Reflux Gastritis The Consequences
of Intestinal Juice in the Stomach
Phillp L. Robbins, MD, Minneapolis, Minnesota Thomas A. Broadie, MD, Minneapolis, Minnesota Henry Sosin, MD, PhD, Minneapolis, Minnesota John P. Delaney, MD, PhD, Minneapolis, Minnesota
Various clinical and experimental observations indicate that chronic gastritis can be induced by topical exposure of the stomach mucosa to intestinal contents. We have previously reported that expo-
sure of the isolated antrum to upper jejunal contents results in antral gastritis with a concomitant rise in parietal cell numbers in the corpus [I]. Similar exposure of an isolated tube of corpus results in more severe gastritis but a diminution of parieta1 cell numbers [2]. The current study was undertaken to determine the consequences of longterm topical exposure of the intact canine stomach to duodenal contents. Material and Methods Six adult female mongrel dogs were operatively equipped with gastric cannulas placed in the anterior distal body of the stomach. After a period of recuperation, acid secretory capacity was standardized. After the secretory rates were established, a modification of the Schmilinsky-McCann procedure [3,4] (hereafter referred to as the Schmilinsky operation) was performed on each dog. The pylorus was excised, separating antrum from duodenum, and the distal stump was oversewn. The duodenum was divided again approximately 6 cm proximal to the ligament of Treitz. The distal cut end was then anastomosed to the gastric antrum and the proximal segment was drained end-to-side into the high gastric corpus. Thus, all duodenal contents, including bile and pancreatic juice, passed through the intact stomach. (Figure 1.) Histology. A generous sample of mucosa near the site of the duodenal-corpus anastomosis and a biopsy of antral mucosa were taken at the time of gastric cannula From the Department of Surgery, University of Minnesota Health Sciences Center, Minneapolis, Minnesota. Reprint requests should be addressed to John P. Delaney. MD, Associate Professor of Surgery, Box 99, University of Minnesota Health Sciences Center, 412 SE &ion Street, Minneapolis, Minnesota 55455. Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas, May 20-21, 1975.
Volume 131, January 1976
placement, again during operative conversion to the Schmilinsky preparation, and at intervals from six to twenty-four months thereafter. Tissues were fixed in formalin, embedded in paraffin, and 7 p sections cut at right angles to the surface so that the gastric glands could be seen throughout their full lengths. Sections were stained with hemotoxylin and eosin and with periodic acid Schifi’ reagent. These specimens were examined microscopically for evidence of histologic gastritis in both antral and corpus mucosa and for quantitation of parietal cells in the tissue taken from the corpus. The microscopist evaluating the slides was unaware of their origin. Gastritis was graded on the basis of three histologic characteristics, each subjectively scored on a scale of 0 to 4. The criteria were: (1) inflammation or round cell infiltration of the lamina propria, (2) disorder in the gastric tubules resulting in a “corkscrew” appearance, and (3) hyperplasia of surface mucus cells leading to a “papillary” configuration [5]. Scores for the three criteria were totaled so that each specimen received a grading between 0 and 12. Corpus mucosal sections were assessed for parietal cell numbers, as previously described [6]. Counts were expressed as the mean number of parieta1 cells per swath of mucosa 0.23 mm in width from muscularis mucosa to the surface. Five areas were counted. The mucus cell population in the corpus was expressed as the per cent of full thickness of mucosa occupied by cells positive on periodic acid Schiff staining. Secretory Studies. Resting and maximal Histalog’” secretory capacities were determined in each dog during the period between implantation of the gastric cannula and conversion to the Schmilinsky operation. From ten to twenty secretory determinations were performed on each animal. After overnight fast, a one hour gastric juice collection was made and then 12 mg per kg of body weight of Histalog were injected subcutaneously. Thirty minutes after the drug was given, another one hour collection was initiated. With use of a pH meter, 5 ml specimens of the gastric juice were titrated against 0.1 normal sodium hydroxide to pH 7. Results were expressed as mEq HCl/hour. Two to three weeks after the dog had undergone the Schmilinsky procedure, resting and max-
23
Robbins et al
Figure 1. ModNkatkn dure.
of the Schmilinsky-McCann proce-
imal acid secretory capacities were again measured at four to ten day intervals for up to two and a half years. The data were pooled in two month periods to analyze trends in acid secretory capacity with time. Acid and pepsin secretions in response to insulin hypoglycemia were also determined during both the control and Schmilinsky periods. An insulin dosage (0.5 units per kg of body weight) previously determined to produce hypoglycemia on a regular basis was given intravenously. After a thirty minute wait, one hour collection was initiated. Pepsin was determined by a modification of the Anson-Mirsky method [7]. Serum Gastrin. Gastrin levels were determined on sera collected during fasting and at 30, 60, 120, and 240 minutes after a standard meat meal. The immunoassay
‘*---I--
-
--
.-
Results Histologic Gastritis. In each of the animals, mucosa obtained after the Schmilinsky operation revealed more gastritis than did control biopsies from the same animals. The mean gastritis grade for the corpus was approximately 2.7 at the time of conversion to the Schmilinsky preparation and increased an average value of 6.0 a year or more
Gastritis Grading - Antrum
Gastritis Grading - Corpus .-.
method of McCuigan [8] was used, before and after conversion to the Schmilinsky preparation. In addition, resting and meat-stimulated serum gastrin levels were determined in six normal control dogs. Data from the experimental animals during the control period and from control animals that never underwent operation were pooled and compared with values from the experimental group of six dogs after chronic exposure of the stomach to duodenal contents. Significance of differences of the means of the pooled data were compared by Student’s t test. Tissue Castrin. Antral and corpus mucosa biopsies were obtained for gastrin assay from four of the dogs a year or more after the Schmilinsky operation and from four control animals. The specimens were homogenized and extracted by the method of Creutzfeldt et al [9]. The extracts were then analyzed for gastrin content, results being expressed in picograms per gram wet weight of tissue. G Cells. Antral mucosa was obtained at the time of the Schmilinsky operation and at eight to twenty-four months thereafter. The tissue was fixed in formalin or paraformaldehyde and embedded in paraffin. Sections measuring 4 p were cut at right angles to the surface. After mounting, the sections were cleared through distilled water and incubated with rabbit antigastrin, after which the slides were washed, overlaid with fluoresceintagged antirabbit IgG, and cover slipped. The slides were examined with a fluorescence microscope equipped with a micrometer. Cells having the characteristic morphology of the G cell and exhibiting fluorescence were counted. Numbers were expressed as G cells per cm length of full thickness mucosa. The technic for G cell identification has been described by Polak et al (101.
