Portal Hypertension and Gastric Lesions in the Rat Thomas O.
Phelps, MS, John
F.
Mullane, MD, PhD
\s=b\ Gastric lesions occurred spontaneously and were increased in number by seven hours of restraint stress in rats with portal vein constriction (PVC). Vagotomy and pyloroplasty protected the congested stomach from erosion formation with stress. Major weight loss occurred two days after PVC, but not thereafter. Platelet counts were decreased in intact and splenectomized rats with portal hypertension, but prothrombin time, partial thromboplastin time, and fibrinogen were unaffected, and histological stains failed to demonstrate thrombin in the gastric blood vessels. The oxygen pressure (Po2) of the gastric luminal fluid was decreased at day 2, but was normal at day 4 after PVC. Hypersecretion of acid and abnormal acid equilibration were not observed in the stomach. Gastric congestion, weight loss, and possibly portasystemic shunting of blood contributed to the higher incidence of gastric erosions with portal hypertension.
groups. The rats
were anesthetized with ether and a midline inci¬ made in the abdomen. The portal vein was exposed and partially constricted by ligating the vein against a hypodermic needle that was subsequently removed. A 23-gauge needle was used for rats weighing between 75 and 100 gm, a 22-gauge needle for those between 101 and 125 gm, and a 21-gauge needle for rats weighing more than 125 gm. Sham surgery consisted of exposing, but not ligating, the portal vein. The incisions were closed in two layers with 4-0 Teflon suture. Sham and experimental rats for each variable studied were prepared on the same day. Experi¬ ments were performed two, four, seven, and 14 days after surgery in nonrestrained sham and portal hypertensive rats. Only the inci¬ dence of gastric lesions after PVC was studied in both the re¬ strained and nonrestrained rat.
sion
was
Gastric Lesions
(Arch Surg 111:190-194, 1976)
patients experimental GastricImpaired hepaticdamage portal hyperten¬ function, gastric congestion,
lesions may occur in or and animals with liver sion.111 shunting of blood past the liver, and jaundice, indepen¬ dently or in concert, might damage the gastric mucosa. The purpose of this study was to evaluate the effect of por¬ tal hypertension without liver damage or jaundice on the incidence of spontaneous and restraint-induced1- gastric lesions in the rat. Gastric lesions were studied following restraint since stress will cause lesions to develop in rats without predilection to spontaneous formation of lesions,11 and stress will increase the incidence of lesions in animal models where lesions spontaneously occur.111214 The dura¬ tion and severity of portal hypertension, gastric secretion and acid equilibration,1315 activity of the vagus nerve, in¬ travascular coagulation, and mucosal oxygen availability determined indirectly by measurement of the gastric luminal oxygen pressure (Po,),u arterial Po,, and in vitro 0, uptake of the gastric mucosa were related to the devel¬ opment of gastric lesions in nonstressed rats with partial ligation of the portal vein. MATERIALS AND METHODS Portal Vein Constriction (PVC) Wistar rats weighing between 75 and 130 gm were paired for and weight before division into sham and experimental
sex
Sham rats and rats with PVC were fasted under the following conditions: (1) for 31 hours prior to being decapitated to quantitate spontaneous gastric lesion formation; and (2) for 24 hours prior to being restrained for seven hours in wire mesh sheets molded to fit the contour of their bodies. After decapitation, the stomachs of restrained and nonrestrained rats were removed and opened along the lesser curvature for quantification of gastric le¬ sions by the same investigator, who was unaware as to which group individual specimens belonged. Lesions occurred in the glandular portion of the stomach and were classified as grade 1 or grade 2 erosions.1'14 Grade 1 erosions varied from minute, super¬ ficial areas of petechial hemorrhage to discrete, punched-out areas with clotted blood in the base. Lesions were classified as grade 2 if they were more than 1 mm in greatest dimension. Grade 2 ero¬ sions were usually linear in shape and were located along the ru¬ gai folds.
Vagotomy A
(Mr Phelps). Reprint requests
to 685 Third
Ave, New York, NY 10017 (Dr Mullane).
Pyloroplasty
subdiaphragmatic,
truncal vagotomy was performed in a group of rats at the time of PVC or sham surgery. The circum¬ ference of the lower part of the esophagus of the rat was stripped of nerve fibers and fascia for a distance of 4 mm. A pyloroplasty was performed to prevent gastric stasis. The rats were then re¬ strained four days later. To assess the effectiveness of surgical vagotomy, vagotomy without pyloroplasty and sham surgery were performed in eight rats each. If vagotomy was effective, stomach size should have in¬ creased.16 Four days later, after a 24-hour fast, rats were anesthe¬ tized with ether and the transverse length of the stomach was measured in situ.
Portal Pressure
Accepted
for publication Nov 13, 1975. From the Division of Surgery, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC (Dr Mullane), and the Department of Biological Sciences, Wright State University, Dayton, Ohio
and
Portal pressures, reported as centimeters of saline, were deter¬ mined acutely and four, seven, and 14 days after PVC. Under ether anesthesia, the abdomen was opened and a Clay Adams PE 50 polyethylene tubing attached to a calibrated pressure trans-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
ducer
was
introduced into
a
branch of the ileocolic vein. For acute
studies, placement of the ileocolic catheter preceded PVC. Liver Function Tests Sham and PVC rats were anesthetized four days after surgery and blood was withdrawn from the abdominal aorta for determi¬ nation of blood ammonia (ftg/100 ml) by a modification of the Conway diffusion method. The plasma was separated from the blood and plasma total bilirubin (mg/100 ml), alkaline phosphatase (in¬ ternational units), and glutamic oxaloacetic transaminase (SGOT [Reitman-Frankel units]) were measured.
