Animal models for male pattern (androgenetic) alopecia

ARTICLE

Alopecia, or hair loss, is a common problem in all mammalian species, not just humans. Therefore, it is logical that other mammals should have diseases similar to those that cause alopecia in humans. This is true for some human diseases such as papular atrichia (the hairless, hr, mutation in the mouse) [1] and alopecia areata (the same disease in mice and other mammals) [2]. The most common form of alopecia in humans is androgenetic alopecia (also called male pattern or just pattern baldness), a disease that affects approximately 80% of men and 40% of women 20+ years of age [3]. Because of the obvious sexual dimorphism in humans, it is logical to look at other mammalian species in which a similar pattern of hair or adnexal structure loss with age is a feature, especially when it affects only one sex. One of the early models proposed was the pattern of feather loss in starlings [4]. This simple observation opened the concept of sexual differences in the plumage of many species of birds and how development (or lack) of feathers for improved mating might correlate to changes in humans (male pattern hair loss), commonly considered to have an adverse effect.

Review of the literature [5] and recent experiments with numerous laboratory mouse strains reveal that androgens have a potent effect on hair biology. Some of these spontaneous or genetically engineered mouse models may be useful to test the effects on hair promotion or blockade of androgenic effects. A summary of the currently available models is presented here with some details on how they are or can be used.

Spontaneous androgenetic alopecia in humans

Androgenetic alopecia (AGA) is the most commonly recognized form of non-scarring alopecia in humans and is known by a number of descriptive terms such as inherited baldness (alopecia), male and female pattern baldness (alopecia), or simply "baldness" [6-8]. As the term implies, AGA has been defined as an autosomal dominant disorder with various degrees of penetrance (probably polygenic mode of inheritance) with a strong predominance in males less than 40 years of age [9]. There may be a bimodal distribution of AGA in males ­ an early onset of severe AGA in young adulthood and a late onset of diffuse and prolonged hair loss in older males and females. Clinically, female AGA usually differs from early and late onset AGA in males by having more diffuse hair loss with retention of the frontal hairline, lacking the typical receding hairline of males. However, females affected by an endocrinopathy may have full expression of the male phenotype when high levels of androgens persist over a prolonged period. In typical male pattern AGA, hair follicles located in the frontal area bitemporally ("widow's peak") and on the vertex ("bald spot") respond to androgens by gradually beginning to produce vellus hairs rather than terminal hairs.

Classic work done by Hamilton [10] showed that none of the 20 men with prepubertal gonadal insufficiency developed male pattern baldness while all four eunuchoids treated with androgens developed the alopecia phenotype. Studies by Van Scott and Ekel [11] revealed a proportional reduction in hair follicle matrix and papilla in early male baldness biopsies compared to normally haired scalp. Montagna described bald areas as not being devoid of hair, but rather having numerous small, colorless, and practically invisible hairs [12]. Density of follicles may be reduced in the alopecic versus haired scalp in patients affected by AGA [13], although this issue is controversial based on the number of visible hairs using nondestructive, semi-invasive techniques [14-16].

Regrowth of new anagen hairs from the secondary hair germ of telogen hairs is the expected pattern in the normal human hair cycle. When telogen hairs are not replaced, tiny bald spots occur in both men and women [16-19]. The number of tiny bald spots and how long they persist correlates with the clinical severity of alopecia reflecting the interval between the end of telogen and anagen induction. Close inspection of areas of thinning reveals short hairs scattered throughout the area. These short hairs are the intermediate "vellus" hairs that represent progressive miniaturization of terminal hairs as AGA increases in severity and chronicity.

The pathological features of acute AGA differ from chronic AGA but there are essentially no differences due to gender in either acute or chronic AGA. Terminal hairs progressively miniaturize and are gradually replaced by vellus hairs in AGA. There is usually no appreciable reduction in the number of hair follicles in AGA although this can occur in 10% of cases. The site of anagen follicle regression to catagen then onto telogen follicles is marked by the presence of residual angiofibrotic tracts commonly known as streamers or follicular stelae. Although there is little or no evidence of dermal scarring in AGA, a mild lymphohistiocytic inflammation may be found around the upper follicle in approximately 1/3 of scalp biopsies from patients with no hair loss and those with AGA. In some cases, this mononuclear cell infiltration may involve the lower portion of the hair follicle, especially in AGA (Van Neste, pers. Obs.). Of interest is the observation that moderate inflammation is found in another 1/3 of cases of AGA, but not in otherwise comparable patients with no hair loss.

