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Normally, an obvious antagonism exists between pain and itch. In normal conditions, painful stimuli suppress itch sensation, whereas pain killers often generate itch. Although pain and itch are mediated by separate pathways under normal conditions, most chemicals are not highly specific to one sensation in chronic pathologic conditions. Notably, in patients with neuropathic pain, histamine primarily induces pain rather than itch, while in patients with atopic dermatitis, bradykinin triggers itch rather than pain. Accordingly, repetitive scratching even enhances itch sensation in chronic itch conditions. Physicians often prescribe pain relievers to patients with chronic itch, suggesting common mechanisms underlying chronic pain and itch, especially peripheral and central sensitization. Rather than separating itch and pain, studies should investigate chronic itch and pain including neuropathic and inflammatory conditions. Here, we reviewed chronic sensitization leading to chronic pain and itch at both peripheral and central levels. Studies investigating the connection between pain and itch facilitate the development of new therapeutics against both chronic dysesthesias based on the underlying pathophysiology.

Despite the high prevalence of chronic dermatitis and the accompanied intractable itch, therapeutics that specifically target itching have low efficacy. Increasing evidence suggests that TLRs contribute to immune activation and neural sensitization; however, their roles in chronic itch remain elusive. Here, we show that the RBL-2H3 mast cell line expresses TLR4 and that treatment with a TLR4 antagonist opposes the LPS dependent increase in mRNA levels of Th2 and innate cytokines. The pathological role of TLR4 activation in itching was studied in neonate rats that developed chronic itch due to neuronal damage after receiving subcutaneous capsaicin injections. Treatment with a TLR4 antagonist protected these rats with chronic itch against scratching behavior and chronic dermatitis. TLR4 antagonist treatment also restored the density of cutaneous nerve fibers and inhibited the histopathological changes that are associated with mast cell activation after capsaicin injection. Additionally, the expression of IL-1β, IL-4, IL-5, IL-10, and IL-13 mRNA in the lesional skin decreased after TLR4 antagonist treatment. Based on these data, we propose that inhibiting TLR4 alleviated itch in a rat model of chronic relapsing itch, and the reduction in the itch was associated with TLR4 signaling in mast cells and nerve fibers.

Background Atopic dermatitis (AD) is a highly pruritic, chronic inflammatory skin disease associated with hyperreactivity to environmental triggers. Among those, outdoor air pollutants such as particulate matter (PM) have been reported to aggravate pre-existing AD. However, underlying mechanisms of air pollution-induced aggravation of AD have hardly been studied. Objective To investigate the molecular mechanisms by which glyoxal, a PM-forming organic compound, exacerbates the symptoms of AD induced by neonatal capsaicin treatment. Methods Naive and AD rats had been exposed to either fresh air or vaporized glyoxal for 5 weeks (2 h/day and 5 days/week) since one week of age. Pruritus and dermatitis were measured every week. The skin and blood were collected and immunological traits such as Staphylococcus aureus skin colonization, production of antimicrobial peptides and immunoglobulin, and mRNA expression of inflammatory cytokines were analyzed. Results Exposure to glyoxal aggravated pruritus and dermatitis in AD rats, but did not induce any symptoms in naive rats. Staphylococcus aureus skin colonization was increased in the skin of both naive and AD rats. Expression of antimicrobial peptides such as LL-37 and β-defensin-2 was also increased by exposure to glyoxal in the skin of both naive and AD rats. The mRNA expression of Th1-related cytokines was elevated on exposure to glyoxal. However, serum immunoglobulin production was not significantly changed by exposure to glyoxal. Conclusion In AD rats, exposure to glyoxal exacerbated pruritus and cutaneous inflammation, which was associated with increased colonization of S. aureus and subsequent immunological alterations in the skin.

Atopic dermatitis (AD) is a complex disease featuring pruritic skin inflammation. Many animal models have been developed. In a rat model, subcutaneous capsaicin injection within 48 hours after birth induces AD-like skin manifestations of dermatitis and scratching behaviour 3 weeks after the injection. When 2- to 4-week-old rats were injected with capsaicin, the lag period was shortened, and the severity of skin manifestations was significantly reduced, suggesting influences of postnatal development. Lgr6 is an epidermal stem cell marker that is normally restricted to the isthmus area of hair follicles at postnatal 2 weeks. Lgr6 persisted in the interfollicular epidermis of capsaicin-injected rats beyond 3 weeks after birth, indicating that capsaicin-induced skin manifestations were influenced by postnatal epidermal development. Capsaicin injection induced alteration of proteolytic processing of filaggrin and corneodesmosin, suggesting epidermal barrier dysfunction. Inappropriate degradation of matriptase was observed. Degrees of proteolysis of these proteins were corelated with the severity of manifestations, suggesting that inappropriate proteolysis might be a possible cause of the skin manifestations. These results strongly suggest that capsaicin may dysregulate the protease system, resulting in alteration of profilaggrin and corneodesmosin proteolysis and skin manifestations. These events may be influenced by postnatal epidermal development.

Isolation stress is a major risk factor for neuropsychiatric disorders such as depressive and anxiety disorders. However, the molecular mechanisms underlying isolation-induced neuropsychiatric disorders remain elusive. In the present study, we investigated the subcellular mechanisms by which long-term isolation elicits depression and anxiety-related behaviors in mice. First, we found that long-term isolation induced depression-related behaviors in the forced swimming test (FST) and the sucrose preference test, as well as anxiety-related behaviors in the elevated zero maze test (EZMT) and the open field test. Next, we showed that intracentral amygdala (CeA) injection of oxytocin (OXT), but not intracerebroventricular injection, attenuated isolation-induced depression and anxiety-related behaviors via oxytocin receptor (OXTR), not vasopressin-1a receptor (V1aR), in the FST and EZMT, respectively. Quantitative real-time polymerase chain reaction analysis revealed that after 5 weeks of isolation, mRNA transcription of OXTR in the CeA, but not that of V1aR, significantly decreased, whereas OXT and vasopressin mRNA transcription in the paraventricular nucleus of hypothalamus did not change significantly. Whole-cell patch clamping of acute brain slices demonstrated that the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in CeA neurons, but not their amplitude, was lower in isolated mice than in group-housed mice. Notably, OXT treatment increased the mIPSC frequency in the CeA neurons, but to a lesser extent in the case of isolated mice than in that of group-housed mice via OXTR. Taken together, our findings suggest that long-term isolation down-regulates OXTR mRNA transcription and diminishes OXT-induced inhibitory synaptic transmission in the CeA and may contribute to the development of depression and anxiety-related behaviors in isolated mice through the enhancement of CeA activity.

