胶原蛋白水解酶对背根神经节细胞亚细胞结构急性损伤的实验

目的：观察胶原蛋白水解酶（胶原酶）对大鼠脊神经背根神经节细胞亚细胞结构急性损伤的影响，以期探讨胶原酶应用的安全性，并进一步论证经皮椎间盘胶原蛋白水解酶化学髓核溶解术（胶原酶髓核溶解术）治疗方法的安全性。方法：实验于2002—07／09在中山大学基础医学院完成。SD健康雄性大鼠28只。随机分为3组：正常组9只；胶原酶急性实验组9只、急性假手术组10只。急性实验组和急性假手术组大鼠经腹腔注射戊巴比妥钠（45mg／kg）麻醉后，分离并辨认大鼠背根神经节，实验组局部滴注胶原酶1mL（4mL含1200U），急性假手术组局部滴注生理盐水1mL。于注药后1h行各组大鼠背根神经节细胞亚细胞结构的电镜检测。结果：各组大鼠背根神经节的神经元细胞、节内神经纤维检测结果：急性实验组背根神经节观测细胞种类、细胞数量、细胞大体形态、胞膜情况、节内神经纤维情况（有髓神经纤维有无肿胀，脱髓鞘，髓鞘松解等改变）、血管等情况与正常组、急性假手术组比较均无明显变化和差别。急性实验组背根神经节细胞亚细胞结构与正常组和急性假手术组比较均有明显变化和差别：①核仁部分偏向一侧。②线粒体大量肿胀，部分嵴断裂、空泡形成。各组均未见节细胞成群细胞坏死、细胞膜早期破裂、凋亡小体形成等节细胞凋亡相关表现和节细胞坏死相关表现。结论：临床应用于胶原酶髓核溶解术治疗浓度的胶原酶对背根神经节细胞是有损伤的。掌握胶原酶的用量和浓度，使胶原酶作用于精确合适的部位，才能提高胶原酶应用的安全性。     

胶原蛋白水解酶对背根神经节细胞亚细胞结构急性损伤的实验

目的:观察胶原蛋白水解酶(胶原酶)对大鼠脊神经背根神经节细胞亚细胞结构急性损伤的影响,以期探讨胶原酶应用的安全性,并进一步论证经皮椎间盘胶原蛋白水解酶化学髓核溶解术(胶原酶髓核溶解术)治疗方法的安全性。方法:实验于2002-07/09在中山大学基础医学院完成。SD健康雄性大鼠28只。随机分为3组:正常组9只;胶原酶急性实验组9只、急性假手术组10只。急性实验组和急性假手术组大鼠经腹腔注射戊巴比妥钠(45mg/kg)麻醉后,分离并辨认大鼠背根神经节,实验组局部滴注胶原酶1mL(4mL含1200U),急性假手术组局部滴注生理盐水1mL。于注药后1h行各组大鼠背根神经节细胞亚细胞结构的电镜检测。结果:各组大鼠背根神经节的神经元细胞、节内神经纤维检测结果:急性实验组背根神经节观测细胞种类、细胞数量、细胞大体形态、胞膜情况、节内神经纤维情况(有髓神经纤维有无肿胀,脱髓鞘,髓鞘松解等改变)、血管等情况与正常组、急性假手术组比较均无明显变化和差别。急性实验组背根神经节细胞亚细胞结构与正常组和急性假手术组比较均有明显变化和差别:①核仁部分偏向一侧。②线粒体大量肿胀,部分嵴断裂、空泡形成。各组均未见节细胞成群细胞坏死、细胞膜早期破裂、凋亡小体形成等节细胞凋亡相关表现和节细胞坏死相关表现。结论:临床应用于胶原酶髓核溶解术治疗浓度的胶原酶对背根神经节细胞是有损伤的。掌握胶原酶的用量和浓度,使胶原酶作用于精确合适的部位,才能提高胶原酶应用的安全性。     

