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Portal hypertension and gastric mucosal injury in rats
(;I~STKOF:~‘I‘F:KOLO(;Y
Portal Hypertension Injury in Rats
1983;84:987-93
and Gastric Mucosal
Effects of Alcohol I. JAMES SARFEH, A. TARNAWSKI, T. MACH, and K.J. IVEY Departments the Veterans
of Surgery and Medicine Administration Medical
A. MALKI,
(Gastroenterology), Center, Long Beach.
The present study was performed primarily in order to determine whether gastric mucosa of rats with portal hypertension has different functional and histologic features when compared with controls, and second to quantitate and compare morphologic and functional changes after exposure to topical ethanol. Portal hypertension was produced by staged portal venous occlusion, and in these animals portal pressure was 32 2 2 cm saline compared with 18 5 2 cm in sham-operated controls (p < 0.005). Before ethanol, portal hypertensive rats compared with controls had significantly higher JuminaJ pH (2.9 i 0.3 vs. 1.9 ? O.l), increased Hi backdiffusion (loss of 138 2 10 vs. 57 * 16 PEq H+/h], lower potential diflerence (8 t 1 mV lower than controls), and extensive submucosal edema (submucosal thickness 325 i 25 vs. 138 -t 18 pm]. After 3 h of exposure to 2 ml intragastric absolute ethanol, the area of macroscopic hemorrhagic mucosal injury was significantly greater in portal hypertensive rats (34.0 i 8.7% vs. 7.6 2 2.1%), than in controls confirmed histologically by the greater number of deep hemorrhagic necrotic lesions and extent of mucosal length involved. Furthermore, after ethanol, portal hypertensive rats compared with controls had significantly increased gastric volume (14.4 +1.5 vs. 8.3 I? 0.6 ml), Na+ (86.6 L 8.0 vs. 64.6 ‘_ 8.0, mEq/L), pH (7.1 t 0.3 vs. 4.3 + 0.41, Ht backdiffusion (loss of 309 2 41 vs. 207 2 33 PEq H+/hJ, and protein and blood loss (100% increases over controls). These results indicate that gastric mucosa ofportal hypertensive rats has distinctive functional and histologic abnormalities that can explain its Keceiveti March 2, IWZ. Amepted November 5, 1982. Address requests for reprints to: I. James Sarfeh. M.D.. Department of Surgery. University of California. Irvine. Mediul Center, 101 City Drive South. Orange. California 92668. ? 1983 by the American (:astroent~rological Association 01~1h-i085~8,~~050987-07S03.00
University California
G.R. MASON. of California,
Irvine.
dntf
increased susceptibility to erosive injury after ethanol. This study quantitatively confirms in an animal model the clinical observations that portal hypertension may predispose to severe gastric mucosal injury. Esophageal varices and erosive gastritis frequently coexist in patients with alcoholic liver disease and (1-s). Analysis of upper gastrointestinal hemorrhage data from a previous retrospective clinical study suggested that the severity of hemorrhagic gastritis depends upon the presence of gastroesophageal varices, and hence portal hypertension (4). These observations, as well as the clinical impression of others (51, would suggest that portal hypertension may render the gastric mucosa more susceptible to injurious agents such as alcohol, and that injury may be more severe. In order to test this hypothesis we first studied whether gastric mucosa of rats with surgically produced portal hypertension has different morphologic and functional features when compared with sham-operated (SO) controls, and, second, we assessed mucosal changes in both groups after exposure to topical alcohol.
Materials Surgical
and Methods Preparation
Male Sprague-Dawley rats (150-200 g) were used in all experiments. Portal hypertension was produced in the following manner: In each of a group of experimental rats under ether anesthesia, the portal vein, immediate11 before its entry into the hepatic hilum, was constricted by approximately half of its original diameter using a fine silk ligature. A second, heavier ligature containing a surgeon’s knot was placed around the same location of the portal vein, but not secured. The ends were exteriorized via the flanks and tied on the dorsum of the animal. All the rats
988
Figure
SARFEH ET AL.
GASTROENTEROLOGY Vol. 84, No. 5, Part
1. Photomicrograph of gastric mucosa submucosal edema (H & E, x6.5).
in PHT rat before ethanol
were then allowed to move freely in their cages with access to rat pelleted diet and water. After 3 days, the heavier externalized ligature was tightened and resecured to the dorsum, resulting in complete portal occlusion. The rats were also lightly anesthetized during this second procedure. All experiments were conducted 48 h later. The sham-operated controls underwent exactly the same procedures, except that both of the ligatures were tied such that portal vein constriction and occlusion were not produced. In order to maintain uniformity, portal hypertensive (PHT) and SO animals were prepared on the same days, and experiments were similarly conducted in parallel. Studies of Rats Not Intragastric Ethanol
Exposed
to Topical
Portal pressure measurements were made in each of 10 SO control and 10 PHT rats after an overnight fast. A PE 50 tubing was inserted via a large pericolonic mesenteric tributory, and pressure was measured from the level of the vena cava to the tip of a column of saline within the tubing. Arterial blood was removed from an aortic cannula, and pH was analyzed using a digital pH meter (Beckman Model 3560, Cleveland, Ohio). Rats were fasted for 24 h in wire-bottom cages. In a group of 8 rats with PHT and 8 SO controls, the stomachs were excised along the greater curvature and inspected for macroscopic damage. Each stomach was then cut into strips obliquely across the entire corpus, and fixed in buffered formalin and stained with hematoxylin and eosin (H & E), and alcian blue and periodic acid-schiff [PAS).
instillation.
