Changes in vascular permeability associated with acetic acid-induced gastrin ulcer in rats.

Eur. surg. Res. 8: 39-50 (1976)

Changes in Vascular Permeability Associated with Acetic Acid-Induced Gastric Ulcer in Rats A nders M0i.ster, K nut Svanes and Stein T0njum Department of General Surgery, University o f Bergen School o f Medicine, Haukeland Hospital, Bergen

Key Words. Experimental gastric ulcer • Inflammation • Capillary permeability • Albumin leakage • Oedema Abstract. Gastric ulcer was induced in rats by the application o f acetic acid to the anterior wall o f the stomach. Leakage o f circulating albumin into the gastric wall was estimated by intravenous injection of radioactive albumin and determina tion of the radioactivity in different samples o f the stomach wall. The protein leakage was found to be markedly increased in the anterior wall o f the stomach, the increase being most pronounced close to the ulcer. The leakage re mained fairly constant during the first 10 h o f the experiment. The protein content o f the posterior wall was about the same as in animals on which a sham operation had been performed. The protein leakage was associated with considerable oedema formation. The protein leakage indicates that inflammatory mediators are released in a wide area around the ulcer. The present experimental model offers an opportunity to find out which mediators are involved in the lesion.

Received: May 12, 1975; accepted: June 16, 1975.

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A gastric ulcer represents a localized tissue defect surrounded by in flamed tissue. An inflammatory process is associated with the local re lease of mediators which cause changes in the local blood flow, increased vascular permeability, oedema formation and emigration of white blood cells [Spector and Willoughby , 1968; W illis, 1969; Di R osa et al., 1971; S vanes and E genberg , 1972]. These mediators (histamine, sero tonin, kinins and prostaglandins) are also known to cause changes in gas tric secretion and to influence the formation and healing of various ex perimental gastric ulcers [Hutson et al., 1969; F uchs et al.. 1972; F er-

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M0lster /Svanes/T0njum

et al., 1973; L i p p m a n n , 1973; W i l s o n , 1973], It therefore appears possible that the inflammatory process might by itself influence the devel opment and maintenance of the ulcer. In an attempt to explore the relationship between the local inflamma tion and the development of a gastric ulcer, it was decided to begin by studying the changes in vascular permeability associated with a gastric ul cer, since these changes make up one of the main characteristics of in flammation. We have chosen the acetic acid ulcer model described by O k a b e and P f e i f f e r [1971] because of its technical simplicity and re producibility, and its tendency to develop into a chronic lesion. guson

Anim als. Male Wistar rats weighing 350-400 g were used. They were fed on pel lets and water ad libitum and were not fasted on the experimental day. Anaesthesia. The animals were given an initial intramuscular injection o f ap proximately 6mg/100g body weight o f pentobarbital (Nembutal-Natrium, Abbot). Minor additional injections were given when needed to keep the animals under light anaesthesia, with respiratory frequencies between 60/min and 80/min. The animals were placed on heating pads, and the rectal temperature was maintained between 37 and 38 ° C during the experiment. Gastric ulcer. Gastric ulcer was induced by the method described by Okabe and Pfeiffer [1971]. The abdomen was shaved with an electric machine clipper, and la parotomy was performed by a midline epigastric incision. The stomach was deliv ered into the wound with the least possible handling, and 100% acetic acid was ap plied to the serosal surface of the anterior gastric fundus by means of a cylindrical metal mould 7 mm in diameter. The acid was removed after 60 sec by gentle suction with cotton wool. The stomach was then slipped back into the abdominal cavity, and the abdominal wall was closed with silk sutures. Sham operation. Laparotomy was performed and the stomach handled in the same way as in the ulcer group. However, no acid was applied to the stomach wall. Tissue samples. The stomach was opened along the major curvature (fig. 1), gen tly rinsed for gastric contents, and mounted on paper with the serosal side down without stretching. Tissue samples including all layers o f the gastric wall were se lected as shown in figure 2. Protein leakage. The accumulation of circulating albumin in the stomach wall was estimated in the following way: iodine-125-labelled human serum albumin with a specific activity o f 50 »Ci/nig albumin was injected into the saphenous vein after isolation of the vein through an incision on the medial aspect of the thigh, the dose being 0.875 t/Ci/100 g rat weight. The animals were killed 20 min later. The different samples o f the stomach were weighed and the radioactivity of the samples was mea sured. The samples were dried at 100 C for 48 h and weighed again. (Preliminary tests showed no further decrease in weight after 2 days.)


