Stress-induced hyperalgesia is associated with a reduced and delayed GABA inhibitory control that enhances post-synaptic NMDA receptor activation in the spinal cord

GABA and glutamate are both affected by stress and are involved in nociception. Thus, we determined whether stress-induced enhancement of inflammatory hyperalgesia is mediated by an imbalance between glutamate and GABA neurotransmission. Male rats were subjected daily to 10 to 20 minutes per day of either forced swimming (FS) or sham swimming for 3 consecutive days; nonconditioned rats served as controls. Some rats were treated i.p. with ketamine (5 mg/kg), diazepam (2 mg/kg), flumazenil (0.1 mg/kg), or vehicle (0.9% NaCl), 30 to 60 minutes before each conditioning session or nociception assessment. Pain behavior, spinal nociceptive neuronal activation and GABA and glutamate release were respectively evaluated by the formalin test, the expression of c-Fos and in vivo microdialysis of superficial laminae of the lumbar spinal cord, 48 hours after the last conditioning session. Nitric oxide metabolites (NO_x) were determined as markers of post-synaptic NMDA receptor activation. FS stress enhanced formalin-induced hyperalgesia, increased pain-elicited c-Fos expression, decreased basal and delayed pain-induced GABA release, and increased basal and induced glutamate release. Hyperalgesia and c-Fos overexpression were blocked only by prestress treatment with diazepam and post-stress treatment with ketamine, whereas changes in GABA and glutamate release were reversed by prestress treatment with diazepam. Diazepam effects were blocked by flumazenil. NO_x increased in lumbar spinal cord of FS rats by a mechanism antagonized by ketamine. Thus, stress-induced hyperalgesia is initiated by a decreased and delayed GABA release and GABA-A receptor activation, whereas it is maintained by increased glutamate release and NMDA glutamate receptor activation at the spinal level.     

Antinociceptive effects of melatonin in a rat model of post-inflammatory visceral hyperalgesia: A centrally mediated process

Previous reports suggest that melatonin may play an important role in visceral nociception and neurogenic inflammation. We aimed to examine the role of melatonin on visceral hypersensitivity and to explore the site of action using a rat model of post-inflammatory visceral hyperalgesia. In all rats, a baseline viscero-motor response (VMR) to graded colorectal distension (CRD; 10–60 mmHg) was recorded prior and 1 week following tri-nitrobenzenesulfonic acid (TNBS) induced colonic inflammation. Melatonin (30, 45 or 60 mg/kg, ip) was given 20 min before testing the VMR in naïve and TNBS-treated rats. Extracellular single-unit recordings were made from CRD-sensitive pelvic nerve afferent (PNA) fibers and lumbosacral (LS) spinal neurons in TNBS-treated animals. The effect of melatonin (60 mg/kg) was examined on responses of PNAs and spinal neurons to graded CRD. In separate experiments, luzindole (non-specific MT₁/MT₂ receptor antagonist) or naltrexone (non-specific opiod receptor antagonist) was injected prior to melatonin. Following TNBS, there was a significant increase in the VMR to CRD compared to baseline. This increase was attenuated by melatonin (60 mg/kg) at pressures >20 mmHg. The same dose of melatonin had no effect on the VMR in naïve animals. In TNBS-treated rats, melatonin significantly attenuated the responses of CRD-sensitive spinal neurons to CRD, but had no effect in spinal transected rats or PNA fibers. Both luzindole and naltrexone blocked melatonin’s effect on the VMR and LS spinal neurons. Results indicate melatonin’s antinociceptive effects are not via a peripheral site of action but rather a supra-spinal process linked to the central opioidergic system.     

