炎症痛引起的猫背根节Aβ神经元自发放电特性的分析

应用角叉菜胶后肢局部致炎作为炎症痛的实验动物模型，通过在细胞内记录技术，观察和分析了猫脊髓背根节Aβ神经元的自发放电特性和诱因。结果表明炎症痛导致猫背根节有自发放电的Aβ神经元数明显增加，并表现为持续高频、簇状和不规则等多种型式的放电；在炎症痛不同时程（4和12h），静息膜电位有一定程度的去极化；大多数有自发放电活动的神经元（91％）对外周机械性刺激表现为兴奋反应，其中有部分神经元（9％）同时具有兴奋性和抑制性感受野。胞体给予TTX能阻断这种自发放电；切断外周神经干能使这种自发放电消失，而切断其背根不影响这种自发放电活动。提示炎症痛猫背根节Aβ神经元的兴奋性增强与炎症部位持续性痛的存在相关，可能是炎症性触诱发痛（Allodynia）形成的基础。     

慢性痛大鼠背根神经元基因表达的研究

目的研究与慢性痛相关基因的特异表达,比较大鼠背根神经节损伤神经元与正常神经元之间基因表达的差异,以寻找构成神经病性疼痛的内在因素。方法应用mRNA差异显示方法从损伤背根神经节中寻找特异表达的基因。结果损伤侧背根节中cDNA条带(25.75±4.7)明显多于对照侧(18.0±5.0)。反向杂交后进行亚克隆得到10个含插入片段的阳性质粒,并对其中4个进行测序。结论神经轴突损伤可导致胞体基因表达改变,其中某些可能与痛觉异常有关,也可能与细胞结构恢复及免疫功能改变有关。     

育亨宾对慢性压迫背根节神经元自发放电的兴奋作用

采用离体灌流背根节 (dorsalrootganglion ,DRG)和单纤维记录神经元自发放电的方法 ,观察到育亨宾对受损DRG神经元的自发放电呈现兴奋作用 ,并初步研究了其发生机制。用外源性育亨宾 (10 μmol/L)灌流损伤的DRG时 ,在 2 2个有自发放电的DRG神经元中 ,有 18个神经元产生明显反应。育亨宾对损伤神经元自发放电的兴奋作用 ,可被α1 肾上腺素能受体拮抗剂哌唑嗪 (5 μmol/L)明显阻断。用 6 羟多巴胺化学性交感神经切断和胍乙啶耗竭交感末梢后 ,育亨宾的兴奋作用均明显减小。结果表明 :育亨宾阻断交感节后神经末梢上的α2 肾上腺素能受体 ,引起去甲肾上腺素 (norepinephrine,NE)的释放 ;释放的NE作用于损伤DRG神经元上的α1 肾上腺素能受体 ,而对受损神经元自发放电呈现兴奋作用。提示交感节后神经末梢可能存在一种持续性抑制NE释放的新机制 ,这种抑制作用不依赖交感节后神经节和动作电位的存在     

急性高血糖对大鼠背根神经节神经元罗丹明荧光强度的影响(英文)

用激光共聚焦技术在急性分离的大鼠背根神经节（DRG）神经元上观察了施加高糖溶液后罗丹明-123（Rhodamine－123）荧光强度的变化，从而推测高血糖对线粒体膜电位的作用及能量代谢的影响。对40个小DRG细胞的观察表明，罗丹明-123荧光强度在细胞的周边最高，而细胞核区的荧光强度最低。当在DRG细胞的培养液（人工脑脊液，ACSF）中滴加30mmol／L的D-葡萄糖溶液后，大部分细胞（8／9）荧光强度迅速降低，在100～400s内，平均降低21．2％±5．4％～22．8％±4．7％。而同体积的ACSF对荧光强度无明显影响（n＝7）。滴加葡萄糖后的罗丹明-123荧光强度变化曲线同滴加ACSF后的曲线相比有显著差异（p＜0．05）。说明急性高血糖可引起初级传入神经元的能且代谢降低。     

面部炎症痛诱发大鼠三叉神经节神经元中辣椒素受体表达的改变(英文)

