
against injury produced by concentrated ethanol and furnished histologic and ultrastructural evidence of the protection and its time sequence. Sequential detailed analysis of the mucosal changes showed that sucralfate did not prevent the initial disruption of the surface epithelium that was seen histologically and by scanning electron microscopy during the first hour after ethanol instillation. Likewise, sucralfate did not prevent the formation of extensive edema of the submucosa and leukocyte infiltration. However, sucralfate did protect against ethanol-induced necrosis of the deeper mucosal layers that contain the mucosal progenitor cells. The gastric surface epithelium was mostly restored within 6 h of ethanol administration in sucralfate-pretreated rats, but not in the controls. The restoration of the surface epithelium was accomplished by migration of protected progenitor cells from the deeper mucosal layers (the proliferative zone). The migration of the cells from the mucosal proliferative zone to replace aged or damaged cells is a common feature of epithelial cell renewal (19-22). After ethanol injury when much of the surface epithelial layer is desquamated, this process of migration is speeded up. The migration of the cells to restore damaged surface epithelium has been postulated by Grant (23) and documented by Svanes et al. (24) in vitro in studies of the frog gastric mucosa and documented by us (25) and by Wallace (26) in studies of the rat gastric mucosa. More detailed discussion on this subject is presented in our previous paper (27). Our data demonstrate that sucralfate provides morphologic as well as functional protection to the gastric mucosa. For example, sucralfate pretreatment reduces changes in the gastric content's volume, hydrogen ion concentration, and sodium concentration produced by ethanol instillation (Table 4). These findings are consistent with previous documentation of sucralfate's reduction of ethanol-induced hydrogen-ion back-diffusion (12) and plasma leakage (13). The morphologic, ultrastructural, and functional features of the protection of the gastric mucosa against ethanol-induced injury by sucralfate are similar to those of prostaglandins or essential fatty acids Table 3. Deep Histologic Necrosis After Alcohol a Time after ethanol instillation 15 min 1h 4h 6h 24 h
Pretreatment Vehicle
(n)
Sucralfate
(n)
± ± ± ± ±
(8) (10) (8) (8) (8)
2 ± lb 1 ± 0.5 b
(8) (10) (8) (6)
55 58 58 60 62
3 5 4 3 5
Db Db
1 ± O.4 b
(8)
n = number of animals. Values are mean ± SE. a Length of necrotic lesions deeper than 0.2 mm per total length of evaluated mucosa in percent. b p < 0.001 vs. vehicle group.
372
HOLLANDER ET AL.
GASTROENTEROLOGY Vol. 88, No. 1, Part 2
Figure 4. Scanning electron micrographs of the gastric mucosa (A, X900; B-D, x 600) . A. Normal control mucosa. Individual surface epithelial cells and the lumina of the gastric glands are clearly visible. Band C. One hour after ethanol instillation in vehiclepretreated (B) and sucraifate-pretreated (C) rats. Disruption of the surface epithelium is clearly present in both groups. Six hours after ethanol in vehicle-pretreated rats (D) the surface epithelium remained disrupted especially in areas of deep necrosis, whereas in sucralfate-pretreated rats (E) the surface epithelium is mostly restored.
found in an identical experimental model (25,26). The postulated mechanisms of the protective action of sucralfate, which have been reviewed and discussed in detail elsewhere (28-30), include formation of a protective "barrier" on eroded mucosal surfaces, deactivation and binding of pepsin, and binding of bile acids. These mechanisms of action cannot explain the protective effect of sucralfate against necrotizing agents such as ethanol as the ulcerations before ethanol administration are absent. These findings suggest that sucralfate influences the
normal gastric mucosa in a manner that enhances the mucosal defense mechanisms. Previously we found that sucralfate does affect the normal gastric mucosa by enhancing prostaglandin and mucus release and the replacement of the old surface epithelial cells with new cells (31). In addition we found that pretreatment with indomethacin (prostaglandin synthetase inhibitor) markedly abolished sucralfate's protection of the gastric mucosa against ethanolinduced necrosis (32) . These data indicate that the protection of the gastric mucosa against ethanol-
Table 4. Volume, pH, and Sodium Concentration in Gastric Contents After Alcohol Instillation Volume Time (h)
Pretreatment
(n)
1
VH SCR VH SCR VH SCR
12 10 8 8 7 8
6 24
Values represent mean ± SE.
a
pH
(ml)
6.3 3.3 10.2 5.5 12.5 4.8
± ± ± ± ± ±
0.7 0.5a 1 O.4 a
1 0.6 a
7.0 4.6 7.2 4.6 7.4 4.8
± ± ± ± ± ±
0.1 O.4 a
0.5 0.6a 0.5 0.5 a
Na (mEq/L) 85 68 96 74 122 86
± ± ± ± ± ±
9 8 6 4a 6 5a
p < 0.05 vs. VH at corresponding time. VH, vehicle. SCR, sucralfate.
K
(mEq/L) 6.3 10.5 12.3 8.8 6.2 5.2
1 3 2 1.8 1 ± 1 ± ± ± ± ±
Cl (mEq/L) 62 69 86 96 110 114
± 3 ± 5 ± 3
± 4 ± 7 ± 5
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
-~I,---~~--~~~------------------------
I SCI
EJDH
-40 1"--T. ~
~
!
