Epithelial proliferation in human fundic mucosa after antrectomy and vagotomy

GASTROENTEROLOGY

79:807-811,

1980

Epithelial Proliferation in Human Fundic Mucosa After Antrectomy and Vagotomy RAMZI

T. ASSAD

and GREGORY

Departments of Medicine, West Roxbury Bent Brigham Hospital, Harvard Medical

L. EASTWOOD

Veterans Administration Hospita1 and Peter School, Boston, Massachusetts

Changes in the gastric mucosa after a partial gastrectomy may predispose to neoplasia, but epithelial proliferation in such patients has not been studied well. Therefore, we obtained one or more suction biopsies of fundic mucosa from 6 volunteers (controls), 5 patients in whom a duodenal ulcer had healed, and 6 patients with antrectomy and vagotomy. Biopsies were organ cultured over tritiated thymidine-containing medium to label proliferating cells, and processed for Iight microscopy and autoradiography. The measurements of proliferation that were used, namely, the total number of cells in the gastric pits, the number of cells in the proliferative zone, the number of labeled cells in the proliferative zone, and proliferation index (number of cells in the proliferative zone/total number of cells in the gastric pit) all indicated that epithelial proliferation is increased signijïcantly in fundic mucosa of patients after antrectomy and vagotomy when compared with fundic mucosa from normal controls or patients with healed duodenal ulcer. Proliferation was increased most in biopsies that showed atrophic gastritis. The enhancement of epithelial proliferation after antrectomy and vagotomy may be due to the development of gastritic changes, but an additional effect of vagotomy cannot be excluded. Further, the expansion of the proliferative zone suggests a disorder of proliferation which may predispose to malignant transformation.

includes chronic gastritis with varying degrees of severity and, in some cases, frank metaplasia. Several studies have suggested that the frequency of carcinoma arising in the gastric remnant many years after surgery for peptic ulcer is higher than in the genera1 population.“-” Because chronic gastritis may predispose to cancer formation,lD.‘l it is reasonable that the development of chronic gastritis in the gastric remnant may account for the observed frequency of cancer. An understanding of epithelial renewal in the residual gastric mucosa after gastric resection may shed more light on the premalignant nature of this condition. Epithelial renewal has been studied in patients with simple atrophic gastritiP3 and atrophic gastritis associated with pernicieus anemia,14 but until recently no comparable studies had been performed in patients after gastric resection.” Hansen et al. reported that epithelial proliferation is increased in gastric mucosa after antrectomy or proximal gastric vagotomy.” Although the measurement of epithelial proliferation which they used, the labeling index, is different from our measurements, their results are similar to our own observations.

The gastric mucosa undergoes marked morphologic changes in most patients after antrectomy and vagotomy.‘-4 The spectrum of mucosal abnormalities

dergone antrectomy and vagotomy for duodenal ulcer disease (ages 45-60 yr, mean age 51 yr) were included in this study. The type of operation and the time between surgery and organ culture study are given in Table 1. Al1 subjects gave written, informed consent. (This study was approved by the Human Studies Committee, West Roxbury VA Hospital, October 1975.) We used the multipurpose suction biopsy tubeX under fluoroscopic control to obtain at least one fundic biopsy from each subject. The biopsies were organ cultured for 3 hr over a medium that contained nine parts Trowel T-8 medium without chloramphenicol (GIBCO Inc., Grand Island, N.Y.), one part fetal calf serum (GIBCO Inc.), crystal-

Methods Six normal 30 yr), 38-59

Received October 15, 1979. Accepted May 15, 1980. Address requests for reprints to: Gregory L. Eastwood, M.D., Gastroenterology Division, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01805. This study was supported by Veterans Administration funds, MRIS #690-2037. The authors thank Emily Fant for her technical assistance these studies. 0 1980 by the American Gastroenterological Association 0016-5085/80/110807-05$02.25

in

male

subjects

(ages 22-38

yr, mean

age

male patients with healed duodenal ulcer (ages yr, mean age 48 yr), and 6 male patients who had un5

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ASSAD AND EASTWOOD

Table

1.

