**12. Hair regrowth studies** *in vivo*

#### **12.1. The need of animal models**

Animals and humans are remarkably similar at physiological and anatomical levels. Also, genetically speaking, we share 67% of our DNA with earthworms and 99% with mice. Almost 90% of the veterinary medicines used to treat animals are similar to the ones developed for human use. Animal models can mimic human responses, but the differences in species and even in individual animals must be taken into consideration [83]. By recreating human diseases in animal models, we can study and understand the physiopathological processes involved in the disease and maybe find an efficient cure. The first Nobel Prize was awarded in 1901 and other 94 prizes were directly dependent on animal research [84].

Laboratory animals are used when human testing is not available for practical or ethical reasons. Animals represent good research subjects as they have a shorter life cycle that enables scientists to observe the animal throughout the entire life and across several generations. Also, animal models can be easily influenced by the environment, which is controlled by the researcher as far as the diet, temperature, lighting, and other factors are concerned.

Researchers use animal models for short-term objectives (to determine how the animal model responds to a stimuli or a treatment) and long-term purposes (development of a new drug, evaluation of bioavailability or toxicity, genetic study). The animal model should be sensitive, appropriate for the studied condition either by using specific evidence of previous studies or using a new animal model with the risk of generating inaccurate results [69]. Besides the similarity with the human response, other key features of the biomedical research on animal models are specificity to the study purpose, validation of the animal model, and improvement for further research. Animal research has brought many benefits not only to humans but also to animals in disease prevention and treatment [47, 48, 69].

For more than a 100 years, almost all the information obtained in the human and animal health research has been the result of studies performed on animal models. The most common aim of animal models use is the development of new methods for the diagnosis and treatment of diseases, through an understanding of the biology and the physiopathological processes involved [47, 69].

Even though animal models remain a necessity, alternatives consist of computer models, tissue and cell cultures, and other nonanimal-related research methods. In order to minimize the harmful effect of research performed on animal models, scientists tend to reduce the number of animals used to obtain valid results, to refine the experimental technique, or replace it with nonanimal research methods.

#### **12.2. Animal models used in hair loss and regrowth**

The data that we now possess about the life and function of the hair follicle in health and disease rely on the successful research performed *in vivo* (experiments on natural animals and genetically manipulated models) and *in vitro* (cultures of a cell type—dermal papilla or organ culture of isolated cell follicles). The preference for one of the two experimental alternatives depends on several factors: the purpose of the research and the advantages and disadvan-

Animals and humans are remarkably similar at physiological and anatomical levels. Also, genetically speaking, we share 67% of our DNA with earthworms and 99% with mice. Almost 90% of the veterinary medicines used to treat animals are similar to the ones developed for human use. Animal models can mimic human responses, but the differences in species and even in individual animals must be taken into consideration [83]. By recreating human diseases in animal models, we can study and understand the physiopathological processes involved in the disease and maybe find an efficient cure. The first Nobel Prize was awarded in

Laboratory animals are used when human testing is not available for practical or ethical reasons. Animals represent good research subjects as they have a shorter life cycle that enables scientists to observe the animal throughout the entire life and across several generations. Also, animal models can be easily influenced by the environment, which is controlled by the

Researchers use animal models for short-term objectives (to determine how the animal model responds to a stimuli or a treatment) and long-term purposes (development of a new drug, evaluation of bioavailability or toxicity, genetic study). The animal model should be sensitive, appropriate for the studied condition either by using specific evidence of previous studies or using a new animal model with the risk of generating inaccurate results [69]. Besides the similarity with the human response, other key features of the biomedical research on animal models are specificity to the study purpose, validation of the animal model, and improvement for further research. Animal research has brought many benefits not only to humans but also

For more than a 100 years, almost all the information obtained in the human and animal health research has been the result of studies performed on animal models. The most common aim of animal models use is the development of new methods for the diagnosis and treatment of diseases, through an understanding of the biology and the physiopathological processes

Even though animal models remain a necessity, alternatives consist of computer models, tissue and cell cultures, and other nonanimal-related research methods. In order to minimize the

researcher as far as the diet, temperature, lighting, and other factors are concerned.

