Laparotomy Stimulates an Endogenous Gastric Mucosal Protective Mechanism in the Rat

GASTROENTEROLOGY 1988;95:600-11

Laparotomy Stimulates an Endogenous Gastric Mucosal Protective Mechanism in the Rat A Microcirculatory and Morphologic Study of Ethanol Injury YOSHIKAZU YONEI and PAUL H. GUTH

Medical and Research Service, Veterans Administration Medical Center, West Los Angeles; UCLA School of Medicine, Los Angeles; and Center for Ulcer Research and Education, Los Angeles, California

In vivo microscopy in the anesthetized rat unexpectedly revealed that the topical application of 75% ethanol to the gastric mucosa had no effect on the mucosal microcirculation. If, however, the synthesis of endogenous prostaglandin was inhibited by indomethacin, the topical application of 75% ethanol resulted in complete superficial mucosal microcirculatory stasis in the majority of rats. In indomethacin-pretreated rats, Rioprostil, asynthetic prostaglandin E1 analogue, reversed this effect of topical ethanol. The degree of ethanolinduced histologic gastric mucosal damage was inversely correlated with time to complete stasis. Ethanol injury studies under various conditions revealed that laparotomy stimulated a gastric protective mechanism that persisted for between 1 and 2 h and that was blocked by indomethacin. In conclusion, (a) laparotomy stimulates a gastric mucosal protective mechanism that probably is mediated by prostaglandin synthesis, and (b) cessation of mucosal blood flow appears to be an important early step in ethanol-induced gastric mucosal injury, and maintenance of mucosal blood flow by endogenous or exogenous prostaglandin may playa major role in prostaglandin protection.

T

he original demonstration by Robert et al. (1) of prostaglandin (PG) cytoprotection, i.e., protection against gastric mucosal injury by a mechanism other than inhibition of acid secretion, has been confirmed in a number of laboratories (2-5). Thus, 50% or higher concentrations of ethanol given intragastrically to conscious rats causes gross and histologic necrotic lesions in the gastric mucosa. Pretreatment with a nonantisecretory dose of 16,16-

dimethyl PGEz, other prostaglandin analogues, or PGEz prevents this ethanol injury (1-5). Recently it has been pointed out that a disturbance of the gastric mucosal microcirculation may be involved in ethanol-induced gastric lesion formation (2,3,6-9). Morphologic studies have revealed the presence of marked engorgement of gastric mucosal microvessels (2,3), platelet thrombi (6), and damage of capillary endothelium (7) in association with these lesions. Leung et al. (8) found that, in conscious rats receiving 100% ethanol intragastrically, gastric mucosal blood flow had ceased in the lesion area, and pretreatment with 16,16-dimethyl PGE z both maintained gastric mucosal blood flow and prevented lesion formation. In contrast, Bou-Abboud et al. (10) reported that, in in vivo microscopic studies in the anesthetized rat, the topical application of 75% ethanol did not stop gastric mucosal blood flow or cause histologic injury. Robert et al. (11) demonstrated that mild irritants given intragastrically, by stimulating endogenous gastric prostaglandin synthesis, protected the stomach against injury by strong irritants. They called this "adaptive cytoprotection." We postulated that preparation of the rat for in vivo microscopy (anesthesia, surgery, and gastric manipulation) stimulated the synthesis of endogenous prostaglandins and so protected the gastric mucosa from damage by 75% ethanol (adaptive cytoprotection). The purpose of the present study was (a) to test the above hypothesis by determining whether 75% eth-

Abbreviations used in this paper: ANOVA, analysis of variance; IHI, index of histologic injury; VRBC ' red blood cell velocity. © 1988 by the American Gastroenterological Association 0016-5085/88/$3.50

September 1988

anol causes gastric mucosal blood flow stasis and mucosal injury when endogenous prostaglandin synthesis is inhibited, (b) to determine-if the above was true-whether exogenous prostaglandin, in a nonantisecretory dose, prevents this, and (c) to determine which aspect of the preparation for in vivo microscopy stimulates this endogenous protective mechanism.

Materials and Methods Animals and Agents Male Sprague-Dawley rats weighing between 180 and 250 g (Simonsen Lab. Inc., Gilroy, Calif.) were fasted overnight, but were allowed free access to water. Experimental groups consisted of 6 or more rats. Indomethacin (Sigma Chemical Co., St. Louis, Mo.) was completely dissolved in 5% sodium bicarbonate (5 mg of indomethacin per milliliter) by heating and stirring for 5 h at 40°C. This solution then was diluted with distilled water so that the final solution contained 1 mg of indomethacin in 1 ml of 1% NaHC0 3 • Indomethacin was injected subcutaneously (5 mg/kg body wt) in conscious rats 1 h before the experiment. Rioprostil (2-decarboxy-2hydroxymethyl-15-deoxy-16RS-hydroxy-16-methyl PGEt ) , a synthetic PGEt analogue (12), was supplied by the Ortho Pharmaceutical Corp., Raritan, N.J. A stock solution of 0.5 mg of Rioprostil per milliliter of 100% ethanol was prepared and then diluted with distilled water. The working solution contained 10 /-Lg of Rioprostil in 1 ml of 2% ethanol. Then, 0.5 ml of this solution per 250 g body wt (20 /-Lg/kg) was given to each rat by orogastric intubation 30 min before the experiment. This dose of Rioprostil was reported to be 10 times less than its antisecretory dose and to prevent gastric lesions in rats induced by a variety of necrotizing agents (12). The vehicle, 2% ethanol, was used as the control for the Rioprostil solution or 75% ethanol.