Results from clinical trials of a selective ionotropic glutamate receptor 5 (iGluR5) antagonist,LY5454694 tosylate,in 2 chronic pain conditions

This article reports results of 2 studies investigating LY545694 in pain due to osteoarthritis (OA) of the knee and diabetic peripheral neuropathic pain (DPNP). Study I randomized patients to either of 2 doses of LY545694 or to placebo, and study II randomized patients to either of 3 doses of LY545694, to pregabalin, or to placebo. No significant differences between LY545694 groups and placebo were observed on the primary (average pain severity) or secondary efficacy measures in either study. Notably, study I lacked an active control, and, in study II, pregabalin, did not separate from placebo. Treatment-emergent nausea, vomiting, and dizziness were significantly more frequent in the LY545694 groups in both trials (P ? .05), and significantly more LY545694-treated patients discontinued because of adverse events (P < .001). Steady-state concentrations of LY545694 were comparable in patients in both studies but were lower than exposures required for efficacy in animal models of pain behavior. Because the active control did not separate from placebo in the DPNP study, the study was potentially failed, rather than negative. Without an active control, it is unknown whether the OA study was negative or failed. Consequently, efficacy of selective ionotropic glutamate receptor antagonism in chronic pain conditions may warrant further investigation. Future trials should consider different pain conditions, contain a positive control with larger patient numbers per arm, and be conducted within a single region.     

Botulinum neurotoxin type A (BoNTA) decreases the mechanical sensitivity of nociceptors and inhibits neurogenic vasodilation in a craniofacial muscle targeted for migraine prophylaxis

The mechanism by which intramuscular injection of BoNTA into the craniofacial muscles decreases migraine headaches is not known. In a blinded study, the effect of BoNTA on the mechanical and chemical responsiveness of individual temporalis muscle nociceptors and muscle neurogenic vasodilation was investigated in female rats. Mechanical threshold was measured for 3 h following intramuscular injection of BoNTA or vehicle, and for 10 min after a subsequent injection of the algogen glutamate. Injection of BoNTA significantly increased the mechanical threshold of muscle nociceptors without altering the muscle surface temperature and blocked glutamate-induced mechanical sensitization and neurogenic vasodilation. None of these effects were reproduced by pancuronium-induced muscle paralysis. Western blot analysis of temporalis muscles indicated that BoNTA significantly decreased SNAP-25. Measurement of interstitial glutamate concentration with a glutamate biosensor indicated that BoNTA significantly reduced glutamate concentrations. The mechanical sensitivity of muscle nociceptors is modulated by glutamate concentration through activation of peripheral NMDA receptors. Immunohistochemical experiments were conducted and they indicated that half of the NMDA-expressing temporalis nerve fibers co-expressed substance P or CGRP. Additional electrophysiology experiments examined the effect of antagonists for NMDA, CGRP and NK1 receptors on glutamate-induced effects. Glutamate-induced mechanical sensitization was only blocked by the NMDA receptor antagonist, but muscle neurogenic vasodilation was attenuated by NMDA or CGRP receptor antagonists. These data suggest that injection of BoNTA into craniofacial muscles acts to decrease migraine headaches by rapidly decreasing the mechanical sensitivity of temporalis muscle nociceptors through inhibition of glutamate release and by attenuating the provoked release of CGRP from muscle nociceptors.     

Co-administration of δ- and μ-opioid receptor agonists promotes peripheral opioid receptor function

Enhancement of peripheral opioid analgesia following tissue injury or inflammation in animal models is well-documented, but clinical results of peripheral opioid therapy remain inconsistent. Previous studies in the central nervous system have shown that co-administration of μ- and δ-opioid receptor agonists can enhance analgesic outcomes; however, less is known about the functional consequences of opioid receptor interactions in the periphery. The present study examines the effects of intraplantar injection of the μ- and δ-opioid receptor agonists, morphine and deltorphin, alone and in combination on behavioral tests of nociception in naïve rats and on potassium-evoked release of CGRP from sciatic nerves of naïve rats. Neither drug alone affected nociceptive behaviors or CGRP release. Two separate measures of mechanical nociceptive sensitivity remained unchanged after co-administration of the two drugs. In contrast, when deltorphin was co-injected with morphine, dose-dependent and peripherally restricted increases in paw withdrawal latencies to radiant heat were observed. Similarly, concentration-dependent inhibition of CGRP release was observed when deltorphin and morphine were administered in sequence prior to potassium stimulation. However, no inhibition was observed when morphine was administered prior to deltorphin. All combined opioid effects were blocked by co-application of antagonists. Deltorphin exposure also enhanced the in vivo and in vitro effects of another μ-opioid receptor agonist, DAMGO. Together, these results suggest that under normal conditions, δ-opioid receptor agonists enhance the effect of μ-opioid receptor agonists in the periphery, and local co-administration of δ- and μ-opioid receptor agonists may improve results of peripheral opioid therapy for the treatment of pain.     