目的探讨辣椒素受体(transient receptor potential vanilloid receptor 1,TRPV1)参与和面部炎症痛相关的热痛觉过敏与冷痛觉感受的可能机制。方法于大鼠面部皮下注射松节油造成面部炎症痛模型,分别应用热测痛和冷测痛装置测量热缩头潜伏期(head withdrawal thermal latency, HWTL)和冷缩头潜伏期(head withdrawal coldlentency, HWCL)的变化,每天测量一次,连续21天。应用免疫组织化学染色,细胞大小频率分析和平均光密度值分析来研究面部炎症痛后第3、5、7、14、21天支配大鼠面部表皮区三叉神经节(trigeminal ganglion,TG)初级感觉神经元、触须部皮肤末梢神经纤维和投射至三叉神经感觉尾侧亚核(trigeminal sensory nuclei caudalis,Vc)中枢突TRPV1表达的改变。结果注射松节油后第1至14天,热退缩反应潜伏期与冷退缩反应潜伏期均明显下降,分别于注射后第5 天和第3 天达到最低,第21 天恢复到正常水平;注射松节油后第1至14天 ,TRPV1表达的细胞数量增加,并于第7天达到最大,第21 天恢复到正常水平。正常大鼠TRPV1主要表达于TG的中小神经元,触须部皮肤以及三叉神经尾侧亚核含丰富的TRPV1阳性末梢;面部炎症痛后2周内,TG的中小神经元,触须部皮肤末梢以及Vc的Ⅰ和Ⅱ外层均可见明显的TRPV1表达增加。结论面部炎症痛可以引起大鼠对伤害性热刺激和冷刺激的痛觉过敏,并导致三叉神经节中TRPV1阳性神经元和外周与中枢阳性神经纤维末梢数目增加,表明TRPV1在三叉神经节的中小神经元和末梢轴突表型的改变可能对松节油引起面部炎症痛时热痛觉过敏和冷痛觉感受的形成与维持起重要作用。     

抑郁状态对伴有自发痛大鼠的热伤害性阈值的调节作用(英文)

目的近年来,人们对抑郁症和疼痛之间的相互作用日益关注。本研究通过观察抑郁对福尔马林注射大鼠的伤害性阈值的影响,探讨抑郁能否降低伴有自发痛的大鼠对伤害性刺激的感受性。方法通过嗅球切除术建立大鼠抑郁模型,并采用旷场测试和体重变化来评价抑郁行为。在大鼠后肢足底注射福尔马林溶液诱导持续性疼痛。在福尔马林注射的对侧后肢足底施加伤害性辐射热刺激,测定其抬脚阈值。结果在非抑郁大鼠中,福尔马林注射肢对伤害性刺激表现出痛觉减退,而对侧肢的伤害性阈值则不受影响。然而,与非抑郁大鼠相比,抑郁大鼠对伤害性热辐射刺激的感受性显著降低,表现为对侧后肢抬脚潜伏期延长。结论上述结果表明,在伴有福尔马林炎症痛的条件下,抑郁同样能降低机体对诱发痛的敏感性。该结果与临床上关于同时患有抑郁与慢性痛患者对实验性痛刺激感受性下降的观察结果一致。     

小鼠背根神经节中三个不同的钙激活氯离子通道基因家族的表达(英文)

目的在背根神经节(dorsal root ganglion,DRG)中等大小感觉神经元中可以观察到钙激活氯离子流(I_(Cl(Ca)))。在坐骨神经损伤模型中,在大多数大中神经元上诱导出类似的氯离子流。本文旨在探讨引起这个离子流的分子基础。方法使用常规的定量RT-PCR方法检测在DRG中三个基因家族的表达,这三个基因家族都具有诱导I_(Cl(Ca))的特点。结果在成年小鼠的DRG中,分别显示了在正常状态和坐骨神经损伤3天后CLCA,Bestrophin和Tweety基因家族成员的转录产物。结论mBestl和Tweety2可能在损伤诱导的DRG神经元I_(Cl(Ca))中发挥作用。     

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。     

Blockade of microglial activation reduces mechanical allodynia in rats with compression of the trigeminal ganglion

The present study investigated the role of microglia and p38 MAPK in the development of mechanical allodynia in rats with compression of the trigeminal ganglion. Male Sprague-Dawley rats weighing 250-260 g were used. Under pentobarbital sodium anesthesia, the animals were mounted onto a stereotaxic frame and given injections of 4% agar solution (10 μL) to compress the trigeminal ganglion. The air-puff thresholds significantly decreased after compression of the trigeminal ganglion. On postoperative day 14, immunoreactivity to both OX-42 and p-p38 MAPK was up-regulated in the medullary dorsal horn as compared to the sham group. P-p38 MAPK was found to be co-localized with OX-42, but not with NeuN, a neuronal cell marker, or with GFAP, an astroglial cell marker. Intracisternal administration of 100 μg of minocycline significantly inhibited both mechanical allodynia and activation of microglia produced by compression of the trigeminal ganglion. Intracisternal administration of 0.1, 1, or 10 μg of SB203580, a p38 MAPK inhibitor, also significantly decreased mechanical allodynia and p38 MAPK activation in the trigeminal ganglion-compressed group. These results suggest that activation of p38 MAPK in the microglia is an important step in the development of mechanical allodynia in rats with compression of the trigeminal ganglion and that the targeted blockade of microglial p38 MAPK pathway is a potentially important new treatment strategy for trigeminal neuralgia-like nociception.     