Study 1: In Vivo Microscopic Study In vivo microscopy preparations. An in vivo microscopic technique was used to study the mucosal microvasculature (13). The rats were anesthetized with 50 mg/kg sodium pentobarbital (Abbott Laboratories, North Chicago, Ill.) intraperitoneally. Rectal temperature was maintained at 37.5°C by a heating pad. The animal was placed on its back on a wooden stage on a heating pad. The abdomen was opened via a 3-cm midline incision and the stomach was exposed. A l-cm incision was made in the anterior wall of the forestomach just proximal and parallel to the limiting ridge. A portion of the posterior wall of the corpus was then everted through the incision, thus making the mucosal surface completely visible. A fiberoptic light carrier rod 5 mm in diameter (American Optical Scientific Instruments, Buffalo, N.Y.) with an attached mirrored prism (Rolyn Optics, Covina, Calif.) was placed beneath the serosa of the everted portion of the corpus. The exposed area was transilluminated by passing a cool light from a 250-W tungsten projection lamp (model EHJ; General Electric, West Lynn, Mass.) through the light carrier

ENDOGENOUS GASTRIC PROTECTION AND LAPAROTOMY 601

rod. A green filter (Kodak Wratten No. 61; Eastman Kodak Co., Rochester, N.Y.) was used to enhance the contrast between the blood-filled microvessels and the background. A concave brass disk (2.5 ern in diameter and 5 mm in depth) with a 5-mm-diameter hole in the center was fixed, water tight, with a silicone plastic adherent (Silly Putty; Binney & Smith Inc., Easton, Pa.) to the area under study for application of different solutions. All solutions were maintained at 37.5°C before application. Krebs' solution (pH 7.4, 37.5°C) was continuously superfused to maintain constant temperature and moisture of the exposed surface. The preparation was allowed to stabilize for 10-15 min before starting the experiment. After a 5-min period of basal observation, the superfusion was stopped and the Krebs' solution remaining in the disk was gently aspirated, then 0.5 ml of 75% ethanol or vehicle was placed in the disk and left there for the duration of the study (10-15 min). The time required for complete stasis after the ethanol treatment was determined using the time display on the screen of the monitor. An American Optical Microstar microscope with a Leitz long working distance x 20/0.33 or a x 32/0.4 objective lens was used for transmitted light in vivo microscopy. The microscope was connected to a closed circuit television system that consisted of a charge-coupled device video camera (TM-34K; Pulnix America Inc., Sunnyvale, Calif.], a television monitor (VM-129U; Hitachi Denshi Ltd., Tokyo, Japan), and a videotape recorder (HR-7100U; JVC, Yokohama, Japan). All in vivo microscopic studies were videotaped for later analysis of red blood cell velocity (VRBc) and vessel diameter. The final magnification on the television screen was X470 or x750. Red blood cell velocity and vessel diameter measurement. The technique described by Holm-Rutili and Obrink (14) was used to measure VRBC in the gastric mucosa. Only those rats studied under high magnification (X750) could be used for VRBC determinations. This explains the difference in numbers of rats in each group in Tables 1 and 2. The gastric superficial microvessels were classified into three orders of postcapillary venules and capillaries according to their branching hierarchy and relative dimensions starting from the collecting venules and moving backward to the capillaries (14). The large branches emptying into collecting venules were designated as first-order postcapillary venules (12-20 /-Lm). Subsequent generations of venous branches upstream from these vessels were called second-order (11-15 /-Lm) and third-order (8-12 /-Lm) postcapillary venules, and finally capillaries (5-9 /-Lm). Measurements of VRBC and vessel diameter were performed on first-order postcapillary venules. RED BLOOD CELL VELOCITY MEASUREMENT. Briefly, on playback of the videotape, using an analog analyzer (Elmore, Tucson, Ariz.) two videophotometric windows were positioned upstream and downstream in the monitored vessel lumen. The voltage output changed as red blood cells passed each window; the resulting signals were fed into a VRBC tracking correlator (model-l02B; Instrumentation for Physiology and Medicine, San Diego, Calif.), which determines the time lag for similar voltage patterns between the upstream and downstream signals. By contin-

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uously dividing this time lag into the preset distance between the two windows,' the correlator continuously outputs VRBC in millimeters per second. Velocities between 0.1 and 1.8 mm/s could be measured. The same vessels were followed throughout the course of an experiment. Measurements were repeated before and 5 min after the topical application of 75% ethanol. As a control, in 6 rats Krebs' superfusion was applied for 2 h, and the average velocity over a lO-min period was determined initially and at the end of 1 and 2 h. VESSEL DIAMETER MEASUREMENT. An image analyzer (VT-I02 with MICRO/PDP-ll; Digital Equipment Corp., Maynard, Mass.) equipped with imaging boards (Imaging Technology Inc., Woburn, Mass.) was used to measure the microvessel diameter in gastric mucosa on playback of the videotapes. The program for diameter measurement was written by Dr. Edward Livingston and Hoang Dao. The program determines the area of an ~30-p.m length of vessel and then divides the area by the length to provide the average diameter of this length of vessel. Diameter measurements were made on first-, second-, and third-order postcapillary venules and capillaries. Blood flow (nanoliters per second) in the first-order postcapillary venules was calculated from these data of VRBC and vessel radius (R) using the following equation (14): Blood flow

=

1TR 2 VRBc ,

Histologic study. Tissue was obtained for histologic study from most, but not all, of the experiments after the period of in vivo microscopic observation (15 min after ethanol application or 10 min after stasis of blood flow). The area under study was marked by topically applying 1% pontamine sky blue solution. The marked piece of tissue then was immediately removed, placed in 10% buffered formalin, and later embedded in paraffin, sectioned (6 p.m thick), and stained with hematoxylin and eosin. Slides were evaluated microscopically in a blinded fashion by coding, so that the examiner was unaware of the treatment group each slide came from. Only after evaluation of all slides was completed were they decoded. Using an eyepiece equipped with a micrometer scale, the length of each type of damage and the total length of mucosa exposed to ethanol were measured and the percentage of each type of damage was determined as described by Lacy and Ito (2). Type of damage was defined using a modification of the criteria established by the latter authors (2) as follows: grade 0 = all gastric mucosal cells appeared intact and had normal shape, location, appearance, and density; grade 1 = surface mucous cell damage--these cells were vacuolated, had pyknotic nuclei, and brightly stained or lysed cytoplasm; grade 2 = extensive surface cell damage plus disruption and exfoliation of cells lining the gastric pits; grade 3 = surface and pit cell damage plus damage extending into the parietal cell area-the parietal cells had pyknotic nuclei and lucent cytoplasm; grade 4 = severe grade 3 lesion with necrosis. Necrosis was defined as loss of parietal cells with the remaining damaged parietal and other cells no longer forming a continuous column of contiguous cells-empty spaces appearing between the

