低能量激光对大鼠背根神经节持续受压后痛觉敏感及TRPV4表达的影响

目的:观察低能量激光对大鼠背根神经节持续受压(chronic compression of the dorsal root ganglion,CCD)后痛觉敏感及脊髓背角内TRPV4表达及分布的影响.方法:选取健康雄性Wistar大鼠36只,分为空白组、CCD组、激光组各12只,各组均给予常规饲养.激光组在CCD后第4...     

大鼠脊髓背角TRPV4在背根神经节持续受压致神经病理性疼痛中的作用

目的:探讨大鼠背根神经节(dorsal root ganglion,DRG)持续受压(chronic compression of right side dorsal root ganglion,CCD)后脊髓背角瞬时感受器电位离子通道4(TRPV4)基因及蛋白变化,明确脊髓背角TRPV4在CCD致神经病理性疼痛中的作用。方法:采用健康成年雄性Wistar大鼠,共36只,随机分为3组,分别为空白对照组、CCD手术组、CCD+钌红组。制备大鼠背根神经节持续受压模型,于术前1天、术后第7天、给药前及给药2h后,测量大鼠机械刺激缩爪反应阈值,观察机械痛阈的变化;利用RT-PCR及Western Blot技术检测各组大鼠手术侧脊髓背角TRPV4基因及蛋白表达的变化。结果:与空白对照组相比,术后第7天,CCD组大鼠术侧机械痛阈值明显下降(P0.001),同侧脊髓背角TRPV4基因及蛋白表达升高(P0.05);与给药前相比,给予钌红2h后,术侧机械痛阈值明显升高(P0.001),同侧脊髓背角TRPV4基因及蛋白表达下降(P0.05)。结论:CCD后大鼠术侧机械痛阈下降,脊髓背角TRPV4基因及蛋白表达升高;钌红可部分逆转CCD后痛觉过敏,部分降低脊髓背角TRPV4基因及蛋白表达。脊髓背角TRPV4参与CCD后大鼠神经病理性疼痛形成。     

慢性背根神经节受压对大鼠脊髓背角Wnt/β-catenin信号通路的影响

目的:探讨慢性背根神经节受压(CCD)对大鼠脊髓背角Wnt/β-catenin信号通路的影响。方法:选取42只成年雄性SD大鼠,按照随机数字表法将其分为假手术组和CCD组,其中假手术组9只,CCD组33只。按照术后时间点不同,将CCD组细分为术后1 d组(6只)、术后3 d组(6只)、术后7 d组(9只)、术后14 d...     

大鼠SNL神经痛模型不同时相背根神经节TRPV1的活化形式

目的:研究大鼠脊神经结扎(spinal nerve ligation,SNL)神经痛模型不同时相背根神经节(dorsal root ganglion,DRG)TRPV1的活化形式。方法:大鼠随机分为正常(D 3)组、模型(D 3)组、正常(D 15)组与模型(D 15)组。采用SNL方法制作大鼠神经痛模型。检测造模后第三天(D 3)与第15天(D 15)大鼠术侧缩足阈值(paw withdrawal threshold,PWT),L4⁶ DRG TRPV1表达与磷酸化水平。结果:SNL模型大鼠早期即出现痛觉敏化现象,直至实验结束。D 3、D 15术侧PWT显著下降(P<0.001)。D 3 L46 DRG TRPV1表达与磷酸化水平。结果:SNL模型大鼠早期即出现痛觉敏化现象,直至实验结束。D 3、D 15术侧PWT显著下降(P<0.001)。D 3 L4⁶DRG TRPV1表达均下降(P<0.05,P<0.01,P<0.01);L5 DRG TRPV1磷酸化水平上升(P<0.001),L4、L6 DRG TRPV1磷酸化水平下降(P<0.01,P<0.05)。D 15 L5 DRG TRPV1表达水平下降(P<0.01),L4、L6 DRG TRPV1表达水平上升(P<0.05);L46DRG TRPV1表达均下降(P<0.05,P<0.01,P<0.01);L5 DRG TRPV1磷酸化水平上升(P<0.001),L4、L6 DRG TRPV1磷酸化水平下降(P<0.01,P<0.05)。D 15 L5 DRG TRPV1表达水平下降(P<0.01),L4、L6 DRG TRPV1表达水平上升(P<0.05);L4⁶ DRG TRPV1磷酸化水平均下降(P<0.01,P<0.001)。结论:SNL神经痛的发生、维持与DRG TRPV1的活化有关。SNL神经痛的早期,TRPV1的活化与损伤DRG TRPV1磷酸化水平升高有关;SNL神经痛的维持阶段,TRPV1的活化与邻近未损伤DRG TRPV1表达增强有关。     

