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Influence of cutaneous nerves on keratinocyte proliferation and epidermal thickness in mice
Neuroscience Vol. 94, No. 3, pp. 965–973, 1999 965 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
Cutaneous nerve degeneration and keratinocyte proliferation
Pergamon PII: S0306-4522(99)00210-9
INFLUENCE OF CUTANEOUS NERVES ON KERATINOCYTE PROLIFERATION AND EPIDERMAL THICKNESS IN MICE I.-T. HUANG,*† W.-M. LIN,*† C.-T. SHUN‡ and S.-T. HSIEH*§k Departments of *Anatomy, ‡Pathology and §Neurology, National Taiwan University College of Medicine, Taipei 10018, Taiwan
Abstract—We evaluated the influence of skin innervation on the epidermis in mice. The rich innervation of skin was demonstrated by immunocytochemistry with protein gene product 9.5, a ubiquitin carboxy hydrolase. Protein gene product-immunoreactive nerve fibers were in the epidermis, subepidermal plexus, dermal nerve trunks, and nerve terminals around sweat glands. Effects of denervation on the plantar surface of the hind foot was assessed by comparing the thickness of the epidermis, which was innervated by the sciatic nerve. Within 48 h after sectioning of the sciatic nerve, protein gene product (1)-nerves in the territory of the sciatic nerve were completely degenerated. There was a significant thinning of the denervated epidermis 72 h post-transection (30.5^1.1 vs 41.4^2.9 mm, 74^4% of the control side). The reduction in epidermal thickness persisted when skin remained denervated (69– 75% of the control side). Incorporation of bromodeoxyuridine was reduced 24 h after denervation (71^6% of the control side). Reduction in bromodeoxyuridine-incorporation was most pronounced within 48 h after denervation (19^6% of the control side). Therefore, the reduction in bromodeoxyuridine-labeling followed a similar temporal course as the thinning of the epidermis (25– 50%). Both epidermal thinning and reduced bromodeoxyuridine-labeling were reversed by epidermal reinnervation three months after denervation. Patterns of keratinocyte differentiation and programmed cell death were unaffected by skin denervation. These findings are consistent with the notion that skin innervation exerts influence on the proliferation of keratinocytes and the thickness of the epidermis, and offers a new look at the interaction between nociceptive nerves and their innervated targets. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: skin innervation, proliferation, keratinocytes, protein gene product 9.5, unmyelinated axons, nerve degeneration.
Sensory innervation of the skin has been known for its afferent capacity of transducing various stimuli applied on the cutaneous structure. The rich innervation of skin is recently appreciated by sensitive immunocytochemistry with various neuronal marker proteins, in particular, protein gene 9.5 (PGP). The existence of PGP(1)-nerves in the epidermis has been confirmed at both light and electron microscopic levels, and in various species including human and rodents. 9,26,29,34,41 PGP is a ubiquitin carboxy hydrolase enriched in the nervous system, encodes an immediate-early gene, and therefore is an excellent candidate for sensory transduction. 25 Nerve fibers terminating in the epidermis are presumably “free nerve endings” and subserve thermal and noxious sensations. Less well appreciated are the efferent potentials of epidermal nerves on various cellular components in the skin. 2,24,27 For example, cutaneous nerves have close associations with mast cells in the dermis and Langerhans cells in the epidermis. 6,12,16,28 The extent of neurogenic inflammatory responses is reduced in patients with defective cutaneous innervation. 4 Taken together, these findings suggest that skin innervation has a broader spectrum of functions than was previously thought. Several lines of evidence suggest that there is a substantial reduction in epidermal thickness after depletion of epidermal nerves. 9,13,29,47 Within one week, the denervated epidermis becomes thinner than the control epidermis by 30–40%. 29
These observations provide supportive evidence for the notion that cutaneous innervation has a profound effect on the epidermis in addition to its functions as transducers of stimuli. Compared with epidermal thinning, degeneration of nerves is usually completed by 48 h after nerve transection. 20,21 Thus understanding temporal changes of epidermal thickness related to that of nerve degeneration should provide new insights into the influence of skin innervation on the epidermis, particularly the keratinocytes. The thickness of epidermis is maintained by a delicate balance between proliferation, differentiation, and cell death of keratinocytes. 14,15 Positive signals for proliferation include calcium ion, and transforming growth factors. 18,51 The balance can be influenced by several mechanisms: a decrease in positive signals, or an increase in the cell loss. Only limited data have explored the influence of skin innervation on the lifespan of keratinocytes. How skin innervation influences keratinocytes and therefore epidermal thickness is equally important in understanding the interactions between the skin and nervous system. This report aimed to examine two issues: temporal changes of epidermal thickness after skin denervation, and the influence of skin innervation on keratinocytes. EXPERIMENTAL PROCEDURES
Animal experiments kTo whom correspondence should be addressed. Fax: 1886-2-2391-5292. E-mail address: [email protected] (S.-T. Hsieh) †Both authors contributed equally to this work. Abbreviations: BrdU, bromodeoxyuridine; CGRP, calcitonin gene-related peptide; DAB, 3,3 0 -diaminobenzidine; Dig, digoxygenin; EDTA, ethylenediaminetetra-acetate; p75-NGFR, p75-nerve growth factor receptor; PGP, protein gene product 9.5; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling.
Groups of male eight-week-old ICR mice obtained from the National Taiwan University Medical Center, Taipei, Taiwan (35– 40 g) were used in the present study. We transected the sciatic nerve to denervate the skin. This is a well-established method with all nerves distal to the site of transection are degenerated in a stereotyped fashion. Details of the procedure were described previously. 9,29 In brief, mice were anesthetized with chloral hydrate (40 mg/kg) for surgery. The right sciatic nerve was transected at the thigh level. A gap (3–5 mm) 965
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in the sciatic nerve was created to prevent the re-connection of the proximal and distal cut ends. For each animal, the left sciatic nerve was sham-operated as control. Five to seven animals were killed at defined intervals after nerve transection: one, two, three, four, five, seven, 10, 14 and 28 days. In mice, there are three pairs of footpads on the plantar side of the hindpaw. 9 Each pair of the pads with the interpad skin was processed together. To determine whether reinnervation of the skin will reverse epidermal thinning, we crushed the right sciatic nerve on another group of mice. In this procedure, the connection between the proximal and distal stumps remained intact. These mice were allowed to survive for three months, when skin reinnervation was completed. 9 Experimental procedures followed the NIH guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985).
footpads along the interfollicular epidermis. 9 Data were analysed according to the status of denervation and the time intervals after sciatic nerve transection.
Epidermal sheet preparation Epidermal sheets were prepared following the established protocol. 29 In brief, a 2-mm punch of fresh skin was incubated in EDTA for 2 h at 378C. Epidermis was removed by a forceps under a dissecting microscope for further immunocytochemistry (see below). The quality of these preparations was assessed in preliminary studies in which the sheets were fixed, sectioned perpendicularly, and stained with hematoxylin.
