Cultured Rat Dorsal Root Ganglion Research Articles

The Effects of IGF-1 on TNF-α-Treated DRG Neurons by Modulating ATF3 and GAP-43 Expression via PI3K/Akt/S6K Signaling Pathway.

Upregulation of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) is involved in the development and progression of numerous neurological disorders. Recent reports have challenged the concept that TNF-α exhibits only deleterious effects of pro-inflammatory destruction, and have raised the awareness that it may play a beneficial role in neuronal growth and function in particular conditions, which prompts us to further investigate the role of this cytokine. Insulin-like growth factor-1 (IGF-1) is a cytokine possessing powerful neuroprotective effects in promoting neuronal survival, neuronal differentiation, neurite elongation, and neurite regeneration. The association of IGF-1 with TNF-α and the biological effects, produced by interaction of IGF-1 and TNF-α, on neuronal outgrowth status of primary sensory neurons are still to be clarified. In the present study, using an in vitro model of primary cultured rat dorsal root ganglion (DRG) neurons, we demonstrated that TNF-α challenge at different concentrations elicited diverse biological effects. Higher concentration of TNF-α (10ng/mL) dampened neurite outgrowth, induced activating transcription factor 3 (ATF3) expression, reduced growth-associated protein 43 (GAP-43) expression, and promoted GAP-43 and ATF3 coexpression, which could be reversed by IGF-1 treatment; while lower concentration of TNF-α (1ng/mL) promoted neurite sprouting, decreased ATF3 expression, increased GAP-43 expression, and inhibited GAP-43 and ATF3 coexpression, which could be potentiated by IGF-1 supplement. Moreover, IGF-1 administration restored the activation of Akt and p70 S6 kinase (S6K) suppressed by higher concentration of TNF-α (10ng/mL) challenge. In contrast, lower concentration of TNF-α (1ng/mL) had no significant effect on Akt or S6K activation, and IGF-1 administration activated these two kinases. The effects of IGF-1 were abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These data imply that IGF-1 counteracts the toxic effect of higher concentration of TNF-α, while potentiates the growth-promoting effect of lower concentration of TNF-α, with the node for TNF-α and IGF-1 interaction being the PI3K/Akt/S6K signaling pathway. This study is helpful for interpretation of the association of IGF-1 with TNF-α and the neurobiological effects elicited by interaction of IGF-1 and TNF-α in neurological disorders.

Assessment of Neuroprotective Effects of Neurotropin in Cultured Rat Dorsal Root Ganglion Neurons Using High- Throughput Imaging

Background: Neurotropin, a non-protein extract derived from inflamed rabbit skin following inoculation with vaccinia virus, is an analgesic agent. The mechanism of action of neurotropin has been partially clarified. No experimental study, however, has been undertaken to assess oxaliplatin-induced neurotoxicity using quantitative in vitro high-throughput image analysis. In the present study, to elucidate the action of neurotropin on dorsal root ganglion (DRG) neurons, we explored the antioxidative and anti-inflammatory activity of neurotropin. We assessed the viability and morphological changes in cultured rat DRG neurons treated with oxaliplatin and neurotropin. Methods: We plated dissociated DRG neurons from female Sprague-Dawley rats (100-120 g) into 96-well plates and added oxaliplatin and/or neurotropin. The cells were examined with a fluorescence microscope, ImageXpress. The images were analyzed with high content image processing software, MetaXpress. Cells treated with neurotropin and oxaliplatin were lysed in Pierce RIPA Buffer and subjected to western blot analysis. Results: Significant increases in neurite length, neurite area, cell body area, and neurite branching were observed in the presence of neurotropin in DRG neurons treated with oxaliplatin. When the highest concentration of neurotropin, 40 neurotropin units (NU) mNU/mL, was applied, a marked effect was seen. Neurotropin did not affect the phosphorylation of c-Jun N-terminal kinase, but suppressed the phosphorylation of p38. This finding suggests that neurotropin reversed oxaliplatin-induced apoptosis via suppression of the p38 mitogenactivated protein kinase signaling pathway. Conclusion: The results of this in vitro study suggest a possible role for neurotropin as a useful neuroprotective and anti-apoptotic agent.

