大麻素受体2参与神经病理性疼痛的研究进展

神经病理性疼痛(Neuropathic Pain)是由于躯体感觉系统产生疾病或受到者损伤后,机体受到的有害或者无害的刺激被病理性的放大后所致.周围神经性痛是起源于周围神经系统受到机械创伤、代谢性疾病、神经化学毒物等影响.而中枢神经性痛一般都是由于脊髓损伤、中风或者多发性硬化引起的[1].然而到目前为止,神经病理性痛确切的发病机制仍不清楚,临床上治疗神经病理性疼痛的措施仍停留在对于原发疾病或损害的处理上,而现有的一些药物治疗(如阿片类药物、抗抑郁药和抗惊厥药物等),存在疼痛缓解不足或一些有害的副作用,药物用量受到限制,治疗效果一直不佳.近年来有研究表明大麻素受体2 (cannabinoid receptor 2,CB2)在选择性激动剂激活下能够有效地抑制急性、炎性疼痛而不产生中枢神经系统的副作用[2-5],因此其可能作为未来治疗神经病理性疼痛的一个治疗靶点.关于大麻素受体2在神经病理性疼痛中的作用的报道也越来越多,本文对其近几年的研究进展综述如下.     

Altered RNA editing of serotonin 2C receptor in a rat model of depression

Altered RNA editing of serotonin 2C receptor (HTR2C) has been suggested to be involved in the pathophysiology of major depression. Here we examined RNA editing status of HTR2C in the learned helplessness (LH) rats, one of well-established animal models of depression. LH rats showed the significantly increased RNA editing of site E, and tendency for increased RNA editing of other editing sites. Treatment with fluoxetine, a selective serotonin reuptake inhibitor, or imipramine, a tricyclic antidepressant, affected the RNA editing status of the LH rats. Although, these antidepressants differentially altered RNA editing status, they commonly reduced RNA editing efficiency of site E. We further revealed that altered RNA editing in the LH rats and by antidepressants was not explained by altered expression of RNA editing enzymes or their substrates (adenosine deaminases that act on RNA, HTR2C, and spliced form of HTR2C). These results suggest that alteration of RNA editing of HTR2C may play a role in the pathophysiology of depression and action of antidepressants.     

GABA_A受体亚基在神经病理性痛大鼠DRG神经元的表达

目的:观察神经病理性痛模型大鼠背根神经节(dorsal root ganglion,DRG)神经元上GABAA受体亚基表达的改变。方法:建立坐骨神经压榨性损伤动物模型(CCI),热板实验检测热刺激缩足反射潜伏期(TWL);并取大鼠L4-L6DRG神经元进行膜片钳实验,观测术后DRG神经元GABA介导的内向电流的变化;分别应用Western Blot技术和免疫荧光技术检测术后GABAA受体γ2和α2亚基表达的变化。结果:(1)CCI模型鼠的TWL比正常组明显缩短(P0.05);(2)膜片钳实验观察到术后14 d CCI术侧DRG神经元GABA介导的内向电流较正常组明显减小(P0.05),对侧电流较正常组明显增强(P0.05);(3)Western Blot检测观察到CCI术侧和对侧DRG神经元上GABAA受体γ2亚基的表达量较对照组均明显下调(P0.05),术侧和对侧磷酸化γ2亚基表达量较对照组明显上调(P0.05);(4)术侧GABAA受体α2亚基表达量下调(P0.05),对侧表达量上调(P0.05)。结论:神经病理性痛时,磷酸化GABAA受体γ2亚基表达的增多以及GABAA受体亚基组合方式的变化,可能参与了痛觉形成的过程。     

