Neuropathic Pain Behavior In Rats Research Articles

LIFU Alleviates Neuropathic Pain by Improving the KCC2 Expression and Inhibiting the CaMKIV-KCC2 Pathway in the L4-L5 Section of the Spinal Cord.

Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC2) in the spinal cords of rats with peripheral nerve injury (PNI) in the lumbar 4–lumbar 5 (L4–L5) section. In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4–L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4–L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU− and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV–KCC2 pathway.

Activation of µ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents.

Several reports support the idea that µ- and δ-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of µ-δ heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on µ-δ heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a µ-δ heteromer-targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats significantly decreased µ-δ heteromer expression in L5 DRG but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC50: 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, subcutaneously). HEK-293T cell surface-labeled with µ- and δ-ORs internalized both receptors after exposure to CYM51010. By contrast, in cells transfected with µ-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats but was markedly attenuated in µ-OR knockout mice. Our studies show that spinal nerve injury may increase µ-δ heterodimerization in uninjured DRG neurons, and that µ-δ heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance.

Effects of ghrelin on pGSK-3\u03b2 and \u03b2-catenin expression when protects against neuropathic pain behavior in rats challenged with chronic constriction injury

Ghrelin has been shown to alleviate neuropathic pain by inhibiting the release of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β/β-catenin signaling in mediating the effect of ghrelin on neuropathic pain and to understand the associated mechanisms. Chronic constriction injury (CCI) of the sciatic nerve was used to establish a rat model of neuropathic pain. Hyperalgesia and allodynia were evaluated by observing the mechanical withdrawal threshold and the thermal withdrawal latency. Wnt3a and β-catenin protein expression and GSK-3β phosphorylation were detected by western blotting analysis. The levels of tumor necrosis factor-α and IL-1β were determined using an enzyme-linked immunosorbent assay. In addition, we used immunohistochemical analysis to determine the levels of GSK-3β phosphorylation in the dorsal horn of the spinal cord. Intrathecal delivery of ghrelin effectively ameliorated CCI-induced mechanical allodynia and thermal hyperalgesia at 7 and 14 days and reduced the levels of tumor necrosis factor-α. Ghrelin inhibited CCI-induced GSK-3β activation and β-catenin overexpression in the spinal dorsal horn. Moreover, intrathecal injection of ghrelin suppressed the activation of GSK-3β in the spinal dorsal horn of CCI rats, as assessed by immunohistochemical analysis. Our data indicated that ghrelin could markedly alleviate neuropathic pain by inhibiting the expression of β-catenin, via the suppression of GSK-3β activation, in the spinal cord of CCI rats.

P38 siRNA-encapsulated PLGA nanoparticles alleviate neuropathic pain behavior in rats by inhibiting microglia activation.

To investigate whether p38 small-interfering RNA-loaded nanoparticles (p38 siRNA NPs) attenuate spinal nerve ligation (SNL)-induced neuropathic pain in rats by suppressing spinal microglia activation via p38 targeting. After synthesizing p38 siRNA NPs with sonication, physical characteristics were measured for size and zeta potential. p38 siRNA NPs were then administrated intrathecally into SNL rats if they could reduce pain behavior excellently. p38 siRNA NPs significantly reduced mechanical allodynia as well as microgliosis in the spinal dorsal horns of SNL rats, consistent with a downregulation of p38-related proinflammatory mediators. As p38 in the spinal microglia plays a critical role in neuropathic pain, we expect that p38 siRNA NPs could be a promising tool for the treatment of neuropathic pain.

The role of hippocampal brain-derived neurotrophic factor in age-related differences in neuropathic pain behavior in rats