Clotting Factors Platelet counts, fibrinogen, prothrombin time, and partial thromboplastin time were determined by standard clinical labora¬ days after surgery. The effect of splenic congestion on the platelet count was studied at the fourth postoperative day in sham, PVC, splenectomized, and PVC-splenectomized rats. tory methods in sham and PVC
rats four
Gastric Luminal Po Rats were fasted 24 hours before gastric luminal Po, was stud¬ ied by a modification of the pyloric and esophageal ligation tech¬ nique.1111 Two hours after surgery, 3 ml of the 4 ml of fluid that had been injected into the stomach was withdrawn through an 18-gauge hypodermic needle and the Po, was measured on a gas analyzer. The time required for Po2 of the injected fluid to equili¬ brate in the stomach has been determined previously.11
Blood Gases
alyzer.
Oxygen Uptake
In vitro tissue respiration of mucosal slices from the gastric fundus was measured with a biological oxygen monitor. The re¬ sults were expressed as microliters of oxygen per milligram of dry weight of tissue per hour.
Acid Secretion and
RESULTS
Mortality of the rats died within the first four hours after PVC. After 96 hours, the cumulative mortality was 45%. Rats that survived more than four days subse¬ quently had less than 2% mortality. The mortality for sham-operated rats was less than 1%.
Twenty percent
Weight Change Rats with PVC gained less weight than sham-operated rats when weights were measured in four groups of rats at two, four, seven, and 14 days (P < .001). At two days, PVC rats lost 16.3 ±4.1 gm and shams lost 1.5 ± 5.9 gm. At four days, PVC rats lost 7.2 ± 2.7 gm and shams gained 17.4 ± 3.9 gm. At seven days, PVC rats had a loss of 0.6 ± 3.9 gm and shams gained 14.6 ± 3.8 gm. At 14 days, PVC rats gained 7.0 ± 5.6 gm and shams gained 24.4 ± 5.5 gm. Gastric Erosions
Unrestrained rats with portal hypertension of four duration had a higher incidence of grade 1 lesions than did sham-operated rats (21.8 ± 14.7 vs 6.5 ± 11.3; 17 and 16; P> .01). The incidence of grade 2 lesions > .01). was similar (0.4 ± 0.3 vs 0.1 ± 0.3; Rats with PVC that were restrained for seven hours to increase the number of gastric lesions had a higher inci¬ dence of grade 1 and grade 2 gastric erosions two, four, and seven days after surgery than did restrained shamoperated rats. At 14 days, the incidence of gastric erosions was similar for PVC and sham rats (Table 1).
days'
Blood pH, carbon dioxide pressure (Pco2), and Po2 were deter¬ mined on awake sham and PVC rats."" Two hours after place¬ ment of a catheter in the carotid artery of sham and PVC rats that had been anesthetized with ether, 2 ml of blood was with¬ drawn and pH and gas values were determined on a blood gas an¬
Gastric Mucosal
standard deviation of the mean. Statistical significance was deter¬ mined by Student t test for unpaired data. When values are given in parentheses, results for the rats with portal hypertension are given before the sham results.
Equilibration
The secretion and the movement of hydrogen ions in the stom¬ ach were determined by a surgical method similar to that for lumi¬ nal Po=.l:' The esophagus and pylorus were ligated and 3 ml of isotonic saline was introduced into the f orestomach through a 27gauge hypodermic needle. Four hours later, the rats were reanesthetized with ether, their stomachs were removed, and the contents were emptied and measured for volume. Acid secretion was determined by titrating a 2-ml aliquot of the gastric fluid with 0.1N sodium hydroxide and phenophthalein as indicator. Other groups of sham and PVC rats had 4 ml of hydrochloric acid in concentrations of approximately 50, 60, or 80 microequivalents per milliliter injected into the forestomach to determine if the movement of hydrogen ions was similar in both groups.
=
Vagotomy The transverse measurement of the vagotomized stom¬ ach was significantly greater than that of sham rats (3.4 ± 0.3 vs 2.7 ± 0.2 cm; < .01). At the fourth day, va¬ gotomy and pyloroplasty protected the stomach of PVC rats from forming gastric erosions associated with stress (grade 1,41 ± 12 vs 14 ± 11; 15 and 14; < .05; grade 2, 10 ± 7 vs 0.1 ± 0.3; < .05). =
Portal Pressures
Sections of stomach taken from sham and PVC rats were stained with hematoxylin-eosin, for thrombin with phosphotungstic acid-hematoxylin, and for mucin with PAS.
Portal pressures were significantly higher in rats with PVC than in sham-operated rats immediately (40 ± 4 cm saline vs 7.6 ± 0.8 cm saline; n 6 and 6; P< .001) and at four days (25 ±7 cm saline vs 8.9 ± 2.2 cm saline; 8 and 9; < .001) after surgery. The portal pressures of PVC and sham rats were similar at seven (13.6 ± 1.4 cm saline vs 12.9 ± 2.6 cm saline; 5 and 5; P>A) and four¬ teen days (5.1 ± 0.4 cm saline vs 5.1 ± 0.2 cm saline; n 5 and 4; < .9) after surgery.
Presentation of Data
Liver Function Tests
Histologie Method
Numerical data
are
expressed
as
the
mean
plus
or
minus
one
=
=
=
=
Glutamic oxaloacetic transaminase (93 ± 10
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
vs
61 ±
2;
Table 1.—Gastric Lesions With Restraint
Days After Portal Vein Constriction (PVC)
Lesions Per Stomach Grade 1 Sham PVC Grade 2 Sham PVC
No.
Day 2
No.
