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

Abstract

The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity.

Effect of dietary resistant starch on inhibition of preneoplasia in azoxymethane-induced rodent models

“…The early onset of these lesions facilitates the use of short-term assays in animal models. The continued use of ACF have identified that ACF were most commonly found in the distal regions of the colon, with the number of ACF decreasing as it reaches the proximal end of the colon [55]. ACF are most often carcinogen-induced but can also be induced by sporadic mutation.…”

“…The foci multiplicity of the MDF is a common characteristic that needs to be recognized when analyzing colon tissues for this lesion. Like ACF, the highest frequency of MDF is found in the distal region of the colon [55]. Artifacts are common in colonic tissue that can be caused by damage by instruments or gut-associated lymphoid tissue (GALT) which can also be devoid of HID-AB stain causing GALT to be mistaken for MDF [55].…”

“…Like ACF, the highest frequency of MDF is found in the distal region of the colon [55]. Artifacts are common in colonic tissue that can be caused by damage by instruments or gut-associated lymphoid tissue (GALT) which can also be devoid of HID-AB stain causing GALT to be mistaken for MDF [55]. progresses toward a tumor growth [53].…”

“…proposed that these two preneoplastic lesions are closely related [53][54][55][56][57][58]. A number of studies have touched on this subject.…”

“…Many researchers have found that the relationship between ACF and tumor formation is not clear[52,53,57,61,[67][68][69] and therefore, additional biomarkers are needed. McKinley et al 2010 note a few issues with the current approach to ACF analysis: 1) Additional steps are required to remove tissue from fixative solutions, 2) Difficult to obtain high quality images from free-floating samples, 3) Fatigue and error of observer, and 4) lack of consistent sample orientation (proximal versus distal)[55,57,[64][65][66]. There is also an issue with the idea that ACF are preneoplastic and their use in short-term assays compared to tumors being the endpoint of long-term assays.…”