Journal of Neuropathology and Experimental Neurology Vol. 59, No. 4 Copyright ᭧ 2000 by the American Association of Neuropathologists April, 2000 pp. 297–307

Pathology of Nerve Terminal Degeneration in the Skin

SUNG-TSANG HSIEH, MD, PHD, HOU-YU CHIANG, MS, AND WHEI-MIN LIN,MS

Abstract. To characterize the pathology of epidermal nerve degeneration and regeneration, we investigated temporal and spatial changes in skin innervation of the mouse footpad. Within 24 hours after sciatic nerve axotomy, terminals of epidermal nerves appeared swollen and there was a mild reduction in epidermal nerve density (5.7 Ϯ 2.8 vs 12.7 Ϯ 2.2 fibers/mm, p Ͻ 0.04). Epidermal nerves completely disappeared by 48 hours (0.2 Ϯ 0.2 vs 14.2 Ϯ 0.9 fibers/mm, p Ͻ 0.001). Concomitant with the disappearance of epidermal nerves, the immunocytochemical pattern of the subepidermal nerve plexus became fragmented. At the electron microscopic level, the axoplasm of degenerating dermal nerves was distended with organelles

and later became amorphous. Beginning from day 28 after axotomy, collateral sprouts from the adjacent saphenous nerve Downloaded from https://academic.oup.com/jnen/article/59/4/297/2609881 by guest on 01 October 2021 territory extended into the denervated area with a beaded appearance. They never penetrated the epidermal-dermal junction to innervate the epidermis. In contrast, 3 months after nerve crushing, the epidermis on the surgery side resumed a normal innervation pattern as the epidermis on the control side (10.3 Ϯ 3.9 vs 10.6 Ϯ 1.5 fibers/mm, p ϭ 0.1). This study demonstrates the characteristics of degenerating and regenerating nerves, and suggests that successful reinnervation mainly originates from regenerating nerves of the original nerve trunks. All these findings provide qualitative and quantitative information for interpreting the pathology of cutaneous nerves.

Key Words: Axonal degeneration; Cutaneous nerves; Free nerve endings; Intraepidermal nerves; Nerve regeneration; Skin innervation; Unmyelinated axons.

INTRODUCTION features of individual nerves in the early phase of degen- eration. Characterization of the pathology of degenerating Evaluation of cutaneous nerve terminals in the epider- and regenerating cutaneous nerves in the aforementioned mis of skin by immunocytochemistry provides a major system should provide important clues for clinical appli- advance in the study of peripheral sensory nerve disor- cation of skin biopsy and new insights into potential ders at the light microscopic level (1–4). Epidermal mechanisms of nerve degeneration. nerves are readily demonstrated with the neuronal marker protein gene product 9.5 (PGP 9.5), a ubiquitin carboxyl hydrolase. This approach offers an important measure- ment complementary to functional studies of cutaneous innervation in both humans and laboratory animals (2, 5–9). By taking advantage of skin biopsy, epidermal nerves can be quantified for diagnosis of sensory neu- ropathy (3, 10, 11). There is a significant reduction in the abundance of epidermal nerves in different types of sen- sory neuropathies (10, 12–14). Quantitation is mainly based on Wallerian degeneration, a final common path- way of nerve degeneration following mechanical, chem- ical, and toxic insults (15, 16). However, only limited studies have explored the issue of qualitative differences between normal degenerating and regenerating cutaneous nerves (3, 7, 13). In rodents, degeneration of nerve terminals is usually completed within days (4, 8). The loss of cutaneous in- nervation following nerve injury has been demonstrated in a well-established experimental animal system (6, 17, Fig. 1. Diagram of foot innervation in mice. This diagram 18). Only limited studies have examined ultrastructural shows the major innervation of the mouse hind foot from the sciatic and saphenous nerves. The sciatic nerve innervates the plantar side of the foot and the lateral aspect of the foot dorsum. From the Departments of Anatomy and Neurology (S-TH, H-YC, W- The saphenous nerve innervates the medial aspect of the foot ML), National Taiwan University College of Medicine, Taipei, Taiwan. dorsum. The shaded area on the foot dorsum is innervated by Correspondence to: Dr. Sung-Tsang Hsieh, Department of Anatomy, both nerves. The circles depict footpads and the numbers in- National Taiwan University College of Medicine, 1 Jen-Ai Road, Sec. dicate the relative positions of each pair of footpads. The first 1, Taipei, 10018 Taiwan. pair (1) is the most distal pair of footpads. The third pair (3) is Supported by a grant from National Health Research Institute, Tai- the most proximal pair near the heel, with the second pair (2) wan, ROC (NHRI-GT-EX89S727C). localized between the first and third pairs.