TRPV4在介导大鼠背根神经节持续受压后机械和热痛敏中的作用

目的:观察持续机械压迫(CCD)对瞬时感受器电位离子通道4(TRPV4)基因、蛋白表达及功能的影响,明确TRPV4是否参与CCD导致的机械和热痛敏。方法:建立CCD模型后,分别于手术前及手术后第7天、第14天及第28天取材前测量运动功能、机械刺激缩爪反应阈值和热辐射刺激缩爪反应潜伏期。为了测量TRPV4反义核苷酸干扰对机械和热痛阈值的影响,在蛛网膜下腔内注入TRPV4寡脱氧核苷酸(ODN)40μg/d,每天1次,第7天后测量大鼠行为学变化。使用实时定量RT-PCR检测TRPV4基因表达的变化,Western blot检测TRPV4蛋白质表达量的变化,激光共聚焦检测低渗溶液和佛波醇(4α-PDD)刺激背根神经节(DRG)神经元后细胞内钙离子浓度的变化。结果:所有动物在损伤前后步态均正常,持续压迫明显降低大鼠的机械和热痛阈,TRPV4干扰可部分逆转该痛敏。持续机械压迫可以明显增加TRPV4基因和蛋白的表达,手术后第7天,第14天和第28天,TRPV4mRNA的表达分别为假手术组大鼠的4.29倍、2.95倍和2.48倍,蛋白表达量分别为假手术组大鼠的4.34倍,3.88倍和2.47倍。持续机械压迫后,对低渗溶液和4α-PDD产生反应的DRG神经元的比例数增加,细胞内钙的峰值增高。这种反应被TRPV4反义ODN所抑制。结论:CCD可以上调TRPV4的基因、蛋白表达,敏化通道的功能;TRPV4参与介导CCD导致的机械和热痛敏。     

神经病理性疼痛大鼠脊髓Ⅱ层突触形态结构的变化

目的:观察神经病理性疼痛大鼠脊髓背角Ⅱ层突触形态结构的可塑性,探讨神经病理性疼痛的发病机制。方法:SD大鼠15只,随机分为正常组、假手术组和模型组各5只,采用坐骨神经慢性压迫性损伤(CCI)建立大鼠神经病理性疼痛模型。应用透射电镜观察和体视学定量分析脊髓Ⅱ层突触形态结构的参数变化。结果:与正常组及假手术组比较,模型组大鼠脊髓背角Ⅱ层的突触后致密物质增厚(P<0.05),突触间隙增宽及突触界面曲率明显增大(P<0.05),凹型棘突触的数密度显著增高(P<0.01),总突触、棘突触和穿孔性突触的数密度增高明显(P<0.05)。结论:脊髓Ⅱ层突触形态结构可塑性变化可能参与神经病理性疼痛的发生。     