... >
i§E
-30
I
____t-------_0---
YH
;.
-20
--------
-10
_-or
--..-------,
o +10
-75 -60
-30
0
60 TIME IMIIIUTESI
120
I.
Figure 5. Gastric mucosal PD recordings (mucosa negative vs. serosa) in baseline period; after vehicle (VH) or sucralfate (SCR) before and after ethanol (ETOH) instillation. Each point represents the mean ± SE from 10 rats. *p < 0.05.
induced necrosis by sucralfate is partly mediated by mucosal prostaglandins. The question of the effect of sucralfate on gastric emptying or augmented gastric secretions as possible mechanisms of its protective action has been addressed in separate experiments in which the pylorus was ligated and gastric contents were aspirated just before ethanol instillation_ In these experiments, sucralfate pretreatment protected the gastric mucosa in a manner and extent similar to our present studies. These findings rule out sucralfate-mediated changes in either gastric emptying or augmented gastric secretions, or both, as explanations for its mechanism of action (Tarnawski A, Hollander D, unpublished observations). Our present study clearly demonstrates that sucralfate is able to minimize gastric mucosal necrosis produced by a necrotizing agent (100% ethanol). Sucralfate's protection has many of the features of the protection furnished by prostaglandins. Thus our experiments suggest that sucralfate has cytoprotective properties. If these protective properties are substantiated in human studies, they would provide an explanation for the mechanism of action of sucralfate in both its prevention of ulcer recurrence and as the therapy of acute injury.
References 1. McHardy GG. A multicenter. double-blind trial of sucralfate and placebo in duodenal ulcer. J Clin Gastroenterol 1981; 3(Suppl 2):147-52. 2. Hollander D. Sucralfate: new agent for treatment of duodenal ulcer. In: Caspary WF. ed. Duodenal ulcer. gastric ulcer: sucralfate. a new therapeutic concept. Baltimore: Urban & Schwarzenberg. 1981:49-53. 3. Hollander D. Efficacy of sucralfate for duodenal ulcers: a multicenter. double-blind trial. J Clin Gastroenterol 1981; 3(SuppI2):153-7. 4. Marks IN. Lucke W. Wright JP. Gridwook AH. Ulcer healing and relapse rates after initial treatment with cimetidine or sucralfate. J Clin GastroenteroI1981;3(Suppl 2):163-5.
373
5. Ishimori A. Arakawa H. A placebo-controlled. double-blind trial using sucralfate for the treatment of peptic ulcer. A multicenter Japanese study. In: Caspary WF. ed. Duodenal ulcer. gastric ulcer: sucralfate. a new therapeutic concept. Baltimore: Urban & Schwarzenberg. 1981:93-100. 6. Miyake T. Ariyoshi J. Suzaki T. Oishi M. Sakai M. Ueda S. Endoscopic evaluation of the effect of sucralfate therapy and other clinical parameters on the recurrence rate of gastric ulcer. Dig Dis Sci 1980; 25:1-7. 7. Shimizu M. Ishii A. Imai T. et al. Experimental studies on anti ulcerous activity of basic aluminum sucrose sulfate (CGA6Jl: I. Assessment of CG-A6J for antiulcerous. antacid. and antipepsin activities in pylorus-ligated rats and effect of CGA6J on blood coagulability in rabbit. Kiso-to-Rinsho (Clinical Report) 1968;2:374-82. 8. Shimizu M. Shibuya E. Ishii A. et al. Experimental studies of an antiulcerous activity of basic aluminum sucrose sulfate (CG-A6Jl: II. Effects of CG-A6J on histamine and stress ulcerations. Kiso-to-Rinsho (Clinical Report) 1968;2:374-82. 9. Shimizu M. Shibuya E. Ishii A. et al. Experimental studies on anti ulcerous activity of basic aluminum sucrose sulfate (CGA6Jl: III. Effects on clamping cortisone ulcers. Kiso-to-Rinsho (Clinical Report) 1968;2:383-93. 10. Hirano T. Takagaki Y. Healing promotion effects of sucrose sulfate aluminum ester (Ulcermin) on acetic acid induced ulcer. Kiso-to-Rinsho (Clinical Report) 1974;8(4):1-12. 11. Okabe S. Takeuchi K. Kunimi H. Kanno M. Kawashima M. Effects of an antiulcer drug. sucralfate (a basic aluminium salt of sulfated disaccharide). on experimental gastric lesions and gastric secretion in rats. Dig Dis Sci 1983;28:1034-42. 12. Harrington SJ, Schlegel JF. Code CF. The protective effect of sucralfate on the gastric mucosa of rats. J Clin Gastroenterol 1981;3(Suppl 2):129-34. 13. Nagashima R. Hoshino E. Hniohara Y. Sakai K. Haa S. Nakano H. Effect of sucralfate on ethanol-induced gastric mucosal damage in the rat. Scand J Gastroenterol 1983;18(Suppl 83):17-20. 14. Hollander D. Tarnawski A. Ivey KJ, et al. Arachidonic acid protection of rat gastric mucosa against ethanol injury. J Lab Clin Med 1982;100:296-308. 15. Meller SM. Coppe MR. Ito S. Transmission electron microscopy of critically point dried tissue after observation in the scanning electron microscope. Anat Rec 1973;176:245-52. 16. Krause WJ. Leeson CR. The postnatal development of the respiratory system of the opossum. Light and scanning electron microscopy. Am J Anat 1973;137:337-56. 17. Tarnawski A. Ivey KJ. Transmucosal potential-difference profile in rat upper gastrointestinal tract. A simple model for testing gastric effects of pharmacological agents. Can J Physiol PharmacoI1978;56:471-3. 18. Conover WJ. Practical nonparametric statistics. New York: John Wiley & Sons. 1971:393. 19. Eastwood G. Gastrointestinal epithelial renewal. Gastroenterology 1977;72:962-75. 20. Willems G. Cell renewal in the gastric mucosa. Digestion 1972;6:46-63. 21. Lipkin M. Proliferation and differentiation of gastrointestinal cells. Phys Rev 1973;53:891-915. 22. Lipkin M. Cell turnover in gastrointestinal mucosa. In: Harmon JW. ed. Basic mechanism of gastrointestinal mucosal cell injury. Baltimore. London: Williams & Wilkins. 1981:31. 23. Grant R. Rate of replacement of the surface epithelial cells of the gastric mucosa. Anat Rec 1945;91:175-85. 24. Svanes K. Ito S. Takeuchi K. Silen W. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Gastroenterology 1982;82:1409-26.