--
Figure 2. Gaotrnts grades for SIX dogs before and stter the Schmliinsky operatkn. Bars stgntty mean scores for the group.
24
The Amerlcan Journal of Surgery
Reflux Gastritis
Figure 3. Gastritk aritral mucosa fifteen months after dlversion of du@enai contents into the stomach. fhere is Increased infiammatkn and hyperpiasia.(Originai magnifkatioti X 40.)
Figure 4. Gastritk corpus mucosa fi&een nbnths after dlvetiiori of dubdenat contents into the stbmach demonstratih gianduiar disorder and corkscrewing.(O&&ai magnificatkn X 4d.)
later. (Figure 2.) The mean gastritis grade of antral mucosa obtained at the time of Schmilinsky operation was 4.6 compared to a mean score of 7.5 in mucosa obtained eight to twenty-four months after the operation. There was no significant change in the percentage of mucosa occupied by cells positive on periodic acid Schiff test in the corpus. Antral mucosa had cells positive on periodic acid Schiff test throughout its entire thickness. Figures 3 and 4 show examples of the gastritic changes. Parietal Cell Counts. Parietal cell density was increased from control values in four of the six stomachs exposed to intestinal contents. In three instances, mean parietal cell density was greater on the first biopsy after the Schmilinsky operation. In a fourth, the parietal cells initially did not change but then were doubled at eighteen months. The two other dogs showed decreases in parietal ceil density six and twenty-four months later. Both of these animals had relatively high control counts. (Table I.)
Secretory Data. Resting acid secretion did not change after prolonged mucosal exposure to duodenal contents. Three of the six dogs had a sustained rise in maximal acid secretoiy capacity in response to Histalog stimulation after the Schmilinsky operation. One animal had diminished measurable maximal acid secretory production at first, but this initial decrease was latei’ reversed and after twelve months secretory caoacity began to increase again to near control values at two years. Two dogs showed no essential change in maximal acid secretory capacity after operation. (Figure 5.) Insulin Hypoglycemia. In response to hypoglycemia, neither acid production nor pepsin output changed as a consequence of the Schmilinsky operation. Serum and Tissue Gastrin. Resting serum gastrifi levels were significantly enhanced by di+ersion of duodenal contents through the stomach. (Table II and Figure 6.) In addition, the increases in serum gastrin levels after a standard meat meal1 were significantly amplified over control levels at
Volume 131, January 1976
25
Robbins et al
TABLE I
Parietal Cell Counts (parietal cells per
0.23 mm swath of mucosa) Months
Control
22 + 52 2 64 f 26 * 133 f 152*
2
4
12
75% 4 95 ?:7
16
14
72 k 1
2” 3 3 3 3 5
**standard
10
8
6
after Operation 20
24
22
107 * 6 75 f 3 lo?
29 * 4
1
45 f 3
82 f 3 22 i 7 error
18
of the mean
81 * 6
85 t 7
89 + 6 of five
counts.
the 30 and 60 minute collection intervals in dogs that had duodenal juices bathing the stomach. Differences were not significant 120 and 240 minutes after feeding. There was no difference in tissue gastrin levels in either antrum or fundus between control animals and animals with Schmilinsky operations. (Table III.) G Cells. Gastrin cell counts ranged from 113 to 320 cells per cm length of antral mucosa in control animals. In antral biopsies taken at six to twentyfour months after the Schmilinsky operation, G cell counts ranged from 105 to 278 cells per cm swath of mucosa. These counts appear fairly consistent in different sections taken from an individual animal. With only a small number of observations, we tentatively conclude that G cell numbers are unaltered by topical bathing with duodenal juices. Comments
An extensive body of information supports the view that exposure of the gastric mucosa to intesti-
nal contents or its components leads to changes in morphology and function. Stomachs bearing gastric ulcers invariably show antral gastritis and often have corpus gastritis [I l]. A number of studies have demostrated increased duodenogastric reflux in patients with gastric ulcer as compared with controls [12]. Gastritis is often seen to develop after simple gastroenterostomy or after partial gastrectomy with anastomosis to the intestine. This condition has been termed “alkaline stoma1 gastritis” and occasionally may cause distressing symptoms. Ritchie and Delaney [13] have demonstrated severe histologic gastritis and diminished parietal cell numbers in canine stomachs after partial gastrectomy and Billroth II gastrojejunal reconstruction, with lesser changes after gastroduodenostomy. Gastritis of varying severity has been reported after the exposure of gastric mucosa to individual components of intestinal contents. Byers and Jordan [14] could not demonstrate gastritic changes in isolated gastric mucosa explanted into the gallbladder. Menguy and Max [15], Lawson
366
4367
’
1 I
I i 4 0 ‘3
Months
16 20 24 26 28 30
Figure 5. Acid secretory response to maximal Hisfa~g stimulation before and after diver&n of duodenal confents through the stomach.
TheAmerican
Journal of Surgery
Reflux TABLE
II
Serum
Gastrin
Levels Minutes Unstimulated
Control dogs Samples Gastrin (pg/ml) Schmilinsky dogs Samples Gastrin (pg/ml) Student’s t test *t standard error f Not significant.
204
19 * 19*
13 272 * 31 P < 0.05 of the
30
289
after a Meat Meal
60
19 r 31
240
10 432 t 20 P < .005
120
17 f 24
251
10 312 ?- 21 P < .05
240
19 + 28
211
11 306 ?- 14 NSt
15 + 25
6 205 + 24 NS
mean.