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Fig. 2. Temporal changes of epidermal nerve degeneration. Normal epidermis (A) and the epidermis denervated at various time points after sciatic nerve injury (B–F) were immunolabeled with protein gene product 9.5 (PGP 9.5). A: In control epidermis there are numerous PGP 9.5(ϩ) nerves forming a dense subepidermal plexus (arrow). PGP 9.5(ϩ) epidermal nerves have typical varicosities extending to the upper portion of granular layers. B: Within 1 day of sciatic nerve axotomy, epidermal nerves retract from the top of granular layers and terminal swelling becomes obvious (arrowheads). C: Epidermal nerves disappear by 2 days after axotomy of the sciatic nerve. PGP 9.5(ϩ)-immunoreactivity in the subepidermal plexus becomes fragmented (open arrow- head). D: Four days after denervation there are only degenerated axons in the subepidermal area. E: By day 7 of nerve injury there is no PGP 9.5(ϩ) epidermal nerve nor PGP 9.5(ϩ) dermal nerve. F: Twenty-eight days after nerve axotomy the epidermis remains denervated and the immunocytochemical pattern is similar to that in (E). Scale bar ϭ 50 ␮m.

Restoration of neural functions after peripheral nerve by sprouting from neighboring nerves (19). Both mech- injury depends on reconnection between nerves and their anisms require different sets of specific molecules (20, targets. Theoretically, reinnervation of targets can be 21). Variables influencing the capacity of regeneration in- achieved by regeneration from original nerve trunks, or clude the types of nerve injury and the environment

