细胞骨架对大鼠背根神经节持续受压后痛觉过敏的调控及机制研究
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摘要
神经性疼痛是由神经系统原发性或继发性损害或功能障碍所引起的疼痛。背根神经节(dorsal root ganglion, DRG)持续受压(chronic compression of DRG,CCD)导致大鼠出现受压侧的自发性疼痛、痛觉过敏和异常疼痛,同时伴随神经元的兴奋性增加,表现为自发性放电增加和电流阈值降低,与临床上腰椎间盘突出症等疾病所导致的症状相似。本课题组的前期研究表明,CCD手术后,大鼠出现手术同侧后肢的机械痛觉过敏和热痛觉过敏,但痛觉过敏的具体机制尚未明确。
微管与微丝是细胞骨架的主要组成部分,参与维持细胞形态、保持细胞内部结构的有序性、转运细胞内物质等生理功能。微管是细胞骨架纤维中最粗的纤维,是由微管蛋白组成的直径约为25nm的中空圆柱状纤维。微管蛋白主要包括α-微管蛋白(a-tubulin, TUBA)、β-微管蛋白(p-tubulin, TUBB)和γ-微管蛋白(γ-tubulin,TUBG)。TUBA和TUBB以非共价键的形式连接形成异二聚体,异二聚体首尾相连则形成微管蛋白原纤维。哺乳动物中,13条微管蛋白原纤维构成1个中空的微管。紫杉醇是微管的稳定剂,稳定微管的聚合状态,抑制微管解聚。秋水仙碱是一种微管解聚剂,抑制微管聚合并促进微管解聚。微丝是细胞骨架结构中最细的纤维,直径7nm左右,主要由肌动蛋白螺旋状聚合形成,包含游离球状肌动蛋白(G-actin)和聚合纤维状肌动蛋白(F-actin)两种形式,只有后者具有生物学作用。细胞松弛素是微丝的解聚剂,可抑制微丝聚合并促进微丝解聚。鬼笔环肽是一种微丝稳定剂,具有稳定聚合微丝、抑制微丝解聚的作用。微管与微丝处于不断的解聚与聚合的动态平衡状态,这种动态平衡是完成生理功能的必要条件。
研究证实,微管和微丝的解聚可降低炎性疼痛。炎性疼痛中,炎症细胞和炎症介质在趋化因子的作用下迁移到病变处发挥作用,其迁移是在微管的作用下完成的,低浓度(10-8mol/L)短时间(30-120分钟)应用微管解聚剂秋水仙碱可抑制炎性细胞的迁移,控制炎症。肾上腺素(epinephrine, EPI)及其下游产物可提高感觉神经元对疼痛的敏感性,而单独应用微管解聚剂或微丝解聚剂均可抑制肾上腺素诱导的痛觉过敏。微管也能够介导神经性疼痛。坐骨神经慢性压迫(sciatic chronic constriction injury, CCI)后,大鼠出现后肢的热痛觉过敏,注射秋水仙碱可缓解CCI所致的热痛觉过敏。因此,我们推测微管、微丝可能介导CCD大鼠机械痛觉过敏和热痛觉过敏,因此,本课题拟利用微管和微丝的解聚剂及聚合剂研究二者在CCD大鼠机械痛敏和热痛敏中的作用。
持续受压后,大鼠的痛觉过敏和DRG神经元的高兴奋性与多种离子通道有关,如电压门控性Na+通道和K+通道、超极化激活性阳离子通道和瞬时感受器电位离子通道(transient receptor potential, TRP)通道等。瞬时感受器电位离子通道香草素受体亚家族4(transient receptor potential vanilloid subtype4, TRPV4)是TRP超家族成员之一,是一种Ca2+通透性较高的阳离子通道。DRG神经元的细胞膜、细胞浆和细胞核上均可检测到TRPV4,细胞膜上表达的TRPV4通道是介导痛觉过敏的主要部分。实验证据支持DRG中表达的TRPV4参与介导多种神经性疼痛和炎性疼痛。前列腺素E2(prostaglandin E2, PGE2)可增强低渗或中度高渗溶液刺激引起的异常疼痛和痛觉过敏,TRPV4反义寡核苷酸干扰和基因敲除后,大鼠和小鼠的异常疼痛和痛觉过敏缓解。TRPV4反义寡核苷酸干扰后,微管稳定剂紫杉醇引起的机械痛觉过敏降低。本课题组的前期研究证实,TRPV4通道参与介导CCD大鼠的机械痛觉过敏和热痛觉过敏。
