Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tissues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.
ABsTRAcrWe investigated the development of early neoplastic lesions preceding the appearance of kidney epithelial tumors in rats treated with a single iv injection of 35 mdkg of streptozotocin (STZ). Most of these lesions were associated with segments of atrophied and regenerative nephrons surrounded by a thick basal membrane that appear precociously in chronic progressive nephropathy. Cytochemical periodic acid-SchilT, Alcian blue, and colloidal iron reactions did not indicate an excessive storage of glycogen or acid mucopolysaccharides in early neoplastic lesions and tumors. Quantitative cytochemistry of mitochondria1 succinate, a-glycerophosphate, and reduced nicotinamide adenine dinucleotide-dehydrogenases revealed a shift in metabolism toward glycolysis in atrophied and regenerative nephrons as well as in early neoplastic lesions and tumors. These correlative cytomorphological and cytochemical findings raise the possibility that changes associated with the initial stages of chronic progressive nephrosis may provide favorable conditions for the selective growth of STZ-initiated cells that generate focal collections of proliferating cells and then progress to tumor growth. Tumor prevalence was remarkably constant in animals sacrificed 33,48, a.nd 54 wk after treatment, suggesting that the prominent inflammatory and scarring reaction later developing in the course of progressive nephrosis might contribute to control the growth of STZ-induced cancer.Keywords. Renal epithelioma; nephropathy; interstitial nephritis; histochemistry; image cytometry; image analysis INTRODU~ION Streptozotocin (STZ), a naturally occurring antibiotic (2-deoxy-2-[([methylnitroso-amino] carbonyl)amino]-D-glucopyranose), is reported to induce kidney adenomas and carcinomas when administered to rats and mice. A single injection of STZ is sufficient to evoke a carcinogenic effect (1 8, 19,23).STZ is also a powerful diabetogenic agent that destroys beta cells of the pancreas (21,36) with profound effects on the metabolism and physiology-of: several organs (4, 11, 16, 27, 28, 30, 31, 34). In STZ-treated rats, epithelial tumors develop in kidneys affected by an extensive clear cell degeneration of distal nephrons (1 6, 30). It is not known whether or not these tumors originate from clear cells. Comparative bioassays conducted in rats with alloxan, a similar diabetogenic agent, suggest that neoplastic growth is due to adirect toxic effect of STZon kidney cells and not to the induced diabetes (23).During the course of experimental studies on diabetic condition conducted on several STZ-treated rats over a period of the last 4 yr, we microscopically followed the appearance of STZ-induced renal tumors.The present study provides a morphological and cytochemical analysis of early neoplastic foci and tumors arising after STZ administration. METHODS Anintals.Male Sprague-Dawley rats (from Charles River, Como, Italy), with initial weights of 300 f 20 g, were housed 4 per cage in polycarbonate cages maintained in an environmentally controlled room with ...
Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tissues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.
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