Material and Methods

Changes in Vascular Permeability in Rats

41

Fig. 2. Schematic drawing of a rat stomach opened along the greater curvature. Tissue specimens were removed from the anterior (A1-A5) and posterior (Pi-P>) walls of the stomach: A i is the ulcer area (diameter 7 mm), A_> the zone adjacent to the ulcer (diameter 14 mm), As the rest of the fundus, A i the antrum, As the forestomach (rumen). P1-P5 are the corresponding areas o f the posterior wall.


Fig. I. Photograph o f rat stomach opened along the greater curvature. The stomach is viewed from the mucosal side; 2-hour acetic acid lesion is seen on the anterior wall; millimetre scale on left side.

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M 0 L ST h R /SV A N F S/T 0 N J L'M

Stomach perfusion. In a group o f animals the stomach was perfused immediately after the animals were sacrificed to clear the specimens o f intravascular albumins. A cannula was inserted into the aorta after clamping at the diaphragm and proximal to the renal arteries. The stomach was perfused with saline until all visible vessels were cleared of blood, except the ones in the ulcer area, which were thrombosed. The tissue samples o f this group were only weighed after drying, and were not used for calculations of water content. Measurement o f radioactivity. The radioactivity of the tissue samples was mea sured with an automatic gamma spectrometer. The gamma-counting head has a 3X3-inch N a l (TI) crystal, viewed by two high photon efficiency bi-alkali photo multipliers in pulse summation. This apparatus yields good energy resolution in conjunction with a high stability o f counting efficiency for our different sample vol umes. Statistics. The Wilcoxon test for two samples (two-tailed and one-tailed) was used for the statistical analyses.

Results

Protein Leakage The leakage of circulating radioactive albumin into the stomach wall of rats was studied in 6 control animals anaesthetized half an hour prior to the injection of albumin, in 21 animals subjected to sham operation (3 at each time interval), and in 42 animals with an acute gastric ulcer (6 at each time interval). The results are recorded in figure 3. In the unoperated control animals the radioactivity was found to be the same in corresponding areas of the anterior and posterior walls of the stomach. In the sham-operated animals the radioactivity was found to be greater in the anterior than in the corresponding posterior areas of the stomach at all time intervals studied, except in area 3 after 1 h. The differences were found to be statistically significant in area 1 after 0.5, 6 and 10 h; in area 2 after 0.5, 2, 6 and 10 h; in area 3 after 0.5, 2. 4, and 10 h; in area 4 af ter 0.5, 2, 4, 6 and 10 h, and in area 5 after h (p^0.05). No significant difference remained after 24 h. The average radioactivity in the posterior wall of the sham-operated animals was usually found to be slightly greater


Gastric Ulcer After application of acetic acid, a standardized ulcer developed in all animals (fig. 2). Free perforation did not occur in any case. There was thrombosis of the serosal vessels in the ulcer area and a few millimetres outside the ulcer towards the major curvature.


43 Unoperated control

A,P,

»

a

2p 2 «

IT

e

a 5p 5

2

4

6

8

10

24

2

4

6

8

1

0

24

Fig.3. Leakage of circulating albumin into the stomach wall of rats with a gastric ulcer, of sham-operated controls, and unoperatcd controls. For the explanation of A i, P i, Aa...........see figure 2. Each point represents the mean o f 6 observations in the ulcer group, 3 observations in the sham-operated group, and 6 observations in the unoperated group. Vertical lines through each point represent standard deviation. — = Anterior w a ll;----- = posterior wall.