微管、微丝与TRPV4直接相连并可调节TRPV4通道的功能。如微丝解聚后,TRPV4通道介导的内向电流和低渗休克导致的TRPV4通道的Ca2+内流受到抑制,TRPV4与肌动蛋白的共表达减少。结合TRPV4在CCD大鼠痛敏中的作用,我们推测,TRPV4可能参与微管、微丝对CCD大鼠痛敏的调节过程。
因此,本课题中,我们拟观察微管、微丝对CCD大鼠痛敏的影响,并研究微管、微丝对CCD大鼠DRG神经元TRPV4通道功能、表达和定位的影响,为微管、微丝对CCD大鼠痛敏的调节提供可能的作用机制。
目的
1.研究微管解聚剂秋水仙碱对CCD大鼠机械痛觉过敏和热痛觉过敏的影响。
2.研究秋水仙碱对TRPV4通道功能和表达的影响,探讨微管解聚影响CCD大鼠痛觉过敏的机制。
材料与方法
1.CCD模型的制备
应用戊巴比妥钠(50mg/kg)行腹腔注射麻醉后,沿L4-S1棘突作后正中偏右切口,暴露横突的根部及L4-L5椎间外孔后,将每侧长4mm,直径0.63mm的“L”形不锈钢棒沿L4-L5椎间外孔的前壁上方水平插入椎管内,另一端位于椎管壁外。通过控制“L”形棒椎管壁外侧端使其椎管内端沿椎骨内壁缓慢下滑至椎管中部,恒定地压迫DRG及邻近神经根。术后用生理盐水冲洗切口,依次缝合肌肉、腰背筋膜和皮肤,腹腔注射青霉素40万单位预防感染,术后密切观察大鼠生命体征变化。
2.神经行为学的测定
对CCD大鼠的手术侧及正常大鼠的同侧后肢进行行为学检测,时间为：术前,术后第4、6、7、14、28天；药物对行为学影响检测时间为：药物处理前测量,排除痛觉表现不明显者(<5%)；药物处理后0.5、1、2、4、8小时分别测量。
大鼠的机械痛觉过敏采用机械缩足反射阈值(Mechanical withdrawal threshold, MWT)测量,使用BME-403型、Von Frey Fibers机械痛刺激仪进行。将不同强度的Von Frey纤维由低到高依次地刺激CCD大鼠手术侧或正常大鼠同侧后肢足底中部并维持5秒,阳性反应为出现快速缩足或舔足现象。每个强度的纤维刺激5次,机械缩足反射阈值为至少能够引发3次缩足反应的纤维强度。
大鼠热痛觉过敏通过热辐射刺激缩爪反应潜伏期(paw withdrawal latency,PWL)来确定,采用BME-410C型热痛刺激仪测量。大鼠安静后,将光源焦距对准CCD大鼠手术侧或正常大鼠同侧后肢足底中心。从开始照射到引起后肢回缩反应所需的时间,即为当次热辐射刺激缩爪反应潜伏期。每肢照射5次,测量结果的均值即为此大鼠的热辐射刺激缩爪反应潜伏期。
3.成年大鼠DRG神经元的培养
取出CCD术后第7天手术侧L4-L5的DRG和正常大鼠同侧L4-L5的DRG,Ⅰ型胶原酶和胰蛋白酶分别消化1小时和30分钟后,应用Neurobasal+1%N2+20ng/mlNGF+1%谷氨酰胺+100U/ml青链霉素培养,培养第4天的DRG神经元用于全细胞膜片钳技术、细胞活性测定及免疫细胞化学检测等后续实验。
4.HEK293细胞的培养和转染
HEK293细胞培养于10%胎牛血清和1%双抗的DMEM培养基中,3-4天传代一次,传代后24小时转染。Lipofectamine2000脂质体和(?)TRPV4-GFP质粒先各自加入一定量的缓冲液OPTI-MEM中,混匀后静置5分钟。将两者再混匀,室温放置20-30分钟。将质粒脂质体混合物加入HEK293细胞培养液中孵育4-6小时,然后将培养液更换为包含血清和抗生素的DMEM培养液。转染后24-48小时进行电生理实验或细胞活性检测。
5.细胞活性检测
