Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method
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.
Figure 1. Ventral View.
Image of the ventral portion of an adult female Sprague Dawley rat, illustrating how to secure the rat on the dissecting surface and the location of the 12 mammary glands, with the nipples circled. * The nipples of glands 6 and 7 are not visible.
- Figure 1 (61 KB)
Figure 10. Intersections Mask.
When the "Create Intersections Mask" option is selected (step 4.3.7), the analysis will output a heat map of the number of intersections across the enclosing radius of the image. This heat map reflects the density of branching intersections throughout the epithelium (red = hot = high density; blue = cold = low density). The entire epithelium would be the same color in a heat map of an image where k = 0.
- Figure 10 (99 KB)
Figure 2. Female Rat Mammary Gland.
Illustration of exposed mammary glands 4 (MG4) and 5 (MG5), with the skin pinned to the dissecting surface above MG4 and just below MG5. The glands should be removed from the skin beginning with MG5 and continuing up and dorsally until MG5 and 4 are completely removed. The nipple is in the distal area of gland 4, and care should be exercised to collect this area. The lymph node is indicated for reference.
- Figure 2 (102 KB)
Figure 3. Mammary Gland Whole-mount.
A whole-mount image of a mammary gland collected from a postnatal day 25 female Sprague Dawley rat. Scale bar =1 mm.
- Figure 3 (83 KB)
Figure 4. Removal of Noise.
The blue color channel of a mammary whole-mount image, with the background subtracted. (A) demonstrates examples with noise. The arrows indicate noise created by blood vessels, and the more heavily shaded region surrounding the ductal ends is an example of noise created by subtracting the background. (B) illustrates the image after the noise has been removed.
- Figure 4 (37 KB)
Figure 5. Image Reconstruction.
Reconstruction of the erased portions of the thresholded image. (A) The red arrows indicate regions where portions of the image were lost due to thresholding. Image reconstruction should be performed at these regions.
(B) Mammary image after reconstructing the deleted regions. Image reconstruction should be conducted carefully and on a minimal basis so as to maintain the integrity of the original image.
- Figure 5 (43 KB)
Figure 6. Overlay of a Skeletonized Image.
Overlay image showing a skeletonized image overlaid onto the original whole-mount image. This image demonstrates that the skeletonized gland reflects the branching of the actual gland with a high degree of accuracy.
- Figure 6 (76 KB)
Figure 7. Enclosing Radius.
Skeletonized image of a mammary whole-mount showing where the enclosing radius is measured (yellow). The line should begin at the base of the epithelial tree (center of analysis) and extend to the most distal point of the epithelium.
- Figure 7 (98 KB)
Figure 8. Mammary Epithelial Area.
Skeletonized image showing a polygon traced around the epithelial tree to determine the MEA.
- Figure 8 (108 KB)
Figure 9. Sholl Plot Output.
Sholl output of linear (A) and semi-log (B) plots of the number of intersections at each radial increment. The red dot in panel (A) is the abscissa of the centroid (geometric center). In panel (B), the blue line is the linear regression over the full range of data, while the red line is the linear regression over the 10th-90th percentile.
- Figure 9 (389 KB)
Table 1. Sholl Analysis Parameters.
- Table 1 (45 KB)
The video component of this article can be found at https://www.jove.com/video/55789/