Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24–48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0–48 h) (r = -0.974, p = 0.001), but not late stage (3–28 days) (r = 0.3395, p = 0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24–48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24–48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3–14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0–48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2–14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.     

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.     

脊神经结扎神经痛大鼠脊髓背角广动力范围神经元的电生理学特性(英文)

目的探讨腰5脊神经结扎(spinal nerve ligation, SNL)后,大鼠脊髓背角的广动力范围(wide dynamic range,WDR)神经元电生理学特性的改变。方法将健康雄性 Sprague-Dawley 大鼠分为正常组和 SNL 组,利用细胞外电生理学方法记录脊髓背角的 WDR 神经元放电。结果与正常大鼠相比,SNL 组大鼠 WDR 神经元兴奋性增加,表现为感受野扩大、有自发放电的神经元比例增加,以及 C 纤维诱发放电的阈值降低、潜伏期缩短、发放时程增加。此外,SNL组大鼠WDR神经元A和C纤维诱发放电数目较正常大鼠降低。结论大鼠腰5脊神经结扎后主要引起WDR神经元的兴奋性增加。WDR神经元的兴奋性增加可能参与神经病理痛的发生。     

Spinal cord stimulation inhibits long-term potentiation of spinal wide dynamic range neurons

It has been suggested that long-term potentiation (LTP) of dorsal horn neurons is a phenomenon that contributes to the development of chronic neuropathic pain. Spinal cord stimulation (SCS) may be an effective tool in alleviating such pain. The aim of this electrophysiological study in rats was to examine if SCS suppresses LTP of dorsal horn wide dynamic range (WDR) neurons. Increased knowledge of the mechanisms behind the effects of SCS may facilitate its further advancement and improve clinical efficacy. As previously shown, intensive, high-frequency electrical stimulation of the sciatic nerve in the rat induces an increased firing response of WDR neurons. Here we report that SCS gradually reduced this increased C-fiber response back to the baseline level. However, A-fiber responses were neither potentiated by the conditioning stimulus used nor were they affected by SCS. These data suggest that SCS affects the C-fiber component of dorsal horn central sensitization which is noteworthy since SCS, based on previous studies, is believed to primarily influence A-fiber functions.     

Spinal AMPA receptor inhibition attenuates mechanical allodynia and neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether a competitive AMPA receptor antagonist, NBQX, attenuates mechanical allodynia and hyperexcitability of spinal neurons in remote, caudal regions in persistent central neuropathic pain following spinal cord injury in rats. Spinal cord injury was produced by unilateral T13 transverse spinal hemisection, from dorsal to ventral, in male Sprague Dawley rats (200-250 g). Mechanical thresholds were measured behaviorally, and the excitability of wide-dynamic-range (WDR) dorsal horn neurons in the lumbar cord (L4-L5) was measured to assess central neuropathicpain. On postoperation day (POD) 28 after spinalhemisection, mechanical thresholds were significantly decreased in both injured (ipsilateral) and noninjured (contralateral) hindpaws compared with preinjury and sham control, respectively (P < 0.05). Intrathecal administration of NBQX (0.25, 0.5, 1 mM) significantly reversed the decreased mechanical thresholds in both hindpaws, dose dependently (P < 0.05). The excitability of WDR neurons was significantly enhanced on both sides of the lumbar dorsal horn 28 days following spinal hemisection (P < 0.05). The hyperexcitability of WDR neurons was attenuated by topical administration of NBQX (0.125, 0.25, 0.5, 1 mM), dose dependently (P < 0.05). Regression analysis indicated that at least three molecules of NBQX bond per receptor complex, and are needed to achieve inhibition of WDR hyperexcitability. In conclusion, our study demonstrates that the AMPA receptor plays an important role in behaviors related to the maintenance of central neuropathic pain below the level of spinal cord injury.     

Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.     