Immunocytochemistry For immunocytochemistry on freezing microtome sections, 36 animals were fixed by intra-cardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. The skin regions innervated by the sciatic nerve, including glabrous skin (the footpads), and pilary skin (areas lateral to the pad regions) were postfixed for 6 h, and then changed to the buffer for storage. After thorough rinsing in buffer, fixed skin samples were cryoprotected with 20% glycerol in 0.1 M phosphate buffer overnight. Vertical sections (30 mm) were cut on a sliding microtome. Sections from each tissue were labeled sequentially and stored with anti-freeze at 2208C. To ensure adequate sampling, every fourth section for each tissue was chosen for PGP immunostaining. Sections were treated with 0.5% Triton X-100 in 0.5 M Tris buffer (Tris) for 30 min and processed for immunostaining. In brief, sections were quenched with 1% H2O2 in methanol, and blocked with 5% normal serum of appropriate species in 0.5% non-fat dry milk in Tris. The sections were incubated with primary antibody in 1% normal serum in Tris for 16–24 h. After rinsing in Tris, sections were incubated with biotinylated secondary antibody made in appropriate species for 1 h, and the avidin–biotin complex (Vector Laboratories, Burlingame, CA) for another hour. The reaction product was demonstrated by 3,3 0 -diaminobenzidine (DAB, Sigma, St Louis, MO, U.S.A.). Primary antibodies to label cutaneous nerves included PGP (1:1000, UltraClone, U.K.), calcitonin gene-related peptide (CGRP, 1:1000, a kind gift of Dr Ian Dickenson, Miami, FL, U.S.A.), p75 nerve growth factor receptor (p75-NGFR, 1:10, Boehringer– Mannheim, Germany). To compare patterns of keratinocyte differentiation, we performed immunocytochemistry on paraffin-embedded sections from the tissues used for the morphometric study (see below). After deparaffinization, and hydration of sections, immunostaining followed a procedure similar to that of immunocytochemistry for freezing microtome sections. Primary antibodies included cytokeratin K10/11 (K8.60, 1:100, Sigma, St Louis, MO, U.S.A.), cytokeratin K13/15/16 (K8.12, 1:50, Sigma, St Louis, MO, U.S.A.), and cytokeratin K14 (No. 199, 1:50, a generous gift of Dr P Coulombe, Johns Hopkins University, Baltimore, MD, U.S.A.). In these experiments, the specificity of each antibody was tested by omitting the primary antibody or substituting the primary antibody with preimmune serum. Morphometry for epidermal thickness For comparison of the epidermal thicknesses, five animals were perfused after nerve transection. The tissues containing the second pair of footpads were postfixed in 4% paraformaldehyde overnight, and then embedded in paraffin. 9,29 Control and denervated tissues were processed at the same time. Care was taken to ensure all the tissue blocks were embedded in the correct orientation, and sectioned perpendicularly. For each tissue, sequential 5-mm-sections were cut and every fifth section was stained with hematoxylin. Sections were photographed at the magnification of ×20 with a Zeiss Axiophot microscope (Carl Zeiss, Germany). Three sections per tissue were used for assessing the thickness of the nucleated layers of epidermis, which was defined as the distance between the epidermal–dermal junction and the top of the outermost granular layer. Epidermal thickness was measured and marked with Image Pro-Plus (Media Cybernetics, Silver Spring, MD, U.S.A.). All the procedures of quantitation, including photography, printing, and measurement were done in a coded fashion. Observers were blinded to the coding information. On each print, five to 10 measurements were made between the two
Incorporation of bromodeoxyuridine and immunocytochemistry To study keratinocyte proliferation, we performed bromodeoxyuridine (BrdU)-labeling, a well-established method of assessing cells in DNA-synthesis phase. 19 Groups of mice were intraperitoneally injected with BrdU (Sigma, St Louis, MO, U.S.A.) in normal saline (100 mg/g), and killed 1 h later by intra-cardiac perfusion with 4% paraformaldehyde. Footpads from both the operated and control sides were postfixed for another 2 h, and sectioned on a sliding microtome. Another group of mice treated with BrdU were killed by overdose of chloral hydrate, and processed for epidermal sheet (2 mm in diameter) preparation. Sliding microtome sections and epidermal sheets were subject to immunocytochemistry with anti-BrdU antibody (1:100, DAKO, Denmark), with the following modifications: pretreatment with trypsin for 15 min and denaturation of the DNA with hydrochloric acid. Procedures of quantitation were performed in a coded fashion, and observers were blind to the information. BrdU-labeled cells on epidermal sheets were then photographed with a ×40 objective lens under a Zeiss Axiophot microscope. We calculated densities of BrdU-labeled cells in the interfollicular epidermis. For each interfollicular area, we assigned consecutive numbers (beginning from 1) as described before. 30 We measured the number of BrdU-labeled cells in a tile of 2500 mm 2 (a square of 50 mm on each side). The size of a tile was 20–30% of the size of each interfollicular area. BrdU(1) cells in the center of each interfollicular area were counted to assure that only labeled cells in the interfollicular area were counted. Sampling of the interfollicular areas was modified from methods described previously. 9 Interfollicular areas were chosen based on random numbers from a statistical table. As the area of an epidermal sheet (2 mm in diameter) is 3.1 mm 2, the sampling would cover more than 50% of the total area. All BrdU(1) cells in the selected tiles were counted, and the extent of BrdU-labeling was expressed as [BrdU(1) cells/mm 2]
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labelling Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL) was modified from the method of Gavrieli et al. 17 Free-floating sections and epidermal sheets were incubated with proteinase K (10 mg/ml, Boehringer–Mannheim, Germany) for 30 min at room temperature. Endogenous peroxidase was quenched with 1% H2O2 for 30 min. End-labeling was then performed with a digoxigenin (Dig)-labeled TUNEL kit (Boehringer–Mannheim, Germany) according to the manufacturer’s instructions. After incubation at 378C for 15 min, sections were rinsed in 2×standard saline citrate for 15 min, blocked with 5% normal serum in Tris buffer. Sections were incubated with anti-Dig–peroxidase antibody (1:100, Boehringer–Mannheim, Germany) at 48C for 2 h, and then demonstrated with DAB as in immunocytochemistry.
Analysis For analysis of epidermal innervation, epidermal thickness, BrdUincorporation, and TUNEL, five to 10 animals were used at each time point after nerve transection. Data are presented as means^S.E.M. and analysed with t-test, and nonparametric Wilcoxon rank sum test to compare the denervated and control groups. Any difference with P,0.05 was considered statistically significant.
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footpads of the hind limb by transecting the sciatic nerve, which innervates the plantar surface of the hind foot. Within 48 h after nerve injury, all epidermal nerves had degenerated (Fig. 1B). In the dermis, the immunocytochemical staining for PGP became faint and discontinuous, reflecting the degeneration of nerve trunks. This picture persisted when the skin remained denervated. To study reinnervation of the skin, we crushed the sciatic nerve of another group of mice. These mice were allowed to survive for three months, when reinnervation was complete based on previous functional studies. 10,29 The overall pattern of skin innervation by PGP immunocytochemistry was similar to that of the control skin with the varicose nerve terminals appearing in the epidermis (Fig. 1C). Epidermal thinning after skin denervation
Fig. 1. Skin innervation. Footpads of control and denervated mice were immunostained with PGP9.5. (A) In the control footpad, PGP-immunoreactive epidermal nerves with varicose appearance reach granular layers of the epidermis. These nerves arise and are perpendicular to the subepidermal plexus. Occasionally, there are nerve terminals of Meissner’s corpuscles in dermal papillae (arrowheads). (B) Denervated footpad (seven days after sciatic nerve transection). Characteristic PGP(1)-epidermal nerves, and subepidermal plexus are lost after denervation. (C) Reinnervated footpad three months after crushing injury of the sciatic nerve. Nerves immunoreactive for PGP with varicose appearance reappear in the epidermis. Scale bar50 mm.
RESULTS
Cutaneous innervation: normal innervation, denervation and reinnervation We demonstrated the rich innervation of skin by PGP immunocytochemistry. In normal mice, nerve fascicles containing PGP-immunoreactive profiles were present in the epidermis and dermis (Fig. 1A). When dermal nerves approached the epidermis, they usually paralleled the epidermal–dermal junction, forming the subepidermal plexus. Single epidermal fibers arising from dermal nerve trunks ascended the epidermis vertically by passing keratinocytes in the basal and suprabasal layers. To eliminate cutaneous innervation, we denervated
Epidermis after denervation had similar organization as the control epidermis, with cuboid cells in the basal layer, and flattened cells in the granular layer (Fig. 2A). A striking difference was the reduction in the epidermal thickness after sciatic nerve transection. One day after nerve transection, there was no appreciable difference in epidermal thickness between the denervated and control sides (45.0^1.9 vs 48.6^1.9 mm, P.0.5) (Fig. 2B). We further compared the difference in epidermal thickness at different time-points after denervation and constructed a plot to demonstrate the temporal changes (Fig. 3A). Within three days, the reduction in epidermal thickness became obvious on the denervated side (30.5^1.1 vs 41.4^2.9 mm, P,0.001) (Fig. 2C–D). Epidermal thinning persisted as long as the skin remained denervated (34.6^1.9 vs 47.5^2.5 mm, P,0.001 at 28 days after denervation). When skin became reinnervated within three months of crushing injury, the thickness of epidermis returned to a comparable level of the control side (43.5^1.7 vs 45.0^2.5 mm, P.0.5). Because epidermal thickness was variable among different animals, we further analysed temporal change by normalizing the denervated epidermal thickness to the control epidermal thickness. The plot of epidermal thickness ratios showed a similar trend (Fig. 3B). Reduced bromodeoxyuridine-incorporation denervation
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To understand how skin innervation influences epidermal thickness, we evaluated keratinocyte proliferation with BrdUlabeling. BrdU-labeled cells were in the basal layer of epidermis (Fig. 4A–D). The extent of BrdU-incorporation was reduced in denervated skin (Fig. 4B, D). To compare the difference in the incorporation of BrdU, we quantified BrdU(1) cells in epidermal sheets (Fig. 4E–H). Within 24 h, there was a substantial decrease in BrdU-labeled cells in the denervated skin (3,952^410 vs 5,576^280 cells/mm 2, P,0.001, Fig. 5A). We also compared the extent of BrdUlabeling by normalizing the density of BrdU(1) cells on the denervated epidermis to that on the control side. The ratio was reduced to 71^6% of the control side within 24 h of denervation (P,0.001, Fig. 5B). The decrease in BrdU-labeling was the most obvious within two days after denervation (932^228 vs 4875^185 cells/mm 2, P,0.0001). During the study period of denervation (three to 28 days), the degree of BrdU-labeling on the denervated side was 25–50% of that
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Fig. 2. Changes in epidermal thickness after denervation. Control and denervated skin was embedded in paraffin. Vertical sections of 5 mm were stained with hematoxylin. The thickness of the nucleated epidermis was quantified in a blinded fashion. (A, C, E, F) are from the control sides, and (B, D, E, F) are from the denervated sides. (A, B) One day after transection; (C, D) three days after transection; (E, F) seven days after transection; (G, H) three months after crushing. Both the control and denervated epidermis show similar organization with strong basophilic keratinocytes in the basal layer, flat keratinocytes in granular layers, and non-nucleated cells in cornified layers. Significant thinning of epidermal thickness is obvious in pairs C–D and E–F. Scale bar: (A–F)50 mm; (G–H)42 mm.
on the control side (Fig. 5B). When the epidermis was reinnervated three months after crushing the sciatic nerve, the reduction in BrdU-incorporation was reversed (4780^400 vs 5152^450 cells/mm 2, P.0.5, Fig. 4G–H).
