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Distinct sensory properties of unmyelinated axons in the isolated rat sciatic nerve have previously been revealed by measuring stimulated neuropeptide (CGRP) release in response to noxious stimuli. Axonal sensitization to heat by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel TRPV1. Recently, we have demonstrated in vitro that heat stimulation of nociceptive axons generates ectopic action potential discharge which resembles the heat response of the corresponding cutaneous nerve endings. It remained however, to be established whether adequate axonal stimulation could also generate projected sensations in a conscious human subject. In a singular human trial, the superficial radial nerve (SR) was exposed and stimulated mechanically as well as with noxious cold (3 degrees C). These stimuli were unable to induce any conscious local or projected sensations. However, controlled radiant heat applied to the nerve resulted in intense slowly adapting burning pain sensations projected into the center of the SR innervation area. No local sensation was reported. Thus, presumably activated nervi nevorum in the sheath of a healthy nerve do not cause conscious sensations, while axons of passage in mid-nerve exhibit a sensory transduction capacity for noxious heat though not for mechanical and cold stimulation. Axonal heat transduction may therefore become a source of ectopic discharge and neuropathic pain when heat threshold drops to body temperature as is the case with peripheral nerve endings in inflamed skin. [ABSTRACT FROM AUTHOR]

Background: Diabetic metabolism causes changes of the chemical milieu including accumulation of reactive carbonyl species, for example, methylglyoxal (MGO). MGO activates chemosensitive TRPA1 on nociceptors, but the contribution to neuronal pathophysiology causing pain and hyperalgesia in diabetic neuropathy is not fully understood., Methods: We employed single-nerve-fiber recordings in type 2 diabetes patients with (spDN) and without cutaneous pain (DN) and in streptozotocin-diabetic and healthy mice. In mice, we measured Ca ++ transients in cultured DRG neurons and stimulated CGRP release from hairy skin., Results: In diabetic patients, we recorded a large proportion of pathologically altered nerve C-fibers (79%). In spDN patients we found a higher percentage (72%) of spontaneously active C-nociceptors than in DN patients (15%). The proportion of spontaneous activity was highest among pathological fibers with mechanoinsensitive fiber properties which are particularly sensitive to MGO in contrast to mechanosensitive fibers. Mouse polymodal nociceptors, in contrast to purely mechanosensitive C-fibers, showed highest prevalence of TRPA1-related chemosensitivity. In diabetic mice about 37% of polymodal nociceptors developed spontaneous activity and exhibited significantly greater MGO responses, indicating sensitized TRPA1 receptors. Low-threshold mechanosensitive Aδ-fibers were vigorously activated by MGO but independently of TRPA1 activation., Interpretation: Our translational findings suggest that TRPA1-expressing C-nociceptors, which in human correspond to mechanoinsensitive and in mice to polymodal nociceptors, are especially vulnerable to develop spontaneous activity. Those two different nociceptor classes might share the functional role as dicarbonyl-sensitive chemosensors and represent the critical nociceptor population that support the development of pain and hyperalgesia in diabetic neuropathy., (© 2023 The Authors. Journal of the Peripheral Nervous System published by Wiley Periodicals LLC on behalf of Peripheral Nerve Society.)

