Dermal Nerve Research Articles

Upregulation of the Axonal Growth and the Expression of Substance P and its NK1 Receptor in Human Allergic Contact Dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.

Characteristic alterations of cutaneous neurogenic factors in photoaged skin

Although it has been recognized that photoageing and chronological ageing differ in various morphological and biological aspects, the characteristic alterations of cutaneous neurogenic factors in photoaged skin are poorly characterized. To characterize cutaneous neurogenic factors, including innervation, neuropeptides, nerve growth factor and interactions of mast cells, in photoaged skin. Paired biopsy specimens were obtained from sun-exposed volar forearm skin and from sun-protected dorsal upper arm skin of 20 elderly subjects. Various cutaneous neurogenic factors, including innervation, neuropeptides, neurokinin receptor, nerve growth factor, neurogenic inflammation and morphology of mast cells, were compared in sun-exposed vs. sun-protected skin quantitatively and qualitatively. Cutaneous neurogenic factors associated with photoageing were characterized by a significant increase in the densities of dermal and intraepidermal nerve fibres, a correlation between epidermal innervation and the severity of photodamage, increases in the number of neuropeptidergic sensory nerve fibres in the dermis and in tissue levels of sensory neuropeptides, increases in the content of nerve growth factor, reduced expression of neurokinin receptor 1 by epidermal keratinocytes and by vascular endothelial cells and a tachykinin-specific reduction of cutaneous neurogenic inflammation. Mast cells in photodamaged skin showed several characteristic morphological features, including various degrees of activation and an intimate association with fibroblasts, which were distinct from those in sun-protected skin. Furthermore, mast cells in photodamaged skin possessed larger amounts of substance P within their granules than did those in sun-protected skin. These findings document for the first time characteristic alterations of cutaneous neurogenic factors in photodamaged skin and suggest that the cutaneous nervous system may be involved in photoageing processes.

EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy

Skin biopsy has become a widely used tool to investigate small calibre sensory nerves including somatic unmyelinated intraepidermal nerve fibres (IENF), dermal myelinated nerve fibres, and autonomic nerve fibres in peripheral neuropathies and other conditions. Different techniques for tissue processing and nerve fibre evaluation have been used. In March 2004, a Task Force was set up under the auspices of the European Federation of Neurological Societies (EFNS) with the aim of developing guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathies. We searched the Medline database from 1989, the year of the first publication describing the innervation of human skin using immunostaining with anti-protein-gene-product 9.5 (PGP 9.5) antibodies, to 31 March 2005. All pertinent papers were rated according to the EFNS guidance. The final version of the guidelines was elaborated after consensus amongst members of the Task Force was reached. For diagnostic purposes in peripheral neuropathies, we recommend performing a 3-mm punch skin biopsy at the distal leg and quantifying the linear density of IENF in at least three 50-mum thick sections per biopsy, fixed in 2% PLP or Zamboni's solution, by bright-field immunohistochemistry or immunofluorescence with anti-PGP 9.5 antibodies (level A recommendation). Quantification of IENF density closely correlated with warm and heat-pain threshold, and appeared more sensitive than sensory nerve conduction study and sural nerve biopsy in diagnosing small-fibre sensory neuropathy. Diagnostic efficiency and predictive values of this technique were very high (level A recommendation). Confocal microscopy may be particularly useful to investigate myelinated nerve fibres, dermal receptors and dermal annex innervation. In future, the diagnostic yield of dermal myelinated nerve fibre quantification and of sweat gland innervation should be addressed. Longitudinal studies of IENF density and regeneration rate are warranted to correlate neuropathological changes with progression of neuropathy and to assess the potential usefulness of skin biopsy as an outcome measure in peripheral neuropathy trials (level B recommendation). In conclusion, punch skin biopsy is a safe and reliable technique (level A recommendation). Training in an established cutaneous nerve laboratory is recommended before using skin biopsy as a diagnostic tool in peripheral neuropathies. Quality control at all levels is mandatory.

Opposite-direction bilateral fracture dislocation of the shoulders after an electric shock

Injuries after an electric shock, such as dermal burns, motor and sensory nerve deficits, fractures and dislocations, are reported in the literature. Posterior dislocation of the shoulder after electric-shock is the common musculoskeletal injury. Bilateral dislocation, either anterior or posterior, is rarely seen and reported. We report a case of bilateral shoulder fracture dislocation in opposite directions following an electric-shock and discuss the mechanism, the diagnosis and the treatment.

