COMPARATIVE EFFECTS OF ANTROCOLIC TRANSPOSITION AND ANTRECTOMY ON FUNDIC MUCOSA AND ACID SECRETION OF THE RAT

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.3 Printed in U.S.A. COMPARATIVE EFFECTS OF ANTROCOLIC TRAPOSITION AND ANTRECTOMY ON FUNDIC MUCOSA AND ACID SECRETION OF THE RAT T. LEHY, C.R., A. BONNEFOND, M.D., M. DUBRAsQuET, C.R., S. NASCA, M.D., M. LEWIN, C.R., AND S. BONFILS, M.D. Unite de Recherches de Gastroenterologie, IERM, Hopital Bichat, Paris, France Three groups of male Wi star rats were surgically prepared with chronic gastric fistulae in the rumen: 15 had a transposition of the whole antrum of the colon, 11 had an antrectomy, and 11 with intact stomachs served as controls. From the 15th to the 45th day after the operation, acid secretory t~sts were made twice weekly. On the 45th day the fundus was prepared for cellular count estimation. The average acid output was not significantly different in controls and antrectomies (112 ± 18 ~Eq per 2 hr and 96 ± 18 ~Eq per 2 hr, respectively) but significantly increased in antrocolic transpositions (384 ± 24 ~Eq per 2 hr). After a postoperative period of 6 weeks there was no significant difference between cell count data for controls and antrectomies. Only the height of the fundic mucosa in the latter decreased significantly. However, antrocolic transposition caused a hyperplasia of parietal (P < 0.001) and chief cell populations, as well as a significant increase in the height and volume of the fundic mucosa. We conclude that high endogenous gastrin levels produced by antrocolic transposition exert a rapid trophic influence on fundic mucosa. Simple removal of the antrum has no evident antitrophic effect within a similar postoperative period. Aside from gastrin's well known role as a secretory stimulant, attempts to demonstrate its trophic role on human and experimental animal gastric mucosa have been made. Hypersecretion of endogenous gastrin in Zollinger-Ellison syndrome provokes a marked increase in parietal 1-3 and also probably chief cell populations. 3 In Received June 5, Accepted October 16, Address requests for reprints to: Dr. T. Lehy, Unite de Recherches de Gastroenterologie, IERM U.10, Hopital Bichat, 170 Bd. Ney, Paris, Cedex 18, France. This study was supported by the Institut de la Sante et de la Recherche Medicale (lerm). T. Lehy, M. Dubrasquet, and M. Lewin are Charges de Recherches, IERM. The authors gratefully acknowledge the technical assistance of Leone Heudes and Francoise Dufougeray. 421 the dog a perfusion of porcine gastrin leads to a significant increase of the labeling index with 3H-thymidine in the gastric mucosa. 4 In the rat, administration of pentagastrin causes hyperplasia of the parietal population 5 and an increase in ribonucleic acid synthesis 6 and protein synthesis. 7 It is also known that duodenal obstruction leads to a general hyperplasia of the gastric mucosa, moflt likely due to stimulation of the antrai gastrin cells. 8, 9 In addition, the relationship between antral gastrin and the acid and pepsin secretory cell populations has recently been confirmed in the rat by the appearance of hypoplasia after antrectomy.10 The aim of this study was to determine in the rat the action of endogenous gastrin released from the antrum on the growth of gastric tissue and on gastric secretion by comparing the effects of antrectomy and

2 422 LEHY ET AL. Vol. 64, No.3 antrocolic transposition with suitable controls. The antrocolic transposition model, the same as used by Dragstedt et al. II in the dog, gave us an acid hypersecretion by chronic antral stimulation. '2 In addition, we checked whether the two parameters, secretion and trophicity, varied correlatively in the two models. Materials and Methods Experimental groups. The experiment was carried out in 37 male Wistar CF rats of the same age (average weight 306 ± 7 g) divided into three groups. The first group of 15 rats had a transposition of the gastric antrum on the colon (subsequently referred to in the text as ACT) according to the technique we have described elsewhere'2 and were equipped with chronic gastric fistulae in the rumen. In our surgical procedures the gastric antrum was transposed in its totality. The