The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

In vivo imaging of the neuronal response to spinal cord injury:a narrative review

Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron imaging.

Effects of Sevoflurane on the discharges of wide dynamic range neurons in spinally transected rats

Objective: To study the effects of clinical concentration of sevoflurane on activity of wide dynamic range neurons. Methods: Eight Spraque-Dawley rats(male) were selected. Their spinal cords were exposed and transected at T 9-10 level. The rate of firings of single neurons in the dorsal horn in response to electrical stimulation of skin was recorded with microelectrodes. The early and late discharges were observed when rats inhaled 0.5%, 1.0%, 1.5%, and 2.0% sevoflurane. Results: Sevoflurane suppressed the early and late discharges at the concentration of 0.5%, 1.0%, 1.5%, and 2.0%. Compared with early discharges, the extent of inhibition of late discharges was wider at the concentration of 1%, 1.5%, and 2.0% of sevoflurane. Conclusion: It is indicated that sevoflurane could suppress the transmission of nociceptive and non-nociceptive stimulation at dorsal horn. The suppression on nociceptive imput is stronger than that on non-nociceptive imput when the concentration of sevoflurane is more than 1%.

THE PROPERTY OF PERIPHERAL INPUT OF PROJECTION NEURONS IN THE SPINAL CORD DORSAL HORN

Antidromic and orthodromic responses of the projection neurons in the dorsal horn ofthe spinal cord have been recorded by a glass microelectrode in anesthetized and paralyz-ed cats. Furthermore, the effect of cervical segment antidromic stimulation to orthodromicresponse of the projection neurons has been observed by way of conditioning-test stimulation. Among all the spinocervical tract neurons (SCT), the dorsal column postsynaptic neu-rons (DCPS) and the spinocervical tract-dorsal column postsynaptic neurons (SCT-DCPS),which were identified by cervical segment antidromic stimulation, 46% are low-thresholdmechanoreceptive (LTM) and 54% are wide-dynamic-range (WDR) neurons. Most LTMneurons can evoke the same response to both 10 times (10 T) and 50 times (50 T) the thresh-old stimulation on the peroneal nerve. Most WDR neurons to 50 T intensity stimulation arestronger than the 10 T stimulation. Under the antidromic-cervical segment conditioningstimulation, the amount of orthodromic-discharging in most WDR and few LTM neuronsreduced significantly. The result shows that both LTM and WDR projection neurons in the spinal cord canrespond to all peripheral Aβ fibers and part of the Aδ fibers; there are some inhibitionaldescending fibers which affect the projection neurons in the cervical segment dorsal col-umn and dorsolateral funiculi.

抑制大鼠大脑皮层SI区对电针抑制持续性痛反应的影响

在大鼠用玻璃微电极细胞外记录的方法，观察了电针同侧前肢“合谷”、“内关”穴对皮下注射福尔马林（５％ ，５０ μｌ）诱发的脊髓背角广动力型（ＷＤＲ）神经元晚时相放电反应的影响。结果表明，低频强电针（５ Ｈｚ， ５～６ ｍ Ａ）和高频弱电针（５０ Ｈｚ， １ ｍ Ａ）都可对背角ＷＤＲ神经元的晚时相反应产生明显的抑制作用，且低频强电针的抑制作用大于高频弱电针的作用。用４％ ＭｇＳＯ４ 抑制皮层体感Ⅰ区（ＳＩ区）的活动后，低频强电针对ＷＤＲ神经元晚时相反应的抑制作用明显减弱，提示ＳＩ区在电针对持续性痛的镇痛机制中具有重要作用。