cells. Sometimes necrosis also would involve the inner third (chief cell area) of the mucosa. To compare differences in histologic damage among groups, a mean damage score or index of histologic injury (IBI), as suggested by the statistician Dr. T. Reedy, was calculated for each section as follows. The percentage of the length of each grade of damage was multiplied by 1, 2, 3, and 4, respectively. Then these figures were added and divided by 100. In effect, the IHI represents the average grade of damage in each section. The experimental groups were as follows. (a) In the non-indomethacin-treated group, 75% ethanol was applied topically to the exposed gastric mucosa under in vivo microscopy. (b) In the indomethacin plus vehicletreated group, rats were pretreated with 5 mg/kg indomethacin subcutaneously 1 h before preparation, and then given vehicle intragastrically 30 min before preparation (for in vivo microscopy). Seventy-five percent ethanol was applied in the same way as in non-indomethacin-treated rats. (c) In the indomethacin plus Rioprostil-treated group, rats were pretreated with 5 mg/kg indomethacin subcutaneously, and 20 p,g/kgRioprostil intragastrically 1 hand 30 min, respectively, before preparation, followed by 75% ethanol application to the exposed gastric mucosa. (d) In the non-indomethacin plus Rioprostil-treated group, rats were pretreated with 20 p,g/kg Rioprostil intragastrically 30 min before preparation, then 75% ethanol was applied to the gastric mucosa. (e) In the non-indomethacin-treated control group, vehicle (2% ethanol) was applied topically to the gastric mucosa under in vivo microscopy. (f) In the indomethacin-treated control group, rats were pretreated with 5 mg/kg indomethacin subcutaneously 1 h before preparation, and then 2% ethanol was applied to the exposed gastric mucosa.

Study 2: Gross Lesion Study To determine which aspect of the preparation of the rat for in vivo microscopy (anesthesia, laparotomy, or gastric manipulation) stimulated an endogenous protective mechanism and to confirm the results of the in vivo microscopic study (see study 1), the following study was performed. Conscious rats or rats anesthetized with 50 mg/kg sodium pentobarbital received 1 ml of 75% ethanol or vehicle (2% ethanol) by orogastric intubation after the various combination of procedures as described above. Fifteen minutes after the instillation of 75% ethanol or vehicle, the rats were killed by cervical dislocation and the stomachs were immediately removed. Each stomach was opened along the greater curvature, then pinned open to expose the mucosa and photographed using a Polaroid pack film camera and Polaroid type 108 film (Polaroid Tech. Photo, Cambridge, Mass.) at 2:1 magnification. The area of all the lesions (long, dark red bands running vertically down the corpus of the stomach) and the total area of the corpus mucosa were measured by tracing on a Hipad digitizer (Houston Instrument, Austin, Tex.) coupled to an Apple lIe computer (Apple Computer Inc., Cupertino, Calif.). The percentage of the corpus mucosa involved with lesions was then determined.

ENDOGENOUS GASTRIC PROTECTION AND LAPAROTOMY 603

Septemb er 1988

Experimental design .

IS ENDOGEl\10US PRO STAGLANDIN RESPO NSIBLE FOR THE

GROSS GASTRIC MUCOS AL IN-

Th e gross lesion study was per formed to confirm whether the endogenous protective mechanism is related to th e endogenous prostaglandin synthesis; Rats were di vided into the following four groups of 6 rat s each; (a) In the anesthetized group (control ), 75% ethanol was given intragastricall y 5 min after anes thesia. (b) In the laparotomized group , anesthetized rats recei ved 75% ethanol intragastrically 15 min after lap arotomy. (c) In the indomethacin plus vehicle-treated and laparotomized group , rats wer e pr etreated with indomethacin and vehicle 1 hand 30 min, respectively, before laparotomy under anesthesia, and then received 75% ethanol intragastrically 15 min after the surgery. (d) In the indomethacin plus Rioprostil-treated and laparotomized group, rats were pretreated with indomethacin and Rioprostil 1 hand 30 min, respectively , before laparotomy under anesthesia, arid then received 75% ethanol intragastrically 15 min after the surge ry . PROTECTIVE MECHANISM?

JURY BY 75% ETHA NOL IN TH E CONSCIOUS , UNOPERATED RAT-

A gross lesion study was performed in conscious , unoperated rat s using experimental groups that mimicked those in study 1. Rats were divided into the following six groups of 6 rats each. (a) In the non-indomethacin-treated group , conscious rats were pretreated with vehicle intragastrically 30 min befor e receiving 1 ml of 75% ethanol by orogastric intubation. (b) In the indomethacin plus vehicle-treated group , conscious rats were pretreated with indomethacin subcutaneously 1 h before, and vehicle intragastrically 30 min before, receiving 75% ethanol intragastrically. (c) In the indomethacin plus Rioprostil-treated group, conscious rats were pretreated with indomethacin subcutaneously 1 h before, and Rioprostil intragastrically 30 min before, receiving 75% ethanol intragastrically. (d) In the non-indomethacin plus Rioprostil-tre ated group , conscious rats were pretr eated with 20 JLg/kg Rioprostil intragastrically 30 min before receiving 75% ethanol intragastrically. (e) In the non-indomethacin-treated contro l group , conscious rats received 1 ml of vehicle (2% ethanol) intragastrically. (f) In th e indomethacin-treated control group , conscious rats were pretreated with 5 mg/kg indomethacin sub cutaneously 1 h before the vehicle wa s in stilled. COMPA RISON WITH THE IN VIVO MICROSCOPIC ST UDY.

DURATIO N OF T HE PROTECTIVE EFFECT OF LAPAROTOM Y.