Ultraviolet-B-induced mechanical hyperalgesia: A role for peripheral sensitisation

Ultraviolet (UV) induced cutaneous inflammation is emerging as a model of pain with a novel sensory phenotype. A UVB dose of 1000 mJ/cm2 produces a highly significant thermal and mechanical hypersensitivity. Here we examined the properties and mechanisms of such hyperalgesia in rats. Significantly, the mechanical hyperalgesia (with ∼60% change in withdrawal thresholds) was restricted to the lesion site with no changes in mechanical threshold in adjacent non-irradiated skin (i.e. no secondary hypersensitivity), suggesting a peripheral mechanism. Consistent with this, we found that primary mechanical hypersensitivity showed no significant changes after intrathecal treatment with 10 μg of the NMDA-receptor antagonist MK-801. Using an in vitro skin–nerve preparation, in the presence and absence of UVB-inflammation, suprathreshold responses to skin displacement stimuli of 6–768 μm of 103 peripheral nociceptors were recorded. At the peak of UVB-induced hyperalgesia we observed that mechanical response properties of Aδ-nociceptors recorded from UVB-inflamed skin (n = 19) were significantly diminished, by ∼50%, compared to those recorded from naïve skin (n = 13). The mechanical response properties of heat-sensitive C-nociceptors were unchanged while their heat responses were significantly increased, by ∼75%, in UVB-inflamed (n = 26) compared to naïve skin (n = 12). Heat-insensitive C-nociceptors, however, demonstrated significantly enhanced (by ∼60%) response properties to mechanical stimulation in UVB-inflamed (n = 21) compared to naïve skin (n = 12). Notably alteration in mechanical responses of Aδ- and heat-insensitive C-nociceptors were particular to stronger stimuli. Spontaneous activity was not induced by this dose of UVB. We conclude that UVB-induced mechanical hyperalgesia may be explained by a net shift in peripheral nociceptor response properties.     

A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel and selective transient receptor potential type V1 receptor antagonist, blocks channel activation by vanilloids, heat, and acid

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC50 = 5 nM). A-425619 showed similar potency (IC50 = 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC50 = 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol ester-sensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA2 = 2.5 nM) of capsaicin-evoked calcium flux. Moreover, A-425619 was 25- to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.     

Sensory Neuron-TRPV4 Modulates Temporomandibular Disorder Pain Via CGRP in Mice

Temporomandibular disorder (TMD) pain that involves inflammation and injury in the temporomandibular joint (TMJ) and/or masticatory muscle is the most common form of orofacial pain. We recently found that transient receptor potential vanilloid-4 (TRPV4) in trigeminal ganglion (TG) neurons is upregulated after TMJ inflammation, and TRPV4 coexpresses with calcitonin gene-related peptide (CGRP) in TMJ-innervating TG neurons. Here, we extended these findings to determine the specific contribution of TRPV4 in TG neurons to TMD pain, and examine whether sensory neuron-TRPV4 modulates TMD pain via CGRP. In mouse models of TMJ inflammation or masseter muscle injury, sensory neuron-Trpv4 conditional knockout (cKO) mice displayed reduced pain. Coexpression of TRPV4 and CGRP in TMJ- or masseter muscle-innervating TG neurons was increased after TMJ inflammation and masseter muscle injury, respectively. Activation of TRPV4-expressing TG neurons triggered secretion of CGRP, which was associated with increased levels of CGRP in peri-TMJ tissues, masseter muscle, spinal trigeminal nucleus, and plasma in both models. Local injection of CGRP into the TMJ or masseter muscle evoked acute pain in naïve mice, while blockade of CGRP receptor attenuated pain in mouse models of TMD. These results suggest that TRPV4 in TG neurons contributes to TMD pain by potentiating CGRP secretion.PerspectiveThis study demonstrates that activation of TRPV4 in TG sensory neurons drives pain by potentiating the release of pain mediator CGRP in mouse models of TMJ inflammation and masseter muscle injury. Targeting TRPV4 and CGRP may be of clinical potential in alleviating TMD pain.     