resection included the whole length of the lesser curvature up to the opening of the esophagus, the pylorus, and the first 2 or 3 mm of the duodenum. A ligature of the pyloric artery was made and vascularization of the antrum was assured by the right branch of the gastroepiploic artery. Total antrectomy was performed on the second group of 11 rats, and they were also equipped with chronic gastric fistulae in the rumen. Since the gastric surgical procedures were the same for both groups, the antrectomy group was the surgical control for the ACT's. It should be noted that in both groups gastroduodenal anastomosis was carried out in the same way, the diameter of the mouth of the anastomosis being greater than that of the pylorus. In preliminary studies, histologic examination of the residual stomach proved that ant recto my was complete in both models. The third group (controls) consisted of 11 rats with intact stomachs; 8 were equipped with chronic gastric fistulae in the rumen. Secretory tests. Basal gastric secretion was studied during the period of stable hypersecretion, 12 that is, from the 15th day after intervention to the 45th day. Secretory tests were repeated twice a week. After an 18-hr fasting period with water ad libitum, the rats were placed in Bollman cages for gastric juice collection for 2 hr. Volume (milliliters) was measured, and hydrochloric acid was titrated to ph 3.5 against 0.01 N NaOH with Topfer reagent as indicator. Acid output was expressed as microequivalents per 2 hr. For each rat, the mean value of all its secretory tests was calculated and considered as representative of the basal secretion of the animal. By establishing a ratio between the basal acid secretory test average and the total parietal population of the stomach for each rat we obtained the standard value of secretion for 10 6 parietal cells. Histologic observations. Removal of the stomach was carried out under ether anesthesia on the 45th day immediately after the last secretory test. In the controls, just before removal, antrectomy was performed on stomachs still in situ according to the same criteria for the other two groups. The residual stomach was immediately opened along the greater curvature. The gastric wall, stretehed to eliminate folds, was pinned, mucosal side up, to a paraffin block and fixed by immersion in Halmi's solution for 11" hr. After fixation, the fundus was separated from the rumen on one side and from the duodenum on the other by cutting along the gastroduodenal suture, and then it was photographed. This photographic outline of the stomach was traced on paper and the fundic surface was then measured by weighing. Four mucosal strips were removed from each fundus parallel to the axis of the curvatures from the rumen to the gastroduodenal suture, two from the anterior wall and two from the posterior wall (fig. 1). Strips A and D, the closest to the greater curvature, were called the "external" strips; strips Band C, the closest to the fundo-fundic suture carried out on the lesser curvature at the time of the operation, were called "internal" strips. The 148 specimens obtained were dehydrated, embedded in paraffin at 58 C, and cut at 3 J.L. Those sections perpendicular to the mucosal surface were stained by the Marks and Drysdale method. I. Mucosal height was determined at intervals of 1 mm and cell counts were made at regular intervals proportional to the mucosal height of the specimen under consideration. Ten to 18 counts were thus made per section with a calibrated ocular grid 165 J.L broad (magnification, x 252). The cell count represented the number of chief or parietal cell nuclei seen in a column of mucosa. The height of this column extended from the base of the glandular tubules to the gastric surface. Its width was equal to that of the ocular grid and its thickness equal to that of.the section. Such a column would underlie a unit of surface area of which the dimensions were the thickness of the section and the width of the ocular grid. Statistical analysis. The cell distribution in the rat gastric mucosa is known to be heterogeneous. '4 We confirmed this result in a preliminary study where the cell counts were made keeping in mind the orientation of the sec-