将成年大鼠DRG神经元和HEK293-TRPV4细胞以1×104／孔的密度接种于96孔培养板,在不同浓度秋水仙碱培养基中培养2小时后,吸去原培养液,每孔加入80μl培养液和20μl5mg/ml的四甲基偶氮唑(3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide, MTT)。经37℃孵箱孵育4小时后,将上清液吸去后应用PBS冲洗后,每孔加入150μl甲基亚砜(Dimethyl sulphoxide, DMSO)溶剂。将孔板放置在摇床上,通过10分钟的低速震荡即可充分溶解结晶。应用酶标检测仪测定波长490nm处的吸光度OD值,各组均以正正常培养基培养的成年大鼠DRG神经元的吸光度作为100%,其他情况下的吸光度数值需与之相比较。
6.全细胞膜片钳记录TRPV4通道的电流
采用常规高阻抗封接的全细胞记录方式,检测DRG神经元及HEK293-TRPV4细胞中TRPV4通道介导的内向电流。电极内液(mmol/L):140CsCl,2NaCl,3MgCl2,10Hepes,5EGTA (290mOsm),用CsOH调节pH至7.25,用针头式滤器(孔径0.22μm)过滤后分装,-20℃保存。电极入水电阻为3-6MΩ。电极外液(mmol/L):124NaCl,5KC1,1.2KH2PO4,1.3MgCl2,2.4CaCl2,26NaHCO3(310mOsm),用NaOH调节pH至7.35,4℃保存。
7.实时定量RT-PCR检测
CCD术后第7天,鞘内注射不同浓度秋水仙碱0.5-8小时后,从各组大鼠手术侧L4-L5DRG中提取RNA,实时定量RT-PCR检测TRPV4mRNA表达的变化。
8. Western blotting检测
CCD术后第7天,鞘内注射不同浓度秋水仙碱0.5-8小时后,从各组大鼠手术侧L4-L5DRG中提取蛋白质,Western blotting检测TRPV4蛋白表达的变化。
9.组织免疫荧光及免疫细胞化学检测TRPV4的定位
将DRG组织切片及培养的DRG神经元用冷丙酮固定10分钟后(-20℃),加入2%BSA室温封闭1小时,然后加入TRPV4一抗4℃孵育过夜。PBS冲洗3次后,加入带有绿色荧光标记的TRPV4二抗室温孵育1小时。使用DAPI细胞核染色后,封片,荧光显微镜下观察TRPV4在DRG神经元的定位区域。
结果
1.鞘内注射微管解聚剂秋水仙碱可缓解CCD大鼠的痛觉过敏
CCD术后,大鼠手术侧机械和热痛觉阈值降低(P＜0.05)。术后7天时,手术侧机械痛觉阈值和热辐射刺激缩爪反应潜伏期降至最低(P＜0.01),此后逐渐升高。
625μg/kg-1250μg/kg的秋水仙碱可部分抑制CCD后大鼠的机械痛敏和热痛敏,此抑制作用(P＜0.05)呈剂量依赖性升高。此抑制作用于鞘注秋水仙碱后30分钟可测得,2小时作用达到最大,此抑制作用可持续至8小时,而312.5μg/kg秋水仙碱则无抑制作用(P＞0.05)。
2.微管解聚剂秋水仙碱对DRG神经元以及HEK293-TRPV4细胞活性的影响
0.1μg/ml秋水仙碱对DRG神经元和HEK293-TRPV4细胞的活性无影响。其中,0.1μg/ml秋水仙碱分别孵育DRG神经元和HEK293-TRPV4细胞0.5小时和2小时,细胞活性均未受影响(P＞0.05)
3.微管解聚剂秋水仙碱对正常大鼠DRG神经元TRPV4通道功能的影响
秋水仙碱孵育后,正常大鼠DRG神经元TRPV4的电流降低。秋水仙碱0.01μ/ml,0.05μg/ml和O.1μg/ml孵育DRG神经元2小时后,TRPV4的内向电流分别为201.62±4.72pA、165.25±7.44pA和128.41±±3.75pA。因此,秋水仙碱对DRG神经元TRPV4电流的抑制呈剂量依赖性,且0.1μg/ml秋水仙碱对TRPV4电流的影响最大(P<0.05)。因此,在本课题中,将0.1μg/ml秋水仙碱作为最适浓度。