The touch dome in human skin is supplied by different types of nerve fibers

Receptor end organs and free-nerve endings in the skin are the peripheral sentinels of the sensorial nervous system encoding for touch, temperature, and pain. Using a novel approach to analyze the outermost nerves of the skin, we visualized for the first time the distinct microanatomical structure of the touch dome of human hairy skin. The dermal nerve fibers of this slowly adapting type 1 mechanoreceptor were embedded in dermal protrusions that could be readily discerned by Laminin-5 staining. Concerning the nerves supplying the touch domes, we found, unexpectedly, that besides Abeta-fibers, Adelta- and C-fibers also were regularly present. The epidermis overlying the nerve convolutes showed a distinctive architecture of the rete ridges clearly demarcated from the surroundings and extending over 0.193 +/- 0.138 mm(2) (mean +/- standard deviation). Within this area, 756 +/- 386 Merkel cells/mm(2) (mean +/- standard deviation) were present compared with less than 50/mm(2) outside the touch dome, demonstrating for the first time a highly discontinuous distribution of these cells in nonglabrous skin. Our findings strongly suggest that the receptive qualities of human touch domes exceed mechanosensation, and that they may serve as multifunctional nerve end organs in human skin.

Skin biopsies in myelin-related neuropathies: bringing molecular pathology to the bedside

Skin biopsy is a minimally invasive procedure and has been used in the evaluation of non-myelinated, but not myelinated nerve fibres, in sensory neuropathies. We therefore evaluated myelinated nerves in skin biopsies from normal controls and patients with Charcot-Marie-Tooth (CMT) disease caused by mutations in myelin proteins. Light microscopy, electron microscopy and immunohistochemistry routinely identified myelinated dermal nerves in glabrous skin that appeared similar to myelinated fibres in sural and sciatic nerve. Myelin abnormalities were observed in all patients with CMT. Moreover, skin biopsies detected potential pathogenic abnormalities in the axolemmal molecular architecture previously undetected in human neuropathies. Finally, myelin gene expression at both mRNA and protein levels was evaluated by real-time PCR and immunoelectron microscopy. Peripheral myelin protein 22 (PMP22) was increased in CMT1A (PMP22 duplication) and decreased in patients with hereditary neuropathy with liability to pressure palsies (PMP22 deletion). Taken together, our data suggest that skin biopsy may in certain circumstances replace the more invasive sural nerve biopsy in the morphological and molecular evaluation of inherited and other demyelinating neuropathies.

Cutis laxa in hereditary gelsolin amyloidosis

Hereditary gelsolin amyloidosis (AGel amyloidosis) is an age-associated systemic disease with global distribution, caused by a G654A or G654T gelsolin gene mutation. Cutis laxa is a principal clinical manifestation of this disease. However, only few data on the dermatological involvement are available, and the pathogenesis of this amyloidosis-associated form of cutis laxa has remained unknown. To elucidate the pathomechanism of this less well-known genodermatosis. We performed systematic clinical, histological, immunohistochemical and ultrastructural skin biopsy studies in 12 patients with a G654A gelsolin gene mutation. For comparison, skin specimens from 10 control subjects were analysed. All patients had clinically characteristic cutis laxa, and frequently other signs of symptomatic skin disease such as increased fragility and risk for intracutaneous bleeding. All patients showed cutaneous deposition of gelsolin amyloid (AGel), mainly attached to basement membranes or basal laminae of various cutaneous structures, dermal nerves and blood vessel walls, and elastic fibres, particularly in the lower reticular dermis. AGel often encircled the elastic fibres, and colocalized with amyloid P component (AP), an elastic fibre microfibrillar sheath-associated protein. Fragmentation and loss of elastic fibres, epidermal atrophy, and reduction of dermal appendages were also common. Antibodies to wild-type gelsolin bound to S-100-positive epidermal dendritic cells, a previously unrecognized immunoreaction. Patients had fewer gelsolin-positive dendritic cells than controls. Widespread skin involvement with AGel deposition and elastic fibre involvement are essential pathological features in AGel amyloidosis, and contribute to the characteristic cutis laxa, dramatic in old age. Codistribution of AGel and AP, with demonstrated specific binding affinity for amyloid fibrils, suggests that elastic fibre-associated AP acts as a matrix for cutaneous amyloid deposition in AGel amyloidosis.

