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Aims: To develop a standardised, automated protocol for detecting protein gene product 9.5 (PGP9.5) positive intra-epidermal nerve fibres (IENFs) in skin biopsies, transitioning from the established manual technique to an automated platform. This automated method, although currently intended for research applications, may improve the accessibility of this diagnostic test for small fibre neuropathy in clinical settings., Methods: Skin biopsies (n = 274) from 100 participants (fibromyalgia syndrome n = 62; idiopathic small fibre neuropathy: n = 16; healthy volunteers: n = 22) were processed using an automated immunohistochemistry platform. IENF quantification was performed by blinded examiners, with reliability assessed via a two-way mixed-effects model to evaluate inter- and intra-observer variability., Results: The automated staining system reproduced intra-epidermal nerve fibre density (IENFD) counts consistent with free-floating sections (mean ± standard deviation: free-floating: 5.6 ± 3.4 fibres/mm; automated: 5.9 ± 3.2 fibres/mm). A median difference of 0.3 with a lower bound 95% Confidence Interval (CI) at -0.00005 established non-inferiority against a margin of -0.4 (p = .08). Specifically, the inter-class correlation coefficient (class denotes consistency in measured observations) was 99% (95% CI: 0.9-1), indicating excellent agreement between free-floating and automated methods. The inter- and intra-class coefficient between examiners were both 99% (95% CI: 0.9-0.1) for IENFD, demonstrating high reliability using sections stained using the automated method., Interpretation: Automated immunohistochemistry provides high-throughput reliable and reproducible intra-epidermal nerve fibre quantification. This method, although currently proof-of-concept, for research use only, may be more widely deployed in histopathology laboratories to increase the adoption of IENFD assessment for the diagnosis of peripheral neuropathies., (© 2024 The Author(s). Journal of the Peripheral Nervous System published by Wiley Periodicals LLC on behalf of Peripheral Nerve Society.)

Epidermal hyperinnervation is a critical feature of pruritus during skin inflammation. However, the mechanisms underlying epidermal hyperinnervation are unclear. This study investigates the role of the transcription factor EGR1 in epidermal innervation by utilizing wild-type (Egr1 +/+ ) and Egr1-null (Egr1 ‒/‒ ) mice topically applied Dermatophagoides farinae extract from dust mite. Our findings revealed that Egr1 ‒/‒ mice exhibited reduced scratching behaviors and decreased density of epidermal innervation compared with Egr1 +/+ mice. Furthermore, we identified artemin, a neurotrophic factor, as an EGR1 target responsible for Dermatophagoides farinae extract-induced hyperinnervation. It has been demonstrated that Dermatophagoides farinae extract stimulates toll-like receptors in keratinocytes. To elucidate the cellular mechanism, we stimulated keratinocytes with Pam3CSK4, a toll-like receptor 1/2 ligand. Pam3CSK4 triggered a toll-like receptor 1/2-mediated signaling cascade involving IRAK4, IκB kinase, MAPKs, ELK1, EGR1, and artemin, leading to increased neurite outgrowth and neuronal migration. In addition, increased expression of EGR1 and artemin was observed in the skin tissues of patients with atopic dermatitis. These findings highlight the significance of the EGR1-artemin axis in keratinocytes, promoting the process of epidermal innervation and suggesting it as a potential therapeutic target for alleviating itch and pain associated with house dust mite-induced skin inflammation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Diagnosing small fiber neuropathies can be challenging. To address this issue, whether serum neurofilament light chain (sNfL) could serve as a potential biomarker of damage to epidermal Aδ- and C-fibers was tested., Methods: Serum NfL levels were assessed in 30 patients diagnosed with small fiber neuropathy and were compared to a control group of 19 healthy individuals. Electrophysiological studies, quantitative sensory testing and quantification of intraepidermal nerve fiber density after skin biopsy were performed in both the proximal and distal leg., Results: Serum NfL levels were not increased in patients with small fiber neuropathy compared to healthy controls (9.1 ± 3.9 and 9.4 ± 3.8, p = 0.83) and did not correlate with intraepidermal nerve fiber density at the lateral calf or lateral thigh or with other parameters of small fiber impairment., Conclusion: Serum NfL levels cannot serve as a biomarker for small fiber damage., (© 2024 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Diabetic neuropathy is a disorder characterized by impaired nerve function and reduction of the number of epidermal nerve fibers per epidermal surface. Additionally, as neuropathy related nerve fiber loss and regrowth progresses over time, the two-dimensional spatial arrangement of the nerves becomes more clustered. These observations suggest that with development of neuropathy, the spatial pattern of diminished skin innervation is defined by a thinning process which remains incompletely characterized. We regard samples obtained from healthy controls and subjects suffering from diabetic neuropathy as realisations of planar point processes consisting of nerve entry points and nerve endings, and propose point process models based on spatial thinning to describe the change as neuropathy advances. Initially, the hypothesis that the nerve removal occurs completely at random is tested using independent random thinning of healthy patterns. Then, a dependent parametric thinning model that favors the removal of isolated nerve trees is proposed. Approximate Bayesian computation is used to infer the distribution of the model parameters, and the goodness-of-fit of the models is evaluated using both non-spatial and spatial summary statistics. Our findings suggest that the nerve mortality process changes as neuropathy advances., (© 2023 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.)

Small fiber neuropathy (SFN) affects unmyelinated and thinly myelinated nerve fibers causing neuropathic pain with distal distribution and autonomic symptoms. In idiopathic SFN (iSFN), 30% of the cases, the underlying aetiology remains unknown. Gadolinium (Gd)-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI). However, side-effects including musculoskeletal disorders and burning skin sensations were reported. We investigated if dermal Gd deposits are more prevalent in iSFN patients exposed to GBCAs, and if dermal nerve fiber density and clinical parameters are likewise affected. 28 patients (19 females) with confirmed or no GBCA exposure were recruited in three German neuromuscular centers. ISFN was confirmed by clinical, neurophysiological, laboratory and genetic investigations. Six volunteers (two females) served as controls. Distal leg skin biopsies were obtained according to European recommendations. In these samples Gd was quantified by elemental bioimaging and intraepidermal nerve fibers (IENF) density via immunofluorescence analysis. Pain phenotyping was performed in all patients, quantitative sensory testing (QST) only in a subset (15 patients; 54%). All patients reported neuropathic pain, described as burning (n = 17), jabbing (n = 16) and hot (n = 11) and five QST scores were significantly altered. Compared to an equal distribution significantly more patients reported GBCA exposures (82%), while 18% confirmed no exposures. Compared to unexposed patients/controls significantly increased Gd deposits and lower z-scores of the IENF density were confirmed in exposed patients. QST scores and pain characteristics were not affected. This study suggests that GBCA exposure might alter IENF density in iSFN patients. Our results pave the road for further studies investigating the possible role of GBCA in small fiber damage, but more investigations and larger samples are needed to draw firm conclusions., (© 2023. The Author(s).)

