Chapter 10 Skin, Hair, and Nails John P. Sundberg, John P. SundbergSearch for more papers by this authorLloyd E. King Jr, Lloyd E. King JrSearch for more papers by this authorRaoul V. Kuiper, Raoul V. KuiperSearch for more papers by this author John P. Sundberg, John P. SundbergSearch for more papers by this authorLloyd E. King Jr, Lloyd E. King JrSearch for more papers by this authorRaoul V. Kuiper, Raoul V. KuiperSearch for more papers by this author Book Editor(s):John P. Sundberg DVM, PhD, Dipl. ACVP, John P. Sundberg DVM, PhD, Dipl. ACVP The Jackson Laboratory, Bar Harbor, ME, USA Department of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USASearch for more papers by this authorPeter Vogel DVM, PhD, Dipl. ACVP, Peter Vogel DVM, PhD, Dipl. ACVP Pathology Department, St. Jude Children's Research Hospital, Memphis, TN, USASearch for more papers by this authorJerrold M. Ward DVM, PhD, Dipl. ACVP, Fellow IATP, Jerrold M. Ward DVM, PhD, Dipl. ACVP, Fellow IATP Global VetPathology, Montgomery Village, MD, USASearch for more papers by this author First published: 16 December 2021 https://doi.org/10.1002/9781119624608.ch10 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter provides examples of spontaneous and induced mutant mice that illustrate general types of abnormalities in skin and its appendages. It describes representative of disease groups to help investigators structure their approach to the complexity of phenotyping the skin and adnexa in newly developed mutants. Skin color can be used as an indicator for hair cycle stage. Different hair follicles producing different hair types develop at different embryologic stages. Mice undergo major changes in their lives that may affect or reflect the phenotype of a mutation. Hair color depends on amount and type of pigment, pigment granule size, shape, distribution, and other structural features influencing reflection and refraction of light. UV light-induced skin cancer has been a long used model for human skin cancer. The bullous or blistering skin diseases of mammals involve proteins that provide adhesion between cells or those that make up the cytoskeletal system. Pathology of Genetically Engineered and Other Mutant Mice RelatedInformation

Topical 17-beta-estradiol (E2) regulates the hair cycle, hair shaft differentiation, and sebum production. Vitamin A also regulates sebum production. Vitamin A metabolism proteins localized to the pilosebaceous unit (PSU; hair follicle and sebaceous gland); and were regulated by E2 in other tissues. This study tests the hypothesis that E2 also regulates vitamin A metabolism in the PSU. First, aromatase and estrogen receptors localized to similar sites as retinoid metabolism proteins during mid-anagen. Next, female and male wax stripped C57BL/6J mice were topically treated with E2, the estrogen receptor antagonist ICI 182,780 (ICI), letrozole, E2 plus letrozole, or vehicle control (acetone) during mid-anagen. E2 or one of its inhibitors regulated most of the vitamin A metabolism genes and proteins examined in a sex-dependent manner. Most components were higher in females and reduced with ICI in females. ICI reductions occurred in the premedulla, sebaceous gland, and epidermis. Reduced E2 also reduced RA receptors in the sebaceous gland and bulge in females. However, reduced E2 increased the number of retinal dehydrogenase 2 positive hair follicle associated dermal dendritic cells in males. These results suggest that estrogen regulates vitamin A metabolism in the skin. Interactions between E2 and vitamin A have implications in acne treatment, hair loss, and skin immunity.

Growth factors and cytokines are small polypeptides which function as intracellular and extracellular signals to regulate the activities of single cells and tissues via their binding to specific receptors. This chapter provides a brief review of the effects of growth factors and, to a lesser extent, of cytokines on mammalian hair growth. It illustrates the growing understanding of how abnormalities in the production and regulation of growth factors and cytokines affect hair follicle formation, hair structure, and the hair cycle to produce abnormal hair or fur. Growth factors, such as epidermal growth factor and transforming growth factor (TFGα), were found to affect the rate of regeneration after burn wounds. Transgenic mice overexpressing TGFβ-l have been produced using a human keratin I promoter vector and a constitutively active mutated TGFβ-1. These transgenic mice had very shiny, tautly stretched skin which restricted the animals' ability to move and breathe.

Primary cicatricial alopecias (PCAs) are a group of skin diseases in which there is progressive and permanent destruction of hair follicles followed by replacement with fibrous tissue. Unfortunately, by the time patients seek clinical evaluation of their hair loss, the skin is already inflamed and/or scarred, so there is little hope for a return to their normal hair growth pattern. Clinical and basic science investigations are now focusing on three forms of human PCA: lichen planopilaris (LPP), frontal fibrosing alopecia (FFA) and central centrifugal cicatricial alopecia (CCCA). Transcriptome, lipidome and other new technologies are providing new insight into the pathogenesis of some of these diseases that are being validated and further investigated using spontaneous and genetically engineered mouse models.

The skin and adnexa are anatomically similar between mice and rats but somewhat different in comparison to humans. Humans have thicker skin and larger hair follicles than rodent and they have apocrine sweat glands which rodents lack. The human hair follicles cycle in a mosaic pattern while rodents cycle in a head to tail wave pattern. Regardless, there are many anatomical similarities making rodents useful models for studying human skin anatomy and disease.