1901 and other 94 prizes were directly dependent on animal research [84].

to animals in disease prevention and treatment [47, 48, 69].

tages involved.

involved [47, 69].

**12. Hair regrowth studies** *in vivo*

242 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**12.1. The need of animal models**

A large variety of animals (mice, rats, hamsters, rabbits, sheep, and even stump-tailed macaque) provide useful models for the *in vivo* study of hair loss and regrowth, but 95% of the animals bred for research purposes are rats and mice [85–89].

Mice represent an excellent model to study the hair cycle for several reasons: the first two cycles of the mouse hair follicle are synchronized; the mouse hair cycle is short, lasting for 3 weeks; hair follicles can be easily harvested and examined at specific time points in the cycle. Most importantly, the stages of the hair cycle have been well characterized in the mouse: anagen being morphologically subdivided into six stages and catagen into eight [22]. The periodic intervals of rodent hair cycles (especially the anagen-growing phase) seem to be less susceptible to iatrogenic influences [90]. The mouse hair cycle does not differ structurally from the human hair follicle cycle, except for the fact that during catagen the hair bulb is remodeled, but the vibrissae follicles do not retract. Scientists have recently discovered that a certain progenitor cell population in mice is analogous to the human cells, encouraging research on this particular animal model.

Besides studying the normal hair cycle on mice, scientists also focussed on the growth waves and hormonal control [91]. Significant differences between species regarding the follicular function and limited androgen-sensitive models were noticed [92]. Spontaneous mutations have been discovered and studied on hairless, nude, and tabby mutants, waved and angora animals, leading to the identification of new genes involved in hair loss and opening the path for transgenic technology research [93, 94].

Transgenic mice, also known as "knockout mice," are mice with altered genome through the use of genetic engineering. This gene-targeting technique has revolutionized the biomedical research by offering researchers the ability to create a specific animal model for the most common human diseases. In order to select the most appropriate immunodeficient mouse models for research purpose, scientists also take into consideration: background strain, behavior, husbandry, disease susceptibility, life span, breeding performance, radiosensitivity, functionality of various endogenous immune system components, and leakiness (tendency to produce functional B and T cells as they age).

Up to the present, immunodeficient mice (with T and B cells deficiencies) were used as models for autoimmune disease mechanisms and androgenetic alopecia studies. The androgen action upon the hair follicles has been studied on spontaneous and genetically engineered nude mutant mice [95].

The C57BL/6 mouse is the most popular laboratory rodent, widely used and studied, having its entire genome published. Research applications using this particular type of mice include immunology, cancer, neurodegerative disease, age-related hearing loss, bone density, diabetes, obesity, and biomarker studies. This black coat mouse has been used for the skin-free pigment and early visible pigmented tips of new anagen regrowth [88]. C57BL/6 represents one of the most well-characterized models available, with a minimum risk of genetic drift. It is also a convenient model for creating transgenic mice, which are recognized by the mixed coat colors.

**12.3. Common study designs in hair loss and regrowth**

*In vivo* hair regrowth studies usually use animals of either sex and weight, kept in experimental rooms that are free of pathogens and opportunistic agents. For 7–14 days prior to the experiment, the animals are housed under specific conditions: room temperature of 23°C, controlled humidity, a 12:12 h light, and dark cycle. In order to avoid licking, individual housing is preferred or a maximum of two animals per cage. Standard laboratory diet and water ad libitum are provided. After completing the experiment, animals are euthanized according to the current regulations. For accurate results, most of the studies on animal models are per-