AimsThis study was aimed to explore the contribution of central brain-derived neurotrophic factor (BDNF) in the neuropathic pain pathogenesis using an aged rodent model. Main methodsAdult and aged rats were randomly assigned to either a sciatic nerve ligation (SNL) group or a control skin sham surgery group. Sensory behavioral testing were performed on the day before surgery and on the 3rd, 7th, 14th, and 21st days after surgery, followed by measurement of BDNF protein levels in different brain regions. In another experiment, the hippocampal BDNF gene expression after SNL surgery was assessed at different time-points. Furthermore, the analgesic effects of intranasal BDNF administration were tested in SNL animals. Key findingsOur behavioral results demonstrated that the hyperalgesia-like behavior after painful nerve injury has a higher incidence in aged rats compared with in adult animals. In particular, the hippocampal BDNF levels were inversely correlated with the probability of hyperalgesia-type behavior, in both brain-region specific and age-dependent manner. Time-course analysis showed that the hippocampal levels of BDNF mRNA in aged and adult rats started to decrease 7 and 14 days after surgery, respectively. However, the decrease was more pronounced in aged animals. Moreover, the repeated intranasal BDNF treatment could restore the central BDNF signaling, counteracting the age-related exacerbation of hyperalgesic behavior. SignificanceOur findings imply that hippocampal BDNF may be related with the pathogenesis of elderly neuropathic pain. Pharmacological data further suggest that brain BDNF may be modifiable in aged neuropathic animals, and therefore, represent a promising target for intervention.

Changes of voltage-gated sodium channels in sensory nerve regeneration and neuropathic pain models.

The present study was conducted to determine changes in the expression of voltage-gated sodium channels (VGSCs) α-subunits after nerve injury and their relation with development of neuropathic pain. We used the crush injury model of regeneration of the sciatic nerve (Crush) and the spared nerve injury (SNI) model of neuropathic pain in the rat. Measurements of thermal and mechanical pain thresholds were performed until 3 months after injury. Real-time PCR and immunohistochemistry of VGSC α-subunits were used to evaluate the mRNA and protein expression in the DRG. Both nerve injuries induced similar alterations in the VGSCs expression at 7 dpi, with upregulation of Nav1.3, and downregulation of Nav1.7, Nav1.8 and Nav1.9. These changes persisted until 28 days, when hyperalgesia was still present in SNI but not in Crush rats. At 90 days, mRNA expression of all analyzed α-subunits returned to basal levels in the Crush group. However, SNI rats still showed altered expression of VGSCs, and neuropathic pain responses. Immunohistochemical staining revealed that Nav1.8 and Nav1.9 were widely expressed in IB4-positive neurons of the DRG, relevant in pain processing. The population of neurons coexpressing each α-subunit and IB4 was also affected by the injury, more markedly after the Crush. Shifts in VGSCs expression occur in parallel to neuropathic pain behavior in rats early after injury, while at later times they appear to be more related to sensory nerve degeneration and regeneration processes.

Dynamic genotype-selective “phenotypic switching” of CGRP expression contributes to differential neuropathic pain phenotype

Using a genetic model we demonstrate the role played by “phenotypic switching” of calcitonin gene related peptide (CGRP) expression in axotomized large Aβ afferents in the development of neuropathic pain behavior in rats. After nerve injury both substance P and CGRP are upregulated in Aβ afferents in the corresponding DRGs. It has been proposed that intraspinal release of these neurotransmitters upon gentle stroking of skin drives ascending pain signaling pathways resulting in tactile allodynia. We reported previously that in rat lines genetically selected for high (HA) vs. low (LA) pain phenotype, SP is upregulated equally in both strains, but that CGRP is upregulated exclusively in the pain prone HA line (Nitzan-Luques et al., 2011). This implicates CGRP as the principal driver of tactile allodynia. Here we confirm this conclusion by showing: 1) that the time of emergence of CGRP-IR in DRG Aβ neurons and their central terminals in HA rats matches that of pain behavior, 2) that following spinal nerve lesion (SNL) selective activation of low threshold afferents indeed drives postsynaptic pain-signaling neurons and induces central sensitization in HA rats, as monitored using c-Fos as a marker. These changes are much less prominent in LA rats, 3) that intrathecal (i.t.) administration of CGRP induces tactile allodynia in naïve rats and 4) that i.t. administration of the CGRP-receptor antagonist BIBN4096BS (Olcegepant) attenuates SNL-evoked tactile allodynia, without blocking baseline nociception. Together, these observations support the hypothesis that genotype-selective phenotypic switching of CGRP expression in Aβ afferents following nerve injury is a fundamental mechanism of neuropathic tactile allodynia.