Keratinocyte differentiation after denervation Keratinocytes are a dynamic population with continuous proliferation and differentiation. An alternative mechanism
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Fig. 3. Temporal changes of epidermal thickness after denervation. Quantitation of epidermal thickness based on paraffin-embedded hematoxylin-stained sections is detailed in Experimental Procedures. Data from five to 10 animals at each time-point are expressed as means^S.E.M., with open circles for the control skin, and closed circles for the denervated skin. (A) Temporal changes of epidermal thickness. Within three days after denervation, there is significant thinning of epidermis (30.5^1.1 vs 41.4^2.9 mm, P,0.01). Epidermal thinning persisted as skin remained denervated during the experimental period (up to 28 days, 34.6^1.9 vs 47.8^2.4 mm, P,0.01). Epidermal thinning was reversed by skin reinnervation three months after crushing of the sciatic nerve (43.5^1.7 vs 45.0^2.5 mm, P.0.5). (B) Trend of epidermal thinning by ratios of denervated epidermal thickness normalized to control epidermal thickness. After denervation, the epidermal thickness on the denervated side became 69–75% of that on the control side. The ratio of epidermal thickness is not different from 1 (the dotted line) after reinnervation (*P,0.01).
for epidermal thinning after skin denervation is an increase of keratinocyte turnover. Cell death of basal keratinocytes could also contribute to the reduction of BrdU-incorporation in the basal layer. To test these hypotheses, we carried out immunocytochemistry for keratin intermediate filaments and TUNEL for programmed cell death on epidermal sheets and vertical sections of the skin. For example, K10 was expressed in suprabasal layers of epidermis (Fig. 6A–B). In vertical sections and epidermal sheets of the control and denervated skin, TUNEL(1) cells were negligible (Fig. 6C–D and G– H). The patterns of TUNEL after adding DNase as positive controls were the same between the control and denervated epidermis (Fig. 6E–F and I–J). Thus, the patterns of keratin expression and TUNEL were similar between the control and denervated sides.
DISCUSSION
Influence of denervation on keratinocytes This report extends the previous observation that depletion of epidermal nerves causes epidermal thinning, and further explores the mechanism of epidermal thinning. Degeneration of epidermal nerves is complete within 48 h. 20,21 Nerve injury is followed by epidermal thinning within 72 h. Any change in the process of keratinocyte differentiation during this interval should provide new insights into the influence of skin innervation. Keratinocytes are a dynamic population in the epidermis. They proliferate in the basal layer and move upwards to the granular layer. During the process, the expression of keratin changes from K5/K14 in the basal layer to K1/K10 in suprabasal layers. 22 After reaching the top of the granular layer, keratinocytes undergo a specific from of programmed cell death, and finally become cornified. 23 To understand potential mechanisms of epidermal thinning after skin denervation, we investigated the time-course of epidermal thinning, the incorporation of BrdU, the expression of keratin proteins, and the programmed cell death of
differentiated keratinocytes. During this period, there is a robust reduction in BrdU-labeling, and it is the only step markedly influenced by skin denervation. The reduction in proliferation index with normal differentiation and unaltered cell loss suggests that sensory innervation influences keratinocyte proliferation and ultimately the thickness of the epidermis. Traditionally, sensory nerves are considered to transduce harmful stimuli. Various types of neuropeptides coexist with a defined type of neurotransmitter in sensory neurons and are potential sources for functions other than sensory transduction. For example, a subset of sensory nerves contain CGRP, which causes vasodilation upon release from nerve terminals. 32 CGRP has a wide range of actions, including modulation of keratinocyte proliferation via an increase in cAMP. 43,49 Various cellular components in the epidermis are influenced by skin innervation in developing and mature animals. The thickness of the epidermis and the height of rete peges are reduced if sensory neurons are deprived during embryonic development. 40 The number of Merkel cells, the mechanoreceptors in the epidermis, decreases after cutaneous nerve degeneration. 1,37,42 Other potential molecules influencing keratinocyte proliferation include neurotrophins, in particular, the nerve growth factor. Nerve growth factor can have an autocrine effect on keratinocytes. 7,11,44,45 A recent study suggests that denervation down-regulates the synthesis of nerve growth factor in the target organs of autonomic nerves. 35 The significance of epidermal innervation on keratinocyte proliferation remains elusive. However, the conclusion may provide therapeutic alternatives. Wound healing is a complicated process after injury, and neuropeptides or neurotrophins promoting reinnervation of the injured skin may speed wound healing. In contrast, antagonists of neuropeptides could be a new strategy for hyperproliferation disorders of keratinocytes, such as psoriasis. These hypotheses will require further investigations on available animal models. 8,46 Collectively, these observations provide a new look at the interactions between the skin and its innervation.
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Fig. 4. Incorporation of BrdU. BrdU-incorporation was demonstrated immunocytochemically after i.p. injection of BrdU. (A, C, E, G) are from the control epidermis. (B, D, F) are from the seven-day denervated epidermis. H is the the reinnervated epidermis three months after denervation. (A, C) On vertical sections of the control epidermis, BrdU-labeled keratinocytes are in the basal layer of the epidermis. (B, D) The pattern of BrdU labeling is similar after denervation. The number of BrdU-labeled cells in the denervated epidermis is substantially smaller than that of the control epidermis. (C, E) For quantitation of BrdU labeling, we immunocytochemically stained epidermal sheets with the anti-BrdU antibody. There is a significant reduction in BrdU-labeling in denervated epidermal sheets. (G, H) The extent of BrdU-labeling is comparable between the control and denervated sides.
Skin innervation and epidermal thinning The influence of cutaneous nerves on the architecture of the skin has become an intriguing issue. 31 Skin denervation by
transection of the sciatic nerve causes epidermal thinning. 13,47,48 In this approach, autonomic and motor nerves are also damaged. Loss of autonomic nerves may alter blood flow with secondary effects on the epidermis. For
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Fig. 5. Temporal changes in incorporation of BrdU related to epidermal denervation. Comparison of BrdU labeling was based on quantitation of BrdU-labeled cells on epidermal sheets as described in Fig. 4. Data from five animals at each time-point are expressed as means^S.E.M., with open circles for control skin and closed circles for denervated epidermis. (A) Density of BrdU(1) cells. In control epidermal sheets, there are variations in BrdU-labeled cells. Within 24 h after denervation, there is a significant reduction in BrdU-labeling on denervated epidermis (3952^410 vs 5576^280 cells/mm 2, P,0.001). The most obvious reduction in BrdUlabeling occurred within two days of denervation (932^228 vs 4875^185 cells/mm 2, P,0.0001). During the period of denervation, the extent of BrdU-labeling persisted, with 2116^388 vs 4556^350 cells/mm 2 (P,0.001) at 28 days after denervation. The extent of BrdU-incorporation on reinnervated epidermis returned to a comparable level as the that of control epidermis three months after sciatic nerve crushing (4780^400 vs 5152^450 cells/mm 2, P.0.5). (B) Relative labeling of BrdU (normalized ratios of denervated/control epidermal sheets). The ratio of BrdUincorporation is 0.71^0.06 at 24 h after denervation, and reaches the nadir within 48 h (0.19^0.06, P,0.0001). During the period of denervation (three to 28 days after denervation), the ratios remain in the range of 0.25–0.50. Three months after reinnervation, the ratio of BrdU-labeling is not different from 1 (the dotted line), 0.93^0.08, P.0.5. (*P,0.01).
