Low-level laser activates Wnt/ß-catenin signaling pathway-promoting hair follicle stem cell regeneration and wound healing: Upregulate the expression of key downstream gene Lef 1.

BACKGROUND: The objective of this study is to investigate the mechanism by which low-level laser stimulation promotes the proliferation of intraepithelial hair follicle stem cells (HFSCs) in wounds. This research aims to expand the applications of laser treatment, enhance wound repair methods, and establish a theoretical and experimental foundation for achieving accelerated wound healing. METHODS: The experimental approach involved irradiating a cell model with low-level laser to assess the proliferation of HFSCs and examine alterations in the expression of proteins related to the Wnt/ß-catenin signaling pathway. A mouse back wound model was established to investigate the effects of low-level laser irradiation on wound healing rate, wound microenvironment, and the proliferation of HFSCs in relation to the Wnt/ß-catenin signaling pathway. RESULTS: The research findings indicate that low-level laser light effectively activates the Wnt signaling pathway, leading to the increased accumulation of core protein ß-catenin and the upregulation of key downstream gene Lef 1. Consequently, this regulatory mechanism facilitates various downstream biological effects, including the notable promotion of HFSC proliferation and differentiation into skin appendages and epithelial tissues. As a result, the process of wound healing is significantly accelerated. CONCLUSION: Low levels of laser activates the Wnt signalling pathway, promotes the regeneration of hair follicle stem cells and accelerates wound healing.

Carbon Dioxide Fractional Laser Treatment Induces Lgr5+ Stem Cell Activation and Hair Regrowth Through the Canonical Wnt/ß-Catenin Pathway.

BACKGROUND: Different types of alopecia have negative impacts on patients. Recently, some kinds of laser or light therapies have been reported to effectively alleviate hair loss. Carbon dioxide fractional laser (CO2FL) treatment is one of the most effective laser treatments, but its beneficial effects and exact mechanism in hair regrowth have not been reported in detail. The purpose of this study was to investigate the effect and molecular mechanism further. METHODS: C57 and Lgr5-Cre: Rosa-mTmG mouse models of hair regrowth were established by CO2FL treatment, and the parameters that induced the best effect were determined. Tissues were harvested on the day prior to the treatment day and on days 3, 5, 7, 10 and 14 after CO2FL. H&E and immunofluorescence staining, RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and related inhibitor were used to determine the molecular mechanism underlying the effect of CO2FL treatment on the hair cycle and hair regrowth. In clinical trial, five participants were treated three sessions at 1-month intervals to obverse the effects. RESULTS: Hair regrew and covered the treatment area on the tenth day after CO2FL treatment with the best parameters, while the control group showed signs of hair growth on the 14th day. H&E and immunofluorescence staining showed that the transition of hair follicles (HFs) from telogen to anagen was accelerated, and the rapid activation and proliferation of Lgr5+ hair follicle stem cells (HFSCs) were observed in the treatment group. The RNA-seq, qPCR and WB results indicated that the Wnt pathway was significantly activated after CO2FL treatment. Improvement achieved with CO2FL treatment in clinical trial. CONCLUSIONS: The results of this study suggest that CO2FL treatment can promote hair regrowth by activating Lgr5+ HFSCs and upregulating the Wnt/ß-catenin pathway. Clinical trial results demonstrated that CO2FL treatment will be a promising therapeutic regimen for alopecia. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing ß-catenin signaling.

Hair loss or alopecia is an unpleasant symptom that exacerbates an individual's self-esteem and requires appropriate treatment. The Wnt/ß-catenin signaling is a central pathway that promotes dermal papilla induction and keratinocyte proliferation during hair follicle renewal. GSK-3ß inactivated by its upstream Akt and ubiquitin-specific protease 47 (USP47) has been shown to inhibit ß-catenin degradation. The cold atmospheric microwave plasma (CAMP) is microwave energy enriched with mixtures of radicals. CAMP has been reported to have antibacterial and antifungal activities with wound healing activity against skin infection; however, the effect of CAMP on hair loss treatment has not been reported. We aimed to investigate the effect of CAMP on promoting hair renewal in vitro and to elucidate the molecular mechanism, targeting ß-catenin signaling and YAP/TAZ, the co-activators in the Hippo pathway, in human dermal papilla cells (hDPCs). We also evaluated plasma effects on the interaction between hDPCs and HaCaT keratinocytes. The hDPCs were treated with plasma-activating media (PAM) or gas-activating media (GAM). The biological outcomes were determined by MTT assay, qRT-PCR, western blot analysis, immunoprecipitation, and immunofluorescence. We found that ß-catenin signaling and YAP/TAZ were significantly increased in PAM-treated hDPCs. PAM treatment also induced ß-catenin translocation and inhibited ß-catenin ubiquitination by activating Akt/GSK-3ß signaling and upregulating USP47 expression. In addition, hDPCs were more aggregated with keratinocytes in PAM-treated cells compared with control. HaCaT cells cultured in a conditioned medium derived from PAM-treated hDPCs exhibited an enhancing effect on activating YAP/TAZ and ß-catenin signaling. These findings suggested that CAMP may be a new therapeutic alternative for alopecic treatment.