Glutamate uptake is attenuated in spinal deep dorsal and ventral horn in the rat spinal nerve ligation model

Alteration of glutamatergic (GLU) neurotransmission within the spinal cord contributes to hyperalgesic and allodynic responses following nerve injury. In particular, changes in expression and efficacy of glutamate transporters have been reported. Excitatory, pain transmitting primary afferent neurons utilizing glutamate as an excitatory neurotransmitter project to both superficial (I-II) and deep (III-V) laminae of the dorsal horn. These experiments were designed to examine changes in glutamate uptake occurring concomitantly within the spinal deep dorsal and ventral horn in situ after experimentally induced neuropathic pain. In vivo voltammetry, using microelectrode arrays configured for enzyme-based detection of GLU were employed. Sprague-Dawley rats had either sham surgery or tight ligation of L5 and L6 spinal nerves (SNL). Four to six weeks later, the L4-L6 spinal cord of chloral hydrate-anesthetized animals was exposed, and ceramic-based glutamate microelectrodes equipped with glass micropipettes 50 microm from the recording surfaces were placed stereotaxically at sites within the spinal cord. Pressure ejection of GLU into the ipsilateral L5-L6 spinal cord resulted in a 72% reduction of GLU uptake in SNL rats compared to sham controls in the ipsilateral L5-L6 deep dorsal horn and a 96% reduction in the ventral horn. In contrast, in the same animals, the contralateral L5-L6 or the ipsilateral L4 spinal cord showed no change in glutamate uptake. The data suggest that spinal nerve ligation produced attenuated glutamate uptake activity extending into the deep dorsal and ventral horn. The study suggests that plasticity related to spinal nerve injury produces widespread alteration in glutamate transporter function that may contribute to the pathophysiology of neuropathic pain.     

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.     

KDM6B epigenetically regulated-interleukin-6 expression in the dorsal root ganglia and spinal dorsal horn contributes to the development and maintenance of neuropathic pain following peripheral nerve injury in male rats

The lysine specific demethylase 6B (KDM6B) has been implicated as a coregulator in the expression of proinflammatory mediators, and in the pathogenesis of inflammatory and arthritic pain. However, the role of KDM6B in neuropathic pain has yet to be studied. In the current study, the neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rats. Immunohistochemistry, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR assays were performed to investigate the underlying mechanisms. Our results showed that SNL led to a significant increase in KDM6B mRNA and protein in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn; and this increase correlated a markedly reduction in the level of H3K27me3 methylation in the same tissue. Double immunofluorescence staining revealed that the KDM6B expressed in myelinated A- and unmyelinated C-fibers in the DRG; and located in neuronal cells, astrocytes, and microglia in the dorsal horn. Behavioral data showed that SNL-induced mechanical allodynia and thermal hyperalgesia were impaired by the treatment of prior to i.t. injection of GSK-J4, a specific inhibitor of KDM6B, or KDM6B siRNA. Both microinjection of AAV2-EGFP-KDM6B shRNA in the lumbar 5 dorsal horn and sciatic nerve, separately, alleviated the neuropathic pain following SNL. The established neuropathic pain was also partially attenuated by repeat i.t. injections of GSK-J4 or KDM6B siRNA, started on day 7 after SNL. SNL also resulted in a remarkable increased expression of interleukin-6 (IL-6) in the DRG and dorsal horn. But this increase was dramatically inhibited by i.t. injection of GSK-J4 and KDM6B siRNA; and suppressed by prior to microinjection of AAV2-EGFP-KDM6B shRNA in the dorsal horn and sciatic nerve. Results of ChIP-PCR assay showed that SNL-induced enhanced binding of STAT3 with IL-6 promoter was inhibited by prior to i.t. injection of GSK-J4. Meanwhile, the level of H3K27me3 methylation was also decreased by the treatment. Together, our results indicate that SNL-induced upregulation of KDM6B via demethylating H3K27me3 facilitates the binding of STAT3 with IL-6 promoter, and subsequently mediated-increase in the expression of IL-6 in the DRG and dorsal horn contributes to the development and maintenance of neuropathic pain. Targeting KDM6B might a promising therapeutic strategy to treatment of chronic pain.     

Spinal cord injury triggers sensitization of wide dynamic range dorsal horn neurons in segments rostral to the injury

A spinal cord injury (SCI) was produced in adult rats by complete spinal cord transection at L6-S1. Neuropathic pain behaviors similar to the chronic central pain (CCP) syndrome in human, such as thermal hyperalgesia, mechanical allodynia and autotomy, were present in these rats after spinal cord injury. Meanwhile, wide dynamic range (WDR) neurons recorded in the spinal dorsal horn rostral to the lesion responded as high frequency of spontaneous activities, long duration of after-discharges to noxious electrical stimuli and an augmented wind-up to 0.5 Hz stimuli. By using bupivacaine powder, a sodium channel blocker, at the locus of transection immediate after nerve injury, the chronic pain behaviors were prevented; the hyperexcitability of WDR neurons was also substantially reduced. It is suggested that spinal cord transection induces the CCP syndromes, which may be evoked and maintained by the hyperexcitability in WDR neurons rostrally. Reducing the neuronal activity at the site of lesion following injury may prevent the development of CCP after SCI.     