Blockage of voltage-gated sodium channels is used to treat neuropathic pain which is chronic and can become debilitating. Sodium channels Nav1.7–1.9 are especially attractive targets for drug discovery because of the broad therapeutic potential of their modulation. For a neuropathic pain therapy, anticonvulsant like lamotrigine, carbamazepine and a topical anesthetic such as Lidocaine are used. A growing number of clinical reports suggest that selective inhibitors of Nav1.7 are likely to be the powerful analgesics for treating a broad range of pain conditions. Therefore we evaluated 108 amide derivatives synthesized on human Nav1.7 (hNav1.7) by VIPR (voltage/ion probe reader), a fluorescence image plate reader (FLIPR) assay that used voltage-sensor fluorescence dye and stable HEK-293 cell lines expressing hNaV1.7. Ten compounds demonstrated inhibitory activity, and the two most active compounds (5 and 6) had IC50 values of 8–10 μM.

Herding with a litter is known to comfort rodents, whereas isolation and grouping with noncagemates provoke stress. The effects of stress induced by isolation and grouping with noncagemates on pain responses, and their underlying mechanisms remain elusive. We assessed the effect of isolation, a common condition during behavioral tests, and of grouping on defecation and pain behaviors of mice. Fecal pellets were counted 2 hours after exposure to the test chamber. It is significantly more in the isolated mice than in the grouped mice. Hindpaw withdrawal threshold and withdrawal latency were adopted as the indicatives of mechanical and thermal pain sensitivities, respectively. Interestingly, isolated mice showed higher pain thresholds than mice grouping with cagemates, and even those with noncagemates, indicating analgesic effects. Such effects were reduced by intrathecal injection of 0.01 mg/kg of naloxone (opioid receptor antagonist), atosiban (oxytocin and vasopressin receptor antagonist), and ketanserin (5-HT receptor antagonist). Intraperitoneal delivery of 1 mg/kg of naloxone and atosiban, but not ketanserin, also alleviated the isolation-induced analgesic effects. In contrast, these drugs at the same dose had no significant effect on the mice grouping with cagemates. In addition, the effect of morphine on thermal pain was more robust in the mice grouping with cagemates than in the isolated mice. These data demonstrated that brief isolation caused analgesia, mediated by endogenous opioidergic, oxytocinergic, and serotonergic pathways. These results indicate that isolation during pain behavioral tests can affect pain responses and the efficacy of drugs; thus, nociception tests should be conducted in grouping.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

Rafael Taeho Han,1,2 Sewon Kim,3 Kyungmin Choi,1,2 Hyeonseok Jwa,1,2 JaeHee Lee,1,2 Hye Young Kim,1,2 Hee Jin Kim,4 Hang-Rae Kim,5 Seung Keun Back,6 Heung Sik Na1,2 1Neuroscience Research Institute, 2Department of Physiology, 3Department of Microbiology, College of Medicine, Korea University, Seoul, 4Division of Biological Science and Technology, Science and Technology College, Yonsei University Wonju Campus, Wonju, 5Department of Anatomy, College of Medicine, Seoul National University, Seoul, 6Department of Pharmaceutics and Biotechnology, College of Medical Engineering, Konyang University, Chungnam, South Korea Abstract: Recent studies have shown that approximately 70% of patients with severe atopic dermatitis (AD) develop asthma. Development of AD in infancy and subsequent other atopic diseases such as asthma in childhood is referred to as atopic march. However, a causal link between the diseases of atopic march has remained largely unaddressed, possibly due to lack of a proper animal model. Recently, we developed an AD rat model showing chronically relapsing dermatitis and scratching behaviors induced by neonatal capsaicin treatment. Here, we investigated whether our model also showed asthmatic changes, with the aim of expanding our AD model into an atopic march model. First, we confirmed that capsaicin treatment (50mg/kg within 24h after birth) induced dermatitis and scratching behaviors until 6 weeks of age. After that, the mRNA expression of Th1 and Th2 cytokines, such as IFN-γ and TNF-α, and IL-4, IL-5, and IL-13, respectively, was quantified with quantitative real-time polymerase chain reaction in the skin and the lungs. The number of total cells and eosinophils was counted in bronchoalveolar lavage (BAL) fluid. The levels of IgE in the serum and BAL fluid were determined with enzyme-linked immunosorbent assay. Paraffin-embedded sections (4μm) were stained with hematoxylin/eosin to analyze the morphology of the lung and the airway. Airway responsiveness was measured in terms of airway resistance and compliance using the flexiVent system. In the capsaicin-treated rats, persistent dermatitis developed, and scratching behaviors increased over several weeks. The levels of IgE in the serum and BAL fluid as well as the mRNA expression of Th2 cytokines, including IL-4, IL-5, and IL-13, in both the skin and the lungs were elevated, and the number of eosinophils in the BAL fluid was also increased in the capsaicin-treated rats compared to control rats. Morphological analysis of the airway revealed smooth muscle hypertrophy and extensive mucus plug in the capsaicin-treated rats. Functional studies demonstrated an increment of the airway resistance and a decrement of lung compliance in the capsaicin-treated rats compared to control rats. Taken together, our findings suggested that neonatal capsaicin treatment induced asthma-like airway inflammation and responses in juvenile rats. Keywords: atopic march, scratching, cytokines, eosinophils, BAL fluid, pruritus, flexiVent, airway hypertrophy and remodelling&nbsp

