持续机械刺激对大鼠背根神经节TRPV4通道的影响和差异蛋白质组学研究
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摘要
第一部分:大鼠背根神经节机械敏感性离子通道电生理性质的研究
背景
当活细胞和有机体受到环境中的机械刺激时,机械信号随即转化成生物信号,使细胞作出适当反应,此过程被称为机械转导。在机械信号转导过程中,机械敏感性离子通道(mechanosensitive channel,MS通道)起了很重要作用。MS通道是一类通道开放概率随细胞膜张力变化呈现相应变化的离子通道,能够将施加在分子膜上的机械信号转换成电信号或生化信号。自从在鸡胚骨骼肌细胞和蛙肌肉上发现MS通道以来,在许多类型的细胞上都发现了MS通道。1999年,McCarter首次在大鼠背根神经节(dorsal root ganglion,DRG)上发现了机械敏感性全细胞电流,随后有研究小组使用不同的刺激方法在DRG上记录到了几种MS通道,由于细胞类型,刺激方法,记录方法和电极液、浴液成分的不同,这些通道的电生理性质不完全相同,也无法确定是否是同样的通道。
DRG是躯体感觉初级传入神经元细胞体的聚集处,由于其特殊的解剖位置和生理结构特点,易于受到机械压迫刺激,从而产生根性疼痛。但这种病理性疼痛的电生理机制尚不明确。研究大鼠DRG神经元细胞膜上的MS通道的生物物理学特性及通道的分布,可为进一步了解DRG神经元细胞电活动的机制提供依据。
目的
研究大鼠DRG神经元细胞膜上的MS通道的生物物理学特性及通道的分布。
方法
将新生大鼠DRG细胞培养4天后,应用细胞贴附式和内面向外式膜片钳技术记录细胞膜上的MS通道电流,分析通道的电生理性质,如压力-电流关系、电压-电流关系,开放动力学,离子选择性,影响因素和通道在DRG细胞上的分布。本试验采用的机械刺激方式为负压抽吸。
结果
1.压力-电流关系
操纵微电极接近DRG神经元,形成细胞贴附式膜片钳记录方式,保持膜电压(membrane voltage,Vm)为-60mV,在未施加负压时,通道的自发性开放很少。逐渐增大所施负压的值,当负压为-12～-15mmHg时,开始出现内向电流。细胞膜能承受的最大负压约为-50mmHg,更大的负压可以破坏细胞膜。P1/2=36.66±0.455mmHg(n=25)。Vm=-60mV时的平均幅度为-3.40±0.04pA(n=125)。同一电压下,随压力的增大,通道开放概率(NPo)逐渐增大(P＜0.01,n=25),但不同压力产生的电流的幅度无差别(P＞0.05,n=25)。同一电压下,细胞贴附式记录方式,Vm=-60mV,在-50mmHg负压下,平均NPo为0.448±0.03(n=25)。
2.电压-电流关系
形成内面向外式膜片钳记录方式,在持续施加负压-40mmHg,在膜电压为正值时,表现为外向电流,+60mV时电流的弦电导为96.15±3.734 pS(+40～+60mV)。当膜电压为负值时,表现为内向电流,同时表现出内向整流特性,内向电流(-60～0mV)的斜率电导为62.47±2.72 pS。平衡溶液中,平均逆转电位Erev=-2.33±0.255mV。
3.通道动力学
形成内面向外式膜片钳记录方式,Vm=-60mV,当负压为-20mmHg时,双指数拟合所得两个开放常数分别为1.338±0.098ms和13.001±0.649ms,关闭常数分别为2.462±0.267ms和23.386±1.206ms。当负压为-40mmHg时,短开放时间常数和长开放时间常数分别变为1.744±0.195ms和17.92±1.623ms,明显高于-20mmHg压力下的时间常数(both P＜0.05)。此时的短关闭时间常数和长关闭时间常数分别变为2.266±0.154 ms和14.071±0.797ms。与负压为-20mmHg时相比,长关闭时间常数明显减少(P＜0.05),短关闭时间变化不大(P=0.545),可见压力是通过增加开放时间和减少长关闭时间来增加通道的活性的。
4.离子选择性
本研究记录到的通道为非选择性阳离子通道,对阴离子没有通透性。
5.影响因素
该通道对机械刺激的反应可被钆和秋水仙素阻断,河豚毒素可阻断大直径神经元上的电流,对小直径细胞无效。阿米洛利对该电流无效。
6.MS通道的分布
该通道多位于小直径(＜20μm,48.41%)和中等直径(20～30μm,36.30%)神经元上,而在大直径神经元上存在较少(＞30μm,15.29%)。
结论
本部分试验研究了大鼠DRG上MS通道的电导、开放动力学、离子选择性等电生理性质及通道在DRG上的分布。机械刺激可使DRG神经元膜上的MS通道开放,产生电流,传导机械信号。
背景
各种原因导致的背根神经节(dorsal root ganglion，DRG)及周围神经根的持续受压被认为是放射性痛最常见的病因之一。但是由于体内环境的复杂性，单纯机械压迫对神经元的影响至今尚不清楚。目前通常使用在体模型来研究持续机械刺激对DRG的形态、功能、电生理性质、相关基因和蛋白的表达的影响。但在体机械压迫通常会伴随继发性的炎症反应，如缺血、水肿及各种炎性细胞的浸润，使我们无法区分出单独机械刺激的作用。因而有必要建立一种简便、可靠、稳定、研究因素单一的细胞模型，以排除其他因素的影响，明确单纯机械压力对DRG神经元的作用。
在目前已有的各种体外加压模型中，静水压模型可以提供持续的机械压迫。这种方法通过向密闭的培养室内注入混合气体，使培养室内的细胞受压。但是由于神经元细胞培养的特殊性，我们必须验证该模型对神经元的形态和活性是否有影响，并确定合适的培养压力和时间。
DRG神经元细胞膜上存在多种可能参与机械觉传导的离子通道，如transient receptor potential vanilloid receptor 1(TRPV1)，transient receptor potential vanilloid receptor 4(TRPV4)，transient receptor potential channel of melastatin type 8(TRPM8)和transient receptor potential subtype ankyrin 1(TRPA1)等。其中，香草素受体亚家族TRPV4是一种多觉感受器，参与了渗透压和伤害性机械刺激信号的传导。但持续的机械刺激对这些离子通道的影响尚不明确。
研究显示持续压迫DRG可以改变多种离子通道的表达或电生理性质，影响神经递质的合成等。对自发性高血压大鼠的研究也显示，持续的血管壁高张力可明显增加血管壁上压力激活性离子通道的密度。所以我们设想，持续的机械压力或许会影响DRG上的机械感受器的表达和性质。
目标
1．建立DRG持续受压体外模型，确定合适的培养压力和时间。
2。观察持续机械压力对DRG神经元细胞膜上各种与机械传导相关的离子通道，特别是TRPV4通道的表达和性质的影响。
方法
1．建立加压培养细胞模型
DRG取材及细胞培养同论文第一部分。37℃培养2天后换1次培养液，然后将细胞分为12h，24h，48h和72h组，分别放入密封加压培养装置中，通过向容器内输入混合气体，使容器内压力高于大气压。分别使用0mmHg(即正常大气压，作为对照)，20mmHg，40mmHg，80 mmHg和120 mmHg压力培养。
2．MTT法测定细胞活性
将DRG神经元以1×10~4／孔的密度接种于96孔培养板，在不同压力下培养不同时间之后，加入MTT，在酶标检测仪上测定波长490nm处吸光度OD值，各组均以0mmHg压力下培养的DRG的吸光度作为100％，其他各压力下的OD值与之相比较。
3．实时定量PCR检测
从各组细胞中提取RNA，使用实时定量PCR检测TRPV4基因表达的变化。
4。Western blotting检测
从各组细胞中提取蛋白质，使用Western blotting分别检测TRPV1、TRPV4、TRPM8和TRPA1的蛋白质表达量的变化。
5．激光共聚焦检测
使用激光共聚焦检测低渗溶液，激动剂和阻断剂引起DRG神经元内钙离子浓度的变化。并比较不同压力和不同培养时间对钙离子浓度变化的影响。
结果
1．加压模型的建立
各个组之间细胞形态未见的明显差异，说明持续机械压力不会影响细胞的形态。MTT分析显示120 mmHg的压力可以明显抑制48h和72h组细胞的活性(bothP<0．01)，而且对72h组的抑制作用更强(P<0．01)。在72h组，40mmHg和80mmHg的压力均可抑制DRG的活性(both P<0．01)。在其他的培养组中，除120 mmHg外，各种压力强度下细胞的活性相类似。而且，由于80mmHg的压力更接近于病理状态下DRG所受的压力，所以我们后面的研究均采用80mmHg作为压力强度，培养时间选用24h和48h。
2．持续压力对TPRV4基因表达的影响
持续压力可明显增加TRPV4 mRNA的表达(P<0．05)。随受压时间的延长，TRPV4 mRNA的表达逐渐增高，与对照组相比，80mmHg压力下培养24h和48h后，TRPV4mRNA的表达分别增加为各自0mmHg压力对照组的2．31和4．04倍，差异有统计学意义(both P<0．05，SNK)。同时，受压48h组TRPV4 mRNA的表达也明显高于受压24h组(P<0．05，SNK)。
3．持续压力对机械感受相关离子通道蛋白质表达的影响
持续压力可明显增加TRPV4蛋白的表达(P<0．05)。随受压时间的延长，TRPV4蛋白的表达逐渐增高，受压24h和48h后分别升高至为对照组的1．80和4．22倍，差异有统计学意义(both P<0．05，Tukey Test)。TRP家族其他离子通道，如TRPV1，TRPM8和TRPA1，受压前后蛋白的表达未见明显变化(all P>0．05)。
4．持续压力对TRPV4通道性质的影响
持续压力可以明显增加低渗溶液和佛波醇引起的细胞内钙浓度的增加fbothP<0．05)。在正常培养组，30％的低渗溶液可以引起37．0％的DRG细胞内钙浓度增加，峰值为1．67+0．02。80mmHg压力下培养24h和48h后，峰值增加至各自对照组的1．74和2．52倍(both P<0．01，SNK)，对低渗溶液产生反应的DRG细胞比例数也升高(48．4％，X~2=1．86，P>0．05 for 24h group and 61．2％，X~2=7．16，P<0．05for 48h group)。与低渗引起的反应相类似，受压培养24h和48h后，佛波醇引起的细胞内钙浓度的峰值分别为对照组的1．48和2．09倍(Fig 2-8 C．both P<0．01．SNK)，产生反应的DRG细胞比例数也升高，从30．92％升高到42．25％(X~2=2．29，P>0．05 for 24 hgroup)和49．18％(X~2=5．30，P<0．05 for48hgroup)。
结论
1．在一定的压力强度和培养时间内，持续加压培养不会对DRG神经元形态和细胞活性产生显著影响，此模型可观察单纯机械刺激因素对神经元的影响，可以成为一种研究机械压力对DRG神经元的影响的有效体外模型。
2．体外单独的机械刺激可以增加TPRV4基因和蛋白的表达，敏化通道功能，而对TRP家族其他离子通道无明显影响。
背景
背根神经节(dorsal root ganglion，DRG)的持续受压(chronic compression of DRG CCD)可以产生自发性疼痛，机械痛觉过敏和异常疼痛，伴随着神经元的自发性放电增加，动作电位和电流阈值降低。多种离子通道被认为与持续受压后DRG的高兴奋性有关，如电压门控性Na~+通道和K~+通道，超极化激活性阳离子通道等，但CCD导致的机械痛觉过敏和异常疼痛的分子基础还不甚清楚。
DRG上存在多种离子通道，其中瞬时感受器电位离子通道家族中香草素受体亚家族TRPV2，TRPV4和锚蛋白亚家族TRPA1都被认为与机械性疼痛有关。其中TRPV4的作用越来越受到关注。TRPV4为多觉感受器，试验证据支持TRPV4参与多种病理状态下，如炎性因子或者化疗导致的炎性痛模型，机械痛敏的传导。不同病理模型中，TRPV4表达和功能的变化也不完全相同。在化疗导致炎性痛模型中，对TRPV4激动剂产生反应的DRG神经元的比例数增加。但在外伤性或糖尿病性神经痛模型中，TRPV4的表达无明显变化。
我们第二部分的试验已经证明持续的机械刺激可以增加TPRV4的表达和功能。CCD模型中，机械压迫通常会伴随继发性的炎症反应，缺血、水肿及各种炎性细胞的浸润，病理生理环境变化比体外单独的械刺激更加复杂。所以，本部分的研究目的是观察CCD对TRPV4基因、蛋白表达及功能的影响，明确TRPV4在CCD导致的机械性异常疼痛中的作用。
目的
1．观察CCD对TRPV4基因、蛋白表达及功能的影响。
2．明确TRPV4是否参与CCD导致的机械性异常疼痛。
方法
1．CCD模型的建立
99只大鼠随机分成7天组，14天组和28天组，每组均33只大鼠。每个组又分成CCD(n=20)和假手术(n=13)2个亚组。大鼠麻醉后，将“U”形棒的一端沿L4及L5椎间外孔的前壁上方水平插入椎管内，使之挤压L4、L5 DRG。假手术组除不插钢棒压迫神经节外，其余操作同手术组。分别于术后7天，14天和28天处死动物。
2．TRPV4翻译核苷酸干扰
为了测量TRPV4反义核苷酸干扰对机械痛阂值的影响，45只大鼠被随机分为正常组(normal mt，n=18)和CCD(CCD rat，n=27)组，每个组又分成对照组(contr01)，反义核苷酸干扰组(antisense ODN group，AS group)和错配组(mismatchODN group，MM group)等3个亚组。正常组大鼠每个亚组n=6，CCD组大鼠每个亚组n=9。正常组中未接受任何处理的大鼠作为对照，CCD组中只接受CCD手术的大鼠作为对照。
ODN注入蛛网膜下腔，40μg／d，每天一次，共7天。CCD组于手术12h后进行第一次注射。7天后处死动物，取出DRG。
3．神经行为学测定
分别于手术前及手术后7天、14天及28天取材前进行。
