NR2B参与脊髓损伤致慢性疼痛的机制研究

目的:研究N-甲基-D-天冬氨酸(NMDA)受体2B型受体(NR2B)参与脊髓损伤后慢性神经病理性痛的机制。方法:制作脊髓半横断大鼠模型,von Frey纤维丝测量机械性刺激缩足阈值变化,Western Blot观察脊髓背角NR2B表达时程变化;同时采用行为药理学方法,鞘内给予NR2B特异性拮抗剂ifenprodil,观察对机械性刺激缩足阈值及NR2B表达的影响。结果:脊髓半横断术后大鼠双侧后足出现触诱发痛状态,NR2B在腰段脊髓双侧背角表达上调。鞘内给予ifenprodil逆转了大鼠的痛敏状态,伴随着NR2B在脊髓背角表达下调。结论:NR2B可能参与脊髓损伤后慢性神经病理性痛的发生发展,特异性拮抗NR2B可能是临床治疗脊髓损伤致慢性神经病理性痛的潜在策略。     

TRPM2通道在星形胶质细胞氨中毒中的作用

 目的：通过建立星形胶质细胞氨中毒的模型,探讨瞬时受体电位M2 (TRPM2)阳离子通道在其中发挥的作用。方法：分离并培养小鼠原代星形胶质细胞。实验分为5组：对照组、氯化铵处理组、氯化铵+3-氨基苯甲酰胺 (3-AB)处理组、氯化铵+PJ-34 处理组和TRPM2基因敲除小鼠+氧化铵处理组。测定细胞的活性、caspase-3 活性、细胞坏死和体积大小,以此来衡量氨中毒的程度。用全细胞膜片钳记录TRPM2通道电流变化。结果：氯化铵引起细胞肿胀伴随着细胞坏死。聚腺苷二磷酸核糖聚合酶(PARP)抑制剂3-AB和PJ-34抑制了氯化铵引起的阳离子电流,并减轻了氯化铵引起的相应细胞伤害。TRPM2基因敲除小鼠组细胞的伤害明显减轻（P<0.01）。结论：TRMP2通道的激活是细胞暴露于氯化铵后发生肿胀、坏死的必要步骤;氯化铵诱导的星形胶质细胞肿胀与坏死密切相关。     

MiR-204-5p减轻大鼠坐骨神经慢性缩窄损伤引起神经病理性疼痛的作用机制

背景:miR-204-5p是神经病理性疼痛的潜在生物标志物,但其在神经病理性疼痛中的作用及作用机制尚未阐明.目的:探讨miR-204-5p对大鼠坐骨神经慢性缩窄性损伤引起的神经病理性疼痛的作用及其分子机制.方法:①将90只雄性SD大鼠随机分为假手术组、模型组、空载体组、miR-204-5p过表达组、杂乱干扰组和AURK...     

局麻药长时间阻滞外周神经对神经源性疼痛发生过程中背根神经节内GAP-43表达的影响

用免疫组织化学方法观察了局麻药长时间阻滞外周神经对神经源性疼痛发生过程中生长相关蛋白(GAP-43)在背根神经节内表达的影响。实验选用SD大鼠35只,随机分成正常对照组、单纯坐骨神经横切组、坐骨神经横切前阻滞组、坐骨神经横切后阻滞组4大组,后三大组再按术后取材时间分为3、7d两个时间组。暴露大鼠右侧坐骨神经,于坐骨结节远端约1cm处横断坐骨神经。根据分组,阻滞组分别从横切前1h和横切后4h开始对神经横切的近心端进行长效局麻药阻滞,持续整个观察期。术后不同时间取与伤侧坐骨神经相连的背根神经节(DRG),应用免疫组织化学和图像分析的方法研究背根神经节中GAP-43的表达并进行定量分析。结果表明:术后3、7d,单纯坐骨神经横切组背根神经节内的GAP-43表达增高,而阻滞组内的GAP-43表达与正常组无差别。本研究结果提示坐骨神经横切前1h或之后4h开始局麻药阻滞神经均能抑制神经源性疼痛发生过程中GAP-43的高表达。     