The flavonoid quercetin suppreses the development of neuropathic pain behavior in rats

The flavonoid quercetin suppreses the development of neuropathic pain behavior in rats

Swim Therapy Reduces Mechanical Allodynia and Thermal Hyperalgesia Induced by Chronic Constriction Nerve Injury in Rats

Neuropathic pain is common and often difficult to treat because it generally does not respond well to the currently available pain medications or nerve blocks. Recent studies in both humans and animals have suggested that exercise may induce a transient analgesia and reduce acute pain in normal healthy individuals. We examined whether swim therapy could alleviate neuropathic pain in rats. Rats were trained to swim over a 2-week period in warm water. After the rats were trained, neuropathic pain was induced by constricting the right sciatic nerve, and regular swimming was resumed. The sensitivity of each hind paw was monitored using the Hargreaves test and von Frey test to evaluate the withdrawal response thresholds to heat and touch. The paw ipsilateral to the nerve ligation expressed pain-like behaviors including thermal hyperalgesia and mechanical allodynia. Regular swim therapy sessions significantly reduced the mechanical allodynia and thermal hyperalgesia. Swim therapy had little effect on the withdrawal thresholds for the contralateral paw. In addition, swim therapy alone did not alter the thermal or mechanical thresholds of normal rats. The results suggest that regular exercise, including swim therapy, may be an effective treatment for neuropathic pain caused by nerve injuries. This study, showing that swim therapy reduces neuropathic pain behavior in rats, provides a scientific rationale for clinicians to test the efficacy of exercise in the management of neuropathic pain. It may prove to be a safe and cost-effective therapy in a variety of neuropathic pain states.

Spinal Cord FAAH in Normal Micturition Control and Bladder Overactivity in Awake Rats

We assessed whether spinal inhibition of the cannabinoid degrading enzyme FAAH would have urodynamic effects in normal rats and rats with bladder overactivity induced by partial urethral obstruction or prostaglandin E2. We also determined the expression of FAAH, and the cannabinoid receptors CB1 and CB2 in the sacral spinal cord. We used 44 rats for functional (cystometry) and Western blot experiments. The FAAH inhibitor oleoyl ethyl amide (3 to 300 nmol) was administered intrathecally (subarachnoidally) or intravenously. The expression of FAAH and CB1/CB2 receptors was determined by Western blot. Oleoyl ethyl amide given intrathecally affected micturition in normal rats and rats with bladder overactivity but effects were more pronounced in the latter. In normal rats oleoyl ethyl amide only decreased micturition frequency, while it decreased frequency and bladder pressures in rats with bladder overactivity. Intravenous oleoyl ethyl amide (3 to 300 nmol) had no urodynamic effect. FAAH and CB1/CB2 receptors were expressed in the rat sacral spinal cord. The expression of CB1/CB2 receptors but not FAAH was higher in obstructed than in normal rats. FAAH inhibition in the sacral spinal cord by oleoyl ethyl amide resulted in urodynamic effects in normal rats and rats with bladder overactivity. The spinal endocannabinoid system may be involved in normal micturition control and it appears altered when there is bladder overactivity.

Comparison of manual and automated filaments for evaluation of neuropathic pain behavior in rats

IntroductionThe most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment. In preclinical research, neuropathic pain models of nerve injury with varied etiology like partial sciatic nerve ligation (PNL), chronic constricted injury (CCI) and spinal nerve ligation (SNL) are employed to screen the analgesic drugs to treat symptoms like allodynia and hyperalgesia. The current study was aimed to validate and compare conventionally used Von Frey monofilaments and automated dynamic plantar aesthesiometer using three different pain models. MethodsPNL, CCI and SNL rats were used to compare and validate the assessment of neuropathic pain using Von Frey monofilaments and automated dynamic plantar aesthesiometer. ResultsMechanical allodynia was assessed at various time points to mimic drug testing conditions in neuropathic pain models and anticipated to observe reliable and reproducible paw withdrawal threshold measurements across these models. Consistent paw withdrawal thresholds were observed in all the three models of neuropathic pain with Von Frey monofilaments, whereas variable paw withdrawal thresholds were noticed in PNL and CCI models but not in SNL model with dynamic plantar aesthesiometer. DiscussionManually used Von Frey filaments can be used in assessment of mechanical allodynia in all the three models, whereas dynamic plantar aesthesiometer is suitable for assessing mechanical allodynia in SNL but not in PNL and CCI models. The reason for variable paw withdrawal thresholds during assessment of mechanical allodynia in PNL and CCI models with dynamic plantar aesthesiometer may be due to the paw deformity and change in foot posture.

