Spontaneous ECLomas in cotton rats (Sigmodon hispidus): tumours occurring in hypoacidic/hypergastrinaemic animals with normal parietal cells
Guanglin Cui,
Gunnar Qvigstad,
Sture Falkmer,
Arne K. Sandvik,
Shiro Kawase1 and
Helge L. Waldum2
Norwegian University of Science and Technology, Faculty of Medicine, N-7006 Trondheim, Norway and
1 Division of Experimental Animals, Hokkaido Institute of Public Health, Sapporo, Japan
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Abstract
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We have identified cotton rats with a high female-predominant occurrence of spontaneous gastric carcinomas localized to the oxyntic mucosa, classified as malignant enterochromaffin-like (ECL) omas. The present study was made to further characterize these ECLomas and surrounding oxyntic mucosa, both morphologically using histochemical and immunohistochemical methods, and for gene expression by northern blot analysis. Among eight female cotton rats, three had an irregularly thickened oxyntic mucosa, increased stomach weight and a high serum gastrin level. Histopathological examination showed adenomatous hyperplasia of the thickened oxyntic mucosa with areas of an invasive neoplastic tumour. Immunohistochemistry, using the general neuroendocrine cell marker chromogranin A (CgA) and the specific ECL cell marker histidine decarboxylase (HDC), showed a considerably increased ECL cell density. These ECL cells displayed active proliferation, with hyperplasia, dysplasia and neoplasia. Parietal cells were not found in the tumour tissue. Parietal cell density was only slightly reduced in the surrounding oxyntic mucosa. The antral mucosa was histopathologically normal with a normal number of gastrin-immunoreactive cells. Likewise, somatostatin-immunoreactive cells did not show any differences in the antral and oxyntic mucosa between rats with pathological and normal oxyntic mucosa. Northern blot analysis revealed increased expression of CgA and HDC mRNA in the thickened oxyntic mucosa, whereas H+/K+ ATPase mRNA was similar in the oxyntic mucosa of those with thickened and normal oxyntic mucosa. Gastrin mRNA in the antral mucosa was high in animals with thickened oxyntic mucosa. Somatostatin mRNA expression was similar in the antral mucosa of control animals and animals with a thickened oxyntic mucosa. We conclude that the spontaneous gastric carcinoma occurring in female cotton rats is an ECLoma developing secondary to hypergastrinaemia due to reduced intragastric pH. The mechanism for reduced acidity is not known, but is not gastric atrophy.
Abbreviations: ABC, avidinbiotinperoxidase complex; AEC, 3-amino-9-ethylcarbazole; CgA, chromogranin A; ECL, enterochromaffin-like; GAPDH, glyceraldehyde 3'-phosphate dehydrogenase; HDC, histidine decarboxylase; HE, haematoxylin and eosin; IHC, immunohistochemistry; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen.
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Introduction
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Enterochromaffin-like (ECL) cells are the predominant neuroendocrine cells of the oxyntic mucosa of the stomach and in man they make up ~35% of the entire population of neuroendocrine cells (1). Their growth and proliferation are both mainly controlled by gastrin (2). In recent years, the development of ECL cell hyperplasia and neoplasia has been the focus of considerable interest and studies in both man and rats have provided evidence to support a relationship between reduced acidity and secondary hypergastrinaemia in the aetiology and pathogenesis of ECL cell hyperplasia and neoplasia (2,3). Previously, we described cotton rats with high, female-predominant occurrence of spontaneous gastric adenocarcinomas (~25% of female rats and only 1% of male rats at 68 months of age) (4). We have recently shown that these spontaneous adenocarcinomas occurred in cotton rats with low acidity and hypergastrinaemia and that the tumours could be classified as ECLomas (5). The present study was made to further characterize this tumour by means of histochemical and immunohistochemical procedures and by northern blot analysis.
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Materials and methods
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Animals
Eight female cotton rats were kept and fed as reported previously until 8 months of age (4,5). The rats were starved for 24 h before they were anaesthetized with ether and weighted, the abdominal cavity was opened and the rats bled from the caval vein. Serum was collected for gastrin measurement by a radioimmunoassay method (6) and total protein measurement by the Bradford method. Mucosa specimens were taken from the antrum and corpus along the greater curvature, fixed immediately in 10% buffered formalin, dehydrated and embedded in paraffin for immunohistochemical examinations.
