Fumiaki Nakayama, M.D., Ph.D., Department of Radiation Emergency Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, Tel.: +81 (0)43 251 2111, Fax: +81 (0)43 206 4094, e-mail: email@example.com
Abstract: Radiation-induced hair loss is a clinically important, but under-researched topic. The aim of the study was to develop an in vivo assay system for radiation-induced apoptosis in hair follicles to promote hair research and exploit new radioprotectors. BALB/c mice received total body irradiation (TBI) with γ-rays at doses in the range from 8 to 16 Gy at 6 days after depilation. Pathological changes were detected progressively in the hair follicles over the time course after TBI and the dystrophy was evaluated on the basis of stage-specific parameters reported previously, which were found to be well-suited for classification of the radiation-induced hair follicle dystrophy. As a result, regression from anagen to catagen was determined in these follicles after irradiation. In addition, radiation-induced apoptosis was a good early dystrophic parameter. In this system, it was found that fibroblast growth factor-1 effectively prevented hair follicle apoptosis in mice.
Radiation therapy is one of the most effective cancer treatments. As radiation technology advances to treat more types of cancer (1), radiation therapy will be prescribed more often. However, irradiation at high doses causes hair loss as a side-effect, and efforts should be made to avoid this complication. Moreover, accidental exposure to radiation occurs occasionally around the world (2) and it is necessary to improve the means to help people suffering from the deleterious effects of radiation.
The availability of mouse models is valuable in the development of more effective strategies against hair damage and to analyse particular molecules in vivo, although several researchers conducting radiation-induced alopecia studies in small animals also use guinea pigs (3,4). Hair plucking during the telogen phase in mice is useful for inducing synchronous anagen phase (5–7) and has been applied in radiation hair damage studies (8,9). Plucking-induced anagen models are relevant to human hair because about 90% of human scalp hairs stay in the anagen phase (10). Moreover, in mice, the stage-specific parameters of hair follicles have been studied to assess alterations in healthy hair follicle cycling (7), and the characteristics of chemotherapy-induced hair follicle dystrophy were identified by applying these parameters (11). Such findings might be valid in radiation-induced hair damage research; however, there are few available references about hair follicle dystrophy caused by ionizing radiation for use in the laboratory.
Fibroblast growth factor-1 (FGF1) is a member of the FGF family (12) and is expressed in the hair follicle and may regulate hair growth (13). Recently, several FGFs have been found to be able to protect against radiation-induced intestinal damage (14–19). In addition, FGF7 increased hair survival during two hair cycles after X-ray irradiation (20). The mechanism of the repair of hair damage is thought to be via the protection of stem cells in the hair follicle. In contrast, the anti-apoptotic effect in the hair follicle is likely to be related to the repair of hair damage because l-carnitine-l-tartrate was found to stimulate human scalp hair growth by the up-regulation of proliferation and down-regulation of apoptosis in follicular keratinocytes in vitro (21). Therefore, the anti-apoptotic effects of FGFs have the potential to reduce hair follicle damage.
The characteristics of radiation-induced hair follicle dystrophy have not been identified previously in mice using plucking-induced anagen hair follicles. The preventative effects of FGF1 on radiation-induced apoptosis of the hair follicles remain unknown.
A portion of the dorsal skin of 7-week-old male BALB/c mice was depilated to induce the anagen phase of the hair growth cycle. The depilated BALB/c mice were given total body irradiation (TBI) with γ-rays at 6 days after depilation (Appendix S1).
Sequential hair follicle dystrophy in depilated skin after radiation exposure
Pathological changes were detected progressively in the anagen hair follicles over the time course after irradiation at a dose of 12 Gy (Fig. 1). In contrast, this dystrophy was characterized by shortened hair shafts without any shedding and did not show any findings of ‘primary recovery’, which comprised the immediate regeneration of new hair shafts as seen in dystrophic anagen induced by chemotherapy (11), indicating that the hair follicle stage was forced to transit from anagen into catagen by irradiation. On the basis of previous studies on chemotherapy-induced hair follicle dystrophy (7,11), each dystrophy during 8–24 h, 48 h and 84 h postexposure was classified as early, mid and late dystrophic catagen respectively. In particular, bulb diameter, apoptosis in the bulb and follicular distortion were good indicators for dystrophic catagen. In addition, club hair, epithelial constriction and DP diameter ratio were the parameters for mid-dystrophic catagen. Finally, the position of DP, length of the epithelial strand and trailing connective tissue sheath (CTS) were the specific parameters for late dystrophic catagen. Apoptotic fragments and a population of TUNEL+ cells were induced in the hair bulbs at a dose of 12 Gy during 8–24 h after depilation (Fig. S3). In addition, the induction of activated caspase-3 was detected in paraffin-embedded sections of the hair follicles at 8 h postexposure (Fig. S3).
Inhibitory effects of FGF1 on radiation-induced apoptosis in anagen hair follicles
The administration of FGF1 significantly reduced the percentage of apoptotic cells in hair bulbs almost in a dose-dependent manner (Fig. 2b). In addition, treatment with 100 μg FGF1 prominently decreased the appearance of activated caspase-3 positive cells in hair bulbs at 8 h after irradiation (Fig. 2a). The anti-apoptotic effect of FGF1 was also observed among irradiation doses ranging from 4 to 16 Gy (Fig. S4a); however, FGF1 did not significantly reduce apoptosis in hair bulbs when given after irradiation (data not shown).
Radiation-induced hair follicle dystrophy was characterized by morphological similarities to dystrophic catagen induced by chemotherapy. Specific stage parameters could be applied to estimate the stage of hair follicle dystrophy. In addition, the diameter of the hair bulb, the length of the follicle and the DP diameter ratio were also useful for classification (Fig. S1). According to these parameters, it was clarified that the radiation dose might promote the rate of the dystrophic process at 24 h after TBI and the extent of hair follicle damage occurred in a radiation dose-dependent manner (Fig. S2).
The induction of anagen by plucking of the hairs was useful for evaluating radiation-induced apoptosis. Expression of the active form of caspase-3 was detected remarkably well in the hair bulb at 8 h after irradiation (Fig. S3a) and it was in accord with the induction of TUNEL+ cells at 6 h after irradiation. FGF1 could prevent radiation-induced hair follicle apoptosis. However, it is known that the structural instability of wild-type FGF1 and its dependence on exogenous heparin for optimal activity may diminish its effects against radiation hair damage and potential for practical use (22,23). Therefore, the improvement of FGF1 is necessary to enable the development of novel protectors against radiation damage (24).
This work was supported in part by a grant from NIRS and also partly by the Budget for Nuclear Research of MEXT of Japan.