Hindpaw Responses Research Articles

Cilostazol Ameliorates Peripheral Neuropathic Pain in Streptozotocin-Induced Type I Diabetic Rats.

Background: Cilostazol is an antiplatelet agent with vasodilating, endothelial function restoration, and anti-inflammatory effects. This study aims to investigate the efficacy of oral cilostazol for preventing the development of diabetic peripheral neuropathy (DPN). Materials and Methods: Ninety adult male Sprague-Dawley rats were divided into five groups: 1) naïve (control); 2) diabetic (DM); 3) DM receiving 10 mg/kg cilostazol (cilo-10); 4) DM receiving 30 mg/kg cilostazol (cilo-30); and 5) DM receiving 100 mg/kg cilostazol (cilo-100). Hindpaw responses to thermal and mechanical stimuli were measured. Activation of microglia and astrocytes in the spinal dorsal horn (SDH) and expression of NaVs in the dorsal root ganglia (DRG) were examined with Western blots and immunofluorescence. Results: DM rats displayed decreased withdrawal thresholds to mechanical stimuli (mechanical allodynia) and blunted responses to thermal stimuli. In addition, the expression of microglia increased, but astrocytes were reduced in the SDH. Upregulation of Nav −1.1, 1.2, −1.3, −1.6, and −1.7 and downregulation of Nav-1.8 were observed in the DRG. The DM rats receiving cilostazol all returned DM-induced decrease in withdrawal threshold to mechanical stimuli and attenuated neuropathic pain. Additionally, all cilostazol treatments suppressed the level of activated microglial cells and ameliorated the DM-induced decline in astrocyte expression levels in the SDH. However, only the rats treated with cilo-100 demonstrated significant improvements to the aberrant NaV expression in the DRG. Conclusion: Oral cilostazol can blunt the responses of mechanical allodynia and has the potential to treat diabetic neuropathy by attenuating NaV and glial cell dysregulation.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8–37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8–37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Acute spatial spread of NO-mediated potentiation during hindpaw ischaemia in mice.

Key points Neuropathic pain spreads spatially beyond the injured sites, and the mechanism underlying the spread has been attributed to inflammation occurring in the spinal cord.However, the spatial spread of spinal/cortical potentiation induced by conduction block of the peripheral nerves can be observed prior to inflammation.In the present study, we found that spreading potentiation and hypersensitivity acutely induced by unilateral hindpaw ischaemia are nitric oxide (NO)‐dependent and that NO is produced by ischaemia and quickly diffuses within the spinal cord.We also found that NO production induced by ischaemia is not observed in the presence of an antagonist for group II metabotropic glutamate receptors (mGluRs) and that neuronal NO synthase‐positive dorsal horn neurons express group II mGluRs.These results suggest strongly that NO‐mediated spreading potentiation in the spinal cord is one of the trigger mechanisms for neuropathic pain. Cortical/spinal responses to hindpaw stimulation are bilaterally potentiated by unilateral hindpaw ischaemia in mice. We tested the hypothesis that hindpaw ischaemia produces nitric oxide (NO), which diffuses in the spinal cord to induce spatially spreading potentiation. Using flavoprotein fluorescence imaging, we confirmed that the spreading potentiation in hindpaw responses was induced during ischaemia in the non‐stimulated hindpaw. This spreading potentiation was blocked by spinal application of l‐NAME, an inhibitor of NO synthase (NOS). Furthermore, no spreading potentiation was observed in neural NOS (nNOS) knockout mice. Spinal application of an NO donor was enough to induce cortical potentiation and mechanical hypersensitivity. The spatial distribution of NO during unilateral hindpaw ischaemia was visualized using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein (DAF‐FM). An increase in fluorescence derived from the complex of DAF‐FM with NO was observed on the ischaemic side of the spinal cord. A similar but smaller increase was also observed on the contralateral side. Somatosensory potentiation after hindpaw ischaemia is known to be inhibited by spinal application of LY354740, an agonist of group II metabotropic glutamate receptors (mGluRs). We confirmed that the spinal DAF‐FM fluorescence increases during hindpaw ischaemia were not observed in the presence of LY354740. We also confirmed that approximately half of the nNOS‐positive neurons in the superficial laminae of the dorsal horn expressed mGluR2 mRNA. These results suggest that disinhibition of mGluR2 produces NO which in turn induces a spreading potentiation in a wide area of the spinal cord. Such spreading, along with the consequent non‐specific potentiation in the spinal cord, may trigger neuropathic pain.