Histopathologic findings are the same in both male and female AGA with terminal hair depletion and a corresponding increase in "vellus" hairs and stelae, although slightly less "vellus" hairs may be found in females with AGA. The severity of AGA appears proportional to the total area of involvement of hair follicles and the ratio of terminal hairs to vellus hairs. The terminal to vellus hair ratio is usually 6:1 or 8:1 and decreases to less than 4:1 or 2:1 in chronic, severe AGA with a corresponding increase in follicular streamers. The perceived cosmetic disability in men appears to be related to the age of onset because until recently men were less likely to wear wigs than women. However, AGA in both sexes may be psychologically threatening because of societal expectations related to aging.

Spontaneous androgenetic alopecia in nonhuman primates

Androgenetic alopecia occurs in chimpanzees (Pan troglodytes), stump-tailed macaques (Macaca arctoides), and South American red uakaris (Cacajao rubicundus) as post adolescence progressive thinning of the scalp (Table I) [20-24]. Of these species, the stump-tailed macaque exhibits the most prominent and greatest incidence (nearly 100%) of AGA-like alopecia [25, 26]. Alopecia in macaques begins at around four years of age when testosterone levels elevate dramatically in males [26]. Female macaques have testosterone levels elevated at this age, although it is one tenth that of males. In spite of the difference, there is no sexual dichotomy for onset of alopecia in macaques [25, 26]. Inhibitors of 5alpha-reductase prevents post adolescent alopecia in macaques in both sexes as well as androgen receptor blockers (e.g. RU58841) [27-29]. Histologic changes in AGA-like alopecia in macaques parallel those observed in humans, i.e. the alopecia is associated with miniaturization of hair follicles; tranformation of terminal to vellus follicles in the frontal scalp [30, 31]. Not normally affected macaque follicles transplanted to alopecic frontal scalp maintain their terminal hair growth for greater than 7 years, indicating, as with humans, that only the frontal scalp is affected [32, 33]. This macaque model has responded positively in clinical trials to various compounds known to induce hair growth in male baldness patients [29, 34-38].

Since several nonhuman primate species have this AGA-like alopecia, it is likely that other species would also have it. During evaluation of a control Macaca mulatta skin biospy for comparison with another in the colony with papular atrichia [39], miniaturized anagen hair follicles were found between large terminal hair follicles (Fig. 1). Careful evaluation of other nonhuman primates in research colonies may yield additional models for androgenetic alopecia.

Testosterone induce alopecia in hamsters. Androgen-induced delay of hair growth in hamsters

Male golden Syrian hamsters implanted with time-release pellets of testosterone propionate had inhibition of hair regrowth for up to 21 days. However, by 28 days after surgically implanting testosterone into skin that had been clipped, the site had regrown hair similar to that of controls [40]. Since these mammals belong to the order Rodentia, it is not surprising that they responded in a manner similar to that of laboratory mice as described below.

Induced androchrongenetic alopecia in B6CBAF1 hybrid mice

Large numbers of spontaneous and induced mouse mutations exist in repositories around the world. While many have been studied in detail, far more mouse mutations remain to be carefully evaluated and compared with specific human diseases. Several hundred mouse mutations exist that have skin or hair abnormalities as part of their clinical phenotype [41-43]. While a spontaneous mutation or complex polygenic trait in an inbred strain may already exist that has the homologous features of AGA, as seen in humans, it has yet to be identified, characterized, and published. Alternatively, the numerous significant biological differences between hair follicle biology and cycling between mice and man may make a true model unrealistic [44].

A testosterone inducible model of alopecia, called the androchronogenetic alopecia (AGA) has been reported in B6CBAF1 mice. This purported mutation arose spontaneously in this hybrid strain and could only be consistently identified when androgen supplementation was given by various routes to mice 12 to 14 weeks of age [45, 46]. The B6CBAF1 mouse is a hybrid cross between a C57BL/6 female and a CBA male mouse.