Histopathological and immunohistochemical (IHC) techniques
From the paraffin blocks, 4 µm thick serial sections were cut and each representative section was stained with haematoxylin and eosin (HE). In addition, sections for IHC examinations were transferred to poly-L-lysine-coated slides and allowed to dry overnight. IHC was then performed with the avidinbiotinperoxidase complex (ABC) method, using Vectastain ABC kits (Vector Laboratories, Burlingame, USA) with rinsing in cold phosphate-buffered saline (PBS) containing 0.25% Triton X-100 (Calbiochem, USA) between each step. The following antibodies were used: polyclonal anti-porcine chromogranin (CgA) at a dilution of 1:500 as a general endocrine cell marker (Dia Sorin, USA); polyclonal anti-recombinant histidine decarboxylase (HDC) at a dilution of 1:3000 as a specific ECL cell marker (Euro-Diagnostica, Sweden); monoclonal anti-hog H+/K+ ATPase at a dilution of 1:2000 as a marker for parietal cells (Affinity Bioreagents, USA); monoclonal anti-rat proliferating cell nuclear antigen (PCNA) at a dilution of 1:100 as a marker for cell proliferation (DAKO, Denmark); polyclonal anti-human gastrin at a dilution of 1:300 (DAKO) as a G cell marker; polyclonal anti-human somatostatin at a dilution of 1:300 (DAKO) as a D cell marker. The antibodies were diluted in PBS containing 0.25% Triton X-100 and 0.25% bovine serum albumin (Sigma, USA) and then incubated either at 4°C overnight (CgA, HDC and H+/K+ ATPase) or at room temperature for 2 h. Antigen retrieval was achieved by boiling the sections for 35 min in 0.01 M citrate buffer, pH 6.0 (CgA and HDC), or with 2 N HCl for 30 min (PCNA). 3-Amino-9-ethylcarbazole (AEC; Vector Laboratories) was used as chromogen and the slides were counterstained with Mayer's haematoxylin for 10 s.
Double IHC with PCNA and HDC on the same slide was achieved by performing the first step with monoclonal anti-PCNA as described above, but using Vector SG (Vector Laboratories) as chromogen. Thereafter, the slides were washed with stirring in glycineHCl buffer (pH 2.2) for 2 h at 4°C. The ABC procedure was then repeated with polyclonal anti-HDC as the primary antibody and AEC as chromogen. No counterstaining was applied.
Histological grading of ECL cell hyperplasia was carried out utilizing the criteria outlined by Mattson et al. (7). Mild: a diffuse increase in ECL cells in the lower half of the oxyntic mucosa. Moderate: the increased ECL cell population expanded up to the foveolar level. Severe: the increased ECL cell population exhibited aggregation and microfocal strands within glands; sometimes micronodular hyperplasia occurred.
Northern blot analysis
Tissue biopsy specimens were homogenized in a denaturing buffer (1 ml/100 mg tissue) with 4 M guanidinium isothiocyanate, 25 mM sodium acetate (pH 6.0) and 0.84% (v/v) ß-mercaptoethanol. Total RNA was extracted by ultracentrifugation on a caesium chloride cushion and precipitated with ethanol. CgA, HDC, H+/K+ ATPase, gastrin and somatostatin cDNA fragments were generated by reverse transcriptionPCR (810) and the fragments ligated into the pCR II vector (Invitrogen TA cloning kit). Probes for hybridization were simultaneously transcribed and 32P-labelled using standard protocols. Total RNA from the corpus and antrum were then analysed by northern blot using established methods; internal controls were cRNA probes complementary to glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) mRNA. Post-hybridization washes were performed twice at room temperature for 20 min with 5x salinesodium phosphateEDTA (SSPE) containing 0.1% SDS and once for 20 min at 65°C with 0.1x SSPE, 0.1% SDS. After washing, the membranes were exposed to a phosphor storage screen and the signal measured and analysed using an Imageqnant phosphorimager and software. For controls, the membranes were stripped with boiling 0.1% SDS and sequentially rehybridized with GAPDH and rat 18S probes.
Serum gastrin radioimmunoassay
Gastrin in serum was measured by a previously described method (6).
Serum total protein assay
Total protein in serum was measured by the Bradford method (BioRad, Richmond, CA).
