Cell Proliferation and Atrophic Gastritis in Explanted Canine Gastric Mucosa Pierre-Paul Casteleyn, M.D., and Gerard Willems, M.D.
1, 2
3,4
SUMMARY-We produced experimental atrophic gastritis in 7 mongrel dogs by explanting a full-thickness wedge of vascularized mucosa from the gastric greater curvature onto the abdominal wall. Autoradiography of mucosal specimens was done after the iv injection of tritiated thymidine. The labeling index, S-phase duration, and cell cycle time were measured in normal and explanted fundic mucosa. The spati'al distribution histograms of the labeled cells showed an enlarged area of proliferation. As in human atrophic gastritis, proliferation was shifted toward the mucosal surface. Significantly higher labeling indices were observed in the atrophic mucosa. Shortening of the S-phase duration and the cell cycle· time was indicated by the curves of the labeled mitoses. Proliferative activity was significantly accelerated in this experimental model of atrophic gastritis.-J Natl Cancer Inst 55: 1383-1387, 1975.
Atrophic gastritis has been experimentally produced by explantation of gastric mucosa onto the ~bdo?li!1aI wall in rats (1 ~ 2) and dogs (3-6), and by the IrradIatIOn of the stomach of guinea pigs (7). A new glandular epithelium develops in the exteriorized canine fundic mucosa with some characteristics reminiscent of human atrophic gastritis. The normal superficial mucous and neck cells are replaced by flattened columnar cells with depleted mucus content. These cells extend to the bottom of the tortuous glands lacking parietal and peptic cells. The histochemical pattern of glycoproteins (8) is modified (9). Diffuse chronic inflammatory infiltrates are frequent. In contrast to human atrophic gastritis, no intestinal metaplasia or apparent reduction in mucosal thickness occurs in the experimental model. Increased cell renewal (10-13) and abnormalities in the spatial distribution of DNA-synthesizing cells in the glands (12) were described in human atrophic gastritis. In contrast to normal fundic mucosa, the proliferative activity of the abnormal gastric glands is not limited to the neck region of the glandular pits but extends to the upper third of the glands (14). Concomitant changes in RNA and protein synthesis are found (14), like those seen in colon epithelial cells during the development of neoplastic lesions (15~ 16). Such gastric-cell maturation disorders suggest that an orderly sequence in the progression of atrophic gastritis to gastric carcinoma could exist (14~ 17~ 18). However, despite extensive epidemiologic (19-22), histologic (17~ 18~ 23), and histochemical (24~ 25) studies, this hypothesis has not yet been firmly established (24-27). In this study, the spatial distribution of DNA-synthesizing cells and the kinetics of cell proliferation were analyzed in the exteriorized fundic mucosa of dogs.
larger fundic explants of approximately 6 X 10 em were prepared. The stomach was closed after the insertion of a stainless-steel gastric cannula. In 2 dogs of group C, a large nylon gastric cannula was placed to allow the biopsy of the gastric wall under direct visualization (8). No gastric explants were prepared in these animals. Four weeks after the operation, the animals were fasted for 36 hours and were given 1 mCi tritiated thymidine (3H-TDR)/kg body weight (sp act 26 ~Ci/mM; Radiochemical Center, Mol, Belgium). The anImals of group A (small explants) were killed with pentobarbital sodium 1 hour after the 3H-TDR injection. Mucosal biopsy specimens were taken from the explant and from the fundic greater curvature. In the animals of group B (large explants), mucosal specimens of the stomach were taken randomly through the cannula. This was repeated every I or 2 hours for 28 hours after injection of 3HTDR. Simultaneous biopsy specimens were taken from the explanted mucosa in such a way as to avoi? the proximity of previous biopsy sites. In the speCImens taken randomly from the stomach, those showing esophageal tissue, antral mucosa, intermediate zone, or hemorrhagic suffusions from a previous .wounding. were discarded. In the animals of group C, bIOPSY spec~me~s from the fundic mucosa were taken under dIrect vIsualIzation every 1 or 2 hours for 28 hours. Mucosal specimens taken a half hour or an hour after the injection of 3H-TDR were fixed in 2.5% glutara~de hyde, embedded in Epon 812, and cut at 1- to 2- fL thIckness. After removal of the Epon (28), the tissues were stained with periodic acid-Schiff and covered with emulsion (Ilford K 5 in gel form). They were developed after 6 weeks and counterstained with 1'% toluidine blue (pH 9.2). Other mucosal specimens were fixed in Carnoy's solution, embedded in paraffin, and cut at 4-fL thickness. They were stained with periodic acid-Schiff, covered with emulsion, developed after 18 days, and counterstained with hematoxylin. Histograms of the spatial distribution of labeled cells in the glandular tubes were constructed. In each gland, the location o£ the cell adjacent to the surface was termed position 1. The positions of all labeled cells under it were thus recorded. The percentage of labeled cells [labeling index (LI)] was estImated on the Epon sections in the area of maximal proliferation of 20 welloriented glands from specimens taken 1 hour after 3HTDR injection in dogs from groups A, B, and C. We constructed the curve of labeled mitoses (29) by counting the number of labeled mitoses in each specimen from groups Band C.