5-HT_(2~7)受体亚型mRNAs在坐骨神经分支选择性损伤模型大鼠背根神经节的表达变化

本研究利用反转录 聚合酶链式反应(RT- PCR)方法,观察了坐骨神经分支选择性损伤(SNI)所致神经病理性痛条件下,大鼠背根神经节(DRG)中 5- HT2~7受体亚型mRNAs的时程表达变化。用RT- PCR方法在正常大鼠DRG中检测到 5-HT2A、5- HT3、5- HT4、5 -HT5A和 5 HT7受体亚型mRNAs的表达,但未检测到 5 -HT2B、5- HT2C、5- HT5B和 5 -HT6受体亚型mRNAs。SNI能诱导 5 -HT2A、5- HT3、5 -HT4 和 5- HT7受体亚型mRNAs在损伤侧DRG的表达上调。其中, 5- HT2A受体亚型mRNA的表达在术后 3d时开始升高,持续增加至 28d; 5- HT3 受体亚型mRNA的表达在术后 4d时明显增加, 14d时达到高峰; 5- HT4受体亚型mRNA于术后 3d的表达明显增加, 21d时达到高峰; 5-HT7 受体亚型mRNA的表达在术后 1d时即显著升高,一直维持高水平的表达至28d。未检测到 5- HT5A受体亚型mRNA的表达变化。在SNI对侧的DRG,各受体亚型mRNAs的表达未出现明显变化。部分 5- HT受体亚型在SNI模型DRG的表达具有不同的时程变化特点,提示它们在SNI所致的神经病理性痛中发挥着不同的作用。     

脊髓背角和背根神经节ERK1/2磷酸化表达在鞘内注射高乌甲素抗大鼠神经病理性疼痛中的作用

目的探讨鞘内注射高乌甲素对神经病理性疼痛大鼠脊髓背角和背根神经节ERK1/2磷酸化(p-ERK1/2)表达的影响。方法将90只SD大鼠随机分为假手术组(S组)、手术组(O组)和高乌甲素(L组)(n=30),建立坐骨神经慢性压迫(chronic constriction injury of sciatic nerve,CCI-SN)模型,术后L组鞘内注射高乌甲素20μg/kg,S组和O组以同样方式注射等体积生理盐水,末次给药后每组又分为术后第4、7和10天三个亚组(n=10);分别于相应时间点测定大鼠机械刺激缩足阈值(paw withdrawal mechanical threshold,PWMT),并采用Western blot技术检测脊髓背角和背根神经节p-ERK1/2的表达。结果与S组相比,O组术后第4、7和10天PWMT值均显著降低(第4、7天:P0.001;第10天:P0.01)。与O组相比,L组术后第4、7和10天PWMT值均明显升高(第4、7天:P0.05;第10天:P0.01)。在脊髓背角和背根神经节中,O组术后第4、7和10天p-ERK1/2表达均明显高于相应S组(脊髓背角:第4、7天,P0.001;第10天,P0.01;背根神经节:第4、10天,P0.01;第7天,P0.001);与O组相比,L组p-ERK1/2水平明显降低(脊髓背角:第4、10天,P0.05;第7天,P0.01;背根神经节:第4、7天,P0.05;第10天,P0.01)。结论鞘内注射高乌甲素能有效缓解CCI-SN大鼠神经病理性疼痛,其机制可能与其抑制脊髓背角和背根神经节中p-ERK1/2的表达有关。     

坐骨神经损伤大鼠模型鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达

背景：坐骨神经损伤模型可测试伤害性的热刺激和机械刺激所引发的痛觉过敏及冷、触觉异常。 目的：观察坐骨神经损伤模型大鼠鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达。 方法：72只SD大鼠随机均分为假手术组、对照组和实验组。对照组和实验组制作坐骨神经损伤模型,假手术组仅暴露坐骨神经,不结扎。分别于造模后第3,10天进行鞘内移植,实验组注入30 μL的神经干细胞悬液,空白组和对照组注入30 μL的细胞培养液。 结果与结论：与假手术组相比,对照组和实验组移植后3 d机械痛阈和热痛阈逐渐降低,至移植后7 d降低至最低点(P < 0.01),于移植后21 d恢复至移植前水平;实验组移植后7,14 d机械痛阈和热痛阈较对照组明显上升(P < 0.01)。与对照组相比,假手术组移植后7,14,21 d各组大鼠脑源性神经营养因子的表达呈低水平(P < 0.05);移植后14,21 d,实验组脑源性神经营养因子的表达量高于对照组(P < 0.05)。提示鞘内移植神经干细胞可提高脊髓背角和背根神经节中脑源性神经营养因子的表达。从而抑制了周围神经损伤产生的神经病理性疼痛。 关键词：脑源性神经营养因子;神经干细胞;慢性限制损伤;脊髓背角;背根神经节 doi:10.3969/j.issn.1673-8225.2012.10.031     