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.

Low-frequency electromagnetic fields promote hair follicles regeneration by injection a mixture of epidermal stem cells and dermal papilla cells.

The bioeffects of low-frequency electromagnetic fields (EMF) on a bio-engineered hair follicle generation had not been fully elucidated. This present study was designed to evaluat the therapeutically effective of low frequency EMF on hair follicles regeneration. In this experiment, epidermal stem cells (ESCs) and dermal papilla (DP) cells were isolated and culture-expanded. Then the mixture containing of ESCs and DP cells was implanted into the epidermal layer or corium layer of nude mice. Those mice were  divided at random into the control group and EMF group, 7 days or 14 days later, the skin specimens were harvested to assess for hair regeneration or a bio-engineered skin formation using H&E staining. After injection of the mixture into the epidermal layer of nude mice for 14 days, H&E staining showed that the new hair formed the correct structure comprising hair matrix, hair shaft, and inner root sheath, outer root sheath, and DP. Comparing to the control, the hair follicles erupted at a higher density in the EMF group. When the mixture was implanted into the corium layer for 7 days, comparing with the characteristics of new hair follicles in the control group, H&E staining also showed the mixture induced to form 4 ~ 6 epidermal layers with a higher density of hair follicle like-structures in the bioengineered epithelial layers after EMF exposure. Our results suggested that the injection of a mixture of ESCs and DP cells in combination with EMF exposure facilitated the induction of hair follicle regeneration and a bioengineered skin formation with hair follicle-like structures.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Dose-response of human follicles during laser-based hair removal: Ex vivo photoepilation model with classification system embracing morphological and histological features.

OBJECTIVES: Photoepilation is a commonly used technology in home-use devices (HUDs) and in professional systems to remove unwanted body hair using pulses of laser or intense pulsed light (IPL). Albeit HUDs and professional systems operate at different fluences and treatment regimes, both demonstrate high hair reduction. The underlying mechanisms, however, remain unknown partly due to high divergence of the existing literature data. The objective of this study was to develop an ex vivo photoepilation model with a set of criteria evaluating response to light pulses; and to investigate dose-response behavior of hair follicles (HFs) subjected to a range of fluences. METHODS: After ex vivo treatment (single pulse, 810 nm, 1.7-26.4 J/cm2 , 4-64 ms pulse) human anagen HFs were isolated and maintained in culture for 7-10 days. Response to light was evaluated based on gross-morphology and histological examination (H&E and TUNEL stainings). RESULTS: HFs treated ex vivo demonstrated a dose-dependent response to light with five distinct classes defined by macroscopic and microscopic criteria. Fluences below 13.2 J/cm2 provoked catagen-like transition, higher fluences resulted in coagulation in HF compartments. CONCLUSION: Observed changes in the HF organ culture model were reflected by clinical efficacy. The developed photoepilation model provides an easy and fast method to predict clinical efficacy and permanency of light-based hair removal devices. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Establishment and characterization of a radiation-induced dermatitis rat model.

Radiation-induced dermatitis is a common and serious side effect after radiotherapy. Current clinical treatments cannot efficiently or fully prevent the occurrence of post-irradiation dermatitis, which remains a significant clinical problem. Resolving this challenge requires gaining a better understanding of the precise pathophysiology, which in turn requires establishment of a suitable animal model that mimics the clinical condition, and can also be used to investigate the mechanism and explore effective treatment options. In this study, a single dose of 90 Gy irradiation to rats resulted in ulceration, dermal thickening, inflammation, hair follicle loss, and sebaceous glands loss, indicating successful establishment of the model. Few hair follicle cells migrated to form epidermal cells, and both the severity of skin fibrosis and hydroxyproline levels increased with time post-irradiation. Radiation damaged the mitochondria and induced both apoptosis and autophagy of the skin cells. Therefore, irradiation of 90 Gy can be used to successfully establish a rat model of radiation-induced dermatitis. This model will be helpful for developing new treatments and gaining a better understanding of the pathological mechanism of radiation-induced dermatitis. Specifically, our results suggest autophagy regulation as a potentially effective therapeutic target.

