Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

Hippocampal synaptic metaplasticity requires the activation of NR2B-containing NMDA receptors

The potential to exhibit synaptic plasticity itself is modulated by previous synaptic activity, which has been termed as metaplasticity. In this paper, we demonstrated that the activation of N-methyl-d-aspartate (NMDA) receptor 2B (NR2B) subunit in NNDA receptors was required for hippocampal metaplasticity at Schaffer collateral-commissural fiber-CA1 synapses. Brief 5 Hz priming stimulation did not cause long-term synaptic plasticity; however, it could result in the inhibition of subsequently evoked long-term potentiation (LTP). Meanwhile, the application of selective antagonists for NR2B subunit of NMDA receptors after delivering priming stimulation could block the metaplasticity. In contrast, LTP induction was not affected by NR2B antagonists in slices without pre-treatment of priming stimulation. These results indicated that the activation of NR2B-containing NMDA receptors was required for metaplasticity.     

CaMKIIalpha enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation-dependent mechanism

Long-term potentiation or depression of synaptic function often requires Ca2+ influx via NMDA-type glutamate receptors (NMDARs) and changes in the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286. Autophosphorylated CaMKII binds directly to NMDAR subunits, co-localizes with NMDARs in the postsynaptic density, and phosphorylates NR2B subunits at Ser1303. Here, we demonstrate that CaMKIIalpha enhances the extent and/or rate of desensitization of NMDA-induced macroscopic currents in HEK293 cells co-expressing NR2B with either the NR1(011) or NR1(101) splice variants, without significantly changing other current parameters. In contrast, the extent of desensitization of NMDARs containing NR2A in place of NR2B is significantly decreased by co-expression of CaMKIIalpha. Kinases harboring K42R (inactive kinase) or T286A (autophosphorylation-deficient) mutations are defective in enhancing the desensitization of NR1/NR2B channels. In addition, the CaMKII-dependent enhancement of NR1/NR2B channel desensitization is abrogated by intracellular loading with BAPTA. These data suggest a novel mechanism for Ca2+-dependent negative-feedback regulation of NR2B-containing NMDARs in a CaMKII activity- and autophosphorylation-dependent manner that may modulate NMDAR-mediated synaptic plasticity.     

Re-expression of NR2B-containing NMDA receptors in vitro by suppression of neuronal activity

N-methyl-D-aspartate receptors (NMDARs) are known to play critical roles in the development of the nervous system, and their expression is regulated in an activity-dependent fashion during development. However, the regulation of NMDAR expression after circuit formation is less well understood. To examine this, we performed patch-clamp recordings from chick cerebral neurons in an activity-controlled culture. Analysis of NMDAR channels from neurons before synapse formation showed that there are two components in channel open kinetics. The major slow component is clearly blocked by ifenprodil, a specific inhibitor of NR2B-containing NMDARs. In contrast, slow component of NMDAR channel opening from neurons after synapse formation became minor and ifenprodil had little effect on the NMDAR channel openings. Furthermore, this change is reversibly regulated by neuronal activity, in that suppression induces the re-expression of NR2B-containing NMDARs, even after circuit formation.     

NR2B-containing NMDA receptors are up-regulated in temporal cortex in schizophrenia

Saturation analyses of [3H]L-689,560, [3H]CGP 39653 and NMDA-specific [3H]ifenprodil binding revealed an equivalent increase (0.7 pmol/mg) in the number of [3H]L-689,560 and [3H]ifenprodil binding sites in superior temporal cortex (BA22) from drug-treated chronic schizophrenic patients and control subjects. No differences were observed between control and schizophrenic subjects for [3H]CGP 39653 binding in BA22, or for any of the radioligands binding to pre-motor cortex (BA6). Since [3H]L-689,560, [3H]CGP 39653 and [3H]ifenprodil label the glycine, glutamate and ifenprodil sites of the NMDA receptor complex, which are associated with NR1, NR1/NR2A and NR1/NR2B subunits respectively, our findings suggest that NR2B-containing receptors are selectively up-regulated in superior temporal cortex in schizophrenia.     