Alopecia areata is an autoimmune disorder characterized by transient, non-scarring hair loss and preservation of the hair follicle. Hair loss can take many forms ranging from loss in well-defined patches to diffuse or total hair loss, which can affect all hair-bearing sites. Patchy alopecia areata affecting the scalp is the most common type. Alopecia areata affects nearly 2% of the general population at some point during their lifetime. Skin biopsies of affected skin show a lymphocytic infiltrate in and around the bulb or the lower part of the hair follicle in the anagen (hair growth) phase. A breakdown of immune privilege of the hair follicle is thought to be an important driver of alopecia areata. Genetic studies in patients and mouse models have shown that alopecia areata is a complex, polygenic disease. Several genetic susceptibility loci were identified to be associated with signalling pathways that are important to hair follicle cycling and development. Alopecia areata is usually diagnosed based on clinical manifestations, but dermoscopy and histopathology can be helpful. Alopecia areata is difficult to manage medically, but recent advances in understanding the molecular mechanisms have revealed new treatments and the possibility of remission in the near future.

A large variety of mouse models for human skin, hair, and nail diseases are readily available from investigators and vendors worldwide. Mouse skin is a simple organ to observe lesions and their response to therapy, but identifying and monitoring the progress of treatments of mouse skin diseases can still be challenging. This chapter provides an overview on how to use the laboratory mouse as a preclinical tool to evaluate efficacy of new compounds or test potential new uses for compounds approved for use for treating an unrelated disease. Basic approaches to handling mice, applying compounds, and quantifying effects of the treatment are presented.

BACKGROUND:There have been repeated initiatives to produce standard nosologies and terminologies for cutaneous disease, some dedicated to the domain and some part of bigger terminologies such as ICD-10. Recently, formally structured terminologies, ontologies, have been widely developed in many areas of biomedical research. Primarily, these address the aim of providing comprehensive working terminologies for domains of knowledge, but because of the knowledge contained in the relationships between terms they can also be used computationally for many purposes.RESULTS:We have developed an ontology of cutaneous disease, constructed manually by domain experts. With more than 3000 terms, DermO represents the most comprehensive formal dermatological disease terminology available. The disease entities are categorized in 20 upper level terms, which use a variety of features such as anatomical location, heritability, affected cell or tissue type, or etiology, as the features for classification, in line with professional practice and nosology in dermatology. Available in OBO flatfile and OWL 2 formats, it is integrated semantically with other ontologies and terminologies describing diseases and phenotypes. We demonstrate the application of DermO to text mining the biomedical literature and in the creation of a network describing the phenotypic relationships between cutaneous diseases.CONCLUSIONS:DermO is an ontology with broad coverage of the domain of dermatologic disease and we demonstrate here its utility for text mining and investigation of phenotypic relationships between dermatologic disorders. We envision that in the future it may be applied to the creation and mining of electronic health records, clinical training and basic research, as it supports automated inference and reasoning, and for the broader integration of skin disease information with that from other domains.

Mouse models of various types of inflammatory skin disease are often accompanied by increased dermal angiogenesis. The C3H/HeJ inbred strain spontaneously develops alopecia areata (AA), a cell mediated autoimmune disorder that can be controllably expanded using full thickness skin grafts to young unaffected mice. This provides a reproducible and progressive model for AA in which the vascularization of the skin can be examined. Mice receiving skin grafts from AA or normal mice were evaluated at 5, 10, 15, and 20 weeks after engraftment. Lymphatics are often overlooked as they are small slit-like structures above the hair follicle that resemble artifact-like separation of collagen bundles with some fixatives. Lymphatics are easily detected using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) by immunohistochemistry to label their endothelial cells. Using LYVE1, there were no changes in distribution or numbers of lymphatics although they were more prominent (dilated) in the mice with AA. Lyve1 transcripts were not significantly upregulated except at 10 weeks after skin grafting when clinical signs of AA first become apparent. Other genes involved with vascular growth and dilation or movement of immune cells were dysregulated, mostly upregulated. These findings emphasize aspects of AA not commonly considered and provide potential targets for therapeutic intervention.

Disease is not limited to humans. Rather, humans are but another mammal in a continuum, and as such, often share similar if not identical diseases with other mammalian species. Alopecia areata (AA) is such a disease. Natural disease occurs in humans, nonhuman primates, many domestic animals, and laboratory rodents. However, to be useful as models of human disease, affected animals need to be readily available to the research community, closely resemble the human disease, be easy to work with, and provide reproducible data. To date, the laboratory mouse (most if not all of the C3H substrains) and the Dundee experimental bald rat fit these criteria. Manipulations using full-thickness skin grafts or specific immune cell transfers have improved the models. New mouse models that carry a variety of genetic-based immunodeficiencies can now be used to recapitulate the human immune system and allow for human full-thickness skin grafts onto mice to investigate human-specific mechanistic and therapeutic questions. These models are summarized here including where they can currently be obtained from public access repositories.

Background: Treatment for adult Philadelphia chromosome+ acute lymphoblastic leukemia includes using dasatinib, a tyrosine kinase inhibitor. Cutaneous squamous cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors of tyrosine kinases. No documentation of dasatinib inducing multiple keratoacanthomas, squamous cell carcinomas type during treatment of Philadelphia chromosome+ acute lymphoblastic leukemia is currently available. Case: A 77-year-old Caucasian male presented to the dermatology clinic two months after starting treatment with dasatinib for Philadelphia chromosome positive+ acute lymphoblastic leukemia. Biopsies confirmed the lesions on the arms, chest, legs and back as keratoacanthoma (KA) type of squamous cell carcinomas (SCCs). The cutaneous lesions were surgically removed and no new or recurrent lesions were detected since their initial rapid onset despite continued dasatinib therapy. Conclusion: This report of the rapid onset of keratoacanthoma type squamous cell carcinomas in a patient with Philadelphia chromosome+ acute lymphoblastic leukemia treated with dasatinib is presumed to be the first due to the rarity of adult Philadelphia chromosome+ acute lymphoblastic leukemia. This report documents another tyrosine kinase inhibitor that is associated with the eruption of keratoacanthomas, and adds to the literature regarding the regularity of this relatively common side effect, which may have treatment other than surgery if only a few lesions are present.

Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been fully studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create further novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis, and while not automated to the level of the physiological phenotyping, histopathology still provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being characterized and developed.

alopecia areata ATP-binding cassette subfamily C, member 6, gene dystrophic cardiac calcinosis 1–4 pseudoxanthoma elasticum quantitative trait loci TO THE EDITOR The proceedings of a recent meeting on alopecia areata (AA) (Bertolini et al., 2012Bertolini M. Gilhar A. Paus R. Alopecia areata as a model for T cell-dependent autoimmune disease.Exp Dermatol. 2012; 21: 477-479Crossref PubMed Scopus (24) Google Scholar) summarized work using the surgically induced C3H/HeJ mouse model for AA (McElwee et al., 1998McElwee K.J. Boggess D. King L.E. et al.Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.J Investig Dermatol. 1998; 111: 797-803Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar; Wang et al., 2013Wang E. Chong K. Yu M. et al.Development of autoimmune hair loss disease alopecia areata is associated with cardiac dysfunction in C3H/HeJ mice.PLOS ONE. 2013; 8: e62935Crossref PubMed Scopus (18) Google Scholar), in which the investigators found enlarged hearts in affected mice, suggesting an association between AA and cardiac findings. However, the heart lesions described are a well-known strain-specific disease not limited to C3H substrains. These lesions have been described by a number of names including epicardial mineralization with fibrosis and dystrophic cardiac calcinosis (Eaton et al., 1978Eaton G.J. Custer R.P. Johnson F.N. et al.Dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences.Am J Pathol. 1978; 90: 173-186PubMed Google Scholar; Frith and Ward, 1988Frith C.H. Ward J.M. Color Atlas of Neoplastic and Non-Neoplastic Lesions in Aging Mice. 1988; : 109Google Scholar). Crosses between C3H/HeJ and C57BL/6J mice have identified four quantitative trait loci (QTLs), designated as dystrophic cardiac calcinosis 1–4 (Dyscalc1–4; Ivandic et al., 2001Ivandic B.T. Kaczmarek P.M. Aherrahrou Z. et al.New Dyscalc loci for myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.Physiol Genomics. 2001; 6: 137-144Crossref PubMed Scopus (39) Google Scholar). Mapping to mouse Chromosome 7 (Ivandic et al., 1996Ivandic B.T. Qiao J.H. Machleder D. et al.A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.Proc Natl Acad Sci USA. 1996; 93: 5483-5488Crossref PubMed Scopus (63) Google Scholar), Dyscalc1 was subsequently identified as being due to non-synonymous single-nucleotide polymorphisms in the ATP-binding cassette, subfamily C (CFTR/MRP), member 6 (Abcc6) gene (Meng et al., 2007Meng H. Vera I. Che N. et al.Identification of Abcc6 as the major causal gene for dystrophic cardiac calcification in mice through integrative genomics.Proc Nat Acad Sci USA. 2007; 104: 4530-4535Crossref PubMed Scopus (102) Google Scholar; Aherrahrou et al., 2008Aherrahrou Z. Doehring L.C. Ehlers E.M. et al.An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.J Biol Chem. 2008; 283: 7608-7615Crossref PubMed Scopus (50) Google Scholar). Mutations in the human ABCC6 gene and targeted mutations in the mouse Abcc6 gene produce pseudoxanthoma elasticum (PXE) (Gorgels et al., 2005Gorgels T.G. Hu X. Scheffer G.L. et al.Disruption of Abcc6 in the mouse: novel insight in the pathogenesis of pseudoxanthoma elasticum.Hum Mol Genet. 2005; 14: 1763-1773Crossref PubMed Scopus (165) Google Scholar; Klement et al., 2005Klement J.F. Matsuzaki Y. Jiang Q.J. et al.Targeted ablation of the Abcc6 gene results in ectopic mineralization of connective tissues.Mol Cell Biol. 2005; 2005: 8299-8310Crossref Scopus (169) Google Scholar), a systemic metabolic disease with cutaneous features distinct from AA (Uitto et al., 2010Uitto J. Li Q. Jiang Q. Pseudoxanthoma elasticum: molecular genetics and putative pathomechanisms.J Invest Dermatol. 2010; 130: 661-670Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In a massive histopathological screening of all organ systems in 31 inbred strains of mice of both genders, dystrophic cardiac calcinosis was diagnosed in eight strains (Berndt et al., in preparation; Sundberg et al., 2011Sundberg J.P. Berndt A. Sundberg B.A. et al.The mouse as a model for understanding chronic diseases of aging: the histopathologic basis of aging in inbred mice.Pathobiol Aging Age Relat Dis. 2011; 1 (e-pub ahead of print 1 June 2011 (doi)https://doi.org/10.3402/pba.v1i0.7179Crossref PubMed Google Scholar). C3H/HeJ and A/J strains were found to develop both heart lesions (Chase et al., 2009Chase T.H. Cox G.A. Burzenski L. et al.Dysferlin deficiency and the development of cardiomyopathy in a mouse model of limb-girdle muscular dystrophy 2B.Am J Pathol. 2009; 175: 2299-2308Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and AA (McElwee et al., 1999McElwee K. Boggess D. Miller J. et al.Spontaneous alopecia areata-like hair loss in one congenic and seven inbred laboratory mouse strains.J Invest Dermatol Symp Proc. 1999; 4: 202-206Abstract Full Text PDF PubMed Scopus (44) Google Scholar) in the aging study, although in both cases more mice with normal skin had heart lesions than those with AA (Table 1a). Three strains were found to develop histologically confirmed AA (MRL/MpJ, SJL/J, and SWR/J), but none of these mice had any type of heart lesion. No correlation was found in a retired breeder study (Table 1b) (Berndt et al., in preparation) or in a large mouse cross (C3H/HeJ × C57BL/6J, C3B6F2; Table 1c) generating F2 females for identifying AA eQTLs. Heart lesions varied in severity and location between the strains (Berndt et al., in preparation). Genome-wide association mapping determined that none of the QTLs for dystrophic cardiac calcinosis corresponded to genomic regions identified to determine AA.Table 1Lack of correlation between histologically confirmed alopecia areata and DCC in 31 inbred strains(a) An aging histopathology study1There was no correlation between alopecia areata and heart lesions in mouse strains in the 31 strain aging study.StrainTotal mice 12 and 20 months and moribund groupsAlopecia areataDCCNormal skinDCCFMFMFMFMFMA/J514601005145238C3H/HeJ28297300212613MRL/MpJ41312000393100SJL/J36102000341000SWR/J24186000181800Total180134174001631302411(b) Evaluation of hearts in retired breeders2Alopecia areata was not diagnosed in any of the strains in the retired breeder survey.StrainTotalAlopecia areataDCCStrainTotalAlopecia areataDCCFMFMFMFMFMFMA/J101000910DBA/2J1010001010BALB/cJ101000910FVB/NJ10100000BALB/cByJ101000810KK/HlJ101000109C3H/HeJ101000106LP/J10100000C57BL/6J10100001PWD/PhJ10100000C57BL/10J101000710SWR/J10100000Total6060004347Total6060002019(c) An F2 hybrid study for mapping quantitative trait loci for alopecia areata3There was no correlation between alopecia areata and heart disease in an F2 hybrid cross used to investigate the genetics of these diseases (P-value=0.651 using a Fisher exact test).StrainAge range (days)GenderAlopecia areataDCCNormal skinDCCC3B6F2195–605F19111454Abbreviations: DCC, dystrophic cardiac calcinosis; F, Female; M, male.1 There was no correlation between alopecia areata and heart lesions in mouse strains in the 31 strain aging study.2 Alopecia areata was not diagnosed in any of the strains in the retired breeder survey.3 There was no correlation between alopecia areata and heart disease in an F2 hybrid cross used to investigate the genetics of these diseases (P-value=0.651 using a Fisher exact test). Open table in a new tab Abbreviations: DCC, dystrophic cardiac calcinosis; F, Female; M, male. Although it is easy to see clinical correlations between seemingly unrelated diseases in small numbers of mice undergoing experimental manipulation, it is critically important to understand strain-specific background lesions. The mineralization and fibrosis phenomena among the inbred strains associated with PXE-like diseases are very complicated. Some are related to each other, whereas others are not. The underlying genetic predisposition can be modified by the genes involved in other diseases. Such appears to be the case for Abcc6 and PXE (Berndt et al., 2013Berndt A. Li Q. Potter C.S. et al.A single nucleotide polymorphism in the Abcc6 gene associates with connective tissue mineralization in mice similar to targeted models for pseudoxanthoma elasticum.J Invest Dermatol. 2013; 133: 833-836Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As the complex genetics of AA in humans and mice continues to be refined, it is possible that some of the genes involved in development of heart lesions may overlap with those that determine AA, but with technologies currently available using large populations of mice it appears that cardiac mineralization and fibrosis phenotypes are not correlated with AA. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R01 AR056635. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Dr Berndt is the recipient of a fellowship by the Parker B. Francis Foundation and recipient of a North American Hair Research Society Mentorship Grant. The Jackson Laboratory Shared Scientific Services were supported in part by a Basic Cancer Center Core Grant from the National Cancer Institute (P30 CA034196).