Effect of dipyrone and thalidomide alone and in combination on STZ-induced diabetic neuropathic pain

Diabetic neuropathy is recognized as one of the most common complications of chronic diabetes, but its pathophysiological mechanism is complex and yet to be completely explored. Monotherapy with conventional analgesics fails to provide adequate pain relief in peripheral diabetic neuropathy. There are a number of evidence suggesting that tumor necrosis factor (TNF-α) plays an important role in the pathogenesis of peripheral diabetic neuropathy. TNF-α up-regulation activates nuclear factor κB, which further up-regulates cyclooxygenase (COX)-2 leading to altered prostaglandin profile. Inhibition of TNF-α and COX-2 provides beneficial effect on diabetic neuropathy by decreasing the oxidative stress level and by preventing neuronal hypersensitivity due to an increased prostaglandin level. The present study was designed to assess the effect of dipyrone and thalidomide on streptozotocin (STZ)-induced neuropathic pain behavior in rats. STZ 50mg/kg, i.p. was administered to induce experimental diabetes in the rats. Three weeks following STZ, dipyrone (300 and 600mg/kg, i.p.) and thalidomide (25 and 50mg/kg, i.p.) alone and subeffective dose combination of dipyrone and thalidomide (300 and 25mg/kg(-1), i.p.) administered daily for 2weeks significantly attenuated thermal hyperalgesia, mechanical allodynia, and formalin-induced phase-2 flinching response. Moreover, the subeffective dose combination of dipyrone and thalidomide and preemptive treatment with thalidomide (50mg/kg) reduces oxidative stress in diabetic rats. In conclusion, the combination of subeffective dose of dipyrone and thalidomide prevented the development and maintenance of experimental diabetic neuropathy. The combination of thalidomide (TNF-α inhibitor) and dipyrone (COX inhibitor) may be used as a potential therapeutic agent for the treatment of diabetic neuropathy.

Chronic neuropathic pain-like behavior correlates with IL-1β expression and disrupts cytokine interactions in the hippocampus

We have proposed that neuropathic pain engages emotional learning, suggesting the involvement of the hippocampus. Because cytokines in the periphery contribute to induction and maintenance of neuropathic pain but might also participate centrally, we used 2 neuropathic pain models, chronic constriction injury (CCI) and spared nerve injury (SNI), to investigate the temporal profile of hippocampal cytokine gene expression in 2 rat strains that show different postinjury behavioral threshold sensitivities. SNI induced long-lasting allodynia in both strains, while CCI induced allodynia with time-dependent recovery in Sprague Dawley (SD) and no allodynia in Wistar Kyoto (WK) rats. In WK rats, only SNI induced sustained upregulation of hippocampal interleukin (IL)-1β, while IL-6 expression was transiently increased and no significant changes in IL-1ra expression were detected. Conversely, in SD rats, SNI resulted in sustained and robust increased hippocampal IL-1β expression, which was only transient in rats with CCI. In this strain, IL-6 expression was not affected in any of the 2 injury models and IL-1ra expression was significantly increased in rats with SNI or CCI at late phases. We found that the degree and development of neuropathic pain depend on the specific nerve injury model and rat strain; that hippocampal IL-1β mRNA levels correlate with neuropathic pain behavior; that, in contrast to sham-operated animals, there are no correlations between hippocampal IL-1β and IL-1ra or IL-6 in neuropathic rats; and that alterations in cytokine expression are restricted to the hippocampus contralateral to the injury side, again implying that the observed changes reflect nociception.

Decoy strategy targeting the brain-derived neurotrophic factor exon I to attenuate tactile allodynia in the neuropathic pain model of rats

The mechanism underlying neuropathic pain is still largely unclear. Recently, much attention has been focused on the role of brain-derived neurotrophic factor (BDNF) as a neuromodulator in the spinal cord. We previously reported that the expression of Bdnf exon I mRNA was remarkably up-regulated in the dorsal root ganglion (DRG) neurons with the rat L5 spinal nerve ligation (SNL) model. In the present study, we investigated whether neuropathic pain response would be reduced by the inhibition of the Bdnf exon I in the rat SNL model. We identified the promoter region of exon I and synthesized the decoy ODNs targeting the region. Reverse transcription-polymerase chain reaction analysis confirmed that the decoy ODN treatment reduced SNL-induced Bdnf exon I mRNA up-regulation in ipsilateral L4 and L5 DRGs. Furthermore, post-treatment with the decoy ODNs significantly attenuated SNL-induced tactile allodynia. This study suggested that decoy ODNs targeting the Bdnf exon I might provide a novel analgesic strategy for the treatment of neuropathic pain.