Atopic dermatitis is a complex disease of heterogeneous pathogenesis, in particular, genetic predisposition, environmental triggers, and their interactions. Indoor air pollution, increasing with urbanization, plays a role as environmental risk factor in the development of AD. However, we still lack a detailed picture of the role of air pollution in the development of the disease. Here, we examined the effect of formaldehyde (FA) exposure on the manifestation of atopic dermatitis and the underlying molecular mechanism in naive rats and in a rat model of atopic dermatitis (AD) produced by neonatal capsaicin treatment. The AD and naive rats were exposed to 0.8 ppm FA, 1.2 ppm FA, or fresh air (Air) for 6 weeks (2 hours/day and 5 days/week). So, six groups, namely the 1.2 FA-AD, 0.8 FA-AD, Air-AD, 1.2 FA-naive, 0.8 FA-naive and Air-naive groups, were established. Pruritus and dermatitis, two major symptoms of atopic dermatitis, were evaluated every week for 6 weeks. After that, samples of the blood, the skin and the thymus were collected from the 1.2 FA-AD, the Air-AD, the 1.2 FA-naive and the Air-naive groups. Serum IgE levels were quantified with ELISA, and mRNA expression levels of inflammatory cytokines from extracts of the skin and the thymus were calculated with qRT-PCR. The dermatitis and pruritus significantly worsened in 1.2 FA-AD group, but not in 0.8 FA-AD, compared to the Air-AD animals, whereas FA didn't induce any symptoms in naive rats. Consistently, the levels of serum IgE were significantly higher in 1.2 FA-AD than in air-AD, however, there was no significant difference following FA exposure in naive animals. In the skin, mRNA expression levels of Th1 cytokines such as TNF-α and IL-1β were significantly higher in the 1.2 FA-AD rats compared to the air-AD rats, whereas mRNA expression levels of Th2 cytokines (IL-4, IL-5, IL-13), IL-17A and TSLP were significantly higher in 1.2 FA-naive group than in the Air-naive group. These results suggested that 1.2 ppm of FA penetrated the injured skin barrier, and exacerbated Th1 responses and serum IgE level in the AD rats so that dermatitis and pruritus were aggravated, while the elevated expression of Th2 cytokines by 1.2 ppm of FA in naive rats was probably insufficient for clinical manifestation. In conclusion, in a rat model of atopic dermatitis, exposure to 1.2 ppm of FA aggravated pruritus and skin inflammation, which was associated with the elevated expression of Th1 cytokines.

In the present study, we investigated whether a novel compound, 2-(2-(4-((4-chlorophenyl)(phenyl)methyl) piperazin-1-yl)-2-oxoethylamino)-N-(3,4,5-trimethoxybenzyl)acetamide (HYP-1), is capable of binding to voltage-gated sodium channels (VGSCs) and evaluated both its inhibitory effect on Na+ currents of the rat dorsal root ganglia (DRG) sensory neuron and its in vivo analgesic activity using rat models of inflammatory and neuropathic pain. HYP-1 showed not only high affinity for rat sodium channel (site 2), but also potent inhibitory activity against the TTX-R Na+ currents of the rat DRG sensory neuron. HYP-1 co-injected with formalin (5%, 50 μl) under the plantar surface of rat hind paw dose-dependently reduced spontaneous pain behaviors during both the early and late phases. This result was confirmed by c-Fos immunofluorescence in the L4-5 spinal segments. A large number of c-Fos-positive neurons were observed in rat injected with a mixture of formalin and vehicle, but not in rat treated with a mixture of formalin and HYP-1. In addition, the effectiveness of HYP-1 (6 and 60 mg/kg, i.p.) in suppression of neuropathic pain, such as mechanical, cold and warm allodynia, induced by rat tail nerve injury was investigated. HYP-1 showed limited selectivity over hERG, N-type and T-type channels.Our present results indicate that HYP-1, as a VGSC blocker, has potential analgesic activities against nociceptive, inflammatory and neuropathic pain.

Novel diphenylpiperazine derivatives were synthesized and evaluated for their inhibitory activity against T-type calcium channel by whole-cell patch clamp recordings on HEK293 cells. Among the test compounds, 2 and 3d were effective in decreasing the response to formalin in both the first and second phases and demonstrated antiallodynic effects in a rat model of neuropathic pain.

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by d(CH2)5[Tyr(Me)2,Dab5] AVP, a vasopressin-1a (V1a) receptor antagonist, but not by desGly-NH2-d(CH2)5[DTyr2, Thr4]OVT, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

Ischemia-reperfusion (I/R) injury caused by abrupt restoration of the circulation after prolonged ischemic insult induces significant morbidity after reconstructive microsurgery. The authors investigated whether a postconditioning (post-con) procedure attenuated skeletal muscle I/R injury and protected muscular function. Three hours of complete ischemia was induced by occluding the muscular branches of rat extensor digitorum longus (EDL) muscle. The post-con procedure was started at the end of ischemia and involved six cycles of 15 seconds of reperfusion followed by 15 seconds of re-occlusion (3 minutes of total intervention) prior to initiating unlimited reperfusion. EDL muscle contractilities were compared with those of normal sides (no ischemic exposure), and experimental group results were also compared with control group results (3 hours of ischemia followed by full reperfusion without post-con) at 3 hours and 5 days postreperfusion. Muscle wet weights, myeloperoxidase (MPO) activities, and histological results were also evaluated. The muscle contractilities in the post-con group were significantly preserved at both 3 hours and 5 days postreperfusion as compared with ischemic controls. Decreased inflammatory cell infiltration, MPO activity, and wet weight of postconditioned EDL muscle suggested that post-con attenuated acute inflammatory reactions induced by I/R. This study demonstrates that post-con provides effective functional protection to skeletal muscles from I/R injury. © 2010 Wiley-Liss, Inc. Microsurgery 2010.

Background: N-type Ca2+ channels (Cav2.2) play an important role in the transmission of pain signals to the central nervous system. ω-Conotoxin (CTx)-MVIIA, also called ziconotide (Prialt®), effectively alleviates pain, without causing addiction, by blocking the pores of these channels. Unfortunately, CTx-MVIIA has a narrow therapeutic window and produces serious side effects due to the poor reversibility of its binding to the channel. It would thus be desirable to identify new analgesic blockers with binding characteristics that lead to fewer adverse side effects. Results: Here we identify a new CTx, FVIA, from the Korean Conus Fulmen and describe its effects on pain responses and blood pressure. The inhibitory effect of CTx-FVIA on N-type Ca2+ channel currents was dose-dependent and similar to that of CTx-MVIIA. However, the two conopeptides exhibited markedly different degrees of reversibility after block. CTx-FVIA effectively and dose-dependently reduced nociceptive behavior in the formalin test and in neuropathic pain models, and reduced mechanical and thermal allodynia in the tail nerve injury rat model. CTx-FVIA (10 ng) also showed significant analgesic effects on writhing in mouse neurotransmitter- and cytokine-induced pain models, though it had no effect on acute thermal pain and interferon-γ induced pain. Interestingly, although both CTx-FVIA and CTx-MVIIA depressed arterial blood pressure immediately after administration, pressure recovered faster and to a greater degree after CTx-FVIA administration. Conclusions: The analgesic potency of CTx-FVIA and its greater reversibility could represent advantages over CTx-MVIIA for the treatment of refractory pain and contribute to the design of an analgesic with high potency and low side effects.