example, shining and thinning of the skin in patients with peripheral nerve disorders was frequently attributed to impaired autonomic regulation and defective vascular supply. 50 However, sympathectomy specifically eliminates autonomic innervation of the skin, but does not cause epidermal thinning. 33 The finding is corroborated with the fact that there is no direct correlation between cutaneous blood flow and epidermal thickness. 38,39 A further issue is that paralysis of the hind limbs alters the pattern of walking and weightbearing. Thus, the contribution of motor denervation to epidermal thinning can not be excluded. 33 Nevertheless, two
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Fig. 6. Changes in keratin immunocytochemistry and TUNEL. Figures on the left panel (A, C, E, G, I) are from the control skin and the right panel (B, D, F, H, J) are from the skin denervated seven days earlier. (A, B) Cytokeratin 10-immunocytochemical patterns in control (A) and denervated (B) epidermis. Cytokeratin 10(1) keratinocytes are in the suprabasal layers. The expression patterns are similar except that the epidermis is thinner on the denervated side. (C–F) TUNEL on epidermal sheets. There is no BrdUlabeled cell in control (C) and denervated (D) epidermal sheets, suggesting no increase in apotopsis in the basal layer of the epidermis. By adding DNase as positive control, all the cells in epidermal sheets become positive for TUNEL. (G–J) TUNEL on vertical sections of the epidermis. TUNEL(1) cells are difficult to find in both control (G) and denervated (H) skin. The positive control for TUNEL was performed after adding DNase. Keratinocytes in all layers of the epidermis become TUNEL(1) in control (I) and denervated (J) skin. Scale bar: (A, B)33 mm; (C– F)25 mm; (G–J)150 mm.
lines of evidence indicate that depletion of epidermal nerves is tightly linked to epidermal thinning. In the null mutant mice for p75-NGFR, there is a robust reduction of epidermal nerves, and a remarkable decrease in epidermal thickness. 5 Finally, ganglionectomy by removing the sensory neurons innervating the find feet, eradicates epidermal nerves, and causes epidermal thinning. 29,33 Taken together, these results strongly suggest that sensory innervation is sufficient to modulate epidermal thickness.
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Potential cellular mechanism of skin innervation The existence of nerve terminals in the epidermis offers support for their roles in transducing noxious stimuli. Epidermal nerves are unmyelinated with the diameter usually in the range of 1–2 mm, and they can only be resolved under electron microscopy by conventional morphological techniques. 26,29 Recent studies by applying immunocytochemistry with various neuronal markers, such as CGRP and substance P, have explored the diversity of epidermal nerves. These peptidergic nerves are presumably nociceptive terminals and may also possess mitogenic potential. Compared with other neuronal proteins, PGP labels the most abundant epidermal nerves. Other researchers as well as ourselves provided ultrastructural evidence of PGP(1)-epidermal nerves by electron microscopic immunocytochemistry. 26,29 In the epidermis, these nerves are in contact with keratinocytes and epidermal Langerhans cells. 16,28 CGRP(1)-epidermal nerves have close contacts with Langerhans cells, and CGRP can alter the ability of antigen presentation of Langerhans cells. 3,28 These
observations suggest that interactions between nerves and various cellular components in the skin are far more complicated than previously thought. Proportions of epidermal nerves labeled with CGRP and substance P are substantially smaller than those of PGP(1)nerves. It remains to be determined exactly how epidermal nerves influence keratinocytes: through synapse-like contact or diffusible factors? Alternatively, epidermal nerves may influence keratinocyte proliferation through diffusible factors. A recent study indicates that there is differential thinning of the epidermis after skin denervation, with the most central part of the denervated skin exhibiting the most profound degree of epidermal thinning. 9 Taken together, these results provide additional support to the notion that skin innervation may play an important role in regulation of keratinocytes. Acknowledgements—The work was supported by the National Health Research Institute, Department of Health, Republic of China (DOH88HR-727).
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Airaksinen M. S., Koltzenburg M., Lewin G. R., Masu Y., Helbig C., Wolf E., Brem G., Toyka K. V., Thoenen H. and Meyer M. (1996) Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation. Neuron 16, 287–295. Ansel J. C., Armstrong C. A., Song I., Quinlan K. L., Olerud J. E., Caughman S. W. and Bunnett N. W. (1997) Interactions of the skin and nervous system. J. invest. Dermatol. Symp. Proc. 2, 23–26. Asahina A., Moro O., Hosoi J., Lerner E. A., Xu S., Takashima A. and Granstein R. D. (1995) Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects. Proc. natn. Acad. Sci. U.S.A. 92, 8323–8327. Baluk P. (1997) Neurogenic inflammation in skin and airways. J. invest. Dermatol. Symp. Proc. 2, 76–81. Bergmann I., Priestley J. V., McMahon S. B., Brockes E.-B., Toyka K. V. and Koltzenburg M. (1997) Analysis of cutaneous sensory neurons in transgenic mice lacking the low affinity neurotrophin receptor p75. Eur. J. Neurosci. 9, 18–28. Bienenstock J., MacQueen G., Sestini P., Marshall J. S., Stead R. H. and Perdue M. H. (1991) Mast cell/nerve interactions in vitro and in vivo. Am. Rev. Respir. Dis. 143, S55–S58. Bothwell M. (1997) Neurotrophin function in skin. J. invest. Dermatol. Symp. Proc. 2, 27–30. Carroll J. M., Romero M. R. and Watt F. M. (1995) Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis. Cell 83, 957–968. Chiang H.-Y., Huang I.-T., Chen W. P., Chien H.-F., Shun C. T., Chang Y. C. and Hsieh S. T. (1998) Regional difference in epidermal thinning after skin denervation. Expl Neurol. 154, 137–145. Devor M., Schonfeld D., Seltzer Z. and Wall P. D. (1979) Two modes of cutaneous reinnervation following peripheral nerve injury. J. comp. Neurol. 185, 211–220. Di Marco E., Marchisio P. C., Bondanza S., Franzi A. T., Cancedda R. and De Luca M. (1991) Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes. J. biol. Chem. 266, 21,718–21,722. Egan C. L., Viglione-Schneck M. J., Walsh L. J., Green B., Trojanowski J. Q., Whitaker-Menezes D. and Murphy G. F. (1998) Characterization of unmyelinated axons uniting epidermal and dermal immune cells in primate and murine skin. J. cutan. Pathol. 25, 20–29. English K. B. (1977) The ultrastructure of cutaneous type I mechanoreceptors (Haarscheiben) in cats following denervation. J. comp. Neurol. 172, 137–164. Fuchs E. (1990) Epidermal differentiation: the bare essentials. J. Cell Biol. 111, 2807–2814. Fuchs E. (1993) Epidermal differentitation and keratin gene expression. J. Cell. Sci. 17, (Suppl.) 197–208. Gaudillere A., Misery L., Souchier C., Claudy A. and Schmitt D. (1996) Intimate associations between PGP9.5-positive nerve fibers and Langerhans cells. Br. J. Dermatol. 135, 343–344. Gavrieli Y., Sherman Y. and Ben-Sasson S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501. Glick A. B., Kulkarni A. B., Tennenbaum T., Hennings H., Flanders K. C., O’Reilly M., Sporn M. B., Karlsson S. and Yuspa S. H. (1993) Loss of expression of transforming growth factor b in skin and skin tumors is associated with hyperproliferation and a high risk for maligant conversion. Proc. natn. Acad. Sci. U.S.A. 90, 6076–6080. Gratzner H. G. (1982) Monoclonal antibody to 5-bromo- and 5-indodeoxyuridine: a new reagent for detection of DNA replication. Science 218, 474–475. Griffin J. W., George E. B., Hsieh S. T. and Glass J. D. (1995) Axonal degeneration and disorders of the axonal cytoskeleton. In The Axon (eds Waxman S. G., Kocsis J. D. and Stys P. K.), pp. 375–390, Oxford University Press, New York. Griffin J. W. and Hoffman P. N. (1993) Degeneration and regeneration in the peripheral nervous system. In Peripheral Neuropathy (eds Dyck P. J., Thomas P. K., Griffin J. W., Low P. A. and Poduslo J.), pp. 361–376, W. B. Saunders, Philadelphia. Griffin J. W., Hsieh S. T., McArthur J. C. and Cornblath D. R. (1996) Laboratory testing in peripheral nerve disease. Neurol. Clin. 14, 119–133. Haake A. R. and Polakowska R. R. (1993) Cell death by apoptosis in epidermal biology. J. invest. Dermatol. 101, 107–112. Hara M., Toyoda M., Yaar M., Bhawan J., Avila E. M., Penner I. R. and Gilchrest B. A. (1996) Innervation of melanocytes in human skin. J. exp. Med. 184, 1385–1395. Hedge A. N., Inokuchi K., Pei W., Casadio A., Ghirardi M., Chain D. G., Martin K. C., Kandel E. R. and Schwartz J. H. (1997) Ubiquitin C-terminal hydrolase is an immediate-early gene essential for long-term facilitation in Aplysia. Cell 89, 115–126. Hilliges M., Wang L. and Johansson O. (1995) Ultrastructural evidence for nerve fibers within all vital layers of the human epidermis. J. invest. Dermatol. 104, 134–137. Holzer P. and Maggi C. A. (1998) Dissociation of dorsal root ganglion neurons into afferent and efferent-like neurons. Neuroscience 86, 389–398. Hosoi J., Murphy G. F., Egan C. L., Lerner E. A., Grabbe S., Asahina A. and Granstein R. D. (1993) Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363, 159–163.