Non-proton ligand-sensing domain of acid-sensing ion channel 3 is required for itch sensation.

Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. However, the molecular mechanisms of itch are unclear. Acid-sensing ion channel 3 (ASIC3) is a sensor of acidic and primary inflammatory pain. The whole-cell patch clamp technique was used to determine the effect of chloroquine (CQ) on ASICs currents in primary sensory neurons or the Chinese hamster ovary cells transfected with rat ASIC1a or ASIC3. Site-directed mutagenesis of plasmid was performed. Scratching behavior was evaluated by measuring the number of bouts during 30min after injection. CQ, an anti-malarial drug defined as a histamine-independent pruritogen, selectively enhanced the sustained phase of ASIC3 current in a concentration-dependent manner either in ASIC3-transfected Chinese hamster ovary cells or in primary cultured rat dorsal root ganglion neurons. Further studies revealed that the effect of CQ on ASIC3 channels depends on the newly identified non-proton ligand-sensing domain. Importantly, CQ-evoked scratching behavior was largely alleviated by APETx2, a selective ASIC3 channel blocker. Like CQ, other compounds such as amiloride, 2-guanidine-4-methylquinazoline and neuropeptide FF, which have been previously reported to be non-proton ligands that activate ASIC3, undoubtedly evoked the scratching response. In conclusion, ASIC3, a proton-gated ion channel critical for pain sensation, also functions as an essential component of itch transduction.

Validating Metal-Organic Framework Nanoparticles for Their Nanosafety in Diverse Biomedical Applications.

Metal-organic frameworks (MOFs) are promising platforms for the synthesis of nanoparticles for diverse medical applications. Their fundamental design principles allow for significant control of the framework architecture and pore chemistry, enabling directed functionalization for nanomedical applications. However, before applying novel nanomaterials to patients, it is imperative to understand their potential health risks. In this study, the nanosafety of different MOF nanoparticles is analyzed comprehensively for diverse medical applications. The authors first evaluate the effects of MOFs on human endothelial and mouse lung cells, which constitute a first line of defense upon systemic blood-mediated and local lung-specific applications of nanoparticles. Second, we validated these MOFs for multifunctional surface coatings of dental implants using human gingiva fibroblasts. Moreover, biocompatibility of MOFs is assessed for surface coating of nerve guidance tubes using human Schwann cells and rat dorsal root ganglion cultures. The main finding of this study is that the nanosafety and principal suitability of our MOF nanoparticles as novel agents for drug delivery and implant coatings strongly varies with the effector cell type. We conclude that it is therefore necessary to carefully evaluate the nanosafety of MOF nanomaterials with respect to their particular medical application and their interacting primary cell types, respectively.

Fabrication and characterization of microspheres encapsulating astrocytes for neural regeneration.

Astrocytes play a critical role in supporting the normal physiological function of neurons. Recent studies have revealed that astrocyte transplantation can promote axonal regeneration and functional recovery after spinal cord injury. Biomaterial can be designed as a growth-permissive substrate and serve as a carrier for astrocyte transplantation into injured spinal cord. In this study, we developed a method to generate collagen microspheres encapsulating astrocytes by injecting a mixture of collagen and astrocytes into a cell culture medium with a syringe controlled by a syringe pump. The collagen microspheres were crosslinked with poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) to reduce the degradation rate. The viability of cells in the crosslinked microspheres was higher than 90%. Astrocytes were transfected with plasmids encoding nerve growth factor (NGF)-ires-enhanced green fluorescent protein (EGFP) genes by electroporation and encapsulated in crosslinked microspheres. The level of NGF released into the cell culture medium was higher than that remaining in the microspheres or astrocytes. When microspheres encapsulating astrocytes transfected with plasmids encoding NGF-ires-EGFP genes were added into the cultured rat dorsal root ganglion, the axonal growth was significantly enhanced. This study shows that the microspheres can be potentially used as a carrier of astrocytes to promote nerve regeneration in injured neural tissue.