Intrathecally administered Sema3A protein attenuates neuropathic pain behavior in rats with chronic constriction injury of the sciatic nerve

Semaphorins, one of the repulsive axonal guidance factors during development, are produced under pathological conditions in adult animals. In the neuropathic pain state associated with peripheral nerve injury, synaptic reorganization occurs in spinal cord dorsal horn. In the present study, we investigated the roles of intrathecal administration of Sema3A, a secreted semaphorin, in the spinal cord of chronic constriction injury (CCI) model rat. Neuropilin 1 (NPR1) and Plexin A (PlexA), co-receptors of Sema3A, were expressed in the dorsal horn of naïve rats. NPR1, and not PlexA, protein expression increased in the dorsal spinal cord of CCI rats. Recombinant Sema3A protein attenuated mechanical allodynia and heat hyperalgesia in CCI rats, whereas heat-inactivated Sema3A had no effect. Immunohistochemistry revealed that Sema3A partially restored the decrease of isolectin B4-positive unmyelinated nerve terminals in lamina II of the ipsilateral dorsal horn of CCI rats. Contrary to our expectations, Sema3A did not change the distribution of myelinated fibers in lamina II at 7 days after CCI. Those results suggested that the suppressive role for Sema3A in the development of neuropathic pain associated with peripheral nerve injury in adult rats, which seemed to be independent from prevention of the myelinated fiber sprouting into lamina II.

Anti-hypersensitivity effects of Shu-jing-huo-xue-tang, a Chinese herbal medicine, in CCI-neuropathic rats

Aim of the study Shu-jing-huo-xue-tang (SJHXT) (Japanese name: Sokei-kakketu-to), a traditional Chinese herbal medicine composed of 17 crude drugs, has been prescribed over hundreds of years for treatment of chronic pain syndromes. We evaluated if oral SJHXT could suppress neuropathic pain behaviors in rats with chronic constriction injury (CCI) of the sciatic nerve. Materials and methods (1) Rats received repeated oral SJHXT 0.5 or 1.0 g/kg once daily for 14 days starting 24 h after CCI surgery, while neuropathic manifestations were evaluated until day 20 post-CCI. (2) Other groups of rats received single oral SJHXT 1.0 g/kg on day 14 post-CCI. (3) Additional groups of rats received oral SJHXT 1.0 g/kg on day 14 post-CCI, concomitantly with intraperitoneal yohimbine 1 mg/kg or methysergide 5 mg/kg. Neuropathic manifestations, including mechanical allodynia and thermal hyperalgesia, were evaluated with paw withdrawal responses to increasing mechanical pressure and radiant heat, respectively. Results Mechanical allodynia and thermal hyperalgesia developed by day 14 post-CCI. Repeated oral SJHXT for 14 days produced anti-allodynic and anti-hyperalgesic effects that outlasted the period of drug administration. Single oral SJHXT on day 14 also produced significant anti-allodynic and anti-hyperalgesic effects, which were inhibited by yohimbine, an alpha-2 adrenoceptor antagonist, but not by methysergide, a serotonin receptor antagonist. Conclusions Oral SJHXT produced anti-hypersensitivity effects by actions on alpha-2 adrenoreceptors in CCI-neuropathic rats, and chronic oral administration of SJHXT could produce the long-lasting anti-hypersensitivity effects.

An electrophysiological method for quantifying neuropathic pain behaviors in rats: measurement of hindlimb withdrawal EMG magnitude

In behavior methods to quantify neuropathic pain, visual observations of limb-withdrawal reflexes to stimuli are not always clear-cut, so this method is partly subjective. Our current data suggest that measurement of electrophysiological EMG magnitudes enables more reliable and objective assessment for quantifying nocifensive behaviors related to neuropathic pain.

