Cholinergic Innervation of the Meibomian Glands'

WILLIAM MONTAGNA AND RICHARD A. ELLIS Arnold Biological Laboratory, Brown University, Providence 12, Rhode Island

No cutaneous nerves containing cholin- esterases have ever been demonstrated near or around sebaceous glands (Hurley et al., '53; Hellmann, '55; Montagna and Ellis, '57; Thies and Galente, '57). The sebace- ous Meibomian glands in the eyelids, how- ever, are encircled by numerous nerve fibers which react strongly for acetylcholin- esterase.

MATERIALS AND METHODS

For this study we have used the upper eyelids of man and several other primates (pottos, lorises, tamarins, several Old and New World monkeys and a chimpanzee), the rat, guinea pig, rabbit, cat, dog and horse. The lids were cut longitudinally and fixed for 4 hours in 4% neutral unbuffered formaldehyde. Frozen sections 40 to 100 u thick were tested for cholinesterases by in- cubating some in acetylthiocholine iodide and some in butyrylthiocholine iodide. For control, physostigmine was added to the in- cubating mixtures at a final total concen- tration of M. The technique used was that of Koelle and Friedenwald ('49), as described by Montagna and Ellis ('57).

OBSERVATIONS

Both the acini and the ducts of the Meibomian glands of all the animals stud- ied, except the rat, are surrounded by nerves that contain histochemically de- monstrable acetylcholinesterase (figs. 1- 6). The sebaceous glands associated with the small hair follicles at the border of the palpebra just above the cilia are also sur- rounded by cholinesterase-reactive nerves; the sebaceous glands farther up the pal- pebra are not. In a few of the primates, as for example the Patas monkey (fig. 6) and in the horse (fig. l ) , nerves from the plexus around the glands continue to the conjunctiva and terminate either freely

in the epithelium, or beneath it in small end-organs which also contain acetylchol- inesterase (figs. 1 and 6) . In most species studied, the nerves around the Meibomian glands show no reaction when sections are incubated with butyrylthiocholine iodide; in some there is slight activity, but in mar- mosets and in the dog, the nerves have a stronger reaction than when acetylthio- choline iodide is used. With physostigmine added to the acetylthiocholine iodide in- cubating medium, the reaction in the nerves is completely abolished; the reaction is inhibited only partially when it is added to the butyrylthiocholine iodide mixture.

Neither acetylthiocholine nor butyryl- thiocholine demonstrates nerves surround- ing the Meibomian glands of the rat. The cytoplasm of the parenchymal cells, how- ever, reacts strongly with both substrates, and both reactions are abolished when physostigmine is added to the incubation mixture.

DISCUSSION

Little is known about the innervation of sebaceous glands (Rothman, '54). A few investigators, using a variety of staining techniques, have succeeded in demonstrat- ing nerves in the vicinity of sebaceous glands and the arrectores pilorum muscles (Boeke, '34; Champy et al., '45-'46; Istbk, '37; John, '42; Nakanishi, '30; Takino, '29; Truffi, '34); most others have failed to demonstrate them. In addition to the pres- ence of cholinesterase in the nerves sur- rounding Meibomian glands, we call at- tention to the method for cholinesterase as a histological technique. Nerves contain- ing cholinesterase are non-myelinated and exceedingly difficult to demonstrate with

1 Supported in part by grants from the National Science Foundation, G-4392, the United States Public Health Service (RG-212548) and the Col- gate-Palmolive Company.

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122 WILLIAM MONTAGNA AND RICHARD A. ELLIS

other techniques. For example, we have tried in vain, as others have, to demon- strate the nerves around eccrine sweat glands with silver, and with osmium methods; yet, the cholinesterase technique reveals them quickly and clearly. We re- commend that this technique be added to the general techniques of the neurohistol- ogist.

Champy et al. ('45-'46) believe that the nerves they demonstrated around the seba- ceous glands of the rat are adrenergic fi- bers. We have no opinion about this other than to doubt that nerves exist there at all. In the hundreds of pieces of skin from man and other mammals which we have ex- amined, we have never succeeded in dem- onstrating nerves around the sebaceous glands. Even the large glands in the face, the perianal regions, and the genital or- gans of man, and the large sebaceous organs in the pelvic and abdominal regions of marmosets are free of nerves that con- tain cholinesterases. A few cholinesterase reactive nerves, however, are found around the parenchyma of the preputial glands of the rat and the white inguinal glands of the rabbit.

The Meibomian glands of the rat are different from those of all other animals studied, in that they are not surrounded by cholinesterase-containing nerves. The glandular parenchyma, however, as is also that of the sebaceous glands of the general body surface (Montagna and Beckett. 'SS), abounds in the enzymes. Such species differences will probably be met frequently when a more systematic study of these glands is completed. A situation similar to that of the Meibomian glands of the rat is found in the sweat glands in the digital pads of the dog. These glands contain cholinesterase, but the nerves around them do not (Hellmann, '55).

The presence of nonspecific cholin- esterase in the nerves of some of the pri- mates and the dog is still another species difference. In these animals not only the nerves around the Meibomian glands, but also those around the eccrine sweat glands in the volar side of the hands and feet and in the digital pads, contain non-specific cholinesterase. Since such striking spe- cies differences occur, the physiology of the

glands of these animals must also be different .

