An efficient, non-invasive approach for in-vivo sampling of hair follicles: design and applications in monitoring DNA damage and aging.

In accordance with the 3 Rs principle (to replace, reduce and refine) animal models in biomedical research, we have developed and applied a new approach for sampling and analyzing hair follicles in various experimental settings. This involves use of a convenient device for non-invasive collection of hair follicles and processing methods that provide sufficient amounts of biological material to replace stressful and painful biopsies. Moreover, the main components of hair follicles are live cells of epithelial origin, which are highly relevant for most types of malignant tumors, so they provide opportunities for studying aging-related pathologies including cancer. Here, we report the successful use of the method to obtain mouse hair follicular cells for genotyping, quantitative PCR, and quantitative immunofluorescence. We present proof of concept data demonstrating its utility for routine genotyping and monitoring changes in quality and expression levels of selected proteins in mice after gamma irradiation and during natural or experimentally induced aging. We also performed pilot translation of animal experiments to human hair follicles irradiated ex vivo. Our results highlight the value of hair follicles as biological material for convenient in vivo sampling and processing in both translational research and routine applications, with a broad range of ethical and logistic advantages over currently used biopsy-based approaches.

Analyses of histological and transcriptome differences in the skin of short-hair and long-hair rabbits.

BACKGROUND: Hair fibre length is an important economic trait of rabbits in fur production. However, molecular mechanisms regulating rabbit hair growth have remained elusive. RESULTS: Here we aimed to characterise the skin traits and gene expression profiles of short-hair and long-hair rabbits by histological and transcriptome analyses. Haematoxylin-eosin staining was performed to observe the histological structure of the skin of short-hair and long-hair rabbits. Compared to that in short-hair rabbits, a significantly longer anagen phase was observed in long-hair rabbits. In addition, by RNA sequencing, we identified 951 genes that were expressed at significantly different levels in the skin of short-hair and long-hair rabbits. Nine significantly differentially expressed genes were validated by quantitative real-time polymerase chain reaction. A gene ontology analysis revealed that epidermis development, hair follicle development, and lipid metabolic process were significantly enriched. Further, we identified potential functional genes regulating follicle development, lipid metabolic, and apoptosis as well as important pathways including extracellular matrix-receptor interaction and basal cell carcinoma pathway. CONCLUSIONS: The present study provides transcriptome evidence for the differences in hair growth between short-hair and long-hair rabbits and reveals that lipid metabolism and apoptosis might constitute major factors contributing to hair length.

Histological and functional comparisons of four anatomical regions of porcine skin with human abdominal skin.

Because of the shortage of human skin for research purposes, porcine skin has been used as a model of human skin. The aim of this study was to identify the region of German Landrace pig skin that could be used as the best possible substitute for human abdominal skin. Porcine samples were collected from the ear, flank, back and caudal abdomen; human abdominal skin samples were excised during plastic surgery. Histological and ultrastructural assessments were carried out on the epidermis and dermis, with emphasis on the dermo-epidermal interface length, dermo-epidermal thickness ratio as well as densities of; hair follicles, arrector pili muscles, blood vessels and sweat glands. In the pig, the barrier function of the four anatomical regions was assessed. Results showed that both histologically and ultrastructurally, all four regions of porcine skin were similar to human skin. These include the shapes of keratinocytes, structure of cell contacts and presence of Weibel Palade bodies in endothelial cells. Other parameters such as the thickness of epidermis, the thickness of stratum basale, spinosum and granulosum and the number of cell layers in the stratum corneum were similar in human abdominal and in all four regions of porcine skin. However, there were also significant differences especially in the thickness of the stratum corneum, the dermo-epidermal interface length and the blood vessel density.

Ultrasound Characteristics of the Hair Follicles and Tracts, Sebaceous Glands, Montgomery Glands, Apocrine Glands, and Arrector Pili Muscles.