1)运动功能(motor function)观察动物自然状态下随意行走的步态，采用记分制：1分=正常步态、足无畸形：2分=正常步态伴明显足畸形；3分=轻度步态障碍伴足下垂；4分=严重步态障碍伴肌无力。
2)机械痛阈值(Mechanical withdrawal threshold，MWT)MWT的测量使用BME-403型VonFrey Monofilaments机械痛刺激仪。将大鼠放在铁丝网上，从低到高依次使用各种强度的Von Frey针丝，刺激动物手术侧足底3、4趾间皮肤，缩爪或舔足为阳性反应。每根针丝测量5次，至少能够引发3次缩爪反应的针丝强度即为机械痛阈值。
4。实时定量PCR检测
从各组大鼠DRG中提取RNA，实时定量PCR检测TRPV4基因表达的变化。
5．Westernblotting检测
从各组大鼠DRG中提取蛋白质，Western blotting检测TRPV4蛋白质表达量的变化。
6．激光共聚焦检测
使用激光共聚焦检测低渗溶液和佛波醇刺激DRG神经元后，细胞内钙离子浓度的变化。并比较各组问的差别。
结果
1．行为学测定
1)运动功能所有动物在损伤前后步态均正常，足无畸形，评分均为1分，损伤前、后各组间无显著性意义。
2)机械痛阈值手术前，各组大鼠的机械缩爪阈基础值无差别(F=1．63，P=-0．16)。与假手术大鼠相比，持续压迫明显降低大鼠的机械痛阈(P<0．001)。手术后7，14和28天的机械痛阈值也存在明显差别(P<0．001)。手术处理与术后天数存在交互作用(P<0．05)。与假手术组相比，手术后机械痛阈值下降，7天达到最低，然后逐渐上升，但术后28天机械痛阈值仍明显低于假手术大鼠(SNK，all P<0．05)。
2．实时定量PCR测量受压前后TRPV4基因的变化
持续机械压迫可以明显增加TRPV4基因的表达(P 3．Western blotting测量TRPV4蛋白质的变化
蛋白质的变化与mRNA的变化相类似，持续机械压迫可以明显增加TRPV4蛋白的表达(P<0．001)。压迫7天后，TRPV4的表达明显增高，为假手术的4．34倍，。随受压时间的延长，TRPV4蛋白的表达逐渐降低，术后14天和28天分别为假手术的3．88和2．47倍，差异有统计学意义(SNK，all P<0．05)。假手术组之间没有明显差别(F=1．21，P=0．412)。
4．持续压迫导致的病理性疼痛与TRPV4
在正常组和CCD组，反义寡脱氧核苷酸可明显抑制TRPV4的合成，但错配寡脱氧核苷酸无明显影响。在正常组，干扰前后大鼠机械痛阈值无变化(尸=0．93)。在CCD组，三个组的基础机械痛阈值无差别(P=0．844)。与基础痛阈值相比，对照组和MM组的机械痛阈值明显下降(Bonferronit-test，both P<0．05)。而在AS组，CCD导致的机械痛阈值降低被部分逆转(Bonferroni t-test，P>0．05)。
5．持续神经节压迫对TRPV4通道功能的影响
TRPV4的基本性质与细胞模型结果相同。持续压迫7天后，30％低渗溶液和佛波醇引起的细胞内钙浓度的峰值产生明显变化(both P<0．01)。与假手术组相比，30％低渗溶液引起的CCD组和MM组钙浓度的峰值明显增加(Tukey Test，both P<0．01)，对低渗溶液产生反应的DRG细胞比例数也明显升高(52．6％，X~2=5．14，P<0．05 for CCD group and 50．8％，X~2=4．32，P<0．01 for MM group)。与CCD组和MM组相比，AS组DRG对低渗溶液的反应明显降低(Tukey Test,bothP<0．05)，产生反应的DRG细胞比例数也明显降低(29．1％，X~2=8．65，P<0．01 vs。CCD group；X~2=7．59，P<0．01 vs．MM group)。与假手术组相比，佛波醇引起的CCD组和MM组钙浓度的峰值明显增加(Tukey Test，both P<0．01)，对佛波醇产生反应的DRG细胞比例数也明显升高(46．1％，X~2=4．25，P<0．05 for CCD group and47．6％，X~2=4．95，P<0．05 for MM group)。佛波醇引起AS组DR(3内钙峰值明显低于CCD组和MM组(Tukey Test，both P<0．05)，AS组起反应的DRG细胞比例数也明显低于CCD组和MM组(26．3％，X~2=7．31，P<0．01 vs．CCD group；X~2=8．22，P<0．01 vs．MM group)。
结论
1．CCD可以上调TRPV4的基因、蛋白表达，敏化通道的功能。
2．TRPV4参与介导CCD导致的机械性异常疼痛。
背景
背根神经节(dorsal root ganglion，DRG)是机体初级感觉传入神经元胞体的聚集处，位于椎间孔内，当椎间盘突出和椎管狭窄等导致椎间孔狭窄时，易于受到压迫。持续压迫DRG(chronic compression of the DRG)可产生损伤侧的异常疼痛，并伴有神经元兴奋性的升高，表现为自发性放电增加和电流阂值降低。但是这种异常疼痛的机制还不清楚。
有研究者使用基因组学和蛋白质组学方法，对病理性神经痛模型的DRG进行了扫查，发现多种与神经元死亡和再生有关的基因和蛋白的表达量在损伤前后产生了变化，包括细胞骨架蛋白，神经递质代谢酶，炎性因子，合成／成熟相关蛋白和髓鞘形成相关蛋白等。这些研究使我们对外周神经损伤后神经退化和微环境改变的机制有了深入的了解。由于不同病理模型的行为学和病理变化不同，其基因和蛋白质的变化也不同。所以研究不同病理状态下的蛋白质的变化，可以帮助我们了解神经病理状态的分子生物学机制。
CCD模型是一种较特别的外周神经损伤模型，其损伤和炎症反应均局限在神经节内。插入钢棒不仅导致机械压迫、DRG缺血、水肿，而且从循环系统、神经元和非神经元释放出的多种炎症因子，使病理环境变得更加复杂。
蛋白质是一切生命活动的基础，蛋白质的结构和功能最终直接影响生命活动的变化。使用蛋白质组学方法从整体角度、高通量分析CCD后DRG蛋白质表达的变化，可以从整体上了解病理过程的发生机制和机体相应的自身保护机制。
目的
使用蛋白质组学方法筛查大鼠DRG持续受压后差异表达的蛋白质，特别是筛查与神经病理性疼痛和组织损伤相关的蛋白。
方法
1 CCD模型的建立
78只Wister大鼠随机分为正常组和CCD组，每组均为39只。CCD模型的建立方法同论文第三部分。28天后处死大鼠，取出DRG。
2行为学测量
测量大鼠运动功能，机械痛阈值(mechanical withdrawal threshold，MWT)和热辐射刺激缩爪反应潜伏期(thermal withdrawl latency)。MWT的测量方法同论文第三部分。热辐射刺激缩爪反应潜伏期使用BME—410A型热痛刺激仪，将热痛刺激仪放在有机玻璃板下方，使光源焦距照射动物后肢足底掌心，电子秒表记录从照射开始到引起后肢回缩反应时的潜伏期作为热痛觉观测指标。
3双向电泳和图像分析
从正常组和CCD组各20只大鼠的L4和L5 DRGs中提取蛋白，Bradford．法定量。双向电泳分离蛋白，银染显色，使用软件进行分析，找出差异表达蛋白。正常组和CCD组之间，斑点体积变化超过3倍的蛋白，为差异表达蛋白。
4蛋白的生物质谱分析鉴定
将选定的蛋白点切下，胰酶消化后，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，选取NCBInr和SwissPmt数据库进行在线检索。
5 Western blotting
使用Western blotting方法验证部分差异表达蛋白质，如膜联蛋白A2，p11，PKCe，GAPDH，热休克蛋白70的变化，方法同论文第二部分。
6 Real-time RT-PCR
使用实时定量PCR方法检测膜联蛋白A2和PKCe基因表达的变化，方法同论文第二部分。
结果
1．行为学测定
CCD明显降低机械痛阈和热辐射刺激缩爪反应潜伏期(both P<0．05)。从第二天开始，机械痛阈下降，到第四天达到最低值，然后逐渐恢复，但直到术后28天，CCD组大鼠的机械痛阈仍明显低于正常对照大鼠(all P<0．05，SNK)。热辐射刺激缩爪反应潜伏期的变化趋势与机械痛阈相似(all P<0．05，SNK)。所有大鼠的运动功能均正常。
2．差异表达蛋白质的鉴定
对照组和CCD组分别得到454±4(n=3)和423±+4(n=3)个蛋白质点。98个蛋白点的表达存在明显差异，质谱分析成功鉴定出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)。
3．Western blotting验证
Westem blotting分析显示膜联蛋白A2，p1 l，PKCe，GAPDH，热休克蛋白70的表达在CCD组均升高，与质谱分析的结果相符。对照组和CCD组p．actin的表达量无明显变化(t=-0．139，P=0．896)，因此适合作为内参。
4．CCD对基因表达的影响
CCD可以明显增加AnnexinA2和PKCε的基因表达，分别增加到对照组的4．55和3．65倍(bothP<0．05)。
结论
我们使用差异蛋白质组学的方法，分析了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PKCe蛋白表达的上调，可能参与了神经性疼痛的发生过程。GAPDH的上调或许参与神经元凋亡，HSP70的表达上调或许和神经保护有关。
PartⅠ: Research of electrophysiological properties of mechanosensitive channels in cultured dorsal root ganglion neurons of neonatal rats
Background
All living organisms face mechanical forces,from the fluid forces around bacterium to the high forces in a human knee during stair climbing.The process of converting physical forces into biochemical signals and integrating these signals into the cellular responses is referred to as mechanotransduction.The mechanosensitive channels(MS channels)play an important role in the mechanotransduction.The idea of MS channels arose originally from studies of specialized mechanosensory neurons. The channel open probability of MS channels changes with the membrane tension and converts mechanical force exerted on the cell membrane into electrical or biochemical signals in physiological processes.Since their discovery in embryonic chick skeletal muscle and frog muscle,MS channels have been found in many cell types.McCarter first identified the whole-cell currents activated by stretch or pressure in dorsal root ganglion(DRG)neurons of rats in 1939.After that,various MS channels activated by various stimuli were reported in DRG.The electrophysiological properties of those MS channels are diverse due to the difference in mechanical stimuli,recording methods,and the composition of pipette solution and bath solution.
DRG neurons are primary afferent neurons that carry sensory signals from the skin,muscles,joints,and visceral organs to the spinal cord.Various mechanical, thermal,or noxious stimuli are known to generate action potentials at peripheral nerve endings of the DRG neurons.DRG is easily mechanical compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae due to the special anatomical position and physiological construction features, resulted in radicular pain.Identification of the biophysics and distribution of the MS channels in the DRG can further our knowledge about the mechanism underlie the electric activity of DRG neurons.