Alopecia occurred spontaneously in a subcolony of B6CBAF1 mice as a perceptible thinning of the dorsal hair coat after sexual maturity in both males and females. Focal alopecia to diffuse thinning of the hair coat was achieved routinely in this colony by administration of testosterone or dihydrotestosterone by subcutaneous injection at various doses. The degree and pattern of testosterone induced alopecia varied between individuals, but after several months defined areas of alopecia developed. Alopecia proceeded from cranial to caudal [47], presumably following the normal hair cycle waves as seen with many other mouse alopecia mutations [41]. If testosterone was not given, less than 1% of the colony developed this form of alopecia [45]. No histologic variations in hair follicle structure were observed between balding and non-balding skin in testosterone treated mice other than that larger sebaceous glands were identified in those receiving testosterone [46]. These changes in hair follicles and hair density were documented and quantitated by fluorescein dye incorporation studies and subjective grading scales [46]. In vitro studies using biopsies from testosterone treated mice that compared alopecia and non-alopecia areas revealed significant variation in the metabolism of testosterone between the two areas [47]. In this hormone induced mouse model of AGA, testosterone appeared to induce alopecia by decreasing the rate of hair growth, decreasing the duration of the anagen phase, and markedly prolonging the duration of the telogen phase [46]. Testosterone-treated mice responded positively with increased hair growth when treated with minoxidil, cyproterone acetate, and diazoxide but there was no effect when mice were treated with dilantin, captopril, or fenoterol [46].

Although the B6CBAF1 is a promising model for AGA, these results are not easily reproduced using the hybrid mice from genetically defined resources (Sundberg and Bascom, unpublished data). Also, the original investigators who reported their AGA model have not made the original stocks available to other laboratories to verify the original observations. The usefulness of this AGA model is thereby limited by its lack of general accessability since B6CBAF1 mice used in these studies apparently do not respond in the same manner as those generally available from commercial vendors.

An alternative approach would be to find a production strain of inbred laboratory mice that had a testosterone inducible alopecia. Systematic evaluation of mice implanted subcutaneously initially with testosterone and later repeated with the more efficacious dihydrotestosterone revealed that 1) there was significant variation in response between inbred strains, 2) there was a sexual dichotomy in some strains but not others in the response, and 3) that rather than developing a progressive alopecia, most mice had a failure of hair to regrow and cover the surgical site where the implant was placed, as was the case with hamsters [40], suggesting the interference was in initiating the anagen phase of the hair cycle rather than changes in the size of the follicles themselves (Sundberg and Bascom, unpublished data). These observations were supported indirectly using the inducible alopecia areata graft model [48]. Recipients neutered prior to receiving histocompatible alopecia areata skin grafts had prolonged onset in a disease dependent upon onset of anagen.

Human to mouse cutaneous xenograft models

Nude (gene symbol: Foxn1^nu) and severe combined immunodeficiency (Prkdc^scid) mouse mutations lack T cells or T and B cells, respectively. These are useful mouse mutations because they readily accept xenografts from unrelated species ranging from humans to reptiles [49, 50]. Human skin grafts onto either or both mutations have been used successfully to study human diseases such as alopecia areata [51, 52], psoriasis vulgaris [53-59], and androgenetic alopecia [60-63] as well as the development of normal human skin [54, 64, 65]. Genetically engineered laboratory mice that have similar severe immunodeficiencies can also be used, such as the Rag1 null mutation. Nude mice have been used primarily for AGA because they have no readily observable surface hair thus making xenographs easy to identify and follow. Nude mice do have hair follicles that function and produce hair fibers, however, the fibers produced are defective, curl at the skin surface, and break off as they emerge [41, 66].

Skin grafts are commonly done to study AGA in nude mice using surplus punch biopsies collected at the time of human autologous grafts. Graft sites are observed for 6 months and used to evaluate various biological parameters [62, 63]. Topical doses of testosterone propionate on human skin grafted onto female nude mice was used to condition the grafts 6 to 8 weeks after surgery. This conditioning reduced the number of second hair cycles in the grafts during a six month observation period [61]. These observations suggest a similar mechanism to that described for the AGA mouse model that also uses testosterone conditioning [45, 46]. It is likely that this is a general phenomenon in mice rather than a true mutation in the AGA mouse model. Further studies will be necessary in order to refine these observations.

Double mutations have been created using mice homozygous for severe combined immunodeficiency (Prkdc^scid/Prkdc^scid), that will accept xenografts, and grafts from homozygous mouse mutations specific for a variety of hormone deficiencies, such as hypogonadal (gene symbol: hpg) and other hormone deficiencies (Table I) [42]. These types of graft models potentially will provide elegant approaches to dissect out how the presence or absence of various hormones affects hair growth, development, hair cycle, and hair loss.

CONCLUSION

Abbreviations

AGA: Androgenetic alopecia in man, also used for androchronogenetic alopecia in B6CBAF1 mice
Foxn1^nu: Gene symbol for mouse nude mutation
Prkdc^scid: Gene symbol for mouse severe combined immunodeficiency mutation
Rag1: Gene symbol for recombination activating gene

Acknowledgements

This work was supported by grants from the National Institutes of Health (CA34196, AR43801, JPS), Procter & Gamble, Inc., and the Bureau of Veterans Affairs (LEK).

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