Characterisation and mechanisms of bradykinin-evoked pain in man using iontophoresis

Bradykinin (BK) is an inflammatory mediator that can evoke oedema and vasodilatation, and is a potent algogen signalling via the B1 and B2 G-protein coupled receptors. In naïve skin, BK is effective via constitutively expressed B2 receptors (B2R), while B1 receptors (B1R) are purported to be upregulated by inflammation. The aim of this investigation was to optimise BK delivery to investigate the algesic effects of BK and how these are modulated by inflammation. BK iontophoresis evoked dose- and temperature-dependent pain and neurogenic erythema, as well as thermal and mechanical hyperalgesia (P < 0.001 vs saline control). To differentiate the direct effects of BK from indirect effects mediated by histamine released from mast cells (MCs), skin was pretreated with compound 4880 to degranulate the MCs prior to BK challenge. The early phase of BK-evoked pain was reduced in degranulated skin (P < 0.001), while thermal and mechanical sensitisation, wheal, and flare were still evident. In contrast to BK, the B1R selective agonist des-Arg9-BK failed to induce pain or sensitise naïve skin. However, following skin inflammation induced by ultraviolet B irradiation, this compound produced a robust pain response. We have optimised a versatile experimental model by which BK and its analogues can be administered to human skin. We have found that there is an early phase of BK-induced pain which partly depends on the release of inflammatory mediators by MCs; however, subsequent hyperalgesia is not dependent on MC degranulation. In naïve skin, B2R signaling predominates, however, cutaneous inflammation results in enhanced B1R responses.     

3H]A-778317 [1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: a novel, stereoselective, high-affinity antagonist is a useful radioligand for the human transient receptor potential vanilloid-1 (TRPV1) receptor

1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 +/- 0.13). The (S)-stereoisomer, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778316), is 6.8-fold less potent (pIC50 = 7.47 +/- 0.07). A-778317 also potently blocks capsaicin and acid activation of native rat TRPV1 receptors in dorsal root ganglion neurons. A-778317 was tritiated ([3H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [3H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (KD = 3.4 nM; Bmax = 4.0 pmol/mg protein). Specific binding of 2 nM [3H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [3H]A-778317 correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A-778317 blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site and that [3H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology.     

Inhibition of glucosylceramide synthase reversibly decreases the capsaicin-induced activation and TRPV1 expression of cultured dorsal root ganglion neurons

Recent studies have demonstrated significant changes in the neuronal ganglioside status associated with altered functional states of nociceptive primary sensory neurons. In the present study, therefore, the effects of the inhibition of glucosylceramide synthase, the key enzyme of ganglioside synthesis, were studied on chemically defined populations and on the activation of TRPV1 of cultured adult rat sensory ganglion neurons. In control cultures, capsaicin resulted in the activation of TRPV1 in 29.7 ± 2.5% of the neurons, as assessed with the cobalt uptake assay. Pretreatment of the cultures for 4 days with an inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP), significantly decreased the proportion of capsaicin-activated neurons to 11.6 ± 1.2%. Immunohistochemistry demonstrated that, in control cultures, 37.5 ± 1.4% of the neurons displayed TRPV1 immunoreactivity, whereas in d-PDMP-treated cultures the proportion of TRPV1-immunoreactive neurons was diminished to 18.2 ± 2.1%. Further experiments disclosed that these effects of d-PDMP were reversible. The capsaicin-, but not the high potassium-induced release of CGRP, was also significantly reduced after d-PDMP treatment, as measured with ELISA. The proportions of IB4- and CGRP-positive neurons were not significantly affected by d-PDMP. The present observations demonstrate that inhibition of neuronal ganglioside synthesis profoundly modulates the expression of the TRPV1 receptor, apparently leaving other markers of nociceptive neurons, such as CGRP and IB4, unaffected. The findings indicate that as yet unidentified ganglioside(s) synthesized by the glucosylceramide synthase pathway may be essential for nociception through mechanisms which may implicate membrane lipid raft function and/or altered nerve growth factor signaling, which are essential for the TRPV1 receptor function.     