2型糖尿病神经病理性痛C57BL/6J小鼠模型的建立

目的利用胃饲酒精和腹腔注射链脲佐菌素(streptozotocin,STZ)建立2型糖尿病神经病理性痛C57BL/6J小鼠模型。方法 80只C57BL/6J小鼠随机分为:对照组(n=15)和实验组(n=65)。对照组每日胃饲生理盐水,12周后单次空腹腹腔注射柠檬酸缓冲液;实验组每日胃饲酒精12周诱导胰岛素抵抗,继以不同剂量链脲佐菌素(40 mg/kg、50 mg/kg、60 mg/kg)腹腔注射1次,于不同时间点分别测体重、血糖、血胰岛素浓度,计算胰岛素抵抗指数、机械缩足阈值和热缩足潜伏期的变化。结果连续胃饲酒精12周后,实验组小鼠体重明显增加,空腹胰岛素浓度升高,胰岛素抵抗指数升高,血糖未升高,机械缩足阈值和热缩足潜伏期无变化。注射STZ后,40 mg/kg剂量组血糖升高但不能长期维持,60 mg/kg剂量组血糖较高,死亡率高;50 mg/kg剂量组血糖中度升高且相对稳定,胰岛素浓度和胰岛素敏感性均降低,其机械缩足阈值和热缩足潜伏期均低于基础值和对照组(P0.05)。结论连续酒精胃饲12周后联合腹腔注射STZ50 mg/kg可以建立理想的2型糖尿病神经病理性痛小鼠模型。     

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.     

Anatomical localization and expression pattern for the NMDA-2D receptor subunit in a rat model of neuropathic pain

The N-methyl-d-aspartate receptor (NMDAR) has been implicated in the etiology of chronic pain. In this regard, this study sought to characterize the localization and expression pattern for the NMDAR-2D subunit in a rat model of neuropathic pain. To this end, one group of rats, 3 weeks post-dorsal root rhizotomy (DRR) and a second group, 3 weeks post-spinal nerve ligation (SNL) and sham surgery, were generated. Dorsal root ganglia (DRG) and/or lumbar spinal cord were excised from DRR, naïve, SNL and sham rats. Both immunohistochemical and real-time PCR analysis confirmed discrete NMDAR-2D subunit expression within the DRG and dorsal horn. However, no overt differences in staining intensity or expression were noted between DRG and spinal cord sections obtained from the different surgical groups. Results also demonstrated that the NMDAR-2D subunit was present within Neu N+ cells in the spinal cord and DRG, but excluded from cells labeled with the astrocytic marker, GFAP, and the microglial maker, OX-42. Lastly, the NMDAR-2D subunit was not co-expressed within neurokinin-1 (NK-1)+ or neurofilament-52 (N-52)+ neurons, but the antibody did co-label a number of isolectin B4+ (IB4) DRG cells. Together, these findings seem to suggest that the NMDAR-2D receptor subunit is present within the cell body region of a population of small diameter sensory afferents and post-synaptically within second order dorsal horn neurons. Although these data suggest that the NMDAR-2D subunit is well poised anatomically to modulate pain neurotransmission, the expression pattern for this subunit is not altered in rats demonstrating the presence of neuropathic-like pain behavior.     

Serotonin receptor mRNA expression in rat dorsal root ganglion neurons

In the present study, we have used in situ hybridization to examine the distribution of serotonin (5-HT) receptors in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, mRNAs for 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3B and 5-HT4 receptors were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. In contrast mRNAs for 5-HT1A, 5-HT1E, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors were undetectable in these neurons. The present study provides an insight into the molecular profile of 5-HT receptor subtypes in neurons responsible for modulating sensory information.     

Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain

Background: Neuropathic pain is characterized by hyperalgesia, allodynia and spontaneous pain. It often occurs as a result of injury to peripheral nerves, dorsal root ganglions (DRG), spinal cord, or brain. Recent studies have suggested that Toll-like receptor 4 (TLR4) might play a role in neuropathic pain. Methodology/Principal Findings: In this study, we investigated the role of TLR4 in a rat chronic constriction injury (CCI) model and explored the feasibility of treating neuropathic pain by inhibiting TLR4. Our results demonstrated that intrathecal siRNA-mediated suppression of TLR4 attenuated CCI-induced mechanical allodynia and thermal hyperalgesia through inhibiting the activation of NF-κB p65 and production of proinflammatory cytokines (e.g., TNF-α and IL-1β). Conclusions/Significance: These findings suggest that suppression of TLR4 mediated by intrathecally administered siRNA may be a new strategy for the treatment of neuropathic pain.     