Functional Selectivity of Lumbosacral Stimulation: Methodological Approach and Pilot Study to Assess Visceral Function in Pigs.

Nearly all spinal cord injured (SCI) individuals lose bladder control and are prone to kidney complications if intermittent catheterization is not applied. Electrical stimulation of the sacral anterior roots with an implantable neuroprosthesis is one means to restore continence and control micturition. However, only a small percentage of the SCI population benefits from this solution because of its drawbacks, e.g., section of sacral posterior roots with loss of spared sensitivity. There has been renewed interest in spinal cord stimulation in recent years, but most studies have focused on locomotion and only few have reported the impact on visceral functions. Moreover, even though the lumbosacral spinal cord is the location of many pre-cabled neural networks (involved in locomotion, bladder, and bowel management), the functional selectivity of spinal stimulations has never been investigated in detail. Here, we present: 1) a methodology designed to study lumbosacral epispinal-intradural stimulation selectivity; 2) preliminary results assessing the impact of epispinal stimulation on bladder and bowel functions in two domestic pigs; and 3) a comparison of these visceral responses with abdominal and lower limb activities. Our experiments resulted in selective bladder and rectal responses, i.e., without hind paw responses, thus reaffirming the rehabilitation potential of spinal cord stimulation.

Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain.

The response of diffuse noxious inhibitory controls (DNIC) is often decreased, or lost, in stress-related functional pain syndromes. Because the dynorphin/kappa opioid receptor (KOR) pathway is activated by stress, we determined its role in DNIC using a model of stress-induced functional pain. Male, Sprague-Dawley rats were primed for 7 days with systemic morphine resulting in opioid-induced hyperalgesia. Fourteen days after priming, when hyperalgesia was resolved, rats were exposed to environmental stress and DNIC was evaluated by measuring hind paw response threshold to noxious pressure (test stimulus) after capsaicin injection in the forepaw (conditioning stimulus). Morphine priming without stress did not alter DNIC. However, stress produced a loss of DNIC in morphine-primed rats in both hind paws that was abolished by systemic administration of the KOR antagonist, nor-binaltorphimine (nor-BNI). Microinjection of nor-BNI into the right, but not left, central nucleus of the amygdala (CeA) prevented the loss of DNIC in morphine-primed rats. Diffuse noxious inhibitory controls were not modulated by bilateral nor-BNI in the rostral ventromedial medulla. Stress increased dynorphin content in both the left and right CeA of primed rats, reaching significance only in the right CeA; no change was observed in the rostral ventromedial medulla or hypothalamus. Although morphine priming alone is not sufficient to influence DNIC, it establishes a state of latent sensitization that amplifies the consequences of stress. After priming, stress-induced dynorphin/KOR signaling from the right CeA inhibits DNIC in both hind paws, likely reflecting enhanced descending facilitation that masks descending inhibition. Kappa opioid receptor antagonists may provide a new therapeutic strategy for stress-related functional pain disorders.

Sciatic Nerve Intrafascicular Lidocaine Injection-induced Peripheral Neuropathic Pain: Alleviation by Systemic Minocycline Administration.

Peripheral nerve block guidance with a nerve stimulator or echo may not prevent intrafascicular injury. This study investigated whether intrafascicular lidocaine induces peripheral neuropathic pain and whether this pain can be alleviated by minocycline administration. A total of 168 male Sprague-Dawley rats were included. In experiment 1, 2% lidocaine (0.1 mL) was injected into the left sciatic nerve. Hindpaw responses to thermal and mechanical stimuli, and sodium channel and activating transcription factor (ATF-3) expression in dorsal root ganglion (DRG) and glial cells in the spinal dorsal horn (SDH), were measured on days 4, 7, 14, 21, and 28. On the basis of the results in experiment 1, rats in experiment 2 were divided into sham, extraneural, intrafascicular, peri-injury minocycline, and postinjury minocycline groups. Behavioral responses, macrophage recruitment, expression changes of myelin basic protein and Schwann cells in the sciatic nerve, dysregulated expression of ATF-3 in the DRG, and activated glial cells in L5 SDH were assessed on days 7 and 14. Intrafascicular lidocaine induced mechanical allodynia, downregulated Nav1.8, increased ATF-3 expression in the DRG, and activated glial cells in the SDH. Increased expression of macrophages, Schwann cells, and myelin basic protein was found in the sciatic nerve. Minocycline attenuated intrafascicular lidocaine-induced neuropathic pain and nerve damage significantly. Peri-injury minocycline was better than postinjury minocycline administration in alleviating mechanical behaviors, mitigating macrophage recruitment into the sciatic nerve, and suppressing activated microglial cells in the spinal cord. Systemic minocycline administration alleviates intrafascicular lidocaine injection-induced peripheral nerve damage.