3 March 1973 AN TROCOLIC TRAPOSITION AND ANTRECTOMY 423 tions. This situation complicates the cell count. Nevertheless (a) in our sampling conditions the average cell count established per strip was assumed to be representative of the gastric area concerned; (b) the cell density was symmetrically distributed in respect to the lesser curvature: there was no significant difference between either the A and D strips, or between the Band C strips. Therefore, for each rat mean counts were calculated for external and for internal strips by averaging strips A and D results and strips Band C results, respectively. In order to make the comparisons as precise as possible we kept the internal and external strips separated in the statistical analysis. But of course the gastric cell population was calculated from the average count obtained by averaging the results of all four strips together. The "average cell count per unit area" was corrected for tissue shrinkage factor due to dehydration and embedding process, 15 the true section thickness, 16 and nuclear overestimation of Abercrombie. 17 To estimate the value of the ratio of chief cell to parietal cell populations w~ averaged the individual values of this ratio established for each cell count. Total population for each rat was calculated by referring the average corrected count per unit area to the total surface area of the fundus. The volume of the fundic mucosa was calculated as the product of the surface area by the mean height of the mucosa. The results of the cell count were compared with, and correlated to, the secretion data. Statistical analysis was performed using Stuent's t-tesl for independent populations. Cell counts relative to the different strips from all the rats of the same group were compared by the paired data test. The significant level was fixed by P < Results Fixation of the mucosa in Halmi's fixative caused no significant tissue contraction, probably owing to the rapidity of fixation process and the topographic quality of the fixative. Embedding in paraffin caused a variable linear tissue contraction (mean value from 2 to 5%) for each strip. Correction of the nuclear count reduced the gross cell count, sometimes by as much as 65%. The mean section thickness determined by the Marengo method was 3.55 /J-. The mean nuclear diameter for parietal and chief cells was 7.51 /J- and 6.13 /J-, respectively. Lelle'_f- Curvature Anterior Wall ~...,...~'r:cl""'l:'_ Iterior Wall FIG. 1. Diagram of rat's stomach cut along the greater curvature after stretching and fixation. Four strips were removed. A and D: external strips; Band C: internal strips. The fundus (dots) was separated from rumen and from duodenum along the gastroduodenal suture. Cell counts were made on the side of the strip indicated by the gray parts. Effects of antrectomy and antrocolic transposition on gastric mucosa. (a) For each group, the combined data from the external strips was significantly different from the internal strips (table 1). Chief cells were concentrated towards the greater curvature and parietal cells towards the lesser curvature. The ratio of chief cell to parietal cell populations was smaller in the internal strips than in the external ones. The corrected average count per unit area for the chief and parietal cells was much greater for the ACT's than for the other two groups in both external and internal strips. However the chief cell-parietal cell ratio was lower since the percentage of increase of pepsin cells was less than that of parietal cells. The greatest variation (32%) in the ratio of chief cell to parietal cell populations between the data of internal and external strips occurred in the antrectomies. It should be noted that in the external strips this ratio increased significantly in the following order: ACT < control < antrectomy. In addition, the mucosal height was much greater in internal strips than in external strips in the ACT's and antrectomies (P <0.05). (b) Table 2 gives the significant differences obtained by comparing the different groups with each other for the various parameters studied in table 1. (c) Table 3 summarizes the data obtained in the three groups after a post-