[16], and Delaney and co-workers [I,21 each found gastritis in bile-bathed gastric mucosa left in continuity with the intestinal tract. Pure pancreatic juice drained into an isolated gastric corpus tube also causes moderate gastritis. Experimentally, a more full-blown picture of gastritis emerges when the mucosa is exposed to whole intestinal contents. Corpus mucosa exposed to whole intestinal contents develops more marked histologic changes than those seen with either bile or pancreatic juice alone [5]. In isolated corpus mucosa so exposed there is a decrease in parietal cells while parietal cell numbers in the parent stomach remain unchanged [2]. From the physiologic standpoint, topical exposure of the antrum to intestinal contents or its components appears to have significant effects on the gastrin mechanism. Studies in dogs by Nahrwold [I 71 and Bedi et al [18] have shown enhanced acid secretion from Heidenhain pouches when antral pouches were irrigated with bile. Friesen et al [19], studying the canine gastrin mechanism in a variety of preparations, found significant resting hypergastrinemia when isolated antral pouches were anastomosed to the biliary system. Although gastrin levels were comparable to those seen in cases of simple antral exclusion, stoma1 ulceration occurred in 40 per cent of animals with cholecystoantrostomy as compared to 16 per cent of those with antral exclusion. We have shown that exposure of the isolated antrum to jejunal contents increases the parietal cell mass in the parent stomach [I], probably due to enhanced gastrin production. McCann [3] in 1929 reported gastric and anastomotic ulcers in 80 per cent of dogs that had undergone drainage of jejunal contents into the intact stomach. At that time, when understanding of gastric physiology was immature, the development of ulcer was attributed to mechanical effects at the stoma. Wangensteen et al [20] were unable to du-
Volume 131, January 1976
Gastritis
plicate McCann’s finding of anastomotic ulcer in dogs undergoing the Schmilinsky operation but reported adverse experiences in three patients, two of whom had severe complications of ulcer disease after the diversion of intestinal contents into the stomach. They attributed production of ulcers to enhancement of the gastric phase of secretion consequent to continuous stimulation of the antrum -
-
L
Minutes
Following
Meat Meal
Figure 6. Serum gastrfn levels after diversion of duodenal contents through the stomach ( l denotes stgnMcance at p CO.05; l * denotes signiffcance at p < 0.005).
TABLE
I II
Tissue wet
Gastrin
weight
(picograms
per gram
of tissue)
Antru m
Corpus
Control
Schmilinskv
Control
Schmilinsky
28,000 20,000 14,000 13,000
26,000 22,000 18,000 1,700
870 1,300 950 3,000
3,100 1,200 430 1,200
Note: pus and 4 normal
Tissue gastrin determinations on extracts of corantral mucosa from 4 normal animals and from animals undergoing Schmilinsky operation.
27
Robbins et al
by alkaline duodenal contents. Lawson [16] noted gastritic changes in dogs 160 and 200 days after the Schmilinsky operation. He remarked on more pronounced histologic changes seen in the antral mucosa compared with the corpus. Gastritis was also produced by exposure to either bile or pancreatic juice separately but was not as severe as that seen with the Schmilinsky operation. Dragstedt et al [4] repeated McCann’s experiments with several modifications. Specifically, a higher intestinal division was made such that duodenal contents were diverted into the antrum, and gastroduodenostomy was used for reestablishment of intestinal continuity. No ulcers developed in these dogs. With drainage of the duodenum into the esophagus and gastroduodenostomy, two of twelve dogs died with perforated anastomotic ulcers. However, when the experiment was repeated, with diversion of a longer intestinal loop into the esophagus and gastrojejunostomy used for distal drainage, six of seven animals had ulcers in the jejunum just beyond the anastomosis. The stomachs were reported grossly normal in appearance and no gastric ulcers occurred. In the discussion of their findings, Dragstedt et al [4] stated that “microscopic examination revealed gastritis of varying degrees in many of the animals.” In the present experiments, chronic exposure of the canine stomach to intestinal contents routinely resulted in histologic gastritis, in agreement with other findings. With regard to the equivocal changes in parietal cell numbers, several factors may have played a role. Whereas direct exposure of corpus to intestinal content causes a diminution in parietal cell mass, similar exposure of antrum appears to have a trophic effect on the parietal cells. It seems probable that these two opposing forces are operational in the Schmilinsky preparation. We can only speculate whether over longer time periods one effect or the other will prevail. We are currently maintaining a small number of animals with Schmilinsky operations in hope of providing further insight into long-term changes. In the clinical situation of a patient with pyloric reflux of duodenal contents into the stomach, many years may be necessary to produce significant gastritis. Interpretations of acid secretory data is limited in this model since gastric juice obtained after the Schmilinsky operation is buffered to an unknown extent by alkaline duodenal contents, particularly pancreatic bicarbonate emptying into the stomach. Thus, the increase in maximal acid secretory capacity seen in three dogs is definitely significant.
28
The lack of secretory enhancement seen in the three other dogs (all of which had values basically equal to control at one year after the Schmilinsky operation) may derive from such buffering. The failure of the experiment to demonstrate’ any changes in acid or pepsin secretion in response to vagal stimulation is not surprising and suggests that no changes take place in the stomach that make it more or less responsive to the cephalic phase of gastric secretion. The direct topical effects of intestinal juice on the antrum appear to cause modifications in the gastrin system. Significantly greater resting and particularly meat-stimulated serum gastrin levels observed after the Schmilinsky operation corroborate this feature and demonstrate an increased proclivity for production and/or release of the hormone by the antrum chronically bathed with duodenal juices. This amplified gastrin response was not explainable by changes in gastrin cell numbers nor was it reflected in discernible changes in antral mucosal gastrin concentration. Since tissue gastrin levels were measured in only a small number of animals, it is not possible to derive any firm conclusions. Summary The consequences of exposure of the intact stomach to intestinal contents were examined in six dogs. Diversion of duodenal contents through the stomach lead to the following changes: histologic gastritis in both antrum and corpus, increase in resting and postprandial serum gastrin levels, increased parietal cell density in four of six animals, and enhanced maximal acid secretory capacity in three of six animals. No significant changes were seen in insulin-stimulated acid secretion, insulin-stimulated pepsin secretion, antral gastrin levels, or G cell numbers. We conclude that chronic exposure of the intact stomach to duodenal contents results in gastritis and an amplified gastrin response to food. Parietal cell numbers and maximal acid secretory capacity are increased in some animals. References 1. Butler BA. Cheng JWB, Ritchie WP, Delaney JP: Antral gastritis and parietal cell hyperplasia. Fed Proc 29: 255, 1970. 2. Delaney JP, Butler BA, Cheng JWB, Broadie TA. Ritchie WP Jr: Gastritis induced by intestinal juices. Bull Sot ht Chir 31: 176. 1972. 3. McCann JC: Experimental peptic ulcer. Arch Surg 19: 600, 1929.