Spinal mechanisms underlying potentiation of hindpaw responses observed after transient hindpaw ischemia in mice.

Transient ischemia produces postischemic tingling sensation. Ischemia also produces nerve conduction block that may modulate spinal neural circuits. In the present study, reduced mechanical thresholds for hindpaw-withdrawal reflex were found in mice after transient hindpaw ischemia, which was produced by a high pressure applied around the hindpaw for 30 min. The reduction in the threshold was blocked by spinal application of LY354740, a specific agonist of group II metabotropic glutamate receptors. Neural activities in the spinal cord and the primary somatosensory cortex (S1) were investigated using activity-dependent changes in endogenous fluorescence derived from mitochondrial flavoproteins. Ischemic treatment induced potentiation of the ipsilateral spinal and contralateral S1 responses to hindpaw stimulation. Both types of potentiation were blocked by spinal application of LY354740. The contralateral S1 responses, abolished by lesioning the ipsilateral dorsal column, reappeared after ischemic treatment, indicating that postischemic tingling sensation reflects a sensory modality shift from tactile sensation to nociception in the spinal cord. Changes in neural responses were investigated during ischemic treatment in the contralateral spinal cord and the ipsilateral S1. Potentiation already appeared during ischemic treatment for 30 min. The present findings suggest that the postischemic potentiation shares spinal mechanisms, at least in part, with neuropathic pain.

Selective antinociceptive effects of a combination of the N-methyl-D-aspartate receptor peptide antagonist [Ser(1)]histogranin and morphine in rat models of pain.

Numerous rather than a few analgesic endogenous neuropeptides are likely to work in concert in vivo in ameliorating pain. Identification of effective neuropeptide combinations would also facilitate the development of gene or cell-based analgesics. In this study, opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and the peptide histogranin analogue [Ser1]histogranin (SHG), which possess activity as an N-methyl-d-aspartate (NMDA) receptor antagonist, were intrathecally (i.t.) injected alone and in combination in rat models of acute and persistent pain. None of the peptides when injected alone altered hind paw responses of uninjured rats to acute noxious stimulation. EM-1 and EM-2 showed divergent efficacies in the persistent pain models. For example, EM-1 injected alone was antinociceptive in rats with neuropathic pain, whereas EM-2 demonstrated no efficacy. Demonstration of synergism was also divergent across the models. For example, while SHG combined with EM-1 did not alter the efficacy of EM-1 in rats with neuropathic pain, SHG significantly increased the efficacy of EM-1 in the formalin test. By contrast, the potency and efficacy of the peptides alone and combinations were much less than those of the reference analgesic morphine. Furthermore, morphine combined with the clinically used NMDA receptor antagonist ketamine showed synergism across a broad range of pain states. While the current set of neuropeptides could serve as a basis for analgesic therapeutics, there could be other neuropeptides with greater efficacy and potency and broader therapeutic application.

Involvement of Nax sodium channel in peripheral nerve regeneration via lactate signaling.

Na(x), a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Na(x) knockout (Na(x)⁻/⁻) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in (Na(x)⁻/⁻) sciatic nerves. The delay in the recovery in Na(x)⁻/⁻ mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)-1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Na(x) was activated by ET-1. The amount of lactate release by ET-1 was lower in Na(x)⁻/⁻ mice than in wild-type mice. These results indicated that Na(x) is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.