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn

OBJECTIVE:To investigate whether the Chinese massage system,Tuina,exerts analgesic effects in a rat model of chronic constriction injury(CCI)by remodeling the synaptic structure in the spinal cord dorsal horn(SCDH).METHODS:Sixty-nine male Sprague–Dawley rats were randomly and evenly divided into the normal group,sham group,CCI group,CCI+Tuina group,CCI+MK-801[an N-methyl D-aspartate receptor subtype 2B(NR2B)antagonist]group,and CCI+MK-801+Tuina group.The neuropathic pain model was established using CCI with right sciatic nerve ligation.Tuina was administered 4 d after CCI surgery,using pressing manipulation for 10 min,once daily.Motor function was observed with the inclined plate test,and pain behaviors were observed by the Von Frey test and acetone spray test.At 19 d after surgery,the L3-L5 spinal cord segments were removed.Glutamate,interleukin 1β(IL-1β),and tumor necrosis factor-α(TNF-α)levels were detected by enzyme-linked immunosorbent assay.The protein expression levels of NR2B and postsynaptic density protein-95(PSD-95)were detected by Western blot,and the synaptic structure was observed by transmission electron microscopy(TEM).RESULTS:CCI reduced motor function and caused mechanical and cold allodynia in rats,increased glutamate concentration and TNF-αand IL-1βlevels,and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH.TEM revealed that the synaptic structure of SCDH neurons was altered.Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801(P<0.05 or<0.01).For the majority of experiments,no significant differences were found between the CCI+MK-801 and CCI+MK-801+Tuina groups.CONCLUSIONS:Chinese Tuina can alleviate pain by remodeling the synaptic structure,and NR2B and PSD-95 receptors in the SCDH may be among its targets.

持续性外周炎症引起脊髓背角浅层nociceptin/or phanin放射性结合位点增加(英文)

采用放射性同位素位点结合法研究了外用持续性炎症对腰段脊髓 (L4 )背角Nociceptin结合位点的影响。大鼠一侧脚掌皮下注射CFA (130 μl)后 ,局部产生明显的炎性水肿 ,并出现行为学的痛觉过敏并有热辐射刺激诱发的脊髓背角浅层No ciceptin结合位点的明显增加。结果提示 ,Nociceptin在脊髓水平抗伤害感受过程中起一定的作用。

Electrophysiological properties of spinal wide dynamic range neurons in neuropathic pain rats following spinal nerve ligation

Objective The present study aimed to investigate the electrophysiological properties of wide dynamic range （WDR） neurons in spinal dorsal horn of rats with neuropathic pain induced by lumber 5 （L5） spinal nerve ligation （SNL） in a large size of samples.Methods Adult Sprague-Dawley rats were divided into normal and SNL groups.Electrophysiological technique was used to record the characteristics of WDR neurons in the spinal dorsal horn.Results Compared with the WDR neurons in normal rats,the WDR neurons in SNL rats showed an increase in excitability,manifested by an enlargement of the receptive field size,an increase in the proportion of neurons that exhibited spontaneous activities,decreases in the Cresponse threshold and latency,and an increase in the C-response duration.In addition,the numbers of A-and C-fiberevoked discharges were smaller in SNL rats than in normal rats.Conclusion The excitability of spinal WDR neurons increased in rats with neuropathic pain induced by L5 SNL.The increase in excitability of WDR neurons may contribute to the development of neuropathic pain.