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adhered to the National Research Council Guide for the care and use of laboratory animals. In mice, there were 3 pairs of footpads on the plantar side of the hindpaw (Fig. 1). The footpads were glabrous skin and were designated as the first, second, and third pairs. The areas immediately exterior to the footpads on the medial and lateral sides were pilary skin. The plantar side and the lateral aspect of the dorsal side (from the third to the fifth toes) were inner- vated by the sciatic nerve, and the medial aspect of the dorsal side was innervated by the saphenous nerve based on previous functional and morphological studies (Fig. 1) (8, 18, 25, 26). When we sampled tissues, each pair of pads with the interpad Fig. 3. Quantitation of epidermal nerve degeneration. Epi- skin was processed together. In preliminary studies, we com-Downloaded from https://academic.oup.com/jnen/article/59/4/297/2609881 by guest on 01 October 2021 dermal innervation was quantified as described in the Materials and Methods section with 5 animals at each time point, and pared results of denervation among the 3 pairs of footpads, and expressed as fibers/mm of linear epidermis. Within 24 h after the results were similar. Therefore, only findings of the first pair sciatic nerve axotomy (Day1 post-Ax), there is a significant of footpads were reported. reduction in epidermal nerve density on the denervated epider- mis (5.7 Ϯ 2.8 fibers/mm, open bar) compared with that on the Immunocytochemistry control side (12.7 Ϯ 2.2 fibers/mm, filled bar). By post-axotomy day 2 (Day2 post-Ax), epidermal nerves no longer exist (0.2 Ϯ For immunocytochemistry on freezing microtome sections 0.2 fibers/mm vs 14.2 Ϯ 0.9 fibers/mm on the control side). On (3), animals were fixed by intra-cardiac perfusion with 4% para- post-axotomy day 7 (Day7 post-Ax), the depletion of epidermal formaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). The skin nerves persists (0.5 Ϯ 0.2 vs 12.3 Ϯ 1.8 fibers/mm). Statistical areas innervated by the sciatic nerve, including the glabrous analysis is based on t-test between the denervated epidermis skin (footpads) and the pilary skin (area lateral to the pads), Ͻ Ͻ and the control epidermis for each time point. * 0.04, ** were fixed for another 6 hours (h), and then changed to PB for 0.001. storage. After thorough rinsing in PB, samples were cryopro- tected with 30% sucrose in PB overnight. Sections perpendic- ular to the dermis of 30 ␮m were cut on a sliding microtome. where nerve regeneration will take place (22–24). In Sections from each tissue were labeled sequentially and stored with anti-freeze at Ϫ20ЊC. To ensure adequate sampling, every mice, reinnervation of the cutaneous field occurs within fourth section for each tissue was chosen for PGP 9.5-immu- weeks following nerve-crushing injuries (8, 18). The nostaining. The sections were treated with 0.5% Triton-X 100 roles of regeneration and sprouting for proper reinner- in 0.5 M Tris buffer (pH 7.6) (Tris) for 30 min and processed vation of the epidermis after denervation of long duration for immunostaining. Briefly, the sections were quenched with remain to be determined. 1% H2O2 in methanol, and blocked with 5% normal serum of To address these issues, we investigated temporal appropriate species in 0.5% nonfat dry milk in Tris. The sec- changes of cutaneous nerve degeneration and regenera- tions were incubated with rabbit antiserum to PGP 9.5 tion, and characterized ultrastructural patterns of cutane- (UltraClone, UK, 1:1,000 diluted in 1% normal serum in Tris) ous nerves at the early stage of nerve degeneration. for 16–24 h. After rinsing in Tris, sections were incubated with biotinylated goat anti-rabbit IgG for 1 h and the avidin-biotin MATERIALS AND METHODS complex (Vector, Burlingame, CA) for another hour. The re- action product was demonstrated by 3,3Ј-diaminobenzidine Animal Surgery (DAB, Sigma, St Louis, MO). We used male 8-week-old ICR mice (35–40 g, National Tai- Quantitation of Epidermal Innervation wan University College of Medicine Animal Center, Taipei, Tai- wan) for the following studies. Mice were anesthetized with Epidermal innervation was quantified according to modified chloral hydrate (40 mg/kg) for surgery. The sciatic nerve on protocols in a coded fashion (3, 8, 27). PGP 9.5-immunoreac- the right side was transected at the thigh level (the sciatic nerve tive nerves in the epidermis of each footpad were counted at a axotomy group). A segment (3–5 mm) of the sectioned sciatic magnification of 40ϫ with an Olympus BX40 microscope (Shi- nerve was removed, and the end of the proximal stump was buya-ku, Japan). Each individual nerve with branchings inside sutured to the overlying muscle fascia and subcutaneous tissues the epidermis was counted as one. For epidermal nerves with to prevent the reconnection of the proximal and distal cut ends branchings in the dermis, each individual nerve was counted (4, 8). For each animal, the left sciatic nerve was sham-operated separately. The total length of epidermis along the upper margin as the control. Three to 5 animals were sacrificed at different of stratum corneum in each footpad was measured with the intervals after sciatic nerve axotomy: 1, 2, 3, 4, 5, 7, 10, 14, Image-Pro PLUS system (Media Cybernetics, Silver Spring, 28, and 90 days. To study reinnervation, additional groups of MD). Epidermal nerve density was therefore derived and ex- 5 mice were subject to crushing injury on the right sciatic nerve pressed as the number of fibers per mm of epidermal length. (the sciatic nerve crush group). These mice were sacrificed 90 Every fourth section for each tissue was quantified, and there days later for comparison with the axotomy group. Experiments were 3 sections for each footpad.