Catecholamine varicosities in cat dorsal root ganglion and spinal ventral roots

Convergence upon reticulospinal neurons which mediate disynaptic, contralateral pyramidal EPSPs to neck motoneurons has been examined in cats with contralateral pyramidal transection at the obex. Conditioning stimuli in the contralateral tectum and ipsilateral mesencephalic tegmentum produced monosynaptic facilitation of the disynaptic pyramidal EPSP, whereas facilitation evoked from the ipsilateral pyramid showed a disynaptic time course. These results show that contralateral pyramidal, tectal and ipsilateral tegmental fibers converge onto common reticulospinal neurons which have direct excitatory connections with neck motoneurons.     

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).     

鞘内注射胶质细胞抑制剂对背根神经节压迫大鼠脊髓星形胶质细胞的影响

BACKGROUND： Astrocytes are considered to provide nutritional support in the central nervous system. However, recent studies have confirmed that astrocytes also play an important role in chronic pain.
OBJECTIVE： To investigate the effects of intrathecal injection of fluorocitrate, minocycline or both on astrocyte activation and proliferation in the spinal dorsal horn of compressed dorsal root ganglion in rats.
DESIGN, TIME AND SETTING： The neurology randomized controlled animal study was performed at the Jiangsu Institute of Anesthesia Medicine, from September 2006 to April 2007. MATERIALS： A total of 96 male Sprague Dawley rats, aged 6-8 weeks, were selected for this study. Following intrathecal catheterization, 80 rats underwent steel bar insertion into the L4-5 intervertebral foramina to make a stable compression on the L4-5 posterior root ganglion. Thus rat models of ganglion compression were established. Minocycline and fluorocitrate were purchased from Sigma, USA.
METHODS： A total of 96 rats were randomly and equally divided into six groups. Rat L4, L5 transverse process and intervertebral foramina were exposed in the sham operation group, but without steel bar insertion. The model group did not receive any manipulations. Rats in the phosphate buffered saline （PBS） group were intrathecally injected with 0.01 mmol/L PBS （20 μL）. Rats in the fluorocitrate group were subjected to 1 μmol/L fluorocitrate （20 μL）. Rats in the minocycline group were intrathecally injected with 5 g/L minocycline （20 μL）. Rats in the minocycline and fluorocitrate group received a mixture （20 μL） of 5 g/L minocycline and 1 μmol/L fluorocitrate. Following model establishment, drugs were administered once a day.
MAIN OUTCOME MEASURES： At 7 and 14 days following model induction, glial fibrillary acidic protein expression in the spinal dorsal horn was measured by immunofluorescence microscopy. Six sections with significant glial fibrillary acidic protein -positive expression were obtained to count astrocytes under an inverted microscope.
RESULTS： No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups （P 〈 0.01）. The decrease in astrocyte count was mainly found in layers Ⅲ–Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups.
CONCLUSION： Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.     

Patterned assembly and neurogenesis in the chick dorsal root ganglion

The birth of small‐diameter TrkA+ neurons that mediate pain and thermoreception begins ≈24 hours after the cessation of neural crest cell migration from progenitors residing in the nascent dorsal root ganglion. Although multiple geographically distinct progenitor pools have been proposed, this study is the first to comprehensively characterize the derivation of small‐diameter neurons. In the developing chick embryo we identify novel patterns in neural crest cell migration and colonization that sculpt the incipient ganglion into a postmitotic neuronal core encapsulated by a layer of proliferative progenitor cells. Furthermore, we show that this outer progenitor layer is composed of three spatially, temporally, and molecularly distinct progenitor zones, two of which give rise to distinct populations of TrkA+ neurons. J. Comp. Neurol. 518:405–422, 2010. © 2009 Wiley‐Liss, Inc.     

