Semi-automated Method of Quantifying Vasculature of 1-Methyl-1-nitrosourea-induced Rat Mammary Carcinomas Using Immunohistochemical Detection

Abstract

S U M M A R YStudies of the vascularization of autochthonous rodent mammary tumors are limited in number, and the majority have used Factor VIII staining for blood vessel detection. Moreover, little effort has been directed at measuring the vascularization of tissue immediately adjacent to a tumor despite its central importance in the process of angiogenesis. Thirty-six chemically-induced mammary carcinomas and tissue immediately adjacent to these carcinomas were used to develop a census counting method for quantitative assessment of intra-and extra-tumor vascularization. Blood vessels were identified using antiserum directed against either CD31 or Factor VIII. Techniques used to create digitized images of all tumors and the semi-automated methods for circumscribing the extra-tumoral region are described. For Factor VIII, CD31 allowed greater discrimination of blood vessels with areas Ͻ 25 m 2 and demonstrated crisp staining of blood vessels, with minimal background and excellent preservation of tissue architecture. Census counting data support the use of CD31 for quantifying both intra-and extra-tumoral vascularization. This method provides a basis for standardizing the approach to evaluation of experimentally induced premalignant and malignant mammary lesions in rodent model systems used to investigate potential anti-angiogenic cancer preventive agents.

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“…Present study exhibited comparison with placebo effect and control. These in vivo breast tissue studies and in vitro literature reports both corroborate on increased intracellular sodium MR image intensity following chemotherapy at subphysiological levels [20,21]. However, intervention of hypoxia or ouabain poisoning further increases [Na] i and IC-Na longitudinal T1 constants [20,21].…”

“…Taxotere injected doses were at the higher end of the maximum tolerable dose (MTD) range (40 nmol/L) for rat above the clinical levels (10 nmol/L) for 24 hours[21,22]. Present study exhibited comparison with placebo effect and control.…”

Effect of Dietary Energy Restriction on Vascular Density during Mammary Carcinogenesis

“…The method for immunostaining of mammary tissue for blood vessel detection has been reported in detail (15). Briefly, paraffin sections from formalin fixed mammary pathologies were cut at 4-m and mounted onto 3-aminopropyltriethoxysilane-treated slides.…”

“…On the basis of the reports from our laboratory (15,16), experiments were conducted to determine how dietary energy restriction modulated blood vessel density during premalignant and malignant stages of mammary carcinogenesis. The effects of dietary energy restriction on blood vessel density prompted the assessment of mammary carcinomas by cDNA microarray analyses to determine whether genes associated with blood vessel development and growth were differentially regulated by dietary energy restriction.…”

A Method for Serial Tissue Processing and Parallel Analysis of Aberrant Crypt Morphology, Mucin Depletion, and Beta-Catenin Staining in an Experimental Model of Colon Carcinogenesis

“…Based on our extensive work in developing techniques appropriate for preparing mammary gland whole mounts [17-19] and carrying out image analysis on digitally captured images of mammary tissue [20,21], we postulated that whole mounts of colon could be prepared by directly adhering the tissue to glass microscope slides and the whole mounted tissue then be used for semi-automated acquisition of contiguous images of the entire colonic mucosa; this would circumvent many of the limitations listed above. Moreover, we undertook the development of approaches to permit methylene blue staining, image acquisition, re-staining with high iron diamine–alcian blue (HID-AB) and further image acquisition in order to address current questions about the relevance of ACF detected by methylene blue versus those in which mucin depletion can be detected as predictors of subsequent development of colon carcinomas [5,9,22-24].…”