Systemic Administration of an Alpha-7 Nicotinic Acetylcholine Agonist Reverses Neuropathic Pain in Male Sprague Dawley Rats

Alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists attenuate pain and inflammation in preclinical models. This study tested whether systemic delivery of an α7 nAChR agonist attenuates neuropathic pain and associated immune-mediated pro-inflammation. Hind paw response thresholds to mechanical stimuli in male Sprague Dawley rats were assessed before and after sciatic chronic constriction injury (CCI) or sham surgery. Osmotic mini-pumps containing TC-7020, an α7 nAChR selective agonist, were implanted 10 to 14 days after surgery. TC-7020 (1, 3, and 10 mg/kg/d; s.c.) significantly attenuated CCI-induced allodynia, which lasted through 2 weeks of test compound administration. Spinal cords were collected after 2 weeks and processed for microglial and astrocyte activation markers within the ipsilateral L4-L6 dorsal horn. In addition, ipsilateral L4-5 dorsal root ganglia (DRGs) were processed for neuronal injury and satellite cell activation markers. CCI-induced central glial cell activation markers were not suppressed by TC-7020, even though TC-7020 is mildly blood-brain barrier permeable. However, TC-7020 downregulated the integrated density of activation transcription factor 3 (ATF3) but not the number of ATF positive cells. TC-7020 also downregulated phosphorylated extracellular signal kinase (p-ERK) and satellite cell activation in the CCI-affected DRGs. Therefore, systemic α7 nAChR agonist may be effective in treating neuropathic pain via reducing neuronal injury and immune cells activation occurring in the periphery. PerspectiveThese studies demonstrated that TC-7020, an alpha7 nicotinic acetylcholine receptor agonist with partial blood-brain barrier permeability, reversed neuropathic pain in rats, likely via attenuation of inflammation in the DRG and/or the site of sciatic injury.

Imaging the Spatio-Temporal Dynamics of Supragranular Activity in the Rat Somatosensory Cortex in Response to Stimulation of the Paws

We employed voltage-sensitive dye (VSD) imaging to investigate the spatio-temporal dynamics of the responses of the supragranular somatosensory cortex to stimulation of the four paws in urethane-anesthetized rats. We obtained the following main results. (1) Stimulation of the contralateral forepaw evoked VSD responses with greater amplitude and smaller latency than stimulation of the contralateral hindpaw, and ipsilateral VSD responses had a lower amplitude and greater latency than contralateral responses. (2) While the contralateral stimulation initially activated only one focus, the ipsilateral stimulation initially activated two foci: one focus was typically medial to the focus activated by contralateral stimulation and was stereotaxically localized in the motor cortex; the other focus was typically posterior to the focus activated by contralateral stimulation and was stereotaxically localized in the somatosensory cortex. (3) Forepaw and hindpaw somatosensory stimuli activated large areas of the sensorimotor cortex, well beyond the forepaw and hindpaw somatosensory areas of classical somatotopic maps, and forepaw stimuli activated larger cortical areas with greater activation velocity than hindpaw stimuli. (4) Stimulation of the forepaw and hindpaw evoked different cortical activation dynamics: forepaw responses displayed a clear medial directionality, whereas hindpaw responses were much more uniform in all directions. In conclusion, this work offers a complete spatio-temporal map of the supragranular VSD cortical activation in response to stimulation of the paws, showing important somatotopic differences between contralateral and ipsilateral maps as well as differences in the spatio-temporal activation dynamics in response to forepaw and hindpaw stimuli.

The atypical cannabinoid O-1602 increases hind paw sensitisation in the chronic constriction injury model of neuropathic pain

O-1602 is an atypical cannabinoid that acts as an agonist at GPR55, a g protein-coupled receptor that previous studies have indicated may have a pronociceptive role in neuropathic pain. We administered O-1602 to both naive rats and rats that had undergone chronic constriction injury surgery. O-1602 did not cause any changes in hind paw responses to Von Frey hair testing in naive rats. However, O-1602 reversed the desensitising effects of ETOH, which was used as an active and opposing vehicle. Our results are consistent with the hypothesis that GPR55 has a pronociceptive role in neuropathic pain.