4 424 LEHY ET AL. Vol. 64, No.3 TABLE 1. Cell count data: average corrected count per unit area in the three experimental groups and according to the location of the strips Average corrected ACT'," count per unit area a (N = 15) Chief cells External d ± 2.81}p < Internal' ± 2.05 Parietal cells External ± 0.91}p < Internal ± 1.42 Chief cells/parietal cells External ± 0.07} P < Internal ± 0.04 Controls' Antrectomy' (N = 11) (N = 11) ± 1.91}p ± 2.23 < ± 0.81}p < ± ± 0.04} P ± 0.03 < ± 1.42} P < ± ± 0.96} P < ± ± 0.07} P < ± 0.09 a Unit area ~ !J. 2., ACT, antrocolic transposition. '" Mean value ± 1 SEM. d Combined data of A and D strips ~ external strips. e Combined data of Band C strips ~ internal strips. TABLE 2. Detection of significant differences between the average corrected cell count per unit area in the three groups and according to the strips location Parameters ACTa-controls ACT-antrectomy Controls-antrectomy External Chief cells P< 0.05 P < 0.05 ' Parietal cells P < P < Chief cells/parietal cells P< P < 0.01 P < 0.05 Internal Chief cells , "... P < 0.05 P < 0.01 Parietal cells P < P < Chief cells/parietal cells... P < P < 0.05 r-;s ACT, antrocolic transposition., not significant. operative period of 6 weeks. The fundic surfaces were not significantly different in the three groups; the mucosal height, however, and therefore the mucosal volume, was much greater in the ACT's. Antrectomy and control results were very similar except for the mucosal height which was smaller in the antrectomies, and for the parietal cell density which was greater in the latter. Antrocolic transposition changed most of the parameter values very significantly (P < 0.05 to P < 0.001). The average corrected counts per unit area and the total chief and parietal cell populations were augmented, whereas the ratio of chief cell to parietal cell populations was greatly reduced (P < 0.001). Total parietal cell population increased by about 30% in comparison to controls and by about 36% in comparison to antrectomies. Total chief cell population increased by 18% in comparison to controls and by 26% in comparison to antrectomies. However, in all groups chief cell density per cubic millimeter did not vary significantly. Effects on gastric secretion. There was no great difference in volume and acid output between antrectomies and controls. However, there was a marked difference in these variables between ACT's and the other groups; acid output was 4 times greater than in antrectomies and 5 times greater than in controls (table 4). The mean ratio of acid output to total parietal population was much greater in the ACT's

5 TABLE 3. Comparative effect of antrocolic transposition (ACT) and antrectomy on the gastric mucosa in the rat Parameters ACT" (N ~ 15) Controls" Antrectomy" (N ~ 11) (N ~ 11) ACT-controls ACT antrectomy Surface area of fundus (cm 2) Height of fundic mucosa (mm) Volume of fundic mucosa (mm').. Parietal cells: average corrected count per unit area' Chief cells: average corrected count per unit area'.. Chief cells/parietal cells Total parietal cell population (x 10 6 ).. Total chief cell population ( x 10 6 ). Parietal cell density ( x 10 ' per mm') Chief cell density ( x 10' per mm') ± ± ± ± ± ± ± ± ± ± ± ± ± ± P < P < ± ± P < 0.01 P < ± ± 0.8 P < P < ± ± 1.2 P < 0.05 P < ± ± 0.07 P< P < ± ± 2.3 P < P < ± ± 4.4 IP < 0.10) P < ± ± 5.6 P < (P < 0.20) ± ± a Mean value ± 1 SEM. b, not significant. 'Unit area ~ !J. '. Controls antrectomy P < 0.05 (P < 0.10) P < I... <0 (::l :t> <: ;; a (") g... (") ;; :t> ~! <: :t> ~ :t> <: ;; t>:l (")..., a ~ '<.;:.. tv en

6 426 LEHY ET AL. Vol. 64, No.3 TABLE 4. Secretory data for the three experimental groups Mean values of gastric secretion ACTa' (N ~ 15) Controls' (N ~ 8) Antrectomy' (N ~ 11) Comparison between ACT and controls (or antrectomy), Volume (ml/2 hr)... Acid output (JLEq/2 hr) Acid output/!0 6 parietal cells (JLEq/hr)..... " ± ± ± ± ± 0.2 P<:: ± ± 18 P< ± ± P < a ACT, antrocolic transposition., Mean value ± 1 SEM. C Controls and antrectomy are not statistically different. than in the other groups. Thus, in this group, 10 6 parietal cells secreted 2.65 times more acid than in antrectomies and 2.25 times more acid than in controls. We found no statistically