Serotonin 2a receptor T102C polymorphism and impaired impulse control

Patients homozygous for the C allele of the T102C serotonin (5-HT) 2a receptor polymorphism have shown increased suicidal ideation or behavior in some reports, but not in others. We conducted a pilot investigation to determine whether this polymorphism might relate more specifically to a dimension of impaired impulse control, which may underlie only a portion of suicides. Rates of commission (impulsive) errors in a variant of the Continuous Performance Test (CPT) were compared across the genotypes of the T102C polymorphism in adults recruited from the community. The 102C/102C genotype was jointly associated with a greater incidence of past mood disorder or substance-use disorder, as well as significantly more commission errors compared to the 102T/102C and 102C/102C genotypes. These preliminary data suggest that the T102C 5-HT2a receptor polymorphism may be a marker for impaired behavior control-perhaps in the context of psychiatric disorder history.     

Electroacupuncture versus celecoxib for neuropathic pain in rat SNL model

Though acupuncture has long been used to treat various kinds of pain, its mechanisms remain partly understood. Our recent study has shown that it may inhibit cyclooxygenase-2 (COX-2) in the spinal dorsal horn where COX-2 is upregulated after the development of neuropathic pain following spinal nerve ligation (SNL). The current study directly compared the effect of acupuncture with COX-2 inhibitor celecoxib in the spinal cord after SNL in rats. After L5 SNL, the rats were treated either with acupuncture applied to Zusanli (ST36) and Sanyinjiao (SP6) bilaterally with or without electrical stimulation (2 Hz, 0.5–1–2 mA) four times over 22 days, and/or celecoxib fed daily. Paw-withdrawal-threshold to mechanical stimulation and paw-withdrawal-latency to thermal test were tested for neuropathic pain at four intervals following the treatments in comparison with the pre-treatment and non-treatment controls. The results demonstrate that electroacupuncture (EA) had a long lasting and better analgesic effect than celecoxib in reducing neuropathic hypersensitivity. Though COX-2 expression in the spinal L4–L6 dorsal horn by immunostaining was significantly reduced by acupuncture just as well as by celecoxib, the superior analgesic mechanism of acupuncture appears well beyond COX-2 inhibition alone.     

Local peripheral effects of mu-opioid receptor agonists in neuropathic pain in rats

Our study was designed to demonstrate peripheral antinociception of the mu-opioid receptor agonists: morphine (MF), [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in Bennett's rat model of neuropathic pain. All the agonists were effective in antagonizing allodynia after their intraplantar (i.pl.) but not subcutaneous (s.c.) administration. Opioid peptides: DAMGO, EM-1 and EM-2 were more effective compared with corresponding doses of morphine (opioid alkaloid) in alleviating chronic pain. Peripheral mu-opioid receptors mediated the observed effects, as was evidenced by the i.pl. treatment with naloxone methiodide (active only at the site of injection) and by cyprodime, a selective mu-opioid receptor antagonist. These results have shown that opioid peptides are effective also after local treatment, and that their peripheral use may be of therapeutic interest in long-term management of chronic pain.     