Day 4
No.
Day
17 24
15.8 ±10.! 31.1 ±16.7*
15 15
18.6 ±9.1 40.5 ± 12.4*
18 19
10.9 ±5.6
17 24
1.7 ±2.8
15
TiT
0.1 ±0.3 10.2 ±7.0*
18 19
0.1 ±0.1
5.8±6.3t
7
Day
No.
22.7±17.5t
14
16 17
18.8 ±4.8 24.1 ± 4.6
16
0.4 ±0.2 0.5 ±0.4
1.3±2.2
*P.1) were not significantly dif¬ ferent in four day PVC and sham-operated rats. The
platelet count was significantly decreased in PVC rats (0.7± 0.1x10e vs 1.2±0.1xl06; n ten pairs; P9
51.0 42.1 ± 2.4 49.8 ±3.0 >.05
204 177±10 208 ±13 >.05
4.0 4.2 ±0.6 4.3 ± 0.2 >.8
64.0 49.0 ±3.1 58.5 ±2.8 >.05
256 212 ±18 251 ±15
4.0 4.2 ±0.2 4.5 ± 0.2
88.0 46.9 ± 3.3 47.3 ± 3.4
352 197± 14 213± 17 >.9
microequivalents/ml
50
0 hr 4 hr sham 4 hr PVC
Pt microequivalents/ml
60
Ohr
4 hr sham 4 hr PVC 80
PÍ microequivalents/ml Ohr 4 hr sham 4hrPVC
Pt *
12 10
10 11
>.1
>.1
Portal vein constriction, hours sham vs PVC.
t Four
is combined with something that affects gastric blood flow.20 By the fourth day after PVC, gastric luminal Po2 was normal, and a residual effect of tissue hypoxia or some other variable may have been the cause of the gastric lesions. Vagotomy has protected the gastric mucosa from restraint-induced erosions.'1·16·*1·22 A similar protection occurred in rats with portal hypertension and stress. This could be due to a favorable effect of vagotomy on gastric blood flow or acid-pepsin secretion. Blood ammonia was elevated and this might reflect portasystemic shunting of blood containing hormones and metabolites that could adversely affect the stomach. In addition, blood ammonia or some other compound shunted past the liver might have affected the respiratory centers and caused respiratory alkalosis.17 Gradual circulatory compensation occurred in response to PVC. At day 4, the gastric luminal Po, was normal, and at day 7, portal pressures were normal. At day 7, the susceptibility to stress gastric lesions was reduced, and by day 14 it was absent. Portal pressure changes correlated with the development and abatement of gastric lesions with PVC and stress. Hypersécrétion of acid, abnormal acid equilibration, ab¬ normal distribution of gastric mucin stain, and thrombi in gastric vessels were not present in rats with PVC. Hyper-
secretion of acid occurred in dogs with PVC23·24 or with liver damage,25 but was not present in this study, nor did it occur in patients2628 or rats10 with liver damage. In addi¬ tion, in situ gastric pH was less acidic in rats with PVC.9 In the latter study, when arterial blood was drawn from anesthetized rats, there was a metabolic acidosis. This contrasts with the respiratory alkalosis present in con¬ scious rats in this study. The metabolic acidosis was not quantified, and this would be of interest since severe aci¬ dosis alone13 or with renal failure29 will cause gastric le¬ sions with stress. It is unlikely that increased back diffu¬ sion of acid masked acid hypersécrétion in the PVC rat, since equilibration of various concentrations of acid was normal. In addition, restraint in the rat3" and mucosal is¬ chemia due to hemorrhage in the dog31 and monkey32 were not associated with back diffusion of acid. If gastric mucus secretion was reduced, the mucosa might have been sus¬ ceptible to injury. Although mucin was not quantitated, the distribution of PAS stain, and hence mucin, in the stomachs of PVC and sham rats was similar. Gastric mu¬ cin staining was reduced with experimental liver damage, but the development of portal hypertension did not reduce mucin staining further,10 However, despite normal results from acid and mucin studies, weight loss and venous con-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
gestion may have lowered local resistance to acid and pep¬ sin so that normal or even subnormal levels of gastric secretion were harmful. Stasis from PVC might have con¬ tributed to intravascular coagulation that could cause fo¬ cal gastric mucosal lesions. Thrombi were not demon¬ strated with histological staining techniques. However, fibrinolysis might have occurred, and this would explain the decreased platelet count, which was not due to splenic activity. Restraint stress increased the number and severity of gastric lesions occurring with portal hypertension. A sim¬ ilar response was observed in rats with carbon tetrachloride-induced cirrhosis.33 Restraint may have augmented
the effect of PVC
on
the stomach because of
venous con¬
gestion occurring with restraint stress,34 a possible action
of PVC on the bacterial flora of the gut,35,36 or the weight loss occurring with PVC, which might have had an effect similar to starvation.12 Gastric congestion due to PVC produced superficial ero¬ sions in the gastric mucosa. It is possible that malnutri¬ tion, venous congestion, and portasystemic shunting of hormones in the patient with liver disease may contribute to gastric erosion formation. Howard Hufnagel, Edward Jenkins, Patricia assisted with the experiments.