Note absence
of erosions
and presence
1
of
Coded specimens were then evaluated by light microscopy. Special attention was paid to the continuity of the surface epithelial layer and presence of mucosal necrosis. Using an eyepiece micrometer, the width of submucosa was measured between mucosa and muscularis of 10 separate points along each gastric strip, each point being -1.5 mm apart from the next. In another group of 8 PHT and 10 SO rats, after 24-h fasts, intragastric luminal pHs were measured in vivo by means of pH microprobe (Microelectrodes, Inc., Londonderry, N.H.) intragastrically and connected to a Beckman digital pH meter. In 6 of the PHT and 6 of the SO rats, ion fluxes were then measured in a similar manner to that previously reported (6). A duodenal cannula was inserted in each rat, and the stomach was washed three times with a solution containing 100 mM HCl and 50 mM NaCl, the contents being discarded each time. An identical solution in 3-ml portions was then introduced intragastrically for three 26-min periods, and after aspiration it was stored for measurement. H+ ion was measured by titration with an automatic titrator (Radiometer, Copenhagen), and K+ and Na+ were measured by flame photometry. For each of the 20-min periods, the contents were assessed for H’, K+, and Na+ fluxes by subtracting the ions instilled from those recovered. In two separate groups of 5 PHT and 5 control rats, in vivo gastric mucosal potential difference changes were recorded by a technique previously described (7,8) using a Keithley 610 B electrometer (Keithley Instruments, Inc., Cleveland, Ohio). Serial measurements were made in one group before and in the other group 3 h after intragastric instillation of 2 ml absolute ethanol.
Mav
PORTAI.
1983
Studies in Rats After of Ethanol
Intragastric
Instillation
and 10 PHT rats, fasted for 24 h, gastric mucosal injury was produced by instillation of absolute alcohol (2 ml) via an orogastric tube. After 3 h, each animal was reanesthetized with nembutal and the esophagus and pylorus ligated, the stomach excised, In a group
of 10 SO control
and the gastric contents removed and saved for analysis. Macroscopic areas of gastric mucosal injury were assessed by an uninformed observer. Photographic slides of the opened stomachs were made and projected onto a small screen. Tracings of areas of hemorrhagic injury were then made on clear plastic sheets. The percent of areas of involvement was measured by weighing the excised tracings and expressing them as fractions of the weight of the whole stomach tracing. Microscopic sections were prepared as before in 7 control and 7 PHT rats 3 h after ethanol instillation. Coded mucosal specimens were evaluated by light microscopy. The lengths of the gastric wall strips were measured with an eyepiece micrometer and mucosal damage was quantitatively assessed. The number of hemorrhagic necrotic lesions deeper than 0.2 mm and >0.4 mm in width were counted and their width was expressed as a percentage of the total mucosal length. Superficial mucosal damage, as assessed by length of surface epithelial denudation, was also expressed as a percentage of total mucosal length. Analysis of gastric contents was made from excised stomachs of 10 control and 10 PHT rats 3 h after ethanol instillation. Intragastric volume was measured by displacement. Sodium and potassium concentrations of gastric contents were measured by flame photometry (Instrumentation Laboratory, Lexington, Mass.]. Gastric pH was analyzed using the digital pH meter. Protein content was measured using the method of Lowry (9). Blood loss was assessed on the basis of spectrophotometric measurements of hematin in the gastric contents treated with 1.0 N HCI. The values were compared with the standard curves of diluted hemolyzed blood treated with 1.0 N HCl. Ion fluxes were measured as before in two groups of 6 PHT and 6 SO control rats 3 h after ethanol instillation and removal of gastric contents.
Statistical
Analysis
Statistical comparisons of the macroscopic hemorrhagic mucosal changes and the result of histologic quantitation were performed with the Kruskall-Wallis nonparametric test. The statistical significance of other data was evaluated using Student’s t-test for paired or unpaired observations where appropriate.
Results Animals with staged portal venous occlusion had gross evidence of PHT with dilated and tortuous mesenteric veins. Their portal pressures measured 32 + 2 cm compared with 18 2 2 cm salink in SO controls (p < 0.001).
Table
HYPERTENSION
1. Analysis
Before Ethanol H+ loss (/&q/h] Na- gain (pEq!h) K- gain (pEqh) 3 h After Ethanol H’ loss (@q/h) Na’ gain (FEq/h) K’ rain (tiEo/h)
of Gastric
AK11 (;ilSTRI(:
ISJURY
~180
Ion Fluxes”
PHT
c:ontro1
I’
137.8 k 10.0
55.7 L 15.h
~-c0.01
48.9 + Y.fi 3.8 !I O.li
309.0 156.3
2 41.2 + 32.4
12.9 i
34.3 i
8.0
i-is
2 8 + O.li
h’s
207.3 + 33.1
149.7
2.1
2 26.2
8.8 f
1.9
~0.05
NS NS
I’All values are mean t SEM. Measurements LLW~ made after ac.icl washing and then instillation of 3 ml of 150 n&l HCI and 50 mbl NaCl. and analysis of contents for thrf:c: 20-min periods, pool~tl. NS = not significant.
Rats Not Exposed
to Ethanol
Morphologic assessment. Neither PHT nor SO control animals exhibited gross or histologic evidence of mucosal necrosis before ethanol administration. Extensive submucosal edema was present in PHT rats (Figure l), but not in SO controls, with the thickness of the submucosa measuring 325 i- 25 pm and 138 2 18 pm, respectively (p < 0.01). LuminaJ pH and ion fluxes. Intragastric luminal pH was 2.9 ? 0.3 in PHT and 1.9 -t 0.1 in SO rats, the differences being significant (p < 0.05). Measurements of gastric ion fluxes are summarized in Table 1. Hi losses were significantly greater in PHT rats compared with SO controls. Na ’ and Kt gains were also moderately, but not significantly. increased. Potential difference. Gastric mucosal potential difference before ethanol instillation was - 40 k 4 mV in PHT rats compared with - 48 i 3 mV in SO controls (p < 0.05). Arterial PH. Systemic arterial pH of SO control and PHT rats were similar, being 7.47 t 0.02 and 7.44 * 0.02, respectively (p > 0.05).