Although numerous animal models for low back pain associated with intervertebral disk (IVD) degeneration have been proposed, insufficient data have been provided to make any conclusions regarding pain. Our aim in this study was to determine the reliability of complete Freund's adjuvant (CFA) injection into the rat spine as an animal model representing human discogenic pain.We studied IVD degenerative changes with pain development after a 10-microL CFA injection into the L5-6 IVD of adult rats using behavioral, histologic, and biochemical studies. Serial histologic changes were analyzed to detect degenerative changes. Expression of calcitonin gene-related peptide (CGRP), prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) were determined using immunohistochemistry or real-time polymerase chain reaction as support data for pain development. In addition, CGRP immunoreactivity (ir) at the IVD was considered indirect evidence of neural ingrowth into the IVD.There was a significant increase of the hindpaw withdrawal response in the CFA group until 7 wk postoperatively (P0.05). Histologic analyses revealed progressive degenerative changes of the disks without any damage in adjacent structures, including nerve roots. In the CGRP-ir staining study, the bilateral dorsal horns and IVD had positive ir after intradiscal CFA injection. CGRP mRNA expression was increased in the dorsal root ganglion (DRG) at 2 and 4 wk, whereas PGE and iNOS mRNAs were markedly increased at 2 wk. The increment of CGRP expression was higher in allodynic rats compared with nonallodynic rats.Intradiscal CFA injection led to chronic disk degeneration with allodynia, which was suggested by pain behavior and expression of pain-related mediators. The increment of CGRP, PGE, and iNOS also suggest pain-related signal processing between the IVD and the neural pathway in this animal model. This animal model may be useful for future research related to the pathophysiology and development of novel treatment for spine-related pain.

The early postnatal period has been suggested to be the vulnerable time for structural and functional reorganization of sensory systems, and painful stimuli at this time may alter neuronal circuits, thereby leading to changes in an individual's response to pain later in life. In the present study, we examined whether inflammatory experience in the early life can affect pain responses to subsequent noxious insults later in life. The two groups of neonatal rats, treated with an inflammatory irritant and untreated, were subjected to inflammation and peripheral nerve injury in adulthood. Neonatal inflammation was induced by injection of complete Freund's adjuvant (CFA, 25 μl) into the hindpaw or tail of newborn rat pups. Adult rats which had suffered from neonatal paw inflammation at P0 were subjected to re-injection of CFA into the paw neonatally exposed to CFA or L5 spinal nerve ligation. Paw thickness and histology of inflamed paw were examined to assess the neonatal inflammation. Adult animals whose tail had been subjected to CFA injection on P3 received tail-innervating nerve injury. The results showed that the neonatal CFA-treated rats suffered from chronic inflammation, confirmed by persistent increase of paw thickness and histological result of inflamed paw. These animals showed enhanced pain responses to re-inflammatory challenge by injection of CFA (200 μl) into the neonatally inflamed paw 8 weeks after birth compared with the neonatally untreated animals. However, neuropathic pain on the hindpaw and the tail which had been induced by peripheral nerve injury in the neonatal CFA-treated group were not different from those of the untreated group. The present data suggest that early neonatal long-lasting inflammation differentially affects pain responses later in life, depending on the types of subsequent noxious insults.

To gain an insight into the developmental characteristics of neuropathic pain induced by peripheral nerve injury during neonatal period, we employed three groups of rats suffering from peripheral nerve injury at different postnatal times, and compared the onset time, severity and persistency of neuropathic pain behaviors, such as mechanical and cold allodynia. The first group (P0 group) was subjected to partial injury of tail-innervating nerves within 24 h after birth, the second group (P10 group) underwent nerve injury at postnatal day (P) 10, and the third group (P60 group) was subjected to injury at P60. Although mechanical allodynia was readily detectable in the P60 group even 1 day after nerve injury, the signs of neuropathic pain were observed from 6 or 8 weeks after nerve injury in the P0 or P10 groups, respectively. Compared with the P60 group, the P0 group showed more robust mechanical and cold allodynia, whereas the P10 group exhibited rather milder pains. In addition, while the P0 and P60 groups showed long-lasting signs of mechanical allodynia, the P10 group exhibited shorter persistency. These results indicate that peripheral nerve injury during neonatal period leads to neuropathic pain with distinct developmental characteristics later in life.

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both Gq/11- and Gs-mediated signaling pathways, whereas NAG activated only the Gq/11-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr97, Gly98, Phe101, and Arg267 of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca2+ levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Low-voltage-activated calcium channels, also known as T-type calcium channels, are widely expressed in various types of neurons. In contrast to high-voltage-activated calcium channels which can be activated by a strong depolarization of membrane potential, T-type channels can be activated by a weak depolarization near the resting membrane potential once deinactivated by hyperpolarization, and therefore can regulate the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Recently, the molecular diversity and functional multiplicity of T-type channels have been demonstrated through molecular genetic studies coupled with physiological and behavioral analysis. Understanding the functional consequences of modulation of each subtype of these channels in vivo could point to the right direction for developing therapeutic tools for relevant diseases.

