Intraepidermal Free Nerve Endings Research Articles

Functional validation of co-culture model of human keratinocytes and neuronal cell line for sensitive skin by using transient receptor potential channel vanilloid subfamily member 1 antagonist.

Sensitive skin is a subjective cutaneous hyper-reactivity that occurs in response to various innocuous stimuli. Keratinocytes have recently been shown to participate in sensory transduction by releasing many neuroactive molecules that bind to intra-epidermal free nerve endings and modulate nociception. In the literature, the characterization of these interactions has been based on the co-culture of keratinocyte and mammalian-origin neuronal cell lines. In this study, we established an in vitro model based on a co-culture of primary human keratinocytes and differentiated SH-SY5Y cells, a human neuronal cell line. Human epidermal keratinocytes and SH-SY5Y cells were monocultured and co-cultured. Changes in calcium influx, substance P, inflammatory cytokines, and neuropeptides between the monoculture and co-culture groups treated with capsaicin only and capsaicin with transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist, trans-4-tert-butylcyclohexanol (TTBC), together. In addition, the difference in stinging sensation was evaluated by applying it to the volunteers. When SH-SY5Y cells were co-cultured with keratinocytes, they had no significant effect on axonal development. Substance P was also released after capsaicin treatment and reduced by TTBC under co-culture conditions. Moreover, the expression of inflammatory cytokines and neuropeptides was significantly increased in co-cultured keratinocytes compared to that under monoculture conditions. In addition, the stinging sensation was significantly induced after the application of capsaicin in vivo and was relieved after the application of the TRPV1 antagonist. We demonstrated that the novel co-culture model is functionally valid through capsaicin and TRPV1 antagonist. We also confirmed that TTBC could be used for the treatment of sensitive skin through a co-culture model and in vivo tests. This co-culture model of keratinocytes and SH-SY5Y cells may be useful in vitro alternatives for studying the close communication between keratinocytes and neuronal cells and for screening therapeutic drugs for sensitive skin.

Emerging roles of keratinocytes in nociceptive transduction and regulation.

Keratinocytes are the predominant block-building cells in the epidermis. Emerging evidence has elucidated the roles of keratinocytes in a wide range of pathophysiological processes including cutaneous nociception, pruritus, and inflammation. Intraepidermal free nerve endings are entirely enwrapped within the gutters of keratinocyte cytoplasm and form en passant synaptic-like contacts with keratinocytes. Keratinocytes can detect thermal, mechanical, and chemical stimuli through transient receptor potential ion channels and other sensory receptors. The activated keratinocytes elicit calcium influx and release ATP, which binds to P2 receptors on free nerve endings and excites sensory neurons. This process is modulated by the endogenous opioid system and endothelin. Keratinocytes also express neurotransmitter receptors of adrenaline, acetylcholine, glutamate, and γ-aminobutyric acid, which are involved in regulating the activation and migration, of keratinocytes. Furthermore, keratinocytes serve as both sources and targets of neurotrophic factors, pro-inflammatory cytokines, and neuropeptides. The autocrine and/or paracrine mechanisms of these mediators create a bidirectional feedback loop that amplifies neuroinflammation and contributes to peripheral sensitization.

The new micropatterned interdigitated electrode for selective assessment of the nociceptive system.

In this neurophysiological study, we aimed at verifying the nociceptive selectivity of the new, micropatterned electrode (150IDE), recently designed to generate an electric field limited to the intraepidermal free nerve endings. Using the new 150IDE, we recorded evoked potentials after stimulation of the face and hand dorsum in 22 healthy participants and in patients with exemplary conditions selectively affecting the nociceptive system. We also measured the peripheral conduction velocity at the upper arm and verified the nociceptive selectivity of 150IDE assessing the effect of a selective block of nociceptive nerve fibres of radial nerve with local anaesthetic infiltration. In healthy participants and in patients, we have also compared the 150IDE-evoked potentials with laser-evoked potentials. In healthy participants, the 150IDE-evoked pinprick sensation and reproducible scalp potentials, with latency similar to laser-evoked potentials. The mean peripheral conduction velocity, estimated at the upper arm, was 12m/s. The selective nociceptive fibre block of the radial nerve abolished the scalp potentials elicited by the 150IDE stimulation. In patients, the 150IDE-evoked potentials reliably detected the selective damage of the nociceptive system. Our neurophysiological study shows that this new 150IDE provides selective information on nociceptive system. 150IDE is a promising new tool for investigating nociceptive system in patients with neuropathic pain.