Nesfatin-1 increases intracellular calcium concentration by protein kinase C activation in cultured rat dorsal root ganglion neurons

Nesfatin-1 is a recently identified anorexigenic hypothalamic polypeptide derived from the posttranslational processing of nucleobindin 2 (NUCB2). Several studies have indicated that this neuropeptide may be participated in somatosensory and visceral transmission including pain signals in addition to energy metabolism. The aim of this study was to explore the possible role of nesfatin-1 in the transmission of peripheral neural signals by investigating the effects of nesfatin-1 on intracellular free calcium levels ([Ca2+]i) in cultured neonatal rat dorsal root ganglion (DRG) neurons. The effects of nesfatin-1 on [Ca2+]i in DRG neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1-or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of nesfatin-1 on [Ca2+]i and role of the protein kinase C (PKC)-mediated pathway in nesfatin-1 effect were assessed. Nesfatin-1 elevated [Ca2+]i in cultured DRG neurons. The response was prevented by pretreating the cells with pertussis toxin. The protein kinase C inhibitor chelerythrine chloride suppressed nesfatin-1-induced rise in [Ca2+]i. The result shows that nesfatin-1 interacts with a G protein-coupled receptor, leading to an increase of [Ca2+]i, which is linked to protein kinase C activation in cultured rat DRG neurons.

Evaluation of Cisplatin Neurotoxicity in Cultured Rat Dorsal Root Ganglia via Cytosolic Calcium Accumulation.

Calcium homeostasis is considered to be important in antineoplastic as well as in neurotoxic adverse effects of cisplatin. This study aimed to investigate the role of Ca(2+) in cisplatin neurotoxicity in cultured rat dorsal root ganglia (DRG) cells. Cell culture study. DRG cells prepared from 1-day old Sprague-Dawley rats were used to determine the role of Ca(2+) in the cisplatin (10-600 μM) neurotoxicity. The cells were incubated with cisplatin plus nimodipine (1-3 μM), dizocilpine (MK-801) (1-3 μM) or thapsigargin (100-300 nM). Toxicity of cisplatinon DRG cells was determined by the MTT assay. The neurotoxicity of cisplatin was significant when used in high concentrations (100-600 μM). Nimodipine (1 μM) but not MK-801 or thapsigargin prevented the neurotoxic effects of 200 μM of cisplatin. Voltage-dependent calcium channels may play a role in cisplatin neurotoxicity.

Acrylamide Retards the Slow Axonal Transport of Neurofilaments in Rat Cultured Dorsal Root Ganglia Neurons and the Corresponding Mechanisms.

Chronic acrylamide (ACR) exposure induces peripheral-central axonopathy in occupational workers and laboratory animals, but the underlying mechanisms remain unclear. In this study, we first investigated the effects of ACR on slow axonal transport of neurofilaments in cultured rat dorsal root ganglia (DRG) neurons through live-cell imaging approach. Then for the underlying mechanisms exploration, the protein level of neurofilament subunits, motor proteins kinesin and dynein, and dynamitin subunit of dynactin in DRG neurons were assessed by western blotting and the concentrations of ATP was detected using ATP Assay Kit. The results showed that ACR treatment results in a dose-dependent decrease of slow axonal transport of neurofilaments. Furthermore, ACR intoxication significantly increases the protein levels of the three neurofilament subunits (NF-L, NF-M, NF-H), kinesin, dynein, and dynamitin subunit of dynactin in DRG neurons. In addition, ATP level decreased significantly in ACR-treated DRG neurons. Our findings indicate that ACR exposure retards slow axonal transport of NF-M, and suggest that the increase of neurofilament cargoes, motor proteins, dynamitin of dynactin, and the inadequate ATP supply contribute to the ACR-induced retardation of slow axonal transport.