Rats After
Intragastric
ElhonoJ
Macroscopic assessment. Macroscopic areas of gastric mucosal injury after ethanol instillation were significantly greater in PHT rats, comprising 34.8 t 8.7% of total glandular mucosal area compared with 7.6 + 2.1% of controls (p < 0.001). These comparisons are illustrated in Figure 2. Histologic assessment. Histologically, two distinct types of mucosal injury after ethanol instillation were present: (a) Superficial damage to surface epithelial cells with their subsequent desquamation, not apparent macroscopically. This was similar in extent for both PHT and controls, comprising 45.4 t
990
SARFEH ET AL.
Rii” Figure
(b)
GASTROENTEROLOGY Vol. 84, No. 5, Part
T1 2. Macroscopic hemorrhagic
3
7 gastric mucosal lesions.
injury
after ethanol
instillation
2.9% and 45.6 2 3.2% of total mucosal length studied, respectively. Deep hemorrhagic mucosal necrosis. This form of mucosal damage closely corresponded with the macroscopic observations, and was significantly more extensive in PHT rats than SO controls. The quantitative data are summarized in Table 2, and illustrated in Figures 3-5.
Submucosal edema in PHT rats was reduced after ethanol instillation, with the thickness of submucosa measuring 250 t 15 ,um, and was now similar to SO rats, whose thickness was 228 t 14 pm (p > 0.05). Analysis of gastric contents. The results of analysis of intragastric contents 3 h after ethanol instillation are summarized in Table 3. Gastric volume and Na+ concentrations were significantly increased in PHT rats, and their gastric acidity was reduced. Intragastric protein content was increased in PHT rats, and hematin content, as a reflection of blood loss was also significantly greater (Table 3). Ion fluxes. The results of measurement of gastric ion fluxes after topical ethanol are summarized in Table 1. As before ethanol instillation, PHT rats had significantly greater net Ht loss than controls, while Nat and Kf gains were slightly, but not significantly, greater in the former group. Potential difference. A sudden drop of gastric potential difference after ethanol instillation was observed in both groups (Figure 61, with absolute
in PHT (left) and control
(right)
rats. Black
areas
correspond
to
potential difference values in PHT rats being significantly lower than in SO controls from 35 to 65 min after instillation.
Discussion The present study demonstrated that gastric mucosa of rats with surgically produced portal hypertension has distinctive morphologic and functional abnormalities when compared with SO controls. These abnormalities include (a) extensive edema of lamina propria and submucosa; (b) increased back-diffusion of H+ ion, which corresponds with and can account for their increased intragastric luminal pH; and (c) reduced electronegativity of gastric mucosal potential difference. When exposed to topical ethanol, rats with portal hypertension
Table
2. Histologic h After
Quantitation” Ethanol
of Mucosal
Necrosis
Length Length of evaluated mucosa per rat (pm) Control PHTb
17,500 + 893 17,570 + 1090
P ('Values are mean significant.
NS’
3
Instillation
Number of deep necrotic lesions per 10,000 pm 2.50 ir 0.1 4.80 2 0.5 co.01
-+ SEM. ” PHT = portal
of deep
necrotic lesions (%, of total mucosal length) 22.8 2 1.4 42.6 2 4.3 -co.01
hypertensive.
NS = not
Ma!’
Figure
1983
:j.
Photomicrograph of PHT rat demonstrating lion of the superficial
mucosal
layers
extensive
after ethanol
mucosal instillation
develop significantly larger areas of mucosal hemorrhagic necrotic lesions than SO controls, 8s demonstrated in our studies by the macroscopic observations, and confirmed by microscopic quantification. These morphologic changes are associated with increased blood loss, protein shedding, intragastric
f;iqurc:
.$ Photomicrograph
of control
SO rat demonstrating
injury
with
deep
necrotic
hemorrhagic
lesion
and desquama-
(H & E, x65).
volume, pH, and sodium, lower gastric mucosal potential difference, and greater H + back-diffusion in PHT rats than in controls after topical ethanol. Since systemic acidosis can influence the development of acute mucosal lesions (lo), we measured arterial pH in both control and PHT rats and found
necrotic
lesion
after ethanol
instillation
IH & 6. Y 1601
992
SARFEH
ET AL
Figure 5 Photomicrograph lesion compared x 140)
GASTROENTEROLOGY
of PHT rat demonstrating with control rat in Figure
a deep necrotic 4, and reduced
them to be similar. The mechanisms whereby PHT rats have increased susceptibility to gastric mucosal injury by alcohol may, therefore, be related to several other underlying factors. Primarily, before ethanol instillation, we found that submucosal edema was significantly greater in PHT animals, presumably as a result of increased hydrostatic pressures of venous hypertension. Such edema would impose greater distances for nutrient transport to mucosal cells and also may cause separation of tight junctions, increasing the possibility for disruption of gastric mucosal barrier (11). As a result, mucosal permeability would be increased, with Ht back-diffusion further rendering the gastric mucosa more susceptible to injury (12,13). These considerations are supported by our findings of lower baseline potential difference, as well as greater H+ loss in PHT rats before topical ethanol, suggesting increased mucosal permeability to Ht ion and impaired active electrogenic transport in these animals. These observations would explain, at least in part, the greater mucosal damage produced by intragastric instillation of ethanol in PHT rats compared with controls. Gastric venous outflow obstruction would also result in reduced arterial inflow due to higher outflow resistance. Other investigators have demonstrated the increased potential
lesion after ethanol submucosal edema
instillation. compared
Vol.
84, No.
5. Part
Note greater width and dep lth of with PHT rat in Figure 1 (H & E,
for gastric mucosal injury in the presence of reduced gastric blood flow (14-17). Delayed gastric emptying could also result in greater gastric mucosal damage after alcohol instillation. This is unlikely, however, since we performed additional studies (Sarfeh IJ and Tarnawski A, unpublished data) in which alcohol was instilled in PHT and control rats, each with previously ligated pylorus. Resultant mucosal hemorrhagic lesions were significantly greater in PHT rats than controls in similar ratios to those reported in this paper. Finally, the local factors influencing mucosal cellular integrity, such as mucus and bicarbonate secretion, local prostaglandin synthesis, and
Table
3.