Evaluation of Animal Models Suitable for Hair Research and Regeneration

http://dx.doi.org/10.5772/intechopen.69698

245

Experimental designs may include one of the depilation methods: shaving, the use of a raisin mixture, or a hair removal cream [91, 103]. The most commonly used is the shaving of a larger skin area (the whole back or body) or of several smaller areas that are denuded for testing. For animal immobilization during procedures, general anesthesia is commonly performed with a combination of ketamine (i.p. 50 mg/kg b.w.) and xylazine (20 mg/kg b.w.) [47, 48, 69]. Some study designs, such as that of Mester et al., required before each successive hair treatment, the shaving of the skin. This procedure can induce mechanic stimulation of hair growth, as previously reported in the literature, and influence the study results. Other experiments done on adult rats point out that after the fur was dyed and shaved, the regrowing hairs formed a system of linear loops that were closely correlated with the shaving process [66, 67]. In order to avoid this effect, it is recommended not to shave the skin of the animal model before each session of therapy. Other factors which influence the hair regrowth are physical

Depilation-induced hair cycle has been studied, and it follows a strict course: nine days after depilation, the hair follicles enter the final stage of the growth cycle (anagen VI). On day 17 after depilation, the follicles enter the regression stage (catagen), while on day 20 follicles get

Efficacy of the treatment is screened by observing the presence, rate, and cosmetic acceptability of hair regrowth. More sophisticated assays include determining how the drug induced

Researchers do not possess standardized methods for *in vivo* hair regrowth assessment. New, accurate, and minimally invasive procedures are still needed as the most commonly used tools are qualitative assessments, limited in number. They include macroscopic assessment with the naked eye (visualization and photographs of the area of interest) based on scales that assess the percentage of hair regrowth on the interest area and tricoscopic evaluation (with a

factors such as low temperature, which triggers fast regrowth after shaving.

*12.3.1. Housing conditions*

formed in triplicate [47, 69].

to the resting stage (telogen) [22].

*12.3.3. Evaluation of hair loss and hair regrowth*

hair regrowth and exploring the pathogenesis of AA.

*12.3.2. Depilation methods*

The C3H/HeJ mouse model was used in a large range of studies: immunology, cancer (especially mammary tumors), inflammation, sensorineural, and cardiovascular disease. This animal model was the most widely reported for hair growth promotion, most possibly due to the fact that C3H/HeJ mice can spontaneously develop alopecia areata (AA) from 6 to 18 months of age. Also, alopecia areata can be surgically induced by skin-grafting from a donor animal with AA onto an isogenic C3H/HeJ recipient (normal haired mice of the same strain) [90, 96].

In 2010, researchers created the first rodent model of AGA, taking into consideration its relationship to androgen metabolism and androgen signaling, mediated by the androgen receptor (AR). They used transgenic mice overexpressing human AR in the skin under control of the keratin 5 promoter and exposed them to high levels of 5-alpha dihydrotestosterone, which led to delayed hair regeneration, mimicking AGA. The scientists concluded that androgen-mediated hair loss is AR-dependent and suggested that AR and beta-catenin mediate this effect [97].

There are many rat strains raised for research purposes, but the albino Wistar Bratislava rat is the most commonly used. Gene knockout techniques are relatively difficult to be applied and successfully achieved in rats. For hair loss and regeneration experiments, the Wistar rats and the Dundee Experimental Bald Rat (DEBR) strain were commonly used. The latter has the ability to spontaneously develop adult onset alopecia areata (AA) at a higher frequency than in the mouse model [98].

In the research field of hair loss and regeneration two major achievements must be mentioned on the rat animal model: coaxing human stem cells to become dermal papilla and producing new hair follicles when transplanted on rat skin [98]. Also, by inhibiting the rejection of foreign skin, human skin grafts were applied and even rat dermal papillae continued to produce hair after reimplantation *in vivo* on a rat model [99, 100].

Research performed on a rabbit animal model, added important data to the field, proving that full thickness transplants, made with full pedicle graft (separated from their original nervous and vascular supply) retain their original *intrinsec* activity and are not modified by the action of the surrounding tissue [101]. Furthermore, the rabbit represents a common animal model used to screen compounds potentially efficient in treating alopecia.