炎症痛模型大鼠背根神经节神经元动作电位改变

目的:探讨炎症痛模型大鼠外周背根神经节(dorsal root ganglion,DRG)神经元细胞动作电位时空特性的改变。方法:大鼠单侧足底皮下注射福尔马林试剂致痛后,使用膜片钳技术对急性分离的大鼠腰5、腰6背根神经节神经元进行动作电位(action potential,AP)记录。结果:福尔马林组DRG神经元动作电位个数与生理盐水对照组相比明显增加,差异有显著统计学意义(P<0.01);福尔马林组神经元动作电位峰值与生理盐水对照组比较显著降低,差异有显著统计学意义(P<0.01);福尔马林组诱导动作电位爆发的阈电位与生理学盐水对照组阈值相比降低,差异有显著统计学意义(P<0.01);静息电位水平及动作电位时程福尔马林组与生理盐水对照组比较差异无统计学意义(P>0.05)。结论:炎性痛的出现是由于初级感觉神经元兴奋性增加导致外周敏化所致。     

坐骨神经震荡伤后腰髓背根神经节病理改变及意义

目的　应用图像分析和免疫组织化学技术观察和分析兔坐骨神经高速弹丸震荡伤后腰髓背根神经节病理改变及意义。方法　大耳白兔 2 5只 (包括正常对照 5只 ) ,致伤靶点为右后肢外侧坐骨神经体表投影线中点 (0 38g钢珠 ,0 6 5g装药量 ) ,观察伤后 1、3、7、14d(n =5 )腰髓背根神经节病理改变及一氧化氮合酶 (nitricoxidesynthase ,NOS)表达变化 (免疫组化法 ) ,并进行神经元计数及神经元截面积图像分析。结果　腰髓背根神经节伤后发生出血、水肿、神经元皱缩、坏死等变化 ,伤后 3dNOS表达显著增强 ,伤后 7d神经元数显著减少 ,神经元平均截面积显著减少。结论　坐骨神经高速弹丸震荡伤后腰髓背根神经节发生了较重的损伤。     

大鼠背根神经节神经元细胞纯化培养的模型建立

目的:建立一种切实可行的新生SD大鼠背根神经节神经元培养及纯化方法.方法:用显微解剖方法获取足够数量新生大鼠背根神经节,通过胰蛋白酶+EDTA消化、交替使用DF-12培养基和加有阿糖胞苷抗有丝分裂的DF-12培养基培养等方法,在体外获得纯化的背根神经节神经元,并采用NSE免疫细胞化学染色方法检测神经元的纯度.结果:获得的背根神经节神经元在体外生长良好,纯度可达到90%以上.结论:本方法可以获得大量高度纯化的大鼠背根神经节神经元.     

硬膜外移植自体髓核大鼠背根神经节的非压迫性损伤

背景:最新的研究表明,髓核所致的炎性反应是导致坐骨神经痛的重要原因之一,在此过程中背根神经节可能起重要作用,但是其病理生理变化目前尚不完全清楚.目的:探讨在无机械压迫情况下,腰椎间盘髓核突出引起坐骨神经痛的发病机制.方法:10月龄雄性SD大鼠24只,随机分成实验组和对照组,每组12只.切开大鼠尾椎椎间盘,髓核呈胶冻样,取5个髓核并加入50 μL生理盐水,充分搅拌稀释成混悬液备用.实验组大鼠行硬膜外穿刺,注射混悬液到腰椎硬膜外腔制作动物模型.对照组硬膜外腔注射生理盐水.测定大鼠后肢机械刺激缩爪阈值并对疼痛相关行为和背根神经节组织形态学进行观察.结果与结论:在无机械压迫情况下,硬膜外移植自体髓核能使大鼠后肢产生明显的痛觉过敏,背根神经节会出现节细胞肿胀、核膜不清晰、核仁变淡或消失、胞浆出现空泡、尼氏小体颜色变浅分布不均匀、内膜间质增宽、充血和水肿等形态学改变.结果表明,移植自体髓核所致的炎性反应是引起大鼠背根神经节损伤和坐骨神经痛的重要原因之一.     