MATERIALS AND METHODS
Nine adult mongrel dogs, between 9 and 15 kg, were used. In 3 dogs of group A, a sheet of full-thickness stomach wall, approximately 3 X 3 cm, was excised from the fundic greater curvature. The blood supply was left intact as a pedicle from the left gastroepiploic vessels. The explant was then exteriorized through a stab incision in the left flank and sutured to the skin as described by Milton and Finck (3). In 4 dogs of group B,
Received April 24, 1975; accepted July 23, 1975. . Supported in part by grants from the Fonds voor Geneeskundlg Wetenschappelijk Onderzoek and the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. 3 Laboratory of Experimental Surgery, Hopltal UmversltaIT~ SalI~t Pierre, rue Haute 322, Vrije Universiteit Brussel and Umverslte Libre de Bruxelles, 1000 Brussels, Belgium. . 4 We thank Mrs. Y. Vansteenkiste and Mrs. M. de Cock for theIr technical assistance, and Dr. S. Refeto ff, University of Chicago, for reviewing the manuscript. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.6, DECEMBER 1975
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1383
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1384
CASTELEYN AND WILLEMS
RESULTS
All histologic characteristics of this experimental model previously described by others (2-6) were observed in our explants at the time of autoradiographic study (fig. I). Poorly differentiated mucous cells occupied the length of the glandular tubes. No intestinal metaplasia occurred. Only exceptional parietal cells were seen. The cell infiltrate of the lamina propria was obvious but moderate. One hour after isotope injection, 96% of the labeled cells were localized between positions 20 and 40 in the normal fundic mucosa. These limits delineated the area of maximal proliferation in the glands. The corresponding area ranged from position 5 to position 50 in the exteriorized mucosa. Significant numbers of labeled cells and mitotic figures were seen in the upper part of the glands and near the mucosal surface in the explants (text-fig. I). Labeling indices (table I) were higher (P
1, 2
3,4
SUMMARY-We produced experimental atrophic gastritis in 7 mongrel dogs by explanting a full-thickness wedge of vascularized mucosa from the gastric greater curvature onto the abdominal wall. Autoradiography of mucosal specimens was done after the iv injection of tritiated thymidine. The labeling index, S-phase duration, and cell cycle time were measured in normal and explanted fundic mucosa. The spati'al distribution histograms of the labeled cells showed an enlarged area of proliferation. As in human atrophic gastritis, proliferation was shifted toward the mucosal surface. Significantly higher labeling indices were observed in the atrophic mucosa. Shortening of the S-phase duration and the cell cycle· time was indicated by the curves of the labeled mitoses. Proliferative activity was significantly accelerated in this experimental model of atrophic gastritis.-J Natl Cancer Inst 55: 1383-1387, 1975.
Atrophic gastritis has been experimentally produced by explantation of gastric mucosa onto the ~bdo?li!1aI wall in rats (1 ~ 2) and dogs (3-6), and by the IrradIatIOn of the stomach of guinea pigs (7). A new glandular epithelium develops in the exteriorized canine fundic mucosa with some characteristics reminiscent of human atrophic gastritis. The normal superficial mucous and neck cells are replaced by flattened columnar cells with depleted mucus content. These cells extend to the bottom of the tortuous glands lacking parietal and peptic cells. The histochemical pattern of glycoproteins (8) is modified (9). Diffuse chronic inflammatory infiltrates are frequent. In contrast to human atrophic gastritis, no intestinal metaplasia or apparent reduction in mucosal thickness occurs in the experimental model. Increased cell renewal (10-13) and abnormalities in the spatial distribution of DNA-synthesizing cells in the glands (12) were described in human atrophic gastritis. In contrast to normal fundic mucosa, the proliferative activity of the abnormal gastric glands is not limited to the neck region of the glandular pits but extends to the upper third of the glands (14). Concomitant changes in RNA and protein synthesis are found (14), like those seen in colon epithelial cells during the development of neoplastic lesions (15~ 16). Such gastric-cell maturation disorders suggest that an orderly sequence in the progression of atrophic gastritis to gastric carcinoma could exist (14~ 17~ 18). However, despite extensive epidemiologic (19-22), histologic (17~ 18~ 23), and histochemical (24~ 25) studies, this hypothesis has not yet been firmly established (24-27). In this study, the spatial distribution of DNA-synthesizing cells and the kinetics of cell proliferation were analyzed in the exteriorized fundic mucosa of dogs.
