Fine morphological changes in the penicillate nerve endings of human hairy skin during prolonged itching.код для вставкиСкачать
Fine Morphological Changes in the Penici late Nerve Endings of Human Hairy Skin During Prolonged Itching NIKOLAJS CAUNA Department of Anatomy and Cell Biology, Uni m i t u o f Pittsburoh School of Medicine, Pittsburgh, Pennsylvania 15261- " - ABSTRACT Morphological changes in cutaneous nerve endings were investigated electron microscopically in patients suffering from certain kinds of urticaria with associated itching and in normal skin in which wheals and local itching were induced either by application of nettle hairs or by intracutaneous injections of a timothy pollen extract. Skin samples were obtained with a high speed dermal punch without anesthesia from the wheal areas. It was found that some subepidermal free nerve endings derived from nonmyelinated nerve fibers (penicillate endings) showed accumulations of extracytoplasmic glycogen which was localized in the distended spaces between the axolemma, the Schwann cell membrane and the nerve basement membrane. In some cases, the glycogen was found to be so abundant that it occupied most of the cross sectional area of the ending. No morphological changes were observed in the pappilary endings, in nerve endings of the hairs or in the autonomic terminals. The conducting segments of all cutaneous nerve fibers showed normal morphology . The unusual morphological changes that occur i n some penicillate nerve endings during the wheal development indicate that these endings are involved in the skin reaction and therefore they may be the specific end organs that are associated with itch. at least in urticaria. The exact function or functions of each form of sensory receptor organ encountered in the skin is still a much debated issue. Only mechanoreceptors have been identified with greater or lesser certainty on the basis of electrophysiological experiments or morphological characteristics of the end organs. Warmth, cold and pain receptors are still escaping definite identification in most cases, and end organs associated with itch and tickle have so far been subject of speculation (Sinclair, '67). The present study is an attempt to identify nerve endings that are associated with itch. MATERIAL A N D METHODS Samples of the hairy skin from the deltoid region or the front of the thigh were obtained from five patients suffering from certain kinds of urticaria associated with itching and from two individuals with normal skin in which itching was induced either by application of nettle hairs (UrtiANAT. REC., 188: 1-12. caria dioica) or by intracutaneous injection of ragweed pollen extract. The urticaria cases included dermographism, pressure urticaria, urticaria pigmentosa and diffuse cutaneous mastocytosis. In the case of pressure urticaria, welts and itching were induced by application of a tourniquet for two to three minutes over the arm or thigh. In the other urticaria cases wheals were produced by stroking the skin over the deltoid region or front of the thigh with a smooth blunt stylus exerting 400 g pressure (Ebken et al., '68). Control samples were obtained from undisturbed skin areas of each urticaria patient and from individuals with normal skin reaction. All samples were obtained with a high speed dermal punch, 3 mm in diameter, without anestheiia. The tissue was immediately cut into strips oriented perpendicularly to the external surface of the epidermis with the use of two stainlessReceived Aug. 19, '76. Accepted Nov. 17, '76. 1 2 NIKOLAJS CAUNA epidermal endings. Figure 3 shows an axon terminal entering the epidermis at the far left. The distended space between the axon and the related keratinocyte is occupied by the granular material (arrows). Similar accumulations of granular material i n penicillate endings were observed in urticaria pigmentosa (fig. 2) and in experimentally induced itching (fig. 4). Occasionally, the granular material was found to be so abundant that it occupied most of the cross sectional area of the ending. Figure 5 shows such a case in which the Schwann sheath is compressed and deformed. The basal lamina of the ending is probably distended but its continuity is not interrupted. However, some of the granular material seems to have escaped through the basal lamina into the interstitial space (on the left of the ending). In most cases, the layer of the deposit extended from the axolemma to the basal OBSERVATIONS lamina. On occasions the granular maThe study revealed that a certain propor- terial was restricted within a space betion (of the free nerve endings of the itch- tween the axolemma and the plasmaleming