374 HOLLANDER ET AL.
25. Tarnawski A, Hollander D, Stachura J, Krause WJ, Gergely H. Prostaglandin protection of the gastric mucosa against alcohol injury-a dynamic time-related process. Role of the mucosal proliferative zone. Gastroenterology 1985;88:334-52. 26. Wallace JL, Morris GP, Krausse EJ, Greaves SE. Reduction by cytoprotective agents of ethanol-induced damage to the rat gastric mucosa: a correlated morphological and physiological study. Can J Physiol Pharmacol 1982;60:1686-99. 27 . Tarnawski A, Hollander D, Stachura J, Krause WJ. Arachidonic acid protection of gastric mucosa against alcohol injury: sequential analysis of morphologic and functional changes. J Lab Clin Med 1983;102:340-51. 28. Bighley D, Giesing D. Mechanism of action studies of sucralfate. In: Caspary WF, ed . Duodenal ulcer, gastric ulcer:
GASTROENTEROLOGY Vol. 88, No.1, Part 2
29. 30. 31.
32.
sucralfate, a new therapeutic concept. Baltimore: Urban & Schwarzenberg, 1981:3-12. Nagashima R. Mechanisms of action of sucralfate. J Clin GastroenteroI1981;3(Suppl 2):117-27. McGraw F, Caldwell G. Sucralfate. Drug intelligence and clinical pharmacy 1981;15:578-80. Tarnawski A, Hollander D, Krause WJ, Zipser RD, Gergely H. Effect of sucralfate on normal gastric mucosa. Histologic, ultrastructural and functional assessment (abstr) . Gastroenterology 1983;84:1331. Hollander D, Tarnawski A, Gergely M, Zipser RD. Sucralfate protection of the gastric mucosa against ethanol induced injury: a prostaglandin mediated process? Scand J GastroenteroI1984;19(Suppl 101):97-102.
Protective Effect of Sucralfate Against Alcohol-Induced Gastric Mucosal Injury in the Rat Macroscopic, Histologic, Ultrastructural, and Functional Time Sequence Analysis DANIEL HOLLANDER, ANDRZEJ T ARNA WSKI, WILLIAM J. KRAUSE, and HELLA GERGELY Veterans Administration Medical Center. Long Beach California; University of California. Irvine; and University of Missouri. Columbia. Missouri
Histologic or ultrastructural evidence of the ability of sucralfate to protect the gastric mucosa against ethanol injury is lacking. Therefore we analyzed morphologic and functional changes in the mucosa of 120 rats receiving, intragastrically, 2 ml of either sucralfate 500 mg/kg body wt or a control solution and 1 h later 2 ml of 100% ethanol. At 15 min, 1,4,6, and 24 h after ethanol instillation, mucosal changes were assessed by macroscopic examination, quantitative histology, scanning electron microscopy, recordings of gastric potential difference, and measurements of volume, pH, and electrolytes in the gastric contents. Between 15 min and 24 h after ethanol instillation, macroscopic necrotic lesions in controls involved > 33% of mucosal area and in the sucralfate-treated group <4% (p < 0 .001 for each period). In controls, ethanol instillation produced surface epithelial cell disruption and deep (>0.2 mm) mucosal necrosis involving >55% ± 3% of the mucosal length. In sucralfate-pretreated animals, disruption of the surface epithelium was present at 15 min, 1 h, and 4 h after ethanol instillation, but deep necrotic lesions were virtually absent (0%-2%; p < 0.001 vs. controls) during the entire study period. The surface epithelium was mostly reestablished by 6 h after ethanol instillation in the sucralfate group but not in the controls. We concluded that sucralfate protects the gastric mucosa against ethanol-induced injury by preventing deep mucosal neReceived April 2, 1984. Accepted Jul y 16, 1984. Address requests for reprints to: Daniel Hollander, M.D ., C340. Medical Science I, Uni versity of California, Irvine, California 92717 . © 1985 by the American Gastroenterological Association 0016-5085/85/$3 .30
crosis and as a consequence the mucosal proliferative zone cells rapidly restitute mucosal integrity. Sucralfate, an aluminum salt of sucrose octasulfate, is effective in the therapy of acute peptic ulcer disease (1-5). In addition to its healing properties, sucralfate effectively reduces the recurrence of gastric ulcers in humans (6) and protects the gastric mucosa against several types of acute experimental injury (7-13) including damage produced by concentrated alcohol (12 ,13). Although the protective effect of sucralfate against ethanol-induced gastric mucosal injury in the rat has been documented (12,13), the histologic or ultrastructural assessment of the protection is lacking and some of the available functional data [e.g., potential difference (PD) changes] are contradictory (12 vs. 13) . Moreover, the mechanisms responsible for the protection against alcohol injury by sucralfate have not been delineated. Therefore , the present study was designed to explore the histologic and ultrastructural aspects of this protection of the gastric mucosa against alcohol injury, and to assess the degree and the time sequence of the protection and its relation to functional changes.