The American Journal of Surgery
Reflux Gastritis
4. DfagstedtLR. Woodward ER, Seito T. lsaza J, Rodriquez JR, Samiiin R: The question of bile regurgitation as a cause of gastric ulcer. Ann Surg 174: 548, 1971. 5. Delaney JP, Broadii TA, Robbins PL: Pyloric reflux gastritis: the offending agent. Surgety 77: 784, 1975. 6. Ritchii WP Jr, Barzilai A. Delaney JP: Mucosal cellular populations and distribution in the normal canine stomach. Anet f?ec 155: 111,1966. 7. Anson ML. Mksky AE: The estimation of pepsin with hemoglobin. J Gen Physio/ 16: 59, 1932. 8. McGuiin JE: lmmunochemical studies with synthetic human gastrin. Gestroenterobgy 54: 1005. 1968. 9. Cruetzfeldt W, Arnold R, Cruetzfekft C, Feurle G. Ketterer H: Gastrin and G-cells in the antral mucosa of patients with pernicious anaemia. acromegaly and hypefiyroldism and in a Zollinger-Ellison tumor of the pancreas. Eur J C/h Invest 1: 461, 1971. 10. Pobk JM. Coulling I, Doe W, Pearse AGE: The G cells in pernicious anaemia. Gut 12: 3 19. 197 1. 11. DuPlessis DJ: Pathogenesis of gastric ulceration. Lancef 1: 974,1965. 12. Rhodes J, Barnard0 DE, Phillips SF, Rovelstad RA. Hofmann AF: Increased reflux of bile into the stomach in patients with gastric ulcer. Gestroenterobgy 57: 241, 1989. 13. Riichie WP Jr, Delaney JP: Parietal cell changes in the stomach after ulcer operations. J Surg Res 12: 17. 1972. 14. Byers FM, Jordan PH: Effect of bile upon gastric mucosa. Pruc Sot Exp Bill 1 IO: 864, 1962. 15. Menguy R, Max MH: Influence of bile on the canine gastric antral mucosa. Am J Surg 119: 177, 1970. 16. Lawson HH: Effect of duodenal contents on the gastric mucosa under experimental condiiions. Lend 1: 469, 1984. 17. Nahrwold DL: Bile as a gastric secretory stimulant. Surgery 71: 157. 1972. 16. Bedi BS, Debas HT, Glliespie G, Gillespie IE: Effects of bile salts on antral gastrin release. Gestroenterobgy 60: 256, 1971. 19. Friesen SR, Crosby I, Boggan MD. Fiillos E, Lees JM, Cudnik DB. Underwood J, Peckler MS. Craig CC, Friesen RH. Bolinger RE, McGuigan JE: An experimental study of the antral gastrin mechanism. Surgery 75: 517, 1974. 20. Wangensteen OH, Varco RL. Hay L, Walpole SH. Trach B: Gastric acidity before and after operative procedure with special reference to the role of the pylorus and antrum. Ann Surg 112: 626. 1940.
Discussion Larry Carey (Columbus, OH): I am a bit puzzled between the relationship of the findings of Doctor Robbins and his coauthors and the condition that we recognize in man as bile reflux gastritis, which is characterized by decreased acid production almost uniformly, weight loss, and a diffuse involvement of the stomach with gastritis, a diminution in both G cells and parietal cells, if the stomach is biopsied and if there is indeed residual antrum present. Furthermore, in seventy-five patients, at the University of Pittsburgh (twenty-five normal, twenty-five with gastric ulcer, and twenty-five with duodenal ulcer), we found that more than 50 per cent of the patients at the time the nasogastric tube was inserted had bile in their stomach. It did not seem to make any difference with regard to their acid secretory capacity; thus, bile and duodenal contents in the stomach is quite a common phenomenon, whether one has ulcer disease or not..
Volume 131, January 1976
The various operations for duodenal ulcer produced
peristomal gastritis almost uniformly, and there are only a few patients, probably less than 1 per cent, who ever have full-blown reflux gastritis involving the entire gatric pouch. This also seems a little hard to understand and suggests that perhaps it is not the quantity of bile but rather the constituents of the bile that may be important. I would like to ask Doctor Robbins why he believes that gastrin levels were increased without evidence of increased tissue gastrin and without evidence of increased G cells, and did the animals lose weight as is quite typical of the patients with bile reflux gastritis?
Arnold L. Flick (San Diego, CA): There are some differences in man as compared with the preparation the authors describe. Human bile gastritis is almost always associated with a substantial element of gastric stasis and usually with low to no acid secretory potential in the stomach. Bacteriologic studies often show bacteria in such a stomach as compared with the absence of bacteria in the normal acid-secreting stomach. Also, the appearance of the bile-stained gastric content on gastric analysis, if done in the morning in the fasting state, often shows the bile to be both very densely colored green, much more so than in the ordinary gastric analysis, and more viscous. Finally, bile solubility is dependent on pH, both as bile acids and as bile salts. I think all of these factors have to be considered before an animal model, which has both a low pH and a flowing gastric juice, is adopted as the human counterpart. J. 0. O’Leary (Gainesville, FA): We have tried to produce gastric ulcers in the remnants of dog stomachs with the same procedure. We have seen gastritis in the individual but we have seen no gastric ulcers. The entity shows all the things that were demonstrated and it would be expected that it might well lead to the production of gastric ulcers. Philip L. Robbins (closing): In the animals demonstrating increased parietal cell numbers or increased acid production, the effects are postulated to be secondary to antral inflammation and release of gastrin, which we feel may have a trophic effect on parietal cells. The reasons for increased serum gastrin levels without demonstrably significant increases in G cell numbers or tissue gastrin levels are obscure at present, and may have to do with the very small number of animals we sampled. It is entirely possible that the increased serum gastrin levels may have no relation to the size of the G cell pool. We did not see any gastric ulcers in any of the preparations nor did we see any anastomotic ulcers. We are looking for them. We are going to continue to watch a number of these animals and are repeating the same experiment in some animals with Heidenhain pouches to see what the even longer-term effects of intestinal juice will be on the stomach.
