THE ANATOMICAL RECORD PART A 272A:467– 473 (2003) Distribution of GAP-43 Nerve Fibers in the Skin of the Adult Human Hand LAURA VERZÉ,1* VIGLIETTI-PANZICA CARLA,1 STELLA MAURIZO,2 SICA MONICA,1 AND PANZICA GIANCARLO1 1 Department of Anatomy, Pharmacology, and Forensic Medicine, Laboratory of Neuroendocrinology, University of Torino, Torino, Italy 2 Plastic Surgery Division and Burn Unit, CTO-CRF, Maria Adelaide Hospital, Torino, Italy ABSTRACT Skin is an important region of somatic sensory input, and is one of the most innervated areas of the human body. In this study, we investigated in human hand skin the distribution of nervous structures immunoreactive for the growth-associated protein 43 (GAP-43) and the protein gene product 9.5 (PGP 9.5). GAP-43 is a neuronal presynaptic membrane protein that is generally considered to be a marker of neuronal plasticity. PGP 9.5 is a neuron-speciﬁc soluble protein that is widely used as general marker for the peripheral nervous system. The entire neural network of the dermis and epidermis was stained with antibody to PGP 9.5. In the dermis, there were fewer GAP-43-immunostained nerve ﬁbers than PGP 9.5-immunostained nerve ﬁbers, whereas in the epidermis the numbers were equal. Only some Merkel cells and Meissner corpuscles were GAP-43-immunoreactive. In conclusion, our results show that GAP-43 protein is expressed in a subset of PGP 9.5-immunoreactive nerve structures. Anat Rec Part A 272A:467– 473, 2003. © 2003 Wiley-Liss, Inc. Key words: hand skin; innervation; growth-associated protein 43; human Growth-associated protein (GAP-43) is a membrane protein that is expressed at high levels during neural development, and is newly produced in injured and regenerating adult nerve tissue. It is considered to be a marker for sprouting, and is usually associated with physiological events such as neuronal growth and synaptic plasticity (Benowitz and Routtenberg, 1987; Skene, 1989; Hoffman, 1989; Gispen et al., 1991). In the adult central nervous system, GAP-43 is present in several types of neurons and in regions of intense synaptic remodeling (Benowitz et al., 1988, 1990). In the normal adult peripheral nervous system, it is expressed at low levels in motor neuron axons (rat: Li and Dahlstrom, 1993), but is highly expressed in sensory nerve ﬁbers (human: Fantini and Johansson, 1992; rat: Verzé et al., 1999). GAP-43-immunoreactivity has also been demonstrated in adult mammalian ganglionic neurons. In the rat it is extensively expressed in the autonomic nervous system (Stewart et al., 1992), whereas in humans it has been demonstrated in trigeminal primary sensory neurons (Del Fiacco et al., 1994). GAP-43 mRNA is expressed in the preganglionic sympathetic neurons of the rat spinal cord (Michael and Priestley, 1995), © 2003 WILEY-LISS, INC. and in prevertebral and paravertebral human ganglia (Schmidt et al., 1991). Axon regeneration following peripheral nerve injury is characterized by a rapid increase of GAP-43 expression in sensory dorsal root ganglia (Sommervaille et al., 1991; Wiese et al., 1992) and in motor neurons (Palacios et al., 1994). After nerve injury, GAP-43 is transported to both the peripheral and central nerve endings of sensory neurons, and may be involved in their peripheral regeneration Grant sponsor: Fondazione Piemontese Studi e Ricerche sulle Ustioni (FPSRU). *Correspondence to: Dr. Laura Verzé, Department of Anatomy, Pharmacology, and Forensic Medicine, Laboratory of Neuroendocrinology, University of Torino, corso Massimo D’Azeglio 52-I10126, Torino, Italy. Fax: ⫹39-011-6707732. E-mail: email@example.com Received 11 June 2002; Accepted 23 January 2003 DOI 10.1002/ar.a.10056 468 VERZÉ ET AL. and central reorganization (Woolf et al., 1990, 1992). Loss of the connections of sensory neurons with their peripheral target tissues after nerve transection or