推拿按揉法对慢性坐骨神经挤压伤大鼠脊髓背角星形胶质细胞和神经元的影响

目的:观察推拿按揉法对慢性坐骨神经挤压伤(CCI)大鼠星形胶质细胞和神经元的影响,探讨其治疗神经病理性疼痛的潜在作用机制。方法:将入组的40只SD大鼠随机平均分成5组(每组8只),分别为空白组、模型组、推拿组、星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组。除空白组外的组别均建立CCI模型,造模后第4天起,推拿组接受按揉法干预,10分钟/次/天,星形胶质细胞抑制剂组鞘内注射氟代柠檬酸(fluorocitrate,FCA),10ul/只/天,星形胶质细胞抑制剂+推拿组进行鞘内注射FCA和按揉委中穴联合干预,连续14天。观察各组大鼠各时间点的机械痛阈值情况,使用免疫荧光检测脊髓背角星形胶质细胞标志物(glialfibrillaryacidicprotein,GFAP)蛋白的表达,尼式染色法观测脊髓背角内神经元的形态、排列和数量的变化,并分析GFAP表达水平与神经元存活数量的相关性。结果:相较空白组,模型组机械痛阈值显著下降(P<0.01);推拿干预后,推拿组阈值较模型组逐渐上升,累计治疗14天后具有显著差异(P<0.01),注射FCA第7天起,星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组疼痛阈值明显上升(P<0.01)。相较空白组,模型组脊髓背角GFAP表达显著升高(P<0.01),神经元形态不规则、排列松散,数量减少(P<0.01),推拿治疗后,GFAP表达下调(P<0.01),神经元形态趋于规则、排列趋于整齐,数量增加(P<0.01)。注射FCA后,星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组的GFAP表达水平较模型组显著降低(P<0.01),神经元形态、排列较模型组更加规整、数量增加(P<0.01)。GFAP平均荧光强度与神经元存活数量在相关性分析中提示负(r=-0.4649,P<0.05)相关。结论:推拿按揉法可以缓解CCI大鼠的疼痛行为,减轻脊髓背角神经元的受损程度,其机制可能与其抑制星形胶质细胞活化相关。

RESPONSES OF RAT SPINAL PROJECTION NEURONS TO SOMATIC AND/OR VISCERAL STIMULATION

Neuroanatomical studies have shown that there are single and double projection neurons in the spinal cord dorsal horn. The cell origin, axon traveling and properties of these neurons have been investigated morphologically and physiologically. The solitary tract nucleus (STN)in the brain stem, as a nucleus responsible for processing visceral sensa-

部分去背根猫备用背根节和脊髓Ⅱ板层NT-3及其mRNA的表达变化

采用免疫组化和原位杂交技术探讨了部分去背根猫备用背根节 (L6 )和 L3、L5脊髓 II板层 NT-3及其 m RNA的表达变化。结果发现 ,正常组 NT-3及其 m RNA阳性产物主要分布于背根节的大型神经元和少数中、小型神经元。部分去背根后 ,3 d和10 d两时相 NT-3 m RNA大型神经元阳性数明显减少 ,而 NT-3阳性大型神经元数术后 10 d时方明显减少 (P<0 .0 1) ;NT-3及其 m RNA阳性小型细胞数在术后两时相均较正常组者增多 (P<0 .0 1) ;而在中型神经元只有 NT-3阳性神经元数有增加。相对地 ,在脊髓 板层 ,两时相 NT-3阳性神经元及胶质细胞百分数均较正常者明显增加 (P<0 .0 1) ,且以 3 d组者为最明显 ,但均未见 NT-3 m RNA阳性信号。结果表明 ,部分去背根不仅导致背根节各类神经元中 NT-3的表达发生了变化 ,且对 板层 NT-3阳性神经元及胶质细胞数量也有明显影响。提示 NT-3可能在脊髓