29
of Intestinal Juice in the Stomach
Phillp L. Robbins, MD, Minneapolis, Minnesota Thomas A. Broadie, MD, Minneapolis, Minnesota Henry Sosin, MD, PhD, Minneapolis, Minnesota John P. Delaney, MD, PhD, Minneapolis, Minnesota
Various clinical and experimental observations indicate that chronic gastritis can be induced by topical exposure of the stomach mucosa to intestinal contents. We have previously reported that expo-
sure of the isolated antrum to upper jejunal contents results in antral gastritis with a concomitant rise in parietal cell numbers in the corpus [I]. Similar exposure of an isolated tube of corpus results in more severe gastritis but a diminution of parieta1 cell numbers [2]. The current study was undertaken to determine the consequences of longterm topical exposure of the intact canine stomach to duodenal contents. Material and Methods Six adult female mongrel dogs were operatively equipped with gastric cannulas placed in the anterior distal body of the stomach. After a period of recuperation, acid secretory capacity was standardized. After the secretory rates were established, a modification of the Schmilinsky-McCann procedure [3,4] (hereafter referred to as the Schmilinsky operation) was performed on each dog. The pylorus was excised, separating antrum from duodenum, and the distal stump was oversewn. The duodenum was divided again approximately 6 cm proximal to the ligament of Treitz. The distal cut end was then anastomosed to the gastric antrum and the proximal segment was drained end-to-side into the high gastric corpus. Thus, all duodenal contents, including bile and pancreatic juice, passed through the intact stomach. (Figure 1.) Histology. A generous sample of mucosa near the site of the duodenal-corpus anastomosis and a biopsy of antral mucosa were taken at the time of gastric cannula From the Department of Surgery, University of Minnesota Health Sciences Center, Minneapolis, Minnesota. Reprint requests should be addressed to John P. Delaney. MD, Associate Professor of Surgery, Box 99, University of Minnesota Health Sciences Center, 412 SE &ion Street, Minneapolis, Minnesota 55455. Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas, May 20-21, 1975.
Volume 131, January 1976
placement, again during operative conversion to the Schmilinsky preparation, and at intervals from six to twenty-four months thereafter. Tissues were fixed in formalin, embedded in paraffin, and 7 p sections cut at right angles to the surface so that the gastric glands could be seen throughout their full lengths. Sections were stained with hemotoxylin and eosin and with periodic acid Schifi’ reagent. These specimens were examined microscopically for evidence of histologic gastritis in both antral and corpus mucosa and for quantitation of parietal cells in the tissue taken from the corpus. The microscopist evaluating the slides was unaware of their origin. Gastritis was graded on the basis of three histologic characteristics, each subjectively scored on a scale of 0 to 4. The criteria were: (1) inflammation or round cell infiltration of the lamina propria, (2) disorder in the gastric tubules resulting in a “corkscrew” appearance, and (3) hyperplasia of surface mucus cells leading to a “papillary” configuration [5]. Scores for the three criteria were totaled so that each specimen received a grading between 0 and 12. Corpus mucosal sections were assessed for parietal cell numbers, as previously described [6]. Counts were expressed as the mean number of parieta1 cells per swath of mucosa 0.23 mm in width from muscularis mucosa to the surface. Five areas were counted. The mucus cell population in the corpus was expressed as the per cent of full thickness of mucosa occupied by cells positive on periodic acid Schiff staining. Secretory Studies. Resting and maximal Histalog’” secretory capacities were determined in each dog during the period between implantation of the gastric cannula and conversion to the Schmilinsky operation. From ten to twenty secretory determinations were performed on each animal. After overnight fast, a one hour gastric juice collection was made and then 12 mg per kg of body weight of Histalog were injected subcutaneously. Thirty minutes after the drug was given, another one hour collection was initiated. With use of a pH meter, 5 ml specimens of the gastric juice were titrated against 0.1 normal sodium hydroxide to pH 7. Results were expressed as mEq HCl/hour. Two to three weeks after the dog had undergone the Schmilinsky procedure, resting and max-
23
Robbins et al
Figure 1. ModNkatkn dure.
of the Schmilinsky-McCann proce-
imal acid secretory capacities were again measured at four to ten day intervals for up to two and a half years. The data were pooled in two month periods to analyze trends in acid secretory capacity with time. Acid and pepsin secretions in response to insulin hypoglycemia were also determined during both the control and Schmilinsky periods. An insulin dosage (0.5 units per kg of body weight) previously determined to produce hypoglycemia on a regular basis was given intravenously. After a thirty minute wait, one hour collection was initiated. Pepsin was determined by a modification of the Anson-Mirsky method [7]. Serum Gastrin. Gastrin levels were determined on sera collected during fasting and at 30, 60, 120, and 240 minutes after a standard meat meal. The immunoassay
‘*---I--
-
--
.-
Results Histologic Gastritis. In each of the animals, mucosa obtained after the Schmilinsky operation revealed more gastritis than did control biopsies from the same animals. The mean gastritis grade for the corpus was approximately 2.7 at the time of conversion to the Schmilinsky preparation and increased an average value of 6.0 a year or more
Gastritis Grading - Antrum
Gastritis Grading - Corpus .-.
method of McCuigan [8] was used, before and after conversion to the Schmilinsky preparation. In addition, resting and meat-stimulated serum gastrin levels were determined in six normal control dogs. Data from the experimental animals during the control period and from control animals that never underwent operation were pooled and compared with values from the experimental group of six dogs after chronic exposure of the stomach to duodenal contents. Significance of differences of the means of the pooled data were compared by Student’s t test. Tissue Castrin. Antral and corpus mucosa biopsies were obtained for gastrin assay from four of the dogs a year or more after the Schmilinsky operation and from four control animals. The specimens were homogenized and extracted by the method of Creutzfeldt et al [9]. The extracts were then analyzed for gastrin content, results being expressed in picograms per gram wet weight of tissue. G Cells. Antral mucosa was obtained at the time of the Schmilinsky operation and at eight to twenty-four months thereafter. The tissue was fixed in formalin or paraformaldehyde and embedded in paraffin. Sections measuring 4 p were cut at right angles to the surface. After mounting, the sections were cleared through distilled water and incubated with rabbit antigastrin, after which the slides were washed, overlaid with fluoresceintagged antirabbit IgG, and cover slipped. The slides were examined with a fluorescence microscope equipped with a micrometer. Cells having the characteristic morphology of the G cell and exhibiting fluorescence were counted. Numbers were expressed as G cells per cm length of full thickness mucosa. The technic for G cell identification has been described by Polak et al (101.