A tim e-course study was performed to determine th e durati on of the protecti ve effect of laparotomy. The rats recei ved 1 ml of 75% ethanol intragastrically 1 , 5, 15 , 3D, 60, and 120 min after laparotomy. As a control , ethanol wa s given to rats just before laparotomy . EFFECT OF ETHANOL VOLUME ON TH E PROTECTI VE EFFECT

DETERMINATIO N OF THE FACTOR RESPONSIBLE FOR S11M·

To determine if th ere still would be a protecti ve effect of laparotomy if the mucosal area in contact with ethanol wa s increased , th e gross lesion study was performed using th e lar ger volume of ethanol, which distended the stomach . Th e rats recei ved 4 ml of 75% eth an ol intragastricall y 15 min after laparotomy. As a control , 4 ml of ethanol was give n to anesthetized , nonlap arotomized rats . Statistical analysis. Data are expressed as mean values ± SEM. Statistical significance of differences was determined by the Kruskal-Wallis analysis of variance (ANOVA) with contrasts or, where appropriate, the paired t-test or the Fisher exact test. A probability level of < 0.05 was considered significant. The correlation between time to stasis and the IHI was analyzed by Spearman's rank OF LAPAROTO MY.

Rats were divided into the following four groups of 6 rats each. (a) In th e conscious group (control) , each rat recei ved 1 ml of 75% ethanol by orogastric intubat ion . (b) In the anesthetiz ed group , 75% ethanol was given 5 min afte r anesthesia . (c) In th e laparotomized group , an esthetized rats recei ved 75% ethanol intragastricall y 15 min after laparotomy (making a 3-cm midline incision in the abdomen). (d) In the gastrotomized group, anesthetized rats received 75% ethanol intragastrically 15 min after th e gastrotomy in the same wa y as performed for in vivo microscopic observation (making an incision in the forestomach and everting the corpus mucosa through the incision , but then restoring it and sutur ing the forestomach incision). ULATING THE ENDOGENOU S PROTECTIVE MECHA NISM .

Table 1. Time to Stasis of Gastric Mucosal Blood Flow After th e Topi cal Application of 75% or 2% Ethanol to the Exposed Mucosa of the Rat Stomach Group 1: non-indomethacintreated 75% Ethanol Indomethacin PGE1 (Rioprostil) Incidence? (stasis/total J Tim e to stas is (min)

+

Group 2: indomethacin + vehicletreated

Group 3: indometh acin + Riopr ostil treated

Group 4: non- indomethacin + Rioprostiltreated

+ +

+ + +

+

0/8

1 2/15 b

NS

6.4 ± l.4 c

4/15 12;7 ± 1.1

+

Group 5: non-indomethacintreated control

Group 6: indometh acintreated control

+

0/6

0/6

0/6

NS

NS

NS

NS, no stasis occurred in the group . a Incidenc e was tested by Fisher exact test. b Significantly different from all the other groups (p < 0.01). Results for time to stasis for all rats in each group are expressed as mean ± SEM of the average rank calculated as follows. Rats were ranked accord ing to the value of time to stasis, and the average rank of each grou p was analyzed by Kruskal-Wallis analysis of variance with contrasts. When no stasis occurred within 15 min, an arbitrary value of 15 min was assigned. C Signific antly different from all the oth er groups (p < 0.01). There wer e no significant differences among the oth er groups.

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Figure 1. Photographs of the gastric superficial mu-

cosa in an indomethacin plus vehicletreated rat taken from the face of the videomonitor (original magnification x470). A. Before the topical application of 75% ethanol, blood was seen to flow through the capillary network and eventually drained into collecting venules. B. After the 75% ethanol application in the same area as A, the flow gradually slowed and finally reached complete stasis. However, the mucosal microvessels remained filled with blood elements. C, capillary; 1, 2, and 3, first-, second-, and third-order venules; CV, collecting venule. Bar represents 100 usn,

correlation as time to stasis was not a continuous variable. The correlation between the calculated blood flow and IHI was analyzed by simple correlation.

Results Study 1: In Vivo Microscopic Study In vivo microscopic observation-time to stasis of blood flow. Before the topical application of 75 % ethanol, red blood cells could be seen flowing through the anastomosing capillary network surrounding the gastric pits and eventually, via postcapillary venules, draining into the collecting venules (Figure 1A). Under basal conditions, there were no apparent differences in the mucosal blood flow between the indomethacin-treated and nonindomethacin-treated groups (group 2 vs, group 1 in Table 1) nor between the Rioprostil-treated and the vehicle-treated groups (group 3 vs. group 2). In rats not pretreated with indomethacin, the topical appli-

cation of 75% ethanol had no effect on the mucosal microcirculation. In contrast, in indomethacin plus vehicle-treated rats (group 2), the topical application of 75% ethanol to the exposed gastric mucosal surface resulted in marked changes in the mucosal microcirculation (Figure 1B). During the first 10-15 s after ethanol application, flow appeared to accelerate transiently. The flow gradually slowed with the appearance of flowing white thrombi; later, white thrombi appeared to stick to the vessel walls and red blood cell rouleaux formation occurred. Finally, all the superficial mucosal microvessels were filled with blood elements but they were no longer flowing. The blood elements filling the microvessels appeared to be mainly red blood cells plus some white thrombi. In 12 of 15 rats thus studied, the flow reached complete stasis (cessation of flow in all vessels in the field observed by in vivo microscopy) in 4.2 ± 0.9 min (range 1.0-10.0 min). In indomethacin plus Rioprostil-treated rats (group 3), stasis

ENDOGENOUS GASTRIC PROTECTION AND LAPAROTOMY

September 1988

605

Table 2. Diameter Change in the Gastric Mucosal Superficial Microvessels After the Topical Application of 75% Ethanol in the Indomethacin-Pretreated Rata Postcapillary venules Before ethanol application (fLm) After ethanol application (/.tm) Percentage change a

First order

Second order

Third order

Capillaries

16.4 ± 1.0 17.2 ± 1.4 6.9 ± 6.4

13.4 ± 0.3 14.6 ± 1.4 9.3 ± 12.5

10.3 ± 0.3 13.2 ± 1.2 b 28.8 ± 8.4

7.7 ± 1.3 12.5 ± 1.6 78.0 ± 37.7

Results for diameter changes are expressed as mean ± SEM. b Significantly different from before ethanol application, p < 0.05 by paired t-test.