The present study was performed to examine the effects of electroacupuncture (EA) on cold allodynia and its mechanisms related to the spinal adrenergic and serotonergic systems in a rat model of neuropathic pain. For the neuropathic surgery, the right superior caudal trunk was resected at the level between S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, EA stimulation (2 or 100 Hz) was delivered to Zusanli (ST36) for 30 min. The behavioral signs of cold allodynia were evaluated by the tail immersion test [i.e., immersing the tail in cold water (4 degrees C) and measuring the latency to an abrupt tail movement] before and after the stimulation. And then, we examined the effects of intrathecal injection of prazosin (alpha1-adrenoceptor antagonist, 30 microg), yohimbine (alpha2-adrenoceptor antagonist, 30 microg), NAN-190 (5-HT1A antagonist, 15 microg), ketanserin (5-HT2A antagonist, 30 microg), and MDL-72222 (5-HT3 antagonist, 12 microg) on the action of EA stimulation. Although both 2 Hz and 100 Hz EA significantly relieved the cold allodynia signs, 2 Hz EA induced more robust effects than 100 Hz EA. In addition, intrathecal injection of yohimbine, NAN-190, and MDL-72222, but not prazosin and ketanserin, significantly blocked the relieving effects of 2 Hz EA on cold allodynia. These results suggest that low-frequency (2 Hz) EA is more suitable for the treatment of cold allodynia than high-frequency (100 Hz) EA, and spinal alpha2-adrenergic, 5-HT1A and 5-HT3, but not alpha1-adrenergic and 5-HT2A, receptors play important roles in mediating the relieving effects of 2 Hz EA on cold allodynia in neuropathic rats.

The dorsal column-medial lemniscal (DC-ML) system is known to be a route of ascending input signals for mechanical allodynia following peripheral nerve injury. We examined whether the pain signals after spinal hemisection were transmitted via the DC-ML system in the induction and maintenance phases of the neuropathic pain. Under enflurane anesthesia, rats were subjected to spinal hemisection at T13 level and bilateral DC lesion was made at T8 level 1 day or 3 weeks after the hemisection. The DC lesion 1 day after the hemisection significantly reduced the mechanical, but not cold, allodynia, whereas the DC lesion 3 weeks after the hemisection did not change both mechanical and cold allodynia. These results suggest that the signals for mechanical allodynia following spinal hemisection should be transmitted via the DC-ML system in the induction, but not maintenance, phase.

To determine what the routes by which mechanical allodynia is transmitted following peripheral nerve injury, we assessed the effects of the dorsal column (DC) lesion performed before and 2 weeks after the partial injury of nerves innervating the tail on mechanical allodynia. Ipsilateral DC lesion 2 weeks after neuropathic surgery significantly, but not completely, attenuated mechanical allodynia. In addition, the DC lesion before peripheral nerve injury did not prevent the generation of mechanical allodynia, which was completely blocked by subsequent contralateral hemisection of the spinal cord. However, unlike mechanical allodynia, DC lesion did not change thermal allodynia. These results suggest that the signals for mechanical allodynia following peripheral nerve injury are transmitted via the ipsilateral DC and the contralateral pathway(s).

We examined whether morphine reduced the behavioral signs of neuropathic pain below level induced by T13 spinal hemisection in rats. In order to examine the effect of morphine on the mechanical allodynia, morphine alone, morphine with naloxone and saline were administered intraperitoneally and intrathecally and behavioral tests were conducted. In systemic injection, mechanical allodynia was reduced only when a higher concentration of morphine (5 mg/kg) was used. Intrathecally injected morphine (0.5, 1, 2, 5 microg) reduced mechanical allodynia dose-dependently. It is suggested that systemic morphine has limited effect on mechanical allodynia but direct spinal administration of morphine is more effective in controlling central pain following spinal cord injury.

The analgesic effects of acupuncture on the mechanical allodynia in the rat model of neuropathic pain have not yet been studied. The aim of the present study is: first, to determine if electroacupuncture (EA) or morphine attenuates the mechanical allodynia; and secondly, to examine if the EA effect may be mediated by endogenous opioids. To produce neuropathic pain, the right superior caudal trunk was resected between the S3 and S4 spinal nerves. Twenty-one days after the neuropathic surgery, low frequency EA stimulation (2 Hz, 0.3 ms, 0.07 mA) delivered to Houxi (S13) for 30 min relieved significantly the signs of mechanical allodynia. Intraperitoneal (i.p.) morphine (0.5 or 1.5 mg/kg) also relieved the signs of mechanical allodynia in a dose-dependent manner. In addition, the antiallodynic effect of Houxi EA was blocked by pretreatment with naloxone (2 mg/kg, i.p.). However, combined application of EA and morphine did not show an obvious synergistic effect. These results suggest that low frequency EA or morphine can relieve the mechanical allodynia signs and the EA effect can be mediated by endogenous opioid systems.

Substance P is known to exert various pro-inflammatory effects that are mediated by neurokinin-1 (NK-1) receptor in peripheral tissues. This study examined the effect of the NK-1 receptor antagonist cis-2-[diphenylmethyl]-N-[(2-iodophenyl)-1-azabicyclo[2.2.2]octan-3-amine] (L-703,606) on nociceptive response following carrageenan injection (2%, 50 microl) into the knee joint cavity of the right hind leg. L-703,606 injection (0.1 or 1 mM, 50 microl) into the same joint cavity immediately before the carrageenan injection significantly reduced the nociceptive response. However, antagonist treatment at 5 h after carrageenan injection was ineffective in alleviating nociception. Neither intraperitoneal injection of the antagonist (1 mM, 50 microl) immediately before the carrageenan injection was effective. These results suggest that local NK-1 receptor contributes to the induction, but not maintenance, of arthritic pain.

Gamma‐aminobutyric acid (GABA) depolarizes dorsal root ganglia (DRG) primary afferent neurons through activation of Cl − permeable GABA A receptors but the physiologic role of GABA A receptors in the peripheral terminals of DRG neurons remains unclear. In this study, we investigated the role of peripheral GABA A receptors in nociception using a mouse model of acute inflammation. In vivo, peripheral administration of the selective GABA A receptor agonist muscimol evoked spontaneous licking behavior, as well as spinal wide dynamic range (WDR) neuron firing, after pre‐conditioning with formalin but had no effect in saline‐treated mice. GABA A receptor‐mediated pain behavior after acute formalin treatment was abolished by the GABA A receptor blocker picrotoxin and cyclooxygenase inhibitor indomethacin. In addition, treatment with prostaglandin E2 (PGE 2 ) was sufficient to reveal muscimol‐induced licking behavior. In vitro, GABA induced sub‐threshold depolarization in DRG neurons through GABA A receptor activation. Both formalin and PGE 2 potentiated GABA‐induced Ca 2+ transients and membrane depolarization in capsaicin‐sensitive nociceptive DRG neurons; these effects were blocked by the prostaglandin E2 receptor 4 (EP4) antagonist AH23848 (10 μ mol/L). Furthermore, potentiation of GABA responses by PGE 2 was prevented by the selective Na v 1.8 antagonist A887826 (100 nmol/L). Although the function of the Na + ‐K + ‐2Cl ‐ co‐transporter NKCC1 was required to maintain the Cl ‐ ion gradient in isolated DRG neurons, NKCC1 was not required for GABA A receptor‐mediated nociceptive behavior after acute inflammation. Taken together, these results demonstrate that GABA A receptors may contribute to the excitation of peripheral sensory neurons in inflammation through a combined effect involving PGE 2 ‐EP4 signaling and Na + channel sensitization.