Intrathecal gabapentin and clonidine synergistically inhibit allodynia in spinal nerve-ligated rats

Aims The objective of this study was to elucidate the interaction between intrathecally administered gabapentin and clonidine on neuropathic pain associated with allodynia in the spinal nerve ligation model in the rat. Main methods Thresholds for hind paw responses to mechanical stimuli were determined by delivering von Frey filaments to the plantar surface. The left L5 spinal nerve was ligated and a fine catheter was intrathecally implanted at the L3–4 interspace under sevoflurane anesthesia. After confirmation of the established allodynia, gabapentin at 10, 30, 60 and 100 μg or clonidine at 5, 15, 30 and 50 μg was injected as a monotherapy in conscious rats through the intrathecal catheter to obtain the dose–response curve of %MPE (maximum possible effect) of the antiallodynic effect and its ED 50. Gabapentin and clonidine were concomitantly administered in a fixed-dose ratio proportional to the predetermined ED 50 of these drugs, thereby obtaining a dose–response curve for the drug combination and its ED 50. The profile of the interaction between these drugs was analyzed using an isobolographic analysis. Key findings The ED 50 for gabapentin and clonidine were 57.3 ± 4.0 and 20.2 ± 1.0 μg, respectively (mean ± SEM). However, the co-administration of gabapentin and clonidine at a ratio of 20:7 contributed to a much smaller experimental ED 50 values (gabapentin 10.1 ± 1.1 μg, and clonidine 3.6 ± 0.3 μg) compared with their theoretical ED 50s on the additive line in the isobologram. Significance In the L5 spinal nerve-ligated rats, the intrathecal co-administration of gabapentin and clonidine exerted a synergistic action on the mechanical antiallodynic effect.

Excrescence of neurotransmitter glutamate from disc material has nociceptive qualities: evidence from a rat model

Background context The authors have previously demonstrated that herniated human lumbar disc is rich in free glutamate from degradation of aggrecan. Prior data have suggested that free glutamate could contribute to a nociceptive state. Purpose Previous behavioral experiments suggested glutamate-related nociception by comparing pre- and postglutamate infusion responses only. This indirectly suggested nociceptive effects of epidural glutamate but was not a definitive evidence. Now, by using larger numbers of subjects, we have demonstrated that lumbar epidural glutamate infusion causes significant left-to-right differences in hind paw response during treatment, demonstrating more directly the focal nociceptive effects of glutamate. Study design Behavioral studies and immunohistochemistry were used to assess for evidence of a nociceptive state. All researchers were blinded to infusion solution. Methods Via an implanted mini osmotic pump, the epidural space of rats was infused with 0.02 mM glutamate or normal saline for 72 hours. Signs of nociception were assessed by von Frey and plantar thermal stimulation testing and by glutamate receptor expression in the corresponding dorsal horn of the spinal cord and dorsal root ganglion. Results Both von Frey mechanical and plantar thermal stimulations showed differences in hind paw reactivity depending on whether it was on the ipsilateral or contralateral side of glutamate infusion. Saline infusion had no significant behavioral effects. Dorsal horn expression of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid and N-methyl- d-aspartic acid receptors was significantly increased in glutamate-infused animals, further indicative of a nociceptive state related to glutamate infusion. Conclusions Elevated epidural glutamate concentrations caused a focal hyperesthetic state. Increased epidural glutamate concentration could be a driving force or “chemical” component of disc-related radiculopathy.

Effect of analgesic standards on persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR)

Various common surgeries such as thoracotomy and inguinal hernia repair involve essential prolonged tissue retraction, often causing persistent postoperative pain. A new model was developed to mimic this clinical scenario, whereby skin/muscle incision and retraction (SMIR) in the medial thigh evoked persistent postoperative pain (Flatters (2008) [Pain 135:119-130]). This study examines the response of SMIR-evoked mechanical hypersensitivity to analgesic standards commonly used as positive controls in behavioural pain studies. Rats were anaesthetised, the skin and superficial muscle of the medial thigh was then incised and retracted for 1 h. In separate experiments, morphine, gabapentin and MK-801 were intraperitoneally administered to SMIR-operated rats, at maximally tolerated doses, on postoperative day 9–13. Mechanical hypersensitivity was measured by withdrawal responses to von Frey stimulation of the plantar hindpaws. Morphine (6 mg/kg) and gabapentin (100 mg/kg) elicited an almost complete reversal of SMIR-evoked mechanical hypersensitivity. In contrast, MK-801 (0.1 mg/kg) did not affect SMIR-evoked mechanical hypersensitivity. Contralateral hindpaw responses to von Frey stimulation were unaffected by SMIR surgery or any drug treatment. In conclusion, the SMIR model displays persistent mechanical hypersensitivity that is reversible by morphine or gabapentin treatment. As previously demonstrated, SMIR-evoked pain is not driven by neuronal damage and these data show that NMDA receptor activation does not play a role in the maintenance of SMIR-evoked pain. This study further demonstrates the value of the SMIR model as a tool to understand persistent postoperative/postsurgical pain mechanisms and evaluate potential treatments.