背根神经节钠通道与疼痛

慢性疼痛是周围神经、组织损伤或炎性刺激所诱导的周围神经性病变的一种主要症状。最近一些研究表明背根神经节（dorsal root ganglion,DRG）钠通道表达及位置的改变与某些病理性疼痛有关，钠通道基因表达的可塑性及电生理的改变导致DRG细胞呈高兴奋性、产生自发动作电位及异常高频电活性，DRG钠通道在疼痛的病理生理中起做重要的作用。通过选择性地影响伤害性神经元产生动作电位及阻滞特异性钠通道可缓解神经性及炎性疼痛，有希望成为疼痛治疗的又一新领域。     

大鼠背根神经节加压培养细胞模型的建立

目的建立大鼠背根神经节（DRG）细胞受压模型，观察在恒定压力下培养24h和48h后神经元形态和活性的变化，评价这种神经元压力培养模型的优缺点。方法将培养的新生大鼠DRG细胞随机分为正常对照组、受压24h组、受压48h组，在80mmHg压力的密闭环境中培养相应时间后观察各组细胞在荧光显微镜和电镜下形态学特点，并用MTT法分析各组神经元细胞生长活性的改变。结果加压培养后，荧光显微镜下DRG神经元形态未发生明显变化，超微结构示48h组和24h组较正常组线粒体肿胀明显，粗面内质网缩短、变少，游离核糖体增多，部分核固缩，神经元代谢水平下降，MTT法比较加压前、后各组细胞活性的差异无统计学意义。结论加压培养未使细胞活性发生显著改变，此模型可观察机械压力单一因素对神经元的影响，有望成为一种在亚细胞和分子水平研究机械压力对DRG神经元的影响的有效细胞模型。     

神经病理性痛大鼠背根神经节细胞电生理学特征的改变

目的:研究神经病理性痛大鼠背根神经节(dorsal root ganglion, DRG)细胞电生理特征的改变.方法:以单侧L5和L6脊神经结扎制备大鼠神经病理性痛模型,运用全细胞电流钳技术进行电生理记录.结果:引起损伤侧DGR中、小直径神经元兴奋的基强度降低,动作电位爆发阈值下降,小直径神经元的动作电位时程变宽,自发放电的细胞比率明显升高.结论:神经损伤诱导初级感觉神经元的兴奋性增强,这可能是引起动物神经病理性痛敏的原因之一.     

胶原酶盘外持续给药治疗腰椎间盘突出症184例随访观察

目的:对胶原酶盘外持续给药治疗腰椎间盘突出症病人进行近期随访,以观察该治疗的临床效果、技术特点及注意事项.方法: 随访2002年1月至2004年3月采用胶原酶盘外持续给药治疗腰椎间盘突出症184例,记录治疗前后患者视觉模拟评分(VAS)、直抬腿高度、腰椎活动度、感觉和肌力等,进行分析总结.结果:治疗之后7天和90天患者疼痛症状明显好转,VAS治疗后7天、90天分别为3.5±0.25和2.7±0.21,与治疗前7.6±0.45比较P<0.05.治疗后7天、90天临床疗效总有效率分别为81.52 %和83.70 %.结论:采用胶原酶盘外持续给药治疗腰椎间盘突出症能够减轻患者疼痛,具有良好疗效.     

Long noncoding RNA BDNF-AS protects local anesthetic induced neurotoxicity in dorsal root ganglion neurons

