A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

The overall goal of this procedure is to provide a quantification of hair density using light absorption. This is accomplished by first photographing animals on a gel imager. The next steps are to define a region of interest on the photograph and determine the light absorption within the region of interest.

Then light absorption is compared to a photographic standard. Ultimately, light absorption can be averaged between similarly treated animals to show the effects of an intervention on hair growth. The main advantages of this technique are that it's unbiased, quantifiable, and reproducible.

This is extremely important because it allows you to use standards, statistical techniques like Inova to determine the statistical significance of the results. For this protocol, use a gel imager with a built-in light source for reflective photography. This ensures good light uniformity, whereas trans illumination creates silhouetting unsuitable for analysis.

Begin by preparing the gel imager, set the focus and the field of view. Using some printed text, the mouse can be slightly out of focus as this actually works. To reduce quantal errors in small regions of interest, then verify the uniformity of light source across the photographed region with the goal of excellent optical averaging at the region of interest.

Next, prepare the animal. Select an anesthetic with rapid onset and short duration of action and deliver just enough to keep the animals still for the photography. Now place the animals on the imager vertically and as close to parallel as possible.

For dorsal photographs, place the animals in a prone position with their limbs extended. For ventral photographs, place the animals in a supine position. Make sure that they are not laterally rotated.

Absorption values can be pulled between images, but this is not what the machine was designed for. So adding a gray scale standard allows us to normalize the absorption values when compiling the data. Now place the gray scale standard in the photographed region.

Then close the chamber and reduce ambient illumination that could enter the chamber on the camera. Set the F stop to an exposure that makes the area of interest not oversaturated or underexposed. Then take a photograph.

If the F stop is chosen such that the image is saturated, the gray scale value will be fixed at the value and B of no use whatsoever. Then change the F.Stop by one, keeping the area of interest and the standard in a linear exposure range, and take a second photograph with the photography completed. Put the animals on a warming table and monitor them until they recover.

Sternal recumbent. Then return the animals to their cages and ultimately to the vivarium for full recovery. To quantify the light absorption using the gel imaging software, mark the regions of interest on the images of the animals for a whole animal dorsal view.

Use a rectangular image, an oval image, or a free hand tool to outline a space from the upper limbs to the lower limbs, extending laterally as much as possible. Keep within the margins of the back of the animal for a whole animal ventral view. Use two rectangles.

One to cover the pelvic region, which is groomed, and one to cover the chest area, which is not groomed. Smaller regions of interest such as where a drug was administered can also be marked as required. Second, mark the region of interest on the gray scale absorption standard.

Then record the absorption from each marked region of interest for comparison between photographs. Normalize the absorption levels to the background standard using a log log relationship of exposure to absorption. To do this plot a curve of the log of exposure against the log of absorption.

Using values obtained from the gray scale absorption standard, then adjust for variations in the standard of each photograph as follows. First, select the F-stop for reading the ROI and the F-stop for the reference and calculate the average absorption of the standard in all the photographs at the reading F-stop. This average is the reference standard value or the RSV.

Next, calculate the difference in absorption between the reading and reference F-Stop settings in each photograph and make the same calculations for the standard and for each defined ROI. Now using data from one F-stop, calculate the corrected absorption for each ROI using the shown formula. Following this, apply standard statistical methods to analyze the data C3 HHEJ engrafted mice, which model alopecia ADA were analyzed using the described method at different exposures.

Variations in non-overlapping ROI were tightly correlated as expected. Similar comparisons produced the same tight correlation over a three week period. The mice were imaged to quantify their expected hair loss.

80%of the imaged mice showed hair loss in this timeframe. By comparison, normal C 57 black six mice did not show any hair loss in the same timeframe. In a second study, chemotherapy induced alopecia was quantified using the described technique.

Mice were treated with cyclophosphamide for three weeks and after two months they showed significant hair loss compared to controls. Once mastered, each image can be analyzed in less than five minutes. After watching this video, you should have a good understanding of how to quantify the amount of hair growth in rodents using a standard gel imaging system.