Activation of TRPV1 and TRPA1 leads to muscle nociception and mechanical hyperalgesia

The involvement of TRPV1 and TRPA1 in mediating craniofacial muscle nociception and mechanical hyperalgesia was investigated in male Sprague–Dawley rats. First, we confirmed the expression of TRPV1 in masseter afferents in rat trigeminal ganglia (TG), and provided new data that TRPA1 is also expressed in primary afferents innervating masticatory muscles in double-labeling immunohistochemistry experiments. We then examined whether the activation of each TRP channel in the masseter muscle evokes acute nocifensive responses and leads to the development of masseter hypersensitivity to mechanical stimulation using the behavioral models that have been specifically designed and validated for the craniofacial system. Intramuscular injections with specific agonists for TRPV1 and TRPA1, capsaicin and mustard oil (MO), respectively, produced immediate nocifensive hindpaw responses followed by prolonged mechanical hyperalgesia in a concentration-dependent manner. Pretreatment of the muscle with a TRPV1 antagonist, capsazepine, effectively attenuated the capsaicin-induced muscle nociception and mechanical hyperalgesia. Similarly, pretreatment of the muscle with a selective TRPA1 antagonist, AP18, significantly blocked the MO-induced muscle nociception and mechanical hyperalgesia. We confirmed these data with another set of selective antagonist for TRPV1 and TRPA1, AMG9810 and HC030031, respectively. Collectively, these results provide compelling evidence that TRPV1 and TRPA1 can functionally contribute to muscle nociception and hyperalgesia, and suggest that TRP channels expressed in muscle afferents can engage in the development of pathologic muscle pain conditions.

Neuropeptide Y receptor-expressing dorsal horn neurons: Role in nocifensive reflex responses to heat and formalin

Lumbar intrathecal injection of neuropeptide Y (NPY) is antinociceptive, particularly in models of nerve injury and inflammation. Intrathecal NPY does not alter nociception in mice null for the Y1 neuropeptide Y receptor (Y1R) and these mice show enhanced nocifensive reflex responses to aversive thermal, mechanical, visceral and chemical stimuli. Y1R and NPY receptor type 2 (Y2R) are present in the spinal dorsal horn presynaptically on primary afferent, and possibly interneuron terminals, but only Y1R is found postsynaptically on dorsal horn neurons. In the present study, we sought to assess the anatomic effects of lumbar intrathecal disulfide conjugate of neuropeptide Y and saporin (NPY-sap) and to determine the role of Y1R-expressing dorsal horn neurons in nocifensive responses to aversive thermal and chemical stimulation. Lumbar intrathecal injection of NPY-sap was used to selectively destroy Y1R-expressing lumbar dorsal horn neurons followed by testing nocifensive reflex responses on the hotplate and after hind-paw formalin injection. NPY-saporin decreased superficial dorsal horn staining for Y1R, but not neurokinin-1 receptor, mu opiate receptor or NPY peptide, and had no effect on Y1R cell counts in fourth lumbar spinal segment dorsal root ganglia. Loss of Y1R-expressing dorsal horn neurons was associated with increased first response latencies on the 44 °C hotplate and reduced total time rats spent licking and guarding hind paws during 600 s trials at 44 °C or 200 s trials at 47 °C. First hind-paw response latencies to high intensity phasic stimulation at 52 °C were unaffected. NPY-sap also reduced formalin-induced nocifensive behaviors during both interphase and phase II. These data demonstrate that selective destruction of Y1R-expressing superficial dorsal horn neurons, probably excitatory interneurons and/or projection neurons, reduces nocifensive reflex responses, particularly to activation of C nociceptors, and suggest a possible role for Y1R-expressing dorsal horn neurons in pain.