Intraepidermal nerve fibres are not the exclusive tranducers of nociception

The goal of this article is to provide an insight of the most recent data on cutaneous nociception. Indeed recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intraepidermal free nerve endings are not exclusive transducers of pain. Therefore, the results of neurophysiological studies should be interpreted in the light of this information.

What about physical contacts between epidermal keratinocytes and sensory neurons?

Recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intra-epidermal free nerve endings are not exclusive transducers of pain. This discovery implies the existence of close afferent communication from keratinocytes to sensory neurons. Although reciprocal interactions between keratinocytes and intra-epidermal free nerve endings via soluble mediators are well established, little attention has been paid to physical contacts between keratinocytes and intra-epidermal free nerve endings. This review proposes to consider the ultrastructural and functional knowledge of these contacts, in both human skin biopsies and keratinocyte-sensory neuron cocultures to speculate on the possible existence of synaptic contacts.

Micropatterned surface electrode for massive selective stimulation of intraepidermal nociceptive fibres

BackgroundNo satisfactory neurophysiological test for nociceptive afferents is available to date. Laser stimuli present risks of skin damage, whilst electrical stimulation through specially designed electrodes is not selective enough. New methodWe present a new electrode designed according to critical issues identified in preliminary computer simulations concerning electric field gradient through the skin. To provide selective stimulation the activating electric field must be limited to intraepidermal free nerve endings. To this end, a new interdigitated electrode (IDE) was made of conductive rails arranged in a comb-like micropattern, situated only 150μm apart from each other (150 IDE) and alternately connected to the opposite poles of the stimulator. ResultsEvoked potentials recorded from the scalp were obtained after stimulation with the 150 IDE and with a similarly designed, but more widely spaced electrode (1000μm, or 1000 IDE). Small amplitude early and medium latency components were recorded with the 1000 IDE, suggesting activation of Aβ fibres. On the other hand, the 150 IDE only evoked late responses, confirming sufficient selectivity in small fibre activation. Comparison with existing method(s)The main differences with existing electrodes are: 1) Microspaced interdigitated conductive rails. 2) The potentially unlimited surface of stimulation and high efficiency per surface unit, resulting in large numbers of activated nociceptors. ConclusionsA new electrode providing selective stimulation of nociceptive nerve free endings is presented. It is non-invasive, and its surface can be enlarged at will. It is expected that it may greatly help in neurophysiological assessment of conditions affecting the nociceptive pathway.

Sodium-Calcium Exchanger and Multiple Sodium Channel Isoforms in Intra-Epidermal Nerve Terminals

BackgroundNociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals.ResultsSmall diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro.ConclusionsNCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

Fine structure of Eimer's organ in the coast mole (Scapanus orarius)

Eimer's organ is a small, densely innervated sensory structure found on the glabrous rhinarium of most talpid moles. This structure consists of an epidermal papilla containing a central circular column of cells associated with intraepidermal free nerve endings, Merkel cell neurite complexes, and lamellated corpuscles. The free nerve endings within the central cell column form a ring invested in the margins of the column, surrounding 1-2 fibers that pass through the center of the column. A group of small-diameter nociceptive free nerve endings that are immunoreactive for substance P surrounds this central ring of larger-diameter free nerve endings. Transmission electron microscopy revealed a high concentration of tonofibrils in the epidermal cells of the central column, suggesting they are more rigid than the surrounding keratinocytes and may play a mechanical role in transducing stimuli to the different receptor terminals. The intraepidermal free nerve endings within the central column begin to degrade 15 microm from the base of the stratum corneum and do not appear to be active within the keratinized outer layer. The peripheral free nerve endings are structurally distinct from their counterparts in the central column and immunocytochemical double labeling with myelin basic protein and substance P indicates these afferents are unmyelinated. Merkel cell-neurite complexes and lamellated corpuscles are similar in morphology to those found in a range of other mammalian skin.

