Acta Path. Jap. 27(6): 799-808, 1977


Hiromitsu WATANABE, Fumio HIROSE,Shoichi TAKIZAWA, and Yoritaka TERADA Department of Cancer Reaserch, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima (Received on Jan. 28, 1977)

In an attempt to produce experimental autoimmunity in small animals the experiment was sought to induce atrophic gastritis in ICR/JCL mice. The stomach antigen of ICR mice was extracted and emulsified with an equal volume of Freund's complete adjuvant. This was subcutaneously injected in 5-week-old ICR/JCL mice a t 1 week intervals for a total of 1 to 4 administrations. The stomach antibody in the serum gradually increased up to 2' until four weeks after the last injection of the stomach antigen. At the same time pyknosis and a decrease in number of the gastric mucosal cells, which ultimately led to the atrophying of gastric mucosa, developed. Thereafter, concomitant with the decrease in serum antibody against mucous cells, regeneration of mucous cells was especially remarkable, but atrophy of the fundic gland continued. ACTA PATH. JAP. 27: 799-808, 1977.


The subject of autoimmune diseases has been extensively discussed in the past few years. Attempts to produce experimental autoimmune gastritis were made by several investigators in dogs,1-6 by injection of autologous, allogenic gastric juice or gastric mucosal homogenate, and in monkeys.' By contrast SIRCUSand c o - w o r k e r ~ ~ ~ ~ have found no effect on the histological appearance of gastric mucosa of dogs of continuous intravenous administration of lyophilized human gastric juice. The injection of an allogenic gastric mucosal homogenate together with Freund's adjuvant has failed to bring about such changes in the gastric mucosa of small animals such as rabbits,1° guinea pigs,1OP rats,10-12 and micelO. A contribution could be made to the study of atrophic gastritis if gastric atrophy could be experimentally produced in small animals. This report describes a successful attempt t o induce gastric atrophy in ICR mice by immunization with allogenic stomach antigen. Materials and Methods 1. Preparation of stomach antigen.

The glandular stomach was excised from 5 to 6-week-old ICR/JCL mice of both sexes

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Dr. Hiromitsu WATANABE:Kasumi 1-2-3, Hiroshima, 734, Japan. This work was supported in part by Grant-in-Aid for Cancer Research (101083, 101086, 101557) from the Ministry of Education, Science and Culture of Japan. 799



Acta Path. Jap.

(purchased from CLEA. Ltd., Osaka, Japan). The stomach was washed several times in ice cold 0.14 M NaC1, homogenized in a Waring blender a t 4°C in equal volume of saline solution by maximum spaed for 5 min and left for 1 hr. The h3mogenate thus obtained was centrifuged a t 3,000 rpm for 15 min, and the supernatant was used as the antigen. All procedures were carried out a t 4°C.

2. Experimental animals. Five-week-old ICR/JCL female mice were used. The animals were given subcutaneous injections of 0.2 ml of the antigen (15 mg of protein/2 ml) in Freund's complete adjuvant (Difco) a t 1 week interval for a total of 1 to 4 administrations. Control animals were immunized only with Freund's adjuvant. The animals were housed in plastic cages, 8 to 10 in each, in an air-conditioned room, and were fed a commercial diet MF (Oriental Yeast Inc., Tokyo) and given water ad libitum. 3. Examination of the animals.

Two or three animals from each group were selected a t random and sacrificed one week after the Ist, 2nd, and 3rd administrations and 1, 2, 4, and 8 weeks after the 4th administration. I n view o f t h e fact that the mitotic index of certain tissues is different during the day and a t night,l33I4 the animals were sacrificed only a t 2:OO pm. The blood of each animal under light ether anaesthesia was drawn from the subclavian artery and immediately thereafter an autopsy was performed. The main organs, including the stomach, Were carefully removed. The stomach was cut open along the greater curvature, stretched and pinned on cardboard with the mucosal surface facing upward. The mucosal surface was immediately washed with 0.14 M NaCl and grossly examined. After gross examination, the main organs, including the liver, kidney, lung and ovary, were fixed in 10% neutral formalin and embedded in paratfin according t o the routine procedure. Serial 3p-thick sections were cut perpendicular t o the mucosal surface along the entire length of the lesser curvature and the anterior wall and also the posterior wall of the greater curvature, and stained with hematoxylin and eosin, Alcian bluePAS stain and van Gieson-Elastic stain, respectively. 4. Antibody titrations. Blood drawn from these animals was kept overnight in an ice box and centrifuged a t 3,000 rpm for 15 min to collect the serum. After serial dilution of the serum, the titer for the stomach antigen was estimated by the Ouchterlony method,15 precipitin testis and immunofluorescence method.lB For the indirect immunofluorescence test, the stomach of normal ICR mice was fixed with 10% neutral formalin and embedded in paraffin. The paraffin was then removed from the sections, the specimens were washed throughly three times with PBS for 20 min and exposed to serial dilution of the serum for 30 min in a moist chamber. After three changes of PBS, the specimens were covered with antimouse rabbit serum conjugated FITC (MBL, Nagoya) for 30 min in a moist chamber, washed three times with PBS again, and mounted. They were then examined under a Olympus fluorescence microscope. 5. Cell count.