Artemisinin and its derivatives are the main therapeutic drugs against Plasmodium protists, the causative agents of malaria. While several putative mechanisms of action have been proposed, the precise molecular targets of these compounds have not been fully elucidated. In addition to their antimalarial properties, artemisinins have been reported to act as anti-tumour agents and certain antinociceptive effects have also been proposed. We investigated the effect of the parent compound, artemisinin, on a number of temperature-gated Transient Receptor Potential ion channels (so called thermoTRPs), given their demonstrated roles in pain-sensing and cancer. We report that artemisinin acts as an agonist of the Transient Receptor Potential Ankyrin type 1 (TRPA1) receptor channel. Artemisinin was able to evoke calcium transients in HEK293T cells expressing recombinant human TRPA1, as well as in a subpopulation of mouse dorsal root ganglion (DRG) neurons which also responded to the selective TRPA1 agonist allyl isothiocyanate (AITC) and these responses were reversibly abolished by the selective TRPA1 antagonist A967079. Artemisinin also triggered whole-cell currents in HEK293T cells transiently transfected with human TRPA1, as well as in TRPA1-expressing DRG neurons, and these currents were inhibited by A967079. Interestingly, using human TRPA1 mutants, we demonstrate that artemisinin acts as a non-electrophilic agonist of TRPA1, activating the channel in a similar manner to carvacrol and menthol. These results may provide a better understanding of the biological actions of the very important antimalarial and anti-tumour agent artemisinin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sorin Tunaru reports financial support was provided by Executive Unit for Financing Higher Education Research Development and Innovation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The most widely used formalin test to screen antinociceptive drug candidates is still apostrophized as targeting inflammatory pain, in spite of strong opposing evidence published. In our rat skin-nerve preparation ex vivo, recording from all classes of sensory single-fibers (n = 32), 30 units were transiently excited by formaldehyde concentrations 1-100 mM applied to receptive fields (RFs) for 3 min, C and Aδ-fibers being more sensitive (1-30 mM) than Aβ-fibers. From 30 mM on, ~1% of the concentration usually injected in vivo, all RFs were defunctionalized and conduction in an isolated sciatic nerve preparation was irreversibly blocked. Thus, formaldehyde, generated a state of 'anesthesia dolorosa' in the RFs in so far as after a quiescent interphase all fibers with unmyelinated terminals developed a second phase of vigorous discharge activity which correlated well in time course and magnitude with published pain-related behaviors. Sural nerve filament recordings in vivo confirmed that higher formalin concentrations (> 42 mM) have to be injected to the skin to induce this second phase of discharge. Patch-clamp and calcium-imaging confirmed TRPA1 as the primary transducer of formaldehyde (10 mM) effects on mouse sensory neurons. However, stimulated CGRP release from isolated skin of TRPA1 +/+ and TRPA1 -/- mice showed a convergence of the saturating concentration-response curves at 100 mM formaldehyde, which did not occur with nerve and trachea preparations. Finally, skin-nerve recordings from C and Aδ-fibers of TRPA1 -/- mice revealed a massive reduction in formaldehyde (30 mM)-evoked discharge. However, the remaining activity was still biphasic, thus confirming additional unspecific excitotoxic actions of the fixative that diffuses along still excitable axons as previously published. The multiplicity of formaldehyde's actions requires extensive discussion and literature review, leading to a fundamental reevaluation of the formalin test., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Painful diabetic neuropathy occurs in approximately 20% of diabetic patients with underlying pathomechanisms not fully understood. We evaluated the contribution of the Ca V 3.2 isoform of T-type calcium channel to hyperglycemia-induced changes in cutaneous sensory C-fiber functions and neuropeptide release employing the streptozotocin (STZ) diabetes model in congenic mouse strains including global knockouts (KOs). Hyperglycemia established for 3-5 weeks in male C57BL/6J mice led to major reorganizations in peripheral C-fiber functions. Unbiased electrophysiological screening of mechanosensitive single-fibers in isolated hairy hindpaw skin revealed a relative loss of (polymodal) heat sensing in favor of cold sensing. In healthy Ca V 3.2 KO mice both heat and cold sensitivity among the C-fibers seemed underrepresented in favor of exclusive mechanosensitivity, low-threshold in particular, which deficit became significant in the diabetic KOs. Diabetes also led to a marked increase in the incidence of spontaneous discharge activity among the C-fibers of wildtype mice, which was reduced by the specific Ca V 3.2 blocker TTA-P2 and largely absent in the KOs. Evaluation restricted to the peptidergic class of nerve fibers - measuring KCl-stimulated CGRP release - revealed a marked reduction in the sciatic nerve by TTA-P2 in healthy but not diabetic wildtypes, the latter showing CGRP release that was as much reduced as in healthy and, to the same extent, in diabetic Ca V 3.2 KOs. These data suggest that diabetes abrogates all Ca V 3.2 functionality in the peripheral nerve axons. In striking contrast, diabetes markedly increased the KCl-stimulated CGRP release from isolated hairy skin of wildtypes but not KO mice, and TTA-P2 reversed this increase, strongly suggesting a de novo expression of Ca V 3.2 in peptidergic cutaneous nerve endings which may contribute to the enhanced spontaneous activity. De-glycosylation by neuraminidase showed clear desensitizing effects, both in regard to spontaneous activity and stimulated CGRP release, but included actions independent of Ca V 3.2. However, as diabetes-enhanced glycosylation is decisive for intra-axonal trafficking, it may account for the substantial reorganizations of the Ca V 3.2 distribution. The results may strengthen the validation of Ca V 3.2 channel as a therapeutic target of treating painful diabetic neuropathy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene-Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca 2+ -dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E 2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease., (© 2020 Becker et al.)

The aminosteroid U73122 is frequently used as a phospholipase C (PLC) inhibitor and as such was used to investigate PLC-dependent activation and modulation of the transient receptor potential ankyrin type 1 (TRPA1) receptor channel. However, U73122 was recently shown to activate recombinant TRPA1 directly, albeit this interaction was not further explored. Our aim was to perform a detailed characterization of this agonistic action of U73122 on TRPA1. We used Fura-2 calcium microfluorimetry and the patch clamp technique to investigate the effect of U73122 on human and mouse wild type and mutant (C621S/C641S/C665S) TRPA1 expressed in HEK293t cells, as well as native TRPA1 in primary afferent neurons from wild type and TRPV1 and TRPA1 null mutant mice. In addition, we measured calcitonin gene-related peptide (CGRP) release from skin isolated from wild-type and TRPA1 null mutant mice. Human and mouse TRPA1 channels were activated by U73122 in the low nanomolar range. This activation was only partially dependent upon modification of the N-terminal cysteines 621, 641, and 665. U73122 also activated a subpopulation of neurons from wild-type and TRPV1 null mutant mice, but this effect was absent in mice deficient of TRPA1. In addition, U73122 evoked marked calcitonin gene-related peptide (CGRP) release from skin preparations of wild type but not TRPA1 null mutant mice. Our results indicate that U73122 is a potent and selective TRPA1 agonist. This effect should be taken into account when U73122 is used to inhibit PLC in TRPA1-expressing cells, such as primary nociceptors. In addition, U73122 may present a novel lead compound for the development of TRPA1-targeting drugs.

The endogenous metabolite methylglyoxal (MG) accumulates in diabetic patients with neuropathic pain. Methylglyoxal could be a mediator of diabetes-induced neuropathic pain through TRPA1 activation and sensitization of the voltage-gated sodium channel subtype 1.8. In this study, we tested the algogenic and sensitizing effect of MG in healthy human subjects using intracutaneous microinjections. The involvement of C fibers was assessed through selective A-fiber nerve block, axon-reflex-erythema, and through single nerve fiber recordings in humans (microneurography). Involvement of the transduction channels TRPA1 and TRPV1 in MG-induced pain sensation was investigated with specific ion channel blockers. We showed for the first time in healthy humans that MG induces pain, axon-reflex-erythema, and long-lasting hyperalgesia through the activation of C nociceptors. Predominantly, the subclass of mechano-insensitive C fibers is activated by MG. A fibers contribute only negligibly to the burning pain sensation. Selective pharmacological blockade of TRPA1 or TRPV1 showed that TRPA1 is crucially involved in MG-induced chemical pain sensation and heat hyperalgesia. In conclusion, the actions of MG through TRPA1 activation on predominantly mechano-insensitive C fibers might be involved in spontaneously perceived pain in diabetic neuropathy and hyperalgesia as well as allodynia.

Nerve terminals of primary sensory neurons are influenced by their environment through target derived trophic factors, like nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF). In mice, subpopulations of DRG neurons express receptors either for NGF or GDNF and therefore differentially respond to these neurotrophic factors. We probed neurite endings from porcine DRG neurons cultured in either NGF or GDNF and examined their shape, elongation and stimulus-evoked CGRP release. A compartmentalized culture system was employed allowing spatial separation of outgrown neurites from their somata and use of different growth factors in the compartments. We show that neurites of GDNF cultured somata extend into lateral compartments without added growth factor, unlike neurites of NGF cultured ones. Neurites of NGF cultured somata extend not only into NGF- but also into GDNF-containing compartments. GDNF at the site of terminals of NGF responsive somata led to a strong neurite arborization and formation of large growth cones, compared to neurites in medium with NGF. Functionally, we could detect evoked CGRP release from as few as 7 outgrown neurites per compartment and calculated release per mm neurite length. CGRP release was detected both in neurites from NGF and GDNF cultured somata, suggesting that also the latter ones are peptidergic in pig. When neurites of NGF cultured somata were grown in GDNF, capsaicin evoked a lower CGRP release than high potassium, compared to those grown in NGF. Our experiments demonstrate that the compartmented culture chamber can be a suitable model to assess neurite properties from trophic factor specific primary sensory neurons. With this model, insights into mechanisms of gain or loss of function of specific nociceptive neurites may be achieved., Competing Interests: The authors have declared that no competing interests exist.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

Unlabelled: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients., Significance Statement: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development., (Copyright © 2016 the authors 0270-6474/16/365264-15$15.00/0.)

The formalin test is the most widely used behavioral screening test for analgesic compounds. The cellular mechanism of action of formaldehyde, inducing a typically biphasic pain-related behavior in rodents is addressed in this study. The chemoreceptor channel TRPA1 was suggested as primary transducer, but the high concentrations used in the formalin test elicit a similar response in TRPA1 wildtype and knockout animals. Here we show that formaldehyde evokes a dose-dependent calcium release from intracellular stores in mouse sensory neurons and primary keratinocytes as well as in non-neuronal cell lines, and independent of TRPA1. The source of calcium is the endoplasmatic reticulum and inhibition of the sarco/endoplasmic reticulum calcium-ATPase has a major contribution. This TRPA1-independent mechanism may underlie formaldehyde-induced pan-neuronal excitation and subsequent inflammation.

The clinical symptoms of diabetic neuropathy (DN) manifest in a time dependent manner as a positive symptoms (i. e. pain, hypersensitivity, tingling, cramps, cold feet etc.) during its early stages and by a loss of function (i. e. loss of sensory perception, delayed wound healing etc.) predominating in the later stages. Elevated blood glucose alone cannot explain the development and progression of DN and the lowering of blood glucose is insufficient in preventing and/or reversing neuropathy in patients with type 2 diabetes. Recently it has been shown that the endogenous reactive metabolite methylglyoxal (MG) can contribute to the gain of function via post-translational modification in DN of neuronal ion channels involved in chemosensing and action potential generation in nociceptive nerve endings. Dicarbonyls, such as MG, that are elevated in diabetic patients, modify DNA as well as extra- and intracellular proteins, leading to the formation of advanced glycation endproducts (AGEs). Increased formation of AGEs leads to increased cellular stress, dysfunction and ultimately cell death. The interaction of AGE-modified proteins through cell surface receptors, such as RAGE, can lead to increased cellular activation and sustained inflammatory responses, which are the molecular hallmarks of the later, degenerative, stages of DN. The direct and indirect effects of dicarbonyls on nerves or neuronal microvascular network provides a unifying mechanism for the development and progression of DN. Targeting the accumulation of MG and/or prevention of RAGE interactions may therefore provide new, more effective, therapeutic approaches for the treatment of DN., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

Vagal sensory afferents innervating airways and abdominal tissues express TRPV1 and TRPA1, two depolarizing calcium permeable ion channels playing a major role in sensing environmental irritants and endogenous metabolites which cause neuropeptide release and neurogenic inflammation. Here we have studied axonal chemosensitivity and control of neuropeptide release from the isolated rat and mouse vagus nerve by using prototypical agonists of these transduction channels - capsaicin, mustard oil and the specific endogenous activators, anandamide (methyl arachidonyl ethanolamide, mAEA), and acrolein, respectively. Capsaicin evoked iCGRP release from the rat vagus nerve with an EC₅₀ of 0.12 μM. Co-application of mAEA had a dual effect: nanomolar concentrations of mAEA (0.01 μM) significantly reduced capsaicin-evoked iCGRP release while concentrations ≥ 1 μM mAEA had sensitizing effects. Only 100 μM mAEA directly augmented iCGRP release by itself. In the mouse, 310 μM mAEA increased release in wildtype and TRPA1-/- mice which could be inhibited by capsazepine (10 μM) and was completely absent in TRPV1-/- mice. CB1-/- and CB1/CB2 double -/- mice equally displayed increased sensitivity to mAEA (100 μM) and a sensitizing effect to capsaicin, in contrast to wildtypes. Acrolein and mustard oil (MO)--at μM concentrations--induced a TRPA1-dependent iCGRP release; however, millimolar concentrations of mustard oil (>1mM) evoked iCGRP release by activating TRPV1, confirming recent evidence for TRPV1 agonism of high mustard oil concentrations. Taken together, we present evidence for functional expression of excitatory TRPV1, TRPA1, and inhibitory CB1 receptors along the sensory fibers of the vagus nerve which lend pathophysiological relevance to the axonal membrane and the control of neuropeptide release that may become important in cases of inflammation or neuropathy. Sensitization and possible ectopic discharge may contribute to the development of autonomic dysregulation in visceral tissues that are innervated by the vagus nerve., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The primary afferent nociceptive neuron has recently attracted major research interest because of the cloning of very selectively expressed and well-conserved ion channel genes. All parts of the neuron, sensory terminals, axon and cell body, are accessible to validated research techniques in vitro using various isolated tissues or cells taken from laboratory animals. Single-unit recording and measuring stimulated calcitonin gene-related peptide (CGRP) release as well as patch-clamping and calcium imaging of cultured sensory neurons provide different kinds of information, and no model alone answers all questions. In combination, however, consistent results and complementary evidence form a solid basis for translational research to follow.

Background: The axonal membrane of unmyelinated sensory nerve fibers is well equipped with different molecular transducer molecules that establish specific sensitivities, the capacity for sensitization by inflammation and generation of ectopic action potentials that contribute to spinal sensitization, leading to projected pain, allodynia and hyperalgesia., Methods: We studied the sensory properties of unmyelinated axons in the midnerve by measuring stimulated neuropeptide release, recording from primary afferents and eliciting projected pain by stimulation of a surgically exposed superficial radial nerve in a conscious human subject., Results: Capsaicin (TRPV1) receptor channels are expressed along the axonal membrane and respond to acidic, thermal and capsaicin stimulation with a graded and calcium-dependent calcitonin gene-related peptide release. These responses can be facilitated by bradykinin or prostaglandin, indicating functional BK and EP receptors along the axonal membrane. Sensitizing effects are lost in preparations from TRPV1 knockout mice. In the isolated vagus nerve, representing visceral innervation, the endovanilloid/endocannabinoid anandamide induced or sensitized calcitonin gene-related peptide release by activation of TRPV1. Our electrophysiological recordings revealed ectopic generation of action potentials. Intact unmyelinated axons showed sensory capacities that resembled those of their individual cutaneous nociceptive terminals, with respect to noxious heat sensitivity. In the human subject, noxious heat stimulation of the exposed skin nerve evoked intense burning pain sensation in the innervation territory., Conclusion: Different lines of evidence indicate that nociceptive axons exhibit essential parts of the signal transduction and spike generation machinery. When amplified (e.g. by inflammatory mediators), this axonal sensitivity may become a source of neuropathic pain., (Copyright 2010 S. Karger AG, Basel.)

Sensory properties of unmyelinated axons in the isolated rat sciatic nerve have been revealed previously by measuring stimulated neuropeptide release in response to noxious stimuli. In addition, axonal sensitization by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel transient receptor potential vanilloid receptor-1. It was unclear whether this responsiveness is accompanied by ectopic generation of action potentials, which may play a crucial role in painful neuropathies. We explored this hypothesis using the isolated mouse skin-nerve preparation. This method enabled us to directly compare the sensory properties of axons in the peripheral nerve with their characterized cutaneous terminals in the receptive field using propagated action potentials as an index of axonal activation. Single-fiber recordings from 51 mechanosensitive mouse C-fibers revealed that a majority of the polymodal nociceptors responded with an encoding discharge rate to graded heating of the cutaneous receptive field (n = 38) as well as of the saphenous nerve carrying the fiber under investigation (n = 25; 66%). Axonal heat responses paralleled those of the receptive fields with regard to thresholds and discharge rates (41.5 +/- 4.3 degrees C; 7.7 +/- 9.6 spikes in a 20 s 32-48 degrees C ranged stimulation). In contrast, axonal mechanosensitivity was poor and noxious cold sensitivity more rarely encountered. In conclusion, peripheral nerve axons exhibit sensory transduction capacities similar to their nociceptive terminals in the skin with respect to noxious heat, although not to mechanical and cold sensitivity. This may become a source of ectopic discharge and pain if axonal heat threshold drops to body temperature, as may be the case during inflammation-like processes in peripheral nerves.

Neuropeptides like calcitonin gene-related peptide (CGRP) and substance P are found in significant proportions of primary afferent neurons. Release of these neuropeptides as well as prostaglandin E(2) is an approved index for the activation of these primary afferents. Previous studies have used cultures of enzyme-treated and mechanically dissociated primary afferent neurons, fresh tissue slices or cubes. In the present study we demonstrate CGRP and prostaglandin E(2) release from intact isolated dorsal root and trigeminal ganglia. Stimulation with noxious heat, low pH, inflammatory mediators and high potassium concentration increased CGRP release. In conclusion, neuropeptide release from intact isolated ganglia is a reliable method to study the responsiveness of sensory neurons in situ in comparison with neuronal cell cultures.

We have previously shown that capsaicin, noxious heat, protons and potassium ions (K(+)) induce a graded, calcium- and receptor-dependent increase of immunoreactive calcitonin gene-related peptide (iCGRP) release from isolated rat sciatic axons. Morphological evidence for axonal vesicular exocytosis has also been presented. Here we determine the differential contribution of voltage-gated calcium and sodium channels to high extracellular potassium and capsaicin-induced iCGRP secretion. Blockade of L-type calcium channels significantly decreased the K(+)-induced axonal response (nimodipine (10 microM) by 66% and methoxyverapamil, D600 (50 microM), by 77%). Interestingly, however, D600 was unable to reduce the capsaicin-induced iCGRP release. Omega-Conotoxin GVIA (1 microM), a N-type blocker, and omega-agatoxin TK (0.1 microM), a P/Q-type blocker, had no significant effect. Also the anticonvulsant gabapentin (50 microM and 100 microM), reported to impede calcium channels, was ineffective. Inhibition of low threshold T-type calcium channels by mibefradil (10 microM) significantly reduced potassium (by 47%) but not capsaicin-stimulated iCGRP release. Reduction of total sodium channel conductance by tetrodotoxin (1 microM), lidocaine (10 microM, 50 microM or 500 microM) or by replacement of extracellular sodium with choline-chloride did not result in a reduction of either potassium- or capsaicin-induced axonal iCGRP release. These results suggest that slow depolarization by high extracellular potassium activates axonal low threshold (T-type) as well as high threshold-activated (L-type) voltage-gated calcium channels to mediate iCGRP release, and that capsaicin-induced release is largely dependent on calcium influx through TRPV1. Action potential generation and propagation are not required for axonal release mechanisms.

Local anesthetics (LAs) block the generation and propagation of action potentials by interacting with specific sites of voltage-gated Na(+) channels. LAs can also excite sensory neurons and be neurotoxic through mechanisms that are as yet undefined. Nonspecific cation channels of the transient receptor potential (TRP) channel family that are predominantly expressed by nociceptive sensory neurons render these neurons sensitive to a variety of insults. Here we demonstrated that the LA lidocaine activated TRP channel family receptors TRPV1 and, to a lesser extent, TRPA1 in rodent dorsal root ganglion sensory neurons as well as in HEK293t cells expressing TRPV1 or TRPA1. Lidocaine also induced a TRPV1-dependent release of calcitonin gene-related peptide (CGRP) from isolated skin and peripheral nerve. Lidocaine sensitivity of TRPV1 required segments of the putative vanilloid-binding domain within and adjacent to transmembrane domain 3, was diminished under phosphatidylinositol 4,5-bisphosphate depletion, and was abrogated by a point mutation at residue R701 in the proximal C-terminal TRP domain. These data identify TRPV1 and TRPA1 as putative key elements of LA-induced nociceptor excitation. This effect is sufficient to release CGRP, a key component of neurogenic inflammation, and warrants investigation into the role of TRPV1 and TRPA1 in LA-induced neurotoxicity.

Threshold tracking of individual polymodal C- and Adelta-fiber terminals was used to assess membrane potential changes induced by de- or hyperpolarizing stimuli in the isolated rat skin-nerve preparation. Constant current pulses were delivered (1 Hz) through a tungsten microelectrode inserted in the receptive field, and the current amplitude was controlled by feedback with a laboratory computer programmed to serially determine the electrical threshold using the method of limits. During threshold tracking, the receptive fields of the fibers were heated (32-46 degrees C in 210 s) or superfused with modified synthetic interstitial fluid containing either 0, 20, 40, 50, or 60 mM [K+], phosphate buffer to pH 5.2 or 6.1, or bradykinin (BK, 10(-8)-10(-5) M). High [K+]e decreased the current threshold for activation by 6-14% over 120 s, whereas K+-free superfusion augmented the threshold by >5%, and after some delay, also induced ongoing discharge in 60% of units. pH 6.1 and 5.2 caused an increase in threshold of 6 and 18%, respectively, and 30% of the fibers were excited by low pH, although the change in threshold of pH responsive and unresponsive fibers did not differ significantly, suggesting a general excitability decrease induced by protons. Heat stimulation increased the mean threshold and conduction velocity of the fibers tested and resulted in activity in 78% of units. Additionally, for these units, activation was preceded by a significant decrease in threshold compared with the tracked thresholds of fibers unresponsive to heat. Bradykinin also led to a significant threshold decrease before activation. In conclusion, the technique of threshold tracking proved suitable to assess changes in excitability resulting from receptor currents evoked by noxious heat and bradykinin in the terminal arborization of cutaneous nociceptors.

Although cutaneous C-fiber nociceptors show dramatic inflammatory sensitization to heat, they do not appear to get sensitized to the mechanical stimulation by von Frey hairs. We employed force-controlled punctate electromechanical stimulation to receptive fields of 61 characterized C-fibers in the isolated rat skin-saphenous nerve preparation. In general: low-in contrast to higher-threshold units showed greater dynamic sensitivity and response magnitude, an earlier onset and a stronger degree of adaptation, the latter due to the linear rise of the force stimulus. On this methodological basis three groups of units were subject to a mix of inflammatory mediators, to flurbiprofen or to control solution. Subsequent mechanostimulation revealed a good reproducibility of the control response and no significant changes in the treatment groups. In conclusion, even refined mechanostimulation was unable to demonstrate sensitization of the predominant nociceptor classes in the rat skin.

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles--presumably on axonal transport--as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10(-6) M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures--the omega structures--lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

We have shown previously that rat sciatic nerve axons in vitro express sensitivity to capsaicin and heat and responded to these stimuli with a Ca2+-dependent and graded immunoreactive calcitonin gene-related peptide release. Morphological evidence for stimulated vesicular exocytosis and for the vanilloid receptor TRPV1 in the axolemma of the unmyelinated nerve fibres has also been presented. Here we used solutions of low pH, high K+ or 47 degrees C to stimulate isolated desheathed sciatic nerves measuring immunoreactive calcitonin gene-related peptide release. pH 6.1 increased immunoreactive calcitonin gene-related peptide release by 31% over baseline and pH 5.2 and 4.3 caused a log-linear concentration-dependent increase of 137 and 265%, respectively. The effect of pH 3.4 was out of the linear range and not reversible. Stimulation in Ca2+-free solutions and under increased intracellular Ca2+ buffering capacity strongly reduced the proton responses. The TRPV1 antagonists capsazepine and ruthenium red substantially reduced the effects of pH 5.2 but not pH 6.1. Combining a stimulus of 60 mm K+ with the subliminal pH 6.3 reduced the axonal immunoreactive calcitonin gene-related peptide response by 88%. The noxious heat response at pH 6.3, however, was only reduced by 39%, suggesting a hidden sensitization to heat by low pH. This was supported by an effect of capsazepine to reduce the combined response to half, indicative of an involvement of TRPV1 in the sensitization but not in the axonal heat response itself that was found to be resistant to capsazepine. Axonal calcitonin gene-related peptide release is thought to play a physiological role in activity-dependent autoregulation of endoneurial blood flow. Axonal sensitivity to and sensitization by protons may be a pathophysiological mechanism involved in certain peripheral neuropathies.

We recently described a novel endogenous mechanism of peripheral antinociception, possibly involving activation of muscarinic M2 acetylcholine receptors that are expressed on nociceptive nerve endings and decrease their sensitivity. In the present study, this mechanism was scrutinized in skin taken from mice with targeted deletions of the muscarinic M2 receptor gene and, for control purposes, of the M4 receptor gene. Two different approaches were taken. Electrophysiologically the effects of muscarine on nociceptive afferents were investigated using the mouse skin-saphenous nerve preparation, in vitro. Muscarine did not excite nociceptors in the wild-type littermates (WT) and M4 knock-out (M4 KO) mice, but almost all fibers exhibited marked desensitization to mechanical and heat stimuli. Surprisingly, in the M2 KO mice, muscarine was able to excite C-nociceptors and to induce a mild sensitization to heat but caused no alteration in mechanical responsiveness tested with von Frey hairs. In the second, neurochemical approach, the heat-induced cutaneous release of calcitonin gene-related peptide (CGRP) was investigated to gain comparative data on the neurosecretory (vasodilatory) functions of the primary afferent neurons. The substantial increase of CGRP release evoked by noxious heat (47 degrees C) was diminished under muscarine by >50% in the WT and M4 KO animals but remained unaltered in the M2 KO mice. Together, these data provide direct evidence that M2 receptors on cutaneous nerve endings mediate effective depression of nociceptive responsiveness. This observation should be of interest for the development of novel classes of analgesic agents.

Noxious heat may act as an endogenous activator of the ionotropic capsaicin receptor (VR1) and of its recently found homologue VRL1, expressed in rat dorsal root ganglion cells and present along their nerve fibres. We have previously reported that capsaicin induces receptor-mediated and Ca++-dependent calcitonin gene-related peptide (CGRP) release from axons of the isolated rat sciatic nerve. Here we extended the investigation to noxious heat stimulation and the transduction mechanisms involved. Heat stimulation augmented the CGRP release from desheathed sciatic nerves in a log-linear manner with a Q10 of approximately 15 and a threshold between 40 and 42 degrees C. The increases were 1.75-fold at 42 degrees C, 3.8-fold at 45 degrees C and 29.1-fold at 52 degrees C; in Ca++-free solution these heat responses were abolished or reduced by 71 and 92%, respectively. Capsazepine (10 microm) and Ruthenium Red (1 microm) used as capsaicin receptor/channel antagonists did not significantly inhibit the heat-induced release. Pretreatment of the nerves with capsaicin (100 microm for 30 min) caused complete desensitization to 1 microm capsaicin, but a significant heat response remained, indicating that heat sensitivity is not restricted to capsaicin-sensitive fibres. The sciatic nerve axons responded to heat, potassium and capsaicin stimulation with a Ca++-dependent CGRP release. Blockade of the capsaicin receptor/channels had little effect on the heat-induced neuropeptide release. We conclude therefore that other heat-activated ion channels than VR1 and VRL1 in capsaicin-sensitive and -insensitive nerve fibres may cause excitation, axonal Ca++ influx and subsequent CGRP release.

The action of cholinergic agonists on modulating basal and heat-induced CGRP release was investigated in isolated rat skin. Nicotine (10(-6), 10(-5) and 10(-4) M) induced a bimodal increase of CGRP release, that was significant for the two larger concentrations (by 113 and 36%, respectively). On the contrary, muscarine (10(-4) M) and arecaidine (10(-5) M) significantly decreased the basal CGRP release (by 16 and 23%, respectively). The substantial increase of CGRP release evoked by noxious heat (47 degrees C) remained unaltered upon co-application of nicotine, but was diminished by 35% upon muscarine. Arecaidine was more effective in this respect causing significant dose-dependent depressions by 30% (at 10(-6) M) and by 60% (at 10(-5) M). These data support a role of muscarinic M2 receptors in nociceptor desensitization.

We have investigated the pro- and anti-inflammatory effects of ricinoleic acid (RA), the main active principle of castor oil, in an experimental model of blepharitis induced by intradermal injection of carrageenan in the guinea-pig eyelid and its possible capsaicin-like mode of action on acutely dissociated rat dorsal root ganglia (DRG) neurons in vitro. Topical treatment with RA (10-100 mg/guinea-pig) or capsaicin (1-10 mg/guinea-pig) caused eyelid reddening and oedema. At lower doses (0.3-3 mg/guinea-pig and 0.009-0.09 mg/guinea-pig for RA and capsaicin, respectively) both drugs significantly potentiated the eyelid oedema induced by carrageenan. The tachykinin NK1 receptor antagonist FK 888 (0.59 mg/kg s.c.) abolished the potentiation of carrageenan-induced eyelid oedema induced by either RA or capsaicin. The neutral endopeptidase inhibitor, thiorphan (1.3 mg/kg i.v.) significantly enhanced the potentiation of carrageenan-induced eyelid oedema produced by RA. This potentiating effect was abolished by FK 888. Repeated (8 days) topical application of RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-pig) inhibited the carrageenan-induced eyelid oedema. This anti-inflammatory effect was accompanied by a reduction (75%-80% of SP and 46%-51% of NKA) in tachykinin content of the eyelids, as determined by radioimmunoassay. In dissociated rat DRG neurons, RA (0.1 mM for 5 min) significantly inhibited the inward currents induced by application of capsaicin (1 microM) and/or low pH (5.8), without inducing any currents by itself or changing voltage-dependent currents. Moreover, after 24-h incubation, RA (0.1 mM) significantly decreased the capsaicin (1 microM)-induced calcitonin gene-related peptide (CGRP) release from rat DRG neurons, whereas acute drug superfusion did not evoke CGRP release by itself. Summarizing, RA possesses capsaicin-like dual pro-inflammatory and anti-inflammatory properties which are observed upon acute and repeated application, respectively. However, unlike capsaicin, RA does not induce inward current in DRG neurons and it is devoid of algesic properties in vivo.