Response properties of primary afferents supplying Eimer's organ

The mole's nose is covered with mechanosensory structures called Eimer's organs. Each organ contains Merkel cell-neurite complexes, Paciniform corpuscles and intraepidermal free nerve endings. The function of Eimer's organ has been the subject of speculation since the 1800s, but responses from the afferents have never been investigated. Our goal was to explore the function of Eimer's organ by recording primary afferent responses to a range of mechanosensory stimuli. Unit activity from the trigeminal ganglion was recorded from coast (Scapanus orarius) and star-nosed (Condylura cristata) moles, while stimulating the nose with a Chubbuck mechanosensory stimulator, a piezo-electric sweeping stimulator, and hand-held probes. Stimuli included static indentations, sinusoidal displacements, different indentation velocities, displacement amplitudes, and directional stimuli across the skin. Receptive fields were small, sometimes restricted to single Eimer's organs. Responses were consistent with a slowly adapting Merkel cell-neurite complex-like receptor class and a dynamically sensitive Pacinian-like rapidly adapting class. A second rapidly adapting class was hypothesized to represent activity of prominent free nerve endings within a central cell column. Some receptors were most sensitive to stimuli applied in particular directions across the skin. Most receptors relayed mechanosensory input with high temporal fidelity. In addition some receptors were tuned to respond best when stimulated at a velocity matching the velocity of the nose during foraging. These results support the hypothesis that Eimer's organ functions to detect small surface features and textures by encoding and integrating deflection information for multiple Eimer's organs during brief touches.

Cutaneous nerves in atopic dermatitis

Although pruritus is the cardinal symptom of atopic dermatitis, its mechanism is not well understood. Free nerve endings in the skin are involved in pruritus as itching receptors. We studied the cutaneous nerve fibres in lichenified lesions of 16 patients with adult atopic dermatitis. On immunohistochemistry, fibres immunoreactive for neurofilament, neuron-specific enolase, and protein gene product 9.5 were observed in the papillary dermis and dermoepidermal junctions as well as in the epidermis. In these areas, no fibres stained positively for substance P, neuropeptide Y, vasoactive intestinal peptide, beta endorphin, somatostatin or serotonin. On electron microscopy, the ultrastructure of subepidermal and intraepidermal free nerve endings appeared to be essentially normal. However, the distribution density of the cutaneous nerve fibres was much higher than in normal controls, and the diameter of these fibres was much larger, because of the large number of axons in each nerve fibre. Degranulation of mast cells was not seen. These findings suggest that pruritus in lichenified atopic skin is probably not caused by damage to the cutaneous free nerve endings. In such lesions, the number of the cutaneous free nerve endings is greatly increased, but they may have a normal function.

Visualization of PGP 9.5 immunoreactive nerve terminals in the mouse snout epidermis.

Intraepidermal free nerve endings were investigated in the mouse snout skin by means of an immunohistochemical procedure using a rabbit antiserum against protein gene product 9.5 (PGP 9.5). Immunoperoxidase reactivity was detected in different subtypes of intraepidermal nerves and cells. The great majority of axons observed in the stratified epithelium were varicose; a small percentage was either smooth (non-varicose) or irregularly shaped. Intraepidermal nerves ended at different levels within the epidermis, often with a terminal knob-like swelling. Various patterns of intraepidermal innervation could be distinguished. Most fibres entering the epidermis originated from large bundles running a horizontal course below the dermo-epidermal junction. Such fibres ascended vertically through the stratified epithelium in a "candelabrum-like" fashion, without emitting collaterals. Other fibres branched profusely and ended in complex intraepidermal neural networks. Less frequently, intraepidermal fibres terminated with large irregularly shaped expansions of different morphologies. Some of these were the intraepidermal continuations of axons within Meissner's corpuscles. Some fibres appeared to come into contact with PGP 9.5-immunoreactive cells (which closely resembled Merkel cells) located in the stratum basale. Rare suprabasal dendritic cells (Langerhans cells?) also became visible.