Red blood cell velocity measurement. The red blood cell velocities were measured to confirm the findings observed by in vivo microscopy and also to compare our data with those of others. With the continuous superfusion of Krebs' solution for 120 min in non-indomethacin-treated rats, VRBCwas constant throughout the first hour. In the last 10 min of the hour, average VRBC was only 3.4% ± 0.1% (mean ± SEM) below initial VRBc. In the second hour, the VRBC gradually slowed and at the end of 2 h was 40%-60% of the initial VRBC' Because VRBCremained uniform throughout the first hour, all experiments were performed within 1 h after preparation. As previously described by Holm-Rutili and Obrink (14), red blood cell flow was pulsatile with regular oscillations of 3-6 cycles/min, but there was no cessation of flow even in the slow phase.

occurred in only 4 of 15 (mean time to stasis in those 4 rats was 6.6 ± 3.2 min with a range of 1.8 to 12 min). Results of the time-to-stasis study are presented in Table 1. Time to stasis was significantly shorter in the indomethacin plus vehicle-treated group than in the non-indomethacin-treated group (p < 0.01, Kruskal-Wallis ANOVA with contrasts) and the indomethacin plus Rioprostil-treated group (p < 0.01). When stasis occurred the superficial mucosal microvessels appeared to be enlarged (Figure lB), but vessel diameter measurements revealed a statistically significant increase in diameter only in thirdorder venules. The changes in vessel diameter before and after treatment with ethanol in the indomethacin plus vehicle-treated group (group 2) are shown in Table 2.

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Figure 2. Red blood cell velocity, vessel diameter, and blood flow in first-order postcapillary venules in the rat gastric mucosa. Results are expressed as mean ± SEM. *, * *, significantly different from indomethacin plus vehicle-treated group (p < 0.05, P < 0.01, respectively, KruskalWallis ANOVA with contrasts). t, significantly different from before ethanol treatment (p < 0.01, paired t-test).

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GASTROENTEROLOGY Vol. 95 , No.3

YONEI AND GUTH

Table 3. Histologic Damage in the Gastric Mucosa After the Topical Application of 75% Ethanol in the Rat

Total tissue length (mm) Grade 0 damage Grade 1 damage Grade 2 damage Grade 3 damage Grade 4 damage IHI

(0/0)

(%) (%) (0/0) (%)

Group 1: non- indomethacintreated (n = 6)

Group 2: indomethacin + vehicletreated (n = 12)

Group 3: indomethacin + Rioprostiltreated (n = 13)

Group 4: non-indomethacin + Rioprostiltreated (n = 6)

Group 5: non-indometha cintreated control (n = 6)

Group 6: indomethacintreated control (n = 6)

4.3 ± 0.4

3.9 ± 0.3

3.9 ± 0.3

4.1 ± 0.1

4.1 ± 0.2

3.9 ± 0.1

20.1 ± 4.8 66.2 ± 4.2 13.7 ± 4.0 0° 0° 0.9 ± o.i->

55.8 ± 11.9 44.3 ± 11.9 0 0° 0° 0.4 ± 0.1°

46.6 ± 6.2 52.2 ± 5.8 1.2 ± 0.8 0° 0° 0.6 ± 0.1°

0.7 ± 0.7 92 .2 ± 4.1 7.2 ± 4.2 0° 0° 1.1 ± O.l°'c

32.7 19.3 15.2 34.3 2,5

0 ± 8.0 ± 3.9 ± 3.0 ± 7.7 ± 0.3

3.7 63.8 16.8 7.5 7.2 1.5

± ± ± ± ± ±

2.5 9.2 4.9

3.3 b 3.6 b

0.2°'c

Results are expressed as mean ± SEM. Index of histologic injury (IHI) is calculated by multiplying the percent length of each grade of damage by 1, 2, 3, and 4; respectivel y. u.b Sign ificantly different from indomethacin + vehicle-treated group, p < 0.01 , P < 0.05, respectively. G Significantly different from both non-indomethacin-treated control group and indomethacin-treated control group, p < 0.01 by Kruskal-Wallis analysis of variance with contrasts.

The re d blood cell velo cities me asured in the six experimental groups and the blood flow calculated based on VRBG and diameter of the first-order postcapillary venules are presented in Figure 2. The VRBC was considered to be a mmls when flow reached complete stasis or was <0.1 mm/s, as it then was out of the measurable range of the VRBG tracking correlator. Initial VRBG an d blood flow was not sta tistically different among the six groups. In nonindomethacin-treated rats (group 1), there was a 5%-21% increase in VRBG above the initial VRBG during the first minute after 75% ethanol application, then VRBC returned to the former level. There

Figure 3. Photomicrograph of a hematoxylin and eosin-stained section from a stomach exposed to 75% ethanol (indomethacin plus vehicle-treated rat) . There is severe histologic injury accompanied by nec rosis . Bar represents 100 ""rn.

was no significant change in average VRBG an d blood flow during the 5-min period after the 75% ethanol application. On the other hand, in the indomethacin plus vehicle-treated group (group 2), there was a marked 89% ± 12% (p < 0.01, Kruskal-Wallis ANOVA with contrasts) decrease in blood flow after ethanol application. Rioprostil pretreatment significantly prevented this reduction of blood flow, blood flow decreasing only 27% ± 21% (p < 0.05). Histologic study. Without indomethacin administration (group 1), 75% ethanol caused only superficial mucosal damage--surlace mucous cell (grade 1) and mucous neck cell (grade 2) damage

ENDOGENOUS GASTRIC PROTECTION AND LAPAROTOMY

September 1988

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to 7.2% ± 3.6% in the Rioprostil-treated group (p < 0.05, Kruskal-Wallis ANaVA with contrasts) as indicated in Table 3.

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In the indomethacin plus vehicle-treated rats, the IHI was significantly higher, 2 .5 ± 0.3 , than in both the non-indomethacin-treated rats [1.1 ± 0.1 (p < 0 .01)] and the indomethacin plus Rioprostil-treated rats [1.5 ± 0.2 (p < 0.01)). The close association between blood flow stasis and mucosal damage is shown in Figure 4. Each dot represents 1 rat. When stasis occurred early after the application of ethanol, the IHI was very high. On the other hand, when blood flow was maintained, the IHI was low. There was a significant correlation between the time to stasis after ethanol application and the IHI, the correlation coefficient being -0.78 (p < 0.01 , Spearman's rank correlation) . It is of interest that in all but 2 of the rats in which blood flow was maintained, the IHI was < 2 .0 , indicating only superficial (grade 1 or 2) mucosal damage. The correlation between the IHI and blood flow (nanoliters per second) after ethanol application also was good, the correlation coefficient being -0.82 (p < 0.01, simple correlation) .