Analvsis
of Gastric
Hypekmske
Contents
" Values are mean instillation.
in Portal
and Sham-Operated PHT rats
Volume (ml) PH Na+ (mEq/L) K+ (mEqiL) Total protein (mg) Hematin content (pg)
1
14.4 -+ 1.5 7.1 k 0.3 86.6 I? 8.0 4.6 -+ 0.5 338 f 34 0.104 t 0.016
t SEM. Measurements NS = not significant.
Rats”
SO controls 8.2 -c 0.6 4.3 r 0.4 64.6 + 8.0 5.2 -c 0.7 176 -+ 10 0.056 + 0.010 made
3 h after
P <0.005
.May 19H3
ALCOHOL
0
Portal
0
Sham-operated
Hypertensive
t
Pd.05
Rats
2
controls
3
4
5 1 ‘;
b
’
210
I BEFORE
Figure
fi.
’ d0
’ TIME
6b
’
810
I 100
I 6
l:D
(minutes)
ALCOHOL
Potential difference c:ontrol rats before
7
changes (mean i- SEM) in PHT and and after ethanol instillation.
energy metabolism, could be altered by gastric venous hypertension. shunting of splanchnic blood, and hepatic portal hypoperfusion. The present study quantitatively confirms in an animal model the clinical observations that portal hypertension may predispose to severe gastric mucosal injury. In a retrospective study, Sarfeh et al. (4) suggested that the clinical severity of hemorrhagic gastritis in cirrhosis is related to the presence of portal hypertension. Others have noted that the incidence of gastric mucosal hemorrhage in cirrhosis is reduced following portacaval shunt for bleeding esophageal varices (18,19). Finally, Lebrec et al. (5) recently found that propranolol therapy in cirrhosis, aimed at reduction of portal pressures, is effective in prevention of not only variceal hemorrhage, but also of that due to hemorrhagic gastritis. All of these clinical observations, as well as the present experimental study, would therefore suggest that the degree of gastric mucosal injury is influenced by portal hypertension. In conclusion the present study showed that gastric mucosa of rats with surgically produced portal hypertension has distinctive histologic and functional abnormalities that can explain its increased susceptibility to erosive injury after ethanol.
a
1976;85:299-303.
9. Lowry
10.
11. 12. 13.
14
15
1
19
References 1. lglesias M. Dourdourekas N, Steigmann F. Prompt
D. Adamavcius J. Villa F. Shobassy endoscopic diagnosis of upper gas-
trointestinal hemorrhage: its value for specific diagnosis and management. Ann Surg 1979:189:90-5. Pitcher JL. Variceal hemorrhage among patients with varices and upper gastrointestinal hemorrhage. South Med J 1977;70:1183-9. McCray RS, hlartin F, Amir-Ahmadi H. Sheahan DC, Zanicheck N. Erroneous diagnosis of hemorrhage from esophageal varices. Am J Dig Dis 1969;14:755-61. Sarfeh IJ, Tabak C, Eugene J. Juler CL Clinic:al significance of erosive gastritis in patients with alcoholic liver disease and upper gastrointestinal hemorrhage. Ann Surg 1981:194:14951. Lebrec D, Poynard T. Hillon P, Benhamou J. Propranolol for prevention of recurrent gastrointestinal bleeding in patients with cirrhosis. N Engl J Med 1981;305:1371-4. Puurunen J. Effect of prostaglandin E, cimetidine, and atropine on ethanol-induced mucosal damage in the rat. Stand J Gastroenterol 1980;35:485-8. Tarnawski A, lvey KJ. Transmucosal potential-difference profile in rat upper gastrointestinal tract: a simple model for testing gastric effects of pharmacologic. agents. Can J Physiol Pharmacol 1978:56:471-X Baskin WN, Ivey KJ, Krause WJ. Jeffrev GE, Gemmell RT. Asprin-induced ultrastructural changes in human gastric mucosa: correlation with potential difference. Ann Intern Med OH, Rosebrough NJ, Farr AL, Kandall RJ. Protein measurement with the Folin phenol reagent. I Biol Chem 1951:193:265-75. Kivilaasko E. Barzilai A, Schiessel R. Crass K, Silen W. Ulceration of isolated amphibian gastric. mu,:osa. Gastroenterology 1979;77:31-7. Moody FG, Zalewsky CA. Larsen KR. Cvtoprotection of the gastric epithelium. World J Surg 1981:5:153-63. Davenport HW. Gastric mucosal injury by fatty and acetylsalicyIic acids. Castrosnterology 1964:46:245-50. Davenport HW. Fluid produced by the gastric mucosa during drainage by acetic and salicylic acids. Castroenterology 1966;50:487-93. Menguy K. Masters YF. Mechanism oi stress ulcer. III. Effects of hemorrhagic shock on energy metabolism in the mucosa of the antrum corpus and fundus of the rabbit stomach. Gastroenterology 1974;66:1166&8. Menguy R, Masters YF. Mechanism of stress ulceration. IV. Influence of fasting on the tolerance of gastric mucosal energy metabolism to ischemia and on the incidence of stress ulceration. Castroenterology 1974:66:1177-82. Menguy R. Gastric mucosal energy metabolism in the etiology of stress ulceration. World J Surg 1981;5:175-9. Kivilaasko E, SiIen W. Pathogenesis of experimental gastric mucosai injury. N Engl J Med 1979::101:364-8. Jackson FC, Perrin EB, Felix WR. Smith AC. A clinical investigation of the portacaval shunt. 1:. Survival analysis of the therapeutic operation. Ann Surg 1971:174:672-701, Resnick RH, Iber FL, lshihara AM. Chalmers TC. Zimmerman H. Boston Interhospital Liver Croup A controlled study of the therapeutic portacaval shunt. (:astrt)rntPrology 1974;67:84:t57.
