Journal of Cutaneous Pathology 1978; 5: 184-192
Surface Architecture of X-lrradiated Hair Follicles A. J. P. KLEIN-SZANTO AND B. M. de REY Division of Radiation Pathology, Department of Radiobiology, National Atomic Energy Commission, Buenos Aires, Argentina The combined use of scanning electron-microscopy and chemical separation of epidermis permitted the study of the irradiated and normal hair follicle surface characteristics. Irradiation reduced the length and density of the hair follieles, produeing at the same time marked alterations and deformities of the individual follicles. As most of these changes were dose- and time-dependent, these struetures, studied by these techniques, provide an interesting model as biological indicators of radiation dose. (Reeeived for publieation January 9, 1978)
Hair follicle radiosensitivity has been extensively studied by numerous techniques. Most of these studies have not directly analyzed the hair follicles but inferred through indirect data, obtained by studying the morphology and the development of the hair shafts, the radiation-induced damage of the hair-forming follicle cells (Malkinson & Griem 1966, 1968, Malkinson et al. 1970, Burlin et al. 1973). Although the histopathology of irradiated hair folhcles has been described previously (Geary 1952, Montagna & Chase 1956, Van Scott & Reinertson 1957) and many studies on the radiation response of the follicle and hair shaft melanin-content have been reported (Moshman & Upton 1954, Potten 1968, Potten & Forbes 1972, Kostanecki et al. 1976) no scanning electron-microscopic analyses of these irradiated structures have, to our knowledge, been published. The aim of this study was to obtain further information on the radiation-induced damage of hair follieles by employing a combination of modern methods of scanning electron-microscopy (SEM) and the splitskin techniques, thus enabling the observation of the undersurfaee of the epidermis
and the complete surface of hair follicles. Material and Methods
The mid-third portion of the tails of 4-dayold Wistar rats were X-irradiated with 2, 4, 8 and 16 Krads in the following conditions — 230 kV, 13 mA using a 0.25 mm Cu and 1 mm Al filter. The proximal portion of the tail which was used as a control, as well as the rest of the animal's body, was shielded with lead plates. The animals were sacrificed 1, 2, 4 and 7 days after irradiation exposure. Fotor to five rats were studied in each group. The irradiated and control skin areas of the taO were separated from the underlying vertebrae by soft traction and immediately incubated in 2NNaBr solution at 37°C for approximately 20 min (Papa & Farber 1971). Working under a dissecting microscope, the epidermis was then separated from the dermis, as described previously (KleinSzanto & Schroeder 1977). The epithelium was stretched out on a blotting paper and fixed in chilled half-strength Karnovsky fixative (Karnovsky 1965). Dehydration in graded ethanol series followed by graded
ISEM OF HAIR FOLLICLES
185
f^ig. 1. Non-irradiated hair follicles of a 9-day-old rat's tail. Note elongated hair follicles and smaller folUcles ofthe underhairs. X 35. follicles as well as smaller ones, probably corresponding to the under hairs of rodents, eould be seen (Figs. 1, 2 and 3). During the 10 days of the animals' life under study the non-irradiated anagen follicle population underwent an active elongation. The non-irradiated follicles showed a partially smooth surface with some undulated areas, especially in the apical part in the proximity of the bulb. In this area it was usually easy to observe a concave depression or an orifice of approximately 30jU, located Results exactly at the center of the bulb's tip In both irradiated and non-irradiated speei- (Fig. 4). This orifice corresponded to the mens the hair follicles were usually distrib- connective tissue bridge communicating the uted in parallel rows in which the main dermal papilla with the dermis. isoamylacetate and subsequent drying by liquid CO2 by the critical point method was carried out. The epidermal samples were mounted in order to show the basal epithelial surface with the attached hair follicles. The speeimens were coated with carbon and gold in a Jeol evaporator and examined with a Jeol JSM-U-3 scanning electron microscope.
186
KLEIN-SZANTO AND de REY
Fig. 2. Hair follieles 2 days after exposure to 2,000 rads, showing few alterations. The numerical density is diminished although the follieles are relatively normal. X 40. Five and 7 days after irradiation these The irradiated follicles showed interesting surfaee and struetural alterations which were structural alterations were seen even more markedly pronounced after 8 and 16 Krads frequently. They consisted of relatively long and somewhat attenuated, although similar "spikes" or "horns" located at the apical in nature, after skin exposure to 2 and portion of the follicle and positioned almost 4 Krads. perpendicularly to the follicle's longitudinal During the first 2 days after irradiation axis (Figs. 6 and 7). the hair follicles showed few modifications. The orifices left by the papillae were very The elongated cylindrical shape was not rarely seen in irradiated follicles. Four and modified, although frequent bending or 7 days after irradiation hairs were also markincurvation of the follicle tips were noted edly shorter and their density per unit area (Fig. 5). The follicles were somewhat shorter decreased. than the controls, especially after exposure to the higher radiation doses. Four days Discussion after radiation exposure the hair follicles were markedly shorter and a few deformities Using conventional morphometric and histocould be noted (Fig. 3). logical techniques, hair follicles have been
SEM OF HAIR FOLLICLES
187
Fig. 3. Four days after exposure to 8,000 rads the follicles are markedly shorter and altered. Note the decrease in the number of follicles per unit area. X 100. previously observed in order to evaluate radiation and drug effects (Van Scott 1958, Argyris & Chase 1966, Smoliar 1966, Albert etal. 1967, Burns etal. 1973). The architectural and surface alterations observed by SEM were in agreetnent with previous histopathological descriptions of these radiosensitive structures but, in addition, follicle deformations were found. Although an artifact cannot be excluded, the presence of these deformations in the irradiated follicles only would suggest that their configuration is real. The procedure whereby the split-skin technique is used in combination with SEM methods has already provided interesting results while studying skin and oral mucosa (Papa & Farber 1971, Klein-Szanto &
Schroeder 1977). In this study the methodology used revealed itself as a potentially useful radiobiological tool for the study of hair follicle radiation-response. The direct SEM observations of hair follicle speeimens dried by the critical point method hopefully overcome most of the preservation and quantitation diffieulties encountered when using conventional preparations. Prelimina'-y experiments in our laboratory have confirmed that SEM specimens are amenable to a precise quantitative analysis (Klein-Szanto & de Rey 1977), and that the architectural alterations as well as the numerical density decrease of hair follicles is dose-dependent.
188
KLEIN-SZANTO AND de REY
Fig. 4. Non-iriadiated hair follicles showing orifice at the center of the bulb's tip. X 200, inset X 1,000.
SEM OF HAIR FOLLICLES
189
Fig. 5. Two days after exposure to 4,000 rads, the follicles began to show incurvations and slight deformities of the bulbar area. X 250.
190
KLEIN-SZANTO AND de REY
Fig. 6. Five days after exposure to 8,000 rads. Marked defoimities of the haii folliele bulbs were frequently seen. X 290.
SEM OF HAIR FOLLICLES
191
Fig. 7. Seven days after exposure to 8,000 rads. Marked deformities of the hair foUicle bulbs were frequently seen. X 290.
KLEIN-SZANTO AND de REY
192 Acknowledgments
Malkinson, F. D. & Griem, M. L. (1966) Reduced growth rates of hair in miee following radiation. We gratefully acknowledge the cooperation Archives of Dermatology 94,491-498. of Dr. J. Tramezzani and the technical staff Malkinson, F. D. & Griem, M. L. (1968) Radiation injury and recovery in anagen and telogen of the SEM Laboratory of the Argentine rodent hairs. Radiation Researeh 33, 554-562. National Research Council (CONICET). Malkinson, E. D., Griem, M. L. & Marianovic, R. (1970) Persistent impairment of hair growth after single layer doses of X-rays. Radiation References Researeh 43, 83-91. Montagna, W. & Chase, H. B. (1956) Histology and Albert, R. E., Burns, E. J. & Heimbaeh, R. D. eytoehemistry of human skin. X. X-irradiation (1967) The assoeiation between chronic radiof the sealp. Atneriean Journal of Anatomy 99 ation damage of the hair follicles and tumor 415-446. formation in the rat. Radiation Research 30, Moshman, J. & Upton, A. C. (1954) Depigmen590-599. tationof hair as a biological radiation dosimeter Argyris, T. S. & Chase, H. B. (1966) Effect of Seienee 119,186-181. X-irradiation on differentiating hair follicles. Papa, C. M. & Farber, B. (1971) Direet scanning The Anatotnieal Record 136, 445-451. electron microscopy of human skin. Arehives Burlin, T. E., Challoner, A. V. J., Magnus, 1. A. & of Dermatology 104, 262-270. Szur, L. (1973) The effect of divided doses of X-rays on the regrowth of hair in the mouse. Potten, C. S. (1968) Radiation depigmentation of mouse hair. A study of follieular melanoeyte International Journal of Radiation Biology 23, populations. Cell and Tissue Kineties 1 239 — 121-131. 