Duration o f experiment, h

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M 0LSTER /SVA N ES/T 0N JU M

than in the posterior wall of the unoperated control animals. However, the differences were statistically significant only in area 1 after 1, 4 and 10 h. in area 3 after 2 h, and in area 4 after 0.5 and I h (p^0.05 to p^Q.025). It is concluded that in the sham-operated animals there is a minimal increase of albumin leakage in the posterior wall and a slight in crease in the anterior wall when compared to the control animals. In animals with gastric ulcer the radioactivity was found to be much higher in the anterior than in the corresponding posterior areas of the stomach wall at all time intervals studied. The difference is statistically significant in all areas except area 4 after 4 h (p^0.025 to p^0.005). The albumin leakage in the anterior wall tended to decrease from 6 to 24 h in areas I and 2, to remain constant over the same intervals in areas


Fig.4. Amount o f radioactive albumin in the stomach wall o f six rats with a 2-hour acetic acid lesion, in which the vessels were flushed with saline immediately after the animals were killed. The curves are drawn through mean values. = Anterior; x = posterior.


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Water Content The water content of tissue samples was studied in the same animals, except for the group with perfused stomachs. The results are recorded in figure 5. In the control group the water content was found to be the same in corresponding areas of the anterior and posterior walls of the stomach. In the sham-operated group the mean water content was found to be greater in the anterior than in the corresponding posterior areas of the stomach at most of the time intervals studied. The differences were found to be statistically significant in area 1 after 0.5, 2, 6 and 10 h; in area 2 after 0.5, 1, 2, 6 and 10 h; in area 3 after 2, 6, and 10 h; in area 4 after 0.5, 1 and 6 h, and in area 5 after 0.5 and 4 h (p^0.05 to p^0.005). There was no significant difference after 24 h. The average water content in the posterior wall of the sham-operated animals was usually found to be slightly greater than in the posterior wall of the unoperated control animals. The difference was significant only af ter 24 h in areas 1, 2, 3 and 5 (p^0.05 to p^0.005). In animals with a gastric ulcer the water content was found to be high er in the anterior than in the corresponding posterior areas of the stomach wall at all time intervals studied. The difference was statistically signifi-


3 and 5 (rumen), but to increase from 6 to 24 h in area 4 (antrum). How ever, these changes were not statistically significant. The radioactivity in the posterior wail of the ulcer stomachs remained remarkably constant during the whole experiment. The mean values were usually slightly higher than the corresponding values in the posterior wall of the control animals. The differences were significant in area 1 after 0.5, 1, 2, 4, 6, 10 and 24 h; in area 2 after 0.5, 1, 2 and 6 h; in area 3 af ter 0.5, 1, 2, 6 and 24 h; in area 4 after 0.5, 1, 2, and 24 h, and in area 5 after 0.5, 1, 2, 4, 6 and 24 h (p^0.05 to p^0.005). The posterior wall of the ulcer stomachs had significantly greater radioactivity than the pos terior wall of the sham-operated stomachs in area 1 after 0.5, 2 and 6 h; in area 2 after 0.5 and 6 h; in area 3 after 0.5 and 6 h; in area 4 after 0.5 and 6 h, and in area 5 after 0.5 and 6 h (p^0.05 to p^0.025). In a group of 6 animals with a 2-hour gastric ulcer, the radioactivity was measured after perfusion of the vessels with saline. The results are recorded in figure 4. The content of radioactive albumin is surprisingly high in the ulcer area (A ,), in the remaining areas the radioactivity is sim ilar to that obtained in the non-perfused specimens.