单一腰6脊神经根结扎延长大鼠神经病理性疼痛发展期的研究

目的：腰5和腰6脊神经根共同结扎是目前最常见的神经病理性疼痛鼠模型,但结扎后大鼠的机械触诱发痛会在24～72 h内迅速发展,时间窗过短,本课题组探索和研究了一种新型的神经病理性疼痛大鼠模型—单一腰6脊神经根结扎鼠模型。方法将36只大鼠随机分为空白对照组、腰5和腰6脊神经根结扎组、单一腰6脊神经根结扎组及假手术组,除单一腰6脊神经根结扎组18只外,其余3组每组6只大鼠;分别检测其50％机械缩爪阈值（50％PWT）。另取54只大鼠,随机分为空白对照组、腰5和腰6脊神经根结扎组、腰6脊神经根结扎组,各组大鼠只数分别为6只、24只、24只。采用蛋白免疫印迹法检测脊髓背角胶质纤丝酸性蛋白（ GFAP）在结扎术后第1 d、第7 d、第14 d和第28 d的表达。结果单一腰6脊神经根结扎组产生了长期发展的机械触诱发痛（1～14 d）;且大鼠同侧脊髓背角GFAP表达随之升高。结论单一腰6脊神经根结扎鼠模型可以作为研究神经病理性疼痛发展期具体机制的有效模型。     

Involvement of spinal cord BDNF in the generation and maintenance of chronic neuropathic pain in rats

Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system. In chronic pain, plastic changes in dorsal horn neurons contribute to a phenomenon of hypersensitivity to pain sensation that is maintained over time, known as central sensitization. This process is accompanied by BDNF overexpression, but the role of BDNF in the generation and maintenance of the hyperalgesic phenomenon is still unclear. The present study was aimed to investigate if exogenous BDNF administered to the rat spinal cord, in addition to trigger pain, participates in the maintenance of the central sensitization process (i.e., pain persistence) and to determine if the pain generated is comparable to that observed in a neuropathic pain model. Results showed that a single intrathecal injection of 0.003 ng of BDNF was able to decrease the nociceptive threshold (Randall-Selitto test) in normal rats, for at least a 42-day period. Furthermore, the hyperalgesia generated was comparable to that observed in rats with a 42-day history of mononeuropathy. Increasing the dose or administering additional doses of BDNF resulted neither in additional effectiveness in reducing the pain threshold nor in the prolongation of the hyperalgesic effect, thus showing that central sensitization induced by BDNF is a dose-independent, all-or-none process. It is concluded that BDNF alone is sufficient for generating a long-lasting neural excitability change in the spinal cord via tyrosine kinase B receptor signaling, similar to that observed in chronic pain models such as neuropathy.     

BDNF released during neuropathic pain potentiates NMDA receptors in primary afferent terminals

NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock‐down of NMDA receptors in primary afferents decreased NMDA‐induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin‐related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75^NTR), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA‐12 but not by the p75^NTR inhibitor TAT‐Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full‐length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr¹⁴⁷² phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch‐clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA‐induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain.     

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。     

Neuroprotective effect of estrogen: Role of nonsynaptic NR2B-containing NMDA receptors

Excessive activation of N-methyl-d-aspartate receptors (NMDARs) has been implicated in the pathophysiology of chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Some studies reported that NR2A and NR2B play different roles in the central nervous system (CNS). The NR2A subunit is primarily found in the synapses and is required for glutamate-mediated neuronal survival. On the other hand, the NR2B subunit is primarily found in the extrasynaptic sites and is required for glutamate-mediated neuronal death in both in vitro and in vivo experiments. Estrogen is a steroid hormone well known for its widespread effects such as neuroprotection in the brain. Classically, estrogen can bind to two kinds of nuclear receptors, namely, estrogen receptor α (ERα) and estrogen receptor β (ERβ), and produce physiological and neuroprotective effects. Aside from nuclear receptors, estrogen has one membrane receptor, which can either be G-protein-coupled receptor 30 (GPR30), Gq-mER, or ER-X. NMDA exposure clearly promotes NR2B subunit phosphorylation at Ser-1303 and causes neuronal cell death. GPR30 mediates rapid non-genomic effects to protect neurons against injury by inhibiting p-DAPK1 dephosphorylation, which inhibits NR2B subunit phosphorylation at Ser-1303. In addition, NMDA exposure and global ischemia activate the autophagy pathway and induce cell death, which are markedly blocked by the NR2B antagonist Ro 25-6981. Thus, NR2B signaling, autophagy induction and cell death may be closely related. Ro 25-6981 inhibits the dissociation of the NR2B-Beclin-1 signaling complex and delays autophagy in vivo, thus confirming the link between NR2B signaling and autophagy. In short, ERα, ERβ, and GPR30 are involved in the neuroprotection of estrogen in the CNS. Additional research must be conducted to reveal the mechanism of estrogen action fully and to identify better targets for the development of more effective drugs.This article is part of a Special Issue entitled ‘Extrasynaptic ionotropic receptors’.     