非谷氨酸受体依赖的钙毒性机制在急性脑缺血损伤中的作用

急性脑缺血神经元损伤的发生机制一直是神经科学研究的重要课题.经典的谷氨酸受体激活导致细胞内钙超载及其毒性学说被认为是脑缺血损伤的重要机制.然而越来越多的证据表明谷氨酸兴奋性毒性理论仅适用于短暂的缺血急性期,在缺血后期运用N-甲基-D-天冬氨酸 (N-methyl-D-aspartate,NMDA) 受体拮抗剂甚至不利于神经元的存活,NMDA受体途径只是介导脑缺血后钙毒性反应的环节之一,可能并不起主导作用.     

大鼠脊髓损伤后不同时间点5-HT2A受体免疫反应性的变化

目的:研究大鼠脊髓骶段(第2骶节)横断伤后5-羟色胺2A受体免疫反应(5-HT2AR-IR)的变化.方法:大鼠随机分为7组,即2h、8h和1d、2d、7d、30 d、60 d组,每组又随机分为手术组和假手术组.手术组在大鼠第2骶髓节段完全横断,假手术组只去除椎板,保留硬脊膜的完整,采用免疫荧光组织化学方法检测脊髓损伤下段5-HT2A-IR的变化.结果:2、8h手术组脊髓前角运动神经元区5-HT 2A-IR密度与假手术组比较无明显不同,1d后与假手术组比较开始增高,5-HT2A-IR密度随时间延长呈逐渐增加的趋势,直到手术后60 d达到最高水平.结论:慢性脊髓骶段完全横断后脊髓前角运动神经元的5-HT2A受体上调可能是5-羟色胺去神经支配后超敏感反应和痉挛发生的潜在机制.     

薄荷醇通过激活瞬时受体电位M8(TRPM8)促进BEAS-2B人支气管上皮细胞的细胞因子分泌

目的 探讨瞬时受体电位M8(TRPM8)在薄荷醇诱导BEAS-2B人支气管上皮细胞表达气道上皮源性细胞因子白细胞介素25(IL-25)、IL-33和胸腺基质淋巴细胞生成素(TSLP)中的作用及其相关信号转导机制.方法 用2mmol/L薄荷醇处理BEAS-2B细胞1、2、3、4h,选择IL-25、IL-33、TSLP或C...     

Proteomic analysis of the dorsal spinal cord in the mouse model of spared nerve injury-induced neuropathic pain

Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th–6th lumbar spinal cord in a mouse model of spared nerve injury (SNI)-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry (MS/MS). After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain, and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.     

Role of postsynaptic density protein-95 in the maintenance of peripheral nerve injury-induced neuropathic pain in rats

Our previous work has demonstrated that postsynaptic density protein-95, a molecular scaffolding protein that binds and clusters N-methyl-D-aspartate receptors at neuronal synapses, plays an important role in the development of peripheral nerve injury-induced neuropathic pain. The current study further investigated the possible involvement of postsynaptic density protein-95 in the maintenance of neuropathic pain. Mechanical and thermal hyperalgesia were induced within 3 days and maintained for 15 days or longer after unilateral injury to the fifth lumbar spinal nerve. The rats injected intrathecally with postsynaptic density protein-95 antisense oligodeoxynucleotide every 24 h for 4 days from day 7 to day 10 post-surgery exhibited not only a marked decrease in spinal cord postsynaptic density protein-95 protein expression but also a significant reduction in mechanical and thermal hyperalgesia on day 11 post-surgery. The rats injected with sense oligodeoxynucleotide did not display these changes. However, in the rats without nerve injury, postsynaptic density protein-95 antisense oligodeoxynucleotide given intrathecally every 24 h for 4 days did not affect responses to mechanical and thermal stimulation. In addition, postsynaptic density protein-95 antisense oligodeoxynucleotide did not change locomotor activity of experimental animals. Our results indicate that the deficiency of postsynaptic density protein-95 protein in the spinal cord significantly attenuates nerve injury-induced mechanical and thermal hyperalgesia during both the development and maintenance of chronic neuropathic pain. These results suggest that postsynaptic density protein-95 might be involved in the central mechanisms of chronic neuropathic pain and provide a novel target for development of new pain therapies.     

