Histopathological features of the proper gastric glands in FVB/N-background mice carrying constitutively-active aryl-hydrocarbon receptor

FVB-CA-AhR mice were created based on C57BL/6-background CA-AhR mice (B6-CA-AhR mice; created by Dr. Hanberg, Karolinska Institute, Stockholm, Sweden) at the National Institutes of Health (NIH; Bethesda, MD, USA) by backcrossing with FVB/N mice over six generations. FVB-CA-AhR mice used in the present study were kindly provided by Dr. Kopp (NIH) and were maintained along with FVB/N mice (CLEA Japan, Inc., Tokyo, Japan) in the Graduate School of Veterinary Medicine, Hokkaido University (Sapporo, Japan) under specific pathogen-free conditions. The gene encoding CA-AhR was genotyped using a PCR-based method with a primer pair (forward, 5′-TTACCTGGGCTTTCAGCAGT-3′; reverse, 5′-AACTGGGGTGGAAAGAATCC-3′), followed by agarose gel electrophoresis. FVB-CA-AhR mice were bred to homozygosity. Wild-type control mice included in this study were of the same mixed genetic background. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals, Graduate School of Veterinary Medicine, Hokkaido University (approved by the Association for Assessment and Accreditation of Laboratory Animal Care International; experimental protocol approval no. 13–0032 and 16–0124).

Male and female FVB-CA-AhR mice and the wild-type mice at 6–33-weeks (wks)-of-age were sacrificed by cervical dislocation under anesthesia (0.3 mg/kg medetomidine, 4.0 mg/kg midazolam and 5.0 mg/kg butorphanol) [26], and systemic organs were observed macroscopically. The stomach was removed from each mouse and kept in cold phosphate-buffered saline (PBS) to investigate histopathological features. After 5–10 min, stomach contents were removed by cutting the greater curvature and by rinsing with cold PBS. Each stomach was weighted, gently flattened, and pinned on a small piece of filter paper. Jejunum, pancrease, gallbladder, and liver were also collected from each mouse for lectin histochemistry. The collected organs were fixed with 4% paraformaldehyde, dehydrated with alcohol, and embedded in paraffin. To detect the proliferating cells, bromodeoxyulidine (BrdU, 100 mg/kg body weight) was intraperitoneally injected in some mice 2 h before euthanasia. For each age group, more than four mice were examined. Details are provided in Additional file 1: Table S1 and Additional file 2: Table S2.

Paraffin sections 3 μm in thickness containing non-glandular and glandular parts of the stomach that crossed the major axis along the lesser curvature were deparaffinized and hydrated. In addition to hematoxylin-eosin (HE) staining, periodic acid Schiff (PAS) or alcian blue pH 2.5 (AB pH 2.5) staining was performed to detect neutral mucins and acidic mucins, respectively.

Immunohistochemistry for cytochrome P450, family 1, subfamily a, polypeptide 1 (CYP1A1) was performed to determine stomach regions with CA-AhR expression-induced activation of AhR signaling. Furthermore, immunohistochemistry for proliferating cell nuclear antigen (PCNA) B220, CD3, pepsinogen, and caudal type homeobox 2 (CDX2) or trefoil factor 2 (TFF2) was done to evaluate the levels of proliferating cells, B-cells, pan T-cells, chief cells, and metaplasia, respectively. BrdU-incorporation was used to detect proliferating cells. Lectin histochemistry was performed by staining the stomach, jejunum, pancrease, gallbladder, and liver of individual mice with 21 types of biotinylated lectins (Vector Laboratories, Inc., Burlingame, CA, USA; Table 1). Details of primary and secondary antibodies, lectins, antigen retrievals, and blocking methods are summarized in Additional file 3: Table S3. Briefly, the paraffin sections were deparaffinized and antigens were retrieved for immunohistochemistry, but not for the lectin histochemistry. Next, slides were soaked in methanol containing 0.3% hydrogen peroxide (H₂O₂) to remove internal peroxidases. After washing with PBS, the sections were blocked with appropriate blocking reagents and were incubated overnight at 4 °C with primary antibodies or biotinylated lectins. Only for immunohistochemistry, after washing with PBS, sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. Next, the sections were incubated with streptavidin–biotin complex (SABPO® kit; Nichirei, Tokyo, Japan) or goat anti-mouse IgG (SouthernBiotech, Birmingham, AL, USA) for 30 min. Colors were developed by incubating with 3, 3-diaminsobenzidine tetrahydrochloride-H₂O₂ solution. Finally, the sections were lightly counterstained with hematoxylin.