OBJECTIVES: To explore the capability of very high-frequency ultrasound (US; 50-71 MHz) to detect the normal morphologic characteristics of the hair follicles and tracts, sebaceous glands, Montgomery glands, apocrine glands, and arrector pili muscles. METHODS: A retrospective study, approved by the Institutional Review Board, evaluated the normal US morphologic characteristics of the hair and adnexal structures in a database of very high-frequency US images extracted from the perilesional or contralateral healthy skin of 1117 consecutive patients who underwent US examinations for localized lesions of the skin and 10 healthy individuals from December 2017 to June 2018. These images were matched with their counterparts from the database of normal histologic images according to the corporal region. The Cohen concordance test and regional mean diameters of the hair follicles and adnexal structures were analyzed. RESULTS: The normal hair follicles and tracts, sebaceous glands, Montgomery glands, apocrine glands, and arrector pili muscles were observed on US images and matched their histological counterparts in all the corporal regions. There was significant US concordance (κ = 0.82; P = .0001) among observers. Regional mean diameters (millimeters) of the hair follicles, sebaceous glands, and apocrine glands are provided. CONCLUSIONS: The hair follicles and tracts, sebaceous glands, Montgomery glands, apocrine glands, and arrector pili muscles are detectable with very high-frequency US, including some regional and anatomic variants. Knowledge of their normal US appearances is a requisite for detecting subclinical changes, understanding the physiopathologic characteristics, and supporting the early diagnosis and management of common dermatologic diseases.

Study on Hair Distribution in Healthy Males for Hair Restoration Design.

BACKGROUND: The incidence of hair loss among Chinese male has increased to 21.3 per hundred. Hair restoration has been an effective technique. Detailed hair distribution has a tremendous impact on the surgery design. OBJECTIVE: To investigate the pattern of hair distribution in Chinese young adult males. METHODS: A total of 1000 males without hair disease were enrolled. We evaluated the locations of the main anatomical marks at different sites on the scalp and analyzed the hair density and follicular unit structure using the standard photographs and trichoscope. RESULTS: The hairline shapes were classified as: linear (48.7%), linear with central protrusion (27.9%), round (9.8%), round with central protrusion (13.7%). The average height of the median line was 6.78 ±â€Š0.75 cm, the ratio of the median line and the forehead height was 0.333. The average distance from the parietal whorl to the vertical bimeatal line was 7.05 ±â€Š3.32 cm, and most of the PWs were on the right (51.4%) and had a clockwise pattern (73.3%). The hair density was 171.12 ±â€Š18.32 hairs/cm in the vertex. 1-hair follicular units were (75.90% and 56.39%) in anterior hairline and temporal area. CONCLUSION: Our study clearly assisted understanding of scalp anatomy and hair distribution in Chinese young adult males.

Hair Biology: Growth and Pigmentation.

Healthy hair is vital to identity. Understanding the intricate anatomy and physiology of hair provides insight into the aging process and the eventual loss of either hair pigmentation or hair shafts. Several biologics are available that have enabled altering or slowing the aging process of hair, but, unfortunately, no agent exists that can reverse the natural course. The commonly used biologics are discussed.

Assessment of markers expressed in human hair follicles according to different skin regions.