crushing is considered to be one of the triggers for induction of GAP-43 expression. Environmental or target-derived factors, as well as their interactions, may regulate GAP-43 expression (Oestreicher et al., 1997). Skin is an ideal model for studying neurochemical markers in the peripheral sensory and autonomic nervous system of adult humans. However, little has been published concerning GAP-43 immunoreactivity in humans (Fantini and Johansson, 1992; McArthur et al., 1998; Kinkelin et al., 2000; Verzé et al., 2000). Immunoreactivity patterns for both GAP-43 and PGP 9.5 have been reported for human skin, but a complete study of the innervation in the human hand has yet to be performed. GAP-43 distribution has been compared with expression of a neuron-speciﬁc marker, the protein gene product 9.5 (PGP 9.5), which is one of the most widely distributed markers of the peripheral nervous system (Gulbenkian et al., 1987; Dalsgaard et al., 1989; Ramieri et al., 1990, 1992a, b; Wang et al., 1990). The aim of the present study was to describe the distribution of GAP-43 and PGP 9.5 immunoreactivity in the skin of the human hand, a highly innervated area. MATERIALS AND METHODS Fixation and Immunocytochemistry Samples of normal hand skin of 10 adults (six males and four females, 20 – 40 years old) were removed during reconstructive procedures, after informed consent was obtained. Three samples were taken from each subject. The specimens (1.5 cm ⫻ 1 cm) were taken from the dorsal (two subjects) and ventral (eight subjects) areas of the hand. Of the latter, three were biopsed in the thumb, three in the palm, and two in the index ﬁnger. The specimens were immediately immersed in Zamboni’s solution (4% paraformaldehyde and 0.2% picric acid in 0.01 M phosphate-buffered saline (PBS), pH 7.2–7.4) and ﬁxed overnight at 4°C. They were washed in PBS containing 15% sucrose for 3 days at 4°C, and then frozen. Serial 20-m-thick sections were cut with a cryostat (Microm, Heidelberg, Germany) and collected on chromealum gelatinized slides for the immunohistochemical procedure. Endogenous peroxidase activity was inhibited by washing the sections in methanol/hydrogen peroxide (Streefkerk, 1972) and incubating them in normal serum (30 min, 15 l/ml PBS) to prevent nonspeciﬁc staining. Adjacent sections were then incubated overnight at room temperature with polyclonal primary antiserum diluted in PBS containing 0.25% Triton X-100, 1:6000 anti-GAP 43 (donated by L. Schrama, Utrecht, The Netherlands; for details of the antibody, see Oestreicher et al. (1983) and 1:8000 anti-PGP 9.5 (Ultraclone, Isle of Man, UK). After the sections were washed brieﬂy, they were incubated for 60 min in a secondary biotinylated antibody, and then in an avidin-biotinylated peroxidase complex (Vectastain Elite Labtec; Vector, Burlingame, CA) for 60 min at room temperature. Peroxidase activity was visualized with a solution containing 0.20 mg/ml 3,3⬘-diaminobenzidine (DAB; Sigma, St. Louis, MO) in 0.05 M Tris-HCl buffer, pH 7.6, as chromogen and 0.35% hydrogen peroxide. The reaction was blocked by rinsing the sections in distilled water. Alcohol-dehydrated sections were then coverslipped with Entellan (Merk, Milano, Italy). To improve the identiﬁcation of positive structures, some sections were counterstained with 1% toluidine blue. Control reactions were performed by replacing the primary antibody with normal serum. The speciﬁcity of the antibodies for human tissues had been veriﬁed in earlier studies (Doran et al., 1983; Benowitz et al., 1989). Photographic Recording Specimens were observed with a bright-ﬁeld microscope (Diaplan; Leitz, Wetzlar, Germany) equipped with a Kodak Wratten no. 75 ﬁlter to enhance DAB product visibility. To increase the number of in-focus structures, we used image reconstruction based on several images of the same ﬁeld digitized at