Objective
The aim of this current study is to explore the electrophysiological properties and distribution of mechanosensitive channels in cultured DRG neurons of neonatal rats.
Methods
After cultured for 4 days,the mechanosensitive channels currents of DRG neurons of neonatal rats were recorded and analyzed using cell-attached and inside-out patch-clamp technique.The electrophysiological properties such as pressure-response relationship,current-voltage relationship,channel kinetics,ion selectivity,influential factor and cell-size distribution were analyzed.Membrane stretch was achieved by the application of negative pressure(suction)to a patch pipette.
Results
1.Pressure-response relationship
In cell-attached patches with·the membrane potential of-60 mV,no single-channel currents were observed before applying suction,except for a few patches,which showed spontaneous openings.The negative current was activated by pressure of-12 to -15mmHg.Those channels activated rapidly when suction was applied,kept stably during sustained application of negative pressure and quickly turned off when the suction was released.When the amplitude of the channel current was measured in cell-attached patches,the mean current amplitude was -3.40±0.04pA(n=125)at -60mV of membrane potential.Channel open possibility(NPo)increased as higher pressures were applied to the patch and reached a maximum approxiamate 50mmHg, with P1/2 was 36.66±0.455mmHg(n=25).A higher pressure would destroy the membrane.The mean NPo was 0.448±0.03(n=25)at-60mV of membrane potential and -50mmHg of pressure.
2.Current-voltage relationship
In inside-out patches with -40mmHg pressure,the MS channels exhibited a nearly linear current-voltage relationship in the symmetrical solution.The outward chord conductance was 96.15±3.73 pS(Vm is between +40mV and +60mV)and the inward slope conductance was 62.47±2.72 pS(Vm is between -60mV and 0mV). This kind of channels appeared to be an inward rectifier.The average reversal potential(Erev)was -2.33±0.255 mV.
3.Channels kinetics
Histograms of open or closed-time durations of MS channels activated by suction pressures were best fitted with two exponential functions.Therefore,the channels exhibited short and long openings and closings.Negative pressure of -20 mmHg activated MS channels with open time constants of 1.338±0.098ms and 13.001±0.649ms,and with closed time constants of 2.462±0.267ms and 23.386±1.206ms.When negative pressure of -40 mmHg was applied to the same channels,the duration of short openings and long openings increased significantly 1.744±0.195ms for short openings and 17.92±1.623ms for long openings(both P＜0.05).In contrast,at -40 mmHg,the duration of long closings 14.071±0.797ms (P＜0.05),but not short closings 2.266±0.154 ms(P=0.545),decreased significantly compared with at -20 mmHg.Therefore,greater pressures could increase the channel activities of MS channels largely by increasing the duration of short openings and long openings and decreasing the duration of long closings.
4.Ion selectivity
The MS channels were non-selective cation channels and were not permeable to anion.
5.Influence factors
The current activated by mechanical stimuli could be blockaded by gadolinium and colchicine.The tetrodotoxin could blockade the current in large diameter DRG neurons,with no effect on the small diameter DRG neurons.
6.Cell-size distribution
The MS channels were founded mainly in small(＜20μm,48.41%)and medium (20～30μm,36.30%)diameter DRG neurons and were rarely found in relatively large diameter(＞30μm,15.29%)DRG neurons.
Conclusion
We identified the electrophysiological properties and the distribution of a kind of MS channels in DRG in rats.The mechanical stimuli could activate the MS channels and transduce the mechanical signals.
Background
Chronic compression of the dorsal root ganglion (DRG) or its near nerve root is considered to be one of the most important causative factors of radicular pain. However, the evidence supporting such an argument is incomplete. The effects of pure mechanical pressure on DRG neurons have not been assessed. In vivo animal models are usually used to study the change of the morphology, function, and expression of related genes and proteins under the mechanical pressure. But the effect of the mechanical pressure on the DRG could be easily confounded by other factors and can not be exclusively evaluated in in vivo models. So it is necessary to establish a convenient, reliable, stable, and single factor in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.
Among the current in vitro models, the model of hydrostatic pressure is able to provide sustained compression at different pressure levels. In such a model, pure mechanical loading is generated by compressing the gas phase within a closed culture chamber that contains the culture dishes. But there is little evidence on whether this model is suitable for the study of neurons, given the specificity for the culture of the neurons.
DRG neurons can transmit polymodal sensations, such as temperature and mechanical sensations. Many ion channels, including transient receptor potential vanilloid receptor 1 (TRPV1), transient receptor potential vanilloid receptor 4 (TRPV4), transient receptor potential channel of melastatin type 8 (TRPM8), and transient receptor potential subtype ankyrin 1 (TRPA1), play a potential role in mechanical nociception. Among these channels, the TRPV4 channel is a polymodal receptor, which plays an important role in nociceptive responses to hypotonic and mechanical stimuli.
Recent studies suggested that the chronic compression of the dorsal root ganglion changed the expression and the electrophysiological property of the voltage-dependent ion channels, as well as the synthesis and transportation of the neurotransmitter. Furthermore, Hoyer et al. found that densities of the pressure-activated channels were significantly higher in spontaneously hypertensive and renovascular hypertensive rats compared with their respective normotensive controls. Therefore, we hypothesized the expression or function of the mechanoreceptors may be changed under direct chronic compression.
Objective
1. To establish an in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.
2. To investigate whether pure chronic mechanical compression of the dorsal root
Methods
1. Establishing the in vitro cell model
DRG neurons were prepared as described previously and were placed in a 37℃incubator in a 95% air-5%CO_2 atmosphere. After 2 days the culture medium was changed and the culture dishes were put into the seal chamber, which was designed for incubating the DRG cells in a range from 0 to 200 mm Hg. Compressive loading is generated by compressing the gas phase within the closed culture chamber and the pressure gas consisted of 95% air and 5%CO_2- Pressure levels are monitored continuously by manometer, with atmospheric pressure (760 mm Hg) calibrated to 0 mm Hg. Dishes were cultured under compressive loading as the test groups and other dishes were cultured without loading as the control group.
2. MTT test
DRGs were cultured on 96-well plates and there were 8 wells in each condition. After cultured under different pressure for different durations, cell morphology was observed under inverted microscope and the cell activation was assessed with MTT test. The original culture medium was then removed, and l00μi 1mg/ ml of MTT was added. After 150μl dimethyl-sulfoxide was added and the plates were oscillated for 10min to fully dissolve the crystal, the absorption at 490 nm was measured with ELISA analyzer. A mean value from 8 wells was calculated as one sample and 3 samples were taken for each condition. Cell viability of control cells was taken as 100%.
3. Real-time PCR
The mRNA levels of TRPV4 in DRG neurons cultured with different pressures and different durations were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
4. Western blotting
Western blotting was performed to investigate the proteins expression of TRPV1, TRPV4, TRPM8, and TRPA1 in DRG neurons cultured with different pressures and different durations.
5. Laser scanning confocal microscope
The responses to the hypotonic solution, TRPV4 agonist, and antagonist were assessed by calcium imaging using the laser scanning confocal microscope.
Results
1. Effect of mechanical pressure on cell morphology and viability
There were no significant changes in cell morphology between the control and the compressed cells. Pressure level only at 120 mmHg significantly inhibited DRG cell growth (P<0..05 vs. 0 mmHg, Bonferroni t-test). The mechanical compression induced a significantly inhibitory effect on DRG viability only in 72h group (P<0.01, Bonferroni t-test), but not in other groups. Therefore, 80 mmHg pressure level was chosen in the current study as the test compression.
2. Effect of mechanical pressure on gene expression
Gene expression was monitored after sustained compression at 0 mmHg (control) and 80 mmHg for 24 hours and 48 hours. The mechanical pressure significantly increased the TRPV4 gene level (P<0.05). Furthermore, gene expression was dependent on the interaction between pressure levels and duration of compressions (P <0.001). Post hoc analysis revealed that compression at 80 mm Hg for 24h and 48h significantly increased the gene transcription of TRPV4 to 2.31-fold and 4.04-fold as compared with the control (both P<0.05, SNK), respectively. Similarly, TRPV4 mRNA level in the 48h compression-exposed cells was significant higher than that in the 24h group (P<0.05, SNK).
3. Effect of mechanical pressure on the protein expression in DRG neurons
The DRG neurons were cultured under 80 mmHg for 0 hour (control), 24 hours, and 48 hours. Mechanical pressure significantly increased the TRPV4 protein level (P<0.05). Post hoc showed compression-exposure (80 mmHg) for 24h and 48h induced a significantly up-regulation in TRPV4 protein levels, which were 1.80-fold and 4.22-fold higher than the control, respectively (both P<0.05, Tukey Test). By contrast, TRPV1, TRPM8 and TRPA1 expression showed no significant changes following sustained compression for 24 and 48h (all P>0.05).
4. Effect of mechanical pressure on TRPV4 Ca~(2+) responses
Hypotonic stimulation increased intracellular Ca~(2+) concentration in an osmolarity-dependent manner in DRG (P<0.001). Pre-treatment with compression caused an increase in the magnitude of Ca~(2+) response, as well as the percentage of cells responding to hypotonic stimulus. When challenged with 30% hypotonic solution, 37.0% neurons showed increase in [Ca~(2+)]_i with the magnitude was 1.67±0.02. In the 24h and 48h groups, 48.4% (x~2=1.86, P>0.05) and 61.2% (X~2=7.16, P<0.05) neurons showed response to hypotonic solution, with the 1.74-fold and 2.52-fold increase in the magnitude of Ca~(2+) response compared to the control (both P<0.01, SNK) respectively. Similar to the hypotonic solution -induced Ca~(2+) response, the response to 4a-PDD showed the increased magnitude of Ca~(2+) response with the longer duration of mechanical compression applied, which increased to 1.48-fold and 2.09-fold compared to the control (both P<0.01, SNK), respectively. The percentage of neurons responding to 4α-PDD was also increased from 30.92% to 42.25 % (X~2=2.29, P>0.05 for 24h group) and 49.18% (X~5.30, P<0.05 for 48h group) . Furthermore, there is an interaction between pressure levels and duration of compressions on the Ca~(2+) response (P <0.001).
Conclusion
1. In a certain culture duration and pressure, pure mechanical pressure alone has no effect on the morphology and cell activation of DRG neurons and the model of hydrostatic pressure is suitable to investigate how the neurons respond to mechanical compression.
2. Pure mechanical pressure alone can induce the upregulation of mRNA and protein expression of TRPV4 without affecting other TRPs. Meanwhile, the mechanical pressure also enhances the function of the TRPV4 channels.
Background
Mechanical compression of the nerve root and the dorsal root ganglion (DRG), by disc herniation or spinal stenosis is one of the major causes of radicular pain in humans. Chronic compression of DRG (CCD) in animals results in spontaneous pain, mechanical hyperalgesia and allodynia. In association with these painful behaviors spontaneous activity, lowered threshold currents, and action potential thresholds have been shown in the neuronal somata of the compressed ganglion. Ion channels such as voltage-gated Na~+ and K~+, and hyperpolarization-activated cation current may contribute to the hyperexcitability of DRG neurons after CCD. However, to date the ionic mechanisms underlying mechanical hyperalgesia and allodynia are not fully understood.
Ion channels in DRG neurons, including the transient receptor potential vanilloidreceptor 2 (TRPV2), transient receptor potential vanilloid receptor 4 (TRPV4), and transient receptor potential subtype ankyrin 1 (TRPA1), may also play a role in mechanical nociception. TRPV4 is a polymodal receptor and it has been shown to mediate nociceptive behaviors to hypotonic and mechanical stimuli under pathological conditions. Chemotherapy (Taxol) has been shown to increase the percentage of the DRG neurons respective to TRPV4 agonists. However, no significant changes in the expression or function of TRPV4 were found in traumatic or diabetic neuropathy pain models. Thus, the role of TRPV4 channel in neuropathic pain is controversial.
We have shown in the second part of the thesis that the pure sustained mechanical pressure can increase the expression levels and sensitized the ion function of TRPV4. In CCD model, the mechanical compression associated with the secondary inflammatory process, such as ischemic, edema, and inflammatory cells infiltrate makes the pathophysiological conditions much more complex. Therefore, the aim of this study was to investigate the effects of CCD on the levels of TRPV4 mRNA and protein expression to determine the role of TRPV4 in CCD-induced mechanical allodynia.