significant correlation between individual total parietal population and individual basal rate of acid secretion in a given experimental group. Discussion Since rat gastric mucosa is very dense in structure, we considered particularly thin sections (3 J-t) necessary for an accurate estimation of nuclear counts. With such thin sections the correction of nuclear overestimation is, of course, much greater (as much as 65% in our data). Strip by strip histologic study of topographic distribution revealed significant cell density variations in the gastric mucosa in the three experimental groups, a fact that underscored the need for a sufficient sampling in this kind of study. In our study, antrectomy seemed to have had no noticeable effect on cell populations after 6 weeks. These results run counter to the data of Martin et al. 10 even though the same resection of antrum was made. Such a discrepancy could be related to the fact that his animals were of a different strain and younger than ours. It is possible that a greater hypoplasia would set in with a much longer postoperative period since we observed, as early as 6 weeks, a significant decrease in mucosal height. In any case, in our surgical procedure we performed antrectomy taking care to maintain entire fundic vagal innervation, 18 for it is well known that vagotomy reduces gastric cell population. 19 The fact that cell count data in controls and antrectomies were identical parallels the fact that there were also no secretory differences in these groups. These results do not agree with those of Svensson 20 who found that antrectomy reduced interdigestive acid secretion from Heidenhain pouch rats by 60%. Svensson explains this reduction by a significant decrease in histamine formation capacity in the pouch mucosa after antrectomy. According to our results it would appear that in a physiologic resting state antral gastrin does not play a chief role. It even seems possible that, in the absence of antral gastrin, acid secretion could still be stimulated, at least within a 6-week postoperative period, by the vagus and/or by a compensatory gastrin release from another source. In support of this hypothesis it was shown than an immunoreactive gastrin component was present in the duodenum and the jejunum in man 21 and in the hog pancreas. 22 In antrocolic transposition our parietal cell data (average cell count and total cell population) are in accord with those obtained by Crean et al.,5 and especially by Stanley et al., 23 by prolonged pentagastrin infusion in the rat. The increase in parietal population in the ACT's followed correlatively the marked increase in their secretory values in comparison to controls and antrectomies. The relative increase of acid output, however, was much greater than the increase in this population: secretion X 4; parietal population x 1.3. Of course, the counting method estimates the real cell population, while gastric secretory level depends on both functioning cell

7 March 1973 ANTROCOLIC TRAPOSITION AND ANTRECTOMY 427 mass and the intensity of the stimulation it undergoes by the action of gastrin hypersecretion. The gastrin hypersecretion was verified by obtaining high blood gastrin levels by radioimmunoassay. 24 In addition, we observed in our ACT's a significant increase (about 18%) in total chief cell population, a phenomenon Crean et al. 5 did not observe after repeated pentagastrin injections. This chief cell growth was related to the increase in the mucosal height since density per cubic millimeter did not change in comparison to controls. The parietal cell growth, on the contrary, appeared to be independent of this parameter. In our ACT experimental model the increase in basal acid secretion was related to the increase in parietal cell population and high gastrinemia. Chief cell population also increased. In addition, in a given experimental group, the plot of parietal cell data for each rat against the acid secretory data leads to no significant correlation. The preceding findings resembled those observed in stomachs from patients wi th the Zollinger-Ellison syndrome previously studied in our laboratory. 