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29. Hsieh S. T., Choi S., Lin W.-M., Chang Y.-C., McArthur J. C. and Griffin J. W. (1996) Epidermal denervation and its effects on keratinocytes and Langerhans cells. J. Neurocytol. 25, 513–524. 30. Hsieh S. T., Kidd G. J., Crawford T. O., Xu Z., Lin W.-M., Trapp B. D., Cleveland D. W. and Griffin J. W. (1994) Regional modulation of neurofilament organization by myelination in normal axons. J. Neurosci. 14, 6392–6401. 31. Hsieh S. T., Lin W.-M., Chiang H.-Y., Huang I.-T. and Chen W. P. (1997) Skin innervation and its influence on the epidermis. J. biomed. Sci. 4, 264–268. 32. Kruger L., Silverman J. D., Mantyh P. W., Sternini C. and Brecha N. C. (1989) Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: cutaneous and deep terminations. J. comp. Neurol. 280, 291–302. 33. Li Y., Hsieh S. T., Chien H.-F., Zhang X., McArthur J. C. and Griffin J. W. (1997) Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin. Expl Neurol. 147, 452–462. 34. Lin W.-M., Hsieh S. T., Huang I.-T., Griffin J. W. and Chen W. P. (1997) Ultrastructural localization and regulation of protein gene product 9.5. NeuroReport 8, 2999–3004. 35. Liu D. T., Reid M. T., Bridges D. C. and Rush R. A. (1996) Denervation, but not decentralization, reduces nerve growth factor content of the mesenteric artery. J. Neurochem. 66, 2295–2299. 36. McCarthy B., Hsieh S. T., Stocks E. A., Hauer P., Macko C., Cornblath D. R., Griffin J. W. and McArthur J. C. (1995) Cutaneous innervation in sensory neuropathies: evaulation by skin biopsy. Neurology 45, 1848–1855. 37. Mills L. R., Nurse C. A. and Diamond J. (1989) The neural dependency of Merkel cell development in the rat: the touch domes and foot pads contrasted. Devl Biol. 136, 61–74. 38. Monterio-Riviere N. A., Banks Y. B. and Birnbaum L. (1991) Laser Doppler measurements of cutaneous blood flow in ageing mice and rats. Toxicol. Lett. 57, 329–338. 39. Monterio-Riviere N. A., Bristol D. G., Manning T. O., Rogers R. A. and Riviere J. E. (1990) Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species. J. invest. Dermatol. 95, 582–586. 40. Morohunfola K. A., Jones T. E. and Munger B. L. (1992) The differentiation of the skin and its appendages. II. Altered development of papillary ridges following neuralectomy. Anat. Rec. 232, 599–611. 41. Navarro X., Verdu E., Wendelscafer-Crabb G. and Kennedy W. R. (1995) Innervation of cutaneous structures in the mouse hind paw: a confocal microscopy immunohistochemical study. J. Neurosci. Res. 41, 111–120. 42. Nurse C. A., Macintyre L. and Diamond J. (1984) Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation. Neuroscience 13, 563–571. 43. Pincelli C., Fntini F., Romualdi P., Sevignani C., Lesa G., Benassi L. and Giannetti A. (1992) Substance P is diminished and vasoactive intestinal peptide is augmented in psoriatic lesions and these peptides exert disparate effects on the proliferation of cultured human keratinocytes. J. invest. Dermatol. 98, 421–427. 44. Pincelli C., Sevignani C., Manfredini R., Grande A., Fantini F., Bracci-Laudiero L., Aloe L., Ferrari S., Cossarizza A. and Giannetti A. (1994) Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes. J. invest. Dermatol. 103, 13–18. 45. Pincelli C. and Yaar M. (1997) Nerve growth factor: its significance in cutaneous biology. J. invest. Dermatol. Symp. Proc. 2, 31–36. 46. Sellheyer K., Bickenbach J. R., Rothnagel J. A., Bundman D., Longley M. A., Krieg T., Roche N. S., Roberts A. B. and Roop D. R. (1993) Inhibition of skin development by overexpression of transforming growth factor b1 in the epidermis of transgenic mice. Proc. natn. Acad. Sci. U.S.A. 90, 5237–5241. 47. Stankovic N., Johansson O. and Hildebrand C. (1996) Occurrence of epidermal nerve endings in glabrous and hairy skin of the rat foot after sciatic nerve regeneration. Cell Tiss. Res. 284, 161–166. 48. Svetikova K. M. and Chumasov E. I. (1987) Changes in the skin epithelium and nerve elements of the rat sole with directed regeneration of the sciatic nerve. Arkhiv Anatomii Gistologii i Embriologii 93, 82–89. 49. Takahashi K., Nakanishi S. and Imamura S. (1993) Direct effects of cutaneous neuropeptides on adenylyl cyclase activity and proliferation in a keratinocyte cell line: stimulation of cyclic AMP formation by CGRP and VIP/PHM, and inhibition by NPY through G protein-coupled receptors. J. invest. Dermatol. 101, 646–651. 50. Thomas P. K. and Ochoa J. (1993) Clinical features and differential diagnosis. In Peripheral Neuropathy (eds Dyck P. J., Thomas P. K., Griffin J. W., Low P. A. and Poduslo J. F.), pp. 749–774, W. B. Saunders, Philadelphia. 51. Vassar R. and Fuchs E. (1991) Transgenic mice provide new insights into the role of TGF-a during epidermal development and differentiation. Genes Dev. 5, 714–727. (Accepted 29 March 1999)
Cutaneous nerve degeneration and keratinocyte proliferation
Pergamon PII: S0306-4522(99)00210-9
INFLUENCE OF CUTANEOUS NERVES ON KERATINOCYTE PROLIFERATION AND EPIDERMAL THICKNESS IN MICE I.-T. HUANG,*† W.-M. LIN,*† C.-T. SHUN‡ and S.-T. HSIEH*§k Departments of *Anatomy, ‡Pathology and §Neurology, National Taiwan University College of Medicine, Taipei 10018, Taiwan
Abstract—We evaluated the influence of skin innervation on the epidermis in mice. The rich innervation of skin was demonstrated by immunocytochemistry with protein gene product 9.5, a ubiquitin carboxy hydrolase. Protein gene product-immunoreactive nerve fibers were in the epidermis, subepidermal plexus, dermal nerve trunks, and nerve terminals around sweat glands. Effects of denervation on the plantar surface of the hind foot was assessed by comparing the thickness of the epidermis, which was innervated by the sciatic nerve. Within 48 h after sectioning of the sciatic nerve, protein gene product (1)-nerves in the territory of the sciatic nerve were completely degenerated. There was a significant thinning of the denervated epidermis 72 h post-transection (30.5^1.1 vs 41.4^2.9 mm, 74^4% of the control side). The reduction in epidermal thickness persisted when skin remained denervated (69– 75% of the control side). Incorporation of bromodeoxyuridine was reduced 24 h after denervation (71^6% of the control side). Reduction in bromodeoxyuridine-incorporation was most pronounced within 48 h after denervation (19^6% of the control side). Therefore, the reduction in bromodeoxyuridine-labeling followed a similar temporal course as the thinning of the epidermis (25– 50%). Both epidermal thinning and reduced bromodeoxyuridine-labeling were reversed by epidermal reinnervation three months after denervation. Patterns of keratinocyte differentiation and programmed cell death were unaffected by skin denervation. These findings are consistent with the notion that skin innervation exerts influence on the proliferation of keratinocytes and the thickness of the epidermis, and offers a new look at the interaction between nociceptive nerves and their innervated targets. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: skin innervation, proliferation, keratinocytes, protein gene product 9.5, unmyelinated axons, nerve degeneration.