Portal Hypertension Injury in Rats
1983;84:987-93
and Gastric Mucosal
Effects of Alcohol I. JAMES SARFEH, A. TARNAWSKI, T. MACH, and K.J. IVEY Departments the Veterans
of Surgery and Medicine Administration Medical
A. MALKI,
(Gastroenterology), Center, Long Beach.
The present study was performed primarily in order to determine whether gastric mucosa of rats with portal hypertension has different functional and histologic features when compared with controls, and second to quantitate and compare morphologic and functional changes after exposure to topical ethanol. Portal hypertension was produced by staged portal venous occlusion, and in these animals portal pressure was 32 2 2 cm saline compared with 18 5 2 cm in sham-operated controls (p < 0.005). Before ethanol, portal hypertensive rats compared with controls had significantly higher JuminaJ pH (2.9 i 0.3 vs. 1.9 ? O.l), increased Hi backdiffusion (loss of 138 2 10 vs. 57 * 16 PEq H+/h], lower potential diflerence (8 t 1 mV lower than controls), and extensive submucosal edema (submucosal thickness 325 i 25 vs. 138 -t 18 pm]. After 3 h of exposure to 2 ml intragastric absolute ethanol, the area of macroscopic hemorrhagic mucosal injury was significantly greater in portal hypertensive rats (34.0 i 8.7% vs. 7.6 2 2.1%), than in controls confirmed histologically by the greater number of deep hemorrhagic necrotic lesions and extent of mucosal length involved. Furthermore, after ethanol, portal hypertensive rats compared with controls had significantly increased gastric volume (14.4 +1.5 vs. 8.3 I? 0.6 ml), Na+ (86.6 L 8.0 vs. 64.6 ‘_ 8.0, mEq/L), pH (7.1 t 0.3 vs. 4.3 + 0.41, Ht backdiffusion (loss of 309 2 41 vs. 207 2 33 PEq H+/hJ, and protein and blood loss (100% increases over controls). These results indicate that gastric mucosa ofportal hypertensive rats has distinctive functional and histologic abnormalities that can explain its Keceiveti March 2, IWZ. Amepted November 5, 1982. Address requests for reprints to: I. James Sarfeh. M.D.. Department of Surgery. University of California. Irvine. Mediul Center, 101 City Drive South. Orange. California 92668. ? 1983 by the American (:astroent~rological Association 01~1h-i085~8,~~050987-07S03.00
University California
G.R. MASON. of California,
Irvine.
dntf
increased susceptibility to erosive injury after ethanol. This study quantitatively confirms in an animal model the clinical observations that portal hypertension may predispose to severe gastric mucosal injury. Esophageal varices and erosive gastritis frequently coexist in patients with alcoholic liver disease and (1-s). Analysis of upper gastrointestinal hemorrhage data from a previous retrospective clinical study suggested that the severity of hemorrhagic gastritis depends upon the presence of gastroesophageal varices, and hence portal hypertension (4). These observations, as well as the clinical impression of others (51, would suggest that portal hypertension may render the gastric mucosa more susceptible to injurious agents such as alcohol, and that injury may be more severe. In order to test this hypothesis we first studied whether gastric mucosa of rats with surgically produced portal hypertension has different morphologic and functional features when compared with sham-operated (SO) controls, and, second, we assessed mucosal changes in both groups after exposure to topical alcohol.
Materials Surgical
and Methods Preparation
Male Sprague-Dawley rats (150-200 g) were used in all experiments. Portal hypertension was produced in the following manner: In each of a group of experimental rats under ether anesthesia, the portal vein, immediate11 before its entry into the hepatic hilum, was constricted by approximately half of its original diameter using a fine silk ligature. A second, heavier ligature containing a surgeon’s knot was placed around the same location of the portal vein, but not secured. The ends were exteriorized via the flanks and tied on the dorsum of the animal. All the rats
988
Figure
SARFEH ET AL.
GASTROENTEROLOGY Vol. 84, No. 5, Part
1. Photomicrograph of gastric mucosa submucosal edema (H & E, x6.5).
in PHT rat before ethanol
were then allowed to move freely in their cages with access to rat pelleted diet and water. After 3 days, the heavier externalized ligature was tightened and resecured to the dorsum, resulting in complete portal occlusion. The rats were also lightly anesthetized during this second procedure. All experiments were conducted 48 h later. The sham-operated controls underwent exactly the same procedures, except that both of the ligatures were tied such that portal vein constriction and occlusion were not produced. In order to maintain uniformity, portal hypertensive (PHT) and SO animals were prepared on the same days, and experiments were similarly conducted in parallel. Studies of Rats Not Intragastric Ethanol
Exposed
to Topical
Portal pressure measurements were made in each of 10 SO control and 10 PHT rats after an overnight fast. A PE 50 tubing was inserted via a large pericolonic mesenteric tributory, and pressure was measured from the level of the vena cava to the tip of a column of saline within the tubing. Arterial blood was removed from an aortic cannula, and pH was analyzed using a digital pH meter (Beckman Model 3560, Cleveland, Ohio). Rats were fasted for 24 h in wire-bottom cages. In a group of 8 rats with PHT and 8 SO controls, the stomachs were excised along the greater curvature and inspected for macroscopic damage. Each stomach was then cut into strips obliquely across the entire corpus, and fixed in buffered formalin and stained with hematoxylin and eosin (H & E), and alcian blue and periodic acid-schiff [PAS).
instillation.