3 Unfortunately, at the time of this experiment, pepsin analysis was not made in the rats but has since been undertaken in our laboratory. We therefore could not compare the ratio of chief cell to parietal cell population with the ratio of pepsin output to acid output which in Zollinger-Ellison stomachs are very closely correlated. In conclusion, our ACT model gave an increased basal acid secretion as is found in the Zollinger-Ellison syndrome, and also provoked an increase in cell populations. It is evident than an excess of endogenous gastrin secreted by a stimulated antrum, without any acid regulation, possesses an important trophic role. If we compare these results with those obtained after antrectomy, it appears that hypoplasia in the antrectomized rats does not set in before 6 weeks, whereas in the ACT's hyperplasia is already clearly established by this time. Therefore, excitation of the antrum of stomach produces much more rapid and marked changes than those brought about by its simple removal. REFERENCES 1. Rosenlud ML, Crean GP, Johnson DG: The Zollinger-Ellison syndrome in a lo-year old boy. J Pediatr 75: , Polacek MA, Ellison EH: Parietal cell mass and gastric acid secretion in the Zollinger Ellison syn drome. Surgery 60: , Neuburger Ph, Lewin M, Bonfils S: Parietal and chief cell populations in four cases of the Zollinger-Ellison syndrome. Gastroenterology 63: , Willems G, Vansteenkiste Y, Limbosch JM: Stimulating effect of gastrin on cell proliferation kinetics in canine fundic mucosa. Gastroenterology 62: , Crean GP, Marshall MW, Rumsey RDE: Parietal cell hyperplasia induced by the administra tion of pentagastrin (lci 50, 123) to rats. Gastroenterology 57: , Johnson LR, Chandler AM: Stimulation of RNA synthesis by pentagastrin in gastric and duodenal mucosa (abstr). Gastroenterology 60:680, Johnson LR, Aures D, Hakanson R: Effect of gastrin on the in vivo incorporation of 14 C-Leucine into protein of the digestive tract. Proc Soc Exp Bioi Med 132: , Crean GP, Hogg DF, Rumsey RDE: Hyperplasia of the gastric mucosa produced by duodenal obstruction. Gastroenterology 56: , Kaye MD: The effect of partial pyloric obstruction on gastric secretion and stomach size in the rat. Am J Dig Dis 16: , Martin F, Macleod lb, Sircus W: Effect of antrectomy on the fundic mucosa of the rat. Gastroenterology.59: , Dragstedt LR, Woodward ER, Oberhelman HA, et al: Effect of transplantation of antrum of stomach on gastric secretion in experimental animals. Am J Physiol 165: , Dubrasquet M, Bonnefond A, Nasca S: Modele d'hypersecretion gastrique acide par transposition totale de I' antre gastrique sur Ie colon chez Ie rat. C R Soc Bioi (Paris) 165: , Marks IN, Drysdale KM: A modification of Zimmerman's method for the differential staining of gastric mucosa. Stain Technol 32:48, ; Bralow SP, Komarov SA: Parietal cell mass and distribution in stomachs of Wistar rats. Am J Physiol 203: , Card WI, Marks IH: The relationship between the acid output of the stomach following "maximal" histamine stimulation and the parietal cell mass. Clin Sci 19: , Marengo NP: Paraffin section thickness, a direct method for measurement. Stain Technol 19: 1-10, Abercrombie M: Estimation of nuclear popula-

8 428 LEHY ET AL. Vol. 64, No.3 tion from microtome sections. Anat Rec 94: , Legros G, Griffith CA: The abdominal vagal system in rats. J Surg Res 9: , Crean GP, Gunn AA, Rumsey RDE: The effect of vagotomy on the gastric mucosa of the rat. Scand J Gastroenterol 4: , Svensson SE: Secretory behaviour and histamine formation in the rat Heidenhain pouch following antrectomy. J Physiol 207: , Berson SA, Yalow RA: Nature of immunoreactive gastrin extracted from tissues of gastrointestinal tract. Gastroenterology 60: , Accary JP, Pointner H, Dubrasquet M, et al: Gastrin immunoreactivity of gastrosecretagogues extracted from hog pancreas, Proceedings of the Fourth International Congress on Endocrinology, Washington, DC. Amsterdam, Excerpta Medica International Congress Series no 256, 1973 (in press) 23. Stanley MD, Coalson RE, Grossman MI, et al: Influence of secretin and pentagastrin on acid secretion and parietal cell number in rats. Gastroenterology 63: Accary JP, Dubrasquet M, Bonfils S: Dosage radioimmunologique de la gastrine chez l'homme et chez l'animal. Presse Med 2:48-49, 1973