Royer, and Robert Wilfong
References 1. Dagradi AE, Mehler R, Tan DT, et al: Sources of upper gastrointestinal bleeding in patients with liver cirrhosis and large esophagogastric varices. Am J Gastroenterol 54:458-463, 1970. 2. Enquist IF, Gliedman ML: The sources of upper gastrointestinal bleeding in patients with cirrhosis. Surg Gynecol Obstet 106:143-149,1958. 3. Merigan TC, Hollister RM, Gryska PF, et al: Gastrointestinal bleeding with cirrhosis: A study of 172 episodes in 158 patients. N Engl J Med 263:579-585, 1960. 4. Palmer ED, Brick IB: Sources of upper gastrointestinal hemorrhage in cirrhotic patients with esophageal varices. N Engl J Med 248:1057-1058, 1953. 5. Zachariae F, Koster KH: Massive gastric and duodenal hemorrhage in patients with liver cirrhosis. Scand J Gastroenterol, suppl 7, pp 143-147, 1969. 6. Baronofsky ID, Wagensteen OH: Obstruction of splenic vein increases weight of stomach and predisposes to erosion or ulcer. Proc Soc Exp Biol Med 59:234-237, 1945. 7. Baronofsky ID: Portal hypertension. Surgery 23:135-163, 1949. 8. Gliedman ML, Girardet R, Karlson KE: Gastroduodenal congestion and the ulcer diathesis. Surgery 58:638-645, 1965. 9. Guerrin F, Latreille JP, Gosselin B: Gastric effects of experimental portal hypertension, in Pfeiffer C (ed): Peptic Ulcer. Philadelphia, JB Lippincott Co, 1971, pp 288-295. 10. Gheorghiu T, Klein HJ, Frotz H, et al: Pathogenesis of gastric ulcer in rats with carbon tetrachloride-induced liver damage, in Pfeiffer C (ed): Peptic Ulcer. Philadelphia, JB Lippincott Co, 1971, pp 267-287. 11. Mullane JF, Ritchie WP Jr, Solis RT, et al: Experimental biliary obstruction and stress ulcer formation. J Surg Res 12:180-184, 1972. 12. Brodie DA, Hanson HM: A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38:353-360, 1960. 13. Mullane JF, Wilfong RG, Phelps TO, et al: Metabolic acidosis, stress, and gastric lesions in the rat. Arch Surg 107:456-459, 1973. 14. Mullane JF, Pyant RL Jr, Wilfong RG, et al: Starvation, glucose, and stress ulcers in the rat. Arch Surg 109:416-419, 1974. 15. Linder AE: Electrolyte changes in rats' stomachs following instillation of acid solutions. Am J Physiol 207:606-612, 1964. 16. Ritchie WP Jr, Roth RR, Fischer RP: Studies on the pathogenesis of stress ulcer: Effect of hemorrhage, transfusion, and vagotomy in the restrained rat. Surgery 71:445-451, 1972. 17. Mullane JF, Popovic NA, Solis RT, et al: Effect of experimental biliary obstruction on respiratory function. J Surg Res 13:197-202, 1972.
18. Mullane JF, Wilfong RG, Phelps TO, et al: Dehydration and factors affecting pulmonary antibacterial defenses. J Surg Res 16:44-49, 1974. 19. Phelps TO, Mullane JF, Wilfong RG, et al: Graded hepatectomy and
stress ulcer formation in the rat, abstracted. Clin Res 20:872, 1972. 20. Mullane JF, Smith JC, Wilfong RG: Hypoxia and stress ulcer formation in the rat. Surgery 74:326-332, 1973. 21. Menguy R: Effects of restraint stress on gastric secretion in the rat. Am J Dig Dis 5:911-916, 1960. 22. Guth PH, Kozbur X: Microcirculatory and mast cell changes in restraint stress: Role of gastric acid. Am J Dig Dis 14:113-117, 1969. 23. Dubuque TJ Jr, Mulligan LV, Neville EC: Gastric secretion and peptic ulceration in the dog with portal obstruction and portacaval anastomosis. Surg Forum 8:208-211, 1958. 24. Gregory RA: Gastric secretory responses after portal venous ligation. J Physiol 144:123-137, 1958. 25. Silen W, Skillman JJ, Hein M, et al: The effect of biliary obstruction upon canine gastric secretory activity. J Surg Res 2:197-200, 1962. 26. Ostrow JD, Timmerman RJ, Gray SJ: Gastric secretion in human hepatic cirrhosis. Gastroenterology 38:303-313, 1960. 27. Scobie BA, Summerskill WH: Reduced gastric acid output in cirrhosis: Quantitation and relationships. Gut 5:422-428, 1964. 28. Tabaqchali S, Dawson AM: Peptic ulcer and gastric secretion in patients with liver disease. Gut 5:417-421, 1964. 29. Fischer RP, Peter ET, Mullane JF: Experimental stress ulcers in acidotic and nonacidotic renal insufficiency. Arch Surg 109:409-411, 1974. 30. Gerety DC, Guth PH: Restraint-induced gastric erosions: Role of acid back diffusion. Am J Dig Dis 17:1012-1018, 1972. 31. Moody FG, Aldrete JS: Hydrogen permeability of canine gastric secretory epithelium during formation of acute superficial erosions. Surgery 70:154-160, 1971. 32. Ritchie WP Jr: Effect of hemorrhage on electrical and pH gradients in the intact stomach of the subhuman primate. Gastroenterology 67:259\x=req-\ 263, 1974. 33. Bergman F, Van Der Linden W: Influence of carbon tetrachloride-induced cirrhosis on the incidence of restraint-stress erosions in the stomach of rats. Acta Chir Scand 129:196-200, 1965. 34. Guth PH, Hall P: Microcirculatory and mast cell changes in restraint\x=req-\
induced gastric ulcer. Gastroenterology 50:562-570, 1966. 35. Goldman H, Rosoff CB: Pathogenesis of acute gastric stress ulcers. Am J Pathol 52:227-236, 1968. 36. Nance FC, Kaufman HJ, Batson RC: The role of the microbial flora in acute gastric stress ulceration. Surgery 72:68-73, 1972.