Note absence
of erosions
and presence
1
of
Coded specimens were then evaluated by light microscopy. Special attention was paid to the continuity of the surface epithelial layer and presence of mucosal necrosis. Using an eyepiece micrometer, the width of submucosa was measured between mucosa and muscularis of 10 separate points along each gastric strip, each point being -1.5 mm apart from the next. In another group of 8 PHT and 10 SO rats, after 24-h fasts, intragastric luminal pHs were measured in vivo by means of pH microprobe (Microelectrodes, Inc., Londonderry, N.H.) intragastrically and connected to a Beckman digital pH meter. In 6 of the PHT and 6 of the SO rats, ion fluxes were then measured in a similar manner to that previously reported (6). A duodenal cannula was inserted in each rat, and the stomach was washed three times with a solution containing 100 mM HCl and 50 mM NaCl, the contents being discarded each time. An identical solution in 3-ml portions was then introduced intragastrically for three 26-min periods, and after aspiration it was stored for measurement. H+ ion was measured by titration with an automatic titrator (Radiometer, Copenhagen), and K+ and Na+ were measured by flame photometry. For each of the 20-min periods, the contents were assessed for H’, K+, and Na+ fluxes by subtracting the ions instilled from those recovered. In two separate groups of 5 PHT and 5 control rats, in vivo gastric mucosal potential difference changes were recorded by a technique previously described (7,8) using a Keithley 610 B electrometer (Keithley Instruments, Inc., Cleveland, Ohio). Serial measurements were made in one group before and in the other group 3 h after intragastric instillation of 2 ml absolute ethanol.
Mav
PORTAI.
1983
Studies in Rats After of Ethanol
Intragastric
Instillation
and 10 PHT rats, fasted for 24 h, gastric mucosal injury was produced by instillation of absolute alcohol (2 ml) via an orogastric tube. After 3 h, each animal was reanesthetized with nembutal and the esophagus and pylorus ligated, the stomach excised, In a group
of 10 SO control
and the gastric contents removed and saved for analysis. Macroscopic areas of gastric mucosal injury were assessed by an uninformed observer. Photographic slides of the opened stomachs were made and projected onto a small screen. Tracings of areas of hemorrhagic injury were then made on clear plastic sheets. The percent of areas of involvement was measured by weighing the excised tracings and expressing them as fractions of the weight of the whole stomach tracing. Microscopic sections were prepared as before in 7 control and 7 PHT rats 3 h after ethanol instillation. Coded mucosal specimens were evaluated by light microscopy. The lengths of the gastric wall strips were measured with an eyepiece micrometer and mucosal damage was quantitatively assessed. The number of hemorrhagic necrotic lesions deeper than 0.2 mm and >0.4 mm in width were counted and their width was expressed as a percentage of the total mucosal length. Superficial mucosal damage, as assessed by length of surface epithelial denudation, was also expressed as a percentage of total mucosal length. Analysis of gastric contents was made from excised stomachs of 10 control and 10 PHT rats 3 h after ethanol instillation. Intragastric volume was measured by displacement. Sodium and potassium concentrations of gastric contents were measured by flame photometry (Instrumentation Laboratory, Lexington, Mass.]. Gastric pH was analyzed using the digital pH meter. Protein content was measured using the method of Lowry (9). Blood loss was assessed on the basis of spectrophotometric measurements of hematin in the gastric contents treated with 1.0 N HCI. The values were compared with the standard curves of diluted hemolyzed blood treated with 1.0 N HCl. Ion fluxes were measured as before in two groups of 6 PHT and 6 SO control rats 3 h after ethanol instillation and removal of gastric contents.
Statistical
Analysis
Statistical comparisons of the macroscopic hemorrhagic mucosal changes and the result of histologic quantitation were performed with the Kruskall-Wallis nonparametric test. The statistical significance of other data was evaluated using Student’s t-test for paired or unpaired observations where appropriate.
Results Animals with staged portal venous occlusion had gross evidence of PHT with dilated and tortuous mesenteric veins. Their portal pressures measured 32 + 2 cm compared with 18 2 2 cm salink in SO controls (p < 0.001).
Table
HYPERTENSION
1. Analysis
Before Ethanol H+ loss (/&q/h] Na- gain (pEq!h) K- gain (pEqh) 3 h After Ethanol H’ loss (@q/h) Na’ gain (FEq/h) K’ rain (tiEo/h)
of Gastric
AK11 (;ilSTRI(:
ISJURY
~180
Ion Fluxes”
PHT
c:ontro1
I’
137.8 k 10.0
55.7 L 15.h
~-c0.01
48.9 + Y.fi 3.8 !I O.li
309.0 156.3
2 41.2 + 32.4
12.9 i
34.3 i
8.0
i-is
2 8 + O.li
h’s
207.3 + 33.1
149.7
2.1
2 26.2
8.8 f
1.9
~0.05
NS NS
I’All values are mean t SEM. Measurements LLW~ made after ac.icl washing and then instillation of 3 ml of 150 n&l HCI and 50 mbl NaCl. and analysis of contents for thrf:c: 20-min periods, pool~tl. NS = not significant.
Rats Not Exposed
to Ethanol
Morphologic assessment. Neither PHT nor SO control animals exhibited gross or histologic evidence of mucosal necrosis before ethanol administration. Extensive submucosal edema was present in PHT rats (Figure l), but not in SO controls, with the thickness of the submucosa measuring 325 i- 25 pm and 138 2 18 pm, respectively (p < 0.01). LuminaJ pH and ion fluxes. Intragastric luminal pH was 2.9 ? 0.3 in PHT and 1.9 -t 0.1 in SO rats, the differences being significant (p < 0.05). Measurements of gastric ion fluxes are summarized in Table 1. Hi losses were significantly greater in PHT rats compared with SO controls. Na ’ and Kt gains were also moderately, but not significantly. increased. Potential difference. Gastric mucosal potential difference before ethanol instillation was - 40 k 4 mV in PHT rats compared with - 48 i 3 mV in SO controls (p < 0.05). Arterial PH. Systemic arterial pH of SO control and PHT rats were similar, being 7.47 t 0.02 and 7.44 * 0.02, respectively (p > 0.05).
