Induction of squamous cell carcinoma of forestomach in diabetic rats by single alloxan treatment

Abstract

Male rats of WBN/Kob strain are one of the diabetic model animals and develop long-lasting diabetic symptoms and some complications from about 40 weeks of age without any treatment. A single intravenous dose of alloxan, a non-genotoxic diabetogenic chemical, frequently induced proliferative lesions of squamous epithelium in tongue, esophagus and forestomach of male and female WBN/Kob rats, and hastened the onset and acceleration of diabetic conditions. Histopathologically, proliferative changes of squamous cell of forestomach varied with the severity of hyperplasia in alloxan-treated rats (100% of 31 males and 94.1% of 17 females) and progressed to SCC in approximately 20% of all rats. Metastasis to regional lymph nodes was also observed in two cases. Proliferative changes were most severe in the forestomach and were constantly accompanied with chronic suppurative inflammation of the mucosal epithelium with infection of filamentous fungi and/or bacterial colonies. In contrast, forestomach of the spontaneously diabetic male rats showed only slight hyperplasia of the mucosal epithelium confined to the limiting ridge in approximately 30% of the cases. All non-diabetic female rats showed neither proliferative changes nor the inflammatory process in the mucosa. Immunohistochemically, COX-2 and iNOS were positive in these chronic suppurative inflammatory lesions accompanied by proliferative squamous epithelium. From these results, it is suggested that chronic inflammatory processes play an important role in the pathogenesis of alloxan-induced SCC. An experimental system of alloxan-induced SCC might serve as a suitable model for the study of the inflammation-related promotion of carcinogenesis.

Figure 1

Experimental protocol, showing timeline of alloxan treatment in WBN/Kob rats with and without spontaneous diabetes.

Figure 2

Hyperplasia of squamous cell epithelium in forestomach. (a) Hyperplastic epithelium of the forestomach increased its cell layer with hyperkeratosis, but papillary protruding into the lumen was not detected. Hyp+, slight hyperplasia; Hyp+ +, moderate hyperplasia; Hyp+ + +, severe hyperplasia; Hyp+ + + +, significantly severe hyperplasia. (b) Invagination of basal layer of squamous epithelium into the lamina propria (Bar: 50 µm). (c) Keratinous cyst formation in submucosa. The frequency of cyst formation increased with age (Bar: 1 m).

Figure 3

SCC in forestomach. (a) Gross findings of gastric mucosa after fixation. Note irregular thickening of gastric wall in lesser curvature. (b) SCC was well‐differentiated but invaded diffusely into submucosal layer (Bar: 500 µm). (c) Adenosquamous cell carcinoma with invasion to muscular and serosal layer (Bar: 100 µm). (d) Metastasis of SCC to the pancreaticoduodenal lymph node (Bar: 100 µm).

Figure 4

Acute and chronic inflammatory changes with bacterial or mycotic infection in the mucosa of forestomach. (a) Acute inflammatory change in superficial mucous membrane characterized by emigration of neutrophils in erosive and ulcerative epithelium and microbial infection (Bar: 100 µm). Inset: Left micrograph is Escherichia coli (Giemsa stain), right micrograph is Candida albicans (Grocott stain). (b) Chronic inflammatory change with severe infiltration of lymphocytes and plasma cells into the lamina propria and deep submucosal layer (Bar: 100 µm).

Figure 5

Immunohistochemistry for COX‐2 and iNOS. (a) COX‐2 expression in mucosal epithelial cells with suppurative inflammation (Bar: 200 µm). Inset: Large magnification of positive cells (Bar: 50 µm). (b) COX‐2 expression in mucosal epithelial cells without inflammation. The degree of overexpression was the same as in the non‐inflammatory area (Bar: 200 µm). (c) Double staining for COX‐2 and vimentin. Vimentin was dyed red in the membrane of fibroblast, and COX‐2 was dyed brown in cytoplasm. Double‐positive cells for COX‐2 and vimentin were considered to be fibroblasts. (d) Double staining for COX‐2 and ED‐1. ED‐1 was dyed red in the membrane of macrophage, and COX‐2 was dyed brown in cytoplasm. Double‐positive cells for COX‐2 and ED1 were considered to be macrophages. (e) iNOS was positive in cytoplasm of mononuclear inflammatory cells in suppurative inflammatory lesion (Bar: 200 µm). Inset: A large magnification of positive cells (Bar: 50 µm).