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.     

Synaptic plasticity in the substantia gelatinosa in a model of chronic neuropathic pain

Chronic neuropathic pain (CNP) is common after peripheral nerve injuries (PNI), but is rather refractory to available anti-pain medication. Advances in neuropathic pain research have identified cellular and molecular cues triggering the onset of neuropathic pain, but the mechanisms responsible for maintenance of chronic pain states are largely unknown. Structural changes such as sprouting of injured A-fibres into the substantia gelatinosa of the dorsal horn in the spinal cord have been proposed to relate to neuropathic pain in partial PNI models. Structural changes in central pain networks may also underlie the more persistent CNP following complete sectioning of a peripheral nerve, because this type of injury results in continuous and spontaneous afferent input to the spinal cord, which can trigger central sensitization. In the present study, the left sciatic nerve was completely sectioned and a 1-cm segment was removed to maintain a chronic pathology, whereas the right sciatic nerve was left intact. Mechanical allodynia was measured up to 84 days after injury, after which synaptic changes were studied in the lumbar substantia gelatinosa. The numbers of larger sized synaptophysin-immunoreactive presynaptic boutons were found to be increased in the substantia gelatinosa ipsilateral to the nerve injury. From these data we conclude that structural synaptic changes within the substantia gelatinosa are present months after complete nerve injury and that this plasticity may be involved in maintaining neuropathic pain states.     

Sleep and EEG patterns in the chronic constriction injury model of neuropathic pain

Chronic neuropathic pain patients often report sleep disturbances such as reduced amount of sleep and excessive daytime tiredness. The aim of this study was to evaluate possible abnormalities in sleep patterns in a widely used animal model of neuropathic pain. Adult male Sprague-Dawley rats were chronically implanted with electrodes for electroencephalogram (EEG) and electromyogram (EMG) registrations to allow continuous 24-h polygraphic recording. Subsequently, a chronic constriction injury (CCI) was inflicted on eight rats in accordance with the CCI model of neuropathic pain and a sham operation was performed on another eight rats. The polygraphic recordings were repeated 13, 27, 55, and 146 days after surgery. Although the CCI animals developed significant mechanical and cold allodynia and heat hyperalgesia, there were no significant differences between the CCI rats and the sham-operated control animals in the spontaneous EEG/EMG in homecage-like conditions. It is concluded that in the chronic phase, this neuropathic pain model does not produce clear sleep disturbances. Such an absence of general suffering from sleep disturbances is advantageous to the CCI model as it makes use of the model more acceptable ethically. Nonetheless, this outcome appears to be in contrast with the clinical situation in neuropathic pain and therefore could also be seen as a disadvantage for the face validity of the CCI model.     

Shoulder muscle co-ordination during chronic and acute experimental neck-shoulder pain. An occupational pain study

Little is known about the mechanisms leading to chronic neck-shoulder musculoskeletal disorders (MSD). The aim of the present study was to investigate and compare motor function during controlled, low load, repetitive work together with chronic or acute experimental neck-shoulder pain. The clinical study was performed on workers with (n?=?12) and without (n?=?6) chronic neck-shoulder pain. In the experimental study, experimental muscle pain was induced in healthy subjects by intra-muscular injection of hypertonic saline into the trapezius muscle (n?=?10). The assessed parameters related to motor performance were: work task event duration, cutting forces, surface electromyogram (EMG) activity in four shoulder muscles, displacement of the centre of pressure, and arm and trunk 3D movements. For controlled cutting force levels, chronic and acute experimental pain provoked a series of changes: a decreased working rhythm and a protective reorganisation of muscle synergy (experimental study), higher EMG frequency contents which may indicate altered motor unit recruitment, and greater postural activity and a tendency towards increased arm and trunk movements. These pain-related changes can play a role in the development of MSD. The present clinical and experimental study demonstrated similar interactions between motor co-ordination and neck-shoulder pain in occupational settings. We therefore suggest that this experimental model can be used to study mechanisms related to MSD. Information on such modulatory processes may help in the design of new strategies aimed at reducing the development of MSD.