The Golden Syrian hamster (*Mesocricetus auratus*) has been previously used for research purposes, even though it is a very common pet. The hamster flank organ has served as a model to study the effect of testosterone (T) upon the hair follicle, the sebaceous glands, and the dermal pigment. This hamster is known to be useful for the specific and quantitative assessment of different substances on hair growth, being also useful for therapy testing in hirsutism. Macroscopic (hair density evaluation) and microscopic (hair diameter analysis) hair growth assessments have been performed on Golden hamsters [102].

#### **12.3. Common study designs in hair loss and regrowth**

#### *12.3.1. Housing conditions*

obesity, and biomarker studies. This black coat mouse has been used for the skin-free pigment and early visible pigmented tips of new anagen regrowth [88]. C57BL/6 represents one of the most well-characterized models available, with a minimum risk of genetic drift. It is also a convenient model for creating transgenic mice, which are recognized by the mixed coat

244 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

The C3H/HeJ mouse model was used in a large range of studies: immunology, cancer (especially mammary tumors), inflammation, sensorineural, and cardiovascular disease. This animal model was the most widely reported for hair growth promotion, most possibly due to the fact that C3H/HeJ mice can spontaneously develop alopecia areata (AA) from 6 to 18 months of age. Also, alopecia areata can be surgically induced by skin-grafting from a donor animal with AA onto an isogenic C3H/HeJ recipient (normal haired mice of the same strain) [90, 96]. In 2010, researchers created the first rodent model of AGA, taking into consideration its relationship to androgen metabolism and androgen signaling, mediated by the androgen receptor (AR). They used transgenic mice overexpressing human AR in the skin under control of the keratin 5 promoter and exposed them to high levels of 5-alpha dihydrotestosterone, which led to delayed hair regeneration, mimicking AGA. The scientists concluded that androgen-mediated hair loss is AR-dependent and suggested that AR and beta-catenin

There are many rat strains raised for research purposes, but the albino Wistar Bratislava rat is the most commonly used. Gene knockout techniques are relatively difficult to be applied and successfully achieved in rats. For hair loss and regeneration experiments, the Wistar rats and the Dundee Experimental Bald Rat (DEBR) strain were commonly used. The latter has the ability to spontaneously develop adult onset alopecia areata (AA) at a higher frequency than

In the research field of hair loss and regeneration two major achievements must be mentioned on the rat animal model: coaxing human stem cells to become dermal papilla and producing new hair follicles when transplanted on rat skin [98]. Also, by inhibiting the rejection of foreign skin, human skin grafts were applied and even rat dermal papillae continued to produce

Research performed on a rabbit animal model, added important data to the field, proving that full thickness transplants, made with full pedicle graft (separated from their original nervous and vascular supply) retain their original *intrinsec* activity and are not modified by the action of the surrounding tissue [101]. Furthermore, the rabbit represents a common animal model

The Golden Syrian hamster (*Mesocricetus auratus*) has been previously used for research purposes, even though it is a very common pet. The hamster flank organ has served as a model to study the effect of testosterone (T) upon the hair follicle, the sebaceous glands, and the dermal pigment. This hamster is known to be useful for the specific and quantitative assessment of different substances on hair growth, being also useful for therapy testing in hirsutism. Macroscopic (hair density evaluation) and microscopic (hair diameter analysis) hair growth

colors.

mediate this effect [97].

in the mouse model [98].

hair after reimplantation *in vivo* on a rat model [99, 100].

used to screen compounds potentially efficient in treating alopecia.

assessments have been performed on Golden hamsters [102].

*In vivo* hair regrowth studies usually use animals of either sex and weight, kept in experimental rooms that are free of pathogens and opportunistic agents. For 7–14 days prior to the experiment, the animals are housed under specific conditions: room temperature of 23°C, controlled humidity, a 12:12 h light, and dark cycle. In order to avoid licking, individual housing is preferred or a maximum of two animals per cage. Standard laboratory diet and water ad libitum are provided. After completing the experiment, animals are euthanized according to the current regulations. For accurate results, most of the studies on animal models are performed in triplicate [47, 69].

