The Adrenergic Innervation of the Apocrine (Ceruminous) Gland of the Human Ear Canal1

Histologic study reveals that, at least morphologically, the ceruminous gland of the human external auditory canal is an apocrine sweat gland (1). It has apocrine secretory cells lining a rather large lumen which apparently acts as a reservoir to hold preformed secretion until the proper stimulus causes contraction of the myoepithelial sheath and consequent emptying of the gland. Recent studies on the physiology of the ceruminous gland have shown that adrenergic drugs such as epinephrine or nor-epinephrine can cause this gland to pour its secretion out onto the skin (2). However, to date, there has been little anatomic study of the innervation of these glands (3). Furthermore new histo-chemical technics (4) permit the demonstration of cholinergic fibers in the skin. In the present study we have shown the presence of nerve fibers about the tubules of the ceruminous gland and have indirect histochemical evidence that these fibers are adrenergic in character. METHOD Biopsies were obtained from the ear canal of four subjects, two males and two females, who were under general anesthesia for major surgery of the ear. (a) Silver staining: The modified Bielschowski stain (5) was used to demonstrate nerve fibers about the glands. Biopsy material was fixed in 10 % formalin before sectioning and deparaffmization. The sections were washed three times in distilled water and then placed in 20 % silver nitrate for one hour. After a quick rinse in distilled water, they were transferred to a 20 % solution of silver nitrate which had been filtered after adding fresh ammonium hydrate drop by drop until the precipitate dissolved. The sections were then washed two times in distilled water and transferred to neutral 10% formalin for one minute. After again washing in distilled water they were toned in gold chloride solution (1 gram of gold chloride to 500 ml. water), and then fixed in sodium thiosulfate (5 grams in 100 ml. water) for one to two minutes. Finally the sections were dehydrated and mounted. (b) Choli nest erase staining: Other excised skin specimens were stored in the deep freeze ( - 1 to - 2°C.) until used (within 72 hours). Frozen sections were cut at 10 micra and placed on glass slides. These sections were then carried through the histochemical method of Koelle, using the latest modifications de- 219 220 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY scribed for the localization of specific and non-specific cholinesterase activity (3). This method provides four sets of slides for the identification of the enzymes as follows : Slides A, which reveal sites of both specific and non-specific cholinesterase activity. Sections of this group were incubated for 45, 75 or 120 minutes at 37.5°C. in a medium containing 0.004 M acetylthiocholine, 0.004 M copper glycinate, 0.04 M MgCl2, 0.01 M sodium maleate buffer (pH 6.0) and 24% Na2S04 saturated with copper thiocholine. Under these conditions, thiocholine which is liberated at the sites of cholinesterase activity as a result of enzymatic hydrolysis of the substrate, is precipitated as a white copper mercaptide. This is subsequently converted to black copper sulfide by immersion in ammonium sulfide. Slides B, which stain sites of specific cholinesterase activity alone. This was accomplished by preliminary incubation of these sections in 1010 M diiso-propylfluorophosphate (DFP) in 24% Na2S04 solution at room temperature for 30 minutes. At this concentration, DFP selectively inhibits non-specific cholinesterase activity. The slides were then treated as were slides A. Slides C, which demonstrate sites of non-specific cholinesterase activity alone. These sections were incubated in a medium similar to that for slides A, but containing 0.004 M butrylthiocholine as substrate for the non-specific cholinesterase. Slides D, for the detection of other esterases and the non-enzymatic adsorption of copper ions. These sections were first treated wTith DFP, then incubated with butrylthiocholine. Fig. 1. Argyrophilic nerve fibers supplying the secretory tubules of a ceruminous gland in man. Note numerous fine beaded dark lines which are the terminal nerve fibrillae. A larger myelinated fiber is seen in the central part of the picture. Magnification 1920X. ADRENERGIC INNERVATION OF APOCRINE GLAND OF HUMAN EAR CANAL 221 Alternate sections were lightly counterstained with eosin or with hematoxylin and eosin. RESULTS On examination of the slides stained with silver, one can discern fine argyro-philic fibers around many of the ceruminous gland tubules. These are interpreted as nerve fibers. The histologic sections which have been stained for specific and non-specific cholinesterase activity reveal no evidence of these enzymes around the tubules of the ceruminous glands. Control specimens of skin from the arm showed abundant specific cholinesterase staining about the eccrine sweat gland tubules. DISCUSSION The slides that have been stained with silver leave little doubt that the ceruminous glands are supplied by nerves. The physiologic data would suggest that these nerves belong to the autonomic nervous system and are motor fibers. The absence of cholinesterase around the tubules of the ceruminous glands indicates that the nerves supplying it are not cholinergic. This is in agreement with the finding that acetylcholine is not an effective stimulus for the ceruminous gland (2). We are therefore led to postulate that fibers of the autonomic nervous system innervate the ceruminous gland, and that these fibers are adrenergic in nature. This is the same finding as for the axillary apocrine gland. SUMMARY Special histologic studies.have revealed that there are nerve fibers coursing about the human ceruminous glands, and that they are probably adrenergic motor fibers of the autonomic nervous system.