Numerous studies have suggested that changes in electrophysiological properties of primary sensory neurons after axonal injury contribute to the generation of neuropathic pain. Presently, however, it is unclear which of the changes is important. To address this issue, we performed behavioral and electrophysiological experiments in a double-blind fashion; we made intracellular recordings in the S1 dorsal root ganglia excised from rats exhibiting cold allodynia behavior after chronic S1 spinal nerve transaction (allodynia-positive group) and from rats lacking such behavior after the same nerve injury (allodynia-negative group) or sham injury (sham group). In this study, we sought which of the membrane property changes produced by the spinal nerve injury in each of C-, Adelta- and Aalpha/beta-cell populations was unique to the allodynia-positive group. Analyses of our data revealed that only some changes in Adelta-cells (e.g. the decrease in resting membrane potential and in the threshold of central process) were more pronounced in or unique to the allodynia-positive group. We concluded that certain membrane property changes in the somata and dorsal root axons of Adelta-cells might be important in the generation of cold allodynia.

Incomplete peripheral nerve injury often leads to neuropathic pains, some of which are relieved by sympathectomy, and results in sympathetic postganglionic nerve fiber sprouting in the dorsal root ganglion (DRG). This study was performed to see whether the sprouting in the DRG plays a key role in the sympathetic dependence of neuropathic pain. To this aim, we compared two groups of rats, both of which were subjected to unilateral transection of the inferior and superior caudal trunks at the levels between the S1 and S2, S2 and S3, and S3 and S4 spinal nerves, with respect to sympathetic fiber sprouting; one group showed neuropathic pain behaviours (i.e. mechanical and cold allodynia signs) which were very sensitive to phentolamine, alpha adrenergic blocker, and the other group exhibited no sensitivity. Immuno-histochemical staining with tyrosine hydroxylase antibody of the S1-S3 DRGs was not correlated with the sensitivity to phentolamine. These results suggest that the degree of sympathetic dependence of neuropathic pain is not a function of the extent of the sympathetic postganglionic nerve fiber sprouting in the DRG.

Previous studies showed that heat-hyperalgesia and mechanical allodynia produced by chronic constrictive injury of the sciatic nerve were differentially sensitive to the NMDA receptor antagonist dextrorphan and to morphine and other opioid receptor agonists. These results support the hypothesis that different kinds of neuropathic pain symptoms are caused by different pathological mechanisms. In the present study we determined whether mechanical and thermal allodynia produced by unilateral transection of the 'superior' caudal trunk which innervates the tail in rats were differentially sensitive to the non-competitive NMDA receptor antagonist MK-801. Injection of MK-801 (0.3 mg/kg, i.p.) prior to nerve injury delayed the emergence of both types of allodynia; the antagonist-treated rats exhibited neither mechanical nor thermal allodynia at least for 4 days after the injury, whereas untreated control rats exhibited clear signs of allodynia from the first day after the injury. MK-801 injection on post-injury day 14, when the allodynia was near peak severity, suppressed temporarily both the mechanical and thermal allodynia. These results suggest that the mechanical and thermal allodynia from partial denervation of the tail are both dependent on NMDA receptors in their induction and maintenance. Thus, our results do not support the notion that different pathological mechanisms underlie different modalities of neuropathic pain from partial peripheral nerve injury.

In neuropathic rats sensitive to phentolamine (alpha-adrenoreceptor antagonist, 2 mg/kg, i.p.), prazosin (alpha1-adrenoreceptor antagonist, 0.5 mg/kg, i.p.) significantly attenuated cold allodynia whereas yohimbine (alpha2-adrenoreceptor antagonist, 0.5 mg/kg, i.p.) had no significant effect. In neuropathic rats insensitive to phentolamine, yohimbine significantly exacerbated cold allodynia whereas prazosin had no significant effect. These results suggest that the individual differences in the sensitivity of cold allodynia to phentolamine may be due to the difference in the alpha-adrenoreceptor subtype predominantly involved in cold allodynia.

Background Electroacupuncture (EA) is used as a prescription to treat pruritus and atopic dermatitis. Whether EA affects experimental itch in rat models of immunologic or neuronal damages, however, is unknown. Objectives The present study was designed to determine the therapeutic effects of high-frequency EA on atopic dermatitis-like lesions in rats. Materials and methods Capsaicin (50 mg/kg) was subcutaneously administered rat pups within 48 h after birth. Rats then underwent 30 min of EA at six acupoints (bilateral BL13, and unilateral LI11, ST36, SP10, SP6) every other day (EA group) for 3 weeks. Measurements of IgE, mast cells, scratching behavior, dynorphin release, skin thickness and dermatitis score were obtained. Results Only the dermatitis score and dynorphin expression were decreased in the EA group compared with the control non-EA group. Conclusion We suggest that high-frequency EA alleviates pruritus of atopic dermatitis-like lesions in rats induced by capsaicin injection, via the release of dynorphin. These findings indicate a new potential therapeutic approach for the amelioration of symptoms of atopic dermatitis.

This study investigated whether and how electroacupuncture (EA) attenuates cold hypersensitivity (allodynia) in a rat model of oxaliplatin-induced neuropathic pain. Cold allodynia [evaluated by immersing the tail into cold water (4 °C) and measuring the withdrawal latency] was induced 3 days after an oxaliplatin administration (6 mg/kg, i.p.). EA stimulation (2/100 Hz, 0.3-ms pulse duration, 0.2–0.3 mA) was delivered to ST36 acupoint or non-acupoint for 20 min. Low-frequency (2 Hz) EA at ST36 relieved cold allodynia more effectively than high-frequency EA at ST36 or low-frequency EA at non-acupoint. Naloxone (opioid antagonist, 2 mg/kg, i.p.) completely blocked such EA-induced anti-allodynia, whereas phentolamine (α-adrenergic antagonist, 2 mg/kg, i.p.) did not. Moreover, plasma β-endorphin levels significantly increased right after the end of EA and subsequently decreased. These results indicate that low-frequency EA at ST36 in rats has a marked relieving effect on oxaliplatin-induced cold allodynia that is mediated by the endogenous opioid, but not noradrenergic, system.