larger fundic explants of approximately 6 X 10 em were prepared. The stomach was closed after the insertion of a stainless-steel gastric cannula. In 2 dogs of group C, a large nylon gastric cannula was placed to allow the biopsy of the gastric wall under direct visualization (8). No gastric explants were prepared in these animals. Four weeks after the operation, the animals were fasted for 36 hours and were given 1 mCi tritiated thymidine (3H-TDR)/kg body weight (sp act 26 ~Ci/mM; Radiochemical Center, Mol, Belgium). The anImals of group A (small explants) were killed with pentobarbital sodium 1 hour after the 3H-TDR injection. Mucosal biopsy specimens were taken from the explant and from the fundic greater curvature. In the animals of group B (large explants), mucosal specimens of the stomach were taken randomly through the cannula. This was repeated every I or 2 hours for 28 hours after injection of 3HTDR. Simultaneous biopsy specimens were taken from the explanted mucosa in such a way as to avoi? the proximity of previous biopsy sites. In the speCImens taken randomly from the stomach, those showing esophageal tissue, antral mucosa, intermediate zone, or hemorrhagic suffusions from a previous .wounding. were discarded. In the animals of group C, bIOPSY spec~me~s from the fundic mucosa were taken under dIrect vIsualIzation every 1 or 2 hours for 28 hours. Mucosal specimens taken a half hour or an hour after the injection of 3H-TDR were fixed in 2.5% glutara~de hyde, embedded in Epon 812, and cut at 1- to 2- fL thIckness. After removal of the Epon (28), the tissues were stained with periodic acid-Schiff and covered with emulsion (Ilford K 5 in gel form). They were developed after 6 weeks and counterstained with 1'% toluidine blue (pH 9.2). Other mucosal specimens were fixed in Carnoy's solution, embedded in paraffin, and cut at 4-fL thickness. They were stained with periodic acid-Schiff, covered with emulsion, developed after 18 days, and counterstained with hematoxylin. Histograms of the spatial distribution of labeled cells in the glandular tubes were constructed. In each gland, the location o£ the cell adjacent to the surface was termed position 1. The positions of all labeled cells under it were thus recorded. The percentage of labeled cells [labeling index (LI)] was estImated on the Epon sections in the area of maximal proliferation of 20 welloriented glands from specimens taken 1 hour after 3HTDR injection in dogs from groups A, B, and C. We constructed the curve of labeled mitoses (29) by counting the number of labeled mitoses in each specimen from groups Band C.
MATERIALS AND METHODS
Nine adult mongrel dogs, between 9 and 15 kg, were used. In 3 dogs of group A, a sheet of full-thickness stomach wall, approximately 3 X 3 cm, was excised from the fundic greater curvature. The blood supply was left intact as a pedicle from the left gastroepiploic vessels. The explant was then exteriorized through a stab incision in the left flank and sutured to the skin as described by Milton and Finck (3). In 4 dogs of group B,
Received April 24, 1975; accepted July 23, 1975. . Supported in part by grants from the Fonds voor Geneeskundlg Wetenschappelijk Onderzoek and the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. 3 Laboratory of Experimental Surgery, Hopltal UmversltaIT~ SalI~t Pierre, rue Haute 322, Vrije Universiteit Brussel and Umverslte Libre de Bruxelles, 1000 Brussels, Belgium. . 4 We thank Mrs. Y. Vansteenkiste and Mrs. M. de Cock for theIr technical assistance, and Dr. S. Refeto ff, University of Chicago, for reviewing the manuscript. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.6, DECEMBER 1975
A
•
• • •
1383
•
1384
CASTELEYN AND WILLEMS
RESULTS
All histologic characteristics of this experimental model previously described by others (2-6) were observed in our explants at the time of autoradiographic study (fig. I). Poorly differentiated mucous cells occupied the length of the glandular tubes. No intestinal metaplasia occurred. Only exceptional parietal cells were seen. The cell infiltrate of the lamina propria was obvious but moderate. One hour after isotope injection, 96% of the labeled cells were localized between positions 20 and 40 in the normal fundic mucosa. These limits delineated the area of maximal proliferation in the glands. The corresponding area ranged from position 5 to position 50 in the exteriorized mucosa. Significant numbers of labeled cells and mitotic figures were seen in the upper part of the glands and near the mucosal surface in the explants (text-fig. I). Labeling indices (table I) were higher (P