Effects of distal nerve injuries on dorsal-horn neurons and glia: Relationships between lesion size and mechanical hyperalgesia

Penetrating limb injuries are common and usually heal without long-lasting effects, even when nerves are cut. However, rare nerve-injury patients develop prolonged and disabling chronic pain (neuralgia). When pain severity is disproportionate to severity of the inciting injury, physicians and insurers may suspect exaggeration and limit care or benefits, although the nature of the relationship between lesion-size and the development and persistence of neuralgia remains largely unknown. We compared cellular changes in the spinal dorsal-horn (the initial CNS pain-processing area) after partial or total tibial-nerve axotomies in male Sprague–Dawley rats to determine if these changes are proportional to the numbers of peripheral axons cut. Unoperated rats provided controls. Plantar hind-paw responses to touch, pin, and cold were quantitated bilaterally to identify hyperalgesic rats. We also compared data from nerve-injured rats with or without hyperalgesic responses to mechanical hind-paw stimulation to evaluate concordance between pain behaviors and dorsal-horn cellular changes. Hyperalgesia was no less prevalent or severe after partial than after total axotomy. L5 spinal-cord sections from rats killed 7 days postoperatively were labeled for markers of primary afferents (substance P calcitonin gene-related peptide isolectin B4, gamma aminobutyric acid, and glial fibrillary acidic protein), then labeled cells were stereologically quantitated in somatotopically defined dorsal-horn regions. Total axotomy reduced markers of primary afferents more than partial axotomy. In contrast, GABA-immunoreactive profiles were similarly reduced after both lesions, and in rats with sensory loss versus hyperalgesia. Numbers of GFAP-immunoreactive astrocytes increased independently of lesion size and pain status. Small nerve injuries can thus have magnified and disproportionate effects on dorsal-horn neurons and glia, perhaps providing a biological correlate for the disproportionate pain of post-traumatic neuralgias (including complex regional pain syndrome-I) that follow seemingly minor nerve injuries. However, the presence of similar dorsal-horn changes in rats without pain behaviors suggests that not all transcellular responses to axotomy are pain-specific.

Assessing mechanical sensitivity of masseter muscle in lightly anesthetized rats: A model for craniofacial muscle hyperalgesia

In this report, we present a simple and reliable way of assessing mechanical sensitivity of masseter muscle as a model for craniofacial muscle hyperalgesia. Mechanical thresholds that evoke nocifensive hindpaw responses following noxious masseter stimulation were assessed. Masseteric injections of widely used sensitizing agents significantly increased mechanical sensitivity of the muscle in a time dependent manner without affecting other muscles and overlying skin. This lightly anesthetized rodent paradigm allows us to provide calibrated and reliable mechanical stimulus, which is not possible in behaving animals. The technique can be applied to study mechanistic bases for craniofacial muscle tenderness.

Effect of acute and chronic administration of caffeine on pain-like behaviors in rats with partial sciatic nerve injury

Caffeine, used in many pain medications as an adjuvant analgesic, is an adenosine A 1 and A 2A receptor antagonist. Here we examined the effects of acute or chronic caffeine administration in rats after partial sciatic nerve injury. The hindpaw response to mechanical or cold stimulation was assessed following photochemically induced sciatic nerve injury which leads to hypersensitivity to these stimuli. Caffeine was administered i.p. acutely or in the drinking water chronically. The mechanical and cold hypersensitivity of sciatic nerve-injured rats was dose-dependently alleviated by acute systemic administration of caffeine (10–80 mg/kg). The effect of caffeine was, however, associated with side effects including locomotor stimulation or depression. Chronic oral administration (average daily doses 27.5 mg/kg/day or 61.5 mg/kg/day for 2 weeks) of caffeine starting at the time of nerve injury did not significantly affect the development of pain-like behaviors. Thus, acute, but not long term, caffeine intake reduced neuropathic pain state in nerve-injured rats, but only at very high doses. The potential hyperalgesic effect of chronic A 1 adenosine receptor blockade may have been compensated for by an antinociceptive effect of caffeine through antagonism of A 2A receptors and tolerance development.