With a cat model of regional cardiac ischemia, we examined whether the incidence of reperfusion-induced ventricular fibrillation (VF) could be reduced by ventricular premature beat (VPB)-driven intermittent reperfusion. In addition, we assessed whether the effect of the intermittent reperfusion was comparable with that of ischemic preconditioning in suppressing the VF. Of 15 cats subjected to uninterrupted reperfusion after 20-minute occlusion of the left anterior descending coronary artery, 13 (86.70%) had VF, whereas only 1 (7.1%) of 14 cats subjected to the VPB-driven intermittent reperfusion had VF. This incidence of VF was significantly lower than that of the animal group subjected to uninterrupted reperfusion. However, it was not statistically different from that (3 of 15) of the group subjected to a 10-minute episode of the coronary artery occlusion before the 20-minute occlusion (i.e., "ischermic preconditioning"). Our results suggest that the VPB-driven intermittent reperfusion (i.e., "postconditioning") is very effective in preventing reperfusion-induced VF and as good as, if not better than, ischemic preconditioning.

The present study examined the effect that aging has on the manifestations of pain behaviors in a rat model of neuropathic pain. Two experiments were conducted. The first experiment compared young, mature and old rats. After tight ligation of the L5 and L6 segmental spinal nerves, young rats displayed much more vigorous behavioral signs of mechanical allodynia and ongoing pain than did either mature or old rats. The second experiment was done using the same rats at two different time points. Spinal nerve ligation was done on the left side of rats when they were young and the same surgery was repeated on the right side 20 weeks later (mature age). Comparison of pain behaviors between the left and right sides confirmed the results of the first experiment. The data suggest that younger rats show much more robust behavioral signs of neuropathic pain compared to older rats.

Background The pathophysiological mechanisms underlying chronic pruritic skin diseases, e.g. atopic dermatitis (AD), and effective therapies remain elusive due to the paucity of animal models. Recently, we rediscovered that injection of capsaicin into rat pups resulted in vigorous scratching behavior and chronically relapsing AD-like cutaneous lesions well into adulthood. Objectives To characterize the chronic pruritic dermatitis induced by neonatal capsaicin treatment. Methods Capsaicin (50mg/kg) was given to rat pups subcutaneously within 48h after birth, and then scratching behavior, dermatitis and pathophysiological changes of rat skin were investigated chronologically. Results Neonatal capsaicin treatment led to not only severe scratching and cutaneous lesions but also a large number of pathophysiological changes in the skin, such as histopathological changes including the deficiency of epidermal filaggrin expression, increases in the number of mast cells, levels of tissue NGF and Th2 cytokine mRNA, impaired skin barrier function and colonization with S. aureus. In addition, we observed the hyperproduction of serum IgE, which is clinically similar to the pathophysiology seen in the patients with atopic dermatitis. During the follow-up observation, the rats showed the alternative periods of relapsing and remitting skin lesions. Conclusion Injection of capsaicin into rat pups results in chronically relapsing pruritic dermatitis, similar to human AD. Therefore, we think neonatal capsaicin treatment could be a useful model for studying human AD and for the development of novel therapeutic drugs.

Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.

Discogenic low back pain has been shown to develop into chronic intractable pain due to an unknown pathogenesis. To study the mechanism of discogenic pain, we analyzed the serial expression of pain-related molecules in the dorsal root ganglia (DRG) and thalamus using a newly developed rat model of disc degeneration.Ten microliters of complete Freund's adjuvant was injected into the L5-6 disc of male Sprague-Dawley rats for 10 minutes using a 26-gauge needle. Using a behavioral test, rats with significant pain were selected and subsequently serial gene expression of pain-related molecules in the DRG and the thalamus was analyzed by reverse transcriptase polymerase chain reaction.The expression of tumor necrosis factor-α and interleukin-1β significantly increased at 4 and 8 weeks in the DRG of rats with pain. Furthermore, interleukin-6 was significantly increased at 4 weeks in the DRG; however, these cytokines did not show a significant change in the thalamus. Calcitonin gene-related peptide and substance P were significantly increased in DRG at 4 and 8 weeks and in the thalamus at 2 and 4 weeks. The level of nerve growth factor-β did not significantly increase in the DRG or thalamus, whereas glial cell line-derived neurotropic factor (GDNF) was significantly increased at 2 weeks and was sustained through 8 weeks in both the DRG and thalamus.The disc degeneration rat model described herein led to significant pain of a chronic nature. The gradual and persistent increase of GDNF in both the thalamus and DRG suggests that GDNF might be a key factor in the development of intractable, chronic discogenic pain.

1. Peripheral nerve injury sometimes leads to the development of neuropathic pain. One of the symptoms of such neuropathic pain is mechanical allodynia, pain in response to normally innocuous mechanical stimuli. We hypothesized that sympathetically driven dysfunction of cutaneous mechanoreceptors is responsible for signaling mechanical allodynia. The present study was undertaken to identify the types of sensory receptors that potentially mediate mechanical allodynia, with the use of a rat neuropathic pain model we have developed. 2. One week to 10 days after tight ligations of the L5 and L6 spinal nerves on one side, the rats fully developed behavioral signs of mechanical allodynia on the affected hindlimb. Various cutaneous mechanoreceptors originating from the neuropathic foot were examined by single-fiber recordings from the L4 dorsal root. 3. Although no particular abnormalities were found in other types of cutaneous mechanoreceptors, an unusual type of mechanoreceptor was found to be innervating the neuropathic foot. The response characteristics of this type of receptor resemble those of rapidly adapting mechanoreceptors (RAs), but with low and irregular static discharges during a maintained mechanical stimulus. We termed this unusual type as a "modified rapidly adapting" mechanoreceptor (MRA). 4. The response characteristics of MRAs change to those of typical RAs after a systemic injection of phentolamine, an alpha-adrenergic receptor blocker. 5. We conclude that many RAs become abnormal under the influence of sympathetic efferents in neuropathic pain, so that their response patterns change to those of MRAs. We propose that this abnormality is responsible for signaling the mechanical allodynia that can be seen in neuropathic pain states such as causalgia.