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
Phelps, MS, John
F.
Mullane, MD, PhD
\s=b\ Gastric lesions occurred spontaneously and were increased in number by seven hours of restraint stress in rats with portal vein constriction (PVC). Vagotomy and pyloroplasty protected the congested stomach from erosion formation with stress. Major weight loss occurred two days after PVC, but not thereafter. Platelet counts were decreased in intact and splenectomized rats with portal hypertension, but prothrombin time, partial thromboplastin time, and fibrinogen were unaffected, and histological stains failed to demonstrate thrombin in the gastric blood vessels. The oxygen pressure (Po2) of the gastric luminal fluid was decreased at day 2, but was normal at day 4 after PVC. Hypersecretion of acid and abnormal acid equilibration were not observed in the stomach. Gastric congestion, weight loss, and possibly portasystemic shunting of blood contributed to the higher incidence of gastric erosions with portal hypertension.
groups. The rats
were anesthetized with ether and a midline inci¬ made in the abdomen. The portal vein was exposed and partially constricted by ligating the vein against a hypodermic needle that was subsequently removed. A 23-gauge needle was used for rats weighing between 75 and 100 gm, a 22-gauge needle for those between 101 and 125 gm, and a 21-gauge needle for rats weighing more than 125 gm. Sham surgery consisted of exposing, but not ligating, the portal vein. The incisions were closed in two layers with 4-0 Teflon suture. Sham and experimental rats for each variable studied were prepared on the same day. Experi¬ ments were performed two, four, seven, and 14 days after surgery in nonrestrained sham and portal hypertensive rats. Only the inci¬ dence of gastric lesions after PVC was studied in both the re¬ strained and nonrestrained rat.
sion
was
Gastric Lesions
(Arch Surg 111:190-194, 1976)
patients experimental GastricImpaired hepaticdamage portal hyperten¬ function, gastric congestion,
lesions may occur in or and animals with liver sion.111 shunting of blood past the liver, and jaundice, indepen¬ dently or in concert, might damage the gastric mucosa. The purpose of this study was to evaluate the effect of por¬ tal hypertension without liver damage or jaundice on the incidence of spontaneous and restraint-induced1- gastric lesions in the rat. Gastric lesions were studied following restraint since stress will cause lesions to develop in rats without predilection to spontaneous formation of lesions,11 and stress will increase the incidence of lesions in animal models where lesions spontaneously occur.111214 The dura¬ tion and severity of portal hypertension, gastric secretion and acid equilibration,1315 activity of the vagus nerve, in¬ travascular coagulation, and mucosal oxygen availability determined indirectly by measurement of the gastric luminal oxygen pressure (Po,),u arterial Po,, and in vitro 0, uptake of the gastric mucosa were related to the devel¬ opment of gastric lesions in nonstressed rats with partial ligation of the portal vein. MATERIALS AND METHODS Portal Vein Constriction (PVC) Wistar rats weighing between 75 and 130 gm were paired for and weight before division into sham and experimental
sex
Sham rats and rats with PVC were fasted under the following conditions: (1) for 31 hours prior to being decapitated to quantitate spontaneous gastric lesion formation; and (2) for 24 hours prior to being restrained for seven hours in wire mesh sheets molded to fit the contour of their bodies. After decapitation, the stomachs of restrained and nonrestrained rats were removed and opened along the lesser curvature for quantification of gastric le¬ sions by the same investigator, who was unaware as to which group individual specimens belonged. Lesions occurred in the glandular portion of the stomach and were classified as grade 1 or grade 2 erosions.1'14 Grade 1 erosions varied from minute, super¬ ficial areas of petechial hemorrhage to discrete, punched-out areas with clotted blood in the base. Lesions were classified as grade 2 if they were more than 1 mm in greatest dimension. Grade 2 ero¬ sions were usually linear in shape and were located along the ru¬ gai folds.
Vagotomy A
(Mr Phelps). Reprint requests
to 685 Third
Ave, New York, NY 10017 (Dr Mullane).
Pyloroplasty
subdiaphragmatic,
truncal vagotomy was performed in a group of rats at the time of PVC or sham surgery. The circum¬ ference of the lower part of the esophagus of the rat was stripped of nerve fibers and fascia for a distance of 4 mm. A pyloroplasty was performed to prevent gastric stasis. The rats were then re¬ strained four days later. To assess the effectiveness of surgical vagotomy, vagotomy without pyloroplasty and sham surgery were performed in eight rats each. If vagotomy was effective, stomach size should have in¬ creased.16 Four days later, after a 24-hour fast, rats were anesthe¬ tized with ether and the transverse length of the stomach was measured in situ.
Portal Pressure
Accepted
for publication Nov 13, 1975. From the Division of Surgery, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC (Dr Mullane), and the Department of Biological Sciences, Wright State University, Dayton, Ohio
and
Portal pressures, reported as centimeters of saline, were deter¬ mined acutely and four, seven, and 14 days after PVC. Under ether anesthesia, the abdomen was opened and a Clay Adams PE 50 polyethylene tubing attached to a calibrated pressure trans-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
ducer
was
introduced into
a
branch of the ileocolic vein. For acute
studies, placement of the ileocolic catheter preceded PVC. Liver Function Tests Sham and PVC rats were anesthetized four days after surgery and blood was withdrawn from the abdominal aorta for determi¬ nation of blood ammonia (ftg/100 ml) by a modification of the Conway diffusion method. The plasma was separated from the blood and plasma total bilirubin (mg/100 ml), alkaline phosphatase (in¬ ternational units), and glutamic oxaloacetic transaminase (SGOT [Reitman-Frankel units]) were measured.