Objective
1. To investigate the effects of CCD on the expression levels of mRNA and protein and the function of TRPV4.
2. To determine the role of TRPV4 in CCD-induced mechanical allodynia.
Methods
1. Establish CCD model
Ninty-nine rats were randomly divided into three groups, corresponding to 7, 14,and 28 days post-CCD. Each group was sub-divided into a sham and a CCD group (n=13 for sham and n=20 for CCD). In CCD rats, under pentobarbital sodium anesthesia, the paraspinal muscles were separated to expose the transverse process and intervertebral foramina of L_4 and L_5 unilaterally as previously described. A stainless steel U-shaped rod (0.63 mm diam and 4 mm length) was inserted into each foramen to compress the DRG, one at L_4 and the other at the L_5 ganglion. The muscle and skin layers were then sutured. Penicillin was injected to prevent infection. Sham group underwent the same surgical procedure as described, but without the insertion of the rods. Animals were sacrificed at 7,14, and 28 days post-CCD, respectively.
2. Antisense oligodeoxynucleotide treatment
To determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced mechanical allodynia and calcium response, 18 normal rats and 27 CCD rats were treated with spinal intrathecal administration of TRPV4 antisense ODN and mismatch ODN, respectively. Each group was sub-divided into control group, TRPV4 antisense ODN group (AS group), and mismatch ODN group (MM group) (n=6 in normal rats and n=9 in CCD rats for each sub-group). ODN was reconstituted in nuclease-free 0.9% NaCl (10μg/μl) and administered into the spinal intrathecal space at a dose of 40μg , once a day for 7 days until the animals were sacrificed.
3. Neuroethology measurement
The motor function and MWT were measured pre-CCD and right before the animals were sacrificed at 7,14, and 28 days post-CCD, respectively.
1) Walk gait pattern was assessed as the index of motor function. "1" indicates normal gait, without foot deformities. "2" indicates normal gait with obvious foot deformities. "3" indicates slight gait disturbance with foot-drop. "4" indicates serious gait disturbance with myashtnia.
2) Mechanical withdrawal threshold (MWT) was measured with a von Frey hair monofilament with logarithmically incremental stiffness (0.09-17.30 g). The von Frey hair was applied through the mesh floor to the plantar skin of the hindpaw in an ascending order. The MWT was defined as the lowest force that evoked a brisk withdrawal response to at least three of five stimuli. A positive response was noted if the paw was immediately withdrawn. The upper limit for testing was 17.30 g hair.
4. Real-time PCR
The levels of TRPV4 gene expression in different groups were assessed using real-time RT-PCR.
5. Western blotting
The levels of TRPV4 protein expression in different groups were assessed using Western blotting.
6. Laser scanning confocal microscope measurementThe calcium responses to hypotonic solution and 4a-phorbol 12, 13-didecanoate (4a-PDD) were assessed following sham surgery, CCD, spinal application of TRPV4 antisense ODN, and mismatch ODN with laser scanning confocal microscope.
Results
1. Neuroethology measurement
1) The walk gait pattern was normal and no foot deformities were found in all rats pre- and post-CCD. The score was "1". There was no significant difference between control and CCD groups.
2) The MWT in CCD group was significantly lower than the sham group (F=39.97, P<0.001). There was also a significant difference in MWT at 7, 14, and 28 days post-CCD (F=8.03, P<0.001). Furthermore, the MWT was dependent on the interaction between the groups and the days post-CCD (F=3.16, P<0.05). The MWT was lower in the CCD group than the sham group at 7, 14, and 28 days post-surgery (SNK, all P<0.05), with the lowest level at 7 days post-CCD.
2. The effect of CCD on the levels of TRPV4 mRNA expression
The levels of TRPV4 mRNA expression were increased significantly in CCD group when compared with the sham group (F=240.02, P<0.001). There were also significant differences in the levels of TRPV4 mRNA expression at different days post-CCD (F=14.77, P<0.001). The levels of TRPV4 mRNA expression were also dependent on the interaction between the groups and the days post-CCD (F=14.22, P<0.001). The levels of gene expression were significantly increased at 7, 14, and 28 days in CCD group when compared with the sham group (SNK, all P<0.05). There was no significant changes in the level of mRNA expression in sham group (SNK, all P>0.05), but the mRNA level was significantly higher at 7 days than that at 14 and 28 days post-CCD (SNK, both P<0.05).
3. The effect of CCD on the levels of TRPV4 protein expression
The levels of TRPV4 protein expression were increased significantly in CCD group when compared with the sham group (F=451.52, P<0.001). There were also significant differences in the levels of TRPV4 protein expression at different days post-CCD (F=25.99, P<0.001). The levels of TRPV4 protein expression were also dependent on the interaction between the groups and the days post-CCD (F=23.99, P<0.001). There were significantly increased at 7, 14, and 28 days in CCD group, compared with the sham group (SNK, all P<0.05). The protein level was significantly higher at 7 days than at 14 and 28 days post-CCD (SNK, both P<0.05).
Furthermore, the sustained increase in the levels of TRPV4 mRNA and protein expression was associated with the decreases in MWT in CCD group.
4. The effect of antisense oligodeoxynucleotide treatment on CCD-induced mechanical allodynia
The antisense ODN, but not the mismatch ODN, significantly inhibited the TRPV4 expression in normal rats (13.75±1.05%, 2.65±0.071%, 13.8±0.7% for control, AS, and MM group, respectively; F=77.79, P<0.005) and CCD rats (42.2±7.19%, 8.75±0.25%, 44.6±6% for control, AS, and MM group, respectively; F=13.93, P<0.05). In CCD rats, the MWTs were similar at baseline (Bonferroni t-test, all P>0.05), but after intrathecal ODN treatment, the MWT was decreased significantly in control group and MM group when compared with the baseline (Bonferronit-test, both P<0.05). In contrast, mechanical allodynia was partly reversed in AS group (Bonferroni t-test, P>0.05 compared with baseline). The MWT of normal rats were not changed following intrathecal AS or MM (F=0.0751, P= 0.93).
5. Laser scanning confocal microscope measurement
The fluorescence ratio of calcium responsive to 30% hypotonic solution and 3μM 4a-PDD were significantly differed between different groups (F=148.46, P<0.01; F=173.26, .P<0.01, respectively). The percentage of the DRG neurons responsive to hypotonicity was upregulated in CCD group (52.6%, X~2=5.14, P<0.05) and in MM group (50.8%, X~2=4.32, P<0.01) when compared with sham group. The hypotonicity-induced increase in fluorescence ratio of calcium was also enhanced in CCD group and MM group when compared with sham group (Tukey Test, both P<0.01). Treatment with TRPV4 antisense significantly decreased the fluorescence ratio when compared with the CCD group and MM group (Tukey Test, both P<0.05). The percentage was also significantly decreased in AS group (29.1%) when compared with the CCD group ( X~2=8.65, P<0.01) and MM group X~2=7.59, P<0.01). Similar to the hypotonic solution-induced Ca~(2+) response, the percentage of DRG neurons responsive to the 4α-PDD was significantly upregulated in CCD group (46.1%, X~2=4.25, P<0.05) and MM group (47.6%, X~2=4.95, P<0.05) when compared with the sham group. The fluorescence ratio of calcium was enhanced in CCD group and MM group compared with the sham group (Tukey Test, both P<0.05). The percentage was significantly decreased in AS group (26.3%, X~2=7.31, P<0.01 vs. CCD group; X~2=8.22, P<0.01 vs. MM group) and the fluorescence ratio reduced significantly compared with CCD group and MM group (Tukey Test, both P<0.05).
Conclusions
1. It was shown that CCD in rats increases the levels of TRPV4 mRNA and protein expression, in addition to an enhancement in the calcium response to hypotonic stimuli and 4α-PDD.
2. TRPV4 plays a crucial role in CCD-induced mechanical allodynia.
Background
The dorsal root ganglion (DRG) in the intervertebral foramen has an important role in the pathogenesis of low back pain and sciatica in patients with disc hemiation and spinal canal stenosis, because primary sensory neurons with their cell bodies are present in this structure. DRG is easily stimulated or compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae. The chronic compression of the DRG produces ipsilateral cutaneous allodynia that is associated with an increased excitability of neuronal somatas in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. But the underlying mechanisms are still not fully elucidated.
Global gene expression and proteomics studies have discovered a large number of genes and proteins that are regulated in vivo after peripheral nerve injury in animal models of neuropathic pain. Many genes and proteins have been identified to be related to neuronal cell death and regeneration, including cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, growth factors, and signal transduction molecules, inflammation factor, protein synthesis/maturation, and myelination in the nerve. These studies have extended our knowledge about cellular and molecular changes in the degenerating nerve and its microenvironment after peripheral nerve injury. Since there are apparent behavioral and morphological differences among the most utilized animal neuropathy models, comparison of protein expression patterns from different types of injuries might help us understand elaborate molecular and cellular mechanisms underlying diverse peripheral neuropathy.
Among the neuropathic pain models, the chronic compression of the dorsal root ganglion (CCD) model is unique in that the injury and inflammation are located in the ganglion. It is reported that in the CCD model, the acute compression to the nerve root induced endoneurial edema and the associated DRG edema. The ischemia along with any mechanical trauma caused by the compression could initiate an inflammatory reaction and the release of a variety of inflammatory mediators derived from the blood circulation, neurons as well as nonneuronal cells in the ganglion. Thus, the molecular and cellular changes that underlie peripheral nerve injury are complex and identification of proteins involved will contribute significantly to our understanding of the mechanisms of neuropathic pain and neuroprotection.
Objective
The aim of the study was to identify the differential protein expressions related to neuropathic pain and neuroprotection in DRG following CCD in rats.
Methods
1. CCD surgery
Seventy-eight adult male Wistar rats weighing 150-180 g (Shandong University) were randomly divided into CCD group and normal control group, 39 in each group. In CCD rats, under pentobarbital sodium anesthesia, the stainless steel U-shaped rods were inserted into the intervertebral foramina of L_4 and L_5 as described in part 3. Animals were sacrificed at 28 days after surgery by decapitation
2. Measurement of mechanical withdrawal threshold and thermal withdrawal latency
Mechanical withdrawal threshold (MWT) was measured with von Frey monofilaments as described in part 3. Thermal allodynia was assessed with paw withdrawal latencies to radiant heat. The radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw when in contact with the floor. The paw withdrawal latencies per animal were obtained for five times with intervals of 5 min.
3. 2-DE and image analysis
The proteins from L_4 and L_5 DRGs of 20 rats in each group were extracted and quantificated with the Bradford method. The differential proteins expression between CCD group and control group were detected using two-dimensional gel electrophoresis (2-DE) followed by silver staining visualization and comparative analysis with the software of ImageMaster 2D Platinum 5.0. Three independent repeats of each sample were performed. The threshold as the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the CCD and control groups.
4. Mass spectrometry for protein identification
Selected spots were located and digested with trypsin. Then the peptide mass fingerprints (PMFs) of differential proteins expressed between the CCD and control groups were generated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and interpreted utilizing Mascot produced by Matrix Science against the NCBInr database.
5. Western blotting
Some of the identified proteins were further examined by Western blotting as described in part 2.
6. Real-time RT-PCR
The gene expression of annexin A2 and PKCεwere measured with real-time RT-PCR as described in part 2.
Results
1. Neuroethology measurement
The chronic compression produced marked mechanical and thermal allodynia, indicated by significantly decreased mechanical thresholds and thermal withdrawal latency compared with the normal control group (F=95.40, P<0.05; F=86.19, P<0.01, respectively). All rats walked normally after surgery which indicated that the surgery did not injury the motor behavior.
2. Identification of the differential proteins
A total of 98 protein spots were detected with significant changes in their expression levels after CCD and 15 protein spots were identified by MALDI-TOF MS analysis. Of these proteins, annexin A2, protein kinase C epsilon (PKCe), glyceraldehyde-3-phosphate dehydrogenases (GAPDH), and heat shock protein 70 (HSP70) were up-regulated significantly compared with the normal control.
3. Confirmation by Western blotting
The results of Western blotting showed consistent data with the results of the proteomic analysis. There was no significant difference in protein levels ofβ-actin between control and CCD groups (t = -0.139, P= 0.896).
4. Effect of mechanical pressure on gene expression
Quantitative real-time RT-PCR experiments indicated that CCD-induced increase the gene levels of annexin A2 and PKCe.
Conclusions
These proteomic results suggest that chronic DRG compression is associated with the upregulation of annexin A2 and PKCεand their related genes, with may be related to the neuropathic pain. The upregulation of GAPDH and HSP70 suggests that there exist concurrent processes of nervous injury and neuroprotection in the course of neuropathic pain.