Materials and Methods Animals One hundred sixty male Sprague-Dawley rats weighing 225-250 g were maintained on regular feed (Wayne Lab-Blox, Allied Mills , Chicago, Ill .) and were fasted in individual wire-bottom cages for 24 h before starting the experiment. Rats received intragastric pretreatment with 2 ml of either solution A, sucralfate 500 mg/kg
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
367
Figure 1. A.Gross appearance of gastric mucosa. The stomach was opened along the greater curvature. The p hotograph shows one-half of a normal glandular mucosa. B. Light micrograph of normal gastric fundic mucosa (H & E, x 160) .
suspended in isotonic saline, or solution B, isotonic saline as a control. The pH and osmolarity of solution B were adjusted (pH of 4.6 and osmolality of 310 mosmol) to be identical to the sucralfate solution (A). One hour after pretreatment, 2 ml of 100% ethanol was instilled intragastrically and the rats were killed after 15 min, 1, 4 , 6, and 24 h.
Studies At the end of the experiment the rat was anesthetized with nembutal (50 mglkg intraperitoneally), the esophagus and pylorus were ligated, and the stomach was removed . The volume of intragastric contents was measured with a calibrated cylinder and contents were stored for pH and electrolyte determinations. The stomach was dissected along the greater curvature and rinsed with cold
saline. The mucosal surface was examined visually and photographed in a standardized fashion described previ· ously (14).
Macroscopic Mucosal Necrosis The areas of gross hemorrhagic necrosis were measured planimetrically and the results were expressed as a percentage of total glandular mucosal area (14) .
Mucosal Histology The gastric wall specimens were cut obliquely through the entire extent of glandular mucosa from the forestomach to the pylorus. They were fixed in 10% buffered formalin and stained with hematoxylin and eosin
Figure 2. Gross appearance of gastric mucosa after ethanol instillation. A. One. hour after ethanol in vehicle-pretreated rat. Hemorrhagic. necrotic, longitudinal lesions (red) are present in the fundic glandular mucosa. B. Six hours after ethanol in vehicle- pretreated group. Hemorrhagic necrosis became more extensive and severe. In sucralfatepretreated rats at 1 h (e) and 6 h (0) after ethanol, necrotic lesions are minimal. Sucralfate incrustations (white) are firmly adherrent to pyloric mucosa and focally to fundic mucosa (C).
w
N
:::.
OJ
"C
~
~
z
CD CD
~
<:
-<
C"l
ot"" o
:::c
tTl
--l
Z
E5 tTl
--l
> CIJ
C"l
r>
--l
tTl
;0
otTl
> Z
t"" t""
o
::r:
CD
'"
January 1985
SUCRALF ATE PROTECTION OF GASTRIC MUCOSA
Table 1. Macroscopic Necrosis After Alcohol (% of total glandular mucosal area) Time after ethanol instillation 15 min 1h 4h 6h 24 h
Vehicle (%)
33 33 37 37
46
± ± ± ± ±
4 2 4 2 5
Pretreatment Sucralfate (n) (%)
(n)
(10) (10) (8) (8) (8)
(10) (12) (12) (8) (8)
n = number of animals. Values are mean vehicle group.
5 ± 2° 4 ± 1° 3 ± 1° 4 ± 1° 3 ± 1° ±
SE. ° p < 0.01 vs.
as well as with alcian blue and periodic acid-Schiff. Coded mucosal specimens were evaluated qualitatively under light microscopy by two investigators unaware of the code. In addition, the extent of deep histologic necrosis (defined as necrotic lesions penetrating into the mucosa deeper than 0.2 mm) was quantitated morphometric ally with the aid of an ocular micrometer by measuring the length of mucosal strips and the total length of necrotic lesions per strip. Similarly, we quantitated disruption of the continuity of the surface epithelial layer by measuring the length of the mucosa devoid of the surface epithelium and expressing it as a percentage of total mucosal length of each studied strip.
Scanning Electron Microscopy Coded gastric mucosal specimens were fixed in 3.5% glutaraldehyde buffered in 0.1 M phosphate buffer. After glutaraldehyde fixation, the specimens were washed in phosphate buffer and then osmicated in 1.0% osmium tetroxide for 2 h. After a second wash in phosphate buffer and dehydration in a graded series of ethanol solutions, the specimens were dried at critical point by liquid carbon dioxide substitution, placed on spinner stubs, and coated with gold to a depth of 20 nm in a polaron SEM coating unit (15,16). Coded specimens were viewed in a JEOL-35 scanning electron microscope operated at 20 kV. Six rats were studied in each group.
Intragastric pH and Electrolytes The pH of gastric contents was measured with a microprobe (model MI-506; Microelectrodes, Inc., Londonderry, N.H.) connected to a Beckman 3560 digital pH meter (Beckman Instruments, Inc., Fullerton, Calif.). Sodium and potassium concentrations in the gastric contents were determined by means of a flame photometer (model 443; Instrumentation Laboratories, Inc., Lexington, Mass.). Chloride concentrations were measured with a BuchlerCotlove chloridometer (Buchler Instruments, Inc., Fort Lee, N.J.).
Gastric Mucosal Potential Difference Measurements Gastric mucosal PD was recorded by using saturated KCI in 5% agar electrodes; one exploring electrode was
369
introduced into the fundic mucosa and a reference electrode was introduced into the peritoneal cavity. Electrodes were connected to the standard calomel reference electrodes, immersed in saturated KCI, and connected to a self-balancing Keithley 610C electrometer (Keithley Instruments Inc., Cleveland, Ohio) (17). The Keithley electrometer was connected to a strip chart recorder (Fisher Recordall 5000; Houston Instrument Co., Austin, Tex.) (17). For this study, fasted rats (10 in each group) were anesthetized intraperitoneally with nembutal (50 mg/kg body wt), their abdomens were opened, and a PD electrode and a polyethylene catheter (for solution instillation and withdrawal) were introduced into the stomach through a small incision in the duodenum (17). The pylorus and lower esophagus were ligated. During the entire period, rats were anesthetized with repeated injections of nembutal. Potential difference measurements were first performed in groups of rats after instillation of 0.9% NaCI to obtain baseline values, then for 1 h after the instillation of pretreatment solutions, and finally for 3 h after the instillation of ethanol.
Statistical Analysis Statistical comparisons of the degree of macroscopic and histologic mucosal necrosis were performed with the Kruskall-Wallis and Wilcoxon tests (18). The statistical significance of other data was evaluated by Student's ttest.
Results Macroscopic Necrosis of the Gastric Mucosa When compared with the normal gastric appearance (Figure 1), intragastric instillation of 2 ml of 100% ethanol to controls produced extensive visible hemorrhagic necrosis occupying >30% of the mucosal area (Figures 2A and 2B, Table 1) seen from 15 min to 24 h after ethanol instillation. In contrast sucralfate pretreatment reduced the extent of deep mucosal necrosis after ethanol instillation to a great extent (Figures 2C and 2D, Table 1).
Histologic Changes Mucosal histology examined between 15 min and 4 h after ethanol instillation revealed three major characteristic changes in the control groups (groups Band C) that included (a) disruption and desquamation of the surface epithelium, (b) extensive necrotic lesions penetrating deeply (>0.2 mm) into the mucosa, and (c) prominent edema of the submucosa as well as leukocyte infiltration (Figures 3A and 3B). In the control group deep necrotic lesions and submucosal edema persisted for 24 h after ethanol instillation, whereas the surface epithelium was restored only in the mucosal areas outside deep necrotic lesions.
Figure 3. Light micrographs of gastric mucosa after ethanol instillation (H & E. x 80). A. One hour after ethanol in vehicle-pretreated rat, disruption of the surface epithelium and necrotic lesions penetrate deeply into the mucosa and extensive edema of submucosa and leukocyte infiltration are present. B. Six hours after ethanol in vehiclepretreated rat. Deep necrotic lesions are better delineated; surface epithelium outside necrotic lesion is partially restored. Submucosal edema persists. C. One hour after ethanol in sucralfate-pretreated rat, disruption of the surface epithelium and extensive edema of the submucosa are present but deep mucosal necrosis is absent. D. Six hours after ethanol in sucralfate-pretreated rat. The surface epithelium is continuous and fully restored; deep mucosal necrosis is absent. The submucosal edema persists.
w
N
~
'1:l
~
~
z
5'"
OJ
<: ~
-<
C'l
o r o
:;<:l
ttl
--l
Z
ottl
:;<:l
--l
(fJ
>-
C'l
r>-
--l
ttl
:;<:l
ttl
(3
>-
r
5r
"o
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
Table 2. Disruption of the Surface Epithelium After Alcohol Instillation a Time after ethanol instillation 15 min 1h 4h 6h 24 h a
Pretreatment Vehicle
(n)
± ± ± ± ±
(7) (10) (8) (8) (8)
96 96 72 65 63
1.3 1.6 5 1.8 5.4
Sucralfate
(n)
± ± ± ± ±
(6) (10) (8) (6) (8)
92 87 50 22 5
Percent of total mucosal length. Mean ± SE. vehicle group. C p < 0.001 vs. vehicle group.
b
1.3 3.4 4.6 b 1.8 e 0.8 e
P < 0.01 vs.