Glycolytic metabolite methylglyoxal inhibits cold and menthol activation of the transient receptor potential melastatin type 8 channel

Methylglyoxal (MG) is a reactive dicarbonyl compound involved in protein modifications linked to diabetes mellitus. The plasma level of MG is elevated in diabetic patients, particularly those with painful diabetic neuropathy. Diabetic neuropathy is often associated with spontaneous pain and altered thermal perception. This study assesses effects of MG on TRPM8, an ion channel involved in innocuous cold sensing and cold allodynia and also in cold-mediated analgesia. Acute treatment with MG inhibited the activation of recombinant rat and human transient receptor potential melastatin type 8 (TRPM8) by cold and chemical agonists. A similar effect was observed when native TRPM8 was investigated in cultured rat dorsal root ganglion (DRG) neurons. DRG neurons treated with MG for 16-24 hr displayed a significant reduction in the fraction of cold- and menthol-sensitive neurons, most likely expressing TRPM8. The fraction of allyl isothiocyanate-sensitive neurons was also reduced, and the coexpression among different neuronal populations was affected. The same prolonged exposure to MG significantly reduced the expression of TRPM8 at the mRNA level. Overall, our data provide evidence for decreased activity and expression level of TRPM8 in the presence of MG, which may be linked to some of the alterations in pain and temperature sensing reported by diabetic patients. © 2015 Wiley Periodicals, Inc.

Effects of apelin-13 in mice model of experimental pain and peripheral nociceptive signaling in rat sensory neurons

Objective: Apelin-13 is an endogenous peptide with potential analgesic action, although the sites of its analgesic effects remain uncertain and the results are even controversial with regard to its pain modulating action. This study evaluated the possible pain-modulating action of peripherally administered apelin-13 using heat-induced, withdrawal latency to the thermal stimuli, acute pain model in mice. Involvement of peripheral mechanisms was tested, by using the intracellular calcium concentrations as a key signal for nociceptive transmission, in cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium-sensitive dye Fura-2 acetoxymethyl ester (1 µM). Intracellular calcium responses in individual DRG neurons were quantified by ratiometric calcium imaging technique. Results: Peripheral injection of a single dose of apelin-13 (100 mg/kg and 300 mg/kg) significantly decreases the latency to painful stimuli in a dose and time-dependent manner (p < 0.01, p < 0.05, respectively, n = 8 each). Apelin-13 (0.1 µM and 1 µM) did not produce a significant effect on cytoplasmic Ca2+ ([Ca2+]i) responses, evoked by membrane depolarization, in cultured rat DRG neurons. Conclusion: Together these results indicate that apelin-13 can cause increased pain sensitivity after peripheral administration, but this effect does not involve calcium mediated signaling in primary sensory neurons.

Inhibitory Activity of Yokukansankachimpihange against Nerve Growth Factor-Induced Neurite Growth in Cultured Rat Dorsal Root Ganglion Neurons.

Chronic pruritus is a major and distressing symptom of many cutaneous diseases, however, the treatment remains a challenge in the clinic. The traditional Chinese-Japanese medicine (Kampo medicine) is a conservative and increasingly popular approach to treat chronic pruritus for both patients and medical providers. Yokukansankachimpihange (YKH), a Kampo formula has been demonstrated to be effective in the treatment of itching of atopic dermatitis in Japan although its pharmacological mechanism is unknown clearly. In an attempt to clarify its pharmacological actions, in this study, we focused on the inhibitory activity of YKH against neurite growth induced with nerve growth factor (NGF) in cultured rat dorsal root ganglion (DRG) neurons because epidermal hyperinnervation is deeply related to itch sensitization. YKH showed approximately 200-fold inhibitory activity against NGF-induced neurite growth than that of neurotropin (positive control), a drug used clinically for treatment of chronic pruritus. Moreover, it also found that Uncaria hook, Bupleurum root and their chemical constituents rhynchophylline, hirsutine, and saikosaponin a, d showed inhibitory activities against NGF-induced neurite growth, suggesting they should mainly contribute to the inhibitory activity of YKH. Further study on the effects of YKH against epidermal nerve density in “itch-scratch” animal models is under investigation.