BACKGROUND: Body region-dependent hair follicle (HF) characteristics are concerned with follicular size and distribution, and have been demonstrated to have characteristics for each region of the body. OBJECTIVES: The aim of the present study was to investigate the expression patterns of the markers called cytokeratin 15 (K15), cytokeratin 6 (K6) and monoclonal antibody Ki-67, and also apoptosis in HFs, which can be observed in different parts of the human body. MATERIAL AND METHODS: In this study, healthy human HFs were taken by biopsy from 5 various donor sites of the human body: the scalp, the leg, the abdomen, the back and waist. HF-containing skin specimens taken using cryosection were stained with hematoxylin & eosin (H&E) and K15, K6, Ki-67 and terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL) immunofluorescence staining protocol was performed. RESULTS: Different skin regions from the human body were examined histologically. While the HFs of scalp tissue showed anatomically obvious hair layers, some hair sections from other regions, like the leg, the abdomen, back and waist, were not as distinct as in the scalp region. According to our findings, K15 expression was highest in the scalp. In addition, the immunoreactivity (IR) intensity of K15 was significantly decreased in the HFs on the waist and abdominal regions, compared to the scalp and back regions (p < 0.001). However, the IR intensity of K6 in the scalp region was statistically significantly higher than the IR intensity of K6 in the abdomen region (p < 0.05). Moreover, we showed intraepithelial apoptosis and proliferation of keratinocytes in the bulge of HF. In the study, Ki-67-positive and TUNEL-positive cell numbers were not statistically significant (p > 0.05). CONCLUSIONS: Our findings are important for further investigation of molecular aspects of the human hair follicle stem cells compartments in health and disease, which might be a promising model for comparative studies with different human diseases.

Retrospective Assessment of Follicular Unit Density in Asian Men With Androgenetic Alopecia.

BACKGROUND: Hair density, together with hair diameter has a tremendous impact on the cosmetic outcome of hair transplantation surgery. OBJECTIVE: This retrospective study aims to assess the average number of follicular unit (FU) grafts in the donor area of Asian men with androgenetic alopecia. METHODS: Seventy patients (ages 24-65 years old) with virgin scalp who had their first hair transplantation were included. During the harvesting procedure, a 2- to 3-cm-wide area of the donor site was shaved from ear to ear with 5 specific areas assigned as reference points for the study namely the central occipital area, left and right parietal and temporal areas. A video microscope was used to capture the field of each reference point. Follicular units in each area were counted and the number of 1-hair, 2-hair, and 3-hair follicular groupings was recorded. RESULTS: East and Southeast Asians had an average of 61.1 FU/cm (111.2 hairs/cm); West Asians had an average density of 63.6 FU/cm (126.8 hairs/cm); South Asians were noted to have an average density of 63.5 FU/cm (126.8 hairs/cm). CONCLUSION: Asian hair density has slight variations in the number of FU grafts in each donor area.

Folliculosebaceous neoplasms: A review of clinical and histological features.

Numerous benign and occasionally malignant tumors arise from the folliculosebaceous apparatus. Confusing terminology, rarity of malignant variants and overlapping histological features can lead to diagnostic uncertainty. This review highlights the clinical and histopathological features that help to classify these entities, as well as the various syndromes associated with certain members of this large family of tumors.

[CO-TRANSPLANTATION OF MOUSE EPIDERMIS AND DERMIS CELLS IN INDUCING HAIR FOLLICLE REGENERATION].

OBJECTIVE: To investigate the co-transplantation of C57-green fluorescent protein (GFP) mouse epidermis and dermis cells subcutaneously to induce the hair follicle regeneration. METHODS: C57-GFP mouse epidermis and dermis were harvested for isolation the mouse epidermis and dermis cells. The morphology of epidermis and dermis mixed cells at ratio of 1:1 of adult mouse, dermis cells of adult mouse, cultured 3rd generation dermis cells were observed by fluorescence microscope. Immunocytochemistry staining was used to detect hair follicle stem cells markers in cultured 3rd generation dermis cells from new born C57-GFP mouse. And then the epidermis and dermis mixed cells of adult mouse (group A), dermis cells of adult mouse (group B), cultured 3rd generation dermis cells of new born mouse (group C), and saline (group D) were transplanted subcutaneously into Balb/c nude mice. The skin surface of nude mice were observed at 4, 5, 6 weeks of transplantation and hair follicle formation were detected at 6 weeks by immunohistochemistry staining. RESULTS: The isolated C57-GFP mouse epidermis and dermis cells strongly expressed the GFP under the fluorescence microscope. Immunocytochemistry staining for hair follicle stem cells markers in cultured 3rd generation dermis cells showed strong expression of Vimentin and α-smooth muscle actin, indicating that the cells were dermal sheath cells; some cells expressed CD133, Versican, and cytokeratin 15. After transplanted for 4-6 weeks, the skin became black at the injection site in group A, indicating new hair follicle formation. However, no color change was observed in groups B, C, and D. Immunohistochemical staining showed that new complete hair follicles structures formed in group A. GFP expression could be only observed in the hair follicle dermal sheath and outer root sheath in group B, and it could also be observed in the hair follicle dermal sheath, outer root sheath, dermal papilla cells, and sweat gland in group C. The expression of GFP was negative in group D. CONCLUSION: Co-transplantation of mouse epidermis and dermis cells can induce the hair follicle regeneration by means of interaction of each other. And transplantation of isolated dermis cells or cultured dermis cells individually only partly involved in the hair follicles formation.