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(Table 3). By blocking the synthesis of endogenous prostaglandins, the ethanol-induced mucosal injury was enhanced, reaching the parietal cell area and sometimes the chief cell area, accompanied by necrosis in 9 of 12 rats (group 2). Figure 3 shows a typical lesion. The surface epithelial cells are completely damaged and partly sloughed, and neck mucous cells have lost their cell outlines. In the parietal cell area, the normal architecture has been lost with a large number of parietal cells and other glandular cells being separated and no longer forming a continuous column of cells. These lesions correspond to grade 4 damage (necrosis). The extent of the mucosal damage was notably less in the Rioprostil-treated rats (group 3). The superficial microvascular engorgement and intramural hemorrhage were mild or moderate compared with that in the vehicle-treated group (group 2). Although necrotic lesions were noted in 4 of 13 rats, the percentage of length of the mucosa with grade 4 severe damage was significantly reduced from 34.3% ± 7.7 % (mean ± SEM) in the vehicle-treated group

Study 2: Gross Lesion Study Gross gastric mucosal injury by 75% ethanol

in the conscious, unoperated rat-comparison with

the in vivo microscopic study. Results are presented in Figure 5. When 75% ethanol was applied intragastrically to non-indomethacin-treated rats, ethanol caused marked gross gastric lesion formation involving 14.6% ± 3.2% (mean ± SEM) of the corpus mucosa. These lesions were characterized by long, dark red bands lying vertically in the corpus mucosa. In addition, some bleeding from the mucosal surface was noted in 5 of 6 ethanol-treated rats. This lesion was significantly blocked by pretreatment with

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Figure 5. Gross corpus mucosal lesions induced by the intragastric instillation of 1 ml of 75% ethanol. Each group consists of 6 rats . Results are expressed as mean ± SEM. The percentage of corpus mucosa with gross lesion was determined from the area of all the lesions and the total area of corpus mucosa measured with the use of a Hipad digitizer. " , significantly different from both the non-indomethacin-treated group and the indomethacin plus vehicle-treated group (p < 0.05, Kruskal-Wallis ANOVA with contrasts).

608

GASTROENTEROLOGY Vol. 95, No.3

YONEI AND GUTH

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Rioprostil (p < 0.05, Kruskal-Wallis ANOVA with contrasts). Pretreatment with indomethacin did not significantly increase ethanol-induced gastric lesions. Rioprostil also reduced gastric lesions in indomethacin-pretreated conscious rats from 18.7% ± 7.4% to 2.6% ± 0.9% (p < 0.05). In contrast to the in vivo microscopic study findings, the intragastric instillation of 75% ethanol caused gastric lesions in non-indomethacin-treated conscious rats. To determine the factor responsible for preventing 75% ethanol injury in rats undergoing in vivo microscopic study, the following study was performed. Determination of the factor responsible for stimulating the endogenous protective mechanism. As presented in Figure 6, the intragastric instillation of 75% ethanol in conscious rats caused marked lesions involving 31.0% ± 5.0% of the corpus mu-

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Figure 8. Duration of protective effect of laparotomy against 75% ethanol-induced gastric corpus mucosal lesions. Laparotomy under anesthesia was performed at various time intervals (from 1 to 120 min) before giving 1 ml of 75% ethanol intragastrically. Control rats were given ethanol just before laparotomy. Animals were killed 15 min after ethanol instillation. The percentage of corpus mucosa with gross lesion was measured by a Hipad digitizer, and data were expressed as mean ± SEM. *, significantly different from control, 1 min, and 120 min after laparotomy (p < 0.05, Kruskal-Wallis ANOVA with contrasts).

cosa. Anesthesia did not affect ethanol-induced gastric mucosal injury. On the other hand, lesions were remarkably and significantly less in both laparotomized rats, 4.7% ± 1.1%, and gastrotomized rats, 3.4% ± 1.0% (both p < 0.01), indicating that laparotomy alone has a protective effect against ethanol-induced gastric lesions. The difference in gross lesion areas between the similarly treated control rats in this section (31.0% ± 5.0%) and group 1 in the previous section (14.6% ± 3.2%) is probably due to the fact that the studies of the four groups in this section were performed on one supply of rats, and those of groups 1-6 in the previous section were performed at another date on another supply of rats. The second

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September 1988

group of rats was more sensitive to ethanol injury. Each batch of rats studied, however, had its own control. Is endogenous prostaglandin responsible for the protective mechanism? The protective effect of laparotomy was demonstrated once more by the marked and significant reduction in the ethanolinduced gross lesion area in the laparotomized group as compared with the control group (Figure 7). This protective effect was significantly blocked by pretreatment with indomethacin in a dose known to inhibit endogenous prostaglandin synthesis (15), the gross lesion area increasing to control level. Pretreatment with Rioprostil, however, significantly protected against this injury in the indomethacinpretreated rats, the gross lesion area returning to laparotomized group levels. Duration of the protective effect of laparotomy. The duration of the protective effect of laparotomy against ethanol-induced injury was determined in the time-course study. Results are presented in Figure 8. The endogenous defense mechanism stimulated by laparotomy began 5 min after laparotomy (p < 0.05), the percentage of lesion area of corpus mucosa being 5.0% ± 0.6% compared with control (22.8% ± 4.8%). This effect lasted for at least 1 h (7.5% ± 2.7%, p < 0.05) but <2 h. Effect of ethanol volume on the protective effect of laparotomy. Results are presented in Figure 9. A protective effect of laparotomy was found in stomachs distended with 4 ml of 75% ethanol, the percentage of lesion area in the corpus mucosa being 15.0% ± 4.4% as compared with that in the anesthetized, nonlaparotomized control of 50.5% ± 5.8% (p < 0.01). The percentage of lesion area in the corpus mucosa induced by 4 ml of 75% ethanol was significantly greater than that induced by 1 ml of 75% ethanol as described in study 2 in anesthetized rats (25.3% ± 4.4%, p < 0.05).