Serotonin receptor binding and mRNA expression in the hippocampus of fearful amygdala-kindled rats

Amygdala kindling in rats increases fear behavior. The neural correlates of this fear are not well understood. In this experiment, we investigated the relation between serotonin receptor binding and mRNA expression and fearful behavior in amygdala-kindled rats. Rats received either 100 kindling stimulations or sham stimulations, and their fear behavior was subsequently assessed in an unfamiliar open field. Then, the rats were sacrificed and 5-HT transporter binding, 5-HT1A and 5-HT2A receptor binding, and 5-HT1A mRNA expression in several brain regions was assessed. The kindled rats were significantly more fearful in the open field than the sham-stimulated rats. They also had significantly more 5-HT1A receptor binding and mRNA expression in the dentate gyrus than the sham-simulated rats, and these increases in 5-HT1A receptor binding and mRNA expression were significantly correlated to the increases in fear. There were no significant differences between the kindled and sham-stimulated rats in 5-HT transporter binding or 5-HT2A receptor binding. These results suggest that alterations in 5-HT1A receptors in the dentate gyrus may play a role in the expression of kindled fear.     

神经病理性疼痛的免疫机制

研究发现,在中枢神经系统及外周神经系统中,免疫细胞和分子在神经病理性疼痛过程中发挥着越来越重要的作用,对传统的以神经元为中心的疼痛理论发起了挑战。神经损伤后外周的肥大细胞、中性粒细胞、巨噬细胞和T淋巴细胞等免疫细胞所产生的级联炎性反应以及胶质细胞的激活是神经病理性疼痛形成和维持的关键因素,这些免疫细胞通过释放大量炎性介质最终导致疼痛的产生。     

Controlling neuropathic pain in HIV

Neuropathic pain is associated with numerous systemic illnesses, including HIV infection. The diagnosis and management of peripheral neuropathy presents diagnostic and therapeutic challenges. Among various forms of HIV-associated peripheral neuropathies, distal symmetrical polyneuropathy (DSP) is the most common. DSP may be caused or exacerbated by neurotoxic antiretrovirals, particularly the dideoxynucleoside analogues (d-drugs). Selection of appropriate pharmacologic intervention for peripheral neuropathy should be based on efficacy, safety, ease of administration, and cost. We review treatment options for painful HIV neuropathy, including experimental agents studied in recent and ongoing clinical trials.     

Intrathecal interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibits an anti-allodynic action in a rat model of neuropathic pain

The expression of interleukin-1beta and tumor necrosis factor has previously been shown to be up-regulated in the spinal cord of several rat mononeuropathy models. This present study was undertaken to determine whether blocking the action of central interleukin-1beta and tumor necrosis factor attenuates mechanical allodynia in a gender-specific manner in a rodent L5 spinal nerve transection model of neuropathic pain, and whether this inhibition occurs via down-regulation of the central cytokine cascade or blockade of glial activation. Interleukin-1 receptor antagonist or soluble tumor necrosis factor receptor was administered intrathecally via lumbar puncture to male Holtzman rats in a preventative pain strategy, in which therapy was initiated 1h prior to surgery. Administration of soluble tumor necrosis factor receptor attenuated mechanical allodynia, while interleukin-1 receptor antagonist alone was unable to decrease allodynia. Interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor, administered to both male and female rats in a preventative pain strategy, significantly reduced mechanical allodynia in a dose-dependent manner (P<0.01). The magnitude of attenuation in allodynia was similar in both males and females. Immunohistochemistry on L5 spinal cord revealed similar astrocytic and microglial activation regardless of treatment. At days 3 and 7 post-transection, animals receiving daily interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibited significantly less interleukin-6, but not interleukin-1beta, in the L5 spinal cord compared to vehicle-treated animals. In an existing pain paradigm, in which treatment was initiated on day 7 post-transection, interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor attenuated mechanical allodynia (P<0.05) in male rats. These findings further support a role for central interleukin-1beta and tumor necrosis factor in the development and maintenance of neuropathic pain through induction of a proinflammatory cytokine cascade.     

