HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN

Transcription

1 HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN XII. CHOLINESTERASES IN THE HAIR FOLLICLES OF THE SCALP* WILLIAM MONTAGNA, PH.D. AND RICHARD A. ELLIS, PH.D. Hair follicles are surrounded by a network of interlocking nerve fibers that converge toward two or more larger nerves (13, 14, 15). Even though other authors have found no cholinesterases in the nerves that surround hair follicles (7, 8), we have found these nerves around the hair follicles of the scalp rich in specific cholinesterase. MATERIALS AND METHODS Specimens of scalp were obtained from surgery and from cadavers dead not longer than four hours. The tissues were first stored in dry ice and then fixed for four hours in 4 % neutral, unbuffered formaldehyde (3). Fixation in formaldehyde stabilizes the localization of the enzyme and improves the histological appearance of the preparations (1). Frozen sections were cut 50 to 100 ft thick; they were washed thoroughly in distilled water and then incubated for one to four hours in the substrate mixture of Koelle and Friedenwald (9) as modified by Gomori (6). The incubation substrate mixture, as outlined by Gomori, was adjusted to ph 6.1; cupric sulfate was replaced by dupric acetate as suggested by Dr. M. A. Gerebtzoff (personal communication). This procedure gives excellent results. Two substrates, acetylthiocholine iodide and butyryithiocholine iodide, were used. The best reaction was obtained after incubation for four hours. For controls, sections were also incubated for one to four hours in each of the substrates containing physostigmine salicylate at a concentration of 1O M. After incubation, the sections were washed repeatedly in saturated sodium sulfate, and enzyme activity was made visible by immersing the sections in dilute ammonium sulfide. After thorough washing in water, the sections were mounted on albuminized slides; they were dried in air, dehydrated in alcohol, cleared in xylene, and mounted in balsam. Some sections were counterstained lightly with paracarmine to show the histological features. OBSERVATIONS The hair follicles of the scalp, at the level of the sebaceous glands, are surrounded by a network of nerve fibers. (Fig. 1) This network forms a collar of nerves that is denser in its upper than in its lower limits (Figs. 3, 4). A few nerve fibers from this plexus extend to the upper part of the pilary canal and form a sparser network around the infundibulum of the follicle (Fig. 5); others are wrapped around the sebaceous glands. Very thin fibers from the follicle plexus * From the Arnold Biological Laboratory, Brown University, Providence, Rhode Island, This work was supported in part by a grant from the United States Public Health Service. RG2125 C7. Received for publication May 17,

2 152 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY rise to the base of the epidermis, divide into barely visible branches, and penetrate the epidermis. The fibers from the plexus around the follicles converge toward two or more nerves that run a straight course along the sides of the fol- [ides from the deeper cutaneous plexuses. Fibers from the network around the follicles also join with the nerves around the eccrine sweat glands (Fig. 6). All of the nerves described here contain cholinesterase demonstrable with acetylthiocholine iodide. To understand the distribution of nerves around resting hair follicles, it is important to remember that when hair follicles become quiescent, the bulb degenerates and the follicle becomes shorter (10). The nerve net around the follicles is closer to the base of quiescent follicles than it is to that of active ones. Smaller quiescent follicles become so short that their base partially withdraws from the nerve plexus (Fig. 7); these plexuses remain intact and rich in cholinesterase, but they collapse around the base of the follicle. The arrectores pilorum muscles, like the nerves that supply them from the plexus around the follicles and sebaceous glands, are very rich in esterase activity. The nerves around the sweat glands are strongly reactive. Even the base of the secretory cells of the glands shows reactivity. This activity is confined to the coiled part of the glands; neither the nerves around the straight part of the duct nor the cells shows enzyme reaction. The numerous nerves that supply the walls of the small arteries and arterioles all have cholinesterase reaction (Fig. 8). No other blood vessel in the scalp shows reactive nerves. When butyrylthiocholine iodide is used as substrate, none of the nerves is reactive (Fig. 2). The arrectores pilorum muscles, however, are intensely stained, and a faint reaction is found in the cells of the sweat glands. Control sections incubated in each of the two substrate mixtures to which physostigmine salicylate is added show a complete elimination of the reaction. The arrectores pilorum muscles in control sections incubated with butyrylthiocholine iodide and physostigmine salicylate still show a residual amount of reactivity. DISCUSSION That the nerves described in this paper contain specific cholinesterase, is indicated by their reactivity with acetylthiocholine iodide and not with butyrylthiocholine iodide. The complete inhibition of the reaction by physostigmine is final proof of this specificity. The arrectores pilorum muscles and the secretory cells of eccrine sweat glands contain pseudocholinesterase in addition to specific cholinesterase. In this investigation we find the presence of demonstrable specific cholinesterase in sensory as well as in some motor nerve fibers. Since the nerves around them subserve touch, hair follicles must be tactile sense organs (13, 14). Both in the distribution of the nerves around them and in the function of these nerves, the hair follicles of the scalp are similar to the follicles of the vibrissae, which are found in the lips of all mammals except man (12); in both cases the nerves are reactive for specific cholinesterase (11). Cholinesterases have also been found in

3 HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN. XII 153 other sensory fibers, such as in Meissner and Pacinian corpuscles of the human finger (2) and in the gustatory cells in the tongue of several laboratory animals. The free nerve endings in the epidermis, which subserve pain (14) are reactive for cholinesterase. This is not easily reconcilable with the concept that the presence of cholinesterase is rare outside of the parasympathetic nervous system, where only the post-ganglionic fibers are always covered with specific cholinesterase along their entire course, from the synapse to their terminations (4, 5). These findings are puzzling, and it remains for the physiologist to redefine the significance of cholinesterase in nerve tissue. When hair follicles are inactive, they are much shorter than when they are active. Thus, they may slip out of the lower part of the nerve plexus that surrounds them. The nerve plexus, however, remains intact, and the follicles grow back into these collapsed plexuses when they become active again. The technic employed here is simple, and it demonstrates these nerves very clearly. The method is useful not only in histochemistry, but also in histology. SUMMARY Using the technic for cholinesterase activity, two nerve plexuses were demonstrated around the hair follicles of the scalp. The larger plexus is situated at the level of the sebaceous glands and its fibers are continuous with the cutaneous plexus and with the fibers surrounding the sebaceous glands and the eccrine sweat glands. The other smaller plexus is at the level of the infundibulum of the follicle; fibers from this plexus also run up to the epidermis. All of these nerves contain specific cholinesterase; the reaction is completely inhibited with physo - stigmine. The follicle plexus is intact even when the hair follicle is inactive. The localization of specific cholinesterase in the nerves investing the hair follicle, which subserve touch, is offered as further evidence of the presence of cholinesterase in some sensory nerve fibers. REFERENCES 1. ALMEIDA, DE U. F. AND CoucEIRo, A.: Resistance to formaldehyde fixation of acetylcholinesterase from the electric tissue and the motor end plate. Acad. Brasiliera CiSncias, 27: 41 47, BECKETT, E. B., BOURNE, G. H. AND MONTAGNA, W.: Histology and cytochemistry of human skin. The distribution of cholinesterase in the finger of the embryo and the adult. J. Physiol., 134: , GEEEBTZOFF, M. A.: Recherches histochimiques sur les acétylcholine et choline estérases. Acta Anat., 19: , Gzitxwrzon, M. A.: Incidence fonctionnelle de la localisation des cholinestérases dans le système nerveaux. XX Congrès internat. Physiol., pp , GEREBTZOFF, M. A.: Recherches sur l'innervation cholinergique comparée du coeur de mammifère et de tortue. Extr. Ann. d'histochim., 1: , 1956a. 6. GoissoEl, G.: Microscopic Histochemistr3r. Principles and Practice. Chicago, University of Chicago Press, HELLMANN, K.: Cholinesterase and amine oxidase in the skin: a histochemical investigation. J. Physiol., 129: , HURLEY, H. J., SHELLEY, W. B., AND KOELLE, G. B.: The distribution of cholinesterases

4 154 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY in human skin, with special reference to eccrine and apocrine sweat glands. J. Invest. Dermat., 21: , KOELLE, G. B. AND FEIEDENwALD, J. S.: A histochemical methcd for localizing cholinesterase activity. Proc. Soc. Exper. Biol. & Med., 70: , MONTAGNA, W.: The Structure and Function of Skin. New York, Academic Press, Inc., MONTAGNA, W. AND BEcKETT, E. B.: Cholinesterases and alpha esterases in the lip of the rat. Acta Anat., (in press), VINCENT, S. B.: The tactile hair of the white rat. J. Comp. Neurol., 23: 1-34, WEnDELL, G.: The pattern of cutaneous innervation in relation to cutaneous sensibility. J. Anat., London, 75: , WEnDELL, G. The anatomy of cutaneous sensibility. Brit. Med. Bull., 3: , WOOLLAED, H. H., WEnDELL, G. AND HAEPMAN, J. A.: Observations on the neurohistological basis of cutaneous pain. J. Anat., London, 74: , PLATE I Explanation of Figures FIG. 1. Frozen section of scalp incubated in acetylthiocholine iodide. The nerves around the hair follicles are shown very clearly. The bracket indicates the entire larger nerve plexus around the follicle in the middle of the field. The straight arrow indicates the nerve plexus around a quiescent hair follicle. The curved arrow points to sweat glands which are very strongly reactive for specific cholinesterase. (X 14). FIG. 2. Frozen section of scalp incubated in butyrylthiocholine iodide. Compare this with figure 1. None of the nerves is reactive. Only the arrectores pilorum muscles (arrows) show a reaction. (X 14). FIG. 3. The entire, larger nerve plexus around the follicle at the level of the sebaceous glands. The upper part of the plexus is much more dense than the lower part. (X 66). FIG. 4. Oblique section through a hair follicle showing just the upper part of the plexus. Frozen section of scalp incubated in acetylthiocholine iodide. (X 66).

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6 I 5G T}IE JOURNAL OF INVESTIGATIVE DERMATOLOGY PLATE II Explanation of Figures FIG. 5. The upper, sparser nerve plexus of a hair follicle is very delicate and barely visible. The arrow points to some of these small nerve fibers. Frozen section of scalp incubated in acetylthioeholine iodide. (X 66). Fio. 6. The black mass on the lower left corner is a nest of strongly reactive sweat glands. The mass on the lower right corner is one of the arrectores pilorum muscles. The arrow points to a fiber which goes from the follicle plexus to the nerves around sweat glands. Frozen section of scalp incubated in acetylthioeholine iodide. (X 66). FIG. 7. The partially collapsed plexus around the base of a quiescent hair follicle. Frozen section of scalp incubated in aeetylthiocholine iodide. (X 132). FIG. 8. Numerous nerves rich in specific eholinesterase in the walls of arterioles in the deep part of the scalp. Frozen section incubated in aeetylthiocholine iodide. (X 66).

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