Clotting Factors Platelet counts, fibrinogen, prothrombin time, and partial thromboplastin time were determined by standard clinical labora¬ days after surgery. The effect of splenic congestion on the platelet count was studied at the fourth postoperative day in sham, PVC, splenectomized, and PVC-splenectomized rats. tory methods in sham and PVC
rats four
Gastric Luminal Po Rats were fasted 24 hours before gastric luminal Po, was stud¬ ied by a modification of the pyloric and esophageal ligation tech¬ nique.1111 Two hours after surgery, 3 ml of the 4 ml of fluid that had been injected into the stomach was withdrawn through an 18-gauge hypodermic needle and the Po, was measured on a gas analyzer. The time required for Po2 of the injected fluid to equili¬ brate in the stomach has been determined previously.11
Blood Gases
alyzer.
Oxygen Uptake
In vitro tissue respiration of mucosal slices from the gastric fundus was measured with a biological oxygen monitor. The re¬ sults were expressed as microliters of oxygen per milligram of dry weight of tissue per hour.
Acid Secretion and
RESULTS
Mortality of the rats died within the first four hours after PVC. After 96 hours, the cumulative mortality was 45%. Rats that survived more than four days subse¬ quently had less than 2% mortality. The mortality for sham-operated rats was less than 1%.
Twenty percent
Weight Change Rats with PVC gained less weight than sham-operated rats when weights were measured in four groups of rats at two, four, seven, and 14 days (P < .001). At two days, PVC rats lost 16.3 ±4.1 gm and shams lost 1.5 ± 5.9 gm. At four days, PVC rats lost 7.2 ± 2.7 gm and shams gained 17.4 ± 3.9 gm. At seven days, PVC rats had a loss of 0.6 ± 3.9 gm and shams gained 14.6 ± 3.8 gm. At 14 days, PVC rats gained 7.0 ± 5.6 gm and shams gained 24.4 ± 5.5 gm. Gastric Erosions
Unrestrained rats with portal hypertension of four duration had a higher incidence of grade 1 lesions than did sham-operated rats (21.8 ± 14.7 vs 6.5 ± 11.3; 17 and 16; P> .01). The incidence of grade 2 lesions > .01). was similar (0.4 ± 0.3 vs 0.1 ± 0.3; Rats with PVC that were restrained for seven hours to increase the number of gastric lesions had a higher inci¬ dence of grade 1 and grade 2 gastric erosions two, four, and seven days after surgery than did restrained shamoperated rats. At 14 days, the incidence of gastric erosions was similar for PVC and sham rats (Table 1).
days'
Blood pH, carbon dioxide pressure (Pco2), and Po2 were deter¬ mined on awake sham and PVC rats."" Two hours after place¬ ment of a catheter in the carotid artery of sham and PVC rats that had been anesthetized with ether, 2 ml of blood was with¬ drawn and pH and gas values were determined on a blood gas an¬
Gastric Mucosal
standard deviation of the mean. Statistical significance was deter¬ mined by Student t test for unpaired data. When values are given in parentheses, results for the rats with portal hypertension are given before the sham results.
Equilibration
The secretion and the movement of hydrogen ions in the stom¬ ach were determined by a surgical method similar to that for lumi¬ nal Po=.l:' The esophagus and pylorus were ligated and 3 ml of isotonic saline was introduced into the f orestomach through a 27gauge hypodermic needle. Four hours later, the rats were reanesthetized with ether, their stomachs were removed, and the contents were emptied and measured for volume. Acid secretion was determined by titrating a 2-ml aliquot of the gastric fluid with 0.1N sodium hydroxide and phenophthalein as indicator. Other groups of sham and PVC rats had 4 ml of hydrochloric acid in concentrations of approximately 50, 60, or 80 microequivalents per milliliter injected into the forestomach to determine if the movement of hydrogen ions was similar in both groups.
=
Vagotomy The transverse measurement of the vagotomized stom¬ ach was significantly greater than that of sham rats (3.4 ± 0.3 vs 2.7 ± 0.2 cm; < .01). At the fourth day, va¬ gotomy and pyloroplasty protected the stomach of PVC rats from forming gastric erosions associated with stress (grade 1,41 ± 12 vs 14 ± 11; 15 and 14; < .05; grade 2, 10 ± 7 vs 0.1 ± 0.3; < .05). =
Portal Pressures
Sections of stomach taken from sham and PVC rats were stained with hematoxylin-eosin, for thrombin with phosphotungstic acid-hematoxylin, and for mucin with PAS.
Portal pressures were significantly higher in rats with PVC than in sham-operated rats immediately (40 ± 4 cm saline vs 7.6 ± 0.8 cm saline; n 6 and 6; P< .001) and at four days (25 ±7 cm saline vs 8.9 ± 2.2 cm saline; 8 and 9; < .001) after surgery. The portal pressures of PVC and sham rats were similar at seven (13.6 ± 1.4 cm saline vs 12.9 ± 2.6 cm saline; 5 and 5; P>A) and four¬ teen days (5.1 ± 0.4 cm saline vs 5.1 ± 0.2 cm saline; n 5 and 4; < .9) after surgery.
Presentation of Data
Liver Function Tests
Histologie Method
Numerical data
are
expressed
as
the
mean
plus
or
minus
one
=
=
=
=
Glutamic oxaloacetic transaminase (93 ± 10
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
vs
61 ±
2;
Table 1.—Gastric Lesions With Restraint
Days After Portal Vein Constriction (PVC)
Lesions Per Stomach Grade 1 Sham PVC Grade 2 Sham PVC
No.
Day 2
No.
Day 4
No.