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research., (© 2023. The Author(s).)

Introduction/aims: Schwann cell clusters have been described at the murine dermis-epidermis border. We quantified dermal Schwann cells in the skin of patients with small-fiber neuropathy (SFN) compared with healthy controls to correlate with the clinical phenotype., Methods: Skin punch biopsies from the lower legs of 28 patients with SFN (11 men, 17 women; median age, 54 [range, 19-73] years) and 9 healthy controls (five men, four women, median age, 34 [range, 25-69] years) were immunoreacted for S100 calcium-binding protein B as a Schwann cell marker, protein-gene product 9.5 as a pan-neuronal marker, and CD207 as a Langerhans cell marker. Intraepidermal nerve fiber density (IENFD) and subepidermal Schwann cell counts were determined., Results: Skin samples of patients with SFN showed lower IENFD (P < .05), fewer Schwann cells per millimeter (P < .01), and fewer Schwann cell clusters per millimeter (P < .05) than controls. When comparing SFN patients with reduced (n = 13; median age, 53 [range, 19-73] years) and normal distal (n = 15, median age, 54 [range, 43-68] years) IENFD, the number of solitary Schwann cells per millimeter (p < .01) and subepidermal nerve fibers associated with Schwann cell branches (P < .05) were lower in patients with reduced IENFD. All three parameters correlated positively with distal IENFD (P < .05 to P < .01), whereas no correlation was found between Schwann cell counts and clinical pain characteristics., Discussion: Our data raise questions about the mechanisms underlying the interdependence of dermal Schwann cells and skin innervation in SFN. The temporal course and functional impact of Schwann cell presence and kinetics need further investigation., (© 2022 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta ) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin , to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Diagnosis of ALS is based on clinical symptoms when motoneuron degeneration is significant. Therefore, new approaches for early diagnosis are needed. We aimed to assess if alterations in appearance and cellular localization of cutaneous TDP-43 may represent a biomarker for ALS. Skin biopsies from 64 subjects were analyzed: 44 ALS patients, 10 healthy controls (HC) and 10 neurological controls (NC) (Parkinson's disease and multiple sclerosis). TDP-43 immunoreactivity in epidermis and dermis was analyzed, as well as the percentage of cells with TDP-43 cytoplasmic localization. We detected a higher amount of TDP-43 in epidermis ( p < 0.001) and in both layers of dermis ( p < 0.001), as well as a higher percentage of TDP-43 cytoplasmic positive cells ( p < 0.001) in the ALS group compared to HC and NC groups. Dermal cells containing TDP-43 were fibroblasts as identified by co-labeling against vimentin. ROC analyses (AUC 0.867, p < 0.001; CI 95% 0.800-0.935) showed that detection of 24.1% cells with cytoplasmic TDP-43 positivity in the dermis had 85% sensitivity and 80% specificity for detecting ALS. We have identified significantly increased TDP-43 levels in epidermis and in the cytoplasm of dermal cells of ALS patients. Our findings provide support for the use of TDP-43 in skin biopsies as a potential biomarker.

Peripheral neuropathy is a condition associated with poor nerve functionality. Epidermal nerve fiber (ENF) counts per epidermal surface are dramatically reduced and the two-dimensional (2D) spatial structure of ENFs tends to become more clustered as neuropathy progresses. Therefore, studying the spatial structure of ENFs is essential to fully understand the mechanisms that guide those morphological changes. In this article, we compare ENF patterns of healthy controls and subjects suffering from mild diabetic neuropathy by using suction skin blister specimens obtained from the right foot. Previous analysis of these data has focused on the analysis and modeling of the spatial ENF patterns consisting of the points where the nerves enter the epidermis, base points, and the points where the nerve fibers terminate, end points, projected on a 2D plane, regarding the patterns as realizations of spatial point processes. Here, we include the first branching points, the points where the nerve trees branch for the first time, and model the three-dimensional (3D) patterns consisting of these three types of points. To analyze the patterns, spatial summary statistics are used and a new epidermal active territory that measures the volume in the epidermis that is covered by the individual nerve fibers is constructed. We developed a model for both the 2D and the 3D patterns including the branching points. Also, possible competitive behavior between individual nerves is examined. Our results indicate that changes in the ENFs spatial structure can more easily be detected in the later parts of the ENFs., (© 2021 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.)

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca 2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca 2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca 2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca 2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca 2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 μM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP + /IB4 - nociceptors in rodents and in CGRP + dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics., (© 2021. The Author(s).)

Intraepidermal nerve fiber density (IENFD) measurements in skin biopsy are performed manually by 1-3 operators. To improve diagnostic accuracy and applicability in clinical practice, we developed an automated method for fast IENFD determination with low operator-dependency. Sixty skin biopsy specimens were stained with the axonal marker PGP9.5 and imaged using a widefield fluorescence microscope. IENFD was first determined manually by 3 independent observers. Subsequently, images were processed in their Z-max projection and the intradermal line was delineated automatically. IENFD was calculated automatically (fluorescent images automated counting [FIAC]) and compared with manual counting on the same fluorescence images (fluorescent images manual counting [FIMC]), and with classical manual counting (CMC) data. A FIMC showed lower variability among observers compared with CMC (interclass correlation [ICC] = 0.996 vs 0.950). FIMC and FIAC showed high reliability (ICC = 0.999). A moderate-to-high (ICC = 0.705) was observed between CMC and FIAC counting. The algorithm process took on average 15 seconds to perform FIAC counting, compared with 10 minutes for FIMC counting. This automated method rapidly and reliably detects small nerve fibers in skin biopsies with clear advantages over the classical manual technique., (© 2021 American Association of Neuropathologists, Inc.)