Discussion In the in vivo microscopic studies, 75% ethanol had no effect on gastric mucosal blood flow unless the rat was pretreated with indomethacin. The latter effect was inhibited by Rioprostil preadministration. These results suggested that the surgical manipulation stimulated a physiologic, natural defense mechanism mediated by endogenous prostaglandin [adaptive cytoprotection (11)]. In addition, exogenous prostaglandin, in a nonantisecretory, socalled cytoprotective dose, prevented the 75% ethanol-induced gastric mucosal blood flow stasis when endogenous prostaglandin synthesis was inhibited (exogenous cytoprotection). Study 2 determined which aspect of the preparation for in vivo micros-

ENDOGENOUS GASTRIC PROTECTION AND LAPAROTOMY

609

copy stimulated this natural defense mechanism. As was found also by Leung et al. (8) and Morris and Wallace (6), anesthesia alone does not protect the gastric mucosa against ethanol injury. Although it might be anticipated that gastric surgery would stimulate gastric prostaglandin production, it was surprising that laparotomy alone, without handling the stomach at all, stimulated a gastric mucosal protective mechanism probably mediated by endogenous prostaglandin synthesis. The protective effect persisted for between 1 and 2 h in this study. This has profound implications for all gastric physiologic studies performed within 1-2 h after laparotomy. Increased endogenous prostaglandin synthesis may playa role in the processes being studied. There are some reports that indicate that laparotomy may stimulate endogenous prostaglandin synthesis in the kidney. Terragno et al. (16) reported that levels of PGE-like materials in renal venous blood of the anesthetized-laparotomized dog were eightfold greater than in conscious dogs, and indomethacin decreased this blood PGE levels. Scherer et al. (17) have shown that PGE2 and PGF 2<> in urine markedly and significantly increased in the anesthetizedlaparotomized rat, and this increase was abolished by pretreatment with indomethacin. Finally Goto et al. (18) reported that the PGE2 excretion rate in urine significantly increased during human surgery. This increase of renal prostaglandin synthesis induced by laparotomy is believed to playa role in maintaining renal blood flow during stressful conditions such as laparotomy and trauma. Morris and Wallace (6), using a gastric chamber technique in the rat, found that 40% ethanol applied to the exposed gastric mucosa produced focal mucosal hyperemia and exfoliation of the surface epithelium within minutes, but that 40% ethanol did not significantly increase the rate of cell loss. In contrast, in this study the topical application of 75% ethanol had no effect on the gastric mucosal microcirculation in non-indomethacin-treated animals. In the former case, ethanol was applied to the entire gastric mucosa 40-60 min after laparotomy, whereas in the latter case the area of contact with ethanol was much smaller (limited to the 5-mm hole in the brass disk) and ethanol was applied 20-40 min after laparotomy. In addition, in the gross lesion study, only 1 ml of 75% ethanol was instilled intragastrically. Therefore the gross lesion study with a larger volume (4 rnl] of ethanol was performed. This volume distended the stomach and increased the area of corpus mucosa involved with lesion from 25.3% ± 4.4% to 50.5% ± 5.8%. Nevertheless, laparotomy still significantly protected against this injury (corpus lesion area being reduced to 15.0% ± 4.4%). Therefore the findings of Morris and Wallace (6) that 40% ethanol

610

YONEl AND GUTH

produced only "focal " hyperemia and did not significantly alter the cell loss may be due to the protective effect of laparotomy. In their gastric chamber study it was not possible for them to control for the effect of laparotomy. Also in contrast with th e findings of the present study, Pihan et a1. (9) observed that the topical application of 50% ethanol to the exposed gastric mucosa caused mucosal blood flow stasis within a few minutes. Those authors applied ethanol to a larger exposed area than that in the present in vivo microscopic study. Th ey also waited until blood flow slowed after the preparation for in vivo microscopy as de scribed by Holm-Rutili and Obrink (14) . The gastric mucosa may be more susceptible to ethanol injury under th ese conditions of larger area of contact with ethanol and decreased mucosal blood flow. In addition, as the time-course study in the present investigation revealed that the protective effect of laparotomy persisted for 1 h , but not 2 h , they ma y ha ve performed their study when this protective effect had worn off. It is of interest that Robert et a1. (11) demonstrated that the stimulation of an endogenous gastric protective mechanism by mild irritants lasted 60-90 min, similar to that stimul ated by laparotomy in the present study. It is possible th at th e more rapid blood flow in the first hour after laparotomy is du e to this stimulation of en dogenous prostaglandin synthesis. However, VRBC and blood flow was th e same in indomethacin- and non-indomethacin-treated rats before ethanol was adm ini stere d (Figure 2). This suggests that th e decrease in VRBC and blood flow after 2 h described by Holm-Rutili and Obrink (14) may be related to deterioration of th e preparation with time due to exposur e of the mucosa rather than to loss of the incre ased endo genous pr ostaglandin synthesi s. In con trast to th e present study, Holm-Rutili (19) has show n th at a bolus intravenous administration of 3 mg/kg bod y wt of indom ethacin significan tly decreased th e VRBC by 23%. There is no clear-cut explanati on of this discrepancy in th e VRBC response to indomethacin. Perhaps it is related to the differences in the time of th e study and basal blood flow , within 1 h after preparing the animal when blo od flow is hi gh vs. > 2 h after animal preparation when flow is low. In the pr esent investigati on blood flow was studied onl y in th e early period whe n flow was high. In the pr esent study, pr etr eatment wit h a cytoprotective do se of Rioprostil did not au gment the initial blood flow as compar ed with th e vehicl e-pretreat ed rats. Wh at Rioprostil did in th e in domethaci npretreated animal was to significantly decrease the ethanol-induced reduction of blood flow from 89% to 27%. Thus the protective effect of th e PGE 1 ana-