#### *12.3.2. Depilation methods*

Experimental designs may include one of the depilation methods: shaving, the use of a raisin mixture, or a hair removal cream [91, 103]. The most commonly used is the shaving of a larger skin area (the whole back or body) or of several smaller areas that are denuded for testing. For animal immobilization during procedures, general anesthesia is commonly performed with a combination of ketamine (i.p. 50 mg/kg b.w.) and xylazine (20 mg/kg b.w.) [47, 48, 69].

Some study designs, such as that of Mester et al., required before each successive hair treatment, the shaving of the skin. This procedure can induce mechanic stimulation of hair growth, as previously reported in the literature, and influence the study results. Other experiments done on adult rats point out that after the fur was dyed and shaved, the regrowing hairs formed a system of linear loops that were closely correlated with the shaving process [66, 67].

In order to avoid this effect, it is recommended not to shave the skin of the animal model before each session of therapy. Other factors which influence the hair regrowth are physical factors such as low temperature, which triggers fast regrowth after shaving.

Depilation-induced hair cycle has been studied, and it follows a strict course: nine days after depilation, the hair follicles enter the final stage of the growth cycle (anagen VI). On day 17 after depilation, the follicles enter the regression stage (catagen), while on day 20 follicles get to the resting stage (telogen) [22].

#### *12.3.3. Evaluation of hair loss and hair regrowth*

Efficacy of the treatment is screened by observing the presence, rate, and cosmetic acceptability of hair regrowth. More sophisticated assays include determining how the drug induced hair regrowth and exploring the pathogenesis of AA.

Researchers do not possess standardized methods for *in vivo* hair regrowth assessment. New, accurate, and minimally invasive procedures are still needed as the most commonly used tools are qualitative assessments, limited in number. They include macroscopic assessment with the naked eye (visualization and photographs of the area of interest) based on scales that assess the percentage of hair regrowth on the interest area and tricoscopic evaluation (with a hand-held dermatoscope, with polarized light and magnification abilities) [41]. Trichoscopy allows a correct hair regrowth evaluation, as it can detect decrease of hair diameter up to ten times or diameter variations. Both macroscopic and microscopic methods assess hair regrowth with the help of personalized hair growth scales or standardized, already published scales.

Usually, the dorsal part of the animals is used for the testing. After being depilated and treated, the animal skin is observed and photographed at specific time intervals (day 1, 7, 14, and 21) to record the start of the hair regrowth period and the pattern of hair regrowth, compared to controls. Several hair regrowth potential scores are mentioned by literature. The one described by Matsuda et al., for instance, ranges from 0 to 5: 0 = no hair growth, 1 = less than 20% of hair growth, 2 = 20–39% of hair growth, 3 = 40–59% of hair regrowth, 4 = 60–79% of hair regrowth, 5 = 80–100% of hair regrowth [54]. Researchers also use self-designed scales of hair regrowth that consider: Type IV (high hair density, full, thick fur), Type III (moderate hair density with no visible skin area), Type II (low hair density, with the visualization of the skin), Type I (uneven hair growth on the test area, skin easily seen) [47, 69].