Day
17 24
15.8 ±10.! 31.1 ±16.7*
15 15
18.6 ±9.1 40.5 ± 12.4*
18 19
10.9 ±5.6
17 24
1.7 ±2.8
15
TiT
0.1 ±0.3 10.2 ±7.0*
18 19
0.1 ±0.1
5.8±6.3t
7
Day
No.
22.7±17.5t
14
16 17
18.8 ±4.8 24.1 ± 4.6
16
0.4 ±0.2 0.5 ±0.4
1.3±2.2
*P.1) were not significantly dif¬ ferent in four day PVC and sham-operated rats. The
platelet count was significantly decreased in PVC rats (0.7± 0.1x10e vs 1.2±0.1xl06; n ten pairs; P9
51.0 42.1 ± 2.4 49.8 ±3.0 >.05
204 177±10 208 ±13 >.05
4.0 4.2 ±0.6 4.3 ± 0.2 >.8
64.0 49.0 ±3.1 58.5 ±2.8 >.05
256 212 ±18 251 ±15
4.0 4.2 ±0.2 4.5 ± 0.2
88.0 46.9 ± 3.3 47.3 ± 3.4
352 197± 14 213± 17 >.9
microequivalents/ml
50
0 hr 4 hr sham 4 hr PVC
Pt microequivalents/ml
60
Ohr
4 hr sham 4 hr PVC 80
PÍ microequivalents/ml Ohr 4 hr sham 4hrPVC
Pt *
12 10
10 11
>.1
>.1
Portal vein constriction, hours sham vs PVC.
t Four
is combined with something that affects gastric blood flow.20 By the fourth day after PVC, gastric luminal Po2 was normal, and a residual effect of tissue hypoxia or some other variable may have been the cause of the gastric lesions. Vagotomy has protected the gastric mucosa from restraint-induced erosions.'1·16·*1·22 A similar protection occurred in rats with portal hypertension and stress. This could be due to a favorable effect of vagotomy on gastric blood flow or acid-pepsin secretion. Blood ammonia was elevated and this might reflect portasystemic shunting of blood containing hormones and metabolites that could adversely affect the stomach. In addition, blood ammonia or some other compound shunted past the liver might have affected the respiratory centers and caused respiratory alkalosis.17 Gradual circulatory compensation occurred in response to PVC. At day 4, the gastric luminal Po, was normal, and at day 7, portal pressures were normal. At day 7, the susceptibility to stress gastric lesions was reduced, and by day 14 it was absent. Portal pressure changes correlated with the development and abatement of gastric lesions with PVC and stress. Hypersécrétion of acid, abnormal acid equilibration, ab¬ normal distribution of gastric mucin stain, and thrombi in gastric vessels were not present in rats with PVC. Hyper-
secretion of acid occurred in dogs with PVC23·24 or with liver damage,25 but was not present in this study, nor did it occur in patients2628 or rats10 with liver damage. In addi¬ tion, in situ gastric pH was less acidic in rats with PVC.9 In the latter study, when arterial blood was drawn from anesthetized rats, there was a metabolic acidosis. This contrasts with the respiratory alkalosis present in con¬ scious rats in this study. The metabolic acidosis was not quantified, and this would be of interest since severe aci¬ dosis alone13 or with renal failure29 will cause gastric le¬ sions with stress. It is unlikely that increased back diffu¬ sion of acid masked acid hypersécrétion in the PVC rat, since equilibration of various concentrations of acid was normal. In addition, restraint in the rat3" and mucosal is¬ chemia due to hemorrhage in the dog31 and monkey32 were not associated with back diffusion of acid. If gastric mucus secretion was reduced, the mucosa might have been sus¬ ceptible to injury. Although mucin was not quantitated, the distribution of PAS stain, and hence mucin, in the stomachs of PVC and sham rats was similar. Gastric mu¬ cin staining was reduced with experimental liver damage, but the development of portal hypertension did not reduce mucin staining further,10 However, despite normal results from acid and mucin studies, weight loss and venous con-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
gestion may have lowered local resistance to acid and pep¬ sin so that normal or even subnormal levels of gastric secretion were harmful. Stasis from PVC might have con¬ tributed to intravascular coagulation that could cause fo¬ cal gastric mucosal lesions. Thrombi were not demon¬ strated with histological staining techniques. However, fibrinolysis might have occurred, and this would explain the decreased platelet count, which was not due to splenic activity. Restraint stress increased the number and severity of gastric lesions occurring with portal hypertension. A sim¬ ilar response was observed in rats with carbon tetrachloride-induced cirrhosis.33 Restraint may have augmented
the effect of PVC
on
the stomach because of
venous con¬
gestion occurring with restraint stress,34 a possible action
of PVC on the bacterial flora of the gut,35,36 or the weight loss occurring with PVC, which might have had an effect similar to starvation.12 Gastric congestion due to PVC produced superficial ero¬ sions in the gastric mucosa. It is possible that malnutri¬ tion, venous congestion, and portasystemic shunting of hormones in the patient with liver disease may contribute to gastric erosion formation. Howard Hufnagel, Edward Jenkins, Patricia assisted with the experiments.