Leukotriene synthases and the receptors induced by peripheral nerve injury in the spinal cord contribute to the generation of neuropathic pain

Leukotrienes (LTs) belong to a large family of lipid mediators, termed eicosanoids, which are derived from arachidonic acids and released from the cell membrane by phospholipases. LTs are involved in the pathogenesis of inflammatory diseases, such as asthma, rheumatoid arthritis, and peripheral inflammatory pain. In the present study, we examined whether LTs were implicated in pathomechanism of neuropathic pain following peripheral nerve injury. Using the spared nerve injury (SNI) model in rats, we investigated the expression of LT synthases (5‐lipoxygenase; 5‐LO, Five lipoxygenase activating protein; FLAP, LTA4 hydrolase; LTA4h and LTC4 synthase; LTC4s) and receptors (BLT1, 2 and CysLT1, 2) mRNAs in the rat spinal cord. Semi‐quantitative RT‐PCR revealed that 5‐LO, FLAP, LTC4s, BLT1, and CysLT1 mRNAs increased following SNI, but not CysLT2 mRNAs. Using double labeling analysis of in situ hybridization with immunohistochemistry, we observed that 5‐LO, FLAP, and CysLT1 mRNAs were expressed in spinal microglia. LTA4h and LTC4s mRNAs were expressed in both spinal neurons and microglia. BLT1 mRNA was expressed in spinal neurons. The p38 mitogen‐activated protein kinase inhibitor, but not MEK inhibitor, reduced the increase in 5‐LO in spinal microglia. Continuous intrathecal administration of the 5‐LO inhibitor or BLT1 and CysLT1 receptor antagonists suppressed mechanical allodynia induced by SNI. Our findings suggest that the increase of LT synthesis in spinal microglia produced via p38 MAPK plays a role in the generation of neuropathic pain. © 2009 Wiley‐Liss, Inc.     

Activation of phosphatidylinositol 3-kinase and protein kinase B/Akt in dorsal root ganglia and spinal cord contributes to the neuropathic pain induced by spinal nerve ligation in rats

Several lines of evidence indicate that phosphatidylinositol 3-kinase (PI3K) and PI3K-protein kinase B/Akt (PKB/Akt) signal pathway mediate the pain hypersensitivity induced by intradermal injection of capsaicin or nerve growth factor. However, the role of PI3K and PI3K-PKB/Akt signal pathway activation in neuropathic pain is still unclear. Using L5 spinal nerve ligation (L5 SNL) and immunohistochemistry, we found that the numbers of phospho-PKB/Akt-immunoreactive (p-PKB/Akt IR) positive neurons were significantly increased in ipsilateral L5 dorsal root ganglia (DRG) and adjacent L4 DRG started at 12 h after surgery and maintained to the 3rd day. Meanwhile, L5 SNL also induced an increased expression of p-PKB/Akt in ipsilateral L5 spinal dorsal horn. Double immunofluorescence staining showed that p-PKB/Akt expressed entirely in DRG neurons, especially in IB4-positive neurons. Intrathecal injection of PI3K inhibitor wortmannin or LY294002 and PKB/Akt inhibitor Akt inhibitor IV or (-)-Deguelin, started before L5 SNL, reduced the behavioral signs of neuropathic pain. Intraperitoneal injection of wortmannin or (-)-Deguelin as above also reduced the pain hypersensitivity. Post-treatment with wortmannin, started at the 1st day or the 3rd day after L5 SNL, decreased abnormal pain behaviors. Whereas the inhibitory effect of Akt inhibitor IV on established neuropathic pain was observed only in those rats that received the drug treatment started at the 1st day. Immunohistochemistry revealed that intrathecal injection of wortmannin significantly inhibited the activation of PKB/Akt in L5 DRG and L5 spinal cord. The data suggested that PI3K and PI3K-PKB/Akt signal pathway activation might contribute to the development of neuropathic pain.     