human skin showed morphological ma Schwann sheath (arrow in the center changes. The endings in question were of fig. 4). Isolated areas of deposit bederived from non-myelinated nerve fibers tween the external aspect of the Schwann in a itypical manner (fig. 7) and have been sheath and the basal lamina were never described as “penicillate” by the author encountered although the granular ma(Cauina, ’73, ’76). The changes were char- terial tended to spread around the Schwann acterized by accumulation of extracyto- cell flaps, occasionally giving a n impresplasmic granular material between the sion of a n isolated pocket (fig. 5: arrows). axon terminals and the Schwann sheath, These findings may signify that the granuusuallly extending as far as the nerve lar material first appears next to the axolembasal lamina. Figure 1 shows a penicillate 1 Penicillate nerve ending cut transversenerve ending from a patient suffering ly.Fig. The Schwann sheath (Sch) enfolds three axon from dermographism and itch. The axolem- terminals (ax). Two areas bounded by the plasmamata of all three axon terminals are ex- lemma of the Schwann cell, axolemmata of the posed to the granular material that has terminals a n d the nerve basal lamina (bl) contain granular material (arrows) which consists of paraccumulated i n two spaces within the ticles that vary in size a n d staining intensity. In ending (arrows i n fig. 1). Such spaces do the area at left, the two layers of the mesaxon have not normally exist in nerve terminals. The been separated by the granular material and the space on the left has apparently been axon terminal has been deformed. In the other the Schwann cell flap has been dislocated created by separation of the two layers of area, away from t h e basal lamina and the axon terminthe mesaxon and by some compression of als. Wheal induced by stroking the skin over the the respective axon terminal. The space on deltoid area. Male, 50 years old suffering from the right has been produced by dislocation dermographism a n d itch. X 50,000. 2 Penicillate nerve ending cut transverseand compression of the Schwann sheath ly Fig. located i n a groove on the deep aspect of the flap that separates the two axon terminals epidermis (Ep). The basal lamina of the nerve and possibly by distension of the basal ending 031) encloses three axon terminals (ax) a n d their Schwann sheath (Sch) along with some lamina in that area. granular material (arrows). Soime intraepidermul axon terminals de- extracytoplasmic Wheal induced by stroking the skin over a macula rived from the penicillate endings also re- at t h e front of the thigh. Male, 11 months old sufvealed features similar to those of the sub- fering from urticaria pigmentosa. X 50,000. steel razor blades mounted parallel and 0.3-0.5 mm apart. The slices were fixed in 1 % osmic acid solution buffered a t pH 7.4 with phosphate buffer for two hours a t 4°C. During the dehydration stages through the alcohol series the strips were divided into blocks which were embedded in a n Epon-Araldite mixture. Serial sections were stained on grids with uranyl acetate and lead citrate solutions. For demonstration of glycogen electron microscopically, osmium fixed sections were mounted on uncoated gold grids and stained with periodic acid-thiosemicarbazide-silver proteinate solutions (Anderson, ’72). Control sections were oxidized with 5 % periodic acid solution for 20 minutes and treated with 0.1 % a-amylase solution in 0.1 M phosphate buffer at pH 7.0 for three hours and then stained for glycogen. CHANGES IN NERVE ENDINGS DURING ITCHING 3 4 NIKOLAJS CAUNA Fig. 3 Intraepidermal axon terminal (ax) cut longitudinally enfolded by a basal keratinocyte (Ker). Some granular material (arrows) is situated between the axolemma of the ending and the cell membrane of the keratinocyte. The basal lamina (bl) of the epidermis is continuous with that of the axon at the far left. Welt induced by application of a tourniquet, front of the thigh. Female, 21 years old suffering from pressure urticaria. X 50,000. ma and then progressively spreads towards the basal lamina. Not every axon terminal within a n ending wits similarly involved. Figure 2 shows an ending with three axon terminals. The one that is nearer to the top of the photographis completelyenfolded by the Schwann sheath and has no relationship to the granular material. Similarly, two axon terminals in figure 4 appear not to be involved. Furthermore, only a certain proportion of the popula.tion of penicillate endings was subject to morphological changes in itching skin. It was esimated that one in every five to six endings showed the presence of granular material. On