鞘内应用普瑞巴林对神经病理性疼痛大鼠的镇痛作用及其电生理学机制研究

目的:研究鞘内应用普瑞巴林(pregabalin,PGB),一种新型抗癫痫药,对神经病理性疼痛大鼠的镇痛作用及其可能的机制。方法:(1)雄性SD大鼠鞘内置管,手术成功后进行坐骨神经分支选择性损伤(spared nerve injury,SNI)手术。术后10 d用von Frey纤维丝测定机械痛敏,将筛选出造模成功的SNI神经病理性疼痛大鼠,按体重随机分为两组(n=10),通过鞘内插管分别鞘内给予(i.t.)普瑞巴林100μg/d或等体积的生理盐水(saline,NS)作阴性对照,连续3 d给药。在每次给药后24 h测定大鼠后爪的50%缩足阈值(paw withdrawal threshold,PWT)及鞘内给药前后大鼠的运动功能。(2)通过在体细胞外记录的电生理学方法,观察脊髓背表面给予普瑞巴林100μg对SNI大鼠脊髓背角广动力范围,(wide dynamic range,WDR)神经元C纤维诱发放电的影响。结果:(1)与NS组相比,鞘内应用普瑞巴林100μg/d,连用3 d,能显著增加SNI大鼠术侧后爪的50%PWT,起到显著的镇痛作用(P<0.001,n=10),给药前后大鼠的运动功能没有明显改变(P>0.05,n=10)。(2)与NS组相比,脊髓背表面给予普瑞巴林100μg可以显著抑制SNI大鼠WDR神经元的C纤维诱发放电(P<0.001,n=9)。结论:鞘内应用普瑞巴林100μg/d可以对SNI大鼠产生显著的镇痛作用而不影响其运动功能,该机制可能与通过WDR神经元的C纤维诱发放电有关。

应用超微结构定量法探讨针剌对猫脊髓后角第Ⅱ板层的可塑性影响

本实验采用双侧备用根模型(切断双侧L_1-L_5,L_7—S_2脊神经背根,保留L_6背根),针刺动物一侧后肢L_6背根支配范围内的足三里、悬钟、伏兔和三阴交四穴,应用电镜超微结构定量方法,观察电针刺激穴位28天后,对脊髓后角第Ⅱ板层终末可塑性的影响。结果显示,第Ⅱ板层单位面积中复合终末的数量,针刺侧比未针刺侧增多32％。提示针刺能促进完好背根纤维更快地侧支出芽和重建突触联系,以代偿损伤的背根纤维终末。本文也对针刺后第Ⅱ板层中复合终末的形态、面积,简单终末的数量以及电针刺激的某些条件进行了初步的探讨。

脊髓背角神经元对胃扩张及电针“足三里”穴的反应

目的 :本实验以猫胸段具有自发活动的背角神经元的放电频率为指标 ,观察了胃扩张和电刺激左侧内脏大神经引起的神经元电活动变化以及电针左侧“足三里”穴和“三阴交”穴的影响。方法 :采用玻璃微电极脑外记录神经元的电活动。工作参数如下 ,电针穴位 :3V ,3 0Hz,方波 ,连续 1 0sec;内脏大神经 :1mA ,双脉冲 ,间隔 4ms,波宽 2 0 0 μs,连续 1 0sec;胃充盈 :8mmHg( 1 0 64kPa)。结果 :①在所观察的 2 4个神经元中 ,1 3个对胃扩张刺激呈增频反应 ,1 1个为减频反应 ;电针“足三里”穴能够翻转或拮抗这种改变 ,即抑制增频神经元 ( 62 .50± 6.52 ,与 2 3 .3 0± 6.86相比 ,P <0 .0 5,脉冲个数 ,M±SE ,下同 ) ,兴奋减频神经元 ( 1 9.1 0± 4.72 ,与 71 .60± 1 7.50相比 ,P <0 .0 5)。电针“三阴交”穴具有类似功能 ,但作用较弱 ,无统计学意义 ;②在 2 1个 ( 2 1 / 2 4)神经元中 ,电刺激左侧内脏大神经可兴奋 1 3个神经元 ,抑制 7个神经元 ,其中 3个为完全抑制 ;③刺激内脏大神经同时电针“足三里”穴可见到后者对前者显著的抑制效应。结论 :①躯体与内脏的信息在脊髓水平汇聚并整合 ,进而通过改变内脏传出神经的活动达到调整治疗的目的 ;