Transplantation of embryonic motor neurons has been shown to improve motor neuron survival and innervation of neuromuscular junctions in peripheral nerves. However, there have been no reports regarding transplantation of sensory neurons and innervation of sensory receptors. Therefore, we hypothesized that the transplantation of embryonic sensory neurons may improve sensory neurons in the skin and innervate Merkel cells and Meissner's corpuscles. We obtained sensory neurons from dorsal root ganglia of 14-day rat embryos. We generated a rat model of Wallerian-degeneration by performing sciatic nerve transection and waiting for one week after. Six months after cell transplantation, we performed histological and electrophysiological examinations in naïve control, surgical control, and cell transplantation groups. The number of nerve fibers in the papillary dermis and epidermal-dermal interface was significantly greater in the cell transplantation than in the surgical control group. The percent of Merkel cells with nerve terminals, as well as the average number of Meissner corpuscles with nerve terminals, were higher in the cell transplantation than in the surgical control group, but differences were not significant between the two groups. Moreover, the amplitude and latency of sensory conduction velocity were evoked in rats of the cell transplantation group. We demonstrated that the transplantation of embryonic dorsal root ganglion cells improved sensory nerve fiber number and innervation of Merkel cells and Meissner's corpuscles in peripheral nerves., (© 2021 John Wiley & Sons Ltd.)

The glabrous skin around the nostrils in mammals is called a rhinarium or planum nasale. Rhinarium skin has multiple epidermal domes that are generally assumed to form a tactile surface. The rhinarium is innervated by a branch of the trigeminal nerve which is associated with stimuli such as touch, chemical irritants and temperature. In this study, our aim was to correlate variation in rhinarium skin sensory innervation with different feeding behaviors while also covering a broad systematic spectrum. Using histological and immunohistological methods, we studied skin morphology, nerve fiber density and nerve fiber distribution in the rhinarium epidermal domes of four species: cow, ring-tailed lemur, brown bear, and dog, that all exhibit different feeding behaviors. All species share similar traits in rhinarium skin morphology, but glands were only found in cow rhinarium skin. The most substantial differences were observed in the innervation pattern. Mechanosensory skin organs were found only in the ring-tailed lemur. Dog epidermal domes possess a pronounced central dermal papilla containing a nerve bundle in its top, close to the skin surface. The abundance of free epidermal nerve fibers in epidermal domes of all species, suggest that the rhinarium skin is a sensory surface, that can be used to detect fine touch, chemical irritants or temperature. In the species where the whole epidermal dome was examined, the intraepidermal nerve fiber density is higher in the central part of the domes. The nerve distribution and the central positioning of a single gland duct in cow and the dermal papilla top organ in dog indicates that each epidermal dome can be considered a functional unit. The observed differences in innervation hint at different sensory functions of rhinaria in mammals that may be correlated to feeding behavior., (© 2021 The Authors. Journal of Morphology published by Wiley Periodicals LLC.)

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein‒coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2‒evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2‒elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons., Methods: Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies., Results: Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release., Interpretation: By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205-1219., (© 2020 American Neurological Association.)

Objective: To investigate small fiber innervation of the skin and its relationships with clinicometry of autism and peripheral afferents for contact heat-evoked potential (CHEP) and psychophysical measures of thermal thresholds., Methods: We recruited 32 men with autism (26.5 ± 5.9 years) and conducted small fiber assessments of skin biopsy with quantifying intraepidermal nerve fiber (IENF) density, CHEP, quantitative sensory testing, and large fiber physiology of nerve conduction studies. Results were compared with age-matched controls and analyzed with clinical measures of autism., Results: Patients with autism showed a lower IENF density than controls (5.53 ± 2.09 vs 11.13 ± 3.49 fibers/mm, p < 0.0001). The IENF density was reduced in 17 (53.1%) men with autism classified as skin denervation group. On psychophysics, 9 (28%) men with autism had elevated thermal thresholds, and the warm threshold of the big toe was negatively correlated with IENF density ( p = 0.0073), indicating functional impairments of small fiber sensory nerves. IENF density was negatively correlated with CHEP amplitude in autism ( p = 0.003), in contrast to the pattern of positive correlation in controls, indicating different processing of nociceptive afferent in autism. Clinically, IENF density was related to distinct tactile symptom patterns in the skin denervation vs normal innervation group, respectively. Furthermore, IENF density was associated with autistic symptoms measured by the Autism Spectrum Quotient in a U-shaped model ( p = 0.014)., Conclusions: These observations indicated that a substantial portion of individuals with autism had small fiber pathology, which was associated with tactile and autistic symptoms, providing structural and physiologic evidence for the involvement of peripheral sensory nerves in autism., (© 2020 American Academy of Neurology.)

Multiple peripheral nerves are known to degenerate after nerve compression injury but the correlation between the extent of nerve alteration and pain severity remains unclear. Here, we used intravital two-photon fluorescence microscopy to longitudinally observe changes in cutaneous fibers in the hind paw of Nav1.8-Cre-tdTomato mice after chronic constriction injury (CCI). Results showed that the CCI led to variable loss of the skin nerve plexus and intraepidermal nerve fibers. The timing of Nav1.8 nerve fiber loss correlated with the development of mechanical hypersensitivity. We compared a scoring approach that assessed whole-paw nerve degeneration with an index that quantified changes in the nerve plexus and terminals in multiple small regions of interest (ROI) from intravital images of the third and fifth toe tips. We found that the number of surviving nerve fibers was not linearly correlated with mechanical hypersensitivity. On the contrary, at 14 days after CCI, the moderately injured mice showed greater mechanical hypersensitivity than the mildly or severely injured mice. This indicates that both surviving and injured nerves are required for evoked neuropathic pain. In addition, these two methods may have the estimative effect as diagnostic and prognostic biomarkers for the assessment of neuropathic pain.

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo . Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD., (Copyright © 2020 Buhl, Ikoma, Kempkes, Cevikbas, Sulk, Buddenkotte, Akiyama, Crumrine, Camerer, Carstens, Schön, Elias, Coughlin and Steinhoff.)