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Fig. 4. Normal and degenerating dermal nerves. Normal (A) and denervated (B–D) footpads were immunocytochemically labeled with protein gene product 9.5 (PGP 9.5) to illustrate dermal nerves. A: In the normal dermis, PGP 9.5-immunoreactive dermal nerve trunks appear with a linear pattern and dense staining intensity. B: Within 24 h of axotomy, PGP 9.5(ϩ) dermal nerves have the same appearance as normal dermal nerves in (A). C: Seventy-two hours after nerve injury, dermal nerves undergo degeneration and have a globular appearance, reflecting degenerated axons and myelin ovoid formation. (D) Seven days post- surgery, all axonal staining disappears. In the dermis there is only faint staining on the membrane surface of denervated Schwann cells along the border of dermal nerves. Scale bar ϭ 20 ␮m.

Ultrastructural Study RESULTS Mice were perfused with 5% glutaraldehyde in PB. The hind Temporal Changes of Cutaneous Nerve Degeneration paws of both control and denervated sides were dissected. Tis- In a normal footpad, typical intraepidermal nerves had sues stayed in the same fixative overnight. After rising in PB, a varicose appearance extending to granular layers of the tissues were postfixed in 2% osmium tetraoxide for 2 h, de- epidermis (Fig. 2A). Terminals of epidermal nerves be- hydrated through graded ethanol, and embedded in Epon (28). came swollen within 24 h after sciatic nerve axotomy, Semithin sections were stained with toluidine blue. Selected and many of the epidermal nerves retracted from the areas were thin-sectioned and observed under a Hitachi electron microscope. granular layers (Fig. 2B). Quantitatively, there was a sig- nificant reduction in epidermal nerve density (5.7 Ϯ 2.8 Statistical Analysis fibers/mm on the denervated side vs 12.7 Ϯ 2.2 fibers/ mm on the control side, p Ͻ 0.04, Fig. 3). Epidermal For quantitation of epidermal innervation, groups of 5 ani- nerves no longer existed by 48 h (Fig. 2C, 0.2 Ϯ 0.2 mals were used at 1, 2, 7 days and 3 months after sciatic nerve fibers/mm vs 14.2 Ϯ 0.9 fibers/mm on the control side, axotomy and 3 months after sciatic nerve crush. The data were Ͻ ϩ presented as mean Ϯ SD. Analyses included t-test and Wilcox- p 0.001, Fig. 3). Only PGP 9.5( ) spots were seen in on rank sum test between the control side and the experimental the subepidermal plexus. Beginning at postaxotomy day side at each time point with SPSS for Windows (version 6.1, 4, immunoreactivity for subepidermal plexuses disap- SPSS Inc., Chicago, IL) and GraphPad Prism (version 2.01, peared altogether (Fig. 2D–F). GraphPad Software Inc., San Diego, CA). Any difference with In the dermis, PGP 9.5(ϩ) dermal nerves looked the p Ͻ 0.05 was considered statistically significant. same as normal dermal nerves within 24 h of sciatic

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Fig. 5. Temporal changes of sweat gland innervation after sciatic nerve axotomy. To investigate sweat gland innervation, we immunostained normal skin (A) and denervated skin (B–F) with protein gene product 9.5 (PGP 9.5). A: In normal sweat glands, the innervation appears interlaced around the ductal and secretory portions of sweat glands. B: Within 24 h of nerve injury, the pattern of sweat gland innervation is similar to that in (A). C: By 48 h, the innervation of sweat glands disappears and the staining for remaining nerve trunks becomes discontinuous. D: The PGP 9.5-immunoreactivity in the sweat gland area is not recognizable 4 days after denervation. E: Seven days after nerve injury, the immunocytochemical pattern of sweat gland inner- vation is similar to that of day 4 denervation. F: Twenty-eight days post-surgery sweat glands remain denervated. There are only some discontinuous PGP 9.5-immunoreactive dots between sweat glands, which may reflect sprouting from neighboring nerve territories. Scale bar ϭ 20 ␮m.