Mechanical hyperalgesia in rats with chronic perfusion of lumbar dorsal root ganglion with hyperglycemic solution

In diabetes, chronic systemic hyperglycemia is associated with pain and other symptoms of peripheral neuropathy. Evaluation of mechanisms causing these symptoms is complicated because of the overlap between the systemic effects of hyperglycemia and its toxic effects within the peripheral nervous system. To address this problem we developed a technique for chronic local in vivo perfusion of rat lumbar dorsal root ganglion (DRG) with a hyperglycemic solution. Osmotic pumps were filled with 30 mM glucose in physiological buffer and implanted in normal adult rats. The output of the catheter attached to the pump was positioned in a hole drilled through the right transverse process of the L₅ vertebrae to perfuse the corresponding DRG. Repetitive tests of foot withdrawal to mechanical stimuli have shown that chronic hyperglycemia localized to the L₅ DRG causes hyperalgesia in the hind limb innervated by perfused ganglion but not in the contralateral limb. Control experiments (DRG perfusion with 5 mM glucose or 5 mM glucose+25 mM mannitol solution) have shown that hyperglycemia-induced hyperalgesia can not be attributed to surgery-related injury or hyperosmolality of the ganglion-perfusing solution. These data demonstrate direct functional toxicity of hyperglycemia in the peripheral nervous system. This technique provides a new approach for in vivo study of chronic effects of physiologically active factors on DRG neuron function.     

Nuclear factor-kappa B mediates TRPV4-NO pathway involved in thermal hyperalgesia following chronic compression of the dorsal root ganglion in rats

The aim of this study was to test the hypothesis that nuclear factor-kappa B (NF-κB) is involved in TRPV4-NO pathway in thermal hyperalgesia following chronic compression of the dorsal root ganglion (DRG) (the procedure hereafter termed CCD) in rat. Intrathecal administration of two NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC; 10⁻¹ to 10⁻² M) and BAY (100-50 μM), both induced significantly dose-dependent increase in the paw withdrawal latency (PWL) and decrease in nitric oxide (NO) content in DRG when compared with control rats. Pretreatment with 4α-phorbol 12,13-didecanoate (4α-PDD, transient receptor potential vanilloid 4 (TRPV4) synthetic activator, 1 nm) attenuated the suppressive effects of PDTC (10⁻¹ M) and BAY (100 μM) on CCD-induced thermal hyperalgesia and NO production. In addition, Western blot analysis indicated that CCD rats exhibited nuclear NF-κB protein expression and low levels of cytoplasmic inhibitory-kappa B (I-κB) expression; the increase in NF-κB expression and decrease in I-κB expression were reversed after intrathecal injection of PDTC. In conclusion, our data suggested that NF-κB could be involved in TRPV4-NO pathway in CCD-induced thermal hyperalgesia.     

去甲肾上腺素对背根神经节神经元P物质释放的调节作用(英文)

目的观察激活或抑制α-肾上腺素受体是否影响体外培养的背根神经节(dorsal root ganglion,DRG)神经元P物质(substance P,SP)的释放。方法胎龄15天的Wistar大鼠DRG神经元培养2天后,分别用去甲肾上腺素(nora-drenaline,NA)(1×10-4mol/L)、α1-受体拮抗剂哌唑嗪(1×10-6mol/L)+NA(1×10-4mol/L)、α2-受体拮抗剂育亨宾(1×10-5mol/L)+NA(1×10-4mol/L)孵育4天。用RT-PCR法检测DRG神经元编码SP蛋白的PPTmRNA表达水平,用Western blot法检测DRG神经元SP蛋白的表达水平,用酶联免疫吸附测定法检测SP的基础释放量和辣椒素刺激后的释放量。结果 NA单独孵育显著增加了DRG神经元辣椒素刺激后的SP释放量,α1-受体拮抗剂哌唑嗪预处理可阻断NA的效应,而α2-受体拮抗剂育亨宾不产生此作用。在各种实验条件下,PPT mRNA水平、SP蛋白表达水平和SP的基础释放量没有显著性差异。结论 NA通过激活α1-受体增加了DRG神经元辣椒素刺激后的SP释放量,这一作用可能与去甲肾上腺素能的疼痛调...     

Opioid regulation of intracellular free calcium in cultured mouse dorsal root ganglion neurons

Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca²⁺]_i) or an inhibition of K⁺ (25 mM)-stimulated increase in [Ca²⁺]_i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser², Leu⁵]enkephalin-Thr (DSLET), U69593, and [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) each induced a transient increase in [Ca²⁺]_i in a small fraction (<30%) of neurons. The increases in [Ca²⁺]_i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca²⁺]_i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca²⁺]_i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K⁺-stimulated increases in [Ca²⁺]_i, which were blocked by naloxone (1 μM). Inhibition of the K⁺-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc.