An important issue in physical rehabilitation is how to protect from or to reduce the effects of peripheral nerve injury. In the present study, we examined whether ankle joint mobilization (AJM) would reduce neuropathic pain and enhance motor functional recovery after nerve injury. In the axonotmesis model, AJM during 15 sessions every other day was conducted in rats. Mechanical and thermal hyperalgesia and motor performance deficit were measured for 5 weeks. After 5 weeks, we performed morphological analysis and quantified the immunoreactivity for CD11b/c and glial fibrillary acidic protein (GFAP), markers of glial activation, in the lumbar spinal cord. Mechanical and thermal hyperalgesia and motor performance deficit were found in the Crush+Anesthesia (Anes) group (P<0.001), which was significantly decreased after AJM (P<0.001). In the morphological analysis, the Crush+Anes group presented reduced myelin sheath thickness (P<0.05), but the AJM group presented enhanced myelin sheath thickness (P<0.05). Peripheral nerve injury increased the immunoreactivity for CD11b/c and GFAP in the spinal cord (P<0.05), and AJM markedly reduced CD11b/c and GFAP immunoreactivity (P<0.01). These results show that AJM in rats produces an antihyperalgesic effect and peripheral nerve regeneration through the inhibition of glial activation in the dorsal horn of the spinal cord. These findings suggest new approaches for physical rehabilitation to protect from or reduce the effects of nerve injury.

Supplemental Digital Content is Available in the Text. Human sodium channel Na V 1.7 in induced pluripotent stem cell–derived sensory neurons sets the action potential threshold but does not support subthreshold depolarizations.

Topically applied camphor elicits a sensation of cool, but nothing is known about how it affects cold temperature sensing. We found that camphor sensitizes a subpopulation of menthol-sensitive native cutaneous nociceptors in the mouse to cold, but desensitizes and partially blocks heterologously expressed TRPM8 (transient receptor potential cation channel subfamily M member 8). In contrast, camphor reduces potassium outward currents in cultured sensory neurons and, in cold nociceptors, the cold-sensitizing effects of camphor and menthol are additive. Using a membrane potential dye-basedscreeningassayandheterologouslyexpressedpotassiumchannels,wefoundthattheeffectsofcamphoraremediatedbyinhibitionofK v 7.2/3 channelssubtypesthatgeneratetheM-currentinneurons.Inlinewiththisfinding,thespecificM-currentblockerXE991reproducedthecold-sensitizing effect of camphor in nociceptors. However, the M-channel blocking effects of XE991 and camphor are not sufficient to initiate cold transduction but require a cold-activated inward current generated by TRPM8. The cold-sensitizing effects of XE991 and camphor are largest in high-threshold cold nociceptors. Low-threshold corneal cold thermoreceptors that express high levels of TRPM8 and lack potassium channels are not affected by camphor. We also found that menthol-like camphor-potently inhibits K v 7.2/3 channels. The apparent functional synergism arising from TRPM8 activation and M-current block can improve the effectiveness of topical coolants and cooling lotions, and may also enhance TRPM8-mediated analgesia.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6-8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/ high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets. Introduction Human induced pluripotent stem cells (hiPSC) are selfrenewing and can differentiate in almost any adult cell type. These characteristics make hiPSC a useful tool for disease modeling in basic research and drug discovery 1. Human iPSC retains the donor genetic background which allows deriving disease-relevant cell types that are most

The trigeminal nerve is comprised of three main divisions, ophthalmic, maxillary and mandibular, each providing somatosensory innervation to distinct regions of the head, face and oral cavity. Recently, a role for endothelins in nociceptive signaling in the trigeminal system has been proposed. The present study aimed to gain better insight into the participation of the endothelin system in trigeminal nociceptive transmission. Herein ET-1 and ET-3 mRNA was detected in the rats' trigeminal ganglion (TG). Fluorescent labeling of TG neurons revealed that ET(A) and ET(B) receptors are distributed along the entire TG, but ET(A) receptor expression slightly predominated within the three divisions. TRPV1 receptors were also detected throughout the entire TG, and a significant proportion of TRPV1-positive neurons (approximately 30%) co-expressed either ET(A) or ET(B) receptors. Our behavioral data showed that ET-1 (3 to 30 pmol/site) induced overt nociceptive responses after injection into the upper lip or temporomandibular joint (TMJ) and hyperalgesic actions when applied to the eye, while ET-3 and the selective ET(B) receptor agonist IRL-1620 (each at 3 to 30 pmol/site) showed only the first two effects. Injection of BQ-123, but not BQ-788 (ET(A) and ET(B) receptor antagonists, respectively, 10 nmol/site each, 30 min beforehand), into the ipsilateral upper lip abolished ET-1 induced facial grooming, but both antagonists markedly reduced the nociceptive responses induced by ET-1 injected into the TMJ. Taken together, these findings suggest that endothelins, acting through ET(A) and/or ET(B) receptors, may play an important role in mediating pain resulting from activation of most trigeminal nerve branches.

Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.

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