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Fig. 6. Ultrastructural characterization of degenerating cutaneous nerves. A: By 24 h after sciatic nerve axotomy, there were occasional degenerating epidermal nerves between adjacent keratinocytes containing electron-dense keratohyaline granules (k). These axons are swollen with disrupted axonal membrane and the axoplasm becomes amorphous (arrow), representing disinte- gration of the axonal cytoskeleton. B: Within 24 h of sciatic nerve axotomy, unmyelinated axons in the subepidermal areas undergo degeneration (arrow) and some have intact axonal membrane (arrowhead). C: By 48 h of axotomy, most of unmyelinated nerves in subepidermal areas are degenerated (arrows). Only scanty of unmyelinated nerves have recognizable axonal membrane (white arrowhead). However, the axoplasm in these axons is amorphous and organelles are swollen. Scale bars: A ϭ 0.67 ␮m; B, C ϭ 1 ␮m. nerve axotomy (Fig. 4A–B). These nerves degenerated later, all axonal staining disappeared, and only faint stain- and transformed into a globular appearance 72 h after ing along the borders of the dermal nerve trunk remained, nerve injury, reflecting degenerated axons and myelin which could be either degenerated nerves or denervated ovoids in Wallerian degeneration (Fig. 4C). Seven days Schwann cells (Fig. 4D).

→ Fig. 7. Reinnervation of skin 3 months after sciatic nerve crushing compared with the denervated skin 3 months after sciatic nerve axotomy. To compare patterns of innervation after different surgical procedures, we immunostained the skin with protein gene product 9.5 (PGP 9.5) from animals in the crushed-nerve group (left panel: A, C, E, and G) and the axotomy group (right panel: B, D, F and H). A: PGP 9.5(ϩ) nerves appear in the epidermis and in the dermis, similar to those in the control footpads. B: There is no sign of reinnervation in the skin 3 months after axotomy. C: In the crushed-nerve group there are epidermal nerves and some of them reach the top of the granular layers. D: In the axotomy group, only discontinuous PGP 9.5-immuno- reactivity parallels the subepidermal area. No epidermal nerve is discernible. E: Sweat gland innervation resumes the interlaced appearance after nerve regeneration. F: After permanent axotomy, only discrete dots of PGP 9.5-immunoreactivity appear near sweat glands. G: In the nerve-crushed group, PGP 9.5(ϩ) dermal nerves appear normal like the control nerves in Figure 4A. H: In the axotomy group, only discontinuous PGP 9.5(ϩ) dots are in the dermis. Scale bars: A, B ϭ 200 ␮m; C, D ϭ 50 ␮m; E– H ϭ 23 ␮m.