Exposure to 50 Hz electromagnetic fields enhances hair follicle regrowth in C57BL/6 mice.

IMPACT STATEMENT: In this study, our experiments confirmed that 50 Hz EMF affected hair follicle regrowth, and 50 Hz EMF enhanced K15+ stem cells proliferation in the hair bulb and follicular outer root sheath of hair follicles. Those results indicated that 50 Hz EMF may be beneficial for functional healing of hair loss.

Promotion of homology-directed DNA repair by polyamines.

Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.

How chemotherapy and radiotherapy damage the tissue: Comparative biology lessons from feather and hair models.

Chemotherapy and radiotherapy are common modalities for cancer treatment. While targeting rapidly growing cancer cells, they also damage normal tissues and cause adverse effects. From the initial insult such as DNA double-strand break, production of reactive oxygen species (ROS) and a general stress response, there are complex regulatory mechanisms that control the actual tissue damage process. Besides apoptosis, a range of outcomes for the damaged cells are possible including cell cycle arrest, senescence, mitotic catastrophe, and inflammatory responses and fibrosis at the tissue level. Feather and hair are among the most actively proliferating (mini-)organs and are highly susceptible to both chemotherapy and radiotherapy damage, thus provide excellent, experimentally tractable model systems for dissecting how normal tissues respond to such injuries. Taking a comparative biology approach to investigate this has turned out to be particularly productive. Started in chicken feather and then extended to murine hair follicles, it was revealed that in addition to p53-mediated apoptosis, several other previously overlooked mechanisms are involved. Specifically, Shh, Wnt, mTOR, cytokine signalling and ROS-mediated degradation of adherens junctions have been implicated in the damage and/or reparative regeneration process. Moreover, we show here that inflammatory responses, which can be prominent upon histological examination of chemo- or radiotherapy-damaged hair follicle, may not be essential for the hair loss phenotype. These studies point to fundamental, evolutionarily conserved mechanisms in controlling tissue responses in vivo, and suggest novel strategies for the prevention and management of adverse effects that arise from chemo- or radiotherapy.

UVB-induced depletion of donor-derived dendritic cells prevents allograft rejection of immune-privileged hair follicles in humanized mice.

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor- and recipient-derived pathways. Donor-derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor-derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti-CD154 antibody (Ab) into recipients in an MHC-mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long-term survival with newly growing pigmented hair shafts in both Ab-treated groups (Ab-only and UV plus Ab) and in the UV-only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3+ T cell and MHC class II+ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV-only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen-specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.

Ultraviolet radiation induces Melan-A-expressing cells in interfollicular epidermis in wild-type mice.

Adult wild-type mice are not supposed to be proper models for ultraviolet radiation (UVR)-induced melanoma since melanocytes are confined to hair follicles and cannot be sufficiently reached by UVR. On the other hand, in mutated mouse models used for melanoma research limitations, including an altered immune system and selection of affected pathways, lead to tumors phenotypically quite different from naturally occurring melanomas. We compared the distribution of epidermal melanocytes in UVR and not-UVR-exposed wild-type C57BL/6 mice. Starting at the age of 8 weeks, mice were exposed to physiologic doses of UVR three times weekly over 16 weeks. Back skin biopsies were taken 4, 8, 12 and 16 weeks after initiation of exposure, and stained for Melan-A, representing a highly selective marker for melanocytes. Surprisingly, after exposure to UVR, Melan-A positive cells were detected also in the interfollicular epidermis of C57BL/6 mice. We conclude that UVR is capable of inducing interfollicular epidermal melanocytes in wild-type mice.

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30-60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0-30 min/day (second group), people use mobile phones for 30-60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.

Activation of Wnt/ß-catenin signaling is involved in hair growth-promoting effect of 655-nm red light and LED in in vitro culture model.