In sucralfate-pretreated rats disruption of the surface epithelium and edema of the submucosa with leukocyte infiltration were found between 15 min and 1 h after ethanol injury (Figure 3C, Table 2). These changes were similar in extent to the changes seen in control rats (Figure 3, Table 2). However, deep necrotic lesions were absent or drastically reduced in sucralfate-pretreated animals through the entire study period (Figures 3C and 3D, Table 3). The surface epithelium was mostly restored (Figure 3, Table 2) in sucralfate-pretreated rats by 6 h after ethanol instillation. Scanning Electron Microscopy Instillation of ethanol produced prominent disruption of the surface epithelium in both sucralfate-pretreated and control rats at 15 min to 1 h after ethanol instillation (Figures 4B and 4C). Unlike controls (Figure 4DJ, the surface epithelium was mostly restored by 6 h in sucralfate-pretreated rats (Figure 4E). Volume, pH, and Electrolytes in Gastric Contents
After ethanol instillation, the gastric volume and pH's were significantly lower in sucralfatepretreated animals at 1-24 h, whereas sodium concentration was lower at 6 and 24 h (Table 4). Gastric Mucosal Potential Difference Instillation of ethanol produced a rapid drop in gastric PD of 47 ± 2 mV in controls and 36 ± 3 mV in sucralfate-pretreated rats (Figure 5). The rapid drop in PD coincided with disruption of the surface epithelium in both groups. Potential difference remained less reduced, however, in the sucralfatepretreated group than in the control group (Figure 5).
Discussion The present study confirmed previous reports indicating that sucralfate protects the gastric mucosa
371
against injury produced by concentrated ethanol and furnished histologic and ultrastructural evidence of the protection and its time sequence. Sequential detailed analysis of the mucosal changes showed that sucralfate did not prevent the initial disruption of the surface epithelium that was seen histologically and by scanning electron microscopy during the first hour after ethanol instillation. Likewise, sucralfate did not prevent the formation of extensive edema of the submucosa and leukocyte infiltration. However, sucralfate did protect against ethanol-induced necrosis of the deeper mucosal layers that contain the mucosal progenitor cells. The gastric surface epithelium was mostly restored within 6 h of ethanol administration in sucralfate-pretreated rats, but not in the controls. The restoration of the surface epithelium was accomplished by migration of protected progenitor cells from the deeper mucosal layers (the proliferative zone). The migration of the cells from the mucosal proliferative zone to replace aged or damaged cells is a common feature of epithelial cell renewal (19-22). After ethanol injury when much of the surface epithelial layer is desquamated, this process of migration is speeded up. The migration of the cells to restore damaged surface epithelium has been postulated by Grant (23) and documented by Svanes et al. (24) in vitro in studies of the frog gastric mucosa and documented by us (25) and by Wallace (26) in studies of the rat gastric mucosa. More detailed discussion on this subject is presented in our previous paper (27). Our data demonstrate that sucralfate provides morphologic as well as functional protection to the gastric mucosa. For example, sucralfate pretreatment reduces changes in the gastric content's volume, hydrogen ion concentration, and sodium concentration produced by ethanol instillation (Table 4). These findings are consistent with previous documentation of sucralfate's reduction of ethanol-induced hydrogen-ion back-diffusion (12) and plasma leakage (13). The morphologic, ultrastructural, and functional features of the protection of the gastric mucosa against ethanol-induced injury by sucralfate are similar to those of prostaglandins or essential fatty acids Table 3. Deep Histologic Necrosis After Alcohol a Time after ethanol instillation 15 min 1h 4h 6h 24 h
Pretreatment Vehicle
(n)
Sucralfate
(n)
± ± ± ± ±
(8) (10) (8) (8) (8)
2 ± lb 1 ± 0.5 b
(8) (10) (8) (6)
55 58 58 60 62
3 5 4 3 5
Db Db
1 ± O.4 b
(8)
n = number of animals. Values are mean ± SE. a Length of necrotic lesions deeper than 0.2 mm per total length of evaluated mucosa in percent. b p < 0.001 vs. vehicle group.
372
HOLLANDER ET AL.
GASTROENTEROLOGY Vol. 88, No. 1, Part 2
Figure 4. Scanning electron micrographs of the gastric mucosa (A, X900; B-D, x 600) . A. Normal control mucosa. Individual surface epithelial cells and the lumina of the gastric glands are clearly visible. Band C. One hour after ethanol instillation in vehiclepretreated (B) and sucraifate-pretreated (C) rats. Disruption of the surface epithelium is clearly present in both groups. Six hours after ethanol in vehicle-pretreated rats (D) the surface epithelium remained disrupted especially in areas of deep necrosis, whereas in sucralfate-pretreated rats (E) the surface epithelium is mostly restored.