GASTROENTEROLOGY

Type of Surgery and Time Between Surgery and Organ Culture Study in the 6 Operated Patients Billroth Billroth Billroth Billroth Billroth Billroth

11 + 1 + 1 + 1 + 1 + 11 +

vagotomy-4 vagotomy_(j vagotomy-6 vagotomy-7 vagotomy-7 vagotomy-14

mo yr yr yr yr yr

line penicillin G (10,000 U/ml), and streptomycine sulfate (10 mg/ml). To label dividing cells, 10 &i of tritiated thymidine (sp act 6.7 Ci/mM, New England Nuclear Corp., Boston, Mass.) were added to each milliliter of medium. The culture dishes were placed in a McIntosh jar (Torsion Balance Co., Clifton, N.J.), gassed with a mixture of 95% 0, and 5% CO,, and placed in an incubator at 37’C. Details of the organ culture method have been described previous1y.‘8”y After 3 hr, biopsies were removed from the organ culture dishes, tïxed in Bouin’s solution, dehydrated in graded alcohol solutions, and embedded in paraffin. Fourmicron-thick sections were cut for autoradiography and mounted on glass slides. The slides were dipped in Kodak NTB-3 photographic emulsion, diluted 1: 1 with distilled water, and stored in lightproof boxes at 2°C for 2 wk. The slides then were developed with Kodak D 19, fixed, and stained with hematoxylin and eosin. After randomization, each slide was classified histologically as showing either normal histology, superficial gastritis, or atrophic gastritis.” Epithelial proliferation in fundic mucosa occurs in the mucous neck region of the gastric pits as wel1 as in the isthmus between the gastric pits and the gastric glands.” Figure 1 is a diagram of a normal gastric pit cut along its longitudinal axis. For the purpose of our study the proliferative zone was defined as the total number of cells, both

Vol. 79, No. 5, Part 1

labeled and unlabeled, between the highest labeled cel1 and the bottom of the pit. To evaiuate proliferation, we screened 6-10 histologie sections and selected a total of 10 well-oriented gastric pits for counting. We screened only every fourth section on any single slide to eliminate the possibility of storing a given cel1 more than once. In each gastric pit we first counted the total number of cells in the pit, then we counted the number of cells in the proliferative zone, and lastly, we counted the number of labeled cells in the proliferative zone. From these data we derived another measurement of proliferation, the proliferation index, which is the fraction of the gastric pit that is occupied by the proliferative zone (number of cells in the proliferative zone/total number of cells in the gastric pit.) Because the background labeling was minimal, we regarded five or more grains overlying a nucleus as indicative of a labeled cell. Only pits approximately in the lateral third of the biopsy sections were used for proliferation measurements since these were most heavily labeled. However, the most latera1 pit was excluded on both sides because epithelial proliferation appeared stimulated at the cut edges of the biopsies.‘” Data were compared for statistical significante between two given groups by use of the t-test for independent means.

Results Histologie Classijïcation (Ta bJe 2)

of Gastric Biopsies

Eleven biopsies were obtained from 6 normal controls; nine of these biopsies had normal histology and two had superficial gastritis. Eight biopsies were obtained from 5 patients with healed duodenal ulcer; four had normal histology, three had superficial gastritis, and one had atrophic gastritis. Seven biopsies were obtained from 6 patients after antrectomy and vagotomy for duodenal ulcer disease; one had superficial gastritis, and six had atrophic gastritis. Proliferation Groups

in the Patient

Measurements

Al1 three primary proliferation measurements (i.e., the number of cells in the gastric pit, the number Table 2.

Histologie Classification

of Gastric Biopsies Histology

No. of subjects Figure 1. Diagram of a gastric pit to illustrate the methods that were used to measure proliferation. The proliferative zone was defined as the total number of cells, both labeled and unlabeled, between the highest Iabeled cel1 and the bottom of the pit. The measurements of proliferation included the total number of cells in the pit, the number of cells in the proliferative zone, and the number of labeled cells.

Normal controls Healed DU A+V Total

6 5 6

No. of biopsies

N

SG

11

9

2

8 7

4

3 1

6

6

7

26

--13

AG

1

DU = duodenal ulcer; A + V = antrectomy and vagotomy; N = normal: SG = superficial gastritis: AG = atrophic gastritis.

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1980

PROLIFERATION

AFI’ER ANTRECTOMY AND VAGOTOMY

809

of cells in the proliferative zone, and the number of labeled cells in the proliferative zone) in patients after antrectomy and vagotomy were increased significantly over the respective measurements in normal controls as wel1 as in patients with healed duodenal ulcer (Figure 2). Further, the derived measurement of proliferation, the proliferation index, also was increased significantly in patients after antrectomy and vagotomy when compared with controls (Figure 3). Examples of autoradiographs of biopsies from a normal control, from a patient with a healed duodena1 ulcer, and from a patient after antrectomy and vagotomy are shown in Figure 4. Proliferation Groups

Measurements

in the Histologie

Al1 three primary proliferation measurements as wel1 as the proliferation index in atrophic gastritis biopsies were significantly increased over respective measurements in biopsies with normal histology (Figures 3 and 5). In superficial gastritis biopsies, only the total number of cells in the pits and the number of labeled cells were increased significantly over biopsies with normal histology.

Discussion We have shown that epithelial proliferation is increased significantly in cultured fundic biopsies from patients after antrectomy and vagotomy when compared to biopsies from normal controls and patients with healed duodenal ulcer, as indicated by

5o1

*

4540-

35NUNBER 30OF CELLS 252015lO-

*

* eb Y

5-

Figure 2.

Measurements of proliferation in the patient groups. 1 = f standard error of the mean, n = number of biopsies, C = controls (n = 11). DU = healed duodenal ulcer (n = 8),A + V = antrectomy and vagotomy (n = 7). asterisk = significantly increased over C or DU (Number of cells in the gastric pit: A + V vs. C, P c 0.05; A + V vs. DU, P < 0.005. Number of cells in the proliferative zone: A + V vs. C, P < 0.005; A + V vs. DU, P < 0.0005. Number of labeled cells: A + V vs. C, P < 0.0005; A + V vs. DU, P < 0.05).

Figure 3. Fraction of the gastric pit occupied by the proliferative zone (proliferation index) in the patient groups and in the histologie groups. 1 = + standard error of the mean, C = control patients, DU = healed duodenal ulcer patien@ A + V = antrectomy and vagotomy patients, N = normal histology, SG = superficial gastritis, AG = atrophic gastritis, asterisk = significantly increased over C or N (Patient groups: A + V vs. C, P < 0.05. Histologie groups: AG vs. N, P c 0.01).

the measurements of proliferation that we used, namely, the total number of cells in the gastric pits, the number of cells in the proliferative zone, the number of labeled cells in the proliferative zone, and the proliferation index. Proliferation was increased most in biopsies that showed atrophic gastritis. The increase in the proliferation index is of particular interest because that indicates that the proliferative zone has expanded to occupy a disproportionately greater fraction of the gastric pit. Our results confirm previous studies that found that the degree of severity of chronic gastritis correlates with the rate of epithelial proliferation.12.‘3 The expansion of the proliferative zone and the increased number of labeled cells in the proliferative zone in patients after antrectomy and vagotomy have special relevante in view of the number of reported cases of carcinoma appearing in the gastric remnant many years after surgery for benign ulcer disease.5-9 The malignancy often occurs in areas of severe atrophic gastritis with metaplasia,‘~” i.e., the carcinoma originates in the areas with the highest rates of epithelial proliferation. Further, expansion of the proliferative zone has been identified in several other pre- and paraneoplastic lesions of the gastrointestinal tract.21-Z4 Several factors could influence epithelial proliferation in fundic mucosa of patients after antrectomy and vagotomy. First is the loss of the antrum and consequent reduction of available gastrin.25 Because gastrin is known to be trophic to fundic epithelium,26

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GASTROENTEROLOGY

Vol. 79, No. 5, Part 1

Figure 4. Light micrographs from biopsies having (A) normal histology, (B) superficial gastritis, and (C) atrophic gastritis. This magnification is intended to illustrate cel1 labeling. Labeled cells are identified by accumulations of black granules which overlie cel1 nuclei (X 800).

a reduction in gastrin could decrease gastrin-stimulated epithelial proliferation. Second, the lack of vagal stimulation secondary to vagotomy could affect epithelial proliferation. Stimulation of the anterior hypothalamus in cats produced hyperplasia of the mucous neck cells, parietal cells, and chief cells.” This trophic effect was obliterated when the vagus was sectioned. However, in vagotomized rats, the rate of proliferation of progenitor cells in fundic mucosa was increased.‘” Further, Hansen et al. found that proximal gastric vagotomy (parietal cel1 vagotomy) in humans was associated with increased epithelial proliferation, which they felt could have been due to the development of gastritis.‘” In support, we have limited information from cultured fundic biopsies from 2 patients after pyloroplasty and vagotomy, which indicates that epithelial proliferation is increased when compared with biopsies from healed duodenal ulcer patients (unpublished observations). This was true when comparison was made between biopsies with the same degree of gastritis.

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1980

PROLIFERATION

50 45 40 35

NUMBER OFCELLS3o

SG

AC

GASTRIC

PIT

N

N

SG

AG

N

SG

AG

P”OLIFERATIVE ZONE

Figure 5. Measurements of proliferation in the histologie groups. 1 = f standard error of the mean, n = number of biopsies, N = normal (n = 13), SG = superficial gastritis (n = 6), AG = atrophic gastritis (n = 7), asterisk = significantly increased over N (Number of cells in the gastric pit: SG vs. N, P < 0.05; AG vs. N, P < 0.05. Number of cells in the proliferative zone: AG vs. N, P < 0.005. Number of labeled cells: SG vs. N, P < O.ooO5;AG vs. N, P < 0.0005).

Third, and probably most important, is the development of chronic gastritis after antrectomy and vagotomy. Chronic gastritis of itself would be expected to enhance epithelial proliferation in fundic mucosa.“-‘* The precise contributions of these three influences wil1 be difficult to ascertain, but, as we have shown in this study, the development of chronic gastritis after antrectomy and vagotomy is common, and it is associated with increased epithelial proliferation. Moreover, the expansion of the proliferative zone, by analogy with other premalignant lesions of the gastrointestinal tract, suggests a disorder of proliferation that may predispose to malignant transformation.

References 1. Lees F, Grandjean LC: The gastric and jejunal mucosa in healthy patients with partial gastrectomy. Arch Intern Med 101:943-951, 1956 2. Gjeruldsen ST, Myren J, Fretheim B: Alterations of gastric mucosa following a graded partial gastrectomy for duodenal ulcer. Stand J Gastroenterol 3:465-470, 1968 3. Aukee S, Krohn K: Occurrence and progression of gastritis in patients operated on for peptic ulcer. Stand J Gastroenterol 7:541-546, 1972 4. Pulimood BM, Knudsen A, Coghill NF: Gastric mucosa after partial gastrectomy. Gut 17:463-470, 1976 5. Helsingen N, Hillestad L: Cancer development in the gastric stump after partial gastrectomy for ulcer. Ann Surg 143:173179, 1956

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6. Kobayashi S, Prolla JC, Kirsner JB: Late gastric carcinoma developing after surgery for benign conditions. Am J Dig Dis 15:905-912, 1970 7. Pack GT, Banner RL: The late development of gastric cancer after gastroenterostomy and gastrectomy for peptic ulcer and benign pyloric stenosis. Surgery 44:1024-1033, 1958 8. Domellof L, Eriksson S, Janunger K-G: Carcinoma and possible precancerous changes of the gastric stump after Billroth 11 resection. Gastroenterology 73:462-468, 1977 9. Schrumpf E, Stadaas J, Myren J, et al: Mucosal changes in the gastric stump 20-25 years after partial gastrectomy. Lancet 2:467-469, 1977 10. Morson BC: Carcinoma arising from areas of intestinal metaplasia in gastric mucosa. Br J Cancer 9:377-385, 1955 ll. Glass GBJ, Pitchumoni CS: Atrophic gastritis. @man Pathol 6:219-250, 1975 12. Castrup HJ, Fuchs K: Cel1 renewal in inflammatory changes of the gastric mucosa. Dtsch Med Wochenschr 99:892-895, 1974 13. Hansen OH, Johansen A, Larsen JK, et al: Relationship between gastric acid secretion, histopathology, and cel1 proliferation kinetics in human gastric mucosa. Gastroenterology 73:453-456, 1977 14. Bel1 B, Almy TP, Lipkin M: Cel1 proliferation kinetics in the gastrointestinal tract of man. 111.Cel1 renewal in esophagus, stomach and jejunum of a patient with treated pernicieus anemia. J Nat1 Cancer Inst 36~615-628, 1967 15. Assad RT, Eastwood GL: Epithelial proliferation in human fundic mucosa after antrectomy and vagotomy (abstr). Gastroenterology 72:1025, 1977 16. Hansen OH, Larsen JK, Svendsen LB: Changes in gastric mucosal cel1 proliferation after antrectomy or vagotomy in man. Stand J Gastroenterol 13:947-952, 1978 17. Brandborg LL, Rubin CE, Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach, smal1 bowel, and colon. Gastroenterology 37:1-16, 1959 16. Browning TH, Trier JS: Organ culture of mucosal biopsies of human smal1 intestine. J Clin Invest 48:1423-1432, 1969 19. Eastwood GL, Trier JS: Organ culture of human recta1 mucosa. Gastroenterology 64:375-382,1973 20. Eastwood GL: Gastrointestinal epithelial renewal. Gastroenterology 72:962-975, 1977 21. Lieb LM, Lisco H: In vitro uptake of tritiated thymidine by carcinoma of the human colon. Cancer Res 26~733-740, 1966 22. Bleiberg H, Mainguet P, Galand P, et al: Cel1 renewal in the human rectum. In vitro autoradiographic study on active ulcerative colitis. Gastroenterology 58:85?-855, 1970 23. Eastwood GL, Trier JS: Epithelial cel1 renewal in cultured rectal biopsies in ulcerative colitis. Gastroenterology 64:383-390, 1973 24. Winawer SJ, Lipkin M: Cel1 proliferation kinetics in the gastrointestinal tract of man. IV. Cel1 renewal in the intestinalized gastric mucosa. J Nat1 Cancer Inst 42:9-17, 1969 25. McGuigan JE, Trudeau WL: Serum gastrin levels before and after vagotomy and pyloroplasty or vagotomy and antrectomy. N Eng1 J Med 286~184-188, 1972 26. Johnson LR: The trophic action of gastrointestinal hormones. Gastroenterology 70:278-288. 1976 27. Pearl JM, Ritchie WP, Gilsdorf RB, et al: Hypothalamic stimulation and feline gastric mucosal cellular populations. Factors in the etiology of the stress ulcer. JAMA 195:281-284, 1966 28. Ley R, Willems G, Vansteenkiste Y: Influence of vagotomy on parietal cel1 kinetics in the rat gastric mucosa. Gastroenterology 65:764-772, 1973