254. Burns, F. J., Albert, R. E., Sinclair, I. P. & Bennet, P. (1973) Tlie effect of fraetionation on tumor Potten, C. S. & Forbes, P. D. (1972) Pigment clumping in growing hair follieles of mice. A induction and hair follicle damage in rat skin. sensitive radiobiological model. Intertiational Radiation Researeh 53, 235-240. Jourtjal of Radiation Biology 22, 337-349. Geary, J. R., Jr. (1952) Effects of roentgen rays Smoliar, V. (1966) Effects of ionizing radiation during various phases of the hair eycle of the on the hair bulbs of young rats. International albino rats. American Journal of Anatomy 91, Journal of Radiation Researeh 11, 21-26. 151-165. Van Scott, E. J. (1958) Response of hair roots to Karnovsky, M. E. (1965) A formaldehyde-glutaralchemical and physical influence. In The biology dehyde fixative of high osmolarity for use in of hair growth, eds. Montagna, W. & Ellis, R. A. eleetron-mieroscopy. Journal of Cell Biology pp. 441-450. New York: Academie Press. 27, 137-138. Van Scott, E. J. & Reinertson, R. P. (1957) DetecKlein-Szanto, A. J. P. & de Rey, B. M. (1977) tion of radiation effeets on hair roots of the Scanning eleetron mieroseopy of irradiated hair human scalp. Journal of Investigative Dermafollieles (Abstract). Radiation Researeh 70, tology 29,205-212. 625. Klein-Szanto, A. J. P. & Schroeder, H. E. (1977) Architecture and density of the connective tissue papillae of the human oral mucosa. Address: Journal of Anatomy (London) 123, 93-109. Dr. B. M. de Rey Kostanecki, W., Radwan, I. & Mroczkowski, T. Departamento de Radiobiologia (1976) The effect of X-ray irradiation upon the Comisidn Nacional de Energia Atdmica epithelial melanin unit of the hah: bulb in Avda del Libertador 8250 hooded rat. Archives for Dermatologieal AR-1429 Buenos Aires Researeh 256, 297-303. Argentina
Surface Architecture of X-lrradiated Hair Follicles A. J. P. KLEIN-SZANTO AND B. M. de REY Division of Radiation Pathology, Department of Radiobiology, National Atomic Energy Commission, Buenos Aires, Argentina The combined use of scanning electron-microscopy and chemical separation of epidermis permitted the study of the irradiated and normal hair follicle surface characteristics. Irradiation reduced the length and density of the hair follieles, produeing at the same time marked alterations and deformities of the individual follicles. As most of these changes were dose- and time-dependent, these struetures, studied by these techniques, provide an interesting model as biological indicators of radiation dose. (Reeeived for publieation January 9, 1978)
Hair follicle radiosensitivity has been extensively studied by numerous techniques. Most of these studies have not directly analyzed the hair follicles but inferred through indirect data, obtained by studying the morphology and the development of the hair shafts, the radiation-induced damage of the hair-forming follicle cells (Malkinson & Griem 1966, 1968, Malkinson et al. 1970, Burlin et al. 1973). Although the histopathology of irradiated hair folhcles has been described previously (Geary 1952, Montagna & Chase 1956, Van Scott & Reinertson 1957) and many studies on the radiation response of the follicle and hair shaft melanin-content have been reported (Moshman & Upton 1954, Potten 1968, Potten & Forbes 1972, Kostanecki et al. 1976) no scanning electron-microscopic analyses of these irradiated structures have, to our knowledge, been published. The aim of this study was to obtain further information on the radiation-induced damage of hair follieles by employing a combination of modern methods of scanning electron-microscopy (SEM) and the splitskin techniques, thus enabling the observation of the undersurfaee of the epidermis
and the complete surface of hair follicles. Material and Methods
The mid-third portion of the tails of 4-dayold Wistar rats were X-irradiated with 2, 4, 8 and 16 Krads in the following conditions — 230 kV, 13 mA using a 0.25 mm Cu and 1 mm Al filter. The proximal portion of the tail which was used as a control, as well as the rest of the animal's body, was shielded with lead plates. The animals were sacrificed 1, 2, 4 and 7 days after irradiation exposure. Fotor to five rats were studied in each group. The irradiated and control skin areas of the taO were separated from the underlying vertebrae by soft traction and immediately incubated in 2NNaBr solution at 37°C for approximately 20 min (Papa & Farber 1971). Working under a dissecting microscope, the epidermis was then separated from the dermis, as described previously (KleinSzanto & Schroeder 1977). The epithelium was stretched out on a blotting paper and fixed in chilled half-strength Karnovsky fixative (Karnovsky 1965). Dehydration in graded ethanol series followed by graded
ISEM OF HAIR FOLLICLES
185
f^ig. 1. Non-irradiated hair follicles of a 9-day-old rat's tail. Note elongated hair follicles and smaller folUcles ofthe underhairs. X 35. follicles as well as smaller ones, probably corresponding to the under hairs of rodents, eould be seen (Figs. 1, 2 and 3). During the 10 days of the animals' life under study the non-irradiated anagen follicle population underwent an active elongation. The non-irradiated follicles showed a partially smooth surface with some undulated areas, especially in the apical part in the proximity of the bulb. In this area it was usually easy to observe a concave depression or an orifice of approximately 30jU, located Results exactly at the center of the bulb's tip In both irradiated and non-irradiated speei- (Fig. 4). This orifice corresponded to the mens the hair follicles were usually distrib- connective tissue bridge communicating the uted in parallel rows in which the main dermal papilla with the dermis. isoamylacetate and subsequent drying by liquid CO2 by the critical point method was carried out. The epidermal samples were mounted in order to show the basal epithelial surface with the attached hair follicles. The speeimens were coated with carbon and gold in a Jeol evaporator and examined with a Jeol JSM-U-3 scanning electron microscope.
186
KLEIN-SZANTO AND de REY
Fig. 2. Hair follieles 2 days after exposure to 2,000 rads, showing few alterations. The numerical density is diminished although the follieles are relatively normal. X 40. Five and 7 days after irradiation these The irradiated follicles showed interesting surfaee and struetural alterations which were structural alterations were seen even more markedly pronounced after 8 and 16 Krads frequently. They consisted of relatively long and somewhat attenuated, although similar "spikes" or "horns" located at the apical in nature, after skin exposure to 2 and portion of the follicle and positioned almost 4 Krads. perpendicularly to the follicle's longitudinal During the first 2 days after irradiation axis (Figs. 6 and 7). the hair follicles showed few modifications. The orifices left by the papillae were very The elongated cylindrical shape was not rarely seen in irradiated follicles. Four and modified, although frequent bending or 7 days after irradiation hairs were also markincurvation of the follicle tips were noted edly shorter and their density per unit area (Fig. 5). The follicles were somewhat shorter decreased. than the controls, especially after exposure to the higher radiation doses. Four days Discussion after radiation exposure the hair follicles were markedly shorter and a few deformities Using conventional morphometric and histocould be noted (Fig. 3). logical techniques, hair follicles have been
SEM OF HAIR FOLLICLES
187
Fig. 3. Four days after exposure to 8,000 rads the follicles are markedly shorter and altered. Note the decrease in the number of follicles per unit area. X 100. previously observed in order to evaluate radiation and drug effects (Van Scott 1958, Argyris & Chase 1966, Smoliar 1966, Albert etal. 1967, Burns etal. 1973). The architectural and surface alterations observed by SEM were in agreetnent with previous histopathological descriptions of these radiosensitive structures but, in addition, follicle deformations were found. Although an artifact cannot be excluded, the presence of these deformations in the irradiated follicles only would suggest that their configuration is real. The procedure whereby the split-skin technique is used in combination with SEM methods has already provided interesting results while studying skin and oral mucosa (Papa & Farber 1971, Klein-Szanto &
Schroeder 1977). In this study the methodology used revealed itself as a potentially useful radiobiological tool for the study of hair follicle radiation-response. The direct SEM observations of hair follicle speeimens dried by the critical point method hopefully overcome most of the preservation and quantitation diffieulties encountered when using conventional preparations. Prelimina'-y experiments in our laboratory have confirmed that SEM specimens are amenable to a precise quantitative analysis (Klein-Szanto & de Rey 1977), and that the architectural alterations as well as the numerical density decrease of hair follicles is dose-dependent.
188
KLEIN-SZANTO AND de REY
Fig. 4. Non-iriadiated hair follicles showing orifice at the center of the bulb's tip. X 200, inset X 1,000.
SEM OF HAIR FOLLICLES
189
Fig. 5. Two days after exposure to 4,000 rads, the follicles began to show incurvations and slight deformities of the bulbar area. X 250.
190
KLEIN-SZANTO AND de REY
Fig. 6. Five days after exposure to 8,000 rads. Marked defoimities of the haii folliele bulbs were frequently seen. X 290.
SEM OF HAIR FOLLICLES
191
Fig. 7. Seven days after exposure to 8,000 rads. Marked deformities of the hair foUicle bulbs were frequently seen. X 290.
KLEIN-SZANTO AND de REY
192 Acknowledgments
Malkinson, F. D. & Griem, M. L. (1966) Reduced growth rates of hair in miee following radiation. We gratefully acknowledge the cooperation Archives of Dermatology 94,491-498. of Dr. J. Tramezzani and the technical staff Malkinson, F. D. & Griem, M. L. (1968) Radiation injury and recovery in anagen and telogen of the SEM Laboratory of the Argentine rodent hairs. Radiation Researeh 33, 554-562. National Research Council (CONICET). Malkinson, E. D., Griem, M. L. & Marianovic, R. (1970) Persistent impairment of hair growth after single layer doses of X-rays. Radiation References Researeh 43, 83-91. Montagna, W. & Chase, H. B. (1956) Histology and Albert, R. E., Burns, E. J. & Heimbaeh, R. D. eytoehemistry of human skin. X. X-irradiation (1967) The assoeiation between chronic radiof the sealp. Atneriean Journal of Anatomy 99 ation damage of the hair follicles and tumor 415-446. formation in the rat. Radiation Research 30, Moshman, J. & Upton, A. C. (1954) Depigmen590-599. tationof hair as a biological radiation dosimeter Argyris, T. S. & Chase, H. B. (1966) Effect of Seienee 119,186-181. X-irradiation on differentiating hair follicles. Papa, C. M. & Farber, B. (1971) Direet scanning The Anatotnieal Record 136, 445-451. electron microscopy of human skin. Arehives Burlin, T. E., Challoner, A. V. J., Magnus, 1. A. & of Dermatology 104, 262-270. Szur, L. (1973) The effect of divided doses of X-rays on the regrowth of hair in the mouse. Potten, C. S. (1968) Radiation depigmentation of mouse hair. A study of follieular melanoeyte International Journal of Radiation Biology 23, populations. Cell and Tissue Kineties 1 239 — 121-131. 254. Burns, F. J., Albert, R. E., Sinclair, I. P. & Bennet, P. (1973) Tlie effect of fraetionation on tumor Potten, C. S. & Forbes, P. D. (1972) Pigment clumping in growing hair follieles of mice. A induction and hair follicle damage in rat skin. sensitive radiobiological model. Intertiational Radiation Researeh 53, 235-240. Jourtjal of Radiation Biology 22, 337-349. Geary, J. R., Jr. (1952) Effects of roentgen rays Smoliar, V. (1966) Effects of ionizing radiation during various phases of the hair eycle of the on the hair bulbs of young rats. International albino rats. American Journal of Anatomy 91, Journal of Radiation Researeh 11, 21-26. 151-165. Van Scott, E. J. (1958) Response of hair roots to Karnovsky, M. E. (1965) A formaldehyde-glutaralchemical and physical influence. In The biology dehyde fixative of high osmolarity for use in of hair growth, eds. Montagna, W. & Ellis, R. A. eleetron-mieroscopy. Journal of Cell Biology pp. 441-450. New York: Academie Press. 27, 137-138. Van Scott, E. J. & Reinertson, R. P. (1957) DetecKlein-Szanto, A. J. P. & de Rey, B. M. (1977) tion of radiation effeets on hair roots of the Scanning eleetron mieroseopy of irradiated hair human scalp. Journal of Investigative Dermafollieles (Abstract). Radiation Researeh 70, tology 29,205-212. 625. Klein-Szanto, A. J. P. & Schroeder, H. E. (1977) Architecture and density of the connective tissue papillae of the human oral mucosa. Address: Journal of Anatomy (London) 123, 93-109. Dr. B. M. de Rey Kostanecki, W., Radwan, I. & Mroczkowski, T. Departamento de Radiobiologia (1976) The effect of X-ray irradiation upon the Comisidn Nacional de Energia Atdmica epithelial melanin unit of the hah: bulb in Avda del Libertador 8250 hooded rat. Archives for Dermatologieal AR-1429 Buenos Aires Researeh 256, 297-303. Argentina