大鼠切口疼痛对脊髓背角Fos蛋白的影响

Ｂｒｅｎｎａｎ等[1] 提出了一种大鼠切口疼痛模型 ,发现该模型与人体术后疼痛状态有某些相似之处。研究显示即刻早期基因产物ｃ Ｆｏｓ可能是神经元被刺激激活的一种标记。为了解大鼠切口疼痛对脊髓背角Ｆｏｓ蛋白表达的影响 ,我们进行了以下研究。材料和方法1.实验动物和分组　健康雄性ＳＤ大鼠 (2 5 0～30 0 ｇ) 8只 ,随机分为 2组。手术组 (ｎ =4 )大鼠在吸入异氟醚麻醉下进行手术 ;对照组 (ｎ =4 )大鼠吸入相同时间的异氟醚 ,但不作手术切口。所有动物均由徐州医学院实验动物中心提供。2 .切口疼痛模型的建立　将大鼠任一后爪进行消毒…     

大鼠背根神经节持续受压后差异蛋白质表达分析

目的:使用蛋白质组学方法筛查大鼠背根神经节(DRG)持续受压(CCD)后差异表达的蛋白质,特别是筛查与疼痛和组织损伤相关的蛋白。方法:78只Wister大鼠随机分为正常组和CCD组,建立CCD模型后,测量行为学指标。28d后处死大鼠,从DRGs中提取蛋白,进行双向电泳分离蛋白,找出差异表达蛋白,使用基质辅助激光解析电离飞行时间质谱分析得到其肽指纹图谱(PMF),进行鉴定。使用Westernblot和实时RT-PCR验证部分差异蛋白。结果:CCD明显降低机械痛阈和热辐射刺激缩爪反应潜伏期。正常组和CCD组98个蛋白点的表达存在明显差异,成功鉴定出15种蛋白,其中8种蛋白的表达在CCD组下调,7种蛋白表达上调(其中1种蛋白只存在于CCD组)。验证显示与质谱分析的结果相符。结论:以差异蛋白质组学的方法分析CCD对DRG蛋白质表达的影响,鉴定出15种差异表达的蛋白。其中膜联蛋白A2、p11和蛋白激酶Cε(PKCε)蛋白表达的上调,可能参与了神经性疼痛的发生过程。三磷酸甘油醛脱氢酶(GAPDH)的上调或许参与神经元凋亡,热休克蛋白70(HSP70)的表达上调或许与神经保护有关。     

ERK-TRPV4途径参与大鼠背根神经节持续受压后痛觉敏感的脊髓中枢敏化机制的研究

摘要 目的：探讨ERK-TRPV4途径参与大鼠背根神经节持续受压(CCD)后痛觉敏感的脊髓中枢敏化的机制。 方法：选取SPF级雄性Wistar大鼠共42只,随机分为空白对照组6只,CCD手术组36只（包括术后2d、4d、7d、10d和14d,每组6只,以及免疫荧光组6只）。利用免疫共沉淀检测正常大鼠脊髓背角中ERK与TRPV4的相互联系。利用免疫组织化学染色法,观察ERK与TRPV4的分布情况。制备CCD模型,利用western blot技术检测空白对照组及术后2d,4d,7d,10d及14d,手术侧及对侧脊髓背角ERK和TRPV4蛋白表达的变化。利用免疫荧光技术观察术后4d脊髓背角中ERK和TRPV4阳性细胞数目、神经元的形态及阳性神经元比率的变化。 结果：CCD术后第2天至第10天,手术侧脊髓背角中ERK及TRPV4表达明显升高（P<0.01）。同时,手术侧脊髓背角中ERK和TRPV4阳性细胞数目、共同阳性表达细胞数及阳性神经元细胞比率均高于非手术侧（P<0.01）。脊髓背角阳性神经元分布于灰质第Ⅰ至Ⅳ层,手术侧的神经元树突棘生成增加,胞体增大。 结论：ERK-TRPV4通路参与了CCD后痛觉敏感的脊髓中枢敏化机制。     

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30μL的神经干细胞悬液,空白组和对照组注入30μL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3d机械痛阈和热痛阈逐渐降低,至移植后7d降低至最低点（P〈0.01）,于移植后21d恢复至移植前水平;实验组移植后7,14d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7,14,21d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14,21d,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。     

Behavioral manifestations of neuropathic pain and mechanical allodynia, and changes in spinal dorsal horn neurons, following L4–L6 dorsal root constriction in rats

