Histochemical Observations on the Elastic Sheath-elastofibril System of the Dermis

It has already been demonstrated that pigmentary changes in both the skin and hair can be induced by changing the levels of certain hormones in the blood-stream (1, 2, 3). However, a hormone which has been shown to have a specific effect on epidermal melanogenesis may have no effect or the opposite effect on hair pigmentation, since the pigmentary systems of hair and skin can act independently as seen for example in the white haired Negro or the black haired Caucasian. Bearing in mind this independence of action of hair and epidermal melanocytes, we have recently devoted out attention to studying the effects of hormones on the hair whose effects upon the skin have already been described. Melatonin, first isolated from the bovine pineal gland by Lerner et al. (4), has been found to aggregate the melanin granules in dermal melanophores in frogs, but its effect on mammalian epidermal melanocytes is questionable and probably nil (5). In this work, we have tried to determine whether melatonin has any definite effect on hair melanogenesis. MATERIALS AND METHODS Six all-red and six-all-black mature male guinea pigs were used. Hair samples were taken from the scalp and anterior abdominal wall of each animal by clipping. The hair was cut close to the skin with standard electric clippers. A group of undisturbed hairs from each clipping was mounted on a slide under a coverslip as described previously (3). Each animal then received intramuscularly once daily for a month 9.6 mgms of melatonin1 dissolved in 0.25 ml of ethyl oleate. A very high dose of hormone was used since it has already been shown in the guinea pig (5) and the human subject (6) that small doses have no effect on skin color. At the end of the treatment a second hair sample was taken from each area. A final hair sample was taken from the same areas one month later. Changes in hair color were assessed by 1) objectively comparing the numbers of hairs of the same colors in the different samples and 2) subjectively comparing the overall color of the different hair samples. Hair counts were performed as follows: In the red animals the hair colors present were divided into two groups which we called light red and dark red. The hairs of the black animals were similarly divided into two main groups, black and grey. A total of 100 hairs was counted for each slide at a magnification of X 200, and the numbers of each hair color present were recorded. To avoid bias, the identity of the samples was not divulged to the person examining the slides. A few hairs of other colors (e.g. white) were seen, but these occurred so infrequently as to be considered negligible. The subjective comparison of the overall hair color of different hair samples was performed with the microscope and the results were recorded using the “+” system. RESULTS The detailed morphology of the light and dark red hairs found in normal untreated red guinea pigs and the grey and black hairs in the 1 The pure melatonin was obtained from Dr. A. B. Lerner, Department of Medicine Yale University. TABLE I Showing the hair counts of hair samples taken from the scalps of animals treated with melatonin No. of animal Color of animal Initial hair sample (A) Hair sample after 1 month of melatonin (B) Hair samplejl month after cessation of melatonin (C) Light red Dark red Light red Dark red Light red Dark red 1984 Red 70 30 69 31 82 18 1985 Red 69 31 71 29 Died Died 1986 Red 58 42 93 7 87 13 1987 Red 93 7 86 14 99 1 1988 Red 80 20 92 8 82 18 1989 Red 95 5 80 20 89 11 Grey Black Grey Black Grey Black 1990 Black 41 59 66 34 84 16 1991 Black 36 64 49 51 Died Died 1992 Black 72 28 34 66 63 37 1993 Black 45 55 8 92 90 10 1994 Black 73 27 63 37 86 14 1995 Black 39 61 75 25 75 25 Bed Animals B minus A = Mean diff. ± S.E. of diff. -4.3 ± 2.3 t, 5 d.f. = 1.8 P > 0.1 C minus B = Mean diff. ± S.E. of diff. -3.8 ± 51, 4 d.f. = 0.76 P > 0.2 C minus A = Mean diff. ± S.E. of diff. -8.6 ± 5.9 t, 4 d.f. = 1.45 P > 0.2 Black Animals B minus A = Mean diff. ± S.E. of diff. +1.8 ± 5.3 t, 5 d.f. = 0.34 P > 0.5 C minus B = Mean diff. ± S.E. of diff. -30.4 ± 14 t, 4 d.f. = 2.2 P > 0.05 C minus A = Mean diff. ± S.E. of diff. -25.6 ± 10.5 t, 4 d.f. = 2.4 P > 0.05 black guinea pigs has been described previously. The relative numbers of these different colored hairs and the arrangement of the melanin granules within them has also been studied (3). The relative numbers of each type of hair found in the initial hair samples are shown in Tables I and II. Effect of Melatonin on Hair Color The hair counts for the red and black animals taken at different times during the experiment are shown in Tables I and II. Scalp. In the red animals, the number of dark red as opposed to light red hairs decreased by an average of four (not significant) following melatonin injections. One month after the melatonin injections had ceased the number of dark red hairs had decreased by a further four (not significant). In the black animals, the number of black as opposed to grey hairs increased by an average of two following melatonin injections. One month later the number of black hairs was reduced by thirty (not signifi-cant). The overall change in hair counts for both the red and black guinea pigs for the two month period showed that in the majority of animals (8 out of 10) the number of dark hairs was reduced; this was not however statistically significant. Anterior abdominal wall. In the red animals practically no change occurred in the numbers of hairs following the melatonin treatment. Furthermore one month later no change had occurred. In the black animals the number of black hairs counted fell by an average of sixteen at the end of the hormone treatment (not significant) and during the following month the number of black hairs decreased by another four. The overall change in the hair counts for both the red and black guinea pigs for the two month period showed that in the majority of the animals (8 out of 10) the number of dark hairs was reduced; in the black animals the decrease in the number of black hairs was significant (P > 0.02). The averaged results of the subjective color mammimm TABLE II Showing the hair counts of hair samples taken from the anterior abdominal walls of animals treated with melatonin No. of animal Color of animal Initial hair sample (A) Hair sample after 1 month of melatonin (B) Hair sample 1 month after cessation of melatonin (C) Light red Dark red Light red Dark red Light red Dark red 1984 Red 94 6 88 12 90 10 1985 Red 76 24 70 30 Died Died 1986 Red 83 17 87 13 85 15 1987 Red 95 5 94 6 90 10 1988 Red 92 8 97 3 98 2 1989 Red 86 14 87 13 91 9 Grey Black Grey Black Grey Black 1990 Black 62 38 99 1 76 24 1991 Black 49 51 80 20 Died Died 1992 Black 45 55 86 14 71 29 1993 Black 57 43 28 72 71 29 1994 Black 49 51 64 36 52 48 1995 Black 60 40 58 42 87 13 Bed Animals B minus A = Mean diff. C minus B = Mean diff. C minus A = Mean diff. Black Animals B minus A = Mean diff. C minus B = Mean diff. C minus A = Mean diff. ± S.E. of diff. +0.5 ± 2 t, 5 d.f. = 0.25 P > 0.5 ± S.E. of diff. -0.2 ± 1.4 t, 4 d.f. = 1.44 P > 0.2 ± S.E. of diff. -0.8 ± 0.9 t, 4 d.f. = 0.9 P > 0.5 ± S.E. of diff. -15.5 ± 11.24 t, 5 d.f. = 1.38 P > 0.2 ± S.E. of diff. -4.4 ± 13.4 t, 4 d.f. = 0.33 P > 0.5 ± S.E. of diff. -16.8 ± 4.5t, 4 d.f. = 3.73 P > 0.02 comparisons of the different hair samples generally concurred with these findings. DISCUSSION Despite the wide variety of hair colors visible to the naked eye, microscopic examination reveals only three basic pigments, black, brown and yellow. The black and brown pigments are produced by the enzymatic oxidation of tyrosine to melanin. The yellow pigment, or pheomela-nin is of unknown origin, although tyrosinase may be required for its formation (7). The relationship of the melanocyte to the other cells of the hair follicle is fully described in a review by Chase (8). Studies of guinea pig hair color, considered from the geneticist's standpoint, have been made by Wright (9) and Russell (10). The guinea pigs used in this work were all-red and all-black, but they were not genetically pure strains. According to Dawson (11) and Strangeways (12) the coat of the guinea pig has no pro-gressive waves of hair growth. The growth period averages four weeks, and may be as short as two weeks, or as long as seven. The average rate of growth varies from 2-5 mm. weekly. The rest period averages twelve weeks (11). Lerner and Case (4) found that melatonin lightens the skin of the frog by causing aggregation of melanin granules within the dermal melanophores. In this respect, its action is similar to that of adrenaline, noradrenaline, acetylcholine, serotonin and triiodothyronine. Melatonin is by far the most potent of these lightening agents, indeed being almost 10° times as effective as noradrenaline. Furthermore, melatonin in as weak a concentration as 10M gm/ml can prevent and partially reverse the darkening actions of the melanocyte stimulating hormone, adrenocorticotrophic hormone, and caffeine on the frog's skin (13). In contrast, Snell (5) showed that in guinea pigs which had received large doses of melatonin no gross or microscopic changes occurred in the pigmentation of the epidermis. The morphological appearances of the epidermal melanocytes in skin sheets removed before and after the hormone treatment were practically identical. In the present investigation melatonin was seen to have no dramatic effect on the hair color of the scalp and anterior abdominal wall of all-red and all-black male guinea pigs. The hair counts showed no statistically significant changes at the end of the hormone treatment. A comparison of the hair counts taken at the end of the hormone treatment with those recorded one month later also showed no significant changes. If the hair counts recorded one month after the cessation of hormone treatment were compared with the initial untreated samples, it was seen that in the majority of the animals the number of dark hairs was reduced; this reduction was only significant, however, in the anterior abdominal wall of the black animals. The occurrence of only one significant result in this experiment might have been explained on the basis that the affected segments of many hairs had not yet emerged sufficiently from the follicle to be harvested after one or even two months of growth. This explanation is not tenable, however, since it has recently been shown, using the same method of assessment, that alpha MSH causes darkening of the hairs after one month of treatment (3). It has also been demonstrated that if hair samples are removed from normal untreated male guinea pigs at monthly intervals, there is a tendency for the proportion of dark colored hairs to rise (3). The explanation given for this phenomenon was that the slight trauma involved in the clipping process might have stimulated melanogenesis. The fact that in the present experiment the proportion of dark colored hairs in many of the animals was lower at the end of the experiment than at the beginning would suggest that melatonin may have a delayed inhibitory ef-fect on follicular melanogenesis. In order to provide a valid answer to this question, it is proposed to extend the experiment using a larger number of animals, different doses of hormone and harvesting the hairs over a longer period of time following the cessation of hormone treatment.