Activation of the Wnt/ß-catenin signaling pathway plays an important role in hair follicle morphogenesis and hair growth. Recently, low-level laser therapy (LLLT) was evaluated for stimulating hair growth in numerous clinical studies, in which 655-nm red light was found to be most effective and practical for stimulating hair growth. We evaluated whether 655-nm red light + light-emitting diode (LED) could promote human hair growth by activating Wnt/ß-catenin signaling. An in vitro culture of human hair follicles (HFs) was irradiated with different intensities of 655-nm red light + LED, 21 h7 (an inhibitor of ß-catenin), or both. Immunofluorescence staining was performed to assess the expression of ß-catenin, GSK3ß, p-GSK3ß, and Lef1 in the Wnt/ß-catenin signaling. The 655-nm red light + LED not only enhanced hair shaft elongation, but also reduced catagen transition in human hair follicle organ culture, with the greatest effectiveness observed at 5 min (0.839 J/cm2). Additionally, 655-nm red light + LED enhanced the expression of ß-catenin, p-GSK3ß, and Lef1, signaling molecules of the Wnt/ß-catenin pathway, in the hair matrix. Activation of Wnt/ß-catenin signaling is involved in hair growth-promoting effect of 655-nm red light and LED in vitro and therefore may serve as an alternative therapeutic option for alopecia.

Laser and Light Treatments for Hair Reduction in Fitzpatrick Skin Types IV-VI: A Comprehensive Review of the Literature.

Unwanted facial and body hair presents as a common finding in many patients, such as females with hirsutism. With advances in laser and light technology, a clinically significant reduction in hair can be achieved in patients with light skin. However, in patients with darker skin, Fitzpatrick skin types (FST) IV-VI, the higher melanin content of the skin interferes with the proposed mechanism of laser-induced selective photothermolysis, which is to target the melanin in the hair follicle to cause permanent destruction of hair bulge stem cells. Many prospective and retrospective studies have been conducted with laser and light hair-removal devices, but most exclude patients with darkly pigmented skin, considering them a high-risk group for unwanted side effects, including pigmentation changes, blisters, and crust formation. We reviewed the published literature to obtain studies that focused on hair reduction for darker skin types. The existing literature for this patient population identifies longer wavelengths as a key element of the treatment protocol and indicates neodymium-doped yttrium aluminum garnet (Nd:YAG), diode, alexandrite, and ruby lasers as well as certain intense pulsed light sources for safe hair reduction with minimal side effects in patients with FST IV-VI, so long as energy settings and wavelengths are appropriate. Based on the findings in this review, safe and effective hair reduction for patients with FST IV-VI is achievable under proper treatment protocols and energy settings.

Activation of mTORC1 Signaling is Required for Timely Hair Follicle Regeneration from Radiation Injury.

Transit amplifying cells (TACs) are highly proliferative in nature and tend to be sensitive to ionizing radiation. Due to the abundance of TACs that support the elongation of hair shafts, growing hair follicles are highly sensitive to radiation injury. How hair follicles repair themselves after radiation injury is unclear. In this study, we observed that in 4 Gy irradiated mice, hair follicle dystrophy was induced with apoptosis-driven loss of hair matrix cells, which are the TACs that fuel hair growth. The dystrophy was repaired within 96 h without significant hair loss, indicating that a regenerative attempt successfully restored the TAC population to resume anagen growth. Soon after irradiation, mTORC1 signaling was activated in the TAC compartment and its activation was maintained until the regeneration process was completed. Inhibition of mTORC1 by rapamycin treatment increased radiation-induced cell apoptosis, reduced cell proliferation and delayed restoration of Wnt signaling in the hair matrix after radiation injury, leading to prolonged dystrophy and hair loss. These results demonstrate that mTORC1 signaling is activated after irradiation and is required for timely regeneration of the TAC pool of hair follicles, so that hair growth can resume after radiation injury.

Wnt/ß-catenin and ERK pathway activation: A possible mechanism of photobiomodulation therapy with light-emitting diodes that regulate the proliferation of human outer root sheath cells.