--
Figure 2. Gaotrnts grades for SIX dogs before and stter the Schmliinsky operatkn. Bars stgntty mean scores for the group.
24
The Amerlcan Journal of Surgery
Reflux Gastritis
Figure 3. Gastritk aritral mucosa fifteen months after dlversion of du@enai contents into the stomach. fhere is Increased infiammatkn and hyperpiasia.(Originai magnifkatioti X 40.)
Figure 4. Gastritk corpus mucosa fi&een nbnths after dlvetiiori of dubdenat contents into the stbmach demonstratih gianduiar disorder and corkscrewing.(O&&ai magnificatkn X 4d.)
later. (Figure 2.) The mean gastritis grade of antral mucosa obtained at the time of Schmilinsky operation was 4.6 compared to a mean score of 7.5 in mucosa obtained eight to twenty-four months after the operation. There was no significant change in the percentage of mucosa occupied by cells positive on periodic acid Schiff test in the corpus. Antral mucosa had cells positive on periodic acid Schiff test throughout its entire thickness. Figures 3 and 4 show examples of the gastritic changes. Parietal Cell Counts. Parietal cell density was increased from control values in four of the six stomachs exposed to intestinal contents. In three instances, mean parietal cell density was greater on the first biopsy after the Schmilinsky operation. In a fourth, the parietal cells initially did not change but then were doubled at eighteen months. The two other dogs showed decreases in parietal ceil density six and twenty-four months later. Both of these animals had relatively high control counts. (Table I.)
Secretory Data. Resting acid secretion did not change after prolonged mucosal exposure to duodenal contents. Three of the six dogs had a sustained rise in maximal acid secretoiy capacity in response to Histalog stimulation after the Schmilinsky operation. One animal had diminished measurable maximal acid secretory production at first, but this initial decrease was latei’ reversed and after twelve months secretory caoacity began to increase again to near control values at two years. Two dogs showed no essential change in maximal acid secretory capacity after operation. (Figure 5.) Insulin Hypoglycemia. In response to hypoglycemia, neither acid production nor pepsin output changed as a consequence of the Schmilinsky operation. Serum and Tissue Gastrin. Resting serum gastrifi levels were significantly enhanced by di+ersion of duodenal contents through the stomach. (Table II and Figure 6.) In addition, the increases in serum gastrin levels after a standard meat meal1 were significantly amplified over control levels at
Volume 131, January 1976
25
Robbins et al
TABLE I
Parietal Cell Counts (parietal cells per
0.23 mm swath of mucosa) Months
Control
22 + 52 2 64 f 26 * 133 f 152*
2
4
12
75% 4 95 ?:7
16
14
72 k 1
2” 3 3 3 3 5
**standard
10
8
6
after Operation 20
24
22
107 * 6 75 f 3 lo?
29 * 4
1
45 f 3
82 f 3 22 i 7 error
18
of the mean
81 * 6
85 t 7
89 + 6 of five
counts.
the 30 and 60 minute collection intervals in dogs that had duodenal juices bathing the stomach. Differences were not significant 120 and 240 minutes after feeding. There was no difference in tissue gastrin levels in either antrum or fundus between control animals and animals with Schmilinsky operations. (Table III.) G Cells. Gastrin cell counts ranged from 113 to 320 cells per cm length of antral mucosa in control animals. In antral biopsies taken at six to twentyfour months after the Schmilinsky operation, G cell counts ranged from 105 to 278 cells per cm swath of mucosa. These counts appear fairly consistent in different sections taken from an individual animal. With only a small number of observations, we tentatively conclude that G cell numbers are unaltered by topical bathing with duodenal juices. Comments
An extensive body of information supports the view that exposure of the gastric mucosa to intesti-
nal contents or its components leads to changes in morphology and function. Stomachs bearing gastric ulcers invariably show antral gastritis and often have corpus gastritis [I l]. A number of studies have demostrated increased duodenogastric reflux in patients with gastric ulcer as compared with controls [12]. Gastritis is often seen to develop after simple gastroenterostomy or after partial gastrectomy with anastomosis to the intestine. This condition has been termed “alkaline stoma1 gastritis” and occasionally may cause distressing symptoms. Ritchie and Delaney [13] have demonstrated severe histologic gastritis and diminished parietal cell numbers in canine stomachs after partial gastrectomy and Billroth II gastrojejunal reconstruction, with lesser changes after gastroduodenostomy. Gastritis of varying severity has been reported after the exposure of gastric mucosa to individual components of intestinal contents. Byers and Jordan [14] could not demonstrate gastritic changes in isolated gastric mucosa explanted into the gallbladder. Menguy and Max [15], Lawson
366
4367
’
1 I
I i 4 0 ‘3
Months
16 20 24 26 28 30
Figure 5. Acid secretory response to maximal Hisfa~g stimulation before and after diver&n of duodenal confents through the stomach.
TheAmerican
Journal of Surgery
Reflux TABLE
II
Serum
Gastrin
Levels Minutes Unstimulated
Control dogs Samples Gastrin (pg/ml) Schmilinsky dogs Samples Gastrin (pg/ml) Student’s t test *t standard error f Not significant.
204
19 * 19*
13 272 * 31 P < 0.05 of the
30
289
after a Meat Meal
60
19 r 31
240
10 432 t 20 P < .005
120
17 f 24
251
10 312 ?- 21 P < .05
240
19 + 28
211
11 306 ?- 14 NSt
15 + 25
6 205 + 24 NS
mean.