Changes of Epidermal Mu-Opiate Receptor Expression and Nerve Endings in Chronic Atopic Dermatitis

There is increasing evidence that neuropeptides such as a substance P, neurotrophins or β-endorphin, an endogenous agonist for µ-opioid receptor, are involved in the pathogenesis of atopic dermatitis in which mental stress and scratching deteriorate the disease. µ-Opioid receptor, a G-protein-coupled receptor, can be downregulated and internalized by agonists and other factors in vitro. In this study, we investigated the regulation of µ-opioid receptor and nerve endings in atopic dermatitis patients. Skin biopsies from atopic dermatitis patients revealed a significant downregulation of µ-opiate receptor expression in epidermis of atopic dermatitis. Permeabilization of the skin showed that the receptor in keratinocytes from atopic dermatitis is internalized. The mRNA expression pattern of the µ-opiate receptor is different in epidermis taken from patients with chronic atopic dermatitis compared to normal skin. In atopic dermatitis, the mRNA is concentrated in the subcorneal layers of the epidermis and in normal skin in the suprabasal layers. Staining of the nerve endings using protein gene product 9.5 shows a different pattern of epidermal nerve endings in normal skin compared to atopic dermatitis. In normal skin, the epidermal nerve endings are rather thick. However, in atopic dermatitis, the epidermal nerve endings are thin and run straight through the epidermis. Based on these observations and combining the ‘intensity’ and ‘pattern’ hypothesis, we propose a new theory especially for histamine-unrelated, peripheral induction of chronic pruritus. We suggest that ‘itch’ is elicited in the epidermal unmyelinated nerve C-fibers and ‘pain’ in the dermal unmyelinated nerve fibers. The downregulation of the opioid receptor in the epidermis contributes to the chronic itching. We call this new hypothesis the ‘layer hypothesis’.

Cutaneous field stimulation with moderate intensity current induces nerve proliferation in rat skin but has no effect on dorsal root ganglia.

Cutaneous field stimulation is used to treat localized itch. The aim of the present study was to determine whether such treatment induces neurochemical changes in the dorsal root ganglia in 30 rats using a pan-neuronal marker protein gene-product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). Electrical stimulation using the currents of either 0.13 mA or 0.53 mA--was given under general anaesthesia for 30 min per day for 10 days. Punch biopsies from the thoracal skín and the corresponding dorsal root ganglia were collected upon sacrifice. Both stimulation regimens induced proliferation of epidermal and dermal nerve fibers in the skin. The mean number of all cutaneous PGP-immunoreactive (IR) nerve fibers after the electrical stimulation with 0.13 mA was increased by 49% (p<0.001), the mean number of epidermal PGP-IR nerve fibers was increased by 25% (p = 0.001) and the mean number of all CGRP-IR nerve fibers was increased by 65% (p<0.001) compared with controls. The mean number of all PGP-IR nerve fibers after the electrical stimulation with 0.53 mA was increased by 39% (p<0.001), the mean number of PGP-IR epidermal nerve fibers was increased by 30% (p = 0.001) and the mean number of CGRP-IR nerve fibers was increased by 65% (p<0.001) compared with controls. Only stimulation with 0.53 mA induced an up-regulation of sensory neuron markers in the dorsal root ganglia. The ratio of positive/negative PGP-IR cells was increased by 17% (p = 0.002), the ratio of positive/negative CGRP-IR cells was increased by 12% (p = 0.003) and the ratio of positive/negative VR1-IR cells was likewise increased by 10% (p = 0.008) as compared with the control ganglia. We conclude that serial cutaneous electrical stimulation by a moderate current in rat does not induce neurochemical changes in the dorsal root ganglia.

Subungual neurothekeoma

Neurothekeomas are benign tumors probably of nerve sheath origin and are also known as dermal nerve sheath myxomas. They are commonly found on the face, arm, or shoulder and less frequently the lower limbs. To our knowledge, this is the first case of a subungual neurothekeoma affecting the big toe. Histology confirmed a well-circumscribed, multilobulated tumor composed of bland stellate and spindle cells in a copious myxoid matrix staining positive with S100 protein.