Pharmacological evaluation of the selective spinal nerve ligation model of neuropathic pain in the rat

Rodent models of neuropathic pain are used to investigate the underlying mechanisms of pain associated with damage to peripheral nerves and to evaluate the efficacy of novel compounds. However, few models have been adequately characterized and the validity of many models remains unclear. The present experiment examined the activity of known anti-allodynic compounds in the L5 spinal nerve ligation (SNL) model of peripheral mononeuropathy in the rat, a modified version of the L5/L6 SNL model [S.H. Kim, J.M. Chung, An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat, Pain 50 (1992) 355-363]. Tactile sensitivity was measured 7-21 days post-surgery using von Frey monofilaments before and following treatment with gabapentin (30, 60 and 120 mg/kg), morphine (1, 3 and 6 mg/kg), amitriptyline (1.5, 3 and 10 mg/kg), fluoxetine (3, 10 and 30 mg/kg), WIN55,212-2 (0.5, 1 and 2.5 mg/kg), indomethacin (1 and 5 mg/kg) or U-50,488H (3 and 6 mg/kg). Compared to sham-operated control animals, L5 SNL animals displayed significant tactile allodynia in the ipsilateral hindpaw that was completely reversed by treatment with gabapentin, morphine, and WIN55,212-2, partially reversed by amitriptyline and fluoxetine, and unaffected by U-50,488H or indomethacin. The robust effects of the non-selective cannabinoid receptor agonist WIN55,212-2 and morphine support reports in the literature that systemic cannabinoid receptor agonists and opioids are active in neuropathic pain. These results suggest that the L5 SNL model can be utilized to determine the anti-allodynic activity of novel compounds.     

Targeting the NMDA receptor subunit NR2B for the treatment of neuropathic pain

Neuropathic pain is generally defined as a chronic pain state resulting from peripheral or central nerve injury, or both. An effective treatment for neuropathic pain is still lacking. The NMDA receptor, one type of the ionotropic glutamate receptors, is known to be important for triggering long-lasting changes in synapses. NMDA receptor-dependent synaptic plasticity plays roles not only in physiological functions such as learning and memory, but also in unwanted pathological conditions such as chronic pain. This review addresses recent progress on NMDA receptors in neuropathic pain, with particular emphasis on the NR2B-subunit-containing receptors. The expression and function of NMDA receptors in synaptic plasticity in the pain transmission pathway from dorsal root ganglia to the anterior cingulate cortex is reviewed, and preclinical and clinical investigations of selective NMDA receptor in neuropathic pain are discussed. The NMDA receptors, in particular NR2B-containing NMDA receptors, serve as promising targets for treatment of neuropathic pain.     

The influence of thiamin on the efficacy of pregabalin in rats with spinal nerve ligation (SNL)-induced neuropathic pain

Objective: The thiamin is often used in the treatment of neuropathy, and pregabalin is often used to treat neuropathic pain. Our study examined the influence of thiamin on the efficacy of pregabalin in a rat model of spinal nerve ligation (SNL)-induced neuropathic pain.

Methods: Sprague-Dawley male rats were randomly divided into six groups. The neuropathic pain-relieving properties were measured by plantar test, cold plate test, and hot plate test after administration of pregabalin (i.v) and/or thiamin (i.p) in SNL rats 14 days after operation.

Results: In the therapy period, pregabalin, or thiamin alone all produced antinociceptive effects in rats with neuropathic pain. And combination treatment of thiamin and pregabalin resulted in an enhanced pain relief compared to the administration of pregabalin or thiamin alone.

Conclusion: Combination of thiamin and pregabalin produces an additive antinociceptive effect in neuropathic pain rats, this drug combination may offer a bene?cial treatment option for neuropathic pain.     

不同剂量神经生长因子对神经源性疼痛大鼠脊髓Fos蛋白的影响

背景：神经生长因子对神经元的存活、生长发育、分化、再生和功能维持起到调控作用。 目的：进一步验证不同剂量神经生长因子对神经源性痛大鼠的治疗作用以及对脊髓中Fos蛋白的影响。 设计、地点：随机对照动物实验，在上海第九人民医院完成。 材料：健康成年雄性Wistar大鼠72只,体质量180~200 g，随机分为4组，模型组及腹腔注射神经生长因子4， 8，16 μg组，每组18只。 方法：72只大鼠结扎左侧坐骨神经制备坐骨神经慢性缩窄性损伤模型；腹腔注射神经生长因子4， 8，16 μg组，造模后分别腹腔注射神经生长因子4，8，16 μg/(kg•d)。 主要观察指标：①于术前和术后第1，2，3，5，7，10，14天进行行为学观察和机械性痛阈测定。②于术后第2，7，14天各组分别处死大鼠各6只，取脊髓，应用免疫组织化学方法和图像分析系统检测脊髓中Fos蛋白的表达。 结果：模型组大鼠术后出现自发抬足、舔足等自发痛表现，术后第3天起开始出现痛阈下降，第14天达最低值，而注射神经生长因子组大鼠没有自发痛表现，没有出现机械性痛阈的低常期，第10，14天模型组大鼠痛阈与其余各组比较差异有显著性意义(P < 0.05)。4组大鼠术后第1天机械性痛阈普遍升高；注射神经生长因子16 μg组大鼠术后第1天机械性痛阈比其他各组低(P < 0.01)，第2天机械性痛阈恢复至术前水平，低于注射神经生长因子4 μg组(P < 0.05)。术后模型组大鼠脊髓Fos蛋白表达进行性升高，而其他各组大鼠脊髓中仅有少量Fos阳性神经元，模型组与其余各组比较差异显著(P < 0.01)。术后第2天注射神经生长因子16 μg组大鼠脊髓Fos蛋白表达最低，与模型组和注射神经生长因子4 μg组比较，差异有显著性意义(P < 0.01)。 结论：神经生长因子对大鼠神经源性痛有治疗作用，且大剂量较小剂量作用更为明显，其机制可能与抑制脊髓中Fos蛋白表达有关。     