Ocular and cutaneous sarcoidosis is a chronic manifestation of sarcoidosis that remains difficult to treat. Recent investigations demonstrating efficacy with antimicrobial therapy in pulmonary and cutaneous sarcoidosis have been reported. Here, we report dual clinical improvement in cutaneous and ocular sarcoidosis following administration of oral antimycobacterial therapy.

alopecia areata alopecia universalis TO THE EDITOR Alopecia areata (AA), the second most common form of hair loss in humans, is characterized by waxing and waning focal hair loss. Alopecic foci can expand to encompass the entire head (alopecia totalis) or body (alopecia universalis, AU). This cell-mediated autoimmune disease is characterized histologically by the “swarm of bees”, CD4+ and CD8+ T cells, natural killer cells, and fewer numbers of other types of inflammatory cells in and around actively growing, anagen-stage, hair follicles. Treatment of persistent, moderate to severe AA is palliative at best, often with a poor outcome (Harris et al., 2010Harris M.J. Sun J. Paus R. et al.Management of alopecia areata.Br Med J. 2010; 341: c3671Crossref PubMed Scopus (84) Google Scholar). Investigation of AA in humans and in nonhuman animal models, primarily C3H/HeJ mice (Sundberg et al., 1994Sundberg J.P. Cordy W.R. King L.E. Alopecia areata in aging C3H/HeJ mice.J Invest Dermatol. 1994; 102: 847-856Abstract Full Text PDF PubMed Scopus (174) Google Scholar), has provided new insights. Spontaneous AA in C3H/HeJ mice is sporadic, of low frequency, and most commonly affects older female mice. Full-thickness skin grafts of AA donor skin to young unaffected, immunocompetent, histocompatible recipients provides a highly reproducible model (McElwee et al., 1998McElwee K.J. Boggess D. King L.E. et al.Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.J Invest Dermatol. 1998; 111: 797-803Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) useful in preclinical trials (Sun et al., 2008Sun J. Silva K.A. McElwee K.J. et al.The C3H/HeJ mouse and DEBR rat models for alopecia areata: preclinical drug screening tools.Exp Dermatol. 2008; 17: 793-805Crossref PubMed Scopus (52) Google Scholar). Heat shock was used to induce a type of AA in C3H/HeJ mice (Wikramanayake et al., 2010Wikramanayake T.C. Alvarez-Connelly E. Simon J. et al.Heat treatment increases the incidence of alopecia areata in the C3H/HeJ mouse model.Cell Stress Chaperones. 2010; 15: 985-991Crossref PubMed Scopus (16) Google Scholar) and was used to determine whether a commercially available laser comb induced hair regrowth (Wikramanayake et al., 2012Wikramanayake T.C. Rodriguez R. Choudhary S. et al.Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata.Lasers Med Sci. 2012; 27: 431-436Crossref PubMed Scopus (68) Google Scholar). The laser comb received a 501(k) clearance as a device safe for marketing by the United States Food and Drug Administration for the treatment of male pattern baldness (Leavitt et al., 2009Leavitt M. Charles G. Heyman E. et al.HairMax LaserComb laser phototherapy device in the treatment of male androgenetic alopecia: a randomized, double-blind, sham device-controlled, multicentre trial.Clin Drug Invest. 2009; 29: 283-292Crossref PubMed Scopus (159) Google Scholar). Laser comb treatment induced cosmetic regrowth of the very small, focal areas of heat shock–induced AA after 6 weeks. To test whether the laser comb would induce regrowth of hair in older C3H/HeJ mice with spontaneous or graft-induced AU, a similar treatment regimen was used. All protocols were approved by The Jackson Laboratory Animal Care and Use Committee. Only C3H/HeJ female mice (JR# 659, The Jackson Laboratory, Bar Harbor, ME) were used. Retired breeders were obtained and aged until approximately 20% developed spontaneous AU. Twelve of these spontaneous AU mice were used directly. Others were used as donors for full-thickness skin grafts to female recipients using protocols previously described in detail (McElwee et al., 1998McElwee K.J. Boggess D. King L.E. et al.Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.J Invest Dermatol. 1998; 111: 797-803Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Twelve grafted mice were aged until they developed AU. Each group was divided into cohorts of six mice each and treated. One set of spontaneous and one of graft-induced AU were held manually, and their ventral abdominal area was exposed for 20 seconds to the laser comb (Hairmax Lux 9, professional 12, laser comb, Lexington International, Boca Raton, FL, wavelength 655 nm, beam diameter <5 mm, divergence 57 mrad three times per week for 6 weeks). At 6 weeks, no change in the pelage was noted, and thus treatments were continued for an additional 6 weeks (12 weeks total). The second group of spontaneous or graft-induced mice with AA were treated in the same manner, but the laser was not turned on (controls). After 12 weeks, mice were euthanized by CO2 asphyxiation, and ventral skin was collected, fixed in Fekete’s acid alcohol formalin, and processed