[16], and Delaney and co-workers [I,21 each found gastritis in bile-bathed gastric mucosa left in continuity with the intestinal tract. Pure pancreatic juice drained into an isolated gastric corpus tube also causes moderate gastritis. Experimentally, a more full-blown picture of gastritis emerges when the mucosa is exposed to whole intestinal contents. Corpus mucosa exposed to whole intestinal contents develops more marked histologic changes than those seen with either bile or pancreatic juice alone [5]. In isolated corpus mucosa so exposed there is a decrease in parietal cells while parietal cell numbers in the parent stomach remain unchanged [2]. From the physiologic standpoint, topical exposure of the antrum to intestinal contents or its components appears to have significant effects on the gastrin mechanism. Studies in dogs by Nahrwold [I 71 and Bedi et al [18] have shown enhanced acid secretion from Heidenhain pouches when antral pouches were irrigated with bile. Friesen et al [19], studying the canine gastrin mechanism in a variety of preparations, found significant resting hypergastrinemia when isolated antral pouches were anastomosed to the biliary system. Although gastrin levels were comparable to those seen in cases of simple antral exclusion, stoma1 ulceration occurred in 40 per cent of animals with cholecystoantrostomy as compared to 16 per cent of those with antral exclusion. We have shown that exposure of the isolated antrum to jejunal contents increases the parietal cell mass in the parent stomach [I], probably due to enhanced gastrin production. McCann [3] in 1929 reported gastric and anastomotic ulcers in 80 per cent of dogs that had undergone drainage of jejunal contents into the intact stomach. At that time, when understanding of gastric physiology was immature, the development of ulcer was attributed to mechanical effects at the stoma. Wangensteen et al [20] were unable to du-
Volume 131, January 1976
Gastritis
plicate McCann’s finding of anastomotic ulcer in dogs undergoing the Schmilinsky operation but reported adverse experiences in three patients, two of whom had severe complications of ulcer disease after the diversion of intestinal contents into the stomach. They attributed production of ulcers to enhancement of the gastric phase of secretion consequent to continuous stimulation of the antrum -
-
L
Minutes
Following
Meat Meal
Figure 6. Serum gastrfn levels after diversion of duodenal contents through the stomach ( l denotes stgnMcance at p CO.05; l * denotes signiffcance at p < 0.005).
TABLE
I II
Tissue wet
Gastrin
weight
(picograms
per gram
of tissue)
Antru m
Corpus
Control
Schmilinskv
Control
Schmilinsky
28,000 20,000 14,000 13,000
26,000 22,000 18,000 1,700
870 1,300 950 3,000
3,100 1,200 430 1,200
Note: pus and 4 normal
Tissue gastrin determinations on extracts of corantral mucosa from 4 normal animals and from animals undergoing Schmilinsky operation.
27
Robbins et al
by alkaline duodenal contents. Lawson [16] noted gastritic changes in dogs 160 and 200 days after the Schmilinsky operation. He remarked on more pronounced histologic changes seen in the antral mucosa compared with the corpus. Gastritis was also produced by exposure to either bile or pancreatic juice separately but was not as severe as that seen with the Schmilinsky operation. Dragstedt et al [4] repeated McCann’s experiments with several modifications. Specifically, a higher intestinal division was made such that duodenal contents were diverted into the antrum, and gastroduodenostomy was used for reestablishment of intestinal continuity. No ulcers developed in these dogs. With drainage of the duodenum into the esophagus and gastroduodenostomy, two of twelve dogs died with perforated anastomotic ulcers. However, when the experiment was repeated, with diversion of a longer intestinal loop into the esophagus and gastrojejunostomy used for distal drainage, six of seven animals had ulcers in the jejunum just beyond the anastomosis. The stomachs were reported grossly normal in appearance and no gastric ulcers occurred. In the discussion of their findings, Dragstedt et al [4] stated that “microscopic examination revealed gastritis of varying degrees in many of the animals.” In the present experiments, chronic exposure of the canine stomach to intestinal contents routinely resulted in histologic gastritis, in agreement with other findings. With regard to the equivocal changes in parietal cell numbers, several factors may have played a role. Whereas direct exposure of corpus to intestinal content causes a diminution in parietal cell mass, similar exposure of antrum appears to have a trophic effect on the parietal cells. It seems probable that these two opposing forces are operational in the Schmilinsky preparation. We can only speculate whether over longer time periods one effect or the other will prevail. We are currently maintaining a small number of animals with Schmilinsky operations in hope of providing further insight into long-term changes. In the clinical situation of a patient with pyloric reflux of duodenal contents into the stomach, many years may be necessary to produce significant gastritis. Interpretations of acid secretory data is limited in this model since gastric juice obtained after the Schmilinsky operation is buffered to an unknown extent by alkaline duodenal contents, particularly pancreatic bicarbonate emptying into the stomach. Thus, the increase in maximal acid secretory capacity seen in three dogs is definitely significant.
28
The lack of secretory enhancement seen in the three other dogs (all of which had values basically equal to control at one year after the Schmilinsky operation) may derive from such buffering. The failure of the experiment to demonstrate’ any changes in acid or pepsin secretion in response to vagal stimulation is not surprising and suggests that no changes take place in the stomach that make it more or less responsive to the cephalic phase of gastric secretion. The direct topical effects of intestinal juice on the antrum appear to cause modifications in the gastrin system. Significantly greater resting and particularly meat-stimulated serum gastrin levels observed after the Schmilinsky operation corroborate this feature and demonstrate an increased proclivity for production and/or release of the hormone by the antrum chronically bathed with duodenal juices. This amplified gastrin response was not explainable by changes in gastrin cell numbers nor was it reflected in discernible changes in antral mucosal gastrin concentration. Since tissue gastrin levels were measured in only a small number of animals, it is not possible to derive any firm conclusions. Summary The consequences of exposure of the intact stomach to intestinal contents were examined in six dogs. Diversion of duodenal contents through the stomach lead to the following changes: histologic gastritis in both antrum and corpus, increase in resting and postprandial serum gastrin levels, increased parietal cell density in four of six animals, and enhanced maximal acid secretory capacity in three of six animals. No significant changes were seen in insulin-stimulated acid secretion, insulin-stimulated pepsin secretion, antral gastrin levels, or G cell numbers. We conclude that chronic exposure of the intact stomach to duodenal contents results in gastritis and an amplified gastrin response to food. Parietal cell numbers and maximal acid secretory capacity are increased in some animals. References 1. Butler BA. Cheng JWB, Ritchie WP, Delaney JP: Antral gastritis and parietal cell hyperplasia. Fed Proc 29: 255, 1970. 2. Delaney JP, Butler BA, Cheng JWB, Broadie TA. Ritchie WP Jr: Gastritis induced by intestinal juices. Bull Sot ht Chir 31: 176. 1972. 3. McCann JC: Experimental peptic ulcer. Arch Surg 19: 600, 1929.