A combined effect of dextromethorphan and melatonin on neuropathic pain behavior in rats

Previous study has shown that administration of melatonin into the anterior cingulate cortex contralateral to peripheral nerve injury prevented exacerbation of mechanical allodynia with a concurrent improvement of depression-like behavior in Wistar–Kyoto (WKY) rats, a genetic variation of Wistar rats. In the present study, we examined the effect of the individual versus combined treatment of melatonin and/or dextromethorphan (DM), a clinically available N-methyl- d-aspartate (NMDA) receptor antagonist, on pain behaviors in WKY rats with chronic constriction sciatic nerve injury (CCI). Pain behaviors (thermal hyperalgesia and mechanical allodynia) were established at one week after CCI. WKY rats were then treated intraperitoneally with various doses of melatonin, DM or their combination once daily for the following week. At the end of this one-week treatment, behavioral tests were repeated in these same rats. While DM alone was effective in reducing thermal hyperalgesia at three tested doses (15, 30 or 60 mg/kg), it reduced mechanical allodynia only at high doses (30 or 60 mg/kg). By comparison, administration of melatonin alone was effective in reducing thermal hyperalgesia only at the highest dose (120 mg/kg, but not 30 or 60 mg/kg) tested in this experiment. Melatonin alone failed to reverse allodynia at all three tested doses (30, 60 and 120 mg/kg). However, the combined intraperitoneal administration of melatonin (30 mg/kg) and DM (15 mg/kg) effectively reversed both thermal hyperalgesia and mechanical allodynia although each individual dose alone did not reduce pain behaviors. These results suggest that a combination of melatonin with a clinically available NMDA receptor antagonist might be more effective than either drug alone for the treatment of neuropathic pain.

Spinal leptin contributes to the pathogenesis of neuropathic pain in rodents

Pain after nerve injury, a phenomenon referred to as neuropathic pain, is a debilitating clinical condition, but the underlying mechanisms remain unclear. As leptin, an adipocytokine produced mainly by nonneuronal tissue, has been implicated in the regulation of neuronal functions, we examined the role of leptin in neuropathic pain using a rat model of the condition chronic constriction sciatic nerve injury (CCI). We report that leptin critically contributed to pain behaviors following CCI. Specifically, spinal administration of a leptin antagonist prevented and reversed neuropathic pain behaviors in rats. Further examination revealed that levels of both leptin and the long form of the leptin receptor (Ob-Rb) were substantially increased within the ipsilateral spinal cord dorsal horn after peripheral nerve injury. Mechanistic studies showed that leptin upregulated the expression of both the spinal NMDA receptor and IL-1beta through the JAK/STAT pathway. Furthermore, these CCI-induced behavioral and cellular responses were diminished in leptin-deficient mice and mimicked by spinal administration of exogenous leptin in naive rats. Our findings reveal a critical role for spinal leptin in the pathogenesis of neuropathic pain and suggest what we believe to be a novel form of nonneuronal and neuronal interactions in the mechanisms of pathological pain.

Low-dose methotrexate reduces peripheral nerve injury-evoked spinal microglial activation and neuropathic pain behavior in rats

Peripheral nerve injuries that provoke neuropathic pain are associated with microglial activation in the spinal cord. We have investigated the characteristics of spinal microglial activation in three distinct models of peripheral neuropathic pain in the rat: spared nerve injury (SNI), chronic constriction injury, and spinal nerve ligation. In all models, dense clusters of cells immunoreactive for the microglial marker CD11b formed in the ipsilateral dorsal horn 7 days after injury. Microglial expression of ionised calcium binding adapter molecule 1 (Iba1) increased by up to 40% and phosphorylation of p38 mitogen-activated protein kinase, a marker of microglial activity, by 45%. Expression of the lysosomal ED1-antigen indicated phagocytic activity of the cells. Unlike the peripheral nerve lesions, rhizotomy produced only a weak microglial reaction within the spinal gray matter but a strong activation of microglia and phagocytes in the dorsal funiculus at lumbar and thoracic spinal cord levels. This suggests that although degeneration of central terminals is sufficient to elicit microglial activation, it does not account for the inflammatory response in the dorsal horn after peripheral nerve injury. Early intrathecal treatment with low-dose methotrexate, beginning at the time of injury, decreased microglial activation, reduced p38 phosphorylation, and attenuated pain-like behavior after SNI. In contrast, systemic or intrathecal delivery of the glucocorticoid dexamethasone did not inhibit the activation of microglia or reduce pain-like behavior. We confirm that microglial activation is crucial for the development of pain after nerve injury, and demonstrates that suppression of this cellular immune response is a promising approach for preventing neuropathic pain.