BACKGROUND: Outer root sheath cells (ORSCs) play important roles in maintaining hair follicle structure and provide support for the bulge area. The hair growth promoting effects of photobiomodulation therapy (PBMT) have been reported, but the mechanisms for this in human ORCs (hORSCs) have rarely been studied. OBJECTIVE: The aim of this study was to investigate the effect of various wavelengths of light-emitting diode (LED) irradiation on human ORSCs (hORSCs). METHODS: LED irradiation effects on hORSC proliferation and migration were examined with MTT assay, BrdU incorporation assay and migration assays. hORSCs were irradiated using four LED wavelengths (415, 525, 660, and 830 nm) with different low energy levels. LED irradiation effects on the expression of molecules associated with the Wnt/ß-catenin signaling and ERK pathway, hair stem cell markers, and various growth factors and cytokines in hORSCs were examined with real-time PCR and Western blot assay. The effect of the LED-irradiated hORSCs on cell proliferation of human dermal papilla cells (hDPCs) was examined with co-culture and MTT assay. RESULTS: PBMT with LED light variably promoted hORSC proliferation and suppressed cell apoptosis depending on energy level. LED irradiation induced Wnt5a, Axin2, and Lef1 mRNA expression and ß-catenin protein expression in hORSCs. Phosphorylation of ERK, c-Jun, and p38 in hORSCs was observed after LED light irradiation, and ERK inhibitor treatment before irradiation reduced ERK and c-Jun phosphorylation. Red light-treated hORSCs showed substantial increase in IL-6, IL-8, TNF-a, IGF-1, TGF-ß1, and VEGF mRNA. Light irradiation at 660 and 830 nm projected onto hORSCs accelerated in vitro migration. LED-irradiated hORSCs increased hDPCs proliferation when they were co-cultured. The conditioned medium from LED-irradiated hORSCs was sufficient to stimulate hDPCs proliferation. CONCLUSION: These results demonstrate that LED light irradiation induced hORSC proliferation and migration and inhibited apoptosis in vitro. The growth-promoting effects of LEDs on hORSCs appear to be associated with direct stimulation of the Wnt5a/ß-catenin and ERK signaling pathway. Lasers Surg. Med. 49:940-947, 2017. © 2017 Wiley Periodicals, Inc.

The Feather Model for Chemo- and Radiation Therapy-Induced Tissue Damage.

Chemo- and radiation therapy are the main modalities for cancer treatment. A major limiting factor is their toxicity to normal tissue, thus reducing the dose and duration of the therapy. The hair follicle, gastrointestinal tract, and hematopoietic system are among the target organs that often show side effects in cancer therapy . Although these organs are highly mitotic in common, the molecular mechanism of the damage remains unclear. The feather follicle is a fast-growing mini-organ, which allows observation and manipulation on each follicle individually. As a model system, the feather follicle is advantageous because of the following reasons: (1) its complex structure is regulated by a set of evolutionarily conserved molecular pathways, thus facilitating the effort to dissect the specific signaling events involved; (2) its morphology allows the continuity of normal-perturbed-normal structure in a single feather, thus "recording" the damaging effect of chemo- and radiation therapy; (3) further histological and molecular analysis of the damage response can be performed on each plucked feather; thus, it is not necessary to sacrifice the experimental animal. Here, we describe methods of applying the feather model to study the molecular mechanism of chemo- and radiation therapy-induced tissue damage.

Accelerated senescence in skin in a murine model of radiation-induced multi-organ injury.

Accidental high-dose radiation exposures can lead to multi-organ injuries, including radiation dermatitis. The types of cellular damage leading to radiation dermatitis are not completely understood. To identify the cellular mechanisms that underlie radiation-induced skin injury in vivo, we evaluated the time-course of cellular effects of radiation (14, 16 or 17 Gy X-rays; 0.5 Gy/min) in the skin of C57BL/6 mice. Irradiation of 14 Gy induced mild inflammation, observed histologically, but no visible hair loss or erythema. However, 16 or 17 Gy radiation induced dry desquamation, erythema and mild ulceration, detectable within 14 days post-irradiation. Histological evaluation revealed inflammation with mast cell infiltration within 14 days. Fibrosis occurred 80 days following 17 Gy irradiation, with collagen deposition, admixed with neutrophilic dermatitis, and necrotic debris. We found that in cultures of normal human keratinocytes, exposure to 17.9 Gy irradiation caused the upregulation of p21/waf1, a marker of senescence. Using western blot analysis of 17.9 Gy-irradiated mice skin samples, we also detected a marker of accelerated senescence (p21/waf1) 7 days post-irradiation, and a marker of cellular apoptosis (activated caspase-3) at 30 days, both preceding histological evidence of inflammatory infiltrates. Immunohistochemistry revealed reduced epithelial stem cells from hair follicles 14-30 days post-irradiation. Furthermore, p21/waf1 expression was increased in the region of the hair follicle stem cells at 14 days post 17 Gy irradiation. These data indicate that radiation induces accelerated cellular senescence in the region of the stem cell population of the skin.