Sensory innervation of the skin has been known for its afferent capacity of transducing various stimuli applied on the cutaneous structure. The rich innervation of skin is recently appreciated by sensitive immunocytochemistry with various neuronal marker proteins, in particular, protein gene 9.5 (PGP). The existence of PGP(1)-nerves in the epidermis has been confirmed at both light and electron microscopic levels, and in various species including human and rodents. 9,26,29,34,41 PGP is a ubiquitin carboxy hydrolase enriched in the nervous system, encodes an immediate-early gene, and therefore is an excellent candidate for sensory transduction. 25 Nerve fibers terminating in the epidermis are presumably “free nerve endings” and subserve thermal and noxious sensations. Less well appreciated are the efferent potentials of epidermal nerves on various cellular components in the skin. 2,24,27 For example, cutaneous nerves have close associations with mast cells in the dermis and Langerhans cells in the epidermis. 6,12,16,28 The extent of neurogenic inflammatory responses is reduced in patients with defective cutaneous innervation. 4 Taken together, these findings suggest that skin innervation has a broader spectrum of functions than was previously thought. Several lines of evidence suggest that there is a substantial reduction in epidermal thickness after depletion of epidermal nerves. 9,13,29,47 Within one week, the denervated epidermis becomes thinner than the control epidermis by 30–40%. 29
These observations provide supportive evidence for the notion that cutaneous innervation has a profound effect on the epidermis in addition to its functions as transducers of stimuli. Compared with epidermal thinning, degeneration of nerves is usually completed by 48 h after nerve transection. 20,21 Thus understanding temporal changes of epidermal thickness related to that of nerve degeneration should provide new insights into the influence of skin innervation on the epidermis, particularly the keratinocytes. The thickness of epidermis is maintained by a delicate balance between proliferation, differentiation, and cell death of keratinocytes. 14,15 Positive signals for proliferation include calcium ion, and transforming growth factors. 18,51 The balance can be influenced by several mechanisms: a decrease in positive signals, or an increase in the cell loss. Only limited data have explored the influence of skin innervation on the lifespan of keratinocytes. How skin innervation influences keratinocytes and therefore epidermal thickness is equally important in understanding the interactions between the skin and nervous system. This report aimed to examine two issues: temporal changes of epidermal thickness after skin denervation, and the influence of skin innervation on keratinocytes. EXPERIMENTAL PROCEDURES
Animal experiments kTo whom correspondence should be addressed. Fax: 1886-2-2391-5292. E-mail address: [email protected] (S.-T. Hsieh) †Both authors contributed equally to this work. Abbreviations: BrdU, bromodeoxyuridine; CGRP, calcitonin gene-related peptide; DAB, 3,3 0 -diaminobenzidine; Dig, digoxygenin; EDTA, ethylenediaminetetra-acetate; p75-NGFR, p75-nerve growth factor receptor; PGP, protein gene product 9.5; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling.
Groups of male eight-week-old ICR mice obtained from the National Taiwan University Medical Center, Taipei, Taiwan (35– 40 g) were used in the present study. We transected the sciatic nerve to denervate the skin. This is a well-established method with all nerves distal to the site of transection are degenerated in a stereotyped fashion. Details of the procedure were described previously. 9,29 In brief, mice were anesthetized with chloral hydrate (40 mg/kg) for surgery. The right sciatic nerve was transected at the thigh level. A gap (3–5 mm) 965
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in the sciatic nerve was created to prevent the re-connection of the proximal and distal cut ends. For each animal, the left sciatic nerve was sham-operated as control. Five to seven animals were killed at defined intervals after nerve transection: one, two, three, four, five, seven, 10, 14 and 28 days. In mice, there are three pairs of footpads on the plantar side of the hindpaw. 9 Each pair of the pads with the interpad skin was processed together. To determine whether reinnervation of the skin will reverse epidermal thinning, we crushed the right sciatic nerve on another group of mice. In this procedure, the connection between the proximal and distal stumps remained intact. These mice were allowed to survive for three months, when skin reinnervation was completed. 9 Experimental procedures followed the NIH guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985).
footpads along the interfollicular epidermis. 9 Data were analysed according to the status of denervation and the time intervals after sciatic nerve transection.
Epidermal sheet preparation Epidermal sheets were prepared following the established protocol. 29 In brief, a 2-mm punch of fresh skin was incubated in EDTA for 2 h at 378C. Epidermis was removed by a forceps under a dissecting microscope for further immunocytochemistry (see below). The quality of these preparations was assessed in preliminary studies in which the sheets were fixed, sectioned perpendicularly, and stained with hematoxylin.
Immunocytochemistry For immunocytochemistry on freezing microtome sections, 36 animals were fixed by intra-cardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. The skin regions innervated by the sciatic nerve, including glabrous skin (the footpads), and pilary skin (areas lateral to the pad regions) were postfixed for 6 h, and then changed to the buffer for storage. After thorough rinsing in buffer, fixed skin samples were cryoprotected with 20% glycerol in 0.1 M phosphate buffer overnight. Vertical sections (30 mm) were cut on a sliding microtome. Sections from each tissue were labeled sequentially and stored with anti-freeze at 2208C. To ensure adequate sampling, every fourth section for each tissue was chosen for PGP immunostaining. Sections were treated with 0.5% Triton X-100 in 0.5 M Tris buffer (Tris) for 30 min and processed for immunostaining. In brief, sections were quenched with 1% H2O2 in methanol, and blocked with 5% normal serum of appropriate species in 0.5% non-fat dry milk in Tris. The sections were incubated with primary antibody in 1% normal serum in Tris for 16–24 h. After rinsing in Tris, sections were incubated with biotinylated secondary antibody made in appropriate species for 1 h, and the avidin–biotin complex (Vector Laboratories, Burlingame, CA) for another hour. The reaction product was demonstrated by 3,3 0 -diaminobenzidine (DAB, Sigma, St Louis, MO, U.S.A.). Primary antibodies to label cutaneous nerves included PGP (1:1000, UltraClone, U.K.), calcitonin gene-related peptide (CGRP, 1:1000, a kind gift of Dr Ian Dickenson, Miami, FL, U.S.A.), p75 nerve growth factor receptor (p75-NGFR, 1:10, Boehringer– Mannheim, Germany). To compare patterns of keratinocyte differentiation, we performed immunocytochemistry on paraffin-embedded sections from the tissues used for the morphometric study (see below). After deparaffinization, and hydration of sections, immunostaining followed a procedure similar to that of immunocytochemistry for freezing microtome sections. Primary antibodies included cytokeratin K10/11 (K8.60, 1:100, Sigma, St Louis, MO, U.S.A.), cytokeratin K13/15/16 (K8.12, 1:50, Sigma, St Louis, MO, U.S.A.), and cytokeratin K14 (No. 199, 1:50, a generous gift of Dr P Coulombe, Johns Hopkins University, Baltimore, MD, U.S.A.). In these experiments, the specificity of each antibody was tested by omitting the primary antibody or substituting the primary antibody with preimmune serum. Morphometry for epidermal thickness For comparison of the epidermal thicknesses, five animals were perfused after nerve transection. The tissues containing the second pair of footpads were postfixed in 4% paraformaldehyde overnight, and then embedded in paraffin. 9,29 Control and denervated tissues were processed at the same time. Care was taken to ensure all the tissue blocks were embedded in the correct orientation, and sectioned perpendicularly. For each tissue, sequential 5-mm-sections were cut and every fifth section was stained with hematoxylin. Sections were photographed at the magnification of ×20 with a Zeiss Axiophot microscope (Carl Zeiss, Germany). Three sections per tissue were used for assessing the thickness of the nucleated layers of epidermis, which was defined as the distance between the epidermal–dermal junction and the top of the outermost granular layer. Epidermal thickness was measured and marked with Image Pro-Plus (Media Cybernetics, Silver Spring, MD, U.S.A.). All the procedures of quantitation, including photography, printing, and measurement were done in a coded fashion. Observers were blinded to the coding information. On each print, five to 10 measurements were made between the two
Incorporation of bromodeoxyuridine and immunocytochemistry To study keratinocyte proliferation, we performed bromodeoxyuridine (BrdU)-labeling, a well-established method of assessing cells in DNA-synthesis phase. 19 Groups of mice were intraperitoneally injected with BrdU (Sigma, St Louis, MO, U.S.A.) in normal saline (100 mg/g), and killed 1 h later by intra-cardiac perfusion with 4% paraformaldehyde. Footpads from both the operated and control sides were postfixed for another 2 h, and sectioned on a sliding microtome. Another group of mice treated with BrdU were killed by overdose of chloral hydrate, and processed for epidermal sheet (2 mm in diameter) preparation. Sliding microtome sections and epidermal sheets were subject to immunocytochemistry with anti-BrdU antibody (1:100, DAKO, Denmark), with the following modifications: pretreatment with trypsin for 15 min and denaturation of the DNA with hydrochloric acid. Procedures of quantitation were performed in a coded fashion, and observers were blind to the information. BrdU-labeled cells on epidermal sheets were then photographed with a ×40 objective lens under a Zeiss Axiophot microscope. We calculated densities of BrdU-labeled cells in the interfollicular epidermis. For each interfollicular area, we assigned consecutive numbers (beginning from 1) as described before. 30 We measured the number of BrdU-labeled cells in a tile of 2500 mm 2 (a square of 50 mm on each side). The size of a tile was 20–30% of the size of each interfollicular area. BrdU(1) cells in the center of each interfollicular area were counted to assure that only labeled cells in the interfollicular area were counted. Sampling of the interfollicular areas was modified from methods described previously. 9 Interfollicular areas were chosen based on random numbers from a statistical table. As the area of an epidermal sheet (2 mm in diameter) is 3.1 mm 2, the sampling would cover more than 50% of the total area. All BrdU(1) cells in the selected tiles were counted, and the extent of BrdU-labeling was expressed as [BrdU(1) cells/mm 2]
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labelling Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL) was modified from the method of Gavrieli et al. 17 Free-floating sections and epidermal sheets were incubated with proteinase K (10 mg/ml, Boehringer–Mannheim, Germany) for 30 min at room temperature. Endogenous peroxidase was quenched with 1% H2O2 for 30 min. End-labeling was then performed with a digoxigenin (Dig)-labeled TUNEL kit (Boehringer–Mannheim, Germany) according to the manufacturer’s instructions. After incubation at 378C for 15 min, sections were rinsed in 2×standard saline citrate for 15 min, blocked with 5% normal serum in Tris buffer. Sections were incubated with anti-Dig–peroxidase antibody (1:100, Boehringer–Mannheim, Germany) at 48C for 2 h, and then demonstrated with DAB as in immunocytochemistry.
Analysis For analysis of epidermal innervation, epidermal thickness, BrdUincorporation, and TUNEL, five to 10 animals were used at each time point after nerve transection. Data are presented as means^S.E.M. and analysed with t-test, and nonparametric Wilcoxon rank sum test to compare the denervated and control groups. Any difference with P,0.05 was considered statistically significant.
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footpads of the hind limb by transecting the sciatic nerve, which innervates the plantar surface of the hind foot. Within 48 h after nerve injury, all epidermal nerves had degenerated (Fig. 1B). In the dermis, the immunocytochemical staining for PGP became faint and discontinuous, reflecting the degeneration of nerve trunks. This picture persisted when the skin remained denervated. To study reinnervation of the skin, we crushed the sciatic nerve of another group of mice. These mice were allowed to survive for three months, when reinnervation was complete based on previous functional studies. 10,29 The overall pattern of skin innervation by PGP immunocytochemistry was similar to that of the control skin with the varicose nerve terminals appearing in the epidermis (Fig. 1C). Epidermal thinning after skin denervation
Fig. 1. Skin innervation. Footpads of control and denervated mice were immunostained with PGP9.5. (A) In the control footpad, PGP-immunoreactive epidermal nerves with varicose appearance reach granular layers of the epidermis. These nerves arise and are perpendicular to the subepidermal plexus. Occasionally, there are nerve terminals of Meissner’s corpuscles in dermal papillae (arrowheads). (B) Denervated footpad (seven days after sciatic nerve transection). Characteristic PGP(1)-epidermal nerves, and subepidermal plexus are lost after denervation. (C) Reinnervated footpad three months after crushing injury of the sciatic nerve. Nerves immunoreactive for PGP with varicose appearance reappear in the epidermis. Scale bar50 mm.
RESULTS
Cutaneous innervation: normal innervation, denervation and reinnervation We demonstrated the rich innervation of skin by PGP immunocytochemistry. In normal mice, nerve fascicles containing PGP-immunoreactive profiles were present in the epidermis and dermis (Fig. 1A). When dermal nerves approached the epidermis, they usually paralleled the epidermal–dermal junction, forming the subepidermal plexus. Single epidermal fibers arising from dermal nerve trunks ascended the epidermis vertically by passing keratinocytes in the basal and suprabasal layers. To eliminate cutaneous innervation, we denervated
Epidermis after denervation had similar organization as the control epidermis, with cuboid cells in the basal layer, and flattened cells in the granular layer (Fig. 2A). A striking difference was the reduction in the epidermal thickness after sciatic nerve transection. One day after nerve transection, there was no appreciable difference in epidermal thickness between the denervated and control sides (45.0^1.9 vs 48.6^1.9 mm, P.0.5) (Fig. 2B). We further compared the difference in epidermal thickness at different time-points after denervation and constructed a plot to demonstrate the temporal changes (Fig. 3A). Within three days, the reduction in epidermal thickness became obvious on the denervated side (30.5^1.1 vs 41.4^2.9 mm, P,0.001) (Fig. 2C–D). Epidermal thinning persisted as long as the skin remained denervated (34.6^1.9 vs 47.5^2.5 mm, P,0.001 at 28 days after denervation). When skin became reinnervated within three months of crushing injury, the thickness of epidermis returned to a comparable level of the control side (43.5^1.7 vs 45.0^2.5 mm, P.0.5). Because epidermal thickness was variable among different animals, we further analysed temporal change by normalizing the denervated epidermal thickness to the control epidermal thickness. The plot of epidermal thickness ratios showed a similar trend (Fig. 3B). Reduced bromodeoxyuridine-incorporation denervation
after
skin
To understand how skin innervation influences epidermal thickness, we evaluated keratinocyte proliferation with BrdUlabeling. BrdU-labeled cells were in the basal layer of epidermis (Fig. 4A–D). The extent of BrdU-incorporation was reduced in denervated skin (Fig. 4B, D). To compare the difference in the incorporation of BrdU, we quantified BrdU(1) cells in epidermal sheets (Fig. 4E–H). Within 24 h, there was a substantial decrease in BrdU-labeled cells in the denervated skin (3,952^410 vs 5,576^280 cells/mm 2, P,0.001, Fig. 5A). We also compared the extent of BrdUlabeling by normalizing the density of BrdU(1) cells on the denervated epidermis to that on the control side. The ratio was reduced to 71^6% of the control side within 24 h of denervation (P,0.001, Fig. 5B). The decrease in BrdU-labeling was the most obvious within two days after denervation (932^228 vs 4875^185 cells/mm 2, P,0.0001). During the study period of denervation (three to 28 days), the degree of BrdU-labeling on the denervated side was 25–50% of that
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Fig. 2. Changes in epidermal thickness after denervation. Control and denervated skin was embedded in paraffin. Vertical sections of 5 mm were stained with hematoxylin. The thickness of the nucleated epidermis was quantified in a blinded fashion. (A, C, E, F) are from the control sides, and (B, D, E, F) are from the denervated sides. (A, B) One day after transection; (C, D) three days after transection; (E, F) seven days after transection; (G, H) three months after crushing. Both the control and denervated epidermis show similar organization with strong basophilic keratinocytes in the basal layer, flat keratinocytes in granular layers, and non-nucleated cells in cornified layers. Significant thinning of epidermal thickness is obvious in pairs C–D and E–F. Scale bar: (A–F)50 mm; (G–H)42 mm.
on the control side (Fig. 5B). When the epidermis was reinnervated three months after crushing the sciatic nerve, the reduction in BrdU-incorporation was reversed (4780^400 vs 5152^450 cells/mm 2, P.0.5, Fig. 4G–H).
Keratinocyte differentiation after denervation Keratinocytes are a dynamic population with continuous proliferation and differentiation. An alternative mechanism
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Fig. 3. Temporal changes of epidermal thickness after denervation. Quantitation of epidermal thickness based on paraffin-embedded hematoxylin-stained sections is detailed in Experimental Procedures. Data from five to 10 animals at each time-point are expressed as means^S.E.M., with open circles for the control skin, and closed circles for the denervated skin. (A) Temporal changes of epidermal thickness. Within three days after denervation, there is significant thinning of epidermis (30.5^1.1 vs 41.4^2.9 mm, P,0.01). Epidermal thinning persisted as skin remained denervated during the experimental period (up to 28 days, 34.6^1.9 vs 47.8^2.4 mm, P,0.01). Epidermal thinning was reversed by skin reinnervation three months after crushing of the sciatic nerve (43.5^1.7 vs 45.0^2.5 mm, P.0.5). (B) Trend of epidermal thinning by ratios of denervated epidermal thickness normalized to control epidermal thickness. After denervation, the epidermal thickness on the denervated side became 69–75% of that on the control side. The ratio of epidermal thickness is not different from 1 (the dotted line) after reinnervation (*P,0.01).
for epidermal thinning after skin denervation is an increase of keratinocyte turnover. Cell death of basal keratinocytes could also contribute to the reduction of BrdU-incorporation in the basal layer. To test these hypotheses, we carried out immunocytochemistry for keratin intermediate filaments and TUNEL for programmed cell death on epidermal sheets and vertical sections of the skin. For example, K10 was expressed in suprabasal layers of epidermis (Fig. 6A–B). In vertical sections and epidermal sheets of the control and denervated skin, TUNEL(1) cells were negligible (Fig. 6C–D and G– H). The patterns of TUNEL after adding DNase as positive controls were the same between the control and denervated epidermis (Fig. 6E–F and I–J). Thus, the patterns of keratin expression and TUNEL were similar between the control and denervated sides.
DISCUSSION
Influence of denervation on keratinocytes This report extends the previous observation that depletion of epidermal nerves causes epidermal thinning, and further explores the mechanism of epidermal thinning. Degeneration of epidermal nerves is complete within 48 h. 20,21 Nerve injury is followed by epidermal thinning within 72 h. Any change in the process of keratinocyte differentiation during this interval should provide new insights into the influence of skin innervation. Keratinocytes are a dynamic population in the epidermis. They proliferate in the basal layer and move upwards to the granular layer. During the process, the expression of keratin changes from K5/K14 in the basal layer to K1/K10 in suprabasal layers. 22 After reaching the top of the granular layer, keratinocytes undergo a specific from of programmed cell death, and finally become cornified. 23 To understand potential mechanisms of epidermal thinning after skin denervation, we investigated the time-course of epidermal thinning, the incorporation of BrdU, the expression of keratin proteins, and the programmed cell death of
differentiated keratinocytes. During this period, there is a robust reduction in BrdU-labeling, and it is the only step markedly influenced by skin denervation. The reduction in proliferation index with normal differentiation and unaltered cell loss suggests that sensory innervation influences keratinocyte proliferation and ultimately the thickness of the epidermis. Traditionally, sensory nerves are considered to transduce harmful stimuli. Various types of neuropeptides coexist with a defined type of neurotransmitter in sensory neurons and are potential sources for functions other than sensory transduction. For example, a subset of sensory nerves contain CGRP, which causes vasodilation upon release from nerve terminals. 32 CGRP has a wide range of actions, including modulation of keratinocyte proliferation via an increase in cAMP. 43,49 Various cellular components in the epidermis are influenced by skin innervation in developing and mature animals. The thickness of the epidermis and the height of rete peges are reduced if sensory neurons are deprived during embryonic development. 40 The number of Merkel cells, the mechanoreceptors in the epidermis, decreases after cutaneous nerve degeneration. 1,37,42 Other potential molecules influencing keratinocyte proliferation include neurotrophins, in particular, the nerve growth factor. Nerve growth factor can have an autocrine effect on keratinocytes. 7,11,44,45 A recent study suggests that denervation down-regulates the synthesis of nerve growth factor in the target organs of autonomic nerves. 35 The significance of epidermal innervation on keratinocyte proliferation remains elusive. However, the conclusion may provide therapeutic alternatives. Wound healing is a complicated process after injury, and neuropeptides or neurotrophins promoting reinnervation of the injured skin may speed wound healing. In contrast, antagonists of neuropeptides could be a new strategy for hyperproliferation disorders of keratinocytes, such as psoriasis. These hypotheses will require further investigations on available animal models. 8,46 Collectively, these observations provide a new look at the interactions between the skin and its innervation.
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Fig. 4. Incorporation of BrdU. BrdU-incorporation was demonstrated immunocytochemically after i.p. injection of BrdU. (A, C, E, G) are from the control epidermis. (B, D, F) are from the seven-day denervated epidermis. H is the the reinnervated epidermis three months after denervation. (A, C) On vertical sections of the control epidermis, BrdU-labeled keratinocytes are in the basal layer of the epidermis. (B, D) The pattern of BrdU labeling is similar after denervation. The number of BrdU-labeled cells in the denervated epidermis is substantially smaller than that of the control epidermis. (C, E) For quantitation of BrdU labeling, we immunocytochemically stained epidermal sheets with the anti-BrdU antibody. There is a significant reduction in BrdU-labeling in denervated epidermal sheets. (G, H) The extent of BrdU-labeling is comparable between the control and denervated sides.
Skin innervation and epidermal thinning The influence of cutaneous nerves on the architecture of the skin has become an intriguing issue. 31 Skin denervation by
transection of the sciatic nerve causes epidermal thinning. 13,47,48 In this approach, autonomic and motor nerves are also damaged. Loss of autonomic nerves may alter blood flow with secondary effects on the epidermis. For
Cutaneous nerve degeneration and keratinocyte proliferation
Fig. 5. Temporal changes in incorporation of BrdU related to epidermal denervation. Comparison of BrdU labeling was based on quantitation of BrdU-labeled cells on epidermal sheets as described in Fig. 4. Data from five animals at each time-point are expressed as means^S.E.M., with open circles for control skin and closed circles for denervated epidermis. (A) Density of BrdU(1) cells. In control epidermal sheets, there are variations in BrdU-labeled cells. Within 24 h after denervation, there is a significant reduction in BrdU-labeling on denervated epidermis (3952^410 vs 5576^280 cells/mm 2, P,0.001). The most obvious reduction in BrdUlabeling occurred within two days of denervation (932^228 vs 4875^185 cells/mm 2, P,0.0001). During the period of denervation, the extent of BrdU-labeling persisted, with 2116^388 vs 4556^350 cells/mm 2 (P,0.001) at 28 days after denervation. The extent of BrdU-incorporation on reinnervated epidermis returned to a comparable level as the that of control epidermis three months after sciatic nerve crushing (4780^400 vs 5152^450 cells/mm 2, P.0.5). (B) Relative labeling of BrdU (normalized ratios of denervated/control epidermal sheets). The ratio of BrdUincorporation is 0.71^0.06 at 24 h after denervation, and reaches the nadir within 48 h (0.19^0.06, P,0.0001). During the period of denervation (three to 28 days after denervation), the ratios remain in the range of 0.25–0.50. Three months after reinnervation, the ratio of BrdU-labeling is not different from 1 (the dotted line), 0.93^0.08, P.0.5. (*P,0.01).
example, shining and thinning of the skin in patients with peripheral nerve disorders was frequently attributed to impaired autonomic regulation and defective vascular supply. 50 However, sympathectomy specifically eliminates autonomic innervation of the skin, but does not cause epidermal thinning. 33 The finding is corroborated with the fact that there is no direct correlation between cutaneous blood flow and epidermal thickness. 38,39 A further issue is that paralysis of the hind limbs alters the pattern of walking and weightbearing. Thus, the contribution of motor denervation to epidermal thinning can not be excluded. 33 Nevertheless, two
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Fig. 6. Changes in keratin immunocytochemistry and TUNEL. Figures on the left panel (A, C, E, G, I) are from the control skin and the right panel (B, D, F, H, J) are from the skin denervated seven days earlier. (A, B) Cytokeratin 10-immunocytochemical patterns in control (A) and denervated (B) epidermis. Cytokeratin 10(1) keratinocytes are in the suprabasal layers. The expression patterns are similar except that the epidermis is thinner on the denervated side. (C–F) TUNEL on epidermal sheets. There is no BrdUlabeled cell in control (C) and denervated (D) epidermal sheets, suggesting no increase in apotopsis in the basal layer of the epidermis. By adding DNase as positive control, all the cells in epidermal sheets become positive for TUNEL. (G–J) TUNEL on vertical sections of the epidermis. TUNEL(1) cells are difficult to find in both control (G) and denervated (H) skin. The positive control for TUNEL was performed after adding DNase. Keratinocytes in all layers of the epidermis become TUNEL(1) in control (I) and denervated (J) skin. Scale bar: (A, B)33 mm; (C– F)25 mm; (G–J)150 mm.
lines of evidence indicate that depletion of epidermal nerves is tightly linked to epidermal thinning. In the null mutant mice for p75-NGFR, there is a robust reduction of epidermal nerves, and a remarkable decrease in epidermal thickness. 5 Finally, ganglionectomy by removing the sensory neurons innervating the find feet, eradicates epidermal nerves, and causes epidermal thinning. 29,33 Taken together, these results strongly suggest that sensory innervation is sufficient to modulate epidermal thickness.
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Potential cellular mechanism of skin innervation The existence of nerve terminals in the epidermis offers support for their roles in transducing noxious stimuli. Epidermal nerves are unmyelinated with the diameter usually in the range of 1–2 mm, and they can only be resolved under electron microscopy by conventional morphological techniques. 26,29 Recent studies by applying immunocytochemistry with various neuronal markers, such as CGRP and substance P, have explored the diversity of epidermal nerves. These peptidergic nerves are presumably nociceptive terminals and may also possess mitogenic potential. Compared with other neuronal proteins, PGP labels the most abundant epidermal nerves. Other researchers as well as ourselves provided ultrastructural evidence of PGP(1)-epidermal nerves by electron microscopic immunocytochemistry. 26,29 In the epidermis, these nerves are in contact with keratinocytes and epidermal Langerhans cells. 16,28 CGRP(1)-epidermal nerves have close contacts with Langerhans cells, and CGRP can alter the ability of antigen presentation of Langerhans cells. 3,28 These
observations suggest that interactions between nerves and various cellular components in the skin are far more complicated than previously thought. Proportions of epidermal nerves labeled with CGRP and substance P are substantially smaller than those of PGP(1)nerves. It remains to be determined exactly how epidermal nerves influence keratinocytes: through synapse-like contact or diffusible factors? Alternatively, epidermal nerves may influence keratinocyte proliferation through diffusible factors. A recent study indicates that there is differential thinning of the epidermis after skin denervation, with the most central part of the denervated skin exhibiting the most profound degree of epidermal thinning. 9 Taken together, these results provide additional support to the notion that skin innervation may play an important role in regulation of keratinocytes. Acknowledgements—The work was supported by the National Health Research Institute, Department of Health, Republic of China (DOH88HR-727).
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