GASTROENTEROLOGY Vol. 95 , No.3

logue was to maintain the mucosal blood flow in the face of an ethanol challenge. Similar findings have been reported with other prostaglandins by Leung et a1. (8) and Pihan et al. (9). The me chanism of the ethanol-induced stasis , which appears to be some form of intravascular coagulation, is unknown. In the hi stologic study, th e prostaglandin analogue did not protect the surface or neck mucous cells against ethanol-induced injury, but did significan tly reduce the length of deep damage (grad e 3 and 4), i.e., it protects cells lying deeper in the gland area against injury. Also, in study 2, Rioprostil pretreatment significantly reduced the ethanol-induced gross lesions. The preventive effect of Rioprostil in these histologic and gross lesion studies is similar to that of 16,16-dimethyl PGE2 against ethanol-induced gastric mucosal lesions in the study of Lacy and Ito (2). There was a close in verse corre lation between time to stasis of gastric mucosal blood flow and the deep damage, the IHI being low when flow was maintained and high when stasis occurred. These studies support th e concept that maintenance of blood flow plays a significant role in the prostaglandin cytoprotective mechanism. Bou-Abboud et al. (10) showed that stasis precedes histologic necrosis. The implication is that an earl y st ep in the pathogenesis of th e ethanol-induced necrotic lesion is cessation of blood flow. Th e parenchymal cells thus are rendered more susceptible to ethanol injury due to (a) failure of blood flow to dilute and carry away back-diffusing alcohol and (b) lack of delivery of ess ential nutrients and oxygen (3), or releas e of a mediator of cell injury from the static blood (10). Pretreatment with exogenous prostaglandin (8,9) or stimulation of endogenous prostaglandin synthesis by laparotomy (this study) does not in crease blood flow but prevents th e ethanol-induced cessation of flow , and thus helps protect th e gastric mucosa against etha nol injury.

References 1. Robert A. Nezamis JE. Lan cast er C, Han ch ar AJ. Cyt op rot ec-

2.

3.

4.

5.

tion by prostaglandins in rat s: pr even ti on of gastric necrosis produced by alcohol, HCI, NaOH, hyperto n ic NaCl, an d therm al in jur y. Gastroent erology 19 79 ;77 :433-43. Lacy ER, lto S. Microscopic analysis of etha nol damage to rat gast ric m uc osa aft er treatment with a prost aglan din . Gastroente ro logy 1982;83:619-25. Guth PH , Paulsen G, Naga ta H. Histol ogic an d mi cro ci rculatory changes in alcoho l-ind uced gas tric lesi on s in th e rat: effec t of prostagland in cy to pro tecti on. Gas t roe n tero logy 1984; 8 7:1083-90 . Tarnawski A, Hollan der D, Stac h ura J, Krau se WI. Gergel y H. Pro st agland in cy to protection of th e gastric mu cosa agai ns t alcoho l in jury: a d yn ami c tim e-rel at ed process. Rol e of th e mu cosal pro liferative zone. Gastro enterology 1985 ;88:334-52. Schmidt KL, Henagan JM, Smith GS, Hilburn PI. Miller TA. Prostaglandin protecti on again st ethanol-induced gastri c in jur y in the rat: a histolo gic an d cytologic st udy . Gastroen terol ogy 1985;88:649-59 .

September 1988

6. Morris GP, Wallace J1. The role of ethanol and of acid in the production of gastric mucosal erosions in the rats. Virchows Arch [B] 1981;38:23-38. 7. Szabo S, Trier JS, Brown A, Schnoor J. Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 1985;88:228--36. 8. Leung F, Robert A, Guth PH. Gastric mucosal blood flow in rats after 16,16-dimethyl PGE2 given at a cytoprotective dose. Gastroenterology 1985;88:1948-53. 9. Pihan G, Majzoubi D, Haudenschild C, Trier JS, Szabo S. Early microcirculatory stasis in acute gastric mucosal injury in the rat and prevention by 16,16-dimethyl prostaglandin E2 or sodium thiosulfate. Gastroenterology 1986;91:1415-26. 10. Bou-Abboud CF, Paulsen G, Guth PH. Blood flow stasis is an important factor in ethanol-induced gastric mucosal necrosis (abstr). Clin Res 1986;34:910. 11. Robert A, Nezamis JE, Lancaster C, Davis JP, Field SO, Hanchar AJ. Mild irritants prevent gastric necrosis through "adaptive cytoprotection" mediated by prostaglandins. Am J Physiol 1983;245:Gl13-21. 12. Shriver DA, Rosenthale ME, Kluender HC, Schut RN, McGuire JL, Hong E. Pharmacology of rioprostil, a new gastric cytoprotective/antisecretory agent. Arzneimittelforschung 1985;35:839-43. 13. Guth PH, Rosenberg A. In vivo microscopy of the gastric microcirculation. Am J Dig Dis 1972;17:391-8. 14. Holm-Rutili L, Obrink KJ. Rat gastric mucosal microcirculation in vivo. Am J Physiol1985;248:G471-6. 15. Ligumsky M, Hansen DG, Kauffman GL Jr. Salicylic acid

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16. 17.

18. 19.

blocks indomethacin- and aspirin-induced cycle-oxygenase inhibition in rat gastric mucosa. Gastroenterology 1982;83: 1043-6. Terragno NA, Terragno DA, McGiff JC. Contribution of prostaglandins to the renal circulation in conscious, anesthetized, and laparotomized dogs. Circ Res 1977;40:590-5. Scherer B, Schnermann 1. Sofroniev M, Weber PC. Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide. Prostaglandins 1978; 15:255-66. Goto F, Kato S, Ishikura H, Tanaka A, Fujita T. Effect of inhibition of prostaglandin synthesis on renal function in laparotomized patients. Anaesthetist 1982;31:387-91. Holm-Rutili L. Effects of prostaglandin E" E2 and 16,16dimethyl-E, on rat gastric microcirculation. Acta Physiol Scand 1986;127:313-21.

Received December 29, 1986. Accepted March 12, 1988. Address requests for reprints to: Paul H. Guth, M.D., Veterans Administration Wadsworth Medical Center, WlllC, Los Angeles, California 90073. This work was supported by National Institutes of Health grant AM25891, Veterans Administration research funds, and a grant from the Ortho Pharmaceutical Corp. The authors thank Dr. H. Wayland for valuable advice, Dr. T. Reedy for statistical assistance, G. Paulsen for histologic assistance, and D. Claus for secretarial assistance.