Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method
Summary
Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.
Abstract
An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.
Introduction
Mammary gland branching is a characteristic that is commonly assessed as an indicator of gland development, but it is difficult to objectively quantify. In 1953, Sholl1 described a method for measuring neuronal dendritic arborization in the visual and motor cortices of the cat, and a plugin for this technique was developed by Ferriera et al2. Because both neurons and mammary glands exhibit a similar tree-like structure, the plugin was employed to quantify mammary epithelial branching densities in 2D images of the peripubertal rat mammary gland. The peripubertal stage was chosen for analysis because weaning is a life stage that is often assessed in academic laboratories and test guideline studies. The Sholl analysis is a plugin distributed with FIJI, which is the open-source image processing package ImageJ, with additional plugins included. The plugin creates a series of concentric rings encircling a predefined center (typically the soma of a neuron or the origin of the primary duct of a mammary gland) and extending out to the distal-most portion of the object (the enclosing radius). It then counts the number of intersections (N) that occur on each of the rings. The plugin also returns a Sholl regression coefficient (k), which is a measurement of the rate of decay of epithelial branching.
Using ImageJ, a skeletonized image of a mammary gland whole-mount is created and the mammary epithelial area (MEA) is measured. The image is analyzed using the Sholl analysis plugin, and values for N and k, among other values not utilized here, are returned. Mammary epithelial branching density is determined by calculating N/MEA. The extent to which branching continues in the outer regions of the glandular epithelium is the branching complexity and is an indicator of uniform distal epithelial growth. As k is a measure of the distal decrease in epithelial branching, it is an effective measure of the branching complexity and a reliable indicator of mammary development.
This protocol describes a computer-assisted method for creating skeletonized images of mammary gland whole-mounts and quantitatively evaluating mammary branching characteristics in peripubertal male and female rats. This method is relatively rapid and does not require the use of specialized microscopy equipment. Development and validation of this method are described in Stanko et al. (2015)3. This report also describes preparation of rat mammary gland whole=mounts. Similar mammary whole-mount procedures have been described in de Assis et al. (2010)4 and Plante et al. (2011)5.
Protocol
Representative Results
Discussion
From birth until puberty, mammary gland growth is allometric. After puberty, the mammary gland develops through extensive ductal branching and elongation, which continue until the mammary epithelium occupies the entire fat pad. Branching characteristics are an important aspect of mammary gland development, and the ability to objectively quantify these characteristics can be highly useful for assessing normal mammary development and for identifying abnormal development following early life exposures to mammary toxicants.<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Dr. Michael Easterling (Social and Scientific Systems, Inc., Durham, NC) for his assistance with the validation of this method and Dr. Tiago Ferreira (McGill University, Montreal, Quebec, Canada) for his continual assistance with the Sholl application.
Materials
| Dissecting board | NA | NA | A piece of styrofoam roughly 10"x12" is suitable. |
| Dissecting T-Pins | Daigger | EF7419A | |
| Spray bottle with ethanol | NA | NA | 70% ethanol is suitable. |
| Curved dissecting scissors | Fine Science Tools | 14569-09 | |
| Straight dissecing scissors | Fine Science Tools | 14568-09 | |
| Curved forceps | Fine Science Tools | 11003-12 | |
| Superfrost Plus 24x75x1 mm microscope slides | ThermoFisher Scientific | 4951PLUS-001 | Thermo Scientific Superfrost Plus & Colorfrost Plus slides hold tissue sections on permanently without the need for expensive coatings in IHC and Anatomical Pathology applications. This treatment reduces tissue loss during staining as well as hours of slide preparation. Slides electro-statically attract frozen tissue sections and cytology preparations and feature a chemistry similar to silane, although optimized to improve application performance. https://www.thermofisher.com/order/catalog/product/4951PLUS4. |
| Fisherfinest Premium Cover Glass 24x60x1 mm | Fisher scientific | 12-548-5P | |
| Bemis Parafilm M Laboratory Wrapping Film | Fisher scientific | 13-374-12 | |
| Chloroform | Sigma-Aldrich | C2432 | |
| Glacial acetic acid | Sigma-Aldrich | A9967 | |
| Ethanol absolute, ≥99.8% (GC) | Sigma-Aldrich | 24102 | |
| Xylene | Sigma-Aldrich | 214736 | |
| Carmine alum | Sigma-Aldrich | C1022 | |
| Aluminum potassium sulfate | Sigma-Aldrich | A6435 | |
| Permount mounting media | Fisher Scientific | SP15 | |
| Macroscope | Leica | Z16 APO | This is the image capturing hardware and software used in this laboratory. As there are many different options, the methods and applications may vary between laboratories. |
| Digital camera | Leica | DFC295 | |
| Camera software | Leica | Leica Application Suite v3.1 | |
| ImageJ software | Open source | http://imagej.net/Welcome | |
| Sholl analysis | Open source | http://imagej.net/Sholl_Analysis |
References
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