46

M 0 L ST T R/SV A NES/T0NJ UM Unoperated

control

n

3

p3

W a

4

Duration of experiment,h

cant in most areas (p^0.05 to p^0.005). Exceptions are area 1 after 1, 6, 10 and 24 h; area 4 after 0.5, 6 and 24 h, and area 5 after 0.5, 1 and 4 h. The water content in the posterior wall of the ulcer stomachs was usually slightly higher than the corresponding values in the posterior wall of the control animals. The differences were statistically significant in area 1 after 1, 10 and 24 h; in area 2 after 2 and 24 h, in area 3 after


Fig. 5. Water content in the stomach wall o f rats with a gastric ulcer, in sham-operated animals, and in unoperated controls. For the explanation of A t, Pi, A a , . . see figure 2. Same number of observations as in figure 3. — = Anterior wall; ----- = posterior wall.


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24 h; in area 4 after 1, 2, 6, 10 and 24 h, and in area 5 only after 10 h (p^0.05 to p^0.005). The absolute values of the differences are, how ever, small. The posterior wall of the ulcer stomach had a significantly greater wa ter content than the posterior wall of the sham-operated stomachs in area 1 after 10 h; in area 3 after 10 h; in area 4 after 6 and 10 h, and in area 5 after 10 and 24 h (p^0 .04 to p^0.025). Here also the differences were small in absolute values.

After application of acetic acid to the serosal side of the anterior wall of the stomach, a standardized ulcer developed in all cases. There was no free perforation. Our experience with this kind of ulcer appears to be in good agreement with that of O k a b e and P f e i f f e r [19711. In the present investigation a marked accumulation of radioactive al bumin occurred in the stomach wall surrounding the ulcer. The radioac tivity remained high after perfusion of the gastric blood vessels with sa line. This supports the view that the major portion of the radioactive al bumin in the tissue samples was located in the extravascular compart ment. This is in good agreement with results obtained in inflammatory re actions in other organs [ A s c h e i m . 1964; J o n e s el al., 1970; W i l l ia m s and M o r l e y , 1974], The protein leakage was found to be largest close to the ulcer, and de creased with increasing distance from the ulcer. Increased leakage was found in the whole anterior wall of the stomach. Some of the curves in figure 3 might suggest that the leakage was diphasic. However, the scatter is considerable and on the whole the protein leakage remained fairly con stant in this time interval. The amount of albumin in the posterior wall of the stomach in the ul cer group did not differ much from that of the sham-operated animals. Consequently, the posterior wall may serve as a fair control of the changes taking place in the anterior wall. Similarly, the water content of the posterior wall can be used as a fair control of that of the anterior wall. In the sham-operated animals a moderate protein leakage was found dur ing the first 10 h after the operation. It is interesting to find that gentle handling of the stomach for a couple of minutes is followed by inflamma tory changes that last for many hours.


Discussion

M0lster /Svanes/T0njum

Albumin leakage into the gastric wall does not appear to have been much investigated earlier [Davenport, 1970|. D avenport [ 19661 showed that secretion of albumins into the gastric juice from the normal mucosa of dogs was independent of stimulation with histamine or bethanechol. B rassine [1974] found this also in hu mans, and he also found that gastric or duodenal ulcers in man did not appear to influence the albumin content of the gastric juice. Davenport [1966] found that the damage caused by acids in the gas tric mucosa of dogs increased the albumin leakage into the gastric juice, and that subsequent stimulation with histamine or bethanechol would raise the albumin content in gastric juice. The albumins in the gastric juice are considered to represent only a fraction of the total amount leak ing from the damaged capillaries into the interstitial fluid; the greater part of the albumin here has to be removed by lymphatic drainage [Dav enport, 1966, 1970J. Measured amounts of albumin and water are net values representing the differences between input and output in our tissue samples. The con stant high levels of protein leakage combined with a fairly constant water content observed in the present investigation suggest a simultaneous drainage of protein from the area, probably by the lymphatics, our results here supporting the concept of Davenport. It is not only vascular permeability that is of importance for the rate of extravasation of protein. Also intra- and extravascular hydrostatic pressure, blood flow and area of available endothelial surface will influ ence the protein leakage, and their relative roles cannot be evaluated by this method [ A s c h e i m , 1964]. However, the increase in albumin accumu lation is highly significant in the areas near the lesion, and the controls in dicate that almost all the increase is due to the application of the acetic acid. We have studied the protein leakage in the acute phase of a lesion which, according to O kabe and Pfeiffer [1971] (and to our own pilot studies), will develop into a chronic ulcer. The acute changes must be ex pected to be of significance for the further development of the ulcer. We believe that the experimental model described in this presentation can be used for further studies concerning the inflammatory changes associated with gastric ulcers. The protein leakage is at least partly due to the release of inflammatory mediators. The present experimental model offers an op portunity to find out which mediators are involved in the different phases of the lesion.