背景
当活细胞和有机体受到环境中的机械刺激时,机械信号随即转化成生物信号,使细胞作出适当反应,此过程被称为机械转导。在机械信号转导过程中,机械敏感性离子通道(mechanosensitive channel,MS通道)起了很重要作用。MS通道是一类通道开放概率随细胞膜张力变化呈现相应变化的离子通道,能够将施加在分子膜上的机械信号转换成电信号或生化信号。自从在鸡胚骨骼肌细胞和蛙肌肉上发现MS通道以来,在许多类型的细胞上都发现了MS通道。1999年,McCarter首次在大鼠背根神经节(dorsal root ganglion,DRG)上发现了机械敏感性全细胞电流,随后有研究小组使用不同的刺激方法在DRG上记录到了几种MS通道,由于细胞类型,刺激方法,记录方法和电极液、浴液成分的不同,这些通道的电生理性质不完全相同,也无法确定是否是同样的通道。
DRG是躯体感觉初级传入神经元细胞体的聚集处,由于其特殊的解剖位置和生理结构特点,易于受到机械压迫刺激,从而产生根性疼痛。但这种病理性疼痛的电生理机制尚不明确。研究大鼠DRG神经元细胞膜上的MS通道的生物物理学特性及通道的分布,可为进一步了解DRG神经元细胞电活动的机制提供依据。
目的
研究大鼠DRG神经元细胞膜上的MS通道的生物物理学特性及通道的分布。
方法
将新生大鼠DRG细胞培养4天后,应用细胞贴附式和内面向外式膜片钳技术记录细胞膜上的MS通道电流,分析通道的电生理性质,如压力-电流关系、电压-电流关系,开放动力学,离子选择性,影响因素和通道在DRG细胞上的分布。本试验采用的机械刺激方式为负压抽吸。
结果
1.压力-电流关系
操纵微电极接近DRG神经元,形成细胞贴附式膜片钳记录方式,保持膜电压(membrane voltage,Vm)为-60mV,在未施加负压时,通道的自发性开放很少。逐渐增大所施负压的值,当负压为-12～-15mmHg时,开始出现内向电流。细胞膜能承受的最大负压约为-50mmHg,更大的负压可以破坏细胞膜。P1/2=36.66±0.455mmHg(n=25)。Vm=-60mV时的平均幅度为-3.40±0.04pA(n=125)。同一电压下,随压力的增大,通道开放概率(NPo)逐渐增大(P＜0.01,n=25),但不同压力产生的电流的幅度无差别(P＞0.05,n=25)。同一电压下,细胞贴附式记录方式,Vm=-60mV,在-50mmHg负压下,平均NPo为0.448±0.03(n=25)。
2.电压-电流关系
形成内面向外式膜片钳记录方式,在持续施加负压-40mmHg,在膜电压为正值时,表现为外向电流,+60mV时电流的弦电导为96.15±3.734 pS(+40～+60mV)。当膜电压为负值时,表现为内向电流,同时表现出内向整流特性,内向电流(-60～0mV)的斜率电导为62.47±2.72 pS。平衡溶液中,平均逆转电位Erev=-2.33±0.255mV。
3.通道动力学
形成内面向外式膜片钳记录方式,Vm=-60mV,当负压为-20mmHg时,双指数拟合所得两个开放常数分别为1.338±0.098ms和13.001±0.649ms,关闭常数分别为2.462±0.267ms和23.386±1.206ms。当负压为-40mmHg时,短开放时间常数和长开放时间常数分别变为1.744±0.195ms和17.92±1.623ms,明显高于-20mmHg压力下的时间常数(both P＜0.05)。此时的短关闭时间常数和长关闭时间常数分别变为2.266±0.154 ms和14.071±0.797ms。与负压为-20mmHg时相比,长关闭时间常数明显减少(P＜0.05),短关闭时间变化不大(P=0.545),可见压力是通过增加开放时间和减少长关闭时间来增加通道的活性的。
4.离子选择性
本研究记录到的通道为非选择性阳离子通道,对阴离子没有通透性。
5.影响因素
该通道对机械刺激的反应可被钆和秋水仙素阻断,河豚毒素可阻断大直径神经元上的电流,对小直径细胞无效。阿米洛利对该电流无效。
6.MS通道的分布
该通道多位于小直径(＜20μm,48.41%)和中等直径(20～30μm,36.30%)神经元上,而在大直径神经元上存在较少(＞30μm,15.29%)。
结论
本部分试验研究了大鼠DRG上MS通道的电导、开放动力学、离子选择性等电生理性质及通道在DRG上的分布。机械刺激可使DRG神经元膜上的MS通道开放,产生电流,传导机械信号。
背景
各种原因导致的背根神经节(dorsal root ganglion，DRG)及周围神经根的持续受压被认为是放射性痛最常见的病因之一。但是由于体内环境的复杂性，单纯机械压迫对神经元的影响至今尚不清楚。目前通常使用在体模型来研究持续机械刺激对DRG的形态、功能、电生理性质、相关基因和蛋白的表达的影响。但在体机械压迫通常会伴随继发性的炎症反应，如缺血、水肿及各种炎性细胞的浸润，使我们无法区分出单独机械刺激的作用。因而有必要建立一种简便、可靠、稳定、研究因素单一的细胞模型，以排除其他因素的影响，明确单纯机械压力对DRG神经元的作用。
在目前已有的各种体外加压模型中，静水压模型可以提供持续的机械压迫。这种方法通过向密闭的培养室内注入混合气体，使培养室内的细胞受压。但是由于神经元细胞培养的特殊性，我们必须验证该模型对神经元的形态和活性是否有影响，并确定合适的培养压力和时间。
DRG神经元细胞膜上存在多种可能参与机械觉传导的离子通道，如transient receptor potential vanilloid receptor 1(TRPV1)，transient receptor potential vanilloid receptor 4(TRPV4)，transient receptor potential channel of melastatin type 8(TRPM8)和transient receptor potential subtype ankyrin 1(TRPA1)等。其中，香草素受体亚家族TRPV4是一种多觉感受器，参与了渗透压和伤害性机械刺激信号的传导。但持续的机械刺激对这些离子通道的影响尚不明确。
研究显示持续压迫DRG可以改变多种离子通道的表达或电生理性质，影响神经递质的合成等。对自发性高血压大鼠的研究也显示，持续的血管壁高张力可明显增加血管壁上压力激活性离子通道的密度。所以我们设想，持续的机械压力或许会影响DRG上的机械感受器的表达和性质。
目标
1．建立DRG持续受压体外模型，确定合适的培养压力和时间。
2。观察持续机械压力对DRG神经元细胞膜上各种与机械传导相关的离子通道，特别是TRPV4通道的表达和性质的影响。
方法
1．建立加压培养细胞模型
DRG取材及细胞培养同论文第一部分。37℃培养2天后换1次培养液，然后将细胞分为12h，24h，48h和72h组，分别放入密封加压培养装置中，通过向容器内输入混合气体，使容器内压力高于大气压。分别使用0mmHg(即正常大气压，作为对照)，20mmHg，40mmHg，80 mmHg和120 mmHg压力培养。
2．MTT法测定细胞活性
将DRG神经元以1×10~4／孔的密度接种于96孔培养板，在不同压力下培养不同时间之后，加入MTT，在酶标检测仪上测定波长490nm处吸光度OD值，各组均以0mmHg压力下培养的DRG的吸光度作为100％，其他各压力下的OD值与之相比较。
3．实时定量PCR检测
从各组细胞中提取RNA，使用实时定量PCR检测TRPV4基因表达的变化。
4。Western blotting检测
从各组细胞中提取蛋白质，使用Western blotting分别检测TRPV1、TRPV4、TRPM8和TRPA1的蛋白质表达量的变化。
5．激光共聚焦检测
使用激光共聚焦检测低渗溶液，激动剂和阻断剂引起DRG神经元内钙离子浓度的变化。并比较不同压力和不同培养时间对钙离子浓度变化的影响。
结果
1．加压模型的建立
各个组之间细胞形态未见的明显差异，说明持续机械压力不会影响细胞的形态。MTT分析显示120 mmHg的压力可以明显抑制48h和72h组细胞的活性(bothP<0．01)，而且对72h组的抑制作用更强(P<0．01)。在72h组，40mmHg和80mmHg的压力均可抑制DRG的活性(both P<0．01)。在其他的培养组中，除120 mmHg外，各种压力强度下细胞的活性相类似。而且，由于80mmHg的压力更接近于病理状态下DRG所受的压力，所以我们后面的研究均采用80mmHg作为压力强度，培养时间选用24h和48h。
2．持续压力对TPRV4基因表达的影响
持续压力可明显增加TRPV4 mRNA的表达(P<0．05)。随受压时间的延长，TRPV4 mRNA的表达逐渐增高，与对照组相比，80mmHg压力下培养24h和48h后，TRPV4mRNA的表达分别增加为各自0mmHg压力对照组的2．31和4．04倍，差异有统计学意义(both P<0．05，SNK)。同时，受压48h组TRPV4 mRNA的表达也明显高于受压24h组(P<0．05，SNK)。
3．持续压力对机械感受相关离子通道蛋白质表达的影响
持续压力可明显增加TRPV4蛋白的表达(P<0．05)。随受压时间的延长，TRPV4蛋白的表达逐渐增高，受压24h和48h后分别升高至为对照组的1．80和4．22倍，差异有统计学意义(both P<0．05，Tukey Test)。TRP家族其他离子通道，如TRPV1，TRPM8和TRPA1，受压前后蛋白的表达未见明显变化(all P>0．05)。
4．持续压力对TRPV4通道性质的影响
持续压力可以明显增加低渗溶液和佛波醇引起的细胞内钙浓度的增加fbothP<0．05)。在正常培养组，30％的低渗溶液可以引起37．0％的DRG细胞内钙浓度增加，峰值为1．67+0．02。80mmHg压力下培养24h和48h后，峰值增加至各自对照组的1．74和2．52倍(both P<0．01，SNK)，对低渗溶液产生反应的DRG细胞比例数也升高(48．4％，X~2=1．86，P>0．05 for 24h group and 61．2％，X~2=7．16，P<0．05for 48h group)。与低渗引起的反应相类似，受压培养24h和48h后，佛波醇引起的细胞内钙浓度的峰值分别为对照组的1．48和2．09倍(Fig 2-8 C．both P<0．01．SNK)，产生反应的DRG细胞比例数也升高，从30．92％升高到42．25％(X~2=2．29，P>0．05 for 24 hgroup)和49．18％(X~2=5．30，P<0．05 for48hgroup)。
结论
1．在一定的压力强度和培养时间内，持续加压培养不会对DRG神经元形态和细胞活性产生显著影响，此模型可观察单纯机械刺激因素对神经元的影响，可以成为一种研究机械压力对DRG神经元的影响的有效体外模型。
2．体外单独的机械刺激可以增加TPRV4基因和蛋白的表达，敏化通道功能，而对TRP家族其他离子通道无明显影响。
背景
背根神经节(dorsal root ganglion，DRG)的持续受压(chronic compression of DRG CCD)可以产生自发性疼痛，机械痛觉过敏和异常疼痛，伴随着神经元的自发性放电增加，动作电位和电流阈值降低。多种离子通道被认为与持续受压后DRG的高兴奋性有关，如电压门控性Na~+通道和K~+通道，超极化激活性阳离子通道等，但CCD导致的机械痛觉过敏和异常疼痛的分子基础还不甚清楚。
DRG上存在多种离子通道，其中瞬时感受器电位离子通道家族中香草素受体亚家族TRPV2，TRPV4和锚蛋白亚家族TRPA1都被认为与机械性疼痛有关。其中TRPV4的作用越来越受到关注。TRPV4为多觉感受器，试验证据支持TRPV4参与多种病理状态下，如炎性因子或者化疗导致的炎性痛模型，机械痛敏的传导。不同病理模型中，TRPV4表达和功能的变化也不完全相同。在化疗导致炎性痛模型中，对TRPV4激动剂产生反应的DRG神经元的比例数增加。但在外伤性或糖尿病性神经痛模型中，TRPV4的表达无明显变化。
我们第二部分的试验已经证明持续的机械刺激可以增加TPRV4的表达和功能。CCD模型中，机械压迫通常会伴随继发性的炎症反应，缺血、水肿及各种炎性细胞的浸润，病理生理环境变化比体外单独的械刺激更加复杂。所以，本部分的研究目的是观察CCD对TRPV4基因、蛋白表达及功能的影响，明确TRPV4在CCD导致的机械性异常疼痛中的作用。
目的
1．观察CCD对TRPV4基因、蛋白表达及功能的影响。
2．明确TRPV4是否参与CCD导致的机械性异常疼痛。
方法
1．CCD模型的建立
99只大鼠随机分成7天组，14天组和28天组，每组均33只大鼠。每个组又分成CCD(n=20)和假手术(n=13)2个亚组。大鼠麻醉后，将“U”形棒的一端沿L4及L5椎间外孔的前壁上方水平插入椎管内，使之挤压L4、L5 DRG。假手术组除不插钢棒压迫神经节外，其余操作同手术组。分别于术后7天，14天和28天处死动物。
2．TRPV4翻译核苷酸干扰
为了测量TRPV4反义核苷酸干扰对机械痛阂值的影响，45只大鼠被随机分为正常组(normal mt，n=18)和CCD(CCD rat，n=27)组，每个组又分成对照组(contr01)，反义核苷酸干扰组(antisense ODN group，AS group)和错配组(mismatchODN group，MM group)等3个亚组。正常组大鼠每个亚组n=6，CCD组大鼠每个亚组n=9。正常组中未接受任何处理的大鼠作为对照，CCD组中只接受CCD手术的大鼠作为对照。
ODN注入蛛网膜下腔，40μg／d，每天一次，共7天。CCD组于手术12h后进行第一次注射。7天后处死动物，取出DRG。
3．神经行为学测定
分别于手术前及手术后7天、14天及28天取材前进行。
1)运动功能(motor function)观察动物自然状态下随意行走的步态，采用记分制：1分=正常步态、足无畸形：2分=正常步态伴明显足畸形；3分=轻度步态障碍伴足下垂；4分=严重步态障碍伴肌无力。