For lectin histochemistry, the staining results were semi-quantitatively classified according to the intensity of positive staining reactions as follows: −, negative staining; ±, positive as well as negative staining; +, weak positive staining with light brown color; ++, positive staining with brown color; and +++, strong positive staining with dark brown color.

The proximal mucosal region of the stomach within 3 mm from the limiting ridge, which is the border between the non-glandular and glandular regions [24], was analyzed using three semi-serial sections a minimum interval of 30 μm. Rounded or oval expansion in the glandular lumen was defined as an “intramucosal cyst (IM cyst)”. The total number of IM cystss was determined in HE-stained sections. Cysts penetrating the muscularis propria were defined as “extramucosal cysts (EM cysts)”, and their incidence was calculated in all histologically examined mice.

The relative position of PCNA-positive (PCNA⁺) cells in the gastric mucosa was determined using ImageJ software (https://​imagej.​nih.​gov/​ij/​) by calculating the area from the lamina muscularis to the limiting line showing the highest position of PCNA⁺ cells in each gastric pit divided by the area of the examined gastric mucosa. The calculated relative area (%) indicated the relative position of PCNA⁺ cells. The numbers of PCNA⁺ cells in the gastric mucosa and parietal cells in HE-stained sections, and the relative positive areas of PAS or AB pH 2.5, were analyzed using a model BZ-II Analyzer (Keyence, Osaka, Japan).

For scanning electron microscopy (SEM), the glandular part of the stomach besides the lesser curvature (approximately 1 cm²) was fixed with 2.5% glutaraldehyde for 2–4 h. After washing in 0.1 M phosphate buffer, the specimens were kept in 0.5% tannic acid for 10 min at 4 °C and in 1% for 1 h at 4 °C. The specimens were dehydrated through graded alcohol, transferred into 3-methylbutyl acetate, and dried using a model HCP-2 critical point dryer (Hitachi, Tokyo, Japan). The dried specimens were sputter-coated using a model E-1020 ion sputter coater (Hitachi), and examined using a model S-4100 microscope (Hitachi) with an acceleration voltage of 10 kV.

All numerical results are presented as mean ± standard error (SE). Differences between groups were analyzed using a non-parametric method (Mann–Whitney U test). Correlation between the number of EM cysts and AB pH 2.5⁺ areas was analyzed using Spearman’s correlation coefficient test (ρ). In the analyses, significance was indicated by P < 0.05.

No FVB-CA-AhR mice died during the observation period (6–33 wks). The mice were fertile and produced an equal sex ratio in pups. Body weights of FVB-CA-AhR and wild-type mice increased comparably with age, and significant sex-related differences were observed in both FVB-CA-AhR (at 9 and 16 wks) and wild-type mice (at 16 wks) (Fig. 1a). Macroscopic observation of systemic organs revealed severe pathological changes only in the stomachs of FVB-CA-AhR mice. No macroscopic changes were observed in the stomachs of wild-type mice (Fig. 1b). In contrast, stomachs of FVB-CA-AhR mice showed cystic structures beneath the serosa on the parietal and visceral surfaces (Fig. 1c); moreover, these structures were only localized around the lesser curvature. These cystic structures appeared from 8 wks in males and 10 wks in females, and increased in size and number with age (Fig. 1d). Hemorrhagic lesions were observed in a few aged mice (Fig. 1d). Relative stomach weight to body weight was significantly increased in FVB-CA-AhR mice compared with wild-type mice. However, significant sex-related differences were observed only in wild-type mice at 9 and 13 wks (Fig. 1e). Although macroscopic examination showed normal gastric mucosa in all the examined mice (Fig. 1f), microscopic examination of the mucous surface yielded different results for FVB-CA-AhR and wild-type mice. In wild-type mice, each gastric pit was clearly observed, and the border between each mucous secreting cell was clear and well-organized (Fig. 1g). In contrast, FVB-CA-AhR mice showed obscure gastric pits and cell borders, with partially fused surfaces of some cells (Fig. 1 g).