Presence of calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity in intraepidermal free nerve endings of cat skin

Immunocytochemistry against neuropeptides contained within primary afferent neurons was used to study the morphology and distribution of intraepidermal free nerve endings in cat facial skin. Calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity was found in similar intraepidermal nerve endings of cat glabrous and hairy skin epithelia. The greatest density of immunoreactive intraepidermal nerve endings was located in the nose epidermis. Small limited areas with immunoreactive intraepidermal nerve endings were also found in facial hairy skin.

Intraepidermal free nerve endings in guinea pigs: their relationship to epidermal keratinocytes.

ABSTRACTUsing transmission electron microscopy, the intraepidermal free nerve endings in the back skin of spotted black and white guinea pigs were examined. Many intraepidermal free nerve endings were present in both the black areas and the white areas. They were located in the spaces between the basal lamina of the epidermis and the cytomembrane of the basal keratinocyte or in the intercellular spaces between the basal keratinocytes, but never beyond the Malphigian layers. Most of them appeared to belong to sensory nerves and some to adrenergic nerves. They seemed to be related intimately to epidermal keratinocytes.

Intraepidermal free nerve endings relating to epidermal melanocytes in spotted guinea pigs. A statistical and electron microscopic study.

ABSTRACTIntraepidermal free nerve endings were studied in spotted (black and white) guinea pigs. Specimens were obtained from black, gray, and white areas. Twenty blocks were made from each area and sections were examined by electron microscopy. Intraepidermal free nerve endings were found in the interfollicular epidermis of all three areas. The distribution density for the black areas was about twice that for the white areas. The frequency in the gray areas was intermediate between that for the black areas and that for the white areas. Intraepidermal free nerve endings contacted the melanocytes directly in the interspaces between the basal lamina of the epidermis and the cytomembrane of the melanocyte or the dendrite of the melanocyte in the intercellular spaces between basal keratinocytes. In most cases, their axons were ensheathed by Schwann cells. In some cases, a part of the axon expanded like a balloon about 200–300 nm in diameter. The ballooning structure contained several vacuoles about 40–60 nm in diameter. These vacuoles seemed to have been secreted into the extracellular spaces. In such cases, an accumulation of dense materials was observed along the opposed membrane of the vacuole and the cytomembrane of the melanocyte. In other cases, the cytomembranes of axons and those of melanocytes seemed to form synapse‐like structures.

Biological changes of human cutaneous nerves caused by ultraviolet irradiation: an ultrastructural study.

Three white male volunteers were irradiated by long wave ultraviolet (UV-A) and by solar simulating radiation (SSR). An acute change of cutaneous nerves was found following a single exposure of UV-A irradiation. Non-myelinated Schwann cells and perineural cells in the papillary and reticular dermis were degenerated. Axoplasms appeared to be electron-dense, but were less affected than Schwann cells. After long-term repeated exposure, many free nerve endings were found in the dermo-epidermal junction above the basal lamina and some of them made a terminal enlargement. In one instance an axon made a swelling. Dermal free nerve endings also seemed to be increased in number and some of them were situated immediately beneath the melanocytes which were active in melanogenesis. Multiplication of basal lamina of the Schwann cells and perineural cells was observed. Amorphous material was precipitated around the non-myelinated Schwann cells. After a single exposure of SSR irradiation the degeneration of Schwann cells and axons in the dermo-epidermal junction was less severe than after UV-A and changes were minor in the reticular dermis. After repeated exposure, intra-epidermal proliferation of free nerve endings was detected. Dermal nerves were slightly affected. No intra-epidermal free nerve endings were observed in controls. The intra-epidermal proliferation of free nerve endings was confirmed following repeated UV exposures. Melanocyte-nerve association is suggested to be the cause of stimulating melanocyte activity.

Free Nerve Endings of the Epidermis in Hairy and Hairless Mice

The fine structure of the intraepidermal free nerve endings and subepidermal nerves of the back and auricle of hairy and hairless mice were studied. The nerves appeared similar in both areas and no significant differences were seen between the hairy and hairless mouse. The intraepidermal free nerve endings were observed in the form of either Schwann cell-axon complexes or denuded axon fibers, which were enveloped by the epidermal cells.