氯胺酮对慢性坐骨神经损伤大鼠脊髓背角异位放电的影响

目的探讨氯胺酮静脉注射对慢性坐骨神经损伤(CCI)大鼠脊髓背角异位放电的影响。方法雄性SD大鼠36只,随机分为假手术组、CCI组和CCI-氯胺酮组。按Bennett等方法制作CCI模型,术后14d以von-Freyfilaments(vFFs)和冷水分别测定触痛和冷刺激反应,采用细胞外微电极记录技术,记录各组大鼠脊髓背角广动力(WDR)神经元的异常电活动情况。结果CCI组脊髓背角WDR细胞的基础放电水平明显高于假手术组,对轻刷、vFFs刺激放电频率与假手术组比较分别增加276·2%、245·1%和144·5%(P<0·01);氯胺酮对CCI大鼠WDR细胞基础放电和轻刷、vFFs刺激诱发放电产生剂量依赖性抑制作用。结论脊髓背角神经元的异位放电是形成中枢敏化的重要原因之一,可能与N-甲基-D-天冬氨酸(NMDA)受体有关。

鞘内注射氯胺酮对甲醛炎性疼痛大鼠脊髓背角nNOS表达的影响

目的：观察甲醛炎性疼痛大鼠脊髓背角神经元型一氧化氮合酶（nNOS）的表达，以及鞘内注射氯胺酮对甲醛炎性痛大鼠脊髓背角nNOS表达的影响。方法：32只SD大鼠采用改良Yaksh法进行鞘内置管，随机分为4组（n=8）：对照组（C组）、鞘内注射生理盐水组（NS组）、氯胺酮50μg组（K1组）和氯胺酮100μg组（K2组）。NS，K1和K2组于置管5d后，复制甲醛炎性疼痛模型。采用疼痛加权评分法（PIS）评估大鼠甲醛致痛后1h内的疼痛行为，24h后用免疫组织化学法观察大鼠腰5节段水平脊髓背角nNOS的表达。结果：与NS组比较，K1组和K2组在甲醛炎性痛第2时相（15～60min）的PIS值明显降低（P〈0．01）；NS组大鼠脊髓背角nNOS免疫反应阳性细胞数量及免疫组织化学评分均较C组明显增加（P〈0．01），而K1和K2组则明显低于Ns组，差异有统计学意义（P〈0．01）。结论：鞘内注射氯胺酮对甲醛炎性痛大鼠具有明显的抗伤害作用；鞘内注射氯胺酮可明显抑制甲醛炎性痛引起的脊髓背角nNOS表达的增加，表明nNOS在脊髓水平伤害性信息的传递和调制中可能发挥重要作用。

刺激大脑皮层体感Ⅰ区和大脑脚对大鼠脊髓背角神经元伤害感受性反应的影响

在大鼠用玻璃微电极细胞外记录的方法,观察了刺激皮层体感Ⅰ区(SI区)和大脑脚(CP)对皮肤强电刺激诱发的脊髓背角广动力范围(WDR)神经元长潜伏期反应(C-反应)的影响。结果表明刺激SI区对背角WDR神经元C-反应的影响以抑制为主,刺激CP的作用与刺激SI区的作用相似,但刺激CP更为有效。抑制作用的持续时间在不同神经元差别很大,短者在刺激停止后仅持续400ms,长者可达10min以上。静注纳洛酮对抑制作用无明显影响,静注二甲麦角新碱在部分神经元可使抑制作用明显减弱或完全消失,提示5-HT部分参与皮层下行抑制作用的实现,而内鸦片肽则否。