We do not known the origin of the nerve plexus around the Meibomian glands. The sensory nerve fibers from the conjunctiva found in some species join the plexus around the glands, and are probably con- nected proximally to the infratrochlear and lacrimal branches of the ophthalmic divi- sion of the trigeminus. The motor fibers to the glands could come from the oculo- motor, or they could be postganglionic fi- bers from the superior cervical ganglion. If they should originate from the cervical ganglion, their relation to the Meibomian and sebaceous glands at the palpebral border would be similar to the relation of the nerves to the eccrine sweat glands of man, and since in most cases they also contain only specific cholinesterase, they are probably also cholinergic. In this re- spect the sebaceous glands at the free edge of the palpebrae are singularly different from the sebaceous glands elsewhere in the skin, which are supposed to receive ad- renergic fibers from the autonomic system (Champy et al., '45-'46), but which most likely have no intrinsic innervation.

SUMMARY

1. In a large number of primates, in the rabbit, guinea pig, cat, dog, and in the horse, the sebaceous parenchyma of the Meibomian glands is surrounded by a plexus of nerve fibers that contain specific cholinesterase. These fibers are probably cholinergic.

2. In some exceptional species of pri- mates and in the dog, these nerves also contain non-specific cholinesterase.

3 . The large sebaceous glands at the border of the palpebra immediately behind the cillia are also surrounded by cholin- esterase-rich nerve fibers.

4. The Meibomian glands of the rat are not surrounded by cholinesterase-con- taining nerves. The glandular cells, how- ever, abound in both specific and non- specific cholinesterase.

5. Other than the Meibomian glands we have found cholinesterase-containing nerves only in the preputial glands of the rat and in the white inguinal glands of the rabbit.

CHOLINESTERASES IN MEIBOMIAN GLANDS 123

LITERATURE CITED Boeke, J . 1934 Le plexus sympathique fonda-

mental situ6 dans le tissue conjonctif et ses rapports avec les 616ments glandulaires. Bull. d'histol. appliq. A la physiol., 11: 221-240.

Champy C., R. Coujard and Ch. Coujard-Champy 1945-'46 L'innervation sympathiques des glandes. Acta Anat., I: 233-283.

HelImann, K. 1955 Cholinesterase and amine oxidase in the skin: A histochemical investiga- tion. J. Physiol., 129: 454-463.

Hurley, H. J., W. B. Shelley and G. B. Koelle 1953 The distribution of cholinesterases in human skin with special reference to eccrine and apo- crine sweat glands. J. Invest. Dermatol., 21: 139-147. ~~~

Istbk, J. 1937 Nerve endings of sebaceous glands. Orvosi hetil. (Hungarian), 81: 348449.

John, F. 1942 Zur vegetativen Innervation der Talgdriisen. Arch. f. Dermat. J. Syph., 182: 402-411.

Koelle, G. B., and Y. S. Friedenwald 1949 A histochemical method for localizing cholin- esterase activity. Proc. SOC. Exp. Biol. Med., 70: 617-622.

Montagna, W., and E. Beckett 1958 Choline- sterases and alpha esterases in the lip of the rat. Acta Anat., 32: 256-261.

Montagna, W., and R. A. Ellis 1957 Histology and cytochemistry of human skin. XII. Chol- inesterases in the hair follicles of the scalp. J. Invest. Dermatol., 29: 151-157.

Nakanishi, K. 1930 A contribution to the histo- logic study of the nerve fibers in the sebaceous gland and the arrector muscle of hair of the human body. Jap. J. Dermat. Urol., 30: 1134-1 137.

Rothman, S. 1954 Physiology and Biochemistry of Skin. The University of Chicago Press, Chicago, Illinois.

Takino, M. 1929 Die Innervation der menschli- chen Haut, besonders iiber die Musculi erectores pilorum, der Talgdriisen, der Schweissdriisen, und der kleinen Haare. Acta scholae med.

Thies, W., and L. F. Galente 1957 Zur histo- chemischen Darstellung der Cholinesterasen im vegetativen Nervensystem der Haut. Der Hautarzt, 8: 69-75.

Truffi, G. 1934 Innervazione degli annessi cutanei. Arch. 1st. biochim. ital., 6: 409-442.

univ. imp. Kioto., 12: 281-294.

PLATE 1

EXPLANATION OF FIGURES

All of these figures are of tissues incubated in acetylthiocholine iodide for three hours.

Upper eyelid of the horse. The Meibomian gland, cut longitudinally, is completely sur- rounded by reactive nerves. The white arrow indicates the conjunctiva containing many reactive free nerve endings; the black arrow indicates end-organs underneath the conjunctiva. x 55.

Enlarged detail of the nerves in figure 1. The sebaceous glandular material is not stained and appears as empty spaces. X 250.

Longitudinal section through the upper eyelid of the Slow Loris, showing the Meibomian gland cut longitudinally. The duct opens to the left. X 80.

None of the preparation is counterstained.

1

2

3

124

CHOLINESTERASES IN MEIBOMIAN GLANDS William Montagna and Richard A. Ellis

PLATE 1

125

PLATE 2

EXPLANATION OF FIGURES

All of these figures are of tissues incubated with acetylthiocholine iodide for three hours. None of the preparation is counterstained. x 250.

4

5

6

Enlarged detail of the nerves around the Meibomian gland in figure 3.

Low power view of a Meibomian gland in the l id of Cercopithecus neglectus. X 80.

Detail of the nerves around a Meibomian gland in the lid of the Patas monkey. The white arrow indicates the conjunctiva, containing reactive free nerve endings. The black arrow points to end-organs underneath the conjunctiva. X 400.

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CHOLINESTERASES IN MEIBOMIAN GLANDS William Montagna and Richard A. Ellis

PLATE 2

127