The Cav2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch-evoked pain). Evidence exists for nerve damage-induced plasticity in both transmission and modulatory systems, including changes in voltage-dependent calcium channel (VDCC) expression and function; however, the role of Ca(v)2.3 calcium channels has not clearly been defined. Here, the effects of SNX-482, a selective Ca(v)2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [Kim & Chung, (1992)Pain, 50, 355-363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham-operated animals. Spinal SNX-482 (0.5-4 microg/50 microL) exerted dose-related inhibitions of noxious C-fibre- and Adelta-fibre-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX-482. In contrast, non-noxious Abeta-mediated responses were not affected by SNX-482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX-482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX-482-sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in Ca(V)2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.     

The direction of synaptic plasticity mediated by C-fibers in spinal dorsal horn is decided by Src-family kinases in microglia: The role of tumor necrosis factor-α

Previous studies have shown that Src-family kinases (SFKs) are selectively activated in spinal microglia following peripheral nerve injury and the activated SFKs play a key role for the development of neuropathic pain. To investigate the underlying mechanism, in the present study the effect of SFKs on long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, which is believed as central mechanism of neuropathic pain, was investigated in adult rats. Electrophysiological data revealed that pretreatment with either microglia inhibitor (minocycline, 200 μM) or SFKs inhibitors (PP2, 100 μM and SU6656, 200 μM) reversed the effect of high frequency stimulation (HFS), that is, HFS, which induces long-term potentiation (LTP) normally, induced long-term depression (LTD) after inhibition of either microglia or SFKs. Western blotting analysis showed that the level of phosphorylated SFKs (p-SFKs) in ipsilateral spinal dorsal horn was transiently increased after LTP induced by HFS, starting at 15 min and returning to control level at 60 min after HFS. Double-labeled immunofluorescence staining demonstrated that p-SFKs were highly restricted to microglia. Furthermore, we found that the inhibitory effects of minocycline or SU6656 on spinal LTP were reversed by spinal application of rat recombinant tumor necrosis factor-α (TNF-α 0.5 ng/ml, 200 μl). HFS failed to induce LTP of C-fiber evoked field potentials in TNF receptor-1 knockout mice and in rats pretreated with TNF-α neutralization antibody (0.6 μg/ml, 200 μl). The results suggested that in spinal dorsal horn activation of SFKs in microglia might control the direction of plastic changes at C-fiber synapses and TNF-α might be involved in the process.     

Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naïve rats and in rat models of inflammatory and neuropathic pain

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH-133 (5 and 15 microg/50 microL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan-induced hindpaw inflammation, sham operated rats and spinal nerve-ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6-80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan-inflamed, sham-operated and SNL rats. Intraplantar injection of JWH-133 (15 microg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 microg/50 microL), attenuated the inhibitory effects of JWH-133 (15 microg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH-133 on these responses. Intraplantar preadministration of JWH-133 also inhibited (P < 0.05) carrageenan-induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous- and noxious-evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.     

Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472

Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a key mediator of long‐term potentiation (LTP), which can be triggered by N‐methyl‐d ‐aspartate (NMDA) receptor‐mediated Ca²⁺ influx. We previously demonstrated that Fyn kinase‐mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F‐KI mice with a knock‐in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F‐KI mice among the major phosphorylation sites. There was no difference in the Ca²⁺ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild‐type and Y1472F‐KI mice, and the Ca²⁺ response to glutamate was attenuated in the Y1472F‐KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F‐KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase Cγ at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild‐type and Y1472F‐KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor‐mediated CaMKII signaling in Y1472F‐KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.     

Induction of long-term potentiation in single nociceptive dorsal horn neurons is blocked by the CaMKII inhibitor AIP

Neuronal events leading to development of long-term potentiation (LTP) in the nociceptive pathways may be a cellular mechanism underlying central hyperalgesia. Here, we examine whether induction of LTP in nociceptive dorsal horn neurons at depths of 80-500 microm from the cord surface can be affected by spinal application of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIP. Extracellular recordings from single neurons in intact urethane anesthetized Sprague-Dawley rats were performed, and the neuronal A-fiber and C-fiber responses after sciatic nerve test pulses were defined according to latencies. A clear LTP of the nociceptive transmission following sciatic nerve high-frequency stimulation (HFS) was observed in single neurons in laminae I-IV of the dorsal horn. The increase in the C-fiber response after HFS was blocked in the presence of 2.0 mM AIP (P < 0.05 HFS group versus AIP + HFS group 2 h after conditioning). However, the C-fiber response was not affected by 2.0 mM AIP alone or by vehicle. Thus, our data show that the neuronal process leading to the induction of LTP in the dorsal horn induced by HFS is clearly inhibited by the specific CaMKII inhibitor AIP. It is concluded that CaMKII may be important for the induction of LTP in single nociceptive dorsal horn neurons.     