found in an identical experimental model (25,26). The postulated mechanisms of the protective action of sucralfate, which have been reviewed and discussed in detail elsewhere (28-30), include formation of a protective "barrier" on eroded mucosal surfaces, deactivation and binding of pepsin, and binding of bile acids. These mechanisms of action cannot explain the protective effect of sucralfate against necrotizing agents such as ethanol as the ulcerations before ethanol administration are absent. These findings suggest that sucralfate influences the
normal gastric mucosa in a manner that enhances the mucosal defense mechanisms. Previously we found that sucralfate does affect the normal gastric mucosa by enhancing prostaglandin and mucus release and the replacement of the old surface epithelial cells with new cells (31). In addition we found that pretreatment with indomethacin (prostaglandin synthetase inhibitor) markedly abolished sucralfate's protection of the gastric mucosa against ethanolinduced necrosis (32) . These data indicate that the protection of the gastric mucosa against ethanol-
Table 4. Volume, pH, and Sodium Concentration in Gastric Contents After Alcohol Instillation Volume Time (h)
Pretreatment
(n)
1
VH SCR VH SCR VH SCR
12 10 8 8 7 8
6 24
Values represent mean ± SE.
a
pH
(ml)
6.3 3.3 10.2 5.5 12.5 4.8
± ± ± ± ± ±
0.7 0.5a 1 O.4 a
1 0.6 a
7.0 4.6 7.2 4.6 7.4 4.8
± ± ± ± ± ±
0.1 O.4 a
0.5 0.6a 0.5 0.5 a
Na (mEq/L) 85 68 96 74 122 86
± ± ± ± ± ±
9 8 6 4a 6 5a
p < 0.05 vs. VH at corresponding time. VH, vehicle. SCR, sucralfate.
K
(mEq/L) 6.3 10.5 12.3 8.8 6.2 5.2
1 3 2 1.8 1 ± 1 ± ± ± ± ±
Cl (mEq/L) 62 69 86 96 110 114
± 3 ± 5 ± 3
± 4 ± 7 ± 5
January 1985
SUCRALFATE PROTECTION OF GASTRIC MUCOSA
-~I,---~~--~~~------------------------
I SCI
EJDH
-40 1"--T. ~
~
!
... >
i§E
-30
I
____t-------_0---
YH
;.
-20
--------
-10
_-or
--..-------,
o +10
-75 -60
-30
0
60 TIME IMIIIUTESI
120
I.
Figure 5. Gastric mucosal PD recordings (mucosa negative vs. serosa) in baseline period; after vehicle (VH) or sucralfate (SCR) before and after ethanol (ETOH) instillation. Each point represents the mean ± SE from 10 rats. *p < 0.05.
induced necrosis by sucralfate is partly mediated by mucosal prostaglandins. The question of the effect of sucralfate on gastric emptying or augmented gastric secretions as possible mechanisms of its protective action has been addressed in separate experiments in which the pylorus was ligated and gastric contents were aspirated just before ethanol instillation_ In these experiments, sucralfate pretreatment protected the gastric mucosa in a manner and extent similar to our present studies. These findings rule out sucralfate-mediated changes in either gastric emptying or augmented gastric secretions, or both, as explanations for its mechanism of action (Tarnawski A, Hollander D, unpublished observations). Our present study clearly demonstrates that sucralfate is able to minimize gastric mucosal necrosis produced by a necrotizing agent (100% ethanol). Sucralfate's protection has many of the features of the protection furnished by prostaglandins. Thus our experiments suggest that sucralfate has cytoprotective properties. If these protective properties are substantiated in human studies, they would provide an explanation for the mechanism of action of sucralfate in both its prevention of ulcer recurrence and as the therapy of acute injury.
References 1. McHardy GG. A multicenter. double-blind trial of sucralfate and placebo in duodenal ulcer. J Clin Gastroenterol 1981; 3(Suppl 2):147-52. 2. Hollander D. Sucralfate: new agent for treatment of duodenal ulcer. In: Caspary WF. ed. Duodenal ulcer. gastric ulcer: sucralfate. a new therapeutic concept. Baltimore: Urban & Schwarzenberg. 1981:49-53. 3. Hollander D. Efficacy of sucralfate for duodenal ulcers: a multicenter. double-blind trial. J Clin Gastroenterol 1981; 3(SuppI2):153-7. 4. Marks IN. Lucke W. Wright JP. Gridwook AH. Ulcer healing and relapse rates after initial treatment with cimetidine or sucralfate. J Clin GastroenteroI1981;3(Suppl 2):163-5.