routinely for histology; next, 5-μm sections were stained with hematoxylin and eosin, and the slides were examined. Mice treated with the laser on or off and examined weekly for 6 weeks and then continued for 6 more weeks had no gross (Figure 1a) or histological differences in skin for either the spontaneous or graft-induced AA mice. Histologic features of AA were confirmed in donor mice and in all 24 mice used in this study (Figure 1b). There was mild epidermal acanthosis with moderate to marked infiltrates of mostly lymphocytes and fewer granulocytes in and around late anagen-stage hair follicles with disrupted outer root sheath architecture. All 24 mice in this study had diffuse AA with generalized hair loss and only remnants of hair whether or not the laser comb was turned on or off (Table 1).Table 1Groups of six mice with spontaneous or full-thickness skin graft–induced AA were treated with the laser comb for a total of 12 weeksTest group6 Weeks alopecia12 Weeks alopeciaAA histopathologyLaser on Spontaneous AA6/66/66/6 Graft-induced AA6/66/66/6Laser off Spontaneous AA6/66/66/6 Graft-induced AA6/66/66/6Abbreviation: AA, alopecia areata.There were no differences between those treated with the comb turned on (test) or off (controls). All mice were confirmed to have alopecia areata by histopathology. Open table in a new tab Abbreviation: AA, alopecia areata. There were no differences between those treated with the comb turned on (test) or off (controls). All mice were confirmed to have alopecia areata by histopathology. Failure to reproduce the earlier laser comb study to induce regrowth of heat shock–induced AA in dorsal skin of C3H/HeJ mice (Wikramanayake et al., 2012Wikramanayake T.C. Rodriguez R. Choudhary S. et al.Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata.Lasers Med Sci. 2012; 27: 431-436Crossref PubMed Scopus (68) Google Scholar) may be due to the heat-induced lesions being very small, as seen in early-stage AA, which may also wax and wane in human AA. In the initial laser comb study, the AA lesions were assumed to regrow, as only laser-treated mice responded. These results are in contrast to the current studies using AU mice rather than early-onset, less severely affected AA mice. Alternatively, treating dorsal skin in the initial report versus ventral skin in this study may be an issue. However, dorsal and ventral lesions in both spontaneous and graft-induced lesions respond similarly in most other studies, and thus this seems unlikely (King et al., 2008King L.E. McElwee K.J. Sundberg J.P. Alopecia areata.in: Nickoloff B.J. Nestle F.O. Dermatologic Immunity: Current Directions in Autoimmunity vol. 10. 2008: 280-312Crossref Scopus (29) Google Scholar). An alternative issue is whether heat shock–induced AA is representative of human AA. The initial report showed gross and histologic evidence that it was indeed quite similar to the established mouse models (Wikramanayake et al., 2010Wikramanayake T.C. Alvarez-Connelly E. Simon J. et al.Heat treatment increases the incidence of alopecia areata in the C3H/HeJ mouse model.Cell Stress Chaperones. 2010; 15: 985-991Crossref PubMed Scopus (16) Google Scholar). However, in the subsequent laser comb study, the gross lesion illustrated was totally different. Although the authors stated that they confirmed histologic lesions of AA in two mice before treatment, their descriptions and figures after treatment show no evidence of AA. Rather, their sham-treated mice (laser turned off) show normal mouse skin in telogen, the common persistent stage found in adult mouse skin. The representative mouse treated with the laser on had normal hair follicles in late anagen (lacking clear evidence of inflammation in and around the follicles; Wikramanayake et al., 2012Wikramanayake T.C. Rodriguez R. Choudhary S. et al.Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata.Lasers Med Sci. 2012; 27: 431-436Crossref PubMed Scopus (68) Google Scholar), which is found, although infrequently, in adult mice (Sundberg and Silva, 2012Sundberg J.P. Silva K.A. What color is the skin of a mouse?.Vet Pathol. 2012; 49: 142-145Crossref PubMed Scopus (17) Google Scholar). Alopecia in small foci in C3H/HeJ mice can be due to AA but also is not less commonly due to grooming behavior abnormalities (barbering, a form of trichotillomania). The laser comb, as used in the original study, potentially could induce anagen activation in the heat shock–induced AA of the normally quiescent mouse telogen follicles. In summary, laser comb treatment did not induce hair growth in C3H/HeJ mice with extensive AA in well-established spontaneous arising or full-thickness skin graft mouse models of human AA. More work on the heat shock–induced AA model needs to be done to define its value in preclinical trials. This work was supported by a grant from the National Institutes of Health (AR056635) and the National Alopecia Areata Foundation (model development). Core facilities at The Jackson Laboratory were supported by the National Cancer Institute (CA34196).