Spinal interleukin-6 (IL-6) inhibits nociceptive transmission following neuropathy

Interleukin-6 (IL-6) is a neuropoietic cytokine which is dramatically upregulated following peripheral nerve injury at the site of injury, in the dorsal root ganglion (DRG) and in the spinal cord. The functional effects of IL-6 in nociception in normal conditions and following nerve injury are unclear. Thus the aim of this study was to assess the effect of spinal IL-6 administration on nociceptive transmission in naive, sham-operated and neuropathic (spinal nerve ligation, SNL) rats using in vivo electrophysiology to elucidate the possible role of IL-6 in neuropathic pain. In anaesthetised rats, extracellular recordings were made from individual convergent dorsal horn neurones following electrical and natural (mechanical and thermal) stimulation of peripheral receptive fields. Exogenous spinal IL-6 (100-500 ng) had no significant effect on electrically evoked neuronal responses in naive rats. In contrast, following neuropathy, spinal IL-6 produced a dose-related inhibition of the electrically evoked C-fibre, initial C-fibre and measures of neuronal hyperexcitability (post discharge and wind-up). In addition, spinal IL-6 markedly inhibited mechanical neuronal responses in neuropathic rats. Higher doses of spinal IL-6 also inhibited, to a lesser degree, the initial C-fibre, post discharge and wind-up responses in sham-operated rats. These studies show that following nerve injury the actions of the cytokine alter so that spinal administration of IL-6 elicits anti-nociceptive effects not observed under normal conditions. Moreover, the inhibitory effects of IL-6 on C-fibre activity and neuronal hyperexcitability, suggest IL-6 to be a potential modulator of neuropathic pain.     

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.     

Spinal nerve ligation increases alpha2-adrenergic receptor G-protein coupling in the spinal cord

Intrathecal and epidural administration of the alpha2-adrenergic receptor agonist clonidine in humans results in analgesia to both acute nociceptive and chronic neuropathic pain. The potency of clonidine increases with hypersensitivity to mechanical stimuli after nerve injury, although the reasons for this change are unknown. In the present study, we tested the hypothesis that peripheral nerve injury alters either spinal alpha2-adrenergic receptor-mediated G-protein activity or alpha2-adrenergic receptor number. Rats were randomized to left spinal nerve ligation (SNL) or sham surgery. Tactile hypersensitivity in the hindpaw was confirmed and lumbar spinal cords were removed for binding assays. To examine agonist-induced G-protein coupling, [35S]GTP gamma S binding experiments were performed in spinal cord membranes and sections using norepinephrine as an alpha2-adrenergic agonist. SNL was associated with an increase in maximal efficacy, but not potency, of norepinephrine-stimulated [35S]GTP gamma S binding in dorsal horn. SNL had no effect on basal [35S]GTP gamma S binding or on muscarinic cholinergic-stimulated [35S]GTP gamma S binding. [35S]GTP gamma S autoradiography showed that this increase in alpha2-adrenergic-activated G-proteins occurred both ipsilateral and contralateral to SNL surgery. SNL did not alter total alpha2-adrenergic receptor number or affinity to [3H]-rauwolscine binding, and displacement studies with the alpha2A-adrenergic antagonist BRL44408 revealed that most of the binding was associated with the alpha2A-adrenergic subtype. These data suggest that the increased potency of clonidine in neuropathic pain could reflect increased efficiency of G-protein coupling from spinal alpha2-adrenergic receptors.     

Ionotropic glutamate receptors contribute to maintained neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 µg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.5 and 1 µg) significantly attenuated neuronal activity in all three phenotypes of neurons (*p < 0.05). However, MCPG (group I/II metabotropic glutamate receptor antagonist, 100 µg) had no effect. The present study, in the context of previous work, suggests that ionotropic glutamate receptor activation play critical roles in the maintenance of neuronal hyperexcitability and neuropathic “below-level” pain behavior following spinal hemisection injury.