The hair growth cycle, consisting of three phases (anagen, catagen, and telogen) is used by both practitioners and researchers to diagnose the hair growth condition and to decide on the hair growth-promoting agent. In human subjects, digital trichoscopy is available, with auto-

**Figure 2.** Classification of the hair regrowth effect (type 0, type 1, type 2, type 3, type 4, type 5) for macroscopic and

Evaluation of Animal Models Suitable for Hair Research and Regeneration

http://dx.doi.org/10.5772/intechopen.69698

247

Several studies focused on the validation of Minoxidil 2% treatment on the animal model used, as this topical treatment is thought to be the gold standard treatment for hair loss. This substance affected the normal hair cycle by shortening telogen, causing premature entry of

The limitation of using an animal model while studying hair regeneration can be briefly summarized as follows. First, synchronized hair cycles generate waves of new hair regrowth, which make the interpretation of result a hard task. Second, the lack of independence of the hair follicles, since they have coordinated regrowth pattern on a precise time scale, as described by Muller-Rover et al. [22]. Third, young mice present the drawback of patchy growth after the second wave of hair growth is completed. Lastly, the increased hair density on an animal model leads to difficulties of assessment by densitometry or cross-section trichometer [69].

The results of our hair growth research performed on Wistar rats showed, besides a normal hair growth in the majority of the animal models, a lack of hair regrowth on the tested area. Other studies performed on black-and-white mice reported that no further hair growth was

We also noticed a diffuse hair regrowth in some study groups, while in others, the hair appeared to make some specific linear loops that were observed macroscopically. Literature date confirmed our findings. In similar situations, researchers experienced a diffuse hair growth in some animals with an uncharacteristic, diagonal strip [66]. Li-Yaun Liu et al. described four main linear hair regrowth patterns noticed on a rat model: the dorsal loop and the lateral dorsal loop (running along the dorsum and hind limb) and the ventral loop and lateral ventral loop (traveling along the thorax, abdomen, and forelimb). These hair-loop-lines create cranio-caudally-oriented waves of regrowth 2–15 mm wide, symmetrically on both sides of the body, running from the head through the torso to the limbs [105]. Li-Yaun Liu et al. concluded that after shaving the skin, the hair follicles from these new hair lines were

matic assessment of the number of follicles in each hair growth phase.

microscopic assessments—personal study performed on New Zealand Rabbits.

the resting follicles into anagen phase [103, 104].

observed on half of the control animals.

always in an anagen phase [106].

**12.4. Limitations of animal models regarding hair regrowth**

The hair regrowth potential scores can be applied for both macroscopic and microscopic assessments (**Figures 1** and **2**).

On the other hand, quantitative methods, such as hair weight determinations, hair density measurements, or histopathological examination offer more accurate results. For hair weight determination, the regrown hair from an area of 1 cm2 of skin is cut and weighed with an analytical balance [48].

In order to analyze the histological features at the end of the treatment period, the animals are sacrificed and a skin biopsy is isolated for histopathological examination. The thickness of the skin and the location of hair follicles in the dermis can be assessed by microscopic photography.

Also the hair cycle can be assessed, as the anagen induction can be calculated with the formula: (number of follicles in hypodermis) × 100/(number of follicles in dermis). Literature data showed an association of increasing skin thickness, follicle count, and macroscopic development of skin pigmentation with anagen induction [18, 23]. The study by Liu et al. found that in the anagen phase the bulb of the hair follicles was enlarged and deeply inserted into the dermis. The research also revealed that the hair follicles in the shaved, bare areas were short, small, and in the phases of telogen, anagen, or catagen [69].

**Figure 1.** Classification of the hair regrowth effect (type I, type II, type III, type IV) for macroscopic and microscopic assessments—personal study performed on Wistar Bratislava rats. The control area is marked with red (left side of the picture), the test area with blue (right side).

Evaluation of Animal Models Suitable for Hair Research and Regeneration http://dx.doi.org/10.5772/intechopen.69698 247

**Figure 2.** Classification of the hair regrowth effect (type 0, type 1, type 2, type 3, type 4, type 5) for macroscopic and microscopic assessments—personal study performed on New Zealand Rabbits.

The hair growth cycle, consisting of three phases (anagen, catagen, and telogen) is used by both practitioners and researchers to diagnose the hair growth condition and to decide on the hair growth-promoting agent. In human subjects, digital trichoscopy is available, with automatic assessment of the number of follicles in each hair growth phase.

