Name: a new technique for quantitative analysis of hair loss in mice using grayscale analysis
Uploaded: 2015-03-09T13:00:00.000+00:00
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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. Verify uniformity of light source across photographed region. To help ensure uniformity, use a gel imager with a built in light source for reflective photography. Do not use a transillumination light source, as this would create a silhouette of the animal which is unsuitable for further grayscale analysis. 
	NOTE: This will place the mouse slightly out of focus, which provides optical averaging across the region of interest (ROI) and will help reduce quantal errors for very small (i.e., &#60;10 pixel) regions of interest. Larger regions of interest will not be affected by this minor focusing adjustment, nor will they be affected by differences in the size of the animal.</li>
	<li>Anesthetize animals using ketamine (100 mg/ml)/xylazine (20 mg/ml) (2:1), as this provides rapid anesthesia effect and rapid recovery and is optimal for photographing multiple animals. 
	NOTE: Anesthesia is confirmed when the animal is still enough to permit photography. As animals typically recover from this light anesthesia within 10-15 min, the veterinarian has not recommended use of eye ointments.</li>
	<li>Place anesthetized animals on gel imager in vertical alignment (as close to parallel as possible).
	<ol>
		<li>For dorsal photographs, place the animals in prone position with limbs extended.</li>
		<li>For ventral photographs, place animals in the supine position, taking care that the animals are not laterally rotated.</li>
	</ol>
	</li>
	<li>Place grayscale standard in photographed region.</li>
	<li>Close access door. Important: Ambient light can introduce variations in exposure.</li>
	<li>Set F-stop to an exposure which places the region of interest within the linear range of acquisition (reading F-stop). 
	NOTE: Most systems will display where the image is saturated.</li>
	<li>Take photograph.</li>
	<li>Change F-stop to another exposure setting which places the region of interest within the linear range by increasing or decreasing the F-stop by 1, such that both the standard and the region of interest remain in a linear range of acquisition (reference F-stop).</li>
	<li>Take photograph.</li>
	<li>Once photography is completed, place animals on a warming table and monitor until they can maintain sternal recumbency. Return animals to their cages. Return the group of animals to the vivarium once all animals are fully recovered.</li>
</ol>
2. Quantification of Light Absorption
<ol>
	<li>Mark regions of interest on the images of the animals using provided software for gel imager.
	<ol>
		<li>For whole animal dorsal view, use a rectangular or oval image extending from the upper limbs to the lower limbs, extending laterally as much as possible such that no part of the box extends beyond the back of the animal as shown in Figure 1A. 
		NOTE: One could also mark the area of interest using a free-hand drawing tool.</li>
		<li>For whole animal ventral view, use 2 rectangles: one covering the pelvic region and one covering the chest area as shown in Figure 1B.</li>
		<li>For a smaller region of interest, i.e., location of drug administration, mark as appropriate.</li>
	</ol>
	</li>
	<li>Mark region(s) of interest on grayscale absorption standard.</li>
	<li>Record absorption from each of the marked regions of interest.</li>
</ol>
3. Analysis
Absorption levels obtained for the regions of interest may need to be normalized to the background standard for comparison between photographs. The relationship of exposure to absorption is log-log (i.e., the relationship is linear between log(exposure) and log(absorption)). Using this relationship, the absorption of the region of interest can be normalized to a standard background value, which will allow absorptions to be compared directly between different photographs, including those taken at different time points (i.e., serial measurements within a study protocol). The procedure for performing these corrections is detailed in optional steps 3.1 and 3.2.
<ol>
	<li>(Optional) Plot curve of log (exposure) vs. log(absorption) using values obtained from grayscale absorption standard.</li>
	<li>(Optional) Adjust for variations in the standard of each photograph as follows:
	<ol>
		<li>Select F-stop for reading (as determined in step 1.6) and F-stop for reference (as determined in step 1.8).</li>
		<li>Calculate average absorption of the standard in all photographs at the reading F-stop. This average is the Reference Standard Value (RSV).</li>
		<li>Calculate difference in absorption between the reading and reference F-stop settings in each photograph for the standard (delta-S) and for each defined ROI (delta-ROI-1, delta-ROI-2 &#8230;..delta-ROI-X)</li>
		<li>Calculate the corrected absorption for each ROI as follows: Corrected absorption (ROI-X) = Absorption (ROI-X) at reading F-stop + (RSV - absorption of standard at reading F-stop) * (delta-ROI-X /delta-S)</li>
	</ol>
	</li>
	<li>Compile experimental data and perform data analysis using standard statistical techniques for continuous variables (i.e., ANOVA, T-test).</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alopecia:+effect+on+cancer+patients'+body+image.">Alopecia: effect on cancer patients' body image.</a> Cancer Nurs. 7 (6), 499-503 (1984).</li><li>Stough, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+effect,+pathophysiology,+and+management+of+androgenetic+alopecia+in+men.">Psychological effect, pathophysiology, and management of androgenetic alopecia in men.</a> Mayo Clin Proc. 80 (10), 1316-1322 (2005).</li><li>Ogunmakin, K. O., Rashid, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alopecia:+the+case+for+medical+necessity.">Alopecia: the case for medical necessity.</a> Skinmed. 9 (2), 79-84 (2011).</li><li>Olsen, E. 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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+evaluating+hair+growth.">Methods of evaluating hair growth.</a> Australas J Dermatol. 44 (1), 10-18 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Kodak/Tiffen Q-13 Gray Scale*. , .</li><li>Freyschmidt-Paul, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+alopecia+areata+in+C3H/HeJ+mice+with+the+topical+immunosuppressant+FK506+(Tacrolimus).">Treatment of alopecia areata in C3H/HeJ mice with the topical immunosuppressant FK506 (Tacrolimus).</a> Eur J Dermatol. 11 (5), 405-409 (2001).</li><li>Katikaneni, R., Ponnapakkam, T., Matsushita, O., Sakon, J., Gensure, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prevention+of+chemotherapy-induced+alopecia+with+PTH-CBD,+a+collagen-targeted+parathyroid+hormone+analog,+in+a+non-depilated+mouse+model.">Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.</a> Anticancer Drugs. 25 (1), 30-38 (2014).</li><li>Peters, E. M., Foitzik, K., Paus, R., Ray, S., Holick, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+modulating+chemotherapy-induced+alopecia,+using+PTH/PTHrP+receptor+agonist+and+antagonist.">A new strategy for modulating chemotherapy-induced alopecia, using PTH/PTHrP receptor agonist and antagonist.</a> J Invest Dermatol. 117 (2), 173-178 (2001).</li><li>Katikaneni, R., Ponnapakkam, T., Seymour, A., Sakon, J., Gensure, R. 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Authors have nothing to disclose.

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><tbody><tr><td>KODAK Gel Logic 100 Imaging System</td><td>Eastman Kodak Company, Rochester, NY, USA</td><td></td><td>Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td></tr><tr><td>The Kodak/Tiffen Q-13 Gray Scale</td><td><a href="http://www.imatest.com/docs/q13/" target="_blank"><img src="/img/new_window.png" /> Imatest</a></td><td></td><td>Greyscale standard</td></tr><tr><td>C57BL/6J Mice</td><td>Jackson Laboratories, Bar Harbor, Maine</td><td></td><td>Mice for representative study</td></tr><tr><td>C3H/HeJ engrafted mouse</td><td>Jackson Laboratories, Bar Harbor, Maine</td><td></td><td>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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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.9K Views.  Children's Hospital at Montefiore. 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.

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

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Neuroscience

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Medicine

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

Developmental Biology

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

Bioengineering

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic.
Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

Technika przeszczepiania ludzkiej skóry w modelu mysim

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

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Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

Technika przeszczepiania ludzkiej skóry w modelu mysim