The American Journal of Surgery
Reflux Gastritis
4. DfagstedtLR. Woodward ER, Seito T. lsaza J, Rodriquez JR, Samiiin R: The question of bile regurgitation as a cause of gastric ulcer. Ann Surg 174: 548, 1971. 5. Delaney JP, Broadii TA, Robbins PL: Pyloric reflux gastritis: the offending agent. Surgety 77: 784, 1975. 6. Ritchii WP Jr, Barzilai A. Delaney JP: Mucosal cellular populations and distribution in the normal canine stomach. Anet f?ec 155: 111,1966. 7. Anson ML. Mksky AE: The estimation of pepsin with hemoglobin. J Gen Physio/ 16: 59, 1932. 8. McGuiin JE: lmmunochemical studies with synthetic human gastrin. Gestroenterobgy 54: 1005. 1968. 9. Cruetzfeldt W, Arnold R, Cruetzfekft C, Feurle G. Ketterer H: Gastrin and G-cells in the antral mucosa of patients with pernicious anaemia. acromegaly and hypefiyroldism and in a Zollinger-Ellison tumor of the pancreas. Eur J C/h Invest 1: 461, 1971. 10. Pobk JM. Coulling I, Doe W, Pearse AGE: The G cells in pernicious anaemia. Gut 12: 3 19. 197 1. 11. DuPlessis DJ: Pathogenesis of gastric ulceration. Lancef 1: 974,1965. 12. Rhodes J, Barnard0 DE, Phillips SF, Rovelstad RA. Hofmann AF: Increased reflux of bile into the stomach in patients with gastric ulcer. Gestroenterobgy 57: 241, 1989. 13. Riichie WP Jr, Delaney JP: Parietal cell changes in the stomach after ulcer operations. J Surg Res 12: 17. 1972. 14. Byers FM, Jordan PH: Effect of bile upon gastric mucosa. Pruc Sot Exp Bill 1 IO: 864, 1962. 15. Menguy R, Max MH: Influence of bile on the canine gastric antral mucosa. Am J Surg 119: 177, 1970. 16. Lawson HH: Effect of duodenal contents on the gastric mucosa under experimental condiiions. Lend 1: 469, 1984. 17. Nahrwold DL: Bile as a gastric secretory stimulant. Surgery 71: 157. 1972. 16. Bedi BS, Debas HT, Glliespie G, Gillespie IE: Effects of bile salts on antral gastrin release. Gestroenterobgy 60: 256, 1971. 19. Friesen SR, Crosby I, Boggan MD. Fiillos E, Lees JM, Cudnik DB. Underwood J, Peckler MS. Craig CC, Friesen RH. Bolinger RE, McGuigan JE: An experimental study of the antral gastrin mechanism. Surgery 75: 517, 1974. 20. Wangensteen OH, Varco RL. Hay L, Walpole SH. Trach B: Gastric acidity before and after operative procedure with special reference to the role of the pylorus and antrum. Ann Surg 112: 626. 1940.
Discussion Larry Carey (Columbus, OH): I am a bit puzzled between the relationship of the findings of Doctor Robbins and his coauthors and the condition that we recognize in man as bile reflux gastritis, which is characterized by decreased acid production almost uniformly, weight loss, and a diffuse involvement of the stomach with gastritis, a diminution in both G cells and parietal cells, if the stomach is biopsied and if there is indeed residual antrum present. Furthermore, in seventy-five patients, at the University of Pittsburgh (twenty-five normal, twenty-five with gastric ulcer, and twenty-five with duodenal ulcer), we found that more than 50 per cent of the patients at the time the nasogastric tube was inserted had bile in their stomach. It did not seem to make any difference with regard to their acid secretory capacity; thus, bile and duodenal contents in the stomach is quite a common phenomenon, whether one has ulcer disease or not..
Volume 131, January 1976
The various operations for duodenal ulcer produced
peristomal gastritis almost uniformly, and there are only a few patients, probably less than 1 per cent, who ever have full-blown reflux gastritis involving the entire gatric pouch. This also seems a little hard to understand and suggests that perhaps it is not the quantity of bile but rather the constituents of the bile that may be important. I would like to ask Doctor Robbins why he believes that gastrin levels were increased without evidence of increased tissue gastrin and without evidence of increased G cells, and did the animals lose weight as is quite typical of the patients with bile reflux gastritis?
Arnold L. Flick (San Diego, CA): There are some differences in man as compared with the preparation the authors describe. Human bile gastritis is almost always associated with a substantial element of gastric stasis and usually with low to no acid secretory potential in the stomach. Bacteriologic studies often show bacteria in such a stomach as compared with the absence of bacteria in the normal acid-secreting stomach. Also, the appearance of the bile-stained gastric content on gastric analysis, if done in the morning in the fasting state, often shows the bile to be both very densely colored green, much more so than in the ordinary gastric analysis, and more viscous. Finally, bile solubility is dependent on pH, both as bile acids and as bile salts. I think all of these factors have to be considered before an animal model, which has both a low pH and a flowing gastric juice, is adopted as the human counterpart. J. 0. O’Leary (Gainesville, FA): We have tried to produce gastric ulcers in the remnants of dog stomachs with the same procedure. We have seen gastritis in the individual but we have seen no gastric ulcers. The entity shows all the things that were demonstrated and it would be expected that it might well lead to the production of gastric ulcers. Philip L. Robbins (closing): In the animals demonstrating increased parietal cell numbers or increased acid production, the effects are postulated to be secondary to antral inflammation and release of gastrin, which we feel may have a trophic effect on parietal cells. The reasons for increased serum gastrin levels without demonstrably significant increases in G cell numbers or tissue gastrin levels are obscure at present, and may have to do with the very small number of animals we sampled. It is entirely possible that the increased serum gastrin levels may have no relation to the size of the G cell pool. We did not see any gastric ulcers in any of the preparations nor did we see any anastomotic ulcers. We are looking for them. We are going to continue to watch a number of these animals and are repeating the same experiment in some animals with Heidenhain pouches to see what the even longer-term effects of intestinal juice will be on the stomach.
29