Several studies focused on the validation of Minoxidil 2% treatment on the animal model used, as this topical treatment is thought to be the gold standard treatment for hair loss. This substance affected the normal hair cycle by shortening telogen, causing premature entry of the resting follicles into anagen phase [103, 104].

#### **12.4. Limitations of animal models regarding hair regrowth**

hand-held dermatoscope, with polarized light and magnification abilities) [41]. Trichoscopy allows a correct hair regrowth evaluation, as it can detect decrease of hair diameter up to ten times or diameter variations. Both macroscopic and microscopic methods assess hair regrowth with the help of personalized hair growth scales or standardized, already published scales.

Usually, the dorsal part of the animals is used for the testing. After being depilated and treated, the animal skin is observed and photographed at specific time intervals (day 1, 7, 14, and 21) to record the start of the hair regrowth period and the pattern of hair regrowth, compared to controls. Several hair regrowth potential scores are mentioned by literature. The one described by Matsuda et al., for instance, ranges from 0 to 5: 0 = no hair growth, 1 = less than 20% of hair growth, 2 = 20–39% of hair growth, 3 = 40–59% of hair regrowth, 4 = 60–79% of hair regrowth, 5 = 80–100% of hair regrowth [54]. Researchers also use self-designed scales of hair regrowth that consider: Type IV (high hair density, full, thick fur), Type III (moderate hair density with no visible skin area), Type II (low hair density, with the visualization of the

The hair regrowth potential scores can be applied for both macroscopic and microscopic

On the other hand, quantitative methods, such as hair weight determinations, hair density measurements, or histopathological examination offer more accurate results. For hair weight

In order to analyze the histological features at the end of the treatment period, the animals are sacrificed and a skin biopsy is isolated for histopathological examination. The thickness of the skin and the location of hair follicles in the dermis can be assessed by microscopic photography. Also the hair cycle can be assessed, as the anagen induction can be calculated with the formula: (number of follicles in hypodermis) × 100/(number of follicles in dermis). Literature data showed an association of increasing skin thickness, follicle count, and macroscopic development of skin pigmentation with anagen induction [18, 23]. The study by Liu et al. found that in the anagen phase the bulb of the hair follicles was enlarged and deeply inserted into the dermis. The research also revealed that the hair follicles in the shaved, bare areas were

**Figure 1.** Classification of the hair regrowth effect (type I, type II, type III, type IV) for macroscopic and microscopic assessments—personal study performed on Wistar Bratislava rats. The control area is marked with red (left side of the

of skin is cut and weighed with an

skin), Type I (uneven hair growth on the test area, skin easily seen) [47, 69].

determination, the regrown hair from an area of 1 cm2

246 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

short, small, and in the phases of telogen, anagen, or catagen [69].

assessments (**Figures 1** and **2**).

picture), the test area with blue (right side).

analytical balance [48].

The limitation of using an animal model while studying hair regeneration can be briefly summarized as follows. First, synchronized hair cycles generate waves of new hair regrowth, which make the interpretation of result a hard task. Second, the lack of independence of the hair follicles, since they have coordinated regrowth pattern on a precise time scale, as described by Muller-Rover et al. [22]. Third, young mice present the drawback of patchy growth after the second wave of hair growth is completed. Lastly, the increased hair density on an animal model leads to difficulties of assessment by densitometry or cross-section trichometer [69].

The results of our hair growth research performed on Wistar rats showed, besides a normal hair growth in the majority of the animal models, a lack of hair regrowth on the tested area. Other studies performed on black-and-white mice reported that no further hair growth was observed on half of the control animals.