different focal planes. The reconstruction was performed using NIH-IMAGE 1.61 software (National Institutes of Health, Bethesda, MD) according to the method of Verzé et al. (1999). The resulting images were then sharpened, contrast-enhanced, and stored for printing with Adobe Photoshop 5.1. Quantiﬁcation The frequency of PGP 9.5- and GAP-43-immunoreactive (-ir) structures was evaluated in ﬁve sections for each specimen. We applied a semiquantitative method, with scores expressed as ⫹⫹⫹ ⫽ high frequency; ⫹⫹ ⫽ medium frequency; ⫹ ⫽ low frequency; and –⫽ absence. RESULTS All of the nerve ﬁbers were PGP 9.5-ir. GAP-43-ir was observed in the branches of nerves in the inner layers of the dermis, from which several nerve ﬁbers entered the dermal plexus. Moreover, ﬁner unmyelinated ﬁbers ran toward the skin surface and branched further into a subepithelial plexus. From this network thin immunoreactive ﬁbers reached the epidermal layers, where they ended as free ﬁbers and occasionally as ﬁbers associated with immunopositive GAP-43-ir Merkel cells. Free nerve endings were observed at all levels of the dermis, where other anatomical structures that received sensory or autonomic innervation were found. In particular, the sweat glands, hair follicles, and blood vessels were surrounded by GAP43-ir nerve ﬁbers. The distribution of GAP-43-ir nerve ﬁbers in comparison with the distribution of PGP 9.5-ir elements is summarized in Table 1. Epidermis In the epidermis, GAP-43-ir nerve ﬁbers were widely distributed in the basal, spinosum, and granulosum layers, and their density was comparable to that of PGP 9.5-ir elements (Fig. 1a– d; Table 1). GAP-43-ir nerve ﬁbers were most frequent in the basal layer of the epidermis, where they appeared thin and probably unmyelinated, and they ran among keratinocytes up to the stratum lucidum. These ﬁbers were in tight contact with the keratinocytes of the basal layer of the epidermis, which they sometimes encircled (Figs. 1c and 2e). In the basal area of the epidermis, isolated Merkel cells were GAP-43-positive. They were present only in the hairy skin of the back of the hand (Fig. 2c, Table 1). PGP 9.5-ir Merkel cells were present in all specimens (Fig. 2b and d, Table 1). No nerve ﬁbers or endings were observed in the stratum corneum. 469 GAP-43 NERVE FIBERS IN HUMAN HAND SKIN TABLE 1. Semi-quantitative analysis of GAP-43 and PGP 9.5 immunopositivity in the skin of the human hand GAP-43 Ventralis Index (2) Thumb (3) Palm (3) Dorsalis Hairy skin (2) PGP 9.5 Ventralis Index Thumb Palm Dorsalis Hairy skin Epidermis Merkel Dermis Meissner Vessels Sweat glands Hairs ⫹⫹ ⫹ ⫹⫹ – – – ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ – – – ⫹⫹⫹ ⫹ ⫹⫹ – ⫹⫹ – ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ – – – ⫹⫹⫹ ⫹ ⫹⫹⫹ – ⫹⫹⫹ – ⫹⫹⫹ ⫹⫹⫹, high frequency; ⫹⫹, medium frequency; ⫹, low frequency; –, absence of immunoreactive structures. Fig. 1. Palmar area of hand: nerve ﬁbers within the skin immunostained for (a) GAP-43 and (b) PGP 9.5. Note intense immunostaining in some ﬁbers of the dermis (short arrows) and deeper dermis (short arrows), and in thinner nerve ﬁbers running into the epidermis (asterisk). Higher magniﬁcation of (c) GAP-43-ir and (d) PGP-9.5-ir nerve ﬁbers (asterisk) in the epidermis and subepidermal plexus (short arrows). D, dermis; E, epidermis. Bars: (a and b) 50 m; (c and d) 100 m. Dermis the deeper layers. Both types of ﬁber were GAP-43-immunopositive (Fig. 2a and b, Table 1). A complex of nerve ﬁbers supplied the hair follicles, and all components were immunoreactive to both GAP-43 and PGP 9.5 (Fig. 2g and h, Table 1). GAP-43-ir nerve ﬁbers of small diameter were evident around the sweat glands and near the vessel walls (Fig. 2f). However, a more restricted pattern was observed for GAP-43 than for PGP 9.5 (Table 1). Meissner corpuscles were occasionally GAP-43-immunostained