2)机械痛阈值(Mechanical withdrawal threshold，MWT)MWT的测量使用BME-403型VonFrey Monofilaments机械痛刺激仪。将大鼠放在铁丝网上，从低到高依次使用各种强度的Von Frey针丝，刺激动物手术侧足底3、4趾间皮肤，缩爪或舔足为阳性反应。每根针丝测量5次，至少能够引发3次缩爪反应的针丝强度即为机械痛阈值。
4。实时定量PCR检测
从各组大鼠DRG中提取RNA，实时定量PCR检测TRPV4基因表达的变化。
5．Westernblotting检测
从各组大鼠DRG中提取蛋白质，Western blotting检测TRPV4蛋白质表达量的变化。
6．激光共聚焦检测
使用激光共聚焦检测低渗溶液和佛波醇刺激DRG神经元后，细胞内钙离子浓度的变化。并比较各组问的差别。
结果
1．行为学测定
1)运动功能所有动物在损伤前后步态均正常，足无畸形，评分均为1分，损伤前、后各组间无显著性意义。
2)机械痛阈值手术前，各组大鼠的机械缩爪阈基础值无差别(F=1．63，P=-0．16)。与假手术大鼠相比，持续压迫明显降低大鼠的机械痛阈(P<0．001)。手术后7，14和28天的机械痛阈值也存在明显差别(P<0．001)。手术处理与术后天数存在交互作用(P<0．05)。与假手术组相比，手术后机械痛阈值下降，7天达到最低，然后逐渐上升，但术后28天机械痛阈值仍明显低于假手术大鼠(SNK，all P<0．05)。
2．实时定量PCR测量受压前后TRPV4基因的变化
持续机械压迫可以明显增加TRPV4基因的表达(P
蛋白质的变化与mRNA的变化相类似，持续机械压迫可以明显增加TRPV4蛋白的表达(P<0．001)。压迫7天后，TRPV4的表达明显增高，为假手术的4．34倍，。随受压时间的延长，TRPV4蛋白的表达逐渐降低，术后14天和28天分别为假手术的3．88和2．47倍，差异有统计学意义(SNK，all P<0．05)。假手术组之间没有明显差别(F=1．21，P=0．412)。
4．持续压迫导致的病理性疼痛与TRPV4
在正常组和CCD组，反义寡脱氧核苷酸可明显抑制TRPV4的合成，但错配寡脱氧核苷酸无明显影响。在正常组，干扰前后大鼠机械痛阈值无变化(尸=0．93)。在CCD组，三个组的基础机械痛阈值无差别(P=0．844)。与基础痛阈值相比，对照组和MM组的机械痛阈值明显下降(Bonferronit-test，both P<0．05)。而在AS组，CCD导致的机械痛阈值降低被部分逆转(Bonferroni t-test，P>0．05)。
5．持续神经节压迫对TRPV4通道功能的影响
TRPV4的基本性质与细胞模型结果相同。持续压迫7天后，30％低渗溶液和佛波醇引起的细胞内钙浓度的峰值产生明显变化(both P<0．01)。与假手术组相比，30％低渗溶液引起的CCD组和MM组钙浓度的峰值明显增加(Tukey Test，both P<0．01)，对低渗溶液产生反应的DRG细胞比例数也明显升高(52．6％，X~2=5．14，P<0．05 for CCD group and 50．8％，X~2=4．32，P<0．01 for MM group)。与CCD组和MM组相比，AS组DRG对低渗溶液的反应明显降低(Tukey Test,bothP<0．05)，产生反应的DRG细胞比例数也明显降低(29．1％，X~2=8．65，P<0．01 vs。CCD group；X~2=7．59，P<0．01 vs．MM group)。与假手术组相比，佛波醇引起的CCD组和MM组钙浓度的峰值明显增加(Tukey Test，both P<0．01)，对佛波醇产生反应的DRG细胞比例数也明显升高(46．1％，X~2=4．25，P<0．05 for CCD group and47．6％，X~2=4．95，P<0．05 for MM group)。佛波醇引起AS组DR(3内钙峰值明显低于CCD组和MM组(Tukey Test，both P<0．05)，AS组起反应的DRG细胞比例数也明显低于CCD组和MM组(26．3％，X~2=7．31，P<0．01 vs．CCD group；X~2=8．22，P<0．01 vs．MM group)。
结论
1．CCD可以上调TRPV4的基因、蛋白表达，敏化通道的功能。
2．TRPV4参与介导CCD导致的机械性异常疼痛。
背景
背根神经节(dorsal root ganglion，DRG)是机体初级感觉传入神经元胞体的聚集处，位于椎间孔内，当椎间盘突出和椎管狭窄等导致椎间孔狭窄时，易于受到压迫。持续压迫DRG(chronic compression of the DRG)可产生损伤侧的异常疼痛，并伴有神经元兴奋性的升高，表现为自发性放电增加和电流阂值降低。但是这种异常疼痛的机制还不清楚。
有研究者使用基因组学和蛋白质组学方法，对病理性神经痛模型的DRG进行了扫查，发现多种与神经元死亡和再生有关的基因和蛋白的表达量在损伤前后产生了变化，包括细胞骨架蛋白，神经递质代谢酶，炎性因子，合成／成熟相关蛋白和髓鞘形成相关蛋白等。这些研究使我们对外周神经损伤后神经退化和微环境改变的机制有了深入的了解。由于不同病理模型的行为学和病理变化不同，其基因和蛋白质的变化也不同。所以研究不同病理状态下的蛋白质的变化，可以帮助我们了解神经病理状态的分子生物学机制。
CCD模型是一种较特别的外周神经损伤模型，其损伤和炎症反应均局限在神经节内。插入钢棒不仅导致机械压迫、DRG缺血、水肿，而且从循环系统、神经元和非神经元释放出的多种炎症因子，使病理环境变得更加复杂。
蛋白质是一切生命活动的基础，蛋白质的结构和功能最终直接影响生命活动的变化。使用蛋白质组学方法从整体角度、高通量分析CCD后DRG蛋白质表达的变化，可以从整体上了解病理过程的发生机制和机体相应的自身保护机制。
目的
使用蛋白质组学方法筛查大鼠DRG持续受压后差异表达的蛋白质，特别是筛查与神经病理性疼痛和组织损伤相关的蛋白。
方法
1 CCD模型的建立
78只Wister大鼠随机分为正常组和CCD组，每组均为39只。CCD模型的建立方法同论文第三部分。28天后处死大鼠，取出DRG。
2行为学测量
测量大鼠运动功能，机械痛阈值(mechanical withdrawal threshold，MWT)和热辐射刺激缩爪反应潜伏期(thermal withdrawl latency)。MWT的测量方法同论文第三部分。热辐射刺激缩爪反应潜伏期使用BME—410A型热痛刺激仪，将热痛刺激仪放在有机玻璃板下方，使光源焦距照射动物后肢足底掌心，电子秒表记录从照射开始到引起后肢回缩反应时的潜伏期作为热痛觉观测指标。
3双向电泳和图像分析
从正常组和CCD组各20只大鼠的L4和L5 DRGs中提取蛋白，Bradford．法定量。双向电泳分离蛋白，银染显色，使用软件进行分析，找出差异表达蛋白。正常组和CCD组之间，斑点体积变化超过3倍的蛋白，为差异表达蛋白。
4蛋白的生物质谱分析鉴定
将选定的蛋白点切下，胰酶消化后，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，选取NCBInr和SwissPmt数据库进行在线检索。
5 Western blotting
使用Western blotting方法验证部分差异表达蛋白质，如膜联蛋白A2，p11，PKCe，GAPDH，热休克蛋白70的变化，方法同论文第二部分。
6 Real-time RT-PCR
使用实时定量PCR方法检测膜联蛋白A2和PKCe基因表达的变化，方法同论文第二部分。
结果
1．行为学测定
CCD明显降低机械痛阈和热辐射刺激缩爪反应潜伏期(both P<0．05)。从第二天开始，机械痛阈下降，到第四天达到最低值，然后逐渐恢复，但直到术后28天，CCD组大鼠的机械痛阈仍明显低于正常对照大鼠(all P<0．05，SNK)。热辐射刺激缩爪反应潜伏期的变化趋势与机械痛阈相似(all P<0．05，SNK)。所有大鼠的运动功能均正常。
2．差异表达蛋白质的鉴定
对照组和CCD组分别得到454±4(n=3)和423±+4(n=3)个蛋白质点。98个蛋白点的表达存在明显差异，质谱分析成功鉴定出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)。
3．Western blotting验证
Westem blotting分析显示膜联蛋白A2，p1 l，PKCe，GAPDH，热休克蛋白70的表达在CCD组均升高，与质谱分析的结果相符。对照组和CCD组p．actin的表达量无明显变化(t=-0．139，P=0．896)，因此适合作为内参。
4．CCD对基因表达的影响
CCD可以明显增加AnnexinA2和PKCε的基因表达，分别增加到对照组的4．55和3．65倍(bothP<0．05)。
结论
我们使用差异蛋白质组学的方法，分析了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PKCe蛋白表达的上调，可能参与了神经性疼痛的发生过程。GAPDH的上调或许参与神经元凋亡，HSP70的表达上调或许和神经保护有关。
PartⅠ: Research of electrophysiological properties of mechanosensitive channels in cultured dorsal root ganglion neurons of neonatal rats
Background
All living organisms face mechanical forces,from the fluid forces around bacterium to the high forces in a human knee during stair climbing.The process of converting physical forces into biochemical signals and integrating these signals into the cellular responses is referred to as mechanotransduction.The mechanosensitive channels(MS channels)play an important role in the mechanotransduction.The idea of MS channels arose originally from studies of specialized mechanosensory neurons. The channel open probability of MS channels changes with the membrane tension and converts mechanical force exerted on the cell membrane into electrical or biochemical signals in physiological processes.Since their discovery in embryonic chick skeletal muscle and frog muscle,MS channels have been found in many cell types.McCarter first identified the whole-cell currents activated by stretch or pressure in dorsal root ganglion(DRG)neurons of rats in 1939.After that,various MS channels activated by various stimuli were reported in DRG.The electrophysiological properties of those MS channels are diverse due to the difference in mechanical stimuli,recording methods,and the composition of pipette solution and bath solution.
DRG neurons are primary afferent neurons that carry sensory signals from the skin,muscles,joints,and visceral organs to the spinal cord.Various mechanical, thermal,or noxious stimuli are known to generate action potentials at peripheral nerve endings of the DRG neurons.DRG is easily mechanical compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae due to the special anatomical position and physiological construction features, resulted in radicular pain.Identification of the biophysics and distribution of the MS channels in the DRG can further our knowledge about the mechanism underlie the electric activity of DRG neurons.