Activation of descending pain-facilitatory pathways from the rostral ventromedial medulla by cholecystokinin elicits release of prostaglandin-E₂ in the spinal cord

Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and “anti-opioid” by actions on pain-modulatory cells within the rostral ventromedial medulla (RVM). One consequence of activation of RVM CCK₂ receptors may be enhanced spinal nociceptive transmission; but how this might occur, especially in states of pathological pain, is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold after ligation of L₅/L₆ spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time-related increase in (prostaglandin-E₂) PGE₂ measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE₂ was approximately 5-fold and was observed at approximately 80 minutes post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-hydroxyindoleacetic acid (5-HIAA), a 5-hydoxytryptophan (5-HT) metabolite, as compared with controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT₃ antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE₂ and 5-HT in the spinal cord.     

Mechanistic Insights Into the Analgesic Efficacy of A-1264087, a Novel Neuronal Ca2+ Channel Blocker That Reduces Nociception in Rat Preclinical Pain Models

Voltage-gated Ca²⁺ channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca²⁺ channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca²⁺ channels with similar potency (IC₅₀ = 1–2 μM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene–related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant–induced inflammatory, and chronic constrictive injury of sciatic nerve—induced, neuropathic pain models with ED₅₀ values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval = 2.2–3.5, 3.7–10, and 5.5–12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal–regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant–inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations.PerspectiveThe present results demonstrate that the neuronal Ca²⁺ channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.     

Modulation of inhibitory neurotransmission by the vanilloid receptor type 1 (TRPV1) in organotypically cultured mouse substantia gelatinosa neurons

The vanilloid receptor type 1 (TRPV1) plays a pivotal role in modulating thermal, chemical, and inflammatory pain. TRPV1s are expressed in some dorsal horn (DH) neurons, but their contribution, if any, to central pain processing still remains unclear. We studied the effects of 2 μM capsaicin-induced TRPV1 activation in organotypically cultured substantia gelatinosa neurons from post-natal (8–12) mice. Capsaicin affected sIPSC frequency (272 ± 60% of control, n = 14, P < 0.02), but not amplitude (131 ± 12% of control, n = 14, P > 0.05) in patch clamp recordings, also in the presence of 50 μM AP-5 (frequency: 265 ± 69% of control; n = 8, P < 0.05; amplitude: 156 ± 28% of control; n = 8, P > 0.05). The frequency increase was reduced by TTX (181 ± 21% of control; n = 12, P < 0.05). Pre-administration of I-RTX (1 μM), a TRPV1 antagonist, prevented the capsaicin effect (frequency: 149 ± 28% of control, P > 0.05, n = 12; amplitude: 97 ± 4% of control, P > 0.05, n = 12). NADA (1 μM), an endovanilloid/endocannabinoid agonist of TRPV1, induced a significant increase of sISPC frequency (191 ± 40% of control; n = 8, P < 0.05) without affecting the amplitude (102 ± 6% of control; n = 8, P > 0.05), and the co-application of two naturally occurring N-acyldopamines, PALDA (5 μM) and STEARDA (5 μM) that facilitate the effect of TRPV1 agonists, also induced a significant increase of sIPSC frequency (278 ± 67% of control, n = 6, P < 0.05). The presence of TRPV1 protein and mRNA in DH neurons was confirmed by histological (immunocytochemistry, in situ PCR) and biochemical (Western blotting, PCR) procedures. These data show that TRPV1 modulates inhibitory neurotransmission in cultured substantia gelatinosa neurons, and suggest that endogenous agonists can activate the spinal receptors in vivo.     

Peripheral nerve injury produces a sustained shift in the balance between glutamate release and uptake in the dorsal horn of the spinal cord