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Acknowledgements We would like to thank Inger V ik 0yr and V igdis B erge Helle for technical as sistance. The statistical analyses were performed by statistician S iri N ome E ikhom, The Medical Faculty, University o f Bergen. The figures were produced at the Photo Department, University o f Bergen. This study was supported by grants from the Norwegian Research Council for Science and Humanities, Nasjonalforeningen, Det Norske R&d for Hjerte- og Karsykdommer, and L . Meltzers H0yskolefond.

References Determination o f vascular permeability. Nature, Lond. 201: 1291-1292 (1964). Brassine, A .: Gastric clearance o f serum albumin in normal man and in certain gas troduodenal disorders. G ut 15: 194-199 (1974). Davenport, H . W .: Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50: N o . 4, 487^199 (1966). Davenport, H . W .: Back diffusion o f acid through the gastric mucosa and its phy siological consequences. Prog. Gastroent. «7: 42-56 (1970). D i R osa, M .; G iroud, J . P., and Willoughby, D . A .: Studies o f the mediators of the acute inflammatory response induced in rats in different sites o f carrageenan and turpentine. J . Path. Bact. 104: 15-29 (1971). F erguson, W . W .; E dmond, A . W .; Starling, J . R ., and Wangensteen, S. L.: Pro tective effect of prostaglandin E , (P G E ,) on lysosomal enzyme release in seroton in-induced gastric ulceration. Ann. Surg. 177: N o. 6 (1973). F uchs, K.; B ecker, H . D.; B ôrger, H . W. und E ydt, M .: Untersuchungen zur Entstehung der Stress-Ulcera. Res. exp. Med. 157: 203-205 (1972). Hutson, D . G .; Z eppa, R ., and L evine, V .: Effect of vagotomy on the content of serotonin and histamine in the gastrointestinal tract. Surg. Forum 20: 332-334 (1969). J ones, T .; M orley, J ., and Williams, T. J.: Use o f radioactive isotopes for contin uous recording o f increased vascular permeability in the rabbit ear. J . Physiol., Lond. 207: 10P-11P (1970). L ippmann, W.: Oral antigastric acid secretory activity of synthetic prostaglandin an alogues (9-oxyprotanoic acids). Experientia 9: 990-991 (1973). Okabe, S. and Pfeiffer , C . J.: The acetic acid ulcer model-A. Procedure for chron ic duodenal or gastric ulcer; in Pfeiffer Peptic ulcer, pp. 13-20 (Munksgaard, Copenhagen 1971). S pector, W . G . and Willoughby, D . A .: The pharmacology of inflammation (English Universities Press, London 1968). Svanes, K . and E genberg, K . E .: Diphasic increase o f blood flow in turpentine in duced inflammation in skin and musculature of mice. Eur. surg. Res. 4: 445-457 (1972).


A scheim, E .:

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Williams, T. J . and Morley , J.: Measurement o f rate o f extravasation o f plasma protein in inflammatory responses in guinea-pig skin using a continous recording method. Br. J . exp. Path. 55: 7-12 (1974). Willis , A . L .: Release o f histamine, kinin and prostaglandines during carrageenin-induced inflammation in the rat; in prostaglandines, peptides and amines, pp. 31-38 (Academic Press, London 1969). Wilson, D . E .: Prostaglandines: therapy for peptic ulcer? Ann. intern. Med. 79: 269-270 (1973).

Bergen (Norway)


A nders M0lster , Department o f General Surgery, Haukeland sykehus, N-5000

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