cycle threshold lymph node microphthalmia-associated transcription factor quantitative real-time reverse transcriptase–PCR pigmented epithelioid melanocytoma tyrosine-related protein 1 TO THE EDITOR The transplantable B16 melanoma model has been used for decades and continues to be used with various degrees of reproducibility in mice (Fidler, 1975Fidler I.J. Biological behavior of malignant melanoma cells correlated to their survival in vivo.Cancer Res. 1975; 35: 218-224PubMed Google Scholar). Melanocytic tumors or nevus-like lesions were induced in two-stage cutaneous chemical oncogenesis experiments in various inbred strains of mice (Bannasch and Goessner, 1994Bannasch B. Goessner W. Pathology of Neoplasia and Preneoplasia in Rodents, Vol. 2. Vol. 2. Schattauer, Stuttgart1994Google Scholar; Sundberg et al., 1997Sundberg J.P. Sundberg B.A. Beamer W.G. Comparison of chemical carcinogen skin tumor induction efficacy in inbred, mutant, and hybrid strains of mice: morphologic variations of induced tumors and absence of a papillomavirus cocarcinogen.Mol Carcinog. 1997; 20: 19-32Crossref PubMed Scopus (33) Google Scholar; Maronpot et al., 1999Maronpot R. Boorman G.A. Gaul B.W. Pathology of the Mouse, Reference and Atlas. 1999; Google Scholar). Recently a number of genetically engineered mouse models have been generated to recapitulate the major signaling pathways deregulated in human melanoma, namely, the RAS–RAF–MAPK, PI3K–AKT, CDK4–INK4A–RB1, and ARF–TP53 pathways (reviewed in the study by Damsky and Bosenberg, 2010Damsky Jr., W.E. Bosenberg M. Mouse melanoma models and cell lines.Pigment Cell Melanoma Res. 2010; 23: 853-859Crossref PubMed Scopus (23) Google Scholar and Walker et al., 2011Walker G.J. Soyer H.P. Terzian T. et al.Modelling melanoma in mice.Pigment Cell Melanoma Res. 2011; 24: 1158-1176Crossref PubMed Scopus (37) Google Scholar). These preclinical models have been invaluable to delineate the relationship between causative gene mutations and molecularly targeted therapeutics; however, there are no spontaneously occurring melanocytic tumors in laboratory mice to globally discover other disrupted gene networks. Although melanomas are relatively common in humans and domestic animals exposed to sunlight, the scarcity of spontaneous melanomas in laboratory mice might be the result of the mice never being exposed to sunlight or artificial UVR under normal husbandry conditions. Herein, we report the finding of a spontaneous, locally invasive, transplantable malignant melanoma that resembles pigmented epithelioid melanocytoma (PEM), formally known as the “animal/equine-type” in humans (Zembowicz et al., 2004Zembowicz A. Carney J.A. Mihm M.C. Pigmented epithelioid melanocytoma: a low-grade melanocytic tumor with metastatic potential indistinguishable from animal-type melanoma and epithelioid blue nevus.Am J Surg Pathol. 2004; 28: 31-40Crossref PubMed Scopus (234) Google Scholar). We found a 172-day-old female LT.B6 “line E” congenic mouse on routine examination to have multiple raised black nodules on the tail (Figure 1a). Histopathology showed diffusely and heavily pigmented dermal tumors with irregular borders, and consisting of epithelioid and/or spindled melanocytes surrounded by numerous melanophages (Figure 1b and c). These tumor cells infiltrated fascia between collagen bundles in the tail, surrounding nerves, and small arteries, without invasion (Figure 1d and e). We aseptically removed the nodular masses and finely minced them in physiological saline solution and implanted them subcutaneously into small incisions between the scapulae of the dorsal thorax in four 10-week-old histocompatible LT/SvEiJ female mice. Mice were observed weekly and the first signs of tumor growth were observed nearly 12 months post surgery at the incision site. Of the four implanted mice, two died of unrelated reasons, and the remaining two developed tumors at the injection site; at necropsy, one mouse had a 2.7 × 2.0 × 1.2 cm3 tumor and the second had a 3.4 × 2.6 × 2.0 cm3 tumor. Complete necropsies (Silva and Sundberg, 2012Silva K. Sundberg J.P. Necropsy methods.Necropsy methods. Academic Press, London2012: 779-806Crossref Scopus (19) Google Scholar) revealed heavily pigmented non-ulcerated dermal nodules, 5–15 mm in diameter (Figure 2a). Overall, the transplanted tumors morphologically resembled the original donor neoplasms with the exception of occasional amelanotic whirling nests consisting of spindle cells with oval nuclei and abundant eosinophilic cytoplasm (Figure 2b and c). There was no detectable mitotic activity or necrosis in the primary melanomas. Regional cervical and popliteal lymph nodes (LNs) were enlarged, pigmented, and histologically effaced by metastatic tumors resembling the primary lesion (Figure 2d). The nodal metastases were both subcapsular and intraparenchymal, similar to the pattern of nodal metastases in melanoma patients (Dadras, 2011Dadras S.S. Molecular diagnostics in melanoma: current status and perspectives.Arch Pathol Lab Med. 2011; 135: 860-869Crossref PubMed Google Scholar). The lungs contained small numbers of widely scattered individual hyperpigmented epithelioid and spindled cells in the interalveolar space, consistent with pulmonary metastases (Figure 2e). Histopathology of liver sections showed scattered melanophages and melanin throughout sinusoids in the liver parenchyma (Figure 2f). The images shown here, along with additional images, are available in color online on the Mouse Tumor Biology Database (http://tumor.informatics.jax.org; Naf et al., 2002Naf D. Krupke D.M. Sundberg J.P. et al.The Mouse Tumor Biology Database: a public resource for cancer genetics and pathology of the mouse.Cancer Res. 2002; 62: 1235-1240PubMed Google Scholar; Krupke et al., 2008Krupke D.M. Begley D.A. Sundberg J.P. et al.The Mouse Tumor Biology Database.Nat Rev Cancer. 2008; 8: 459-465Crossref PubMed Scopus (57) Google Scholar). Whole-(virtual) slide images are available on Skinbase (http://www.pathbase.net/; Schofield et al., 2010Schofield P.N. Gruenberger M. Sundberg J.P. Pathbase and the MPATH ontology. Community resources for mouse histopathology.Vet Pathol. 