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from that of the axotomy group (Fig. 7A, B). In the crush group, PGP 9.5(ϩ) nerves appeared in the epidermis and dermis, similar to those in the control footpads (Fig. 7A, C, E, G). There was, however, no sign of reinnervation in the epidermis and dermis 3 months after sciatic nerve axotomy (Fig. 7B, D, F, H). In the crush group, some of the epidermal nerves reached the granular layers (Fig. 7C). In the axotomy group, discontinuous PGP 9.5-immunoreactivity ap- peared in the subepidermal area, but these nerves never penetrated the epidermal-dermal junctions (Fig. 7D). Downloaded from https://academic.oup.com/jnen/article/59/4/297/2609881 by guest on 01 October 2021 Fig. 8. Quantitation of epidermal reinnervation. Epidermal Sweat gland innervation resumed and dermal nerves re- reinnervation 3 months after sciatic nerve injury was quantified appeared in the crushed-nerve group, with the same ap- with 5 animals for each group. Epidermal nerve density is ex- pearance as those in the normal skin (Fig. 7E–G). When pressed as fibers/mm of linear epidermis. For the sciatic nerve nerve regeneration was retarded by permanent nerve ax- axotomy group (Axotomy), the epidermis remains denervated otomy, there were much fewer regenerating nerves in the (0.2 Ϯ 0.2 fibers/mm, open bar) compared with the control (11.0 Ϯ 3.5 fibers/mm, filled bar). In the sciatic nerve crush dermis. The reinnervating nerves had a beaded appear- group (Crush), the epidermal nerve density of the previously ance with axonal swellings. These included nerve ter- denervated skin (10.3 Ϯ 3.9 fibers/mm) is similar to that of the minals innervating the sweat glands, dermal nerve trunks, control skin (10.6 Ϯ 1.5 fibers/mm, p ϭ 0.1). Statistical analysis and subepidermal plexuses (Fig. 7D, F, H). is based on t-test between the denervated epidermis and the The influence of sciatic nerve axotomy and sciatic control epidermis for each time point. ** Ͻ 0.001. nerve crush on epidermal reinnervation was demonstrat- ed by quantitation of epidermal nerves (Fig. 8). The num- The time course and pattern of degeneration of nerves ber of epidermal nerves was negligible on the axotomized innervating sweat glands were similar to those of epider- side 3 months after sciatic nerve axotomy (0.20 Ϯ 0.20 mal nerves. There was no distinguishing change within vs 11.0 Ϯ 3.5 fibers/mm, p Ͻ 0.001). In contrast, 3 24 h (Fig. 5A, B). By 48 h, the innervation of sweat months after sciatic nerve crush, epidermal nerve densi- glands disappeared and the staining for remaining nerve ties were similar between the control side and the exper- trunks became discontinuous (Fig. 5C). The PGP 9.5- imental side (10.6 Ϯ 1.5 vs 10.3 Ϯ 3.9 fibers/mm, p ϭ immunoreactivity in the area of sweat glands was no lon- 0.1). ger recognizable during the period of denervation (Fig. We further investigated clues of unsuccessful reinner- 5D–F). vation of the epidermis after permanent sciatic nerve ax- otomy. This was based on the results from 28 days and Ultrastructural Appearance of Cutaneous Nerve 3 months after permanent nerve axotomy (Fig. 9). Grow- Degeneration ing nerves originated from the neighboring saphenous To study ultrastructural characteristics of degeneration nerve and extended into the adjacent footpad in the sci- in the terminal region of cutaneous nerves, we examined atic nerve territory (Fig. 9A). At higher magnification, epidermal nerves and subepidermal plexuses in the early these nerves approached the epidermis of the footpad phase of degeneration. By 24 h of axotomy, the axoplasm (Fig. 9B, C). None of the collateral sprouts extended be- of epidermal nerves became amorphous with disrupted yond the epidermal-dermal junction after permanent axonal membrane, representing disintegration of the ax- nerve injury (Fig. 9C, c.f. Fig. 7D). onal cytoskeleton (Fig. 6A). In subepidermal area, some of unmyelinated axons had intact axonal membrane and DISCUSSION others were undergoing degeneration (Fig. 6B). Within The present study demonstrates the early pathology of 48 h, disintegration of axonal cytoskeleton and swelling cutaneous nerve terminal degeneration, as well as the ul- of organelles became prominent features. Only scanty un- trastructural characteristics of degenerating nerves. Tem- myelinated axons continuing degeneration were recog- poral and spatial patterns of nerve degeneration provide nizable (Fig. 6C). important information not only for understanding the mechanisms of Wallerian degeneration, but also for in- Reinnervation of the Skin after Sciatic Nerve Crush terpreting the findings of skin biopsies. To investigate pathological characteristics of regener- ating nerves, we compared the results of sciatic nerve Degeneration of Nerve Terminals in the Epidermis crush with those of permanent sciatic nerve axotomy. The depletion of intraepidermal nerves within 24–48 Three months after nerve injury, the immunocytochemi- h suggests that nerve terminal degeneration is an early cal pattern of the crush group was substantially different event, consistent with the finding of motor nerve terminal