The contributing role of CD14 in toll-like receptor 4 dependent neuropathic pain

We have previously demonstrated that CNS toll-like receptor 4 (TLR4) plays a key role in the development of behavioral hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). TLR4 is a well-known receptor for lipopolysaccharide (LPS) in innate immune responses. In the current study, we further investigated the role of CD14, an accessory molecule in the LPS-TLR4 signaling pathway, in the development of L5Tx-induced neuropathic pain. CD14 knockout (KO) mice displayed significantly decreased behavioral sensitivity (mechanical allodynia and thermal hyperalgesia) as early as day 1 post-L5Tx, indicating a nociceptive role of CD14. By flow cytometric analyses, we observed significantly elevated microglial surface CD14 expression in the ipsilateral lumbar spinal cord 3 days post-L5Tx, as well as remarkable increases in microglial size (via forward scatter (FSC)) and granularity (via side scatter (SSC)). Further, intrathecal injection of soluble CD14 induced significantly greater mechanical hypersensitivity in wild type (C3H/HeN) mice compared with TLR4-deficient (C3H/HeJ) mice. Together, these data demonstrate that CD14 plays a contributing role in TLR4-dependent nerve injury-induced neuropathic pain.     

mRNA expression and RNA editing (2451 C-to-U) of IL-12 receptor beta2 in adult atopic patients

Interleukin (IL)-12 activates T helper (Th) 1 cells to produce interferon (IFN)-gamma which inhibits atopic inflammation. IL-12 acts through interaction with its receptor, especially beta(2) subunit. In several studies, the low production of IFN-gamma in peripheral mononuclear cells of atopic patients on response to IL-12 stimulation has been reported. Therefore we investigated the IL-12 receptor beta(2) (IL-12R beta(2)) mRNA expression and RNA editing, nucleotide 2451 C-to-U conversion, to find the cause of low responsiveness to IL-12 in atopy. Quantitative real time PCR for mRNA expression and sequence analysis for RNA editing were performed in 80 atopic patients and 54 healthy controls. The expression of IL-12R beta(2) mRNA was significantly lower in atopic patients than healthy controls (p<0.05). In sequence analysis, RNA editing on nucleotide 2451 was not found from either atopic patients or healthy controls. In additional evaluation, there was no relationship between expression of IL-12R beta(2) mRNA and serum total IgE or blood eosinophil count. Reduced IL-12R beta(2) mRNA expression in atopic patients indicate the reduced capacity to respond to IL-12 which induce IFN-gamma production and this may contribute to Th2-skewed immune response in atopy.     

Anti-allodynic efficacy of botulinum neurotoxin A in a model of neuropathic pain

Fyn, a member of the Src-family protein tyrosine kinase (PTK), is an essential factor in myelination in the CNS and is involved in murine embryonic stem (ES) cell growth and differentiation. Although dysfunctions of Fyn have been comparatively studied, the gain of function by ectopic expression, especially using ES cells, has seldom been investigated. In this article, we give the first report of the involvement of Fyn alteration in the sphere formation ability of murine ES cells. First, transient transfection of Fyn hardly affected multiplication and specialization. Then, we investigated Fyn overexpression using ES cells, which stably express Fyn. As a result, altered sphere formation capability was observed in all clones stably expressing Fyn. These results may provide important information for reproduction medical treatment using ES cells.     

Suppression of enriched environment-induced neurogenesis in a rodent model of neuropathic pain

Exposure to an enriched environment (EE) enhances neurogenesis and regulates emotionality. Previous reports have revealed that the rate of neurogenesis can be influenced by various environmental, endocrine, and pharmacologic stimuli. Chronic pain is a debilitating disease state characterized by complex alterations in both peripheral and central nociceptive pathways. In the present study, we evaluated the effect of chronic pain on environmental enrichment-induced hippocampal neurogenesis. Nerve-ligated mice were housed either in a standard environment or in the EE for 4 weeks. EE increased the immunoreactivity for doublecortin (DCX), a marker for immature neuron-positive cells, in the dentate gyrus (DG). Furthermore, the number of NeuroD (a neurogenic basic helix-loop-helix factor)-positive cells, in the DG was clearly increased by EE. Under these conditions, chronic pain suppressed enriched environment-mediated induction of both DCX- and NeuroD-labeled cells. These results suggest that chronic pain has stress-like damaging modulatory effects on hippocampal neurogenesis.