Calcitonin gene-related peptide (CGRP) and adrenomedullin are structurally similar neuropeptides acting as potent vasodilators of blood pressure and mediator of inflammation in skin. Revealing the expression pattern of their common receptor-Calcitonin gene-related peptide receptor (Calcrl) in their targeted cells is important to explain the functions of CGRP and adrenomedullin in skin. Our immunostaining results showed that Calcrl is enriched in hair follicles bulge stem cells and differentially expressed in basal stem cells of interfollicular epidermis. In addition, Calcrl expression in interfollicular epidermis is dependent on presence of nerve fibers. Long-term ablation of the murine cutaneous nerve leads to loss of Calcrl expression in interfollicular epidermis but not in hair follicle bulge stem cells. Our results demonstrate a tight interaction between neuronal components and epidermis, and indicates potential roles of Calcrl in epidermal stem cells.

Small fiber neuropathy (SFN) is being recognized with increasing frequency in neuromuscular practice due to improved diagnostic techniques. Although there are some common etiologies, up to one-third of cases are considered idiopathic. In recent years, several disorders have unexpectedly been reported in association with SFN, on clinical grounds and complementary investigations, including quantitative sensory testing, intraepidermal nerve fiber density and confocal corneal microscopy. Knowledge of these disorders is important in clinical practice as increased awareness enables prompt diagnosis of SFN in these settings and early optimal therapeutic management of affected patients. Furthermore, these new developments may lead to a better understanding of the pathophysiologic mechanisms underlying SFN in these different disorders as well as, in some cases, an expanded spectrum of affected organs and systems. This article reviews these reported associations, their possible pathophysiologic bases, and the potential resulting management implications., (© 2020 Wiley Periodicals, Inc.)

Meissner corpuscles are mechanosensory end organs that densely occupy mammalian glabrous skin. We generated mice that selectively lacked Meissner corpuscles and found them to be deficient in both perceiving the gentlest detectable forces acting on glabrous skin and fine sensorimotor control. We found that Meissner corpuscles are innervated by two mechanoreceptor subtypes that exhibit distinct responses to tactile stimuli. The anatomical receptive fields of these two mechanoreceptor subtypes homotypically tile glabrous skin in a manner that is offset with respect to one another. Electron microscopic analysis of the two Meissner afferents within the corpuscle supports a model in which the extent of lamellar cell wrappings of mechanoreceptor endings determines their force sensitivity thresholds and kinetic properties., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Pruritus is a major symptom of atopic dermatitis (AD) and is transmitted by a subpopulation of non-myelinated C-type free nerve endings in the epidermis and upper dermis. Stimulation of these nerve terminals is affected by histamine, neurotrophins and physical factors. Eosinophils of patients with AD are a source of neurotrophins, including brain-derived neurotrophic factor (BDNF), levels of which correlate with disease severity., Objective: The purpose of this study was to determine the anatomical localization of eosinophils in the skin of patients with AD with regard to peripheral nerves and to investigate whether eosinophils induce sprouting and neurite outgrowth in murine sensory neurons., Methods: Cryosections of skin derived from AD and control (NA) patients were subjected to immunofluorescence analysis with markers for eosinophils, BDNF and neuronal cells. Stimulated eosinophil supernatants were used for the treatment of cultured peripheral mouse dorsal root ganglia (DRG) neurons followed by morphometric analysis., Results: Dermal axon density and the proximity of eosinophils to nerve fibres were significantly higher in AD patients vs NA. Both neuronal projections and eosinophils expressed BDNF. Furthermore, activated eosinophil supernatants induced BDNF-dependent mouse DRG neuron branching., Conclusions and Clinical Relevance: Our results indicate that BDNF-positive eosinophils are also localized in close proximity with nerve fibres in AD, suggesting a functional relationship between BDNF-expressing eosinophils and neuronal projections. These observations suggest that eosinophils may have considerable impact on pruritus by supporting sensory nerve branching., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Cleaning products such as soaps, shampoos, and detergents are comprised mainly of surfactants, agents known to cause dermatitis and cutaneous hypersensitivity characterized by itching, stinging, and burning of the skin and scalp. However, the mechanisms underlying surfactant-induced cutaneous hypersensitivity remain unclear. In the present study, we investigated the mechanisms of cutaneous hypersensitivity in mice treated with the detergent sodium dodecyl sulfate (SDS). Repeated SDS application to the skin induced inflammation, xeroderma, and elongation of peripheral nerves into the epidermis. The number of neurons immunopositive for c-Fos, a well known marker of neural activity, was substantially higher (+441%) in spinal dorsal horn (SDH) lamina I-II (but not lamina III-VI) of SDS-treated mice compared to vehicle-treated mice. In vivo extracellular recording revealed enhanced spontaneous (+64%) and non-noxious mechanical stimulation-evoked firing (+139%) of SDH lamina I-II neurons in SDS-treated mice, and stimulation-evoked neuronal firing was sustained (+5333%) even after stimulation. The number of GFAP-positive (activated) astrocytes, but not Iba1-positive microglia, was also elevated (+137%) in SDH lamina I-II of SDS-treated mice compared to vehicle-treated mice. Peripheral nerve elongation and hyperexcitability of afferent or SDH neurons, possible associated with the activation of spinal astrocytes, may underlie cutaneous hypersensitivity induced by surfactants., Competing Interests: Declaration of Competing Interest Dr. Inami, Yosuke Mano, and Miki Fukushima are employed by Hoyu Co., Ltd. who financially supported the research., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Small fiber neuropathies (SFN) are associated with a reduction in quality of life. In adults, epidermal nerve fiber density (END) analysis is recommended for the diagnosis of SFN. In children, END assessment is not often performed. We analyzed small nerve fiber innervation to elucidate the potential diagnostic role of skin biopsies in young patients with pain., Methods: Epidermal nerve fiber density and sudomotor neurite density (SND) were assessed in skin biopsies from 26 patients aged 7 to 20 years (15 female patients) with unexplained chronic pain. The results were compared with clinical data., Results: Epidermal nerve fiber density was abnormal in 50% and borderline in 35% of patients. An underlying medical condition was found in 42% of patients, including metabolic, autoimmune, and genetic disorders., Discussion: Reduction of epidermal nerve fibers can be associated with treatable conditions. Therefore, the analysis of END in children with pain may help to uncover a possible cause and guide potential treatment options., (© 2019 The Authors. Muscle & Nerve published by Wiley Periodicals, Inc.)