Rats After
Intragastric
ElhonoJ
Macroscopic assessment. Macroscopic areas of gastric mucosal injury after ethanol instillation were significantly greater in PHT rats, comprising 34.8 t 8.7% of total glandular mucosal area compared with 7.6 + 2.1% of controls (p < 0.001). These comparisons are illustrated in Figure 2. Histologic assessment. Histologically, two distinct types of mucosal injury after ethanol instillation were present: (a) Superficial damage to surface epithelial cells with their subsequent desquamation, not apparent macroscopically. This was similar in extent for both PHT and controls, comprising 45.4 t
990
SARFEH ET AL.
Rii” Figure
(b)
GASTROENTEROLOGY Vol. 84, No. 5, Part
T1 2. Macroscopic hemorrhagic
3
7 gastric mucosal lesions.
injury
after ethanol
instillation
2.9% and 45.6 2 3.2% of total mucosal length studied, respectively. Deep hemorrhagic mucosal necrosis. This form of mucosal damage closely corresponded with the macroscopic observations, and was significantly more extensive in PHT rats than SO controls. The quantitative data are summarized in Table 2, and illustrated in Figures 3-5.
Submucosal edema in PHT rats was reduced after ethanol instillation, with the thickness of submucosa measuring 250 t 15 ,um, and was now similar to SO rats, whose thickness was 228 t 14 pm (p > 0.05). Analysis of gastric contents. The results of analysis of intragastric contents 3 h after ethanol instillation are summarized in Table 3. Gastric volume and Na+ concentrations were significantly increased in PHT rats, and their gastric acidity was reduced. Intragastric protein content was increased in PHT rats, and hematin content, as a reflection of blood loss was also significantly greater (Table 3). Ion fluxes. The results of measurement of gastric ion fluxes after topical ethanol are summarized in Table 1. As before ethanol instillation, PHT rats had significantly greater net Ht loss than controls, while Nat and Kf gains were slightly, but not significantly, greater in the former group. Potential difference. A sudden drop of gastric potential difference after ethanol instillation was observed in both groups (Figure 61, with absolute
in PHT (left) and control
(right)
rats. Black
areas
correspond
to
potential difference values in PHT rats being significantly lower than in SO controls from 35 to 65 min after instillation.
Discussion The present study demonstrated that gastric mucosa of rats with surgically produced portal hypertension has distinctive morphologic and functional abnormalities when compared with SO controls. These abnormalities include (a) extensive edema of lamina propria and submucosa; (b) increased back-diffusion of H+ ion, which corresponds with and can account for their increased intragastric luminal pH; and (c) reduced electronegativity of gastric mucosal potential difference. When exposed to topical ethanol, rats with portal hypertension
Table
2. Histologic h After
Quantitation” Ethanol
of Mucosal
Necrosis
Length Length of evaluated mucosa per rat (pm) Control PHTb
17,500 + 893 17,570 + 1090
P ('Values are mean significant.
NS’
3
Instillation
Number of deep necrotic lesions per 10,000 pm 2.50 ir 0.1 4.80 2 0.5 co.01
-+ SEM. ” PHT = portal
of deep
necrotic lesions (%, of total mucosal length) 22.8 2 1.4 42.6 2 4.3 -co.01
hypertensive.
NS = not
Ma!’
Figure
1983
:j.
Photomicrograph of PHT rat demonstrating lion of the superficial
mucosal
layers
extensive
after ethanol
mucosal instillation
develop significantly larger areas of mucosal hemorrhagic necrotic lesions than SO controls, 8s demonstrated in our studies by the macroscopic observations, and confirmed by microscopic quantification. These morphologic changes are associated with increased blood loss, protein shedding, intragastric
f;iqurc:
.$ Photomicrograph
of control
SO rat demonstrating
injury
with
deep
necrotic
hemorrhagic
lesion
and desquama-
(H & E, x65).
volume, pH, and sodium, lower gastric mucosal potential difference, and greater H + back-diffusion in PHT rats than in controls after topical ethanol. Since systemic acidosis can influence the development of acute mucosal lesions (lo), we measured arterial pH in both control and PHT rats and found
necrotic
lesion
after ethanol
instillation
IH & 6. Y 1601
992
SARFEH
ET AL
Figure 5 Photomicrograph lesion compared x 140)
GASTROENTEROLOGY
of PHT rat demonstrating with control rat in Figure
a deep necrotic 4, and reduced
them to be similar. The mechanisms whereby PHT rats have increased susceptibility to gastric mucosal injury by alcohol may, therefore, be related to several other underlying factors. Primarily, before ethanol instillation, we found that submucosal edema was significantly greater in PHT animals, presumably as a result of increased hydrostatic pressures of venous hypertension. Such edema would impose greater distances for nutrient transport to mucosal cells and also may cause separation of tight junctions, increasing the possibility for disruption of gastric mucosal barrier (11). As a result, mucosal permeability would be increased, with Ht back-diffusion further rendering the gastric mucosa more susceptible to injury (12,13). These considerations are supported by our findings of lower baseline potential difference, as well as greater H+ loss in PHT rats before topical ethanol, suggesting increased mucosal permeability to Ht ion and impaired active electrogenic transport in these animals. These observations would explain, at least in part, the greater mucosal damage produced by intragastric instillation of ethanol in PHT rats compared with controls. Gastric venous outflow obstruction would also result in reduced arterial inflow due to higher outflow resistance. Other investigators have demonstrated the increased potential
lesion after ethanol submucosal edema
instillation. compared
Vol.
84, No.
5. Part
Note greater width and dep lth of with PHT rat in Figure 1 (H & E,
for gastric mucosal injury in the presence of reduced gastric blood flow (14-17). Delayed gastric emptying could also result in greater gastric mucosal damage after alcohol instillation. This is unlikely, however, since we performed additional studies (Sarfeh IJ and Tarnawski A, unpublished data) in which alcohol was instilled in PHT and control rats, each with previously ligated pylorus. Resultant mucosal hemorrhagic lesions were significantly greater in PHT rats than controls in similar ratios to those reported in this paper. Finally, the local factors influencing mucosal cellular integrity, such as mucus and bicarbonate secretion, local prostaglandin synthesis, and
Table
3.