We investigated behavioral symptoms of neuropathic pain, and associated changes in dorsal horn neurons, in a rat model involving loose ligation of lumbar dorsal roots. The L4–L6 dorsal roots were exposed unilaterally and loosely constricted central to the respective ganglia with one (1-ligation) or two (2-ligation) silk 7-O ligatures. In control groups the dorsal roots were exposed but not ligated (sham-operated), or sutures were placed lengthwise between the dorsal roots (suture control). There was a significant reduction in mechanical withdrawal threshold on the operated side in both 1- and 2-ligation groups which began at 3 days, peaked at 2–5 week, and gradually recovered. A delayed threshold reduction was also seen on the non-operated side. Immediately post-surgery there was a significant increase (hypoalgesia) in thermal paw withdrawal latency (Hargreaves test) in 1- and 2-ligation groups on the operated (but not non-operated) side that recovered after 1 week. Significantly less weight was borne by the operated limb 1–5 weeks post-operatively in 1- and 2-ligation groups. The force of hind limb withdrawals elicited by graded noxious heat pulses (38–52°C) was significantly lower 1 week post-surgery on the operated side (1-ligation group) followed by recovery. Withdrawal forces were higher 5–9 week post-surgery on the non-operated side in 1- and 2-ligation groups. We found no evidence of cold allodynia. Neither sham-operated nor suture controls showed any signs of allodynia or hyperalgesia. Following behavioral testing, rats were anesthetized with halothane for single-unit recordings from lumbar wide dynamic range-type (WDR) neurons. At 22 week post-surgery, the mean area of mechanosensitive receptive fields was significantly larger for units on the operated side in 1- and 2-ligation groups compared with those on the non-operated side or with those from sham-operated rats. Mean stimulus-response functions to graded noxious heat pulses (38–52°C, 5 s) were not significantly different between operated and non-operated sides for 1- or 2-ligation groups, or compared with the 22-week sham-operated group. At 5 week post-surgery, the mean area of cutaneous receptive fields, and stimulus-response functions to graded noxious heat, were not significantly different between units recorded on operated versus non-operated sides, or compared with units from 5-week sham-operated rats. Spontaneous unit activity was significantly higher on the operated versus non-operated side in the 2-ligation (22-week) and sham (5-week) groups. Enlarged cutaneous receptive fields of dorsal horn neurons may contribute to mechanical allodynia associated with dorsal root constriction. However, the slow (>5 week) development of receptive field enlargement does not match the rapid development of allodynia. The lack of effect of dorsal root constriction on thermal sensitivity of dorsal horn units ipsilaterally corresponds to the lack of marked thermal hyperalgesia observed behaviorally.     

Relationship between mechano-receptive fields of dorsal horn convergent neurons and the response to noxious immersion of the ipsilateral hindpaw in rats

This study examines the relationship between mechano-receptive fields (inhibitory and excitatory, located on the ipsilateral hindpaw) of convergent dorsal horn neurons, and the responses of the neurons to noxious immersion of an entire paw in noxious hot water. In pentobarbital anesthetized rats with intact spinal cords and in unanesthetized decerebrate-spinalized rats, rat hindpaws were immersed in 50°C water for 10 s after the mechano-receptive fields had been delineated using 5-s noxious pinches. Convergent neurons were either excited or inhibited by noxious immersion of the hindpaw. In both groups, a significant association (χ², P<0.01) was found between the make-up of the mechano-receptive field and the response of the neuron to immersion. Immersion-inhibited neurons (intact=27, spinalized=13), always had both an excitatory and an inhibitory mechano-receptive field on the same hindpaw. Additionally, when the hindpaw was removed from the noxious water, these immersion-inhibited cells displayed a strong afterdischarge which was immediately inhibited once the paw was reimmersed. Pinch-induced and immersion-induced inhibition were found in both spinalized and intact rats suggesting spinal mechanisms were sufficient to mediate this effect. The majority of immersion-excited cells showed only an excitatory mechano-receptive field on the hindpaw (intact rats=18/23 or 78.3%, spinalized rats=24/36 or 66.7%). However, other immersion-excited cells had both an inhibitory and an excitatory mechano-receptive field on the hindpaw (intact rats=5/23 or 21.7%, spinalized rats=12/36 or 33.3%). The response of a convergent neuron, which has its excitatory receptive field located on a paw, to noxious immersion of the entire paw can be predicted by the make-up of the mechano-receptive fields. Additionally, since noxious paw immersion affects ipsilateral convergent neurons in two opposite manners, it suggests that other effects, such as heterotopic actions, might also not be uniform.     