Statistics
Differences in plasma gastrin and in the expression of CgA, HDC, H+/K+ ATPase, gastrin and somatostatin mRNA were evaluated by the MannWhitney U-test, whereas total protein concentration in serum were analysed by Student's t-test. P values <0.05 were regarded as significant.
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Results
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Animals
Among the eight female cotton rats, three had an irregularly and diffusely thickened gastric corpus mucosal wall and the stomach weight was increased 4-fold in these rats when compared with controls with normal oxyntic mucosa.
Histopathological findings
In HE staining, the grossly thickened lesions were diffuse, but restricted to the oxyntic mucosa. The antral mucosa was never involved. The thickened oxyntic mucosa represented diffuse adenomatous hyperplasia (Figure 1
) and had a close association with the neoplasia. In neoplastic lesions, ECL tumour cells were found. In some areas, neoplastic tissue had penetrated into the muscle layer, showing the invasive character of the neoplasia (Figures 2 and 5
).

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Fig. 1. Medium power photomicrograph of the grossly thickened oxyntic mucosa in a female cotton rat showing diffuse hyperplasia. The small inset in the upper left corner shows the thickness of a normal mucosa. HE staining.
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Fig. 2. Low power photomicrograph of a neoplastic lesion in the oxyntic mucosa (ECLoma) with invasion of the muscular layer (left). HE staining.
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Fig. 5. High power photomicrograph showing CgA immunoreactivity (red/brown) in the ECLoma. Neoplastic ECL cells are found both in the oxyntic mucosal glands (right) and in the muscular layer (middle and left), thus displaying a clear-cut infiltrative growth pattern. Counterstaining with haematoxylin.
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IHC findings
In the thickened oxyntic mucosa surrounding the neoplastic lesions, the number of CgA-immunoreactive cells was considerably higher than that in the controls. When using HDC antibodies, it was confirmed that the CgA-immunoreactive cells were predominantly ECL cells. Thus, the ECL cell density was greatly increased in the thickened oxyntic mucosa (Figure 3
). Moreover, a considerable fraction of these ECL cells was PCNA immunoreactive, indicating active proliferation (Figure 4
). In the basal parts of the hyperplastic glands, the ECL cells displayed a clear-cut hyperplasia, mostly of linear type. In neoplastic areas, many of the tumour cells were immunoreactive for CgA (Figure 5
) as well as HDC (Figure 6
), in agreement with ECL cell origin of the tumour cells. Tumour cells with ECL cell phenotype were also found in the submucosa and muscular layer (Figure 5
). The tumour cells were not immunoreactive for somatostatin. The number of somatostatin-immunoreactive cells in the thickened oxyntic mucosa outside the neoplastic areas was normal. The tumour cells were not immunoreactive for H+/K+ ATPase. However, H+/K+ ATPase-containing cells (parietal cells) were present in the thickened oxyntic mucosa outside the neoplastic areas. The number of parietal cells in the thickened oxyntic mucosa was not different from that of the control animals (Figure 7
). The G cell number in the antral mucosa was also similar in the two groups.

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Fig. 3. Medium power photomicrograph showing HDC immunoreactivity (red/brown) in the hyperplastic mucosa. Linear ECL cell hyperplasia is clearly visible in the thickened oxyntic mucosa. Counterstaining with haematoxylin.
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Fig. 4. High power photomicrograph showing PCNA immunoreactivity (red/brown) in the hyperplastic oxyntic mucosa of a female cotton rat. Counterstaining with haematoxylin. The small insets in the upper left corner show double immunohistochemistry for HDC (red/brown) and PCNA (blue). Cells showing immunoreactivity for both HDC and PCNA are found, indicating proliferation of ECL cells (no counterstaining used).
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Fig. 6. Medium power photomicrograph showing HDC immunoreactive (red/brown) tumour cells in the grossly thickened neoplastic mucosa (ECLoma) of a female cotton rat. Counterstaining with haematoxylin.
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Fig. 7. Medium power photomicrograph of the oxyntic mucosa outside the neoplastic area from a female cotton rat with grossly thickened hyperplastic mucosa, showing H+/K+ ATPase immunoreactivity (red/brown).
