This study identified mucus granules, determined mode of release and quantified their volume in the gallbladder epithelium of Richardson's ground squirrels (Spermophilus richardsonii) fed a lithogenic diet of 2% cholesterol to experimentally induce gallstone formation. Tissue was examined using light microscopy histochemistry, scanning and transmission electron microscopy, as well as autoradiography and morphometry at the electron microscopic level. Autoradiography demonstrated incorporation of a glycoprotein precursor, [3H]galactose, within the membrane-bound granules localized in the supranuclear region of the epithelial cells. Exocytosis of granule contents was by merocrine secretion. Morphometry indicated a significant increase in the amount of intracellular mucin granules as early as 18 hr on the lithogenic diet, a feature that continued throughout the experimental period of 20 weeks. Mucus synthesis/secretion rates returned to control values within 3 weeks after removal from the diet. Scanning electron microscopy observations revealed a thick sludge-like layer overlying the epithelium at a time in the chronology of the cholelithiasic model that correlated well with the initial phases of stone formation. Histochemistry showed this layer to be a mixture of acidic mucins. Neutral mucins were not observed. The hypersecretion of mucus and formation of this sludge-like layer appear to be critical nucleating factors in the formation of cholesterol gallstones.

Richardson's ground squirrels (Spermophilus richardsonii) of both sexes were fed a 2 per cent cholesterol-enriched diet for intervals of 12, 18, and 24 h; 3, 5, and 7 days; and 2, 3, 10, and 20 weeks. It was shown that free (unesterified) cholesterol, phospholipid, and cholesterol ester accumulated in specific regions of the gallbladder mucosa during cholelithiasis. Electron microscopy revealed the presence of lipids inter- and intracellularly as early as 12 h after ingestion. By 7 days, lipids were seen in dilated endoplasmic reticulum, as well as in supranuclear and basal regions of epithelial cells. Histochemical localization revealed free cholesterol in dilated endoplasmic reticulum and residual bodies at the ultrastructural level. Neutral lipid was observed by light microscopy in the supranuclear and basal regions of the cells. In 10- and 20-week treated animals, lipid droplets were also seen in the lamina propria and macrophages. The lesion induced by cholesterol ingestion persisted throughout the experimental period, and while different from that in human tissue, it was similar to those observed in experimental canine cholesterosis.

Richardson's ground squirrels (Spermophilus richardsonii) of both sexes were fed a 2 per cent cholesterol-enriched rat chow diet for intervals of 1, 2, 10 and 20 weeks. Light microscopy and 3H-thymidine autoradiography revealed an increase in cell proliferation prior to the occurrence of macroscopically visible stones, but in the presence of crystals and microliths. Transmission electron microscopy studies showed that columnar epithelial cells undergo mitosis rather than basal cells and that oedematous cells were extruded from the epithelial sheet. Transmission and scanning electron microscopy studies on gallbladders of animals fed the lithogenic diet for 10 and 20 weeks revealed damaged epithelial cells either singly or in groups. Neighbouring cells often slide under the basal aspects of cells being extruded. Hypertrophy, hyperplasia, Rokitansky-Aschoff sinuses, thickening of the lamina propria around the muscle bundles and inflammatory cells in the lamina propria began to occur about the time macroscopically visible stones were present. The epithelium of the Richardson's ground squirrel gallbladder is damaged more slowly than that of other animal models by a cholesterol-enriched, lithogenic diet and may more accurately reflect changes occurring in human cholecystitis.

No abstract

The cholesterol-fed Richardson's ground squirrel (Spermophilus richardsonii) has proven to be an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium. Ground squirrels of either sex, fed a 2% cholesterol-enriched diet, exhibit cholesterol monohydrate crystal precipitation within 24 hours and macroscopically visible cholesterol stones by 3 weeks. Data on bile chemistry, biliary cholesterol precipitation, and various mucosal alterations occurring prior to, during, and after stone formation were collected using sampling intervals from 6 hours to 20 weeks on the diet. The results indicate that mucin hypersecretion appears to be more closely related to the initiation of nucleation than does either bile calcium of pH. Mucus hypersecretion begins within 18 hours of diet initiation and continues throughout the 20 week experimental period. Apical excrescences became more common and were larger in size during the early stages of cholelithiasis. Administration of aspirin during the experimental period demonstrated an inhibition of mucin synthesis and release. Gallstones were not formed in these aspirin-treated animals. A lectin-binding panel for 10 epithelial glycoprotein-related sugars indicated the mucin secreted by the gallbladder epithelium of 7 day experimental animals differed from that of controls. The most obvious difference was the abolition of WGA binding in the experimental animals, suggesting an absence of sialic acid expression in the mucin during the lithogenic process. Ultrastructural histochemistry indicated that both sulphomucin and sialomucin were present in the secretory granules and within the surface mucus layer of both experimental and control animals. Experimental animals, however, exhibited a significant predominance for sulphomucin. This pattern varies from that typically seen in other regions of the gastrointestinal tract where sialomucins predominate during pathologic processes.