Analvsis
of Gastric
Hypekmske
Contents
" Values are mean instillation.
in Portal
and Sham-Operated PHT rats
Volume (ml) PH Na+ (mEq/L) K+ (mEqiL) Total protein (mg) Hematin content (pg)
1
14.4 -+ 1.5 7.1 k 0.3 86.6 I? 8.0 4.6 -+ 0.5 338 f 34 0.104 t 0.016
t SEM. Measurements NS = not significant.
Rats”
SO controls 8.2 -c 0.6 4.3 r 0.4 64.6 + 8.0 5.2 -c 0.7 176 -+ 10 0.056 + 0.010 made
3 h after
P <0.005
.May 19H3
ALCOHOL
0
Portal
0
Sham-operated
Hypertensive
t
Pd.05
Rats
2
controls
3
4
5 1 ‘;
b
’
210
I BEFORE
Figure
fi.
’ d0
’ TIME
6b
’
810
I 100
I 6
l:D
(minutes)
ALCOHOL
Potential difference c:ontrol rats before
7
changes (mean i- SEM) in PHT and and after ethanol instillation.
energy metabolism, could be altered by gastric venous hypertension. shunting of splanchnic blood, and hepatic portal hypoperfusion. The present study quantitatively confirms in an animal model the clinical observations that portal hypertension may predispose to severe gastric mucosal injury. In a retrospective study, Sarfeh et al. (4) suggested that the clinical severity of hemorrhagic gastritis in cirrhosis is related to the presence of portal hypertension. Others have noted that the incidence of gastric mucosal hemorrhage in cirrhosis is reduced following portacaval shunt for bleeding esophageal varices (18,19). Finally, Lebrec et al. (5) recently found that propranolol therapy in cirrhosis, aimed at reduction of portal pressures, is effective in prevention of not only variceal hemorrhage, but also of that due to hemorrhagic gastritis. All of these clinical observations, as well as the present experimental study, would therefore suggest that the degree of gastric mucosal injury is influenced by portal hypertension. In conclusion the present study showed that gastric mucosa of rats with surgically produced portal hypertension has distinctive histologic and functional abnormalities that can explain its increased susceptibility to erosive injury after ethanol.
a
1976;85:299-303.
9. Lowry
10.
11. 12. 13.
14
15
1
19
References 1. lglesias M. Dourdourekas N, Steigmann F. Prompt
D. Adamavcius J. Villa F. Shobassy endoscopic diagnosis of upper gas-
trointestinal hemorrhage: its value for specific diagnosis and management. Ann Surg 1979:189:90-5. Pitcher JL. Variceal hemorrhage among patients with varices and upper gastrointestinal hemorrhage. South Med J 1977;70:1183-9. McCray RS, hlartin F, Amir-Ahmadi H. Sheahan DC, Zanicheck N. Erroneous diagnosis of hemorrhage from esophageal varices. Am J Dig Dis 1969;14:755-61. Sarfeh IJ, Tabak C, Eugene J. Juler CL Clinic:al significance of erosive gastritis in patients with alcoholic liver disease and upper gastrointestinal hemorrhage. Ann Surg 1981:194:14951. Lebrec D, Poynard T. Hillon P, Benhamou J. Propranolol for prevention of recurrent gastrointestinal bleeding in patients with cirrhosis. N Engl J Med 1981;305:1371-4. Puurunen J. Effect of prostaglandin E, cimetidine, and atropine on ethanol-induced mucosal damage in the rat. Stand J Gastroenterol 1980;35:485-8. Tarnawski A, lvey KJ. Transmucosal potential-difference profile in rat upper gastrointestinal tract: a simple model for testing gastric effects of pharmacologic. agents. Can J Physiol Pharmacol 1978:56:471-X Baskin WN, Ivey KJ, Krause WJ. Jeffrev GE, Gemmell RT. Asprin-induced ultrastructural changes in human gastric mucosa: correlation with potential difference. Ann Intern Med OH, Rosebrough NJ, Farr AL, Kandall RJ. Protein measurement with the Folin phenol reagent. I Biol Chem 1951:193:265-75. Kivilaasko E. Barzilai A, Schiessel R. Crass K, Silen W. Ulceration of isolated amphibian gastric. mu,:osa. Gastroenterology 1979;77:31-7. Moody FG, Zalewsky CA. Larsen KR. Cvtoprotection of the gastric epithelium. World J Surg 1981:5:153-63. Davenport HW. Gastric mucosal injury by fatty and acetylsalicyIic acids. Castrosnterology 1964:46:245-50. Davenport HW. Fluid produced by the gastric mucosa during drainage by acetic and salicylic acids. Castroenterology 1966;50:487-93. Menguy K. Masters YF. Mechanism oi stress ulcer. III. Effects of hemorrhagic shock on energy metabolism in the mucosa of the antrum corpus and fundus of the rabbit stomach. Gastroenterology 1974;66:1166&8. Menguy R, Masters YF. Mechanism of stress ulceration. IV. Influence of fasting on the tolerance of gastric mucosal energy metabolism to ischemia and on the incidence of stress ulceration. Castroenterology 1974:66:1177-82. Menguy R. Gastric mucosal energy metabolism in the etiology of stress ulceration. World J Surg 1981;5:175-9. Kivilaasko E, SiIen W. Pathogenesis of experimental gastric mucosai injury. N Engl J Med 1979::101:364-8. Jackson FC, Perrin EB, Felix WR. Smith AC. A clinical investigation of the portacaval shunt. 1:. Survival analysis of the therapeutic operation. Ann Surg 1971:174:672-701, Resnick RH, Iber FL, lshihara AM. Chalmers TC. Zimmerman H. Boston Interhospital Liver Croup A controlled study of the therapeutic portacaval shunt. (:astrt)rntPrology 1974;67:84:t57.