Objective
The aim of this current study is to explore the electrophysiological properties and distribution of mechanosensitive channels in cultured DRG neurons of neonatal rats.
Methods
After cultured for 4 days,the mechanosensitive channels currents of DRG neurons of neonatal rats were recorded and analyzed using cell-attached and inside-out patch-clamp technique.The electrophysiological properties such as pressure-response relationship,current-voltage relationship,channel kinetics,ion selectivity,influential factor and cell-size distribution were analyzed.Membrane stretch was achieved by the application of negative pressure(suction)to a patch pipette.
Results
1.Pressure-response relationship
In cell-attached patches with·the membrane potential of-60 mV,no single-channel currents were observed before applying suction,except for a few patches,which showed spontaneous openings.The negative current was activated by pressure of-12 to -15mmHg.Those channels activated rapidly when suction was applied,kept stably during sustained application of negative pressure and quickly turned off when the suction was released.When the amplitude of the channel current was measured in cell-attached patches,the mean current amplitude was -3.40±0.04pA(n=125)at -60mV of membrane potential.Channel open possibility(NPo)increased as higher pressures were applied to the patch and reached a maximum approxiamate 50mmHg, with P1/2 was 36.66±0.455mmHg(n=25).A higher pressure would destroy the membrane.The mean NPo was 0.448±0.03(n=25)at-60mV of membrane potential and -50mmHg of pressure.
2.Current-voltage relationship
In inside-out patches with -40mmHg pressure,the MS channels exhibited a nearly linear current-voltage relationship in the symmetrical solution.The outward chord conductance was 96.15±3.73 pS(Vm is between +40mV and +60mV)and the inward slope conductance was 62.47±2.72 pS(Vm is between -60mV and 0mV). This kind of channels appeared to be an inward rectifier.The average reversal potential(Erev)was -2.33±0.255 mV.
3.Channels kinetics
Histograms of open or closed-time durations of MS channels activated by suction pressures were best fitted with two exponential functions.Therefore,the channels exhibited short and long openings and closings.Negative pressure of -20 mmHg activated MS channels with open time constants of 1.338±0.098ms and 13.001±0.649ms,and with closed time constants of 2.462±0.267ms and 23.386±1.206ms.When negative pressure of -40 mmHg was applied to the same channels,the duration of short openings and long openings increased significantly 1.744±0.195ms for short openings and 17.92±1.623ms for long openings(both P＜0.05).In contrast,at -40 mmHg,the duration of long closings 14.071±0.797ms (P＜0.05),but not short closings 2.266±0.154 ms(P=0.545),decreased significantly compared with at -20 mmHg.Therefore,greater pressures could increase the channel activities of MS channels largely by increasing the duration of short openings and long openings and decreasing the duration of long closings.
4.Ion selectivity
The MS channels were non-selective cation channels and were not permeable to anion.
5.Influence factors
The current activated by mechanical stimuli could be blockaded by gadolinium and colchicine.The tetrodotoxin could blockade the current in large diameter DRG neurons,with no effect on the small diameter DRG neurons.
6.Cell-size distribution
The MS channels were founded mainly in small(＜20μm,48.41%)and medium (20～30μm,36.30%)diameter DRG neurons and were rarely found in relatively large diameter(＞30μm,15.29%)DRG neurons.
Conclusion
We identified the electrophysiological properties and the distribution of a kind of MS channels in DRG in rats.The mechanical stimuli could activate the MS channels and transduce the mechanical signals.
Background
Chronic compression of the dorsal root ganglion (DRG) or its near nerve root is considered to be one of the most important causative factors of radicular pain. However, the evidence supporting such an argument is incomplete. The effects of pure mechanical pressure on DRG neurons have not been assessed. In vivo animal models are usually used to study the change of the morphology, function, and expression of related genes and proteins under the mechanical pressure. But the effect of the mechanical pressure on the DRG could be easily confounded by other factors and can not be exclusively evaluated in in vivo models. So it is necessary to establish a convenient, reliable, stable, and single factor in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.
Among the current in vitro models, the model of hydrostatic pressure is able to provide sustained compression at different pressure levels. In such a model, pure mechanical loading is generated by compressing the gas phase within a closed culture chamber that contains the culture dishes. But there is little evidence on whether this model is suitable for the study of neurons, given the specificity for the culture of the neurons.
DRG neurons can transmit polymodal sensations, such as temperature and mechanical sensations. Many ion channels, including transient receptor potential vanilloid receptor 1 (TRPV1), transient receptor potential vanilloid receptor 4 (TRPV4), transient receptor potential channel of melastatin type 8 (TRPM8), and transient receptor potential subtype ankyrin 1 (TRPA1), play a potential role in mechanical nociception. Among these channels, the TRPV4 channel is a polymodal receptor, which plays an important role in nociceptive responses to hypotonic and mechanical stimuli.
Recent studies suggested that the chronic compression of the dorsal root ganglion changed the expression and the electrophysiological property of the voltage-dependent ion channels, as well as the synthesis and transportation of the neurotransmitter. Furthermore, Hoyer et al. found that densities of the pressure-activated channels were significantly higher in spontaneously hypertensive and renovascular hypertensive rats compared with their respective normotensive controls. Therefore, we hypothesized the expression or function of the mechanoreceptors may be changed under direct chronic compression.
Objective
1. To establish an in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.
2. To investigate whether pure chronic mechanical compression of the dorsal root
Methods
1. Establishing the in vitro cell model
DRG neurons were prepared as described previously and were placed in a 37℃incubator in a 95% air-5%CO_2 atmosphere. After 2 days the culture medium was changed and the culture dishes were put into the seal chamber, which was designed for incubating the DRG cells in a range from 0 to 200 mm Hg. Compressive loading is generated by compressing the gas phase within the closed culture chamber and the pressure gas consisted of 95% air and 5%CO_2- Pressure levels are monitored continuously by manometer, with atmospheric pressure (760 mm Hg) calibrated to 0 mm Hg. Dishes were cultured under compressive loading as the test groups and other dishes were cultured without loading as the control group.
2. MTT test
DRGs were cultured on 96-well plates and there were 8 wells in each condition. After cultured under different pressure for different durations, cell morphology was observed under inverted microscope and the cell activation was assessed with MTT test. The original culture medium was then removed, and l00μi 1mg/ ml of MTT was added. After 150μl dimethyl-sulfoxide was added and the plates were oscillated for 10min to fully dissolve the crystal, the absorption at 490 nm was measured with ELISA analyzer. A mean value from 8 wells was calculated as one sample and 3 samples were taken for each condition. Cell viability of control cells was taken as 100%.
3. Real-time PCR
The mRNA levels of TRPV4 in DRG neurons cultured with different pressures and different durations were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.
4. Western blotting
Western blotting was performed to investigate the proteins expression of TRPV1, TRPV4, TRPM8, and TRPA1 in DRG neurons cultured with different pressures and different durations.
5. Laser scanning confocal microscope
The responses to the hypotonic solution, TRPV4 agonist, and antagonist were assessed by calcium imaging using the laser scanning confocal microscope.
Results
1. Effect of mechanical pressure on cell morphology and viability
There were no significant changes in cell morphology between the control and the compressed cells. Pressure level only at 120 mmHg significantly inhibited DRG cell growth (P<0..05 vs. 0 mmHg, Bonferroni t-test). The mechanical compression induced a significantly inhibitory effect on DRG viability only in 72h group (P<0.01, Bonferroni t-test), but not in other groups. Therefore, 80 mmHg pressure level was chosen in the current study as the test compression.
2. Effect of mechanical pressure on gene expression
Gene expression was monitored after sustained compression at 0 mmHg (control) and 80 mmHg for 24 hours and 48 hours. The mechanical pressure significantly increased the TRPV4 gene level (P<0.05). Furthermore, gene expression was dependent on the interaction between pressure levels and duration of compressions (P <0.001). Post hoc analysis revealed that compression at 80 mm Hg for 24h and 48h significantly increased the gene transcription of TRPV4 to 2.31-fold and 4.04-fold as compared with the control (both P<0.05, SNK), respectively. Similarly, TRPV4 mRNA level in the 48h compression-exposed cells was significant higher than that in the 24h group (P<0.05, SNK).
3. Effect of mechanical pressure on the protein expression in DRG neurons
The DRG neurons were cultured under 80 mmHg for 0 hour (control), 24 hours, and 48 hours. Mechanical pressure significantly increased the TRPV4 protein level (P<0.05). Post hoc showed compression-exposure (80 mmHg) for 24h and 48h induced a significantly up-regulation in TRPV4 protein levels, which were 1.80-fold and 4.22-fold higher than the control, respectively (both P<0.05, Tukey Test). By contrast, TRPV1, TRPM8 and TRPA1 expression showed no significant changes following sustained compression for 24 and 48h (all P>0.05).
4. Effect of mechanical pressure on TRPV4 Ca~(2+) responses
Hypotonic stimulation increased intracellular Ca~(2+) concentration in an osmolarity-dependent manner in DRG (P<0.001). Pre-treatment with compression caused an increase in the magnitude of Ca~(2+) response, as well as the percentage of cells responding to hypotonic stimulus. When challenged with 30% hypotonic solution, 37.0% neurons showed increase in [Ca~(2+)]_i with the magnitude was 1.67±0.02. In the 24h and 48h groups, 48.4% (x~2=1.86, P>0.05) and 61.2% (X~2=7.16, P<0.05) neurons showed response to hypotonic solution, with the 1.74-fold and 2.52-fold increase in the magnitude of Ca~(2+) response compared to the control (both P<0.01, SNK) respectively. Similar to the hypotonic solution -induced Ca~(2+) response, the response to 4a-PDD showed the increased magnitude of Ca~(2+) response with the longer duration of mechanical compression applied, which increased to 1.48-fold and 2.09-fold compared to the control (both P<0.01, SNK), respectively. The percentage of neurons responding to 4α-PDD was also increased from 30.92% to 42.25 % (X~2=2.29, P>0.05 for 24h group) and 49.18% (X~5.30, P<0.05 for 48h group) . Furthermore, there is an interaction between pressure levels and duration of compressions on the Ca~(2+) response (P <0.001).
Conclusion
1. In a certain culture duration and pressure, pure mechanical pressure alone has no effect on the morphology and cell activation of DRG neurons and the model of hydrostatic pressure is suitable to investigate how the neurons respond to mechanical compression.
2. Pure mechanical pressure alone can induce the upregulation of mRNA and protein expression of TRPV4 without affecting other TRPs. Meanwhile, the mechanical pressure also enhances the function of the TRPV4 channels.
Background
Mechanical compression of the nerve root and the dorsal root ganglion (DRG), by disc herniation or spinal stenosis is one of the major causes of radicular pain in humans. Chronic compression of DRG (CCD) in animals results in spontaneous pain, mechanical hyperalgesia and allodynia. In association with these painful behaviors spontaneous activity, lowered threshold currents, and action potential thresholds have been shown in the neuronal somata of the compressed ganglion. Ion channels such as voltage-gated Na~+ and K~+, and hyperpolarization-activated cation current may contribute to the hyperexcitability of DRG neurons after CCD. However, to date the ionic mechanisms underlying mechanical hyperalgesia and allodynia are not fully understood.
Ion channels in DRG neurons, including the transient receptor potential vanilloidreceptor 2 (TRPV2), transient receptor potential vanilloid receptor 4 (TRPV4), and transient receptor potential subtype ankyrin 1 (TRPA1), may also play a role in mechanical nociception. TRPV4 is a polymodal receptor and it has been shown to mediate nociceptive behaviors to hypotonic and mechanical stimuli under pathological conditions. Chemotherapy (Taxol) has been shown to increase the percentage of the DRG neurons respective to TRPV4 agonists. However, no significant changes in the expression or function of TRPV4 were found in traumatic or diabetic neuropathy pain models. Thus, the role of TRPV4 channel in neuropathic pain is controversial.
We have shown in the second part of the thesis that the pure sustained mechanical pressure can increase the expression levels and sensitized the ion function of TRPV4. In CCD model, the mechanical compression associated with the secondary inflammatory process, such as ischemic, edema, and inflammatory cells infiltrate makes the pathophysiological conditions much more complex. Therefore, the aim of this study was to investigate the effects of CCD on the levels of TRPV4 mRNA and protein expression to determine the role of TRPV4 in CCD-induced mechanical allodynia.