2010; 47: 1016-1020Crossref PubMed Scopus (40) Google Scholar).Figure 2Histopathology and melanocyte-specific gene expression analysis of transplanted melanomas. Subcutaneously transplanted melanomas grew in the interscapular area of all four mice, giving rise to heavily pigmented dermal nodules (a), scale bar=5 mm, with amelanotic nests and nodules (b), scale bar=200 μm; showing oval nuclei and abundant eosinophilic cytoplasm (c), scale bar=25 μm. After 524 days of transplantation, necropsy showed metastases to the cervical (d), scale bar=50 μm, and popliteal (not shown) lymph nodes (LNs) and lungs (e), scale bar=25 μm. Only melanin (no tumor cells) was found in the liver sinusoids (f), scale bar=25 μm. Quantitative real-time reverse transcriptase–PCR (qRT-PCR) showed specific expression of Tyrp1 and Mitf in the B16 melanoma (positive control) and the LT.B6 tumor but not in chemically-induced squamous cell carcinoma (SCC, negative control) (g). Decreased expression of Tyrp1 and Mitf was detected in the metastatic LN but not in two liver samples with sinusoidal melanin. Decreased expression of Prkar1a was detected in LT.B6 and B16 tumors compared with SCC. All reaction assays were performed in triplicate on an ABI 7500 Fast system (Applied Biosystems, Carlsbad, CA) using Sdha as an endogenous control. The qRT-PCR was repeated showing the same results. Cycle threshold (Ct) values for each mRNA were normalized to Sdha (ΔCT) and represented as RQ=2-ΔCT. For comparison, fold differences of all samples were compared with the B16 melanoma.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate melanocyte-specific gene expression, we subjected acid-alcohol-fixed, paraffin-embedded blocks from the transplanted primary dermal tumors, cervical LN metastasis, two separate liver blocks, chemically induced squamous cell carcinoma (negative control), and B16 transplant (positive control) to quantitative real-time reverse transcriptase PCR (qRT-PCR). Total RNA was extracted from the blocks as described (Chakraborty et al., 2013Chakraborty N.G. Yadav M. Dadras S.S. et al.Analyses of T cell-mediated immune response to a human melanoma-associated antigen by the young and the elderly.Hum Immunol. 2013; 74: 640-647Crossref PubMed Scopus (4) Google Scholar). Using 15 ng total RNA, the quantification of mouse tyrosine-related protein 1 (Tyrp1), microphthalmia-associated transcription factor (Mitf), protein kinase, cAMP-dependent regulatory type 1 alpha (Prkar1a), and housekeeping gene succinate dehydrogenase complex subunit A (Sdha) transcripts was accomplished by qRT-PCR amplification of a cDNA using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes (Life Technologies, New York, NY). The following catalog numbers represent the respective genes analyzed (Life Technologies): Tyrp1 (Mm00453201_m1), Mitf (Mm00434954_m1), Prkar1a (Mm00660315_m1), and Sdha (Mm01352366_m1). The qRT-PCR results clearly showed that both Tyrp1 and Mitf are expressed in the LT.B6 mouse primary dermal tumor and B16, but not in the squamous cell carcinoma (SCC; Figure 2g). Lower expression levels of Tyrp1 and Mitf were detected in the LN metastasis but not in either of the liver sections, which was consistent with tumor metastasis to the LN but not to the liver. The expressions of Tyrp1 and Mitf were only detected in the melanocytic tumors (B16, primary dermal tumor, and LN metastasis) but not in the SCC or liver sections. The lost expression of Prkar1a has been reported as a molecular event in 28 of the PEMs (82%; Zembowicz et al., 2007Zembowicz A. Knoepp S.M. Bei T. et al.Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions.Am J Surg Pathol. 2007; 31: 1764-1775Crossref PubMed Scopus (100) Google Scholar). qRT-PCR for Prkar1a showed >2-fold decrease in expression in the primary dermal tumor and LN metastasis, compared with the control (SCC); however, a similar reduction was also detected in B16 tumor (Figure 2g). Historically, the LT strain is light brown (a, Blt) and was developed from a mutation at the brown locus in strain C58 in 1950 and then outcrossed to BALB/c, and is commonly used to study spontaneous ovarian teratomas (Damjanov et al., 1975Damjanov I. Katic V. Stevens L.C. Ultrastructure of ovarian teratomas in LT mice.Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1975; 83: 261-267Crossref PubMed Scopus (2) Google Scholar). The LT.B6 “line E” congenic strain was developed to resolve the genetic interval containing the ovarian teratoma susceptibility locus (Ots1; Lee et al., 1997Lee G.H. Bugni J.M. Obata M. et al.Genetic dissection of susceptibility to murine ovarian teratomas that originate from parthenogenetic oocytes.Cancer Res. 1997; 57: 590-593PubMed Google Scholar). Comparing the pathologic features of LT.B6 melanomas with human counterparts showed a remarkable resemblance to the histopathology of PEM (Zembowicz et al., 2004Zembowicz A. Carney J.A. Mihm M.C. Pigmented epithelioid melanocytoma: a low-grade melanocytic tumor with metastatic potential indistinguishable from animal-type melanoma and epithelioid blue nevus.Am J Surg Pathol. 2004; 28: 31-40Crossref PubMed Scopus (234) Google Scholar): (1) heavily pigmented dermal tumor without junctional proliferation or epidermal hyperplasia, (2) blue nevus-like architecture, (3) infiltrative borders with extension into subcutaneous tissue along peri-adnexal connective tissue of hair follicles, muscle, or neurovascular bundles, (4) dendritic and epithelioid tumor cell cytology, and (5) lack of tumor necrosis. We detected no mitoses in LT.B6 melanomas, whereas low mitotic activity (1–3 mm-2) has been reported for PEM. The biological behavior of LT.B6 melanoma is similar to the clinical behavior of PEM: (1) slow growth, (2) common metastases to regional LNs, and (3) rare metastasis to liver; however, lung metastasis in these mice is a different feature than PEM. The finding of a naturally occurring melanoma in the laboratory LT.B6 mouse strain with successful tumor transplantation in two out of two mice followed by metastases could represent a clinically relevant mouse model for melanoma. Therefore, this mouse neoplasm resembles a variant of human melanoma of limited metastatic potential and could provide the opportunity to globally investigate the genetic and epigenetic alterations associated with metastasizing melanoma. We thank Elizabeth Fleming (Dadras Laboratory) for excellent technical assistance. We also thank Linda Washburn and Eva M. Eicher, for providing the mice with melanoma from their research colony. This work was supported in part by grants from the National Institutes of Health (CA34196, pilot project grant, and CA0897123, Mouse Tumor Biology Database).