Peripheral nerve injury provokes heightened excitability of primary sensory afferents including nociceptors, and elicits ectopic activity in lesioned and neighboring intact nerve fibers. The major transmitter released by sensory afferents in the superficial dorsal horn of the spinal cord is glutamate. Glutamate is critically involved in nociceptive signaling and the development of neuropathic pain. We recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II of the rat dorsal horn to assess spontaneous synaptic activity after spared nerve injury (SNI), a model of chronic neuropathic pain. Following SNI, the frequency of mEPSCs doubled, indicating heightened glutamate release from primary afferents or spinal interneurons. Consistent with this finding, glutamate concentrations in the cerebrospinal fluid were elevated at 1 and 4 weeks after SNI. Transmitter uptake was insufficient to prevent the rise in extracellular glutamate as the expression of glutamate transporters remained unchanged or decreased. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride, an antagonist of metabotropic glutamate receptor 5 (mGluR5), reduced the frequency of mEPSCs to its preinjury level, suggesting a positive feedback mechanism that involves facilitation of transmitter release by mGluR5 activation in the presence of high extracellular glutamate. Treatment with the β-lactam antibiotic ceftriaxone increased the expression of glutamate transporter 1 (Glt1) in the dorsal horn after SNI, raised transmitter uptake, and lowered extracellular glutamate. Improving glutamate clearance prevented the facilitation of transmitter release by mGluR5 and attenuated neuropathic pain-like behavior. Balancing glutamate release and uptake after nerve injury should be an important target in the management of chronic neuropathic pain.     

辣椒素受体-1、一氧化氮和降钙素基因相关肽在大鼠上颈段脊髓刺激诱导脑血流增加中的作用

目的:探讨辣椒素受体-1(TRPV1)、一氧化氮(NO)和降钙素基因相关肽(CGRP)在上颈段脊髓刺激(cSCS)诱导脑血流(CBF)增加中的作用和相互关系。方法:将雄性SD大鼠在全麻下行C1-2椎板切除暴露颈段脊髓,刺激电极放置在左侧C2脊髓背柱上。切开颅骨暴露左侧大脑皮质,CBF变化由激光多普勒血流仪测量。分析90%运动阈值(MT)cSCS在静脉注射辣椒素类似物树胶脂毒素(RTX)2μg/kg(n=9)、CGRP8-37(CGRP受体拮抗剂)2.5mg/kg(n=8)、L-NAME(一氧化氮合酶抑制剂)5.0mg/kg(n=7)前和20min后同侧脑血流变化(%△CBF)及脑血管阻力变化(%△CVR)。结果:60%和90%MT的cSCS使同侧大脑皮质血流增加(P0.05,n=9)。静脉注射含有TRPV1纤维脱敏化的RTX(2μg/kg)后,90%MT的cSCS引起%△CBF由65.0%±9.5%减少到27.4%±7.2%(P0.05,n=9),%△CVR由-28%±7.6%增加到-14.8%±4.2%(P0.05,n=9);静脉注射CGRP8-37(2.5mg/kg)使90%MT的cSCS引起增加的CBF降低(%△CBF:63.4%±10.4%vs.22.7%±5.3%,P0.05,n=8;%△CVR:-27.9%±5.9%vs.-14.8%±3.2%,P0.05,n=8);静脉注射L-NAME(5.0mg/kg)使90%MT的cSCS引起增加的CBF显著下降(%△CBF:58.1%±9.5%vs.14.0%±2.5%,P0.01,n=7;%△CVR:-20.4%±4.3%vs.-7.6%±0.6%,P0.01,n=7)。结论:TRPV1、NO和CGRP参与cSCS引起显著的脑血流增加或脑血管扩张,它可能为cSCS治疗缺血性脑血管病提供新的理论依据。     

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation

Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC₅₀: 51 nmol/L, electrophysiology, 67 nmol/L, Ca²⁺ assay) and rat TRPA1 (IC₅₀: 101 nmol/L, electrophysiology, 289 nmol/L, Ca²⁺ assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED₅₀: 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.     

Paclitaxel-loaded polymeric microparticles: Quantitative relationships between in vitro drug release rate and in vivo pharmacodynamics