J Neuropathol Exp Neurol, Vol 59, April, 2000 NEUROPATHOLOGY OF CUTANEOUS NERVE DEGENERATION 305 degeneration (24, 29). Epidermal nerve terminals become swollen within 24 h of axotomy at the light microscopic level. This early sign of degeneration may reflect a block- ing of axonal transport, and offers an ultrastructural il- lustration of the degenerating nerves in neuropathy (3, 13). Within 48 h of sciatic nerve axotomy, no more epi- dermal fibers exist. Some of the dermal nerves remain visible immunocytochemically with intact organization at the ultrastructural level. These findings indicate that the disappearance of PGP 9.5(ϩ)-epidermal nerves is not simply due to a difference in the clearance of PGP 9.5 between epidermal nerves and dermal nerves. Several Downloaded from https://academic.oup.com/jnen/article/59/4/297/2609881 by guest on 01 October 2021 mechanisms account for the faster degeneration of nerve terminals. For example, the temperature in the nerve ter- minal regions is lower than that of the proximal part of damaged nerves (16, 30, 31). Alternatively, calcium in- flux may mediate earlier nerve degeneration in nerve ter- minals (32). The presence of calcium channels in motor nerve terminals and calcium-dependent proteolysis may facilitate nerve terminal degeneration (16, 33). Axonal degeneration after rhizotomy illustrates different speeds of degeneration in different portions of the same nerve. Degeneration of nerve terminals in the region of the nu- cleus gracile is faster than that in the region of the dorsal column; both are the same central processes of dorsal root ganglion neurons (34, 35). Whether degeneration of sen- sory terminals shares the same mechanism remains an open issue. The presence of calcium-binding proteins in the dorsal root ganglion, whose peripheral processes con- stitute the cutaneous nerves, suggests that calcium may play a role in nerve degeneration and regeneration (36– 38). Cutaneous Reinnervation by Regenerating Axons Both axonal regeneration and collateral sprouting con- tribute to reinnervation of the denervated skin. The over- all outcome depends on types of nerves and lesions (23, 24, 39) and guidance molecules (19, 40–43). Collateral sprouts from neighboring saphenous nerves appear as early as 2 weeks after nerve injury. However, growth cones of these sprouts only extend laterally into their neighboring area for a limited distance. Despite ap- proaching the epidermal-dermal junction, collateral sprouts fail to cross the basement membrane of the epi- dermis and ascend in the epidermis as normal intraepi- dermal nerves do. These findings extend previous obser- vations (22) and provide a morphological basis for investigating the territory of nerves complementary to functional dermatome mapping (44). After the nerve- from the neighboring saphenous nerve territory was immuno- stained with protein gene product 9.5 (PGP 9.5). The tissue was crushing injury, regenerating nerves from the sciatic sampled 28 days after nerve axotomy. A: PGP 9.5(ϩ) epidermal and dermal nerves are in the epidermis of the saphenous nerve territory. Dermal nerves extend into the neighboring dermis. B: → At higher magnification, dermal nerves approach the epidermis of the footpad. C: Collateral sprouts parallel the epidermal-der- Fig. 9. Collateral sprouting after sciatic nerve axotomy. mal junction but never ascend in the epidermis. Scale bars: A The footpad in the sciatic nerve territory with adjacent skin ϭ 200 ␮m; B, C ϭ 20 ␮m.