occasions it was possible to visualize all endings derived from a single penicillus. This happened frequently in blocks in which the plane of sections was oriented perpendicularly to the longitudinal distribution of the endings. Sections cut through the penicillus immediately distal to the terminal Schwann cell made it possible to examine all its components before they had splayed out over their distribution areas (fig. 7 cf. with CHANGES IN NERVE ENDINGS DURING ITCHING 5 Fig. 4 Penicillate nerve ending cut transversely showing four axon terminals, (ax), their Schwann sheath (Sch) and extracytoplasmic granular material (arrows). Itching induced by touching normal skin of the deltoid area with nettle hairs. Male, 24 years old. x 50,000. fig. 6). In such cases it was evident that only one or two endings of the whole penicillus were involved (arrow in fig. 7). The changes described in the penicillate endings were not observed in the preterminal nerve fibers from which the endings were derived. Nor did the changes occur in the papillary endings which were derived from myelinated nerve fibers, or in the nerve terminals associated with the shafts of the fine hairs. The autonomic nerve terminals related to blood vessels, arrectores pilorum muscles or sweat glands also showed normal morphology. Nochanges in any nerve endings were observed in the 20 urticaria cases in which itching was absent or minimal. Morphologically, the granular material resembled glycogen particles. When stained histochemically, the extracytoplasmic granular material encountered in the penicillate nerve endings gave a strong positive reaction for glycogen. Figure 8 (arrows) shows the heavily stained particles in the intercellular space. These can be compared with a cluster of glycogen particles that are present in the axoplasm of one of the terminals (ax: right bottom in fig. 8). Following treatment of the osmium fixed sections with a-amylase, the glycogen stain gave a negative reaction in the tissues of the skin in general and also in the spaces previously occupied by the granular material (arrows in fig. 9). Although extracytoplasmic location of glycogen particles in nerve endings is an 6 NIKOLAJS C A U N A Fig. 5 Penicillate nerve ending cut transversely situated next t o the deep aspect of the epidermis (Ep). The ending contains two axon terminals (ax) and a greatly deformed Schwann sheath (Sch). Most of the cross sectional area of the ending i s filled with granular material. The basal lamina of the nerve ending (bl) is not disrupted. Wheal induced by stroking the skin of the deltoid region. Male, 9 years old suffering from d i f h s e cutaneous mastocytosis. X 50,000. CHANGES IN NERVE ENDINGS DURING ITCHING 7 Fig, 6 Diagram showing the manner of origin of the penicillate nerve endings from a sensory non-myelinated nerve fiber. The perikaryon of the terminal Schwann cell (T Sch) marks the origin of the penicillus, and it provides each ending and its branches with cytoplasmic sheaths throughout their subepidermal course. Arrows indicate the approximate plane of figure 7. intense itching, only some endings derived from a penicillus underwent changes while the majority of the same kind and all other kinds of nerve endings, both sensory and autonomic, as well as the conducting segments of nerve fibers remained unDISCUSSION affected. This may be an indication that The fine reversible changes that occur those penicillate nerve endings that undergo in a sensory end organ during its stimula- changes are associated with itch. It does not tion are not accessible to morphological follow that itch is the only function of observations. Therefore, the transient ac- these endings, nor that other nerve endings cumulation of glycogen in certain penicillate could not be associated with itch under difnerve endings during prolonged itching ferent conditions. While the morphological changes that is an unexpected finding, and to evaluate its significance requires careful considera- occur in certain penicillate nerve endings serve as a marker by which their association. It is fairly evident that the phenomenon tion with itch can be identified, the cytorepresents a byproduct of a rather com- chemical process itself is obscure, and the plex skin reaction of which itching is a source of the glycogen is not evident. Scatpart. In all cases represented in this study tered particles and occasional larger clusters itching was accompanied by wheal or welt of glycogen are normally present in the formation (Cauna and Levine, '70). How- axoplasm as well as in the cytoplasm of ever, wheal induced without itch did not the Schwann sheath of the endings. Thereproduce changes in nerve endings. Fur- fore, i t might be argued that the extracythermore, when wheal was accompanied by toplasmic glycogen had escaped from its unusual finding, extracytoplasmic glycogen occurs naturally in certain areas of the epidermis. In particular, the epithelial root sheath of the fine hairs is rich in intraand extracytoplasmic glycogen (fig. 10). 