Alopecia areata (AA), a cell mediated autoimmune disease, is the second most common form of hair loss in humans. While the autoimmune disease is responsible for the underlying pathogenesis, the alopecia phenotype is ultimately due to hair shaft fragility and breakage associated with structural deficits. Quantitative trait genetic analyses using the C3H/HeJ mouse AA model identified cysteine-rich secretory protein 1 (Crisp1), a hair shaft structural protein, as a candidate gene within the major AA locus. Crisp1 transcripts in the skin at various times during disease development were barely detectable. In situ hybridization identified Crisp1 expression within the medulla of hair shafts from clinically normal strains of mice but not C3H/HeJ mice with AA. Follow-up work with 5-day-old C3H/HeJ mice with normal hair also had essentially no expression of Crisp1. Other non-inflammatory based follicular dystrophy mouse models with similar hair shaft abnormalities also have little or no Crisp1 expression. Shotgun proteomics, used to determine strain difference in hair proteins, confirmed that there was very little CRISP1 within normal C3H/HeJ mouse hair in comparison to 11 other strains. However, mutant mice with hair medulla defects also had undetectable levels of CRISP1 in their hair. Crisp1 null mice had normal skin, hair follicles, and hair shafts indicating that the lack of the CRISP1 protein does not translate directly into defects in the hair shaft or hair follicle. These results suggest that CRISP1 may be an important structural component of mouse hair and that its strain-specific dysregulation may indicate a predisposition to hair shaft disease such as AA.