Glutamine and glutamine synthetase immunoreactivities in Schwann cells of the rat glabrous skin

We have shown recently that glial cells of the peripheral nervous system contain substrates and enzymes related to the glutamine cycle, e.g. glutamine, glutamine synthetase, and glutamate dehydrogenase (Miller et al., Brain Res 2002: 945: 202–11). The present study used immunohistochemistry to determine the cellular distribution of glutamine and glutamine synthetase in the rat glabrous skin. Normal adult Sprague–Dawley rats were used for these studies. Glutamine and glutamine synthetase were localized with both immunoperoxidase and immunofluoresent techniques. Glutamine and glutamine synthetase immunoreactivities were abundant in Schwann cells of large dermal nerve bundles. An enrichment of glutamine and glutamine synthetase immunoreactivies occurred in terminal and near‐terminal Schwann cells in dermal papilla and at the dermal–epidermal interface. We hypothesize that the peripheral glial glutamine cycle is important for supplying neuronal energy demands in dermal nerve fibers. Furthermore, the peripheral glial glutamine cycle in terminal Schwann cells most likely produces glutamine for sensory terminal uptake. This glutamine would be converted to glutamate for release from sensory afferents. Glutamate released from sensory afferents may be important for the development of hyperalgesia and allodynia. Terminal Schwann cells, therefore, are potential targets for modulating sensory afferent sensitivity during acute and chronic pain.Supported by NIH AR47410 (KEM).

Acne and sebaceous gland function

The embryologic development of the human sebaceous gland is closely related to the differentiation of the hair follicle and the epidermis. The number of sebaceous glands remains approximately the same throughout life, whereas their size tends to increase with age. The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is an important formation site of active androgens. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids, and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids. Among other lipid fractions, sebaceous glands have been shown to synthesize considerable amounts of free fatty acids without exogenous influence. Sebaceous lipids are responsible for the three-dimensional skin surface lipid organization. Contributing to the integrity of the skin barrier. They also exhibit strong innate antimicrobial activity, transport antioxidants to the skin surface, and express proinflammatory and anti-inflammatory properties. Acne in childhood has been suggested to be strongly associated with the development of severe acne during adolescence. Increased sebum excretion is a major factor in the pathophysiology of acne vulgaris. Other sebaceous gland functions are also associated with the development of acne, including sebaceous proinflammatory lipids; different cytokines produced locally; periglandular peptides and neuropeptides, such as corticotrophin-releasing hormone, which is produced by sebocytes; and substance P, which is expressed in the nerve endings at the vicinity of healthy-looking glands of acne patients. Current data indicate that acne vulgaris may be a primary inflammatory disease. Future drugs developed to treat acne not only should reduce sebum production and Propionibacterium acnes populations, but also should be targeted to reduce proinflammatory lipids in sebum, down-regulate proinflammatory signals in the pilosebaceous unit, and inhibit leukotriene B 4–induced accumulation of inflammatory cells. They should also influence peroxisome proliferator-activated receptor regulation. Isotretinoin is still the most active available drug for the treatment of severe acne.

The role of neuropeptides and neuropeptide‐degrading enzymes in wound healing

Thirty to 40% of diabetic patients develop sensory neuropathy. Neuropathy is a major causal factor in diabetic ulcers. Only 31% of neuropathic diabetic ulcers heal in 20 weeks. Patients with neuropathy have a 15.5‐fold excess risk of amputation. Diabetic patients with neuropathy particularly lose epidermal and papillary dermal sensory nerves which release neuropeptides such as substance P (SP). Neutral endopeptidase (NEP), the enzyme that degrades SP, is dramatically over expressed in patients with diabetic neuropathy. SP has positive effect on wound healing. Treatment strategies related to the nervous system for prevention and treatment of diabetic ulcers currently being studied include prevention of neuropathy with tight control of blood glucose, application of neuropeptides, nerve growth factors (NGF), and antagonists of NEP.