only in the In both hairy and hairless skin, numerous GAP-43-ir ﬁbers were arranged in a subepithelial plexus. Most of the positive ﬁbers ran parallel to the skin; others crossed the dermo-epidermal junction, entered the epidermis, and gave rise to epidermal free endings (Figs. 1a and c, and 2e). Within the dermis, thinner tortuous, probably unmyelinated ﬁbers terminated in this region as free endings. Thicker probably myelinated ﬁbers were more frequent in 470 VERZÉ ET AL. Fig. 2. Thumb, ventral side: immunostained nerve ﬁbers for (a) GAP-43 and (b) PGP 9.5 in the epidermis (asterisk) and dermis (short arrows). b: PGP 9.5-ir Merkel cells in the basal layer of the epidermis (thin arrows), and positive ﬁbers in the dermis (short arrows). (c) GAP-43-ir and (d) PGP 9.5-ir Merkel cells in the basal layer of the epidermis (thin arrows). e: Anterior area of the index ﬁnger: intraepithelial GAP-43- positive ﬁbers (asterisk). f: Palm: distribution of GAP-43-ir nerve trunks (short arrows) and GAP-43:-ir nerve ﬁbers around blood vessels and sweat glands, in the deeper dermis (thin arrows). Hairy skin (dorsal area of index ﬁnger): (g) GAP 43 and (h) PGP 9.5 thin nerve ﬁbers innervating hair follicles (thin arrows). Bars: (a– d, f, and g) 50 m, and (e) 100 m. GAP-43 NERVE FIBERS IN HUMAN HAND SKIN 471 Fig. 3. Index ﬁnger, ventral side: adjacent sections of a dermal papilla. (a) GAP-43 and (b) PGP 9.5 immunostaining of a Meissner corpuscle (asterisk) and its axon (short arrow). Bar: 50 m. dermis of the ventral area of the index ﬁnger, whereas they were normally positive for PGP 9.5 in the dermal papillae of the anterior regions of the index ﬁnger, thumb, and palm (Fig. 3a and b, Table 1). Gender and Age No gender-based differences in GAP-43 and PGP 9.5 expression or ﬁber distribution were observed in our samples. DISCUSSION The innervation of human skin has traditionally been considered to consist of a plexus of ﬁbers in the reticular dermis and a more superﬁcial plexus in the papillary dermis, with most sensory endings located in the subpapillary dermis. Intraepidermal nerve terminals have been identiﬁed in the basal layers of the epidermis that are mainly associated with Merkel cells. The use of the general neuronal marker PGP 9.5 has enabled easy recognition of intraepidermal ﬁbers by immunocytochemical staining (Ramieri et al., 1990, 1992b; Hilliges et al., 1995; Johansson et al., 1999). This marker is effective for identifying and quantifying unmyelinated intraepidermal nerves, a class that is frequently affected in sensory and sensory-motor neuropathies (Barohn, 1998; McArthur et al., 1998; Verzé et al., 2000). It has been suggested that GAP-43 immunoreactivity is a sign of neuronal plasticity (Benowitz and Routtemberg, 1997; Oestreicher et al., 1997). In this report we compared GAP-43 to PGP 9.5 to determine where remodeling in the periphery nervous system normally takes place. Our immunocytochemical ﬁndings of GAP-43-ir nerve structures in the hand are in agreement with the known anatomical distribution as derived from clinical neuroanatomical (silver and gold staining) and electrophysiological studies. We observed the presence of GAP-43 immunoreactivity in a large population of nerve ﬁbers in this highly innervated region of the human skin. GAP-43-ir ﬁbers were particularly present in the epidermis, excluding the stratum corneum, and the subepidermal layer, where their density was comparable to that of PGP9.5-ir structures. This observation conﬁrms the data of our previous study on the rat lower lip, in which we similarly demonstrated the presence of a large number of GAP-43-ir ﬁbers in the epithelium of both skin and mucosa (Verzé et al., 1999). In humans, GAP-43-ir ﬁbers (Fantini and Johansson, 1992; Del Fiacco et al., 1994; Verzé et al., 2000) and GAP-43 mRNA (Schmidt et al., 1991) have been described in