BackgroundLong noncoding RNA (lncRNA) BDNF-AS was recently identified to regulate neurotrophin signaling pathway. In this study, we examined the functional role of BDNF-AS in regulating local anesthetic-induced neurotoxicity in dorsal root ganglion (DRG) neurons.MethodsNeonatal mouse DRG neurons were cultured in vitro, and treated with local anesthetic, bupivacaine, to induce neurotoxicity. The corresponding change in BDNF-AS expression level in DRG was probed by qRT-PCR. BDNF-AS was knocked down by siRNA in DRG. The effects of BDNF-AS downregulation on neurite regrowth, neuronal apoptosis and activating TrkB signaling pathway in bupivacaine-injured DRG neurons were probed by neurite outgrowth assay, TUNEL assay and western blot assay, respectively.ResultsDuring the process of bupivacaine-induce neurotoxicity in DRG, BDNF-AS was significantly upregulated in both dosage- and time- dependent manners. In DRG neurons, siRNA-mediated BDNF-AS downregulation promoted neurite outgrowth, reduced neuronal apoptosis, and phosphorylated TrkB signaling pathway after bupivacaine-induce neurotoxicity.ConclusionsBDNF-AS downregulation rescued local anesthetic-induce neurotoxicity in DRG neurons, probably through the activation of neurotrophin TrkB signaling pathway.     

大鼠背根神经节机械敏感性离子通道的生物物理学性质

目的探讨新生大鼠背根神经节(DRG)的机械敏感性离子通道(MS通道)生物物理学性质。方法取出新生大鼠DRG细胞,培养2~4d后,应用细胞贴附式和内面向外式膜片钳技术对细胞膜上的MS通道电流进行记录,对通道生物物理学性质,如压力-电流关系、电位-电流关系、动力学和离子选择性等进行了分析。采用的机械刺激方式为负压抽吸。结果压力可引起大鼠背根神经节上的机械敏感性非选择性阳离子通道开放,压力恒定时,电流恒定;去除压力,电流回到基线水平。在平衡溶液中,通道的电位-电流关系近似为直线。在膜电位为正值时,通道电流表现为外向电流,同时表现出外向整流特性,+40~+60mV电流的弦电导为(96.2±3.6)pS;当膜电位为负值时,通道电流表现为内向电流,-60~0mV斜率电导为(62.5±0.4)pS。通道的平均逆转电位为(-2.3±0.8)mV。通道的动力学分析表明压力可使短开放时间和长开放时间都明显升高,长关闭时间明显减少,而短关闭时间变化不大。结论分析了大鼠DRG神经元细胞膜上的MS通道的生物物理学性质,有助于进一步了解大鼠背根神经节神经元细胞电活动的机制。     

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy

EphrinB–EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague–Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB–EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time‐dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium‐ and small‐sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI‐induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB–EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR‐induced analgesia in clinic.     

Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons

BackgroundApplication of general anesthetics may induce neurotoxicity in dorsal root ganglia (DRG) neurons. In this study, we examined the possible protective mechanism and associated signaling pathways of small-molecule glycogen synthase kinase-3 (GSK-3) inhibitor, SB216763, in bupivacaine-injured mouse DRG neurons in vitro.MethodsIn vitro DRG explant of 6-week old mice was treated with 5 mM bupivacaine to induce neurotoxicity. The explants were also pre-treated with SB216763 for 72 h. Neural protection of SB216763 on bupivacaine-injured DRG neurons was investigated by TUNEL assay, neurite outgrowth assay and western blot assay, respectively. Possible downstream gene of GSK-3 signaling pathway, protein kinase C (PKC) was knocked down by siRNA in DRG explant. Its function in regulating GSK-3 inhibition induced DRG neural protection was also examined by TUNEL, neurite outgrowth and western blot assays.ResultsPre-treatment of SB216763 significantly ameliorated bupivacaine induced apoptosis and neurite loss in DRG neurons. Western blot showed that, in addition to the decrease of phosphorylated-GSK-3 α/β protein, SB216763 increased PKC and decreased caspase-3 (Casp-3) in bupivacaine-injured DRG neurons. SiRNA-mediated PKC knockdown was able to reverse the neural protection of SB216763 in bupivacaine-injured DRG neurons. Western blot showed that PKC knockdown increased phosphorylated-GSK-3 α/β and Casp-3 protein in DRG neurons, confirming that PKC was directly involved in GSK-3-inhibition induced neural protection in DRG.ConclusionsGSK-3 inhibitor SB216763, through PKC, is effective in protecting anesthetics-induced neurotoxicity in DRG.     