脊髓损伤后慢性中枢性疼痛与脊髓背角P物质关系研究

目的探讨脊髓损伤 (SCI)后慢性中枢性疼痛 (CCP)与P物质的关系。方法选取SD大鼠 2 8只 ,分为正常组 (A组 )、假手术组 (B组 ) ,以及用WADE法复制出SCI后无CCP组 (C组 )和CCP组 (D组 )。取大鼠T13 和L2 脊髓节段 ,采用免疫荧光组织化学染色法结合激光共聚焦显微镜技术观察脊髓背角P物质 (SP)的变化。结果各组大鼠T13 和L2 节段脊髓背角SP含量比较为 :D组较C组减少 (P <0 .0 5 ) ,较A组和B组明显减少 (P <0 .0 1) ;C组较A组和B组减少 (P <0 .0 5 ) ;A组与B组无显著性差异。结论SCI后CCP大鼠脊髓背角SP可能对CCP有某种程度的抑制作用。     

瞬时感受器电位离子通道香草素受体4下游信号分子的确定及其对大鼠背根神经节慢性压迫后痛觉过敏的作用

目的:探讨大鼠背根神经节慢性压迫CCD后瞬时感受器电位离子通道香草素受体4(TRPV4)下游信号分子及其在痛觉过敏中的机制。方法:鞘内分别注射TRPV4拮抗剂钌红(RR)、TRPV4反义寡脱氧核苷酸(ASODN)和一氧化氮合成酶(NOS)抑制剂L-NAME,检测CCD大鼠背根神经节DRG内一氧化氮(NO)代谢产物亚硝酸盐(nitrite)含量变化,并观测热刺激缩爪反应潜伏期(PWL)的变化。结果:鞘内分别注射RR、TRPV4 AS ODN和L-NAME后,均能够显著降低CCD大鼠DRG内亚硝酸盐含量(P<0.05),CCD大鼠的热痛敏行为也能够显著改善(P<0.05)。结论:TRPV4及其下游信号分子NO参与介导CCD大鼠的热痛觉过敏。     

脊髓背角细胞外信号调节激酶磷酸化参与调控2,4-二硝基氟苯诱导的小鼠慢性痒

目的：探讨脊髓背角细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）磷酸化对2,4-二硝基氟苯（2,4-dinitrofluorobenzene,DNFB）诱导的小鼠慢性痒的调控作用。方法：8～12周ICR雄性小鼠随机分为DNFB诱导组和丙酮对照组。DNFB组小鼠分别于面颊部和背部反复涂抹1.5% DNFB溶液,建立类似特异性皮炎的慢性痒模型。分别观察慢性痒模型建立后小鼠的自发性行为,以及鞘内注射MEK抑制剂U0126后小鼠自发性慢性痒行为的变化。免疫组织化学染色法分析小鼠脊髓组织磷酸化ERK（phosphrylated ERK,pERK）的表达情况。结果：面颊部诱导模型中,DNFB组小鼠的搔抓次数明显高于丙酮对照组（P<0.05）,而擦涂行为无差异,证实DNFB诱发的是痒觉;颈背部诱导模型中,DNFB组小鼠表现出剧烈的搔抓行为,而丙酮对照组的搔抓行为不明显。背部慢性痒模型建立后,分别在第1、3、7和14天鞘内注射MEK抑制剂U0126,小鼠的搔抓行为受到明显抑制（P<0.05）,且脊髓背角pERK的表达水平也明显降低（P<0.05）。pERK活化主要表达于小鼠脊髓背角Ⅰ～Ⅱ层,且与神经元标志物NeuN共标,与星形胶质细胞标志物GFAP和小胶质细胞标志物Iba1不共标。结论：脊髓背角浅层神经元ERK磷酸化可能参与调控DNFB诱导的慢性痒。     