The vast majority of human peripheral nerve injuries occur in the upper limb, whereas the most animal studies have been conducted using the hindlimb models of neuropathic pain, involving damages of the sciatic or lumbar spinal nerve(s). We attempted to develop a rat forelimb model of peripheral neuropathy by partial injury of the median and ulnar nerves. The halves of each nerve were transected by microscissors at about 5mm proximal from the elbow joint and behavioral signs of neuropathic pain, such as mechanical and cold allodynia, and heat hyperalgesia, were monitored up to 126 days following nerve injury. Mechanical allodynia was assessed by measuring the forepaw withdrawal threshold to von Frey filaments, and cold allodynia was evaluated by measuring the time spent in lifting or licking the forepaw after applying acetone to it. Heat hyperalgesia was also monitored by investigating the forepaw withdrawal latencies using the Hargreaves' test. After the nerve injury, the experimental animals exhibited long-lasting clear neuropathic pain-like behaviors, such as reduced forepaw withdrawal threshold to von Frey filaments, the increased response duration of the forepaw to acetone application, and the decreased withdrawal latency to radiant heat stimulation. These behaviors were significantly alleviated by administration of gabapentin (5 or 50mg/kg, i.p.) in a dose-dependent manner. Therefore, these abnormal sensitivities are interpreted as the signs of neuropathic pain following injury of the median and ulnar nerves. Our rat forelimb model of neuropathic pain may be useful for studying human neuropathic pain and screening for valuable drug candidates.

A series of novel non-peptide diamide compounds was synthesized and evaluated as antibradykinin agents by utilizing guinea-pig ileum smooth muscle. Among the final compounds, (Z)-4-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-oxo-N-(4-phenylbutan-2-yl)but-2-enamide showed most favorable bradykinin inhibitory activity and demonstrated analgesic efficacies in the rat models of inflammatory and neuropathic pain.

Impairment in spinal inhibition caused by quantitative alteration of GABAergic elements following peripheral nerve injury has been postulated to mediate neuropathic pain. In the present study, we tested whether neuropathic pain could be induced or reversed by pharmacologically modulating spinal GABAergic activity, and whether quantitative alteration of spinal GABAergic elements after peripheral nerve injury was related to the impairment of GABAergic inhibition or neuropathic pain. To these aims, we first analyzed the pain behaviors following the spinal administration of GABA antagonists (1 microg bicuculline/rat and 5 microg phaclofen/rat), agonists (1 microg muscimol/rat and 0.5 microg baclofen/rat) or GABA transporter (GAT) inhibitors (20 microg NNC-711/rat and 1 microg SNAP-5114/rat) into naïve or neuropathic animals. Then, using Western blotting, PCR or immunohistochemistry, we compared the quantities of spinal GABA, its synthesizing enzymes (GAD65, 67) and its receptors (GABA(A) and GABA(B)) and transporters (GAT-1, and -3) between two groups of rats with different severity of neuropathic pain following partial injury of tail-innervating nerves; the allodynic and non-allodynic groups. Intrathecal administration of GABA antagonists markedly lowered tail-withdrawal threshold in naïve animals, and GABA agonists or GAT inhibitors significantly attenuated neuropathic pain in nerve-injured animals. However, any quantitative changes in spinal GABAergic elements were not observed in both the allodynic and non-allodynic groups. These results suggest that although the impairment in spinal GABAergic inhibition may play a role in mediation of neuropathic pain, it is not accomplished by the quantitative change in spinal elements for GABAergic inhibition and therefore these elements are not related to the generation of neuropathic pain following peripheral nerve injury.

Transient receptor potential vanilloid subtype 1 (TRPV1) and metabotropic glutamate receptor 5 (mGluR5) located on peripheral sensory terminals have been shown to play critical roles in the transduction and modulation of pain sensation. To date, however, very little is known regarding the significance of functional expression of mGluR5 and TRPV1 on the central terminals of sensory neurons in the dorsal horn of the spinal cord. Here we show that TRPV1 on central presynaptic terminals is coupled to mGluR5 in a membrane-delimited manner, thereby contributing to the modulation of nociceptive synaptic transmission in the substantia gelatinosa neurons of the spinal cord. Further, our results demonstrate that TRPV1 is involved in the pain behaviors induced by spinal mGluR5 activation, and diacylglycerol produced by the activation of mGluR5 mediates functional coupling of mGluR5 and TRPV1 on the presynaptic terminals. Thus, mGluR5–TRPV1 coupling on the central presynaptic terminals of nociceptive neurons may be an important mechanism underlying central sensitization under pathological pain conditions.

Spinal cord injury leads to the permanent loss of motor and sensory function in the body. To enhance spinal cord regeneration, we used a hyaluronic acid-based hydrogel as a three-dimensional biomimetic scaffold for peptides and growth factors. Three components were used to provide guidance cues: a matrix metalloproteinase peptide crosslinker, an IKVAV (Ile- Lys-Val-Ala-Val) peptide derived from laminin, and brain-derived neurotrophic factor (BDNF). Human mesenchymal stem cells (hMSCs) were cultured in hydrogels in vitro for 10 days to induce neuronal differentiation of hMSCs. Based on gene-expression data, the matrix metalloproteinase-sensitive peptide, IKVAV peptide, and BDNF were critical in the differentiation of hMSCs. Remodeling activity was found to be a key factor in guiding neural differentiation of stem cells. To test this approach in vivo, we used the spinal cord injured rat model and five different hydrogel compositions. Samples were injected into the intrathecal space, and animals were monitored for 6 weeks. Compared to all other groups, animals injected with BDNF-containing hydrogels showed the greatest improvement on locomotive tests (Basso-Beattie-Bresnahan score) during the initial stage after injury. These results suggest that hyaluronic acid-based hydrogels containing IKVAV and BDNF create microenvironments that foster differentiation of stem cells along the neural cell lineage, and they could be used to facilitate nerve regeneration after spinal cord injury.