Role of TNF-alpha in sensitization of nociceptive dorsal horn neurons induced by application of nucleus pulposus to L5 dorsal root ganglion in rats

Herniation of the nucleus pulposus (NP) from lumbar intervertebral discs commonly results in radiculopathic pain and paresthesia (sciatica). While traditionally considered the result of mechanical compression of the dorsal root ganglion (DRG) and/or spinal nerve root, recent studies implicate pro-inflammatory mediators released from or evoked by NP, a possibility that was presently investigated. Single-unit recordings were made from L5 wide dynamic range dorsal horn neurons in pentobarbital-anesthetized rats. Autologous NP was harvested from a coccygeal disc and placed onto the exposed L5 DRG. A control group had subcutaneous adipose tissue or saline placed similarly. To test involvement of tumor necrosis factor- (TNF-), a third group received autologous NP plus local soluble TNF- receptor type 1 (0.013 μg) which binds TNF- to prevent its action. In each group, neuronal responses to graded heat (38–50 °C) and mechanical (von Frey filaments 4–76 g) stimuli were recorded prior to and at three successive hourly intervals following each treatment. Responses to noxious heat and mechanical stimuli were significantly enhanced 1 h post-NP and remained elevated thereafter. Thermally and mechanically evoked responses were not significantly affected in control rats or those treated with NP+soluble TNF- receptor type 1. These results indicate that sensitization of nociceptive spinal neuronal responses develops quickly following exposure of the DRG to NP, and that TNF- is involved. This electrophysiological model of herniated NP may prove useful in further characterizing the role of inflammatory mediators in hyperalgesia and allodynia resulting from lumbar disc herniation.     

Key role of the dorsal root ganglion in neuropathic tactile hypersensibility.

Cutting spinal nerves just distal to the dorsal root ganglion (DRG) triggers, with rapid onset, massive spontaneous ectopic discharge in axotomized afferent A-neurons, and at the same time induces tactile allodynia in the partially denervated hindlimb. We show that secondary transection of the dorsal root (rhizotomy) of the axotomized DRG, or suppression of the ectopia with topically applied local anesthetics, eliminates or attenuates the allodynia. Dorsal rhizotomy alone does not trigger allodynia. These observations support the hypothesis that ectopic firing in DRG A-neurons induces central sensitization which leads to tactile allodynia. The question of how activity in afferent A-neurons, which are not normally nociceptive, might induce allodynia is discussed in light of the current literature.     

Changes in dorsal root ganglion CGRP expression in a chronic inflammatory model of the rat knee joint: differential modulation by rofecoxib and paracetamol.

Neuropeptide-expressing small diameter sensory neurones are thought to be vital in generating inflammatory hyperalgesic responses. Within the dorsal root ganglion (DRG), both the levels of the neuropeptide calcitonin gene-related peptide (CGRP) and the numbers of CGRP-immunoreactive (CGRP-IR) DRG neurones have been shown to increase in a number of acute adjuvant-induced inflammatory pain models. The aim of this study was to look specifically at changes in numbers of CGRP-IR DRG neurones in a chronic model of inflammatory joint pain following complete Freund's adjuvant (CFA) injection into the rat knee. In this model, there were significant increases in the number of ipsilateral CGRP-IR small DRG neurones at days 1, 16 and 35 following intra-articular CFA, compared to saline-injected sham animals. This correlated with the behavioural readouts of hypersensitivity and knee joint inflammation at the same time points. There was also a significant increase in the number of ipsilateral CGRP-IR medium DRG neurones and contralateral CGRP-IR small DRG neurones at day 1. Following dosing of CFA-injected rats with rofecoxib (Vioxx) or paracetamol, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurones in rofecoxib- but not paracetamol-treated rats. These data also correlated with behavioural readouts where hypersensitivity and knee joint inflammation were significantly reduced by rofecoxib but not paracetamol treatment. In conclusion, these data show that changes in ipsilateral CGRP expression within small DRG neurones are consistent with behavioural readouts in both time course, rofecoxib and paracetamol studies in this model of chronic inflammatory pain.