The actions of different cholinergic agonists and antagonists were investigated on nociceptive afferents using the rat skin-saphenous nerve preparation, in vitro. Nicotine was able to weakly excite C-nociceptors and to induce a mild sensitization to heat stimulation (in 77% of tested fibers) in a dose-dependent manner (10(-)6 to 10(-)5 m), but it caused no alteration in mechanical responsiveness tested with von Frey hairs. Muscarine did not induce a significant nociceptor excitation, but almost all fibers exhibited a marked desensitization to mechanical and heat stimuli in a dose-dependent manner (from 10(-)6 to 10(-)4 m). The muscarinic effects could be prevented by the general muscarinic antagonist scopolamine (10(-)5 m), by the M3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidium oxide (10(-)6 m) co-applied with the M2 antagonist gallamine (10(-)5 m), and by gallamine alone. As positive control we used the relatively M2-selective agonist arecaidine (10(-)6 to 10(-)5 m), obtaining a similar desensitizing effect as with muscarine. Finally, we performed an immunocytochemical study that demonstrated the presence of M2 but not M3 receptors in thin epidermal nerve fibers of the rat hairy skin. Altogether, these data demonstrate opposite effects of nicotinic and muscarinic receptor stimulation on cutaneous nociceptors. M2 receptor-mediated depression of nociceptive responsiveness may convey a therapeutic, i.e., analgesic or antinociceptive, potential.

We investigated the possible neurogenic origin of prostaglandin E2 (PGE2) in the rat skin, in vitro. The hairy skin of one hindpaw was denervated and one week later the dorsal hindpaws were skinned to study the release of calcitonin gene-related peptide (CGRP) and PGE2 using the EIA technique. Stimulation with bradykinin (BK) caused a significant release of CGRP (1.4-fold increase) and PGE2 (3-fold) which was massively augmented under neurokinin I (NKI) receptor antagonist treatment (CGRP: 4-fold, PGE2: 5-fold). In denervated skin the BK-evoked CGRP release was lost whereas the PGE2 release was unchanged. Thus, neither nerve endings nor neuropeptides contribute essentially to BK-induced PGE2 release in the skin. However, excessive neuropeptide levels, as under NKI blockade facilitate PGE2 formation, which may play a role in sustained inflammation.

The presence of an intrinsic afferent innervation of nerves and their connective tissues (nervi nervorum) suggests that these neural elements participate in sensation and pathological processes affecting nerves. Primary afferent nociceptors contain and release neuropeptides including calcitonin gene-related peptide, implicated in inflammatory vasodilatation. We sought to evaluate the ability of different peripheral nerve components, in vitro, to release calcitonin gene-related peptide and prostaglandin E2 in response to electrical and noxious chemical stimuli, using sensitive enzyme immunoassays. We observed significant increases in both calcitonin gene-related peptide and prostaglandin E2 in response to a mixture of inflammatory mediators (bradykinin, histamine, and serotonin; 10(-5) M) applied to the intact nerves (+37% and +700%, respectively) and isolated sheaths (35% and 430%, respectively), but not when this mixture was applied to isolated axons. Proximal (antidromic) but not distal (orthodromic) electrical stimulation also evoked a comparable release of calcitonin gene-related peptide (+30%) from intact nerves. These results suggest that nervi nervorum nociceptors participate in neural inflammation. Capsaicin (10(-6) M) elicited a very large release of calcitonin gene-related peptide when applied to either the intact nerve (+400%), isolated sheaths (+500%), or isolated axons (1400%). The latter effect was substantially but not completely blocked by Ruthenium Red and capsazepine, and was completely blocked using a calcium-free bathing solution. The results support the presence of capsaicin receptors in peripheral nerves that can effect calcitonin gene-related peptide release from axons as well as from terminals.

The excitatory effect of bradykinin (BK) and of low pH on nociceptors appears to partly depend on secondary release of prostaglandins from the surrounding tissue. Rat skin, in vitro, is introduced as a novel model to measure basal and stimulated release of PGE2 and, in future, other substances relevant to nociception, such as neuropeptides. Flaps of hairy skin (n=57) from the rat saphenous region of the hindpaw were subcutaneously excised and fixed on acrylic rods, the corium side exposed. The preparations were equilibrated in carbogen gassed "synthetic interstitial fluid" (SIF) for 30 minutes. The skin flaps were then immersed for 5 minutes each in 9 consecutive glass tubes, which were mounted in a shaking bath at 32 degrees C. Each tube was filled with 5 ml of gassed SIF, the third tube contained inflammatory mediator(s) dissolved in SIF or solutions of low pH. After passage of the skin flap, the eluates were deep frozen (-70 degrees C) and the PGE2 content measured, off-line, using an enzyme immuno-assay. As stimulants, BK at 10(-5) M (n=9) and 10(-6) M (n=4) and BK in equimolar combination with histamine (HA) and serotonin (5-HT; 10(-5) M: n=8, 10(-6) M: n=6, 10(-7) M: n=6) dose-dependently increased PGE2 release. Considering the total amount of PGE2 secreted the combination of inflammatory mediators caused a significantly greater release of PGE2 at 10(-5) and 10(-6) M (p<0.01, Kruskal-Wallis test) than BK stimulation alone. Racemic flurbiprofen caused a profound depression of basal and stimulated release. Solutions of high proton concentration are known to stimulate and sensitize nociceptors. However, phosphate buffered SIF at pH 6.1 and 6.4 caused a substantial and significant decrease of the PGE2 release, probably due to low-pH block of phospholipases. Thus, algogenic potency of mediators does not necessarily match their pro-inflammatory action.