[Establishment of an in vitro culture model of single human hair follicle].

OBJECTIVE: To establish an in vitro culture model of single human hair follicle, and observe their morphological and histological changes. METHODS: Human hair follicles were isolated from the volunteer patients. After dissecting follicles into single, follicles in growth phase were cultured in Williams E without any serum. This experiment included 3 groups: single follicle without sebaceous gland and other surrounding tissue (control group); single follicle with sebaceous gland and without the other surrounding tissue( experiment group A); single follicles with sebaceous gland and the other surrounding tissue (experiment group B). The survival rate, survival time, growth rate, multiplication capacity and apoptosis of cultured follicles and their morphological and histological changes were observed sequentially. RESULTS: The hair follicles in experiment groups showed a better viability and a higher growth rate than those in control group. And the follicles in group B could keep growing for more than 25 days, which was longer than those in group A. Moreover, the sebaceous gland and the other surrounding tissue in group B showed great induction effect on follicle-cell proliferation and anti-apoptosis. CONCLUSION: The in vitro culture model of signal human hair follicles (single follicles including epidermis, sebaceous gland and the other surrounding tissue) had optimized internal environment which is similar to in vivo internal environment.

A Guide to Studying Human Hair Follicle Cycling In Vivo.

Hair follicles (HFs) undergo lifelong cyclical transformations, progressing through stages of rapid growth (anagen), regression (catagen), and relative "quiescence" (telogen). Given that HF cycling abnormalities underlie many human hair growth disorders, the accurate classification of individual cycle stages within skin biopsies is clinically important and essential for hair research. For preclinical human hair research purposes, human scalp skin can be xenografted onto immunocompromised mice to study human HF cycling and manipulate long-lasting anagen in vivo. Although available for mice, a comprehensive guide on how to recognize different human hair cycle stages in vivo is lacking. In this article, we present such a guide, which uses objective, well-defined, and reproducible criteria, and integrates simple morphological indicators with advanced, (immuno)-histochemical markers. This guide also characterizes human HF cycling in xenografts and highlights the utility of this model for in vivo hair research. Detailed schematic drawings and representative micrographs provide examples of how best to identify human HF stages, even in suboptimally sectioned tissue, and practical recommendations are given for designing human-on-mouse hair cycle experiments. Thus, this guide seeks to offer a benchmark for human hair cycle stage classification, for both hair research experts and newcomers to the field.

The effect of parathyroid hormones on hair follicle physiology: implications for treatment of chemotherapy-induced alopecia.

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) influence hair follicles through paracrine and intracrine routes. There is significant evidence that PTH and PTHrP influence the proliferation and differentiation of hair follicle cells. The PTH/PTHrP receptor signalling plays an important role in the hair follicle cycle and may induce premature catagen-telogen transition. Transgenic mice with an overexpression or blockade (PTH/PTHrP receptor knockout mice) of PTHrP activity revealed impaired or increased hair growth, respectively. Some findings also suggest that PTHrP may additionally influence the hair cycle by inhibiting angiogenesis. Antagonists of the PTH/PTHrP receptor have been shown to stimulate proliferation of hair follicle cells and hair growth. A hair-stimulating effect of a PTH/PTHrP receptor antagonist applied topically to the skin has been observed in hairless mice, as well as in mice treated with cyclophosphamide. These data indicate that the PTH/PTHrP receptor may serve as a potential target for new (topical) hair growth-stimulating drugs, especially for chemotherapy-induced alopecia.