A cell count was made to determine the quantity of the level of atrophy. According t o our preliminary study,17 in fundic mucosa the number of parietal cells per crypt is constant at the crypt in height of 250 p t o 400 p. Therefore, the number of parietal, chief and mucous cells and pyknotic figures of each cell in the fundic mucosa was counted in about 100 crypts (about 5,000 cells) a t a magnification of x 200, and the number of each type of cell per crypt was used t o indicate the level of atrophy. On the other hand, the number of pyknotic cells, mitotic figures and inflammatory infiltration cells was counted in about 5,000 t o 10,000 mucous cells of the pyloric gland a t a magnification of x 400, and these were expressed as a percentage of tho total number of mucous cells.

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H. WATANABE e i al.


1. Titration of Antibody against Gastric Mucosal Anteens in the Serum The reaction between the stomach extract used as antigen and the serum of the animals administered the antigen was studied individually. The serum of the animals administered the antigen did not respond a t all to the antigen by the Ouchterlony method. I n the serial dilution precipitation method, the serum 1 week after one administration was positive a t a dilution of 23 against the stomach antigen, after 2 or 3 administrations a t a dilution of 24, 1 week after 4 injections a t a dilution of Z4 t o 25 and after 2 weeks a t a dilution of 25 and after 4 weeks a t a dilution of 25 t o 26. After 8 weeks the titer of the serum decreased a t a dilution of 24 to F. The components of the stomach cell antibody were examined by the immunofluorescence method. The antibody for chief and mucous cells was weak in the serum, but mainly the parietal cell antibody was strong and the antibody did not react in the muscle layer. The titer in the serum against parietal cells was positive a t a dilution of 27 from 1 week after the 3rd injection. The parietal cell antibody of such a high titer was also seen after the last injection (Table I). The serum of the control animals did not respond in all methods used. Table 1. Titration of Antibody against Mucosal Antigen in the Serum by Immunojluorescence Method


A f t e $ ~ ~ ~ Chief ~ t Cell

Parietal Cell Mucous Cell

21 22 22

2. Pathological Changes in the Gastric Mucosa I n 2 of 3 animals 4 weeks after a total of 4 injections and in all 3 animals 8 weeks after the last injection, hemorrhage was observed in the gastric mucosa near the limiting ridge. Histologically, atrophy of the mucosa and edema of intraglandular tissue in the pyloric gland were seen as early as one week after the 1st injection of the stomach antigen (Pig. lb). Atrophy of the mucosa was most severe 1 week after a total of 2 injections (Pig. lc) and continued for 1 week after a total of 3 or 4 injections, but thereafter slight recovery was seen (Pig. Id). These changes were observed in all experimental animals. Atrophy of the mucosa in the fundic gland area appeared later than in the pyloric gland area. Pyknosis of the parietal cells and chief cells developed (Fig. 2b, 2c). Atrophy of the parietal and chief cells continued for four weeks after the last



Acta Path. Jap.

27(6): 1977


H. WATANABE c t al.

injection, but regeneration of the crypt epithelium developed and the mucous cells were replaced by regenerated epithelium which was stained with PAS (Fig. 3). Intraglandular fibrotic reaction was occasionally observed accompanied by degenerative changes in parietal and chief cells. I n general, inflammatory changes were mild. Alcian blue-PAS positive cells and Paneth-like cells containing eosinophilic granules in the pyloric gland appeared 4 and 8 weeks after a total of 4 injections, but the number was small. Intestinal type crypt and goblet cells were not observed. I n the control group, infiltrative cells appeared as in the experimental group, but there were no atrophic changes in the gastric mucosa (Fig. la, 2a). Alcian blue-PAS positive cells and Paneth-like cells were not seen. No remarkable histological changes were found in the liver, kidney, small intestine, lung and ovary of the animals in both groups.

3. Changes in the Number of Cells in the Fundic Mucosa The total number of cells in the fundic mucosa decreased remarkably during administration of the antigen. The number of pyknotic cells increased (p

Induction of atrophic gastritis in ICR mice by the administration of an allogenic antigen.

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