Objective
1. To investigate the effects of CCD on the expression levels of mRNA and protein and the function of TRPV4.
2. To determine the role of TRPV4 in CCD-induced mechanical allodynia.
Methods
1. Establish CCD model
Ninty-nine rats were randomly divided into three groups, corresponding to 7, 14,and 28 days post-CCD. Each group was sub-divided into a sham and a CCD group (n=13 for sham and n=20 for CCD). In CCD rats, under pentobarbital sodium anesthesia, the paraspinal muscles were separated to expose the transverse process and intervertebral foramina of L_4 and L_5 unilaterally as previously described. A stainless steel U-shaped rod (0.63 mm diam and 4 mm length) was inserted into each foramen to compress the DRG, one at L_4 and the other at the L_5 ganglion. The muscle and skin layers were then sutured. Penicillin was injected to prevent infection. Sham group underwent the same surgical procedure as described, but without the insertion of the rods. Animals were sacrificed at 7,14, and 28 days post-CCD, respectively.
2. Antisense oligodeoxynucleotide treatment
To determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced mechanical allodynia and calcium response, 18 normal rats and 27 CCD rats were treated with spinal intrathecal administration of TRPV4 antisense ODN and mismatch ODN, respectively. Each group was sub-divided into control group, TRPV4 antisense ODN group (AS group), and mismatch ODN group (MM group) (n=6 in normal rats and n=9 in CCD rats for each sub-group). ODN was reconstituted in nuclease-free 0.9% NaCl (10μg/μl) and administered into the spinal intrathecal space at a dose of 40μg , once a day for 7 days until the animals were sacrificed.
3. Neuroethology measurement
The motor function and MWT were measured pre-CCD and right before the animals were sacrificed at 7,14, and 28 days post-CCD, respectively.
1) Walk gait pattern was assessed as the index of motor function. "1" indicates normal gait, without foot deformities. "2" indicates normal gait with obvious foot deformities. "3" indicates slight gait disturbance with foot-drop. "4" indicates serious gait disturbance with myashtnia.
2) Mechanical withdrawal threshold (MWT) was measured with a von Frey hair monofilament with logarithmically incremental stiffness (0.09-17.30 g). The von Frey hair was applied through the mesh floor to the plantar skin of the hindpaw in an ascending order. The MWT was defined as the lowest force that evoked a brisk withdrawal response to at least three of five stimuli. A positive response was noted if the paw was immediately withdrawn. The upper limit for testing was 17.30 g hair.
4. Real-time PCR
The levels of TRPV4 gene expression in different groups were assessed using real-time RT-PCR.
5. Western blotting
The levels of TRPV4 protein expression in different groups were assessed using Western blotting.
6. Laser scanning confocal microscope measurementThe calcium responses to hypotonic solution and 4a-phorbol 12, 13-didecanoate (4a-PDD) were assessed following sham surgery, CCD, spinal application of TRPV4 antisense ODN, and mismatch ODN with laser scanning confocal microscope.
Results
1. Neuroethology measurement
1) The walk gait pattern was normal and no foot deformities were found in all rats pre- and post-CCD. The score was "1". There was no significant difference between control and CCD groups.
2) The MWT in CCD group was significantly lower than the sham group (F=39.97, P<0.001). There was also a significant difference in MWT at 7, 14, and 28 days post-CCD (F=8.03, P<0.001). Furthermore, the MWT was dependent on the interaction between the groups and the days post-CCD (F=3.16, P<0.05). The MWT was lower in the CCD group than the sham group at 7, 14, and 28 days post-surgery (SNK, all P<0.05), with the lowest level at 7 days post-CCD.
2. The effect of CCD on the levels of TRPV4 mRNA expression
The levels of TRPV4 mRNA expression were increased significantly in CCD group when compared with the sham group (F=240.02, P<0.001). There were also significant differences in the levels of TRPV4 mRNA expression at different days post-CCD (F=14.77, P<0.001). The levels of TRPV4 mRNA expression were also dependent on the interaction between the groups and the days post-CCD (F=14.22, P<0.001). The levels of gene expression were significantly increased at 7, 14, and 28 days in CCD group when compared with the sham group (SNK, all P<0.05). There was no significant changes in the level of mRNA expression in sham group (SNK, all P>0.05), but the mRNA level was significantly higher at 7 days than that at 14 and 28 days post-CCD (SNK, both P<0.05).
3. The effect of CCD on the levels of TRPV4 protein expression
The levels of TRPV4 protein expression were increased significantly in CCD group when compared with the sham group (F=451.52, P<0.001). There were also significant differences in the levels of TRPV4 protein expression at different days post-CCD (F=25.99, P<0.001). The levels of TRPV4 protein expression were also dependent on the interaction between the groups and the days post-CCD (F=23.99, P<0.001). There were significantly increased at 7, 14, and 28 days in CCD group, compared with the sham group (SNK, all P<0.05). The protein level was significantly higher at 7 days than at 14 and 28 days post-CCD (SNK, both P<0.05).
Furthermore, the sustained increase in the levels of TRPV4 mRNA and protein expression was associated with the decreases in MWT in CCD group.
4. The effect of antisense oligodeoxynucleotide treatment on CCD-induced mechanical allodynia
The antisense ODN, but not the mismatch ODN, significantly inhibited the TRPV4 expression in normal rats (13.75±1.05%, 2.65±0.071%, 13.8±0.7% for control, AS, and MM group, respectively; F=77.79, P<0.005) and CCD rats (42.2±7.19%, 8.75±0.25%, 44.6±6% for control, AS, and MM group, respectively; F=13.93, P<0.05). In CCD rats, the MWTs were similar at baseline (Bonferroni t-test, all P>0.05), but after intrathecal ODN treatment, the MWT was decreased significantly in control group and MM group when compared with the baseline (Bonferronit-test, both P<0.05). In contrast, mechanical allodynia was partly reversed in AS group (Bonferroni t-test, P>0.05 compared with baseline). The MWT of normal rats were not changed following intrathecal AS or MM (F=0.0751, P= 0.93).
5. Laser scanning confocal microscope measurement
The fluorescence ratio of calcium responsive to 30% hypotonic solution and 3μM 4a-PDD were significantly differed between different groups (F=148.46, P<0.01; F=173.26, .P<0.01, respectively). The percentage of the DRG neurons responsive to hypotonicity was upregulated in CCD group (52.6%, X~2=5.14, P<0.05) and in MM group (50.8%, X~2=4.32, P<0.01) when compared with sham group. The hypotonicity-induced increase in fluorescence ratio of calcium was also enhanced in CCD group and MM group when compared with sham group (Tukey Test, both P<0.01). Treatment with TRPV4 antisense significantly decreased the fluorescence ratio when compared with the CCD group and MM group (Tukey Test, both P<0.05). The percentage was also significantly decreased in AS group (29.1%) when compared with the CCD group ( X~2=8.65, P<0.01) and MM group X~2=7.59, P<0.01). Similar to the hypotonic solution-induced Ca~(2+) response, the percentage of DRG neurons responsive to the 4α-PDD was significantly upregulated in CCD group (46.1%, X~2=4.25, P<0.05) and MM group (47.6%, X~2=4.95, P<0.05) when compared with the sham group. The fluorescence ratio of calcium was enhanced in CCD group and MM group compared with the sham group (Tukey Test, both P<0.05). The percentage was significantly decreased in AS group (26.3%, X~2=7.31, P<0.01 vs. CCD group; X~2=8.22, P<0.01 vs. MM group) and the fluorescence ratio reduced significantly compared with CCD group and MM group (Tukey Test, both P<0.05).
Conclusions
1. It was shown that CCD in rats increases the levels of TRPV4 mRNA and protein expression, in addition to an enhancement in the calcium response to hypotonic stimuli and 4α-PDD.
2. TRPV4 plays a crucial role in CCD-induced mechanical allodynia.
Background
The dorsal root ganglion (DRG) in the intervertebral foramen has an important role in the pathogenesis of low back pain and sciatica in patients with disc hemiation and spinal canal stenosis, because primary sensory neurons with their cell bodies are present in this structure. DRG is easily stimulated or compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae. The chronic compression of the DRG produces ipsilateral cutaneous allodynia that is associated with an increased excitability of neuronal somatas in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. But the underlying mechanisms are still not fully elucidated.
Global gene expression and proteomics studies have discovered a large number of genes and proteins that are regulated in vivo after peripheral nerve injury in animal models of neuropathic pain. Many genes and proteins have been identified to be related to neuronal cell death and regeneration, including cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, growth factors, and signal transduction molecules, inflammation factor, protein synthesis/maturation, and myelination in the nerve. These studies have extended our knowledge about cellular and molecular changes in the degenerating nerve and its microenvironment after peripheral nerve injury. Since there are apparent behavioral and morphological differences among the most utilized animal neuropathy models, comparison of protein expression patterns from different types of injuries might help us understand elaborate molecular and cellular mechanisms underlying diverse peripheral neuropathy.
Among the neuropathic pain models, the chronic compression of the dorsal root ganglion (CCD) model is unique in that the injury and inflammation are located in the ganglion. It is reported that in the CCD model, the acute compression to the nerve root induced endoneurial edema and the associated DRG edema. The ischemia along with any mechanical trauma caused by the compression could initiate an inflammatory reaction and the release of a variety of inflammatory mediators derived from the blood circulation, neurons as well as nonneuronal cells in the ganglion. Thus, the molecular and cellular changes that underlie peripheral nerve injury are complex and identification of proteins involved will contribute significantly to our understanding of the mechanisms of neuropathic pain and neuroprotection.
Objective
The aim of the study was to identify the differential protein expressions related to neuropathic pain and neuroprotection in DRG following CCD in rats.
Methods
1. CCD surgery
Seventy-eight adult male Wistar rats weighing 150-180 g (Shandong University) were randomly divided into CCD group and normal control group, 39 in each group. In CCD rats, under pentobarbital sodium anesthesia, the stainless steel U-shaped rods were inserted into the intervertebral foramina of L_4 and L_5 as described in part 3. Animals were sacrificed at 28 days after surgery by decapitation
2. Measurement of mechanical withdrawal threshold and thermal withdrawal latency
Mechanical withdrawal threshold (MWT) was measured with von Frey monofilaments as described in part 3. Thermal allodynia was assessed with paw withdrawal latencies to radiant heat. The radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw when in contact with the floor. The paw withdrawal latencies per animal were obtained for five times with intervals of 5 min.
3. 2-DE and image analysis
The proteins from L_4 and L_5 DRGs of 20 rats in each group were extracted and quantificated with the Bradford method. The differential proteins expression between CCD group and control group were detected using two-dimensional gel electrophoresis (2-DE) followed by silver staining visualization and comparative analysis with the software of ImageMaster 2D Platinum 5.0. Three independent repeats of each sample were performed. The threshold as the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the CCD and control groups.
4. Mass spectrometry for protein identification
Selected spots were located and digested with trypsin. Then the peptide mass fingerprints (PMFs) of differential proteins expressed between the CCD and control groups were generated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and interpreted utilizing Mascot produced by Matrix Science against the NCBInr database.
5. Western blotting
Some of the identified proteins were further examined by Western blotting as described in part 2.
6. Real-time RT-PCR
The gene expression of annexin A2 and PKCεwere measured with real-time RT-PCR as described in part 2.
Results
1. Neuroethology measurement
The chronic compression produced marked mechanical and thermal allodynia, indicated by significantly decreased mechanical thresholds and thermal withdrawal latency compared with the normal control group (F=95.40, P<0.05; F=86.19, P<0.01, respectively). All rats walked normally after surgery which indicated that the surgery did not injury the motor behavior.
2. Identification of the differential proteins
A total of 98 protein spots were detected with significant changes in their expression levels after CCD and 15 protein spots were identified by MALDI-TOF MS analysis. Of these proteins, annexin A2, protein kinase C epsilon (PKCe), glyceraldehyde-3-phosphate dehydrogenases (GAPDH), and heat shock protein 70 (HSP70) were up-regulated significantly compared with the normal control.
3. Confirmation by Western blotting
The results of Western blotting showed consistent data with the results of the proteomic analysis. There was no significant difference in protein levels ofβ-actin between control and CCD groups (t = -0.139, P= 0.896).
4. Effect of mechanical pressure on gene expression
Quantitative real-time RT-PCR experiments indicated that CCD-induced increase the gene levels of annexin A2 and PKCe.
Conclusions
These proteomic results suggest that chronic DRG compression is associated with the upregulation of annexin A2 and PKCεand their related genes, with may be related to the neuropathic pain. The upregulation of GAPDH and HSP70 suggests that there exist concurrent processes of nervous injury and neuroprotection in the course of neuropathic pain.
引文
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