Anatomical evidence that the uninjured adjacent L4 nerve plays a significant role in the development of peripheral neuropathic pain after L5 spinal nerve ligation in rats

Rats develop hyperalgesia and allodynia in the hind paw after L5 spinal nerve ligation. Phosphorylated extracellular regulated kinase (pERK) was used as a pain marker to investigate the potential role of adjacent uninjured L4 nerve in the development of heat hyperalgesia after L5 nerve injury. Left L5 nerve was ligated and sectioned in rats. Three days later, rats were randomly assigned to five groups; each had both hind paws immersed in water at different temperatures (no heat, 37, 42, 47, and 52°C) under sevoflurane anesthesia for 2 minutes. Five minutes after stimulation the rats were sacrificed and sections of L3–L6 spinal segments were stained immunocytochemically with pERK antibody. pERK immunoreactivity, which is not detectable in the normal spinal cord, was discernible in neurons (not glia) of the superficial dorsal horn after noxious heat stimuli. pERK‐positive neurons clearly overlapped in laminae I–II with normal unmyelinated and thin myelinated afferents labeled with calcitonin gene‐related peptide and isolectin B4, and injured unmyelinated afferents labeled with vasoactive intestinal polypeptide. There was a linear increase in pERK immunoreactivity on both sides with an increase in temperature. Importantly, the number of positive pERK neurons was significantly higher in the ipsilateral side of L4 spinal segment, which receives innervation from uninjured L4 nerve, compared with the contralateral control side, which receives both uninjured L4 and L5 spinal nerves. The data demonstrate that the uninjured L4 nerve plays an important role in the development of heat hyperalgesia at the spinal cord level after L5 nerve injury. J. Comp. Neurol. 523:1731–1747, 2015. © 2015 Wiley Periodicals, Inc.     

Blockade of NMDA receptors decreased spinal microglia activation in bee venom induced acute inflammatory pain in rats

Objectives: Microglial cells in spinal dorsal horn can be activated by nociceptive stimuli and the activated microglial cells release various cytokines enhancing the nociceptive transmission. However, the mechanisms underlying the activation of spinal microglia during nociceptive stimuli have not been well understood. In order to define the role of NMDA receptors in the activation of spinal microglia during nociceptive stimuli, the present study was undertaken to investigate the effect of blockade of NMDA receptors on the spinal microglial activation induced by acute peripheral inflammatory pain in rats.

Methods: The acute inflammatory pain was induced by subcutaneous bee venom injection to the plantar surface of hind paw of rats. Spontaneous pain behavior, thermal withdrawal latency and mechanical withdrawal threshold were rated. The expression of specific microglia marker CD11b/c was assayed by immunohistochemistry and western blot.

Results: After bee venom treatment, it was found that rats produced a monophasic nociception characterized by constantly lifting and licking the injected hind paws, decreased thermal withdrawal latency and mechanical withdrawal threshold; immunohistochemistry displayed microglia with enlarged cell bodies, thickened, extended cellular processes with few ramifications, small spines, and intensive immunostaining; western blot showed upregulated expression level of CD11b/c within the period of hyperalgesia. Prior intrathecal injection of MK-801, a selective antagonist of NMDA receptors, attenuated the pain behaviors and suppressed up-regulation of CD11b/c induced by bee venom.

Conclusion: It can be concluded that NMDA receptors take part in the mediation of spinal microglia activation in bee venom induced peripheral inflammatory pain and hyperalgesia in rats.