High follicle density does not decrease sweat gland density in Huacaya alpacas.

When exposed to high ambient temperatures, mammals lose heat evaporatively by either sweating from glands in the skin or by respiratory panting. Like other camelids, alpacas are thought to evaporate more water by sweating than panting, despite a thick fleece, unlike sheep which mostly pant in response to heat stress. Alpacas were brought to Australia to develop an alternative fibre industry to sheep wool. In Australia, alpacas can be exposed to ambient temperatures higher than in their native South America. As a young industry there is a great deal of variation in the quality and quantity of the fleece produced in the national flock. There is selection pressure towards animals with finer and denser fleeces. Because the fibre from secondary follicles is finer than that from primary follicles, selecting for finer fibres might alter the ratio of primary and secondary follicles. In turn the selection might alter sweat gland density because the sweat glands are associated with the primary follicle. Skin biopsy and fibre samples were obtained from the mid-section of 33 Huacaya alpacas and the skin sections were processed into horizontal sections at the sebaceous gland level. Total, primary, and secondary follicles and the number of sweat gland ducts were quantified. Fibre samples from each alpaca were further analysed for mean fibre diameter. The finer-fibred animals had a higher total follicle density (P<0.001) and more sweat glands (P<0.001) than the thicker-fibred animals. The fibre diameter and total follicle density were negatively correlated (R(2)=0.56, P<0.001). Given that the finer-fibred animals had higher follicle density and more sweat glands than animals with thicker fibres, we conclude that alpacas with high follicle density should not be limited for potential sweating ability.

Three dimensional distribution of Propionibacterium acnes biofilms in human skin.

Propionibacterium acnes is regarded as a common member of the human skin microbiota, often occurring in biofilms. Little is known about the size of bacterial biofilms in hair follicles as a few sections of biopsy tissue are routinely evaluated. Transversal sectioning provides a better opportunity for histological analyses of hair follicles which can be followed through the different morphological levels. Direct visualization of P. acnes biofilms in hundreds of consecutive sections allowed insight into the 3D distribution in human hair follicles as well as investigating the depth of biofilm distribution within hair follicles. Four distinct colonization patterns of P. acnes biofilms were revealed. Results have shown that an individual P. acnes biofilm can spread for 1900 µm in a terminal hair follicle. This information can be of help while designing potential antibiofilm treatment.

Evaluation of nerves in Mohs micrographic surgery: histologic mimickers of perineural invasion and nervous tissue on frozen section.

BACKGROUND: Perineural invasion (PNI) is an important histologic finding and may be a negative prognostic factor for squamous cell carcinoma (SCC). It may be associated with more-aggressive tumor behavior. Mohs surgeons encounter microscopic PNI regularly and must be able to diagnose it accurately to guide care decisions. OBJECTIVE: To describe benign histologic mimickers of PNI and neural structures in SCC commonly encountered on frozen, hematoxylin and eosin-stained sections and to review how to differentiate them from PNI. METHODS AND MATERIALS: Review of the literature regarding histologic mimickers of PNI and additional contributions to frozen section PNI and nerve tissue mimickers. RESULTS: We describe benign findings, including arrector pili muscles, eccrine muscles, vessels, granulomatous inflammation, and eddies of SCC, that may each be mistaken for nerves or PNI. We discuss the ways in which they may be distinguished on frozen sections and review other commonly encountered entities that resemble PNI. CONCLUSION: Perineural inflammation and peritumoral fibrosis are common mimickers of PNI on frozen section, although other mimickers exist on permanent sections. Normal structures may appear "neural" by way of frozen tissue orientation, processing, or inflammation and thus must be differentiated from nerve tissue and PNI during Mohs surgery.

Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges.

Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.