早期运动训练对脊髓损伤大鼠痛觉阈值及脊髓后角胶质细胞活化的影响

目的:观察早期运动训练对T10不完全性SCI大鼠机械性及热刺激痛觉阈值、脊髓后角小胶质细胞和星形胶质细胞活化的影响。方法:将24只成年雌性SD大鼠随机分为:假手术组(Sham组)、SCI-对照组(SCI-Sed组)和SCI-运动组(SCI-TT组)。SCI-Sed组和SCI-TT组使用改良Allen’s法制作T10不完全SCI模型,Sham组只暴露脊髓。SCI-TT组于SCI第8天行减重平板训练。于SCI术前、术后第1、7、14、21、28、35天使用Von Frey单丝及热刺激痛觉测试仪对大鼠的痛觉阈值进行评估。SCI 5周后,使用免疫组化技术对所有大鼠L4—5脊髓进行染色,观察脊髓后角小胶质细胞及星形胶质细胞活化情况,并对大鼠痛觉阈值与胶质细胞活化之间的相关性进行分析。结果:机械性痛觉阈值评估结果显示,SCI术后第1天,SCI-Sed组和SCI-TT组阈值均较Sham组增加(P0.05);之后两组阈值均低于Sham组(P0.05);第21—35天,SCI-TT组阈值明显高于SCI-Sed组(P0.05)。热刺激痛觉阈值结果显示,SCI术后第1天,SCI-Sed组和SCI-TT组痛觉阈值较Sham组均增加(P0.05);SCI 7天后,两组大鼠痛觉阈值均低于Sham组(P0.05);术后14—35天,SCI-TT组痛觉阈值明显高于SCI-Sed组(P0.05)。小角质细胞及星形胶质细胞免疫组化结果显示,SCI-Sed组和SCI-TT组脊髓后角内的阳性细胞数量多于Sham组(P0.05);而SCI-TT组明显少于SCI-Sed组(P0.05)。相关性分析显示,SCI后第35天,痛觉阈值与脊髓后角胶质细胞活化数量之间呈负相关(P0.001)。结论:早期运动训练对缓解SCI大鼠NP的发生有一定作用,其机制可能与抑制脊髓后角胶质活化相关。     

低频电疗对周围神经损伤后白鼠的脊髓后角神经细胞活动电位的影响

目的:应用周围神经损伤的动物模型,探讨低频电疗对被机械性刺激和温度刺激所引发的脊髓后角神经细胞活动电位(膜电位)的影响。方法:用Sprague-Dawley雄性白鼠,制作出周围神经损伤疼痛模型作为实验组。用行动学和电生理学方法测定后腿的躲避反应和脊髓后角神经细胞的膜电位,并与对照组进行比较。结果:①实验组被机械性刺激和温度刺激所引发的躲避反应频率明显高于对照组(P<0.01)。②实验组被机械性刺激和温度刺激所引发的脊髓后角神经细胞膜电位明显高于对照组(P<0.01)。③实验组经低频电刺激10min后,其脊髓后角神经细胞的膜电位明显低于低频电刺激前脊髓后角神经细胞的膜电位(P<0.01)。④在实验组低频电刺激的同时静脉注射纳络酮10min后,其脊髓后角神经细胞的膜电位明显高于未注射纳络酮组(P<0.01)。结论:低频电缓解周围神经损伤所致疼痛的作用机理可能与其促进分泌内啡呔等中枢鸦片样物质,作用于脊髓后角神经细胞,降低其活性有关。