Intraperitoneal therapy (IP) has demonstrated survival advantages in patients with peritoneal cancers, but has not become a widely practiced standard-of-care in part due to local toxicity and sub-optimal drug delivery. Paclitaxel-loaded, polymeric microparticles were developed to overcome these limitations. The present study evaluated the effects of microparticle properties on paclitaxel release (extent and rate) and in vivo pharmacodynamics. In vitro paclitaxel release from microparticles with varying physical characteristics (i.e., particle size, copolymer viscosity and composition) was evaluated. A method was developed to simulate the dosing rate and cumulative dose released in the peritoneal cavity based on the in vitro release data. The relationship between the simulated drug delivery and treatment outcomes of seven microparticle compositions was studied in mice bearing IP human pancreatic tumors, and compared to that of the intravenous Cremophor micellar paclitaxel solution used off-label in previous IP studies. Paclitaxel release from polymeric microparticles in vitro was multi-phasic; release was greater and more rapid from microparticles with lower polymer viscosities and smaller diameters (e.g., viscosity of 0.17 vs. 0.67 dl/g and diameter of 5–6 vs. 50–60 μm). The simulated drug release in the peritoneal cavity linearly correlated with treatment efficacy in mice (r² > 0.8, p < 0.001). The smaller microparticles, which distribute more evenly in the peritoneal cavity compared to the large microparticles, showed greater dose efficiency. For single treatment, the microparticles demonstrated up to 2-times longer survival extension and 4-times higher dose efficiency, relative to the paclitaxel/Cremophor micellar solution. Upon repeated dosing, the paclitaxel/Cremophor micellar solution showed cumulative toxicity whereas the microparticle that yielded 2-times longer survival did not display cumulative toxicity. The efficacy of IP therapy depended on both temporal and spatial factors that were determined by the characteristics of the drug delivery system. A combination of fast- and slow-releasing microparticles with 5–6 μm diameter provided favorable spatial distribution and optimal drug release for IP therapy.     

低频电针对选择性神经损伤大鼠神经痛维持期脊髓背角PKA-TRPV1通路及痛敏递质的干预作用

目的 探讨低频电针干预神经病理痛维持期脊髓背角（SCDH）蛋白激酶A（PKA）、辣椒素受体（TRPV1）通路的调控机制。 方法 将大鼠随机分为空白对照组、假手术组、模型对照组、电针干预组4组。采用坐骨神经分支选择性神经损伤（SNI）方法建立神经病理痛模型。电针干预取术侧足三里、昆仑穴,频率2Hz,每日1次,连续干预14d。检测大鼠术侧后足缩足阈值（PWT）、SCDH PKA和TRPV1以及降钙素基因相关肽（CGRP）和P物质（SP）水平。 结果 SNI模型大鼠术侧PWT下降（P<0.01）,术侧SCDH PKA、TRPV1、CGRP、SP水平均上调（P<0.05）;2Hz电针可提高SNI模型大鼠PWT（P<0.01）,降低术侧SCDH PKA、TRPV1、CGRP、SP水平（P<0.05）。 结论 低频电针能改善神经病理痛,可能与其下调SCDH PKA-TRPV1通路以及CGRP、SP痛敏递质水平有关。     

Sensitized peripheral nociception in experimental diabetes of the rat

Painful neuropathy is a common complication of diabetes. Particularly in the early stage of diabetic neuropathy, patients are characterized by burning feet, hyperalgesia to heat, and mechanical stimuli, as if residual nociceptors were sensitized. Such symptoms are barely explained by common pathophysiological concepts of diabetic neuropathy. Diabetes was induced in Wistar rats by streptozotocin (STZ). After 4 weeks behavioral testing (Plantar test, Randall-Selitto) was conducted. Basal and stimulated release of calcitonin gene-related peptide (CGRP), Substance P (SP) and prostaglandin E₂ (PGE₂) from isolated skin and sciatic nerve were assessed by enzyme immunoassays. Electrophysiological properties of identified nociceptors under hyperglycemic, hypoxic, and acidotic conditions were investigated using the skin-nerve preparation. The diabetic rats showed hyperalgesia to heat and pressure stimulation. The basal CGRP/SP release was reduced, but chemical stimulation with bradykinin induced greater release of SP, CGRP and PGE₂ than in control animals. In contrast, capsaicin-stimulated CGRP release was reduced in sciatic nerves. Hypoxia per se lowered von Frey thresholds of most C-nociceptors to half. Hyperglycemic hypoxia induced ongoing discharge in all diabetic but not control C-fibers which was further enhanced under acidosis. Sensory and neurosecretory nociceptor functions are sensitized in diabetes. Diabetic C-fibers show exaggerated sensitivity to hyperglycemic hypoxia with and without additional acidosis, conditions that are thought to mimic ischemic episodes in diabetic nerves. Ongoing C-fiber discharge is known to induce spinal sensitization. Together with altered receptor and ion channel expressions this may contribute to painful episodes in diabetic neuropathy.