the sensory and autonomic nervous system. The detection of PGP 9.5-positive Merkel cells, as well as nerve ﬁbers related to Merkel cells in the human epidermis, is in agreement with previous ﬁndings in different regions of human skin and oral mucosa (Ramieri et al., 1992a, b). There were notable differences between the GAP-43 and the PGP 9.5 immunostaining. We detected GAP-43 immunoreactivity only in proximity to a few Merkel cells and Meissner corpuscles. We suggest that the different expressions of these two neuronal markers indicate different degrees of function in the Merkel cells (Misery and Gaudillere, 1996; Verzé et al., 1999) and Meissner corpuscles. Meissner corpuscles in particular are widely regarded as multiafferent receptor organs that may have nociceptive capabilities, in addition to being low-threshold mechanoreceptors (Pare et al., 2001, 2002). In the dermis, all nerve ﬁbers were found to label with PGP 9.5, whereas a more restricted pattern was observed for GAP-43. The deeper nerve trunks and ﬁbers were less intensely stained for GAP-43 than for PGP 9.5. A basal expression of GAP-43 in autonomic structures innervating the sweat glands and vessels is in agreement with previous ﬁndings in human (Fantini and Johansson, 1992) and rat (Verzé et al., 1999) skin. Complex nervous structures associated with hair follicles showed a wide distribution of GAP-43 immunoreactivity comparable with that of PGP 9.5. The innervation of these nerve structures was previously documented in mice, in which hair-cycle-dependent plasticity was shown to be present in skin and hair follicle innervation. In particular, GAP-43 immunoreactivity ap- 472 VERZÉ ET AL. peared to be related with the early anagen phase of the hair cycle (Botchkarev et al., 1997). It seems, therefore, that epidermal nerve endings should be more involved in remodeling and plasticity than the populations of ﬁbers located in deeper regions, such as the dermis. The high level of basal GAP-43 immunoexpression in the epidermis may be related to continuous migration of the basal cells of the epidermis toward more superﬁcial layers; this migration, which results in a loss of the target for a subpopulation of sensory nerve ﬁbers, could be one of the local stimuli promoting GAP-43 immunoexpression in the most superﬁcial nerve ﬁbers of the skin (Fantini and Johansson, 1992; Verzé et al., 1999). Similar conditions may be involved in the basal expression of GAP-43 in the nerve structures of hairs in adult hairy hand skin, in the absence of any abnormal condition. The molecular nature of the signals that determine the basal expression of GAP-43 is unknown, but the high density of GAP-43-ir nerve ﬁbers in the basal layer of the epidermis suggests the existence of tight contacts, as well as a possible interaction between keratinocytes and nerve structures (Hsieh et al., 1996). Intense nerve growth factor (NGF)-like immunoreactivity has been observed in rat keratinocytes, excluding the stratum corneum (English et al., 1994). In a recent report we demonstrated an almost complete loss of epidermal innervation in a human sensory neuropathy induced by genetically-derived alteration of NGF receptor expression (Verzé et al., 2000). It is possible that NGF is a signaling molecule that induces nerve plasticity in the epidermis and therefore determines the expression of GAP-43. In support of this hypothesis, in vitro studies have shown that NGF increases GAP-43 expression in PC12 cells, as well as in neurons of the superior cervical ganglia (Federoff et al., 1988). LITERATURE CITED Barohn RJ. 1998. Intraepidermal nerve ﬁber assessment: a new window on peripheral neuropathy. Arch Neurol 55:1505–1506. Benowitz LI, Routtenberg A. 1987. A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism and synaptic plasticity. 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