Towards Development of a Dermal Pain Model: In Vitro Activation of Rat and Human Transient Receptor PotentialAnkyrin Repeat 1 and Safe Dermal Injection of o-Chlorobenzylidene Malononitrile to Rat.

During clinical development of analgesics, it is important to have access to pharmacologically specific human pain models. o-Chlorobenzylidene malononitrile (CS) is a selective and potent agonist of the transient receptor potential ankyrin repeat 1 (TRPA1), which is a transducer molecule in nociceptors sensing reactive chemical species. While CS has been subject to extensive toxicological investigations in animals and human beings, its effects on intradermal or subcutaneous injection have not previously been reported. We have investigated the potential of CS to be used as an agonist on TRPA1 in human experimental pain studies. A calcium influx assay was used to confirm the capacity of CS to activate TRPA1 with >100,000 times the selectivity over the transient receptor potential vanilloid receptor 1. CS dose-dependently (EC50 0.9μM) released calcitonin gene-related peptide in rat dorsal root ganglion cultures, supporting involvement in pain signalling. In a local tolerance study, injection of a single intradermal dose of 20mM CS to rats resulted in superficial, circular crusts at the injection sites after approximately 4days. The histopathology evaluation revealed a mild, acute inflammatory reaction in the epidermis and dermis at the intradermal CS injection site 1day after administration. After 14days, the epidermal epithelium was fully restored. The symptoms were not considered to be adverse, and it is suggested that doses up to 20μL of 20mM CS can be safely administered to human beings. In conclusion, our data support development of a CS human dermal pain model.

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by high-K+ (30 mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1 µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p < 0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300 µM) inhibited the Ca2+ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca2+]i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca2+]i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca2+]i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.

Effect of protons on the mechanical response of rat muscle nociceptive fibers and neurons in vitro

Strong exercise makes muscle acidic, and painful. The stimulus that activates muscle nociceptors in such instance may be protons. Reportedly, however, not many afferents are excited by protons alone. We, therefore, posited that protons sensitize muscular nociceptors to mechanical stimuli. We examined effects of protons on mechanical sensitivity of muscle nociceptors by single-fiber recording from rat muscle-nerve preparations in vitro and by whole cell patch-clamp recording of mechanically activated (MA) currents from cultured rat dorsal root ganglion neurons. We recorded 38 Aδ- and C-fibers. Their response magnitude was increased by both pH 6.2 and pH 6.8; in addition the mechanical threshold was lowered by pH 6.2. Decrease in the threshold by pH6.2 was also observed in MA currents. Presently observed sensitization by protons could be involved in several types of ischemic muscle pain, and may also be involved in cardiovascular and respiratory controls during exercise.

Decelerated transport and its mechanism of 2,5-hexanedione on middle-molecular-weight neurofilament in rat dorsal root ganglia cells

Chronic exposure to n-hexane induces peripheral-central axonopathy, mediated by its metabolite 2,5-hexanedione (2,5-HD), in occupational workers and experimental animals, but the underlying mechanism is still unclear. In the current study, we investigated the effects of 2,5-HD on middle-molecular-weight neurofilament (NF-M) axonal transport using live-cell imaging technique in cultured rat dorsal root ganglia (DRG) cells. PA-GFP-NF-M plasmid was transfected into DRG neurons and live-cell imaging was performed to observe the slow axonal transport of NF-M. The levels of cytoskeleton and motor proteins in DRG cells were detected by Western-blot and the concentration of ATP was determined using an ATP Assay Kit. The results showed that 2,5-HD administration resulted in a decrease of NF-M axonal transport and a reduction of three neurofilament subunits levels in DRG cells. Furthermore, 2,5-HD exposure significantly decreased ATP contents and the protein levels of kinesin heavy chain (KHC). These findings indicated that 2,5-HD reduced slow axonal transport, neurofilaments cargoes, motor proteins and ATP energy in rat DRG cells, which may contribute to 2,5-HD-induced neurotoxicity.

Ciliary neurotrophic factor reverses aberrant mitochondrial bioenergetics through the JAK/STAT pathway in cultured sensory neurons derived from streptozotocin-induced diabetic rodents.

Mitochondrial dysfunction occurs in sensory neurons and contributes to diabetic neuropathy. Ciliary neurotrophic factor (CNTF) stimulates axon regeneration in type 1 diabetic rodents and prevents deficits in axonal caliber, nerve conduction, and thermal sensation. We tested the hypothesis that CNTF enhances sensory neuron function in diabetes through JAK/STAT (Janus kinase/signal transducers and activators of transcription) signaling to normalize impaired mitochondrial bioenergetics. The effect of CNTF on gene expression and neurite outgrowth of cultured adult dorsal root ganglia (DRG) sensory neurons derived from control and streptozotocin (STZ)-induced diabetic rodents was quantified. Polarization status and bioenergetics profile of mitochondria from cultured sensory neurons were determined. CNTF treatment prevented reduced STAT3 phosphorylation (Tyr 705) in DRG of STZ-diabetic mice and also enhanced STAT3 phosphorylation in rat DRG cultures. CNTF normalized polarization status of the mitochondrial inner membrane and corrected the aberrant oligomycin-induced mitochondrial hyperpolarization in axons of diabetic neurons. The mitochondrial bioenergetics profile demonstrated that spare respiratory capacity and respiratory control ratio were significantly depressed in sensory neurons cultured from STZ-diabetic rats and were corrected by acute CNTF treatment. The positive effects of CNTF on neuronal mitochondrial function were significantly inhibited by the specific JAK inhibitor, AG490. Neurite outgrowth of sensory neurons from age-matched control and STZ-induced diabetic rats was elevated by CNTF and blocked by AG490. We propose that CNTF's ability to enhance axon regeneration and protect from fiber degeneration in diabetes is associated with its targeting of mitochondrial function and improvement of cellular bioenergetics, in part, through JAK/STAT signaling.

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons.

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knockdown of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150μg/50μL intraplantarly) and D-Ala2-MePhe4-Glyol5 enkephalin (300μg/50μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5mg/kg intraperitoneally) and MK-801 (0.2mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3mg/kg subcutaneously), but not loperamide (3mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca2+]i. However, this drug effect significantly decreased in cells pretreated with loperamide (3μM, 72hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca2+]i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.

Camphor Activates and Sensitizes Transient Receptor Potential Melastatin 8 (TRPM8) to Cooling and Icilin

Camphor is known to potentiate both heat and cold sensations. Although the sensitization to heat could be explained by the activation of heat-sensitive transient receptor potential (TRP) channels TRPV1 and TRPV3, the camphor-induced sensitization to cooling remains unexplained. In this study, we present evidence for the activation of the cold- and menthol-sensitive channel transient receptor potential melastatin 8 (TRPM8) by camphor. Calcium transients evoked by camphor in HEK293 cells expressing human and rat TRPM8 are inhibited by the TRPM8 antagonists 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide and 2-aminoethyl diphenylborinate. Camphor also sensitized the cold-induced calcium transients and evoked desensitizing outward-rectifying currents in TRPM8-expressing HEK293 cells. In the presence of ruthenium red (a blocker of TRPV1, TRPV3, and TRPA1), the camphor sensitivity of cultured rat dorsal root ganglion neurons was highest in a subpopulation of cold- and icilin-sensitive neurons, strongly suggesting that camphor activates native TRPM8. Camphor has a dual action on TRPM8: it not only activates the channel but also inhibits its response to menthol. The icilin-insensitive chicken TRPM8 was also camphor insensitive. However, camphor was able to activate an icilin-insensitive human TRPM8 mutant channel. The activation and sensitization to cold of mammalian TRPM8 are likely to be responsible for the psychophysical enhancement of innocuous cold and "stinging/burning" cold sensations by camphor.

MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway

We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.