Introduction: We characterized dermal innervation in patients with fibromyalgia syndrome (FMS) as potential contribution to small fiber pathology., Methods: Skin biopsies of the calf were collected (86 FMS patients, 35 healthy controls). Skin was immunoreacted with antibodies against protein gene product 9.5, calcitonine gene-related peptide, substance P, CD31, and neurofilament 200 for small fiber subtypes. We assessed two skin sections per patient; on each skin section, two dermal areas (150 x 700 μm each) were investigated for dermal nerve fiber length (DNFL)., Results: In FMS patients we found reduced DNFL of fibers with vessel contact compared to healthy controls (p<0.05). There were no differences for the other nerve fiber subtypes., Discussion: We found less dermal nerve fibers in contact with blood vessels in FMS patients than in controls. The pathophysiological relevance of this finding is unclear, but we suggest the possibility of a relationship with impaired thermal tolerance commonly reported by FMS patients., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine the feasibility of examining intraepidermal nerve fiber density (IENFD) in postmortem skin., Methods: From 12 subjects, 3-mm skin punch biopsies were collected 1-4 days postmortem from the proximal leg and distal leg, with a mean (range) interval from the death of 37 (15-91) hours. Causes of death varied broadly, including hepatocellular carcinoma, chronic lymphocytic leukemia, generalized atherosclerosis, progressive supranuclear palsy, Parkinson disease, emphysema, and obesity. The mean (range) number of sections evaluated from each biopsy was 5.08 (2-6) from the proximal leg and 5.92 (5-6) from the distal leg. Sections were stained with PGP 9.5 for blinded counting using bright field microscopy. Qualitative and quantitative assessment of feasibility included a comparison of fiber staining with that in healthy subjects and mean IENFD in postmortem samples. Interobserver reliability was assessed among 3 blinded raters by calculating intraclass correlation coefficients and percentage variability of IENFD in at least 4 sections from biopsies in 5 healthy subjects., Results: Intraobserver and interobserver correlation coefficients of blinded IENFD counts undertaken by 4 authors were consistently >0.80, and the coefficient of variation was ≤10%. The quality of staining in postmortem samples was comparable with that in healthy subjects and was not substantially affected by time from death to specimen collection of up to nearly 4 days. Mean (range) IENFD from postmortem samples in the proximal and distal leg was 2.73 (0-7.65) and 1.93 (0-4.91) fibers/mm of skin, respectively. Two of 3 patients who had received chemotherapy during life showed a nearly complete absence of intraepidermal nerve fibers., Conclusions: IENFD measurement in postmortem skin is feasible and may be used to study the epidemiology of SFN.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious adverse side effect of cancer treatment with no Food and Drug Administration-approved medication for its prevention or management. Using RNA sequencing analysis of dorsal root ganglia (DRG), we identify critical contributions of histone deacetylase 6 (HDAC6) and mitochondrial damage to the establishment of CIPN in a mouse model of cisplatin-induced neuropathy. We show that pharmacological inhibition of HDAC6 using ACY-1215 or global deletion of HDAC6 is sufficient to prevent cisplatin-induced mechanical allodynia, loss of intraepidermal nerve fibers (IENFs), and mitochondrial bioenergetic deficits in DRG neurons and peripheral nerves in male and female mice. The bioenergetic deficits in the neuronal cell bodies in the DRG are characterized by reduced oxidative phosphorylation, whereas the mitochondrial deficits in the nerves are due to a reduction in axonal mitochondrial content. Notably, deleting HDAC6 in sensory neurons protects against the cisplatin-induced loss of IENFs and the reduction in mitochondrial bioenergetics and content in the peripheral nerve. By contrast, deletion of HDAC6 in sensory neurons only partially and transiently prevents cisplatin-induced mechanical allodynia and does not protect against impairment of mitochondrial function in DRG neurons. We further reveal a critical role of T cells in the protective effects of HDAC6 inhibition on these signs of CIPN. In summary, we show that cisplatin-induced mechanical allodynia is associated with mitochondrial damage in DRG neurons, whereas the loss of IENFs is related to bioenergetic deficits in peripheral nerves. Moreover, our findings identify cell-specific contributions of HDAC6 to mechanical allodynia and loss of IENFs that characterize cisplatin-induced peripheral neuropathy.

Itch, also known as pruritus, is an unpleasant sensation that results in an urge to scratch. We can feel itch at any location where itch occurs from the top of the head to the toes. However, there are topographical differences in itch intensity. Itch is mainly conducted by C nerve fibres from the skin where itch emanates to the central nervous system. However, the abundancy of C fibres does not necessarily lead to higher itch intensity. Interestingly, reduction and/or structural changes in C fibres seem to play a role in itch sensation. In addition, C tactile fibres (CT afferents), which are activated by gentle "affective" touch and seem to be associated with scratching pleasurability and the reward system in the brain, can be involved in itch sensation and topographical differences of itch., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Current strategies to enhance regeneration of peripheral neurons involve broad activation of sensory, autonomic, and motor axons. Peripheral neuron regeneration is limited in persons with damage or disease of peripheral axons. Here, we provide evidence that subtoxic activation of TRPV1 channels in sensory neurons is associated with activation of growth and subtle changes in skin reinnervation. We identify a bidirectional, dose-related impact of capsaicin, a TRPV1 agonist, on sensory neurons and their axons with rises in their outgrowth plasticity at low doses and toxic neurodegeneration at high doses. Moreover, its impact on growth added to that of preconditioning. Neither outcome was observed in TRPV1 null neurons. We confirmed that low dose activation was associated with rises in neuronal calcium, as well as rises in TRPV1 mRNA transcripts. In mice with a sciatic nerve crush followed by a single application of capsaicin directly to the injury site, there was no impact on motor or myelinated axon recovery but there was evidence of better recovery of thermal sensation toward baseline with hyperalgesia. Moreover, skin reinnervation by epidermal axons approached contralateral levels. TRPV1 null mice displayed loss of thermal sensation during later recovery. In sensory axons innervating the pinna of the ear, local capsaicin rendered early axon loss followed by later hyperinnervation. Taken together, TRPV1 activation alters the regenerative behavior of adult neurons and their axons both in vitro and during epidermal reinnervation in vivo. The findings identify a selective manipulation that augments cutaneous innervation by thermosensitive axons.

Objective: To assess the effect of diabetes type on the long-term rate and extent of epidermal nerve regeneration., Methods: Subjects with well controlled type 1 diabetes mellitus (n = 11) or type 2 diabetes mellitus (n = 36), with normal nerve conduction studies and baseline intraepidermal nerve fiber density (IENFD), and healthy controls (n = 10) underwent chemical axotomy of the intraepidermal nerves at the thigh using topical capsaicin. Skin biopsies were performed at 30, 90, 150, and 180 days post-axotomy., Results: After 180 days, IENFD in diabetic subjects remained significantly below baseline levels, while healthy controls returned to normal. At each time point, regeneration rates were significantly slower among diabetic subjects, although type 1 subjects regenerated significantly faster and achieved higher percentages of baseline IENFD compared with type 2., Interpretation: Among diabetic patients, nerve injury recovery is likely to take significantly longer than in healthy individuals, and remains incomplete, particularly among type 2 patients. This may partially explain the progression of neuropathy among diabetic patients: damage accumulates because nerve recovery is slowed and incomplete. Furthermore, these findings support caution when recommending certain procedures, such as carpal tunnel repair, to patients with progressed diabetic disease., (© 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

We introduce the Thomas process in a Bayesian hierarchical setting as a model for point pattern data with a nested structure. This model is applied to a nerve fiber data set which consists of several point patterns of nerve entry points from 47 subjects divided into 3 groups, where the grouping is based on the diagnosed severity of a certain nerve disorder. The modeling assumption is that each point pattern is a realization of a Thomas process, with parameter values specific to the subject. These parameter values are in turn assumed to come from distributions that depend on which group the subject belongs to. To fit the model, we construct an MCMC algorithm, which is evaluated in a simulation study. The results of the simulation study indicate that the group level mean of each parameter is well estimated, but that the estimation of the between subject variance is more challenging. When fitting the model to the nerve fiber data, we find that the structure within clusters appears to be the same in all groups, but that the number of clusters decreases with the progression of the nerve disorder., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The epidermal barrier is thought to protect sensory nerves from overexposure to environmental stimuli, and barrier impairment leads to pathological conditions associated with itch, such as atopic dermatitis (AD). However, it is not known how the epidermal barrier continuously protects nerves for the sensory homeostasis during turnover of the epidermis. Here we show that epidermal nerves are contained underneath keratinocyte tight junctions (TJs) in normal human and mouse skin, but not in human AD samples or mouse models of chronic itch caused by epidermal barrier impairment. By intravital imaging of the mouse skin, we found that epidermal nerve endings were frequently extended and retracted, and occasionally underwent local pruning. Importantly, the epidermal nerve pruning took place rapidly at intersections with newly forming TJs in the normal skin, whereas this process was disturbed during chronic itch development. Furthermore, aberrant Ca 2+ increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca 2+ increases in epidermal nerves and itch. These results suggest that epidermal nerve endings are pruned through interactions with keratinocytes to stay below the TJ barrier, and that disruption of this mechanism may lead to aberrant activation of epidermal nerves and pathological itch.

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long-range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long-term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation., (© 2019 Japanese Society of Developmental Biologists.)

Sensing environmental cues requires well-built neuronal circuits linked to the body surface. Sensory neurons generate dendrites to innervate surface epithelium, thereby making it the largest sensory organ in the body. Previous studies have illustrated that neuronal type, physiological function and branching patterns are determined by intrinsic factors. Perhaps for effective sensation or protection, sensory dendrites bind to or are surrounded by the substrate epidermis. Recent studies have shed light on the mechanisms by which dendrites interact with their substrates. These interactions suggest that substrates can regulate dendrite guidance, arborization and degeneration. In this review, we focus on recent studies of Drosophila and Caenorhabditis elegans that demonstrate how epidermal cells can regulate dendrites in several aspects.

Itch-a major symptom of many chronic skin diseases-can exacerbate inflammation by provoking scratching and subsequent skin damage. Here, we show that activation, via near infrared illumination, of a phototoxic agent that selectively targets itch-sensing cells can reduce itch-associated behaviours in mice. We generated a SNAP-tagged interleukin-31 (IL-31) ligand derivative (IL-31 K138A-SNAP ) that selectively binds receptors on itch-associated cells, without evoking IL-31-receptor signalling or scratching, and conjugated it to the photosensitizer IRDye 700DX phthalocyanine. Subcutaneous injection of IL-31 K138A-SNAP -IR700 in mice followed by near infrared illumination resulted in the long-term reversal of the scratching behaviour evoked by the pruritogenic IL-31, an effect that was associated with the selective retraction of itch-sensing neurons in the skin. We also show that a topical preparation of IL-31 K138A-SNAP -IR700 reversed the behavioural and dermatological indicators of disease in mouse models of atopic dermatitis and of the genetic skin disease familial primary localized cutaneous amyloidosis. Targeted photoablation may enable itch control for the treatment of inflammatory skin diseases.

Nociceptin/orphanin FQ opioid peptide (NOP)-receptor (NOP-R) is a member of the opioid receptor family. NOP-R activation has demonstrated analgesic effects in preclinical pain models without the addiction risks associated with other opiate targets. Pachyonychia congenita (PC) is a palmoplantar keratoderma characterized by neuropathic pain in affected skin. A cohort of KRT6A gene mutation PC patients with no other explanation for their neuropathic pain offered a unique opportunity to assess potential of NOP-R as a therapeutic target. Plantar biopsies from 10 PC patients and 10 age/gender matched controls were performed at the ball (PC-affected) and the arch (PC-unaffected) of the foot. NOP-R expression was assessed by immunohistochemistry. Localization of NOP-R in subsets of epidermal nerve fibers was investigated using the pan-neuronal marker PGP9.5, markers for unmyelinated peptidergic fibers (calcitonin gene-related peptide [CGRP] and substance P [SP]), as well as for myelinated Aδ and Aβ fibers (neurofilament H [NFH]). Robust NOP-R expression was detected in epidermal keratinocytes and in a subset of PGP9.5+ fibers in both epidermis and dermis, confirmed by western blot and absorption experiments with NOP-R peptide. NOP-R expression in keratinocytes was significantly reduced in PC-affected plantar skin compared with PC-unaffected skin. In addition, NOP-R expression occurred in dermal NFH+ myelinated fibers in all groups, although few CGRP+ fibers co-expressed NOP-R. Furthermore, most SP+ fibers also co-expressed NOP-R. These findings indicate that NOP-R is expressed on epidermal keratinocytes, as well as on epidermal and dermal nerve fibers and has potential as a promising target to treat neuropathic pain in PC., (© 2018 Peripheral Nerve Society.)

Objective: Findings regarding small nerve fiber damage in complex regional pain syndrome type I (CRPS-I) are not uniform, and studies have not included a matched healthy control group. The aim was to assess intraepidermal nerve fiber density (IENFD) in relation to thermal sensitivity of the same skin areas in CRPS-I patients and a gender- and age-matched healthy control group., Methods: IENFD was investigated in skin biopsies from the CRPS-affected and contralateral limbs of eight CRPS-I patients and from an equivalent site in eight gender- and age-matched healthy controls (HCs). Thermal thresholds (cold/warm detection, cold- and heat-pain detection) were assessed on the affected limb, the matching contralateral limb, and on the equivalent limbs of HCs, and participants rated the intensity of cold/heat and pain to static thermal stimuli (5 °C and 40 °C)., Results: IENFD was significantly lower in both the affected and contralateral limbs of CRPS-I patients than HCs, but IENFD did not differ between the affected and contralateral limbs of patients. The heat pain threshold was lower in the affected CRPS-I limb than in HCs, but all other thermal thresholds were similar in both groups. CRPS-I patients rated the cold stimulus as colder and more painful in the affected limb, and the warm stimulus as hotter, bilaterally, than the HCs., Conclusions: CRPS-I may be associated with bilateral small fiber damage, and perhaps small fiber neuropathy and bilateral disturbances in thermo-sensory perception. These disturbances could stem from a systemic response to injury or might increase the risk of developing CRPS-I after physical trauma.

Introduction: For sequential and somatotopic assessment of small fiber neuropathy, heat pain (HP) tests of hypoalgesia might be used instead of decreased counts of epidermal nerve fibers (ENFs), but then healthy subject reference values of HP thresholds are needed., Methods: Using the Computer Assisted Sensation Evaluator IVc system, HP thresholds of hypoalgesia were estimated for 10 unilateral sites and counts of ENFs for 4 of them in healthy subjects., Results: In healthy subjects, small but statistically significant differences of both HP thresholds of hypoalgesia and counts of ENFs were observed among tested sites. Significant correlations between HP thresholds and counts of ENFs were not found., Discussion: For the studied somatotopic sites, we provide ≥95th and ≥99th percentile reference limits for HP 0.5 and 5 of 1-10 HP thresholds of hypoalgesia and decreased counts of ENFs at ≤5th and ≤1st percentile levels. Muscle Nerve 58: 509-516, 2018., (© 2018 Wiley Periodicals, Inc.)

Background: Topical capsaicin shows efficacy in the treatment of brachioradial pruritus (BRP); however, its mechanisms of action remain unclear., Objective: The effect of capsaicin on the epidermis (i.e. peripheral expression of non-neuronal sensory receptors on keratinocytes, morphological changes in innervation) is still unknown. We aimed to investigate the effect of topical capsaicin on keratinocyte expression of TRP channels and on the intraepidermal nerve fibre density (IENFD) in patients with BRP., Methods: Thirty-one patients with BRP received an 8% capsaicin patch. Biopsies in lesional and non-lesional skin were taken to assess epidermal morphology, keratinocyte expression of TRP channels and IENFD before and 3 weeks after treatment., Results: Treatment with the capsaicin patch led to a significant decrease in itch and paresthetic symptoms (P < 0.05). Keratinocyte morphology is unaltered after capsaicin therapy. Reduced keratinocyte expression of TRPV1 in lesional skin (P = 0.009; n = 9) normalized 3 weeks after treatment (P = 0.016; n = 10), but not the IENFD, which remained reduced in lesional epidermis., Conclusion: The normalization of the decreased TRPV1 expression may account for the effectiveness of topical capsaicin, which does not reconstitute the reduced IENFD, arguing for a role of epidermal TRPV1 in the maintenance of BRP., (© 2018 European Academy of Dermatology and Venereology.)

The under-reporting of pain and atypical manifestations of painful syndromes within the elderly population have been well documented, however, the specific relationship between pain and aging remains ambiguous. Previous studies have reported degenerative changes in primary afferents with aging. In this study, we questioned whether there is any change in the density of primary afferent endings within the epidermis of aged animals. Rats were categorically assessed in 4 age groups, each representing a key developmental stage across their life span: juvenile (2 months), adult (7 months); aged (18 months), and senescent (24-26 months). The plantar hind paw skin was removed, post-fixed, cut, and immunostained for protein gene product 9.5 and type IV collagen. Rats in the adult aged groups had significantly increased epidermal nerve densities and total lengths of immunoreactive nerve fibers, compared with juvenile as well as senescent rats. However, the paw withdrawal thresholds to punctate mechanical stimulation progressively increased with age, and did not exhibit a clear relationship with epidermal innervation. We conclude a nonlinear, inverted-U shaped relationship between rat plantar epidermal nerve density with aging, which does not correlate with mechanically-induced paw withdrawal behaviors., Perspective: This article presents age-related decreased epidermal innervation in rat hind paw skin, which partly explains mechanisms underlying decreased pain sensitivity in aged subjects. The report may help clinicians to understand that any compromise of pain-sensing pathway can lead to under-reporting of pain, inadequate analgesia, and slower recovery from a painful condition., (Copyright © 2018 The American Pain Society. Published by Elsevier Inc. All rights reserved.)

The mechanisms responsible for painful and insensate diabetic neuropathy are not completely understood. Here, we have investigated sensory neuropathy in the Ins2 +/Akita mouse, a hereditary model of diabetes. Akita mice become diabetic soon after weaning, and we show that this is accompanied by an impaired mechanical and thermal nociception and a significant loss of intraepidermal nerve fibers. Electrophysiological investigations of skin-nerve preparations identified a reduced rate of action potential discharge in Ins2 +/Akita mechanonociceptors compared with wild-type littermates, whereas the function of low-threshold A-fibers was essentially intact. Studies of isolated sensory neurons demonstrated a markedly reduced heat responsiveness in Ins2 +/Akita dorsal root ganglion (DRG) neurons, but a mostly unchanged function of cold-sensitive neurons. Restoration of normal glucose control by islet transplantation produced a rapid recovery of nociception, which occurred before normoglycemia had been achieved. Islet transplantation also restored Ins2 +/Akita intraepidermal nerve fiber density to the same level as wild-type mice, indicating that restored insulin production can reverse both sensory and anatomical abnormalities of diabetic neuropathy in mice. The reduced rate of action potential discharge in nociceptive fibers and the impaired heat responsiveness of Ins2 +/Akita DRG neurons suggest that ionic sensory transduction and transmission mechanisms are modified by diabetes., (© 2018 by the American Diabetes Association.)

We determined whether compensating ceramides in the stratum corneum (SC) may ameliorate the impaired barrier function and subsequently attenuate the enhanced skin sensitivity. Treatment for 4 weeks with the ceramide complex cream or the placebo cream significantly ameliorated the intensity of lactic acid sensations in 39 female subjects with sensitive skin, the degree of which was attenuated to a greater extent at 1 week by the ceramide complex cream compared with the placebo cream. The amelioration of skin sensations was accompanied by a significant increase in total ceramide content in the SC elicited by the ceramide complex cream that was significantly more effective than the placebo cream at 4 weeks. Consistently, TEWL and conductance values were significantly decreased or increased at 1 and 4 weeks, respectively, to a greater extent by the ceramide complex cream than by the placebo cream. TEWL levels were significantly correlated with the increased levels of SC total ceramide in the ceramide complex cream-treated skin but not in the placebo cream-treated skin. Thus, the amelioration of lactic acid sensations by topical application of a ceramide complex cream, provides a deep insight into the pathophysiology of sensitive skin as a reduced barrier function-dependent sub-clinical sensory response.

Intense chronic itch significantly reduces quality of life for atopic dermatitis patients, impairing daily activity. Although abnormal itch sensation can be induced by innocuous stimuli, known as alloknesis, the mechanisms driving this process remain obscure. Psychological and environmental stimuli are known to aggravate atopic dermatitis symptoms. Recently, the enzyme 11β-hydroxysteroid dehydrogenase-1 (HSD11β1), which is expressed in keratinocytes, has been implicated in maintaining homeostasis against environmental stimuli by activating endogenous glucocorticoids. To investigate the role of HSD11β1 in keratinocytes, we generated keratinocyte-specific Hsd11b1-knockout (Hsd11b1 KC-/- ) mice and analyzed skin phenotype. Hsd11b1 KC-/- mice exhibited abnormal cutaneous innervation and skin sensitivity, including light mechanical stimulus-evoked itch (i.e., alloknesis). Attenuated endogenous glucocorticoid activation induced by aberrant artemin production in keratinocytes was involved in alloknesis in Hsd11b1 KC-/- mice. Finally, we observed a significant negative correlation between expression of HSD11β1 and artemin in human skin with and without AD. These results suggest that endogenous glucocorticoids that maintain skin homeostasis in the epidermis affect both skin innervation and cutaneous sensation. Modulation of HSD11β1 activation could be a therapeutic target for sensitive or itchy skin., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic skin diseases with itch and dry skin show increased peripheral nerve fiber elongation into the epidermis. However, the characteristics of the elongated nerve fibers remain unclear. Therefore, we investigated the characteristics of the elongated nerve fibers using a dry skin mouse model with itch. In this mouse model, prepared via repetitive treatments with an acetone/ether mixture and water, the stratum corneum water content was decreased, whereas spontaneous scratching and epidermal hyperplasia were increased. In addition, the number of substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers (C-fibers) was increased in the epidermis of treated mice compared to that in non-treated control mice. However, neurofilament 200-immunoreactive nerve fibers (A-fibers) were not detected in the epidermis of treated mice. These results suggest that the elongated epidermal peripheral nerve fibers comprise SP/CGRP-containing C-fibers but not A-fibers. Thus, these fibers may be involved in the induction of dry skin pruritus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. Substance P (SP) is an 11-amino-acid endogenous neuropeptide that belongs to the tachykinin family and several reports recently have supported the anti-inflammatory and tissue repairing roles of SP., Objective: In this study, we investigated whether SP can improve AD symptoms, especially the impaired skin barrier function, in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced chronic dermatitis of NC/Nga mice or not., Method: AD-like dermatitis was induced in NC/Nga mice by repeated sensitization with TNCB for 5 weeks. The experimental group designations and topical treatments were as follows: vehicle group (AD-VE); SP group (AD-SP); and SP with NK1R antagonist CP99994 (AD-SP-A) group. Histological analysis was performed to evaluate epidermal differentiation, dermal integrity, and epidermal nerve innervation in AD-like lesions. The skin barrier functions and pruritus of NC/Nga mice were evaluated by measuring transepidermal water loss (TEWL) and scratching behavior, respectively., Result: Topical SP treatment resulted in significant down-regulation of Ki67 and the abnormal-type keratins (K) K6, K16, and K17, restoration of filaggrin and claudin-1, marked reduction of TEWL, and restoration of basement membrane and dermal collagen deposition, even under continuous sensitization of low dose TNCB. In addition, SP significantly reduced innervation of itch-evoking nerve fibers, gelatinase activity and nerve growth factor (NGF) expression in the epidermis but upregulated semaphorin-3A (Sema3A) expression in the epidermis, along with reduced scratching behavior in TNCB-treated NC/Nga mice. All of these effects were completely reversed by co-treatment with the NK1R antagonist CP99994. In cultured human keratinocytes, SP treatment reduced expression of TGF-α, but upregulated TGF-β and Sema3A., Conclusion: Topically administered SP can restore normal skin barrier function, reduce epidermal infiltration of itch-evoking nerve fibers in the AD-like skin lesions, and alleviate scratching behavior. Thus, SP may be proposed as a potential medication for chronic dermatitis and AD., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)