8 NIKOLAJS CAUNA Fig. 7 Penicillus cut transversely immediately distal to the terminal Schwann cell (arrows i n fig. 6). It consists of seven nerve endings of various sizes. Only one ending reveals extracytoplasmic granular material (arrow). The other endings show normal morphology. Wheal induced by stroking the skin over a macula, front of the thigh. Male, 21 months old suffering from urticaria pigmentosa. X 40,000. CHANGES IN NERVE ENDINGS DURING ITCHING 9 Fig. 8 Penicillate nerve ending stained for glycogen using the Periodic acid-ThiosemicarbazideSilver proteinate method. The Schwann sheath (Sch) enfolds three axon terminals (ax) one of which (right bottom) contains glycogen particles i n its axoplasm. Extracytoplasmic glycogen is present i n three areas (arrows). Wheal induced by stroking the skin over the deltoid region. Male, 9 years old suffering from diffuse cutaneous mastocytosis. X 50,000. Fig. 9 Penicillate nerve ending treated with a-Amylase (after oxidizing the osmium fixed section with Periodic acid) a n d stained for glycogen (as i n fig. 8). The Schwann sheath (Sch) enfolds three axon terminals (ax). Two empty extracytoplasmic areas (between arrows) bounded by the plasmalemma of the Schwann sheath, axolemmata of the terminals a n d the nerve basal lamina (bl) mark the position previously occupied by glycogen. Wheal induced by stroking the skin over a macula, front of the thigh. Male, 11 months old suffering from urticaria pigmentosa. X 50.000. 10 NIKOLAJS CAUNA Fig. 10 Part of the epithelial root sheath of a small hair follicle cut transversely showing two keratinocytes (Ker) connected by a dermosome (d). The space between the two cells which is separated from the surrounding corium by the basal lamina (bl) contains accumulatiasn of glycogen particles (unlabelled arrows). Skin from the deltoid region. Male, 49 years old. X 50,000 naturallocation. However, the small amount of int racellular glycogen is insufficient to account for the large extracytoplasmic accumul.ations. From a neurophysiological point of view the present findings are of particular interest. This is the first indication that itch may be linked with a particular end organ. Since each penicillus gives origin to a numbler of endings and only some of these undergo changes during itching the findings also tend to support a n earlier suggestio:n that penicillate endings are multimodal receptor organs (Cauna, '73, '76). The set ofsensory non-myelinated axons that gives origin to the penicillate endings (fig. 6) originates from several nerve cell bodies in the sensory ganglion, each of which may be associated with a different modality of sensation. Therefore the penicillus seems to represent a package of receptor organs subserving several sensory modalities rather than a single sensation. This suggestioin appears to be confirmed by recent electrophysiological studies of sensory nonmyelinated nerve fibers (Pearl et al., '76). ACKNOWLEDGMENTS The author wishes to thank Macy I. Levine, M.D., Department of Medicine, University of Pittsburgh, for the supply of biopsy material along with the clinical data, and to Ms. Gloria DeLuiso for her technical assistance and the aid with the photographic work. This study was supported by U. S. Public Health Service Grant NB 04147. LITERATURE CITED Anderson, W. A. 1972 Methods for electron microscopic localization of glycogen. In: Techniques of Biochemical and Biophysical Morphology. Vol. 1. D. Glick and R. M. Rosenbaum, eds. Wiley-Interscience, a division of John Wiley & Sons, Inc., New York, pp. 1-23. Cauna, N. 1973 The free penicillate nerve endings of the h u m a n hairy skin. J. Anat., 1 1 5 : 277-288. Cauna, N. 1976 Morphological basis of sensation in hairy skin. Progress i n Brain Res., 43: 3545. CHANGES IN NERVE ENDINGS DURING ITCHING Cauna, N., and M . I. Levine 1970 The fine morphology of the human skin in dermographism. J. Allergy, 45: 266-285. Ebken, R. K., F. A. Bauschard and M. I. Levine 1968 Dermographism: Its definition, demonstration and prevalence. J. Allergy, 41: 338343. 11 Pearl, E. R., T. Kumazawa, B. Lynn and P. Kenins 1976 Sensitization of high threshold receptors with unmyelinated ( C ) afferent fibers. Progress i n Brain Res., 43: 263-277. Sinclair, D. 1967 Cutaneous sensation. Oxford University Press, London, pp. 104, 110-113, 142-144.