Royer, and Robert Wilfong
References 1. Dagradi AE, Mehler R, Tan DT, et al: Sources of upper gastrointestinal bleeding in patients with liver cirrhosis and large esophagogastric varices. Am J Gastroenterol 54:458-463, 1970. 2. Enquist IF, Gliedman ML: The sources of upper gastrointestinal bleeding in patients with cirrhosis. Surg Gynecol Obstet 106:143-149,1958. 3. Merigan TC, Hollister RM, Gryska PF, et al: Gastrointestinal bleeding with cirrhosis: A study of 172 episodes in 158 patients. N Engl J Med 263:579-585, 1960. 4. Palmer ED, Brick IB: Sources of upper gastrointestinal hemorrhage in cirrhotic patients with esophageal varices. N Engl J Med 248:1057-1058, 1953. 5. Zachariae F, Koster KH: Massive gastric and duodenal hemorrhage in patients with liver cirrhosis. Scand J Gastroenterol, suppl 7, pp 143-147, 1969. 6. Baronofsky ID, Wagensteen OH: Obstruction of splenic vein increases weight of stomach and predisposes to erosion or ulcer. Proc Soc Exp Biol Med 59:234-237, 1945. 7. Baronofsky ID: Portal hypertension. Surgery 23:135-163, 1949. 8. Gliedman ML, Girardet R, Karlson KE: Gastroduodenal congestion and the ulcer diathesis. Surgery 58:638-645, 1965. 9. Guerrin F, Latreille JP, Gosselin B: Gastric effects of experimental portal hypertension, in Pfeiffer C (ed): Peptic Ulcer. Philadelphia, JB Lippincott Co, 1971, pp 288-295. 10. Gheorghiu T, Klein HJ, Frotz H, et al: Pathogenesis of gastric ulcer in rats with carbon tetrachloride-induced liver damage, in Pfeiffer C (ed): Peptic Ulcer. Philadelphia, JB Lippincott Co, 1971, pp 267-287. 11. Mullane JF, Ritchie WP Jr, Solis RT, et al: Experimental biliary obstruction and stress ulcer formation. J Surg Res 12:180-184, 1972. 12. Brodie DA, Hanson HM: A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38:353-360, 1960. 13. Mullane JF, Wilfong RG, Phelps TO, et al: Metabolic acidosis, stress, and gastric lesions in the rat. Arch Surg 107:456-459, 1973. 14. Mullane JF, Pyant RL Jr, Wilfong RG, et al: Starvation, glucose, and stress ulcers in the rat. Arch Surg 109:416-419, 1974. 15. Linder AE: Electrolyte changes in rats' stomachs following instillation of acid solutions. Am J Physiol 207:606-612, 1964. 16. Ritchie WP Jr, Roth RR, Fischer RP: Studies on the pathogenesis of stress ulcer: Effect of hemorrhage, transfusion, and vagotomy in the restrained rat. Surgery 71:445-451, 1972. 17. Mullane JF, Popovic NA, Solis RT, et al: Effect of experimental biliary obstruction on respiratory function. J Surg Res 13:197-202, 1972.
18. Mullane JF, Wilfong RG, Phelps TO, et al: Dehydration and factors affecting pulmonary antibacterial defenses. J Surg Res 16:44-49, 1974. 19. Phelps TO, Mullane JF, Wilfong RG, et al: Graded hepatectomy and
stress ulcer formation in the rat, abstracted. Clin Res 20:872, 1972. 20. Mullane JF, Smith JC, Wilfong RG: Hypoxia and stress ulcer formation in the rat. Surgery 74:326-332, 1973. 21. Menguy R: Effects of restraint stress on gastric secretion in the rat. Am J Dig Dis 5:911-916, 1960. 22. Guth PH, Kozbur X: Microcirculatory and mast cell changes in restraint stress: Role of gastric acid. Am J Dig Dis 14:113-117, 1969. 23. Dubuque TJ Jr, Mulligan LV, Neville EC: Gastric secretion and peptic ulceration in the dog with portal obstruction and portacaval anastomosis. Surg Forum 8:208-211, 1958. 24. Gregory RA: Gastric secretory responses after portal venous ligation. J Physiol 144:123-137, 1958. 25. Silen W, Skillman JJ, Hein M, et al: The effect of biliary obstruction upon canine gastric secretory activity. J Surg Res 2:197-200, 1962. 26. Ostrow JD, Timmerman RJ, Gray SJ: Gastric secretion in human hepatic cirrhosis. Gastroenterology 38:303-313, 1960. 27. Scobie BA, Summerskill WH: Reduced gastric acid output in cirrhosis: Quantitation and relationships. Gut 5:422-428, 1964. 28. Tabaqchali S, Dawson AM: Peptic ulcer and gastric secretion in patients with liver disease. Gut 5:417-421, 1964. 29. Fischer RP, Peter ET, Mullane JF: Experimental stress ulcers in acidotic and nonacidotic renal insufficiency. Arch Surg 109:409-411, 1974. 30. Gerety DC, Guth PH: Restraint-induced gastric erosions: Role of acid back diffusion. Am J Dig Dis 17:1012-1018, 1972. 31. Moody FG, Aldrete JS: Hydrogen permeability of canine gastric secretory epithelium during formation of acute superficial erosions. Surgery 70:154-160, 1971. 32. Ritchie WP Jr: Effect of hemorrhage on electrical and pH gradients in the intact stomach of the subhuman primate. Gastroenterology 67:259\x=req-\ 263, 1974. 33. Bergman F, Van Der Linden W: Influence of carbon tetrachloride-induced cirrhosis on the incidence of restraint-stress erosions in the stomach of rats. Acta Chir Scand 129:196-200, 1965. 34. Guth PH, Hall P: Microcirculatory and mast cell changes in restraint\x=req-\
induced gastric ulcer. Gastroenterology 50:562-570, 1966. 35. Goldman H, Rosoff CB: Pathogenesis of acute gastric stress ulcers. Am J Pathol 52:227-236, 1968. 36. Nance FC, Kaufman HJ, Batson RC: The role of the microbial flora in acute gastric stress ulceration. Surgery 72:68-73, 1972.
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Iowa User on 06/02/2015