J Neuropathol Exp Neurol, Vol 59, April, 2000 306 HSIEH ET AL nerve trunks easily penetrate the basement membrane and degeneration and subsequent reinnervation of epidermal nerve fi- terminate in the epidermis. bers: Correlation with sensory function. J Neurosci 1998;18: 8947–59 Regenerating nerves arising from the original nerve 3. McCarthy B, Hsieh ST, Stocks EA, et al. Cutaneous innervation in trunks appear to be the major source of successful rein- sensory neuropathies: Evaulation by skin biopsy. Neurol 1995;45: nervation of previous denervated epidermis. Why does 1848–55 the sciatic nerve reinnervate the previously sciatic terri- 4. Hsieh ST, Choi S, Lin W-M, Chang Y-C, McArthur JC, GriffinJW. tory better than the saphenous nerves? Several lines of Epidermal denervation and its effects on keratinocytes and Lan- gerhans cells. J Neurocytol 1996;25:513–24 evidence suggest that nerve-target connections and ter- 5. Kennedy WR, Wendelschafer-Crabb G, Breije TC. Innervation and ritory integrity could exert complicated interactions. Neu- vasculature of human sweat glands: An immunocytochemistry-laser rotrophin requirement is different for collateral sprouting scanning confocal fluorescence microscopy study. J Neurosci and regenerating growth. Collateral sprouting is depen- 1994;14:6825–33 dent on endogenous nerve growth factor and regeneration 6. Navarro X, Verdu E, Wendelscafer-Crabb G, Kennedy WR. Inner- Downloaded from https://academic.oup.com/jnen/article/59/4/297/2609881 by guest on 01 October 2021 vation of cutaneous structures in the mouse hind paw: A confocal from original nerve trunks is independent of nerve microscopy immunohistochemical study. J Neurosci Res 1995;41: growth factor (20, 21). In acute stage of denervation, the 111–20 content of nerve growth factor in the skin is up-regulated 7. Hsieh ST, Lin W-M, Chiang H-Y, Huang I-T, Chen WP. Skin in- (45). However, the content of nerve growth factor in the nervation and its influence on the epidermis. J Biomed Sci 1997;4: long-term denervated skin is down-regulated (46). It re- 264–68 8. Chiang H-Y, Huang I-T, Chen WP, et al. Regional difference in mains to be determined whether there are factors inhib- epidermal thinning after skin denervation. Exp Neurol 1998;154: iting the extension of neurites into the aberrant territory 137–45 as documented in the central nervous system, for exam- 9. Johansson O, Wang L, Hilliges M, Liang Y. Intraepidermal nerves ple, the myelin-associated glycoprotein (42, 47–49). The in human skin: PGP 9.5 immunohistochemistry with special ref- establishment of nerve-target connection requires correct erence to the nerve density in skin from different body regions. J Peripher Nerv Syst 1999;4:43–52 guidance cues, for example, attractive and repulsive mol- 10. Kennedy WR, Wendelschafer-Crabb G, Johnson T. Quantitation of ecules (41, 50, 51). These signals are mediated through epidermal nerves in diabetic neuropathy. Neurol 1996;47:1042–48 cyclic nucleotides (52). Neurotrophins modulate the re- 11. McArthur JC, Stocks EA, Hauer P, Cornblath DR, Griffin JW. Epi- sponse of growth cones to guidance molecules (53). Fur- dermal nerve fiber density: Normative reference range and diag- ther studies will be necessary to determine the contribu- nostic efficiency. Arch Neurol 1998;55:1513–20 12. Holland NR, Stocks A, Hauer P, Cornblath DR, Griffin JW, Mc- tion of individual factors and their interactions to Arthur JC. Intraepidermal nerve fiber density in patients with pain- successful reinnervation of the skin after nerve injury. ful sensory neuropathy. Neurol 1997;48:708–11 13. Holland NR, Crawford TO, Hauer P, Cornblath DR, GriffinJW, Characteristics of Degenerating and Regenerating McArthur JC. Small-fiber sensory neuropathies: Clinical course and Nerves neuropathology of idiopathic cases. Ann Neurol 1998;44:47–59 14. Scott LJ, Griffin JW, Luciano C, et al. Quantitative analysis of Recent applications of immunocytochemical staining epidermal innervation in Fabry disease. Neurol 1999;52:1249–54 of skin biopsies open a new field for evaluating sensory 15. Johnson PC, Beggs JL, Olafsen AG, Watkins CJ. Unmyelinated neuropathies. Much of the work has been focused on the nerve fiber estimation by immunocytochemistry. Correlation with quantitative features of intraepidermal nerve fibers (10, electron microscopy. J Neuropathol Exp Neurol 1994;53:176–83 16. Griffin JW, George EB, Hsieh ST, Glass JD. Axonal degeneration 11). A further and critical issue in applying such a tech- and disorders of the axonal cytoskeleton. In: Waxman SG, Kocsis nique is the morphological recognition of degenerating JD, Stys PK, eds. The axon. New York: Oxford University Press, and regenerating nerves. Normal epidermal nerves have 1995:375–90 typical varicosities. Epidermal nerve terminals become 17. 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