We also noticed a diffuse hair regrowth in some study groups, while in others, the hair appeared to make some specific linear loops that were observed macroscopically. Literature date confirmed our findings. In similar situations, researchers experienced a diffuse hair growth in some animals with an uncharacteristic, diagonal strip [66]. Li-Yaun Liu et al. described four main linear hair regrowth patterns noticed on a rat model: the dorsal loop and the lateral dorsal loop (running along the dorsum and hind limb) and the ventral loop and lateral ventral loop (traveling along the thorax, abdomen, and forelimb). These hair-loop-lines create cranio-caudally-oriented waves of regrowth 2–15 mm wide, symmetrically on both sides of the body, running from the head through the torso to the limbs [105]. Li-Yaun Liu et al. concluded that after shaving the skin, the hair follicles from these new hair lines were always in an anagen phase [106].

Also, the behavior of the animals should be taken into consideration, as it can create issues and interfere with the research results [107]. For example, the C57BL/6 mice show barbering behavior, the dominant mouse in a cage selectively removing hair from its subordinate cage mates. Mice that have been barbered have large bald patches on their bodies, especially around the head, snout, and shoulders [108].

[5] Montagna W. The evolution of human skin. Journal of Human Evolution. 1985;**14**:3-10

Evaluation of Animal Models Suitable for Hair Research and Regeneration

http://dx.doi.org/10.5772/intechopen.69698

249

[6] Wheeler PE. The loss of functional body hair in man: The influence of thermal environment, body form and bipedality. Journal of Human Evolution. 1985;**14**:23-28

[7] Jablonski NG, Chaplin G. Human skin pigmentation as an adaptation to UV radiation. Proceedings of the National Academy of Sciences of the United States of America.

[8] Iyengar B. The hair follicle: a specialised UV receptor in the human skin? Biological

[9] Feughelman M. Mechanical Properties and Structure of Alpha-keratin Fibers: Wool,

[10] Goodier M, Hordinsky M. Normal and aging hair biology and structure. Current Pro-

[11] Randall VA, Hibberts NA, Thornton MJ, Merrick AE, Hamada K, Kato S, et al. Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla

[12] Slominski A, Wortsman J, Plonka PM, Schallreuter KU, Paus R, Tobin DJ. Hair follicle

[13] Lin C, Li Y, Ji YC, Keng H, Cai XN, Zhang JK. Micro-encapsulated human hair dermal papilla cells: A substitute for dermal papilla. Archives of Dermatological Research.

[14] Liao YH, Kuo WC, Chou SY, Tsai CS, Lin GL, Tsai MR, et al. Quantitative analysis of intrinsic skin aging in dermal papillae by in vivo harmonic generation microscopy.

[15] Randall VA, Ebling FJG. Seasonal changes in human hair growth. British Journal of Der-

[16] Orentreich N. Biology of the skin. In: Montagna W, Dobson RL, editors. Hair Growth.

[17] Ellis JA, Sinclair R, Harrap SB. Androgenetic alopecia: pathogenesis and potential for

[19] Randall VA. Androgens are the main regulator of human hair growth. In: Camacho F, Randall VA, Price V, editors. Hair and its Disorders: Biology, Pathology and Management.

[20] Paus R. Therapeutic strategies for treating hair loss. Drug Discovery Today: Therapeutic

[18] Dry E. The coat of the mouse (*Mus musculus*). Journal of Genetics. 1926;**1**:287-340

therapy. Expert Reviews in Molecular Medicine. 2002;**4**(22):1-11

pigmentation. Journal of Investigative Dermatology. 2005;**124**(1):13-21

2010;**107**(Suppl 2):8962-8968

Signals and Receptors. 1998;**7**(3):188-194

blems in Dermatology. 2015;**47**:1-9

2008;**300**(9):531-535

matology. 1991;**124**:146-151

Strategies. 2006;**3**(1):101-110

Human Hair and Related Fibers. Sydney: UNSW Press; 1996

cells. European Journal of Dermatology. 2001;**11**:315-320

Biomedical Optics Express. 2014;**5**(9):885-889

Vol. IX. Oxford: Pergamon; 1969. pp. 99-108

London: Martin Dunitz; 2000. pp. 121-136

Regardless of the shortcomings of either animal model, most of them validate their usefulness for drug efficacy and safety testing for humans.