Wild-type mice showed IM cysts in different regions of the proper gastric glands. These cysts were lined with simple cuboidal or columnar epithelial cells (Fig. 2a). In contrast, FVB-CA-AhR mice showed numerous IM cysts (Fig. 2a). The number of IM cysts was significantly higher in FVB-CA-AhR mice than in wild-type mice, with a slight increase in cysts with age (Fig. 2b). The number of IM cysts was significantly higher in female FVB-CA-AhR mice than in male mice at 28 wks (Fig. 2b). Only in FVB-CA-AhR mice, some IM cysts were connected to EM cysts through the lamina muscularis as well as muscularis propria (Fig. 2c). The histopathological structure of EM cysts became complicated with branching, which increased with age (Fig. 2c). However, no sex-related histopathological differences were observed in EM cysts. EM cysts first appeared from 8 wks of age in males and from 10 wks of age in females, and their appearance corresponded with the appearance of macroscopically evident cystic structures (Fig. 1). The incidence of EM cysts increased with age in both sexes of FVB-CA-AhR mice (Fig. 2d). Infiltrations of B220⁺ B-cells or CD3⁺T-cells in the gastric mucosa were not severe during the observation period (Fig. 2e and f). In some FVB-CA-AhR mice with EM cysts, B220⁺ B-cells infiltrated beneath the lamina muscularis (Fig. 2g). However, there were few B220⁺ B-cells around EM cysts in younger mice.

The morphology of epithelial cells in some regions of the proper gastric glands changed to a columnar shape in FVB-CA-AhR and wild-type mice, indicating epithelial metaplasia (Fig. 3a and b). PAS⁺ substances were localized in the superficial regions of the normal mucosa in wild-type mice, and were enlarged toward the basal region of metaplastic lesions in both wild-type and FVB-CA-AhR mice (Fig. 3c). The relative PAS⁺ area was significantly greater in FVB-CA-AhR than in wild-type mice, and no age-related changes were observed in any of the examined mice (Fig. 3d). Male FVB-CA-AhR and wild-type mice showed higher values than the female counterpart mice. However, a significant sex-related difference was observed only in wild-type mice at 13 wks.

A previous report [27] described metaplastic lesions that were positive for PAS and AB pH 2.5. Presently, in the wild-type mice, the PAS⁺ area was negative for AB pH 2.5 staining (Fig. 3e). In contrast, some PAS⁺ areas in FVB-CA-AhR mice displayed AB pH 2.5 positivity, which was pronounced in metaplastic lesions (Fig. 3e). Histoplanimetry revealed that the relative AB pH 2.5⁺ area was significantly greater in FVB-CA-AhR mice than in wild-type mice (Fig. 3f). Characteristically, the relative AB pH 2.5⁺ area was higher in male FVB-CA-AhR mice than in wild-type mice from 9 wks of age, but that in female FVB-CA-AhR mice increased with aging. Moreover, a significant correlation was observed between the number of IM cysts and AB pH 2.5⁺ area in males, females, and all examined mice (Fig. 3g).

Nuclear PCNA positivity was observed from the isthmus to the neck region in proper gastric glands in wild-type mice, and from the body to the base of the glands in FVB-CA-AhR mice (Fig. 4a). Histoplanimetry revealed that the relative positions of PCNA⁺ cells decreased with age in all the examined mice, and were significantly lower in FVB-CA-AhR mice than in wild-type mice (Fig. 4b), indicating a downward shift in the cell proliferation zone. A sex-related difference was observed only in FVB-CA-AhR at 9 wks, with male mice displaying lower values than female mice. However, the number of PCNA⁺ cells in the gastric mucosa was comparable between wild-type and FVB-CA-AhR mice (Fig. 4b). Altered positions of PCNA⁺ cells in FVB-CA-AhR mice were also confirmed by the detection of cells that incorporated BrdU (Fig. 4c). Moreover, the parietal/chief cell zone, which normally occupies the region of mucous two-thirds from the bottom, decreased with a change in the position of the cell proliferation zone (Fig. 4a and d). The number of parietal cells was significantly lower in FVB-CA-AhR mice than in wild-type male mice during the examination period, and the cell number in female mice was significantly decreased with age; however, this was not observed in wild-type mice (Fig. 4e). Significant sex-related difference was detected in FVB-CA-AhR and wild-type mice at 9 and 13 wks, and males displayed lower values than females (Fig. 4e).

We histopathologically examined the EM cysts in aged FVB-CA-AhR mice in detail (Fig. 5). Some EM cysts contained cell debris and seemed to continue from the entrance of gastric pit (Fig. 5a and b). Positive reaction for CYP1A1, indicating the activation of the AhR signaling pathway, was detected in the lining epithelium from the gastric pits to the penetrating EM cysts (Fig. 5b). Some lymphoid clusters were observed around EM cysts (Fig. 5c). Epithelial cells lining the EM cysts were morphologically similar to mucous secreting cells in the proper gastric glands. These cells were columnar shaped (Fig. 5d) and showed both PAS⁺ and AB pH 2.5⁺ reactions (Fig. 5d). Some cells incorporated BrdU (Fig. 5e), indicating the existence of proliferating cells. Fat tissues, blood vessels, and mild CD3⁺T-cell infiltrations were observed around EM cysts, but there were few B220⁺ B-cells (Fig. 5f and g). Lymphoid clusters around EM cysts contained both CD3⁺ T-cells and B220⁺ B-cells (Fig. 5f and g). In contrast, no other cell type, such as parietal cells and chief cells, which are present in the proper gastric glands were observed. No dysplastic features were observed.

To investigate the characteristics of EM cysts, especially their epithelial features, histochemical analysis was done using 21 types of lectins. The staining patterns were compared to the mucous in the healthy stomach as well as in other organs, including the intestine (jejunum), liver (interlobular bile duct), gallbladder, and pancreas (intercalated duct). The results are summarized in Table 1. Figure 6 shows the results of staining with a representative lectin Griffonia simplicifolia lectin II (GSII), which produced strong staining of epithelial cells in the stomach (Table 1). GSII⁺ staining was detected in superficial, neck, parietal, and chief cells in wild-type mice (Fig. 6a–d), with neck cells showing strong staining (Fig. 6c). However, GSII⁺ staining was not detected in the other organs (Table 1 and Fig. 6e–g). In the EM cysts, GSII⁺ staining was clearly observed in lining cells, which morphologically resemble mucous neck cells and which do not secrete much mucous (Fig. 6h). The collective lectin staining patterns in EM cysts closely matched the pattern of glandular cells of the stomach, nearly matched the pattern in the intestine, and did not match patterns in other organs (Table 1).

To clarify the characteristic of metaplasia found in the stomach of FVB-CA-AhR mice at 33 wks, we performed immunohistochemistry for CDX2 (Fig. 6i-k), a marker for intestinal metaplasia. Lesions in FVB-CA-AhR mice were negative for CDX2 but a positive reaction was obtained in the jejunum (Fig. 6i-k).

Immunohistochemistry of serial sections examined pepsinogen and TFF2 to examine the development of spasmolytic polypeptide-expressing metaplasia (SPEM)-like lesions in FVB-CA-AhR mice at 33 wks (Fig. 7). The positive reactions for pepsinogen tended to be weak in FVB-CA-AhR mice compared to the wild-type mice (Fig. 7a and b). Furthermore, FVB-CA-AhR mice showed weakly positive pepsinogen reactions in cells in the apical portion of IM cysts (Fig. 7b). In EM cysts, pepsinogen negative and positive cells were observed (Fig. 7c). TFF2 positive reactions were mainly observed in cells located in the apical region of the neck in wild-type mice. There were more TFF2 positive cells in CA-AhR mice (Fig. 7d and e). In CA-AhR mice, most IM and EM cysts were positive for TFF2 (Fig. 7e and f). In well-developed EM cysts, very few cells showed positive reactions for pepsinogen (Fig. 7g and h). TFF2⁺ reactions were mainly observed at the apical portion of large EM cyst lining cells (Fig. 7i) and some epithelial cells were positive for both pepsinogen and TFF2 (Fig. 7j).