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Northern blot results (Tables I and II
)
By northern blot analysis, the expression of CgA and HDC mRNA was clearly higher, whereas H+/K+ ATPase mRNA was only slightly lower in the oxyntic mucosa of cotton rats with thickened oxyntic mucosa compared with those with normal mucosa. The expression of gastrin mRNA was markedly higher, somatostatin mRNA was possibly lower and CgA mRNA was similar in antral mucosa of rats with thickened oxyntic mucosa compared with that in those without. GAPDH mRNA level was similar in both corpus and antral mucosa in the two groups.
Gastrin in serum (Table III
)
Serum gastrin levels were 882 ± 320 pM (range 3451450) in female cotton rats with ECLomas and 36 ± 7 pM (range 2260) in cotton rats without ECLomas (P = 0.036).
Total protein concentration in serum (Table IV
)
Serum total protein concentration was 56 ± 1 g/l (range 5458) in tumour rats and 53 ± 4 g/l (range 5364) in control rats (P > 0.05).
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Discussion
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The results of the present study confirm those previously obtained regarding the presence of ECL cell hyperplasia and neoplasia in the thickened oxyntic mucosa and marked hypergastrinaemia in the affected animals (5). Previously, we showed that cotton rats with thickened oxyntic mucosa were hypo/anacidic (5), indicating that the hypergastrinaemia was secondary to reduced gastric acidity. In the present study we examined the oxyntic mucosa outside the neoplastic areas and found a normal density of parietal cells, as determined by immunohistochemical techniques using antibodies towards the proton pump H+/K+ ATPase, as well as normal expression of H+/K+ ATPase mRNA. These findings strongly suggest that the oxyntic mucosa was capable of producing acid. Nevertheless, animals with pathological oxyntic mucosa were previously shown to be hypo/anacidic (5). The changes in the oxyntic mucosa in our cotton rats in many ways resembles what is found in patients with Menetrier's disease (11), where a proportion of the patients become anacidic or hypoacidic (12) secondary to the escape of interstitial fluid into the gastric lumen, neutralizing the gastric content. A hallmark of Menetrier's disease is hypoalbuminaemia secondary to gastric loss of albumin (13), manifesting itself as oedema. Therefore, we examined the blood for the concentration of proteins. However, we could not reveal any differences between affected and healthy animals. Nevertheless, not all patients with Menetrier's disease have hypoalbuminaemia and the gastric protein loss may fluctuate. Therefore, it is still possible that the hypo/anacidity and the secondary hypergastrinaemia in the affected cotton rats could be due to a process resembling Menetrier's disease in man. Patients with Menetrier's disease may have a marked hypergastrinaemia (14,15). It is also known that Menetrier's disease predisposes to gastric neoplasms (16,17).
The aetiology and pathogenesis of Menetrier's disease, like the gastropathy in our cotton rats, are unknown. However, an infectious agent could play a role (18). Interestingly, the human pathogen Helicobacter pylori has been reported to induce multiple hyperplastic polyps in Mongolian gerbils (19). It should also be added that cotton rats seem to be especially prone to develop infections (20).
The thickened oxyntic mucosa in our cotton rats consists of glandular hyperplasia in the areas of ECL cell neoplasia. In man, hyperplastic polyps (21) and glandular hyperplasia (22) have been described in the oxyntic mucosa together with diffuse gastric carcinomas. Furthermore, long-term omeprazole treatment, which induces ECL cell hyperplasia secondary to hypo/anacidity and hypergastrinaemia (23,24), has been shown to induce hyperplastic polyps in the oxyntic mucosa (25), indicating that the ECL cell plays an important trophic role (26).
In conclusion, in the pathogenesis of the changes of the oxyntic mucosa in affected cotton rats, hypergastrinaemia plays an important role by inducing ECL cell hyperplasia and neoplasia. Since the parietal cell density and H+/K+ ATPase mRNA concentration were close to normal, it is conceivable that the oxyntic mucosa produces normal quantities of acid. Therefore, the reduced gastric acidity most probably is due to neutralization of the acid by interstitial fluid escaping into the gastric lumen, as is found in Menetrier's disease in man. The aetiology and pathogenesis of these changes remain unknown. Studies on gastric acidity, blood gastrin and gastric mucosal structure in young cotton rats may possibly lead to a better understanding of this process.
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Notes
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2 To whom correspondence should be addressed at: Department of Medicine, Section of Gastroenterology, University Hospital of Trondheim, N-7006 Trondheim, Norway Email: helge.waldum{at}medisin.ntnu.no 
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Received July 23, 1999;
accepted September 22, 1999.