Name: a new technique for quantitative analysis of hair loss in mice using grayscale analysis
Uploaded: 2015-03-09T13:00:00
Description: Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

Ethics Statement: All studies involving animals must be approved by the IACUC of the institution (for the data which follows, protocols were approved by the Montefiore IACUC, protocol #11-6-240 and #13-7-100). Animals are provided light anesthesia as indicated for the sole purpose of keeping them still during photography, there are no painful procedures required for this protocol.
1. Acquisition of Photographs
<ol>
	<li>Set focus and field of view for gel imager using paper with printed text...

<ol>
	<li>McAndrews, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Hair+Loss+Association.">American Hair Loss Association.</a> , (2011).</li><li>Safavi, K. H., Muller, S. A., Suman, V. J., Moshell, A. N., Melton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+alopecia+areata+in+Olmsted+County,+Minnesota,+1975+through+1989.">Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989.</a> Mayo Clin Proc. 70 (7), 628-633 (1995).</li><li>Hussein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy-induced+alopecia:+new+developments.">Chemotherapy-induced alopecia: new developments.</a> South Med J. 86 (5), 489-496 (1993).</li><li>Trueb, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+clinical+trial+of+5%+topical+minoxidil+versus+2%+topical+minoxidil+and+placebo+in+the+treatment+of+androgenetic+alopecia+in+men.">A randomized clinical trial of 5% topical minoxidil versus 2% topical minoxidil and placebo in the treatment of androgenetic alopecia in men.</a> J Am Acad Dermatol. 47 (3), 377-385 (2002).</li><li>Rossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1+mg+daily+administration+on+male+androgenetic+alopecia+in+different+age+groups:+10-year+follow-up.">1 mg daily administration on male androgenetic alopecia in different age groups: 10-year follow-up.</a> Dermatol Ther. 24 (4), 455-461 (2011).</li><li>Gibbons, R. D., Fiedler-Weiss, V. 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In this report a detailed description is provided of a new technique for quantifying hair loss in rodents. This technique utilizes a gel imager for image acquisition and analysis, equipment which is readily available in most laboratories. The measurements have been shown to be robust to minor variations in technique (Figures 4-5), and are well-correlated to the degree of visual hair loss in treatment models (Figure 6). These techniques have been successfully employed in ...

Alopecia (hair loss) can be a psychologically and emotionally distressing event with multiple causes. Male-pattern baldness is the most common cause of alopecia, affecting approximately two thirds of males by age 351. A similar pattern of hair loss can be observed in females with polycystic ovarian syndrome. In both of these disorders, the hair loss is androgen mediated. Alopecia can also occur as an autoimmune disease, alopecia areata, which affects 1.7% of the population2.&#160; Alopecia can occur as a side-effect of some medical treatments, such as chemotherapy3.&#160; A high percentage (65-85%) of chemotherapy patients experience some degree of alopecia4,5. The psychological consequences of hair loss have been well studied in the chemotherapy setting. Chemotherapy-induced alopecia can result in anxiety, depression, a negative body image, lowered self-esteem and a reduced sense of well-being6,7. A high percentage (47-58%) of female cancer patients consider hair loss to be the most traumatic aspect of chemotherapy, and up to 8% decline treatment for fear of hair loss4,6. There is also evidence in androgenic alopecia to support therapy to reduce psychological and even medical consequences of hair loss8,9. Likewise, alopecia areata has been reported to have severe psychological consequences2, and the patchy nature of the hair loss can lead to a more unpleasant cosmetic result than most other causes of hair loss.
While drugs with mild anti-androgenic effects (i.e., spironolactone) had been used with limited success as therapy for alopecia, the first effective medication for alopecia was minoxidil10. This antihypertensive has an observed side-effect of causing hair growth, and is now used as topical therapy for many forms of alopecia. However, responses are often incomplete, with some subjects showing only slowing of hair loss rather than actual regrowth10. Finasteride is a competitive antagonist to type II 5&#945;-reductase which blocks conversion of testosterone to dihydrotestosterone, resulting in improvements in androgenic alopecia at the expense of partial systemic androgen blockade. Response rates with long-term (10 years) therapy are around 50%11. Overall, despite considerable research in this area, there is still no adequate therapy for hair loss.
For decades, scientists and clinicians have examined methods of measuring scalp hair growth in clinical trials. With the development of drugs that treat alopecia, there has been a greater need for reliable, economical and minimally invasive means of measuring hair growth and, specifically, response to therapy. Image analysis technology for a precise quantification of hair density in patients with hair loss disorders yielded consistent and valid results in the past using a variety of techniques, including analysis of digitized images12, image analysis of individual hairs and skin lesions13, and microscopic scanning to quantify hair mass in a defined scalp region14 .
Unfortunately, while the above methodologies have provided improved assessment of efficacy for hair growth-promoting interventions in clinical trials, these methods have not been applied to rodent studies in preclinical investigations. Our goal is to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to more accurately assess and compare new therapeutic approaches for various forms of alopecia. We have developed a methodology using equipment readily available in most laboratories which will allow rapid and reliable quantification of hair density in mice with brown or black hair. This methodology has been tested in mouse models of chemotherapy-induced alopecia, alopecia areata, and alopecia from waxing. A detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. As this technique relies on detecting light absorption from pigments in the hair shaft, it cannot be used to detect hair growth in white mice or albino mice.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

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 male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

This technique was validated using C3H/HeJ engrafted mice, the mouse model of alopecia areata16. These animals develop global hair loss which progresses gradually over time. However, the hair loss occurs in patches, and may vary from one mouse to the next, introducing considerable variability and hampering qualitative assessments. This model provided an opportunity to test correlations between measurements of optical density at different locations on the mouse as an assessment of validity.
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Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

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.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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