嗅鞘细胞对脊髓后角神经元突起生长的影响

目的:观察嗅鞘细胞对胚胎脊髓后角神经元突起生长的影响。方法:分离孕15d胚胎大鼠脊髓,取纵向分开的后半制备成细胞悬液,培养于:(1)原代培养的嗅鞘细胞单细胞层上;(2)嗅鞘细胞培养上清中;(3)原代培养的成纤维细胞单细胞层上;(4)单纯培养。24h后终止培养,行抗神经丝-200免疫细胞化学染色。每组随机取15个神经元,在LeicaQ-570图像分析仪中测量各组神经元的平均总突起长度及平均单个最长突起长度,进行组间比较。结果:神经元平均总突起长度(1)～(4)组分别为408.62±126.91滋m、336.24±106.34滋m、199.37±76.14滋m和64.61±24.49滋m;平均单个最长突起长度(1)～(4)组分别为180.13±83.54滋m、141.22±53.68滋m、107.29±43.35滋m和23.04±5.30滋m。第(1)、(2)组神经元平均总突起长度和平均单个最长突起长度明显高于第(3)、(4)组(P<0.01),而(1)、(2)组之间没有明显差异(P>0.05)。结论:与嗅鞘细胞共培养能明显促进胚胎脊髓后角神经元突起的生长速度。

鞘内注射M_ERK2-shRNA腺病毒对糖尿病神经痛小鼠的影响

目的探讨细胞外调节蛋白激酶2 (ERK2)在糖尿病神经病理性疼痛(DNP)小鼠模型中的作用机制。方法40只小鼠随机分4组:对照组,DNP组,siERK2-DNP组(鞘内注射M_ERK2-shRNA腺病毒),siRNA-DNP组(鞘内注射siRNA腺病毒)。模型制作成功后检测痛阈值;模型制作前、模型制作后2周、 4周及6周检测机械痛阈值;6周后取脊髓组织,利用Western blotting及免疫组织化学法检测脊髓后角ERK2及核因子κB (NFκB)的表达。结果与对照组比较,DNP组和siRNA-DNP组痛阈值显著降低, ERK2、NFκB的表达显著上调(P<0.05),siERK2-DNP组与对照组比较,痛阈值无显著变化, ERK2、NFκB的表达无显著差别(P>0.05)。结论下调ERK2可能是治疗糖尿病神经病理性疼痛的脊髓后角神经元损伤的方法之一。

电针超前镇痛对脊髓背角神经元及星形胶质细胞活化的影响

目的观察电针的超前镇痛效应及对脊髓背角神经元及星形胶质细胞活化的影响。方法将80只SD大鼠随机分为正常组、模型组、预电针组、后电针组,每组20只。模型组、预电针组、后电针组采用4%甲醛右侧足底注射方法构建急性炎性疼痛模型,正常组右侧足底注射等量生理盐水,电针取同侧足三里、阳陵泉穴。预电针组先进行电针刺激30 min后再造模,造模后继续捆绑30 min;后电针组前30 min仅捆绑不进行电针刺激,造模后立即行电针刺激30 min;正常组和模型组先捆绑30 min后造模,再捆绑30 min。观察各组大鼠行为学变化(缩足次数和舔足时间),采用免疫荧光染色及Western blot法观察各组大鼠脊髓背角神经元、星形胶质细胞活化情况及c-fos和GAFP表达情况,ELISA法检测各组大鼠脊髓中干扰素-γ(IFN-γ)、P物质含量。结果与正常组相比,模型组大鼠缩足次数明显增加(P<0.05),舔足时间明显延长(P<0.05);与模型组相比,预电针组、后电针组大鼠缩足次数均明显减少(P均<0.05),舔足时间均明显缩短(P均<0.05),且预电针组效果优于后电针组(P均<0.05)。与正常组相比,模型组大鼠脊髓背角神经元、星形胶质细胞的活化明显增强,脊髓中c-fos和GAFP的表达水平、IFN-γ及P物质含量均明显增高(P均<0.05);与模型组相比,预电针组、后电针组大鼠脊髓背角神经元、星形胶质细胞的活化明显减弱,脊髓中c-fos和GAFP的表达水平、IFN-γ及P物质含量均明显降低(P均<0.05),且预电针组改善情况明显优于后电针组(P均<0.05)。结论电针预刺激可通过抑制脊髓背角神经元、星形胶质细胞的活化,减弱神经炎症以及疼痛放大,从而发挥超前镇痛效应。