Induction of neuropeptides in skin innervating sensory neurons by stress and nerve growth factor as a possible reason for hair growth alteration

Recently, we introduced a mouse model launching experimental evidence for stress‐induced hair growth inhibition (HGI), pointing to the existence of a brain‐hair follicle axis (BFA). We suggested that nerve growth factor (NGF), besides neuropeptide substance P (SP), is a candidate mediator along the BFA. Published data further indicate that stress‐related neuropeptides, e.g. calcitonin gene‐related peptide (CGRP) and SP may be involved in HGI. SP and CGRP are synthesized in dorsal root ganglia (DRG) and released after axonal transport in the skin. Thus, aim of the present study was to investigate the effect of stress or subcutaneous injection of NGF, which mimics stress and regulates neuropeptide genes in sensory neurons, on the expression of SP and CGRP in DRG. Anagen was induced in C57BL/6 mice by depilation and retrograde tracing was employed on day 9 post‐depilation (PD). On day 14 PD, mice were either exposed to sound stress (n = 4) injected subcutaneously with NGF (n = 4) or served as control (n = 4). On day 16 PD, DRG (mean of 30/mouse) were harvested and SP and CGRP in skin‐specific sensory neurons, as identified by the tracer dye, were labelled by immunohistochemistry and counted. Stress exposure as well as NGF injection leads to a significant induction of SP and CGRP in retrograde‐labelled neurons. This allows us to conclude that sensitive dermal nerve fibres are likely to originate from the presently identified neuropeptide‐positive neurons. Peripheral activation of SP‐expressing afferent nerve fibres via NGF‐dependent pathways may cause neurogenic inflammation, eventually resulting in HGI.

Cutaneous and sympathetic denervation in neonatal rats with a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 gene

The mutilated-foot rat ( mf rat) is an autosomal recessive mutant with characteristic digit deformities in adult animals, and this phenotype mimics many aspects of human sensory neuropathy. The genetics of mf rats was recently elucidated. To understand whether the genotype is responsible for cutaneous denervation before clinically overt mutilation in adult mf rats, we investigated skin innervation in postnatal day 7 (P7) mf rats and compared the patterns with P7 wild-type rats. The mf rat carries a G→A mutation in the gene encoding the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 ( Cct4). In the footpad skin of P7 mf rats, there was a >90% loss of epidermal nerves (0.7–7.9% of P7 wild-type rats) as indicated by neuronal markers including protein gene product 9.5 (PGP 9.5), growth-associated protein 43 (GAP43), calcitonin gene-related peptide (CGRP), and substance P (SP). The epidermis of hairy skin in hind feet was completely denervated in mf rats as well. Compared with an approximately 80% reduction in the size of dermal nerve fascicles and a parallel loss of nerve fibers, the nearly complete absence of epidermal innervation suggests further sensory nerve degeneration at the level of nerve terminals in the epidermis. In contrast, the loss of epidermal nerves in the abdominal skin of mf rats was less extensive than that in the footpad skin of mf rats; CGRP (+) and SP (+) fibers were moderately reduced (28.3–56.4% of levels of wild-type rats) with normal amounts of PGP 9.5 (+) and GAP43 (+) nerves. Sympathetic innervation as assessed by tyrosine hydroxylase immunoreactivity was absent from the footpad and abdominal skin of mf rats. In conclusion, there is regional skin denervation with diffuse sympathetic denervation in P7 mf rats. These results suggest that the mutation in Cct4 underlies cutaneous nerve degeneration in mf rats.

Nitrotyrosine localization to dermal nerves in borderline leprosy.

Nerve damage is a common and disabling feature of leprosy, with unclear aetiology. It has been reported that the peroxidizing agents of myelin lipids-nitric oxide (NO) and peroxynitrite-are produced in leprosy skin lesions. To investigate the localization of nitrotyrosine (NT)-a local end-product of peroxynitrite-in leprosy lesions where dermal nerves are affected by a granulomatous reaction. We investigated by immunohistochemistry and immunoelectron microscopy the localization of the inducible NO synthase (iNOS) and NT in biopsies exhibiting dermal nerves from patients with untreated leprosy. There were abundant NT-positive and iNOS-positive macrophages in the borderline leprosy granulomas infiltrating peripheral nerves identified by light microscopy, S-100 and neurofilament immunostaining. Immunoelectron microscopy showed NT reactivity in neurofilament aggregates and in the cell wall of Mycobacterium leprae. Our results suggest that NO and peroxynitrite could be involved in the nerve damage following borderline leprosy.

Merkel cells, a new localization of prepro-orexin and orexin receptors.

Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.

Sheet Preparations Expose the Dermal Nerve Plexus of Human Skin and Render the Dermal Nerve End Organ Accessible to Extensive Analysis

Since vertical tissue sections used for the study of the human cutaneous nervous system inherently allow visualization of only a small part of the mainly horizontally oriented cutaneous nerves, we searched for possibilities to extend this view. We now propose a method based on the immuno-staining of dermal sheet preparations for subsequent analysis by electron-, light- or laser scanning microscopy. Dermal sheet preparations for the first time allowed the imaging of the complex structure of the nerve end organ over several cm2, and facilitated viewing of its topological relationship to other tissue components. We could visualize that the bulk of free ending nerve fibers ramified within 25 microm of the dermo-epidermal junction, whereas below that only larger nerve bundles were present. This method further allowed the detection and quantification of NCAM/CD56+ non-myelinating Schwann cells which envelope terminal axons within the dermis. Depending on the body region, we detected between 140 to over 300 individual terminal Schwann cells per mm2 skin surface. Our method should allow the acquisition of new insights into the highly organized architecture of the skin nerve end organ. Its further application will give new impetus in the investigation of alterations of this skin compartment under pathological conditions.

Presence of β-arrestin-1 immunoreactivity in the cutaneous nerve fibers of rat glabrous skin

β-Arrestin-1 (βArr1) plays a major role in the desensitization and internalization of G protein-coupled receptors. We previously localized βArr1 in the sensory neurons of rat lumbar 4 and 5 dorsal root ganglia (DRG) and reported the predominant presence of βArr1 in the small-diameter DRG neurons that are often implicated with nociception. Because of βArr1’s crucial role in regulating the initiation of cellular signaling, in the current study we evaluated the distribution of βArr1 in the peripheral sensory terminals where various receptors are present. Western blotting confirmed the presence of βArr1 immunoreactivity in the rat skin. Sciatic nerve ligation demonstrated that βArr1 is transported peripherally from the DRG, and immunohistochemistry showed βArr1 immunoreactivity in the glabrous skin of the rat hindpaw. In the glabrous skin, strong βArr1 immunoreactivity was detected in nerve fibers in the dermal nerve plexus and dermal papillae. Fine varicose immunoreactive fibers were found in the epidermis. In addition, βArr1 was observed in specialized sensory receptors such as Meissner corpuscles. Our observations thus indicate that βArr1 may be involved in modulation of specific tactile stimulation from the skin in addition to nociception.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 70

The availability of antibodies against the pan‐axonal marker protein‐gene‐protein 9.5 (PGP) allowed to definitely demonstrate the existence of small‐diameter nerve fibers within the vital layers of the epidermis and prompted to their quantification in sensory neuropathies. However, morphological features of intra‐epidermal nerve fibers (IENF) have not been fully characterized. We performed immunohistochemical and confocal immunofluorescence studies in skin biopsies taken in 15 healthy subjects at the proximal thigh or the distal leg using anti‐PGP, anti‐unique b‐tubulin (TuJ1), anti‐non‐phosphorylated microtubule‐associated protein‐1B (MAP‐1B), anti‐70 and 200 kDa neurofilament (NF), anti‐phosphorylated neurofilament‐312 (SMI), anti‐peripheral myelin protein‐22 (PMP22), anti‐basic myelin protein (MBP), and anti‐myelin associated glycoprotein (MAG) antibodies. IENF density was quantified under light microscope using PGP, TuJ1, MAP‐1B, NF, and SMI immunostaining. In each site, the density of TuJ1‐ and MAP‐1B‐positive IENF was similar to that of PGP‐positive IENF, whereas that of NF‐positive IENF was significantly lower. Only sporadic SMI‐positive IENF were found. Double confocal staining studies confirmed that TuJ1 and MAP‐1B diffusely co‐localized with PGP in IENF. These findings suggest that IENF cytoskeleton is predominantly composed by tubules, whereas neurofilaments are less represented. No IENF showed PMP22, MBP, or MAG labeling. All dermal nerve fibers presented PMP22 immunoreactivity, which labeled Schwann cell membrane, whereas only a few of them were MBP‐ or MAG‐positive, confirming that most fibers are unmyelinated. Double staining confocal microscope studies revealed that PMP22 labeling in dermal nerves stopped at the dermal‐epidermal junction, whereas TuJ1‐positive axons crossed it and run between keratinocytes. This definitely demonstrates that IENF are naked axons, arising from sub‐papillary unmyelinated nerves which loose the Schwann cell at the dermal‐epidermal junction. The presence of naked nerve fibers within the epidermis suggests that functional interactions with the resident cells, even in sensory transduction, might exist.