373
5. Ishimori A. Arakawa H. A placebo-controlled. double-blind trial using sucralfate for the treatment of peptic ulcer. A multicenter Japanese study. In: Caspary WF. ed. Duodenal ulcer. gastric ulcer: sucralfate. a new therapeutic concept. Baltimore: Urban & Schwarzenberg. 1981:93-100. 6. Miyake T. Ariyoshi J. Suzaki T. Oishi M. Sakai M. Ueda S. Endoscopic evaluation of the effect of sucralfate therapy and other clinical parameters on the recurrence rate of gastric ulcer. Dig Dis Sci 1980; 25:1-7. 7. Shimizu M. Ishii A. Imai T. et al. Experimental studies on anti ulcerous activity of basic aluminum sucrose sulfate (CGA6Jl: I. Assessment of CG-A6J for antiulcerous. antacid. and antipepsin activities in pylorus-ligated rats and effect of CGA6J on blood coagulability in rabbit. Kiso-to-Rinsho (Clinical Report) 1968;2:374-82. 8. Shimizu M. Shibuya E. Ishii A. et al. Experimental studies of an antiulcerous activity of basic aluminum sucrose sulfate (CG-A6Jl: II. Effects of CG-A6J on histamine and stress ulcerations. Kiso-to-Rinsho (Clinical Report) 1968;2:374-82. 9. Shimizu M. Shibuya E. Ishii A. et al. Experimental studies on anti ulcerous activity of basic aluminum sucrose sulfate (CGA6Jl: III. Effects on clamping cortisone ulcers. Kiso-to-Rinsho (Clinical Report) 1968;2:383-93. 10. Hirano T. Takagaki Y. Healing promotion effects of sucrose sulfate aluminum ester (Ulcermin) on acetic acid induced ulcer. Kiso-to-Rinsho (Clinical Report) 1974;8(4):1-12. 11. Okabe S. Takeuchi K. Kunimi H. Kanno M. Kawashima M. Effects of an antiulcer drug. sucralfate (a basic aluminium salt of sulfated disaccharide). on experimental gastric lesions and gastric secretion in rats. Dig Dis Sci 1983;28:1034-42. 12. Harrington SJ, Schlegel JF. Code CF. The protective effect of sucralfate on the gastric mucosa of rats. J Clin Gastroenterol 1981;3(Suppl 2):129-34. 13. Nagashima R. Hoshino E. Hniohara Y. Sakai K. Haa S. Nakano H. Effect of sucralfate on ethanol-induced gastric mucosal damage in the rat. Scand J Gastroenterol 1983;18(Suppl 83):17-20. 14. Hollander D. Tarnawski A. Ivey KJ, et al. Arachidonic acid protection of rat gastric mucosa against ethanol injury. J Lab Clin Med 1982;100:296-308. 15. Meller SM. Coppe MR. Ito S. Transmission electron microscopy of critically point dried tissue after observation in the scanning electron microscope. Anat Rec 1973;176:245-52. 16. Krause WJ. Leeson CR. The postnatal development of the respiratory system of the opossum. Light and scanning electron microscopy. Am J Anat 1973;137:337-56. 17. Tarnawski A. Ivey KJ. Transmucosal potential-difference profile in rat upper gastrointestinal tract. A simple model for testing gastric effects of pharmacological agents. Can J Physiol PharmacoI1978;56:471-3. 18. Conover WJ. Practical nonparametric statistics. New York: John Wiley & Sons. 1971:393. 19. Eastwood G. Gastrointestinal epithelial renewal. Gastroenterology 1977;72:962-75. 20. Willems G. Cell renewal in the gastric mucosa. Digestion 1972;6:46-63. 21. Lipkin M. Proliferation and differentiation of gastrointestinal cells. Phys Rev 1973;53:891-915. 22. Lipkin M. Cell turnover in gastrointestinal mucosa. In: Harmon JW. ed. Basic mechanism of gastrointestinal mucosal cell injury. Baltimore. London: Williams & Wilkins. 1981:31. 23. Grant R. Rate of replacement of the surface epithelial cells of the gastric mucosa. Anat Rec 1945;91:175-85. 24. Svanes K. Ito S. Takeuchi K. Silen W. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Gastroenterology 1982;82:1409-26.
374 HOLLANDER ET AL.
25. Tarnawski A, Hollander D, Stachura J, Krause WJ, Gergely H. Prostaglandin protection of the gastric mucosa against alcohol injury-a dynamic time-related process. Role of the mucosal proliferative zone. Gastroenterology 1985;88:334-52. 26. Wallace JL, Morris GP, Krausse EJ, Greaves SE. Reduction by cytoprotective agents of ethanol-induced damage to the rat gastric mucosa: a correlated morphological and physiological study. Can J Physiol Pharmacol 1982;60:1686-99. 27 . Tarnawski A, Hollander D, Stachura J, Krause WJ. Arachidonic acid protection of gastric mucosa against alcohol injury: sequential analysis of morphologic and functional changes. J Lab Clin Med 1983;102:340-51. 28. Bighley D, Giesing D. Mechanism of action studies of sucralfate. In: Caspary WF, ed . Duodenal ulcer, gastric ulcer:
GASTROENTEROLOGY Vol. 88, No.1, Part 2
29. 30. 31.
32.
sucralfate, a new therapeutic concept. Baltimore: Urban & Schwarzenberg, 1981:3-12. Nagashima R. Mechanisms of action of sucralfate. J Clin GastroenteroI1981;3(Suppl 2):117-27. McGraw F, Caldwell G. Sucralfate. Drug intelligence and clinical pharmacy 1981;15:578-80. Tarnawski A, Hollander D, Krause WJ, Zipser RD, Gergely H. Effect of sucralfate on normal gastric mucosa. Histologic, ultrastructural and functional assessment (abstr) . Gastroenterology 1983;84:1331. Hollander D, Tarnawski A, Gergely M, Zipser RD. Sucralfate protection of the gastric mucosa against ethanol induced injury: a prostaglandin mediated process? Scand J GastroenteroI1984;19(Suppl 101):97-102.