4.微管解聚剂秋水仙碱对CCD大鼠DRG神经元TRPV4通道功能的影响
与正常大鼠DRG神经元TRPV4电流相比,CCD大鼠DRG神经元TRPV4通道介导的内向电流峰值增加(CCD大鼠电流峰值为310.19±9.17pA,正常大鼠为230.55±17.23pA,P<0.05)。CCD大鼠DRG神经元TRPV4通道的电流峰值延迟(CCD大鼠为243.6s,正常大鼠为172.4s,P＜0.05)。0.1μg/ml秋水仙碱孵育2小时后,CCD大鼠DRG神经元TRPV4通道电流峰值降低,且峰值时间延迟(230.10±12.10pA,199.1s,P＜0.05)
5.微管解聚剂秋水仙碱对HEK293-TRPV4细胞TRPV4通道功能的影响
0.1μg/ml秋水仙碱孵育2小时后,HEK293-TRPV4细胞的TRPV4通道电流峰值降低,且峰值时间延迟(正常HEK293-TRPV4细胞为412.50±27.15pA,131.6s;秋水仙碱组为219.53±5.24pA和42.7s,P＜0.05)。孵育秋水仙碱后,HEK293-TRPV4细胞的内向电流表现出典型的TRPV4通道的外向整流特性,但逆转电位更倾向于正相电位。
6.微管解聚剂秋水仙碱对TRPV4通道表达的影响
鞘内注射625μg/kg-1250μg/kg秋水仙碱时,TRPV4通道的基因表达明显降低,其中,鞘内注射1250μg/kg秋水仙碱2小时,TRPV4通道的基因表达下降最明显(P＜0.05)。当鞘内注射625μg/kg-1250μg/kg秋水仙碱时,TRPV4通道的蛋白表达显著降低,其中,鞘内注射1250μg/kg秋水仙碱2小时和4小时,TRPV4通道的蛋白表达下降最明显(P＜0.05)
7.微管解聚剂秋水仙碱对TRPV4定位的影响
组织免疫荧光的结果提示,鞘内注射1250μg/kg秋水仙碱未影响CCD大鼠背根神经节TRPV4的定位。DRG神经元的免疫细胞化学结果示,DRG神经元上胞膜表达的TRPV4多于胞浆内表达的TRPV4,并且秋水仙碱并未影响TRPV4的定位。
结论
1.微管解聚剂秋水仙碱缓解CCD大鼠的机械痛敏和热痛敏。
2.秋水仙碱可抑带(?)TRPV4的表达,降低TRPV4通道的电流峰值,因此,TRPV4介导了秋水仙碱对CCD大鼠痛敏的缓解作用。
目的
1.研究微丝在CCD大鼠痛觉过敏中的作用。
2.研究微丝对CCD大鼠DRG神经元TRPV4通道功能和定位的影响,探讨微丝调控CCD大鼠痛觉过敏的机制。
方法
1.CCD模型的制备
大鼠被随机分为CCD组和假手术组。戊巴比妥钠(50mg/kg)麻醉后,沿L4-S1棘突作后正中偏右切口,暴露右侧L4-L5椎间外孔后,将每侧长4mm,外径0.63mm的中空的“L”形不锈钢棒沿L4-L5椎间外孔的前壁上方水平插入椎管内,恒定地压迫DRG及其邻近的神经根。不锈钢棒外套有内径0.51mm,30-40mm长的硅胶管,硅胶管的外端置于椎管壁外,内端随中空钢棒一起插入椎管内,硅胶管内充满肝素。硅胶管的外端是封闭的,只有注射药物时打开。术后用生理盐水冲洗切口,依次用4-0丝线缝合肌肉、筋膜和皮肤,并注射青霉素40万单位以预防感染。假手术组除不插钢棒压迫神经节外,其余操作同手术组。术后大鼠没有发生伤侧肢体自噬现象。
2.神经行为学的测定
分别于手术后7天注射化学药物前及注射药物后1、2、4、6、8、24小时测量,排除痛觉表现不明显者(<5%)；所有的测量在一个安静的环境内进行,室温保持在26±0.5℃,且均使用单盲法。药物通过与压迫DRG的中空不锈钢管相连的硅胶管直接注射。
大鼠机械缩足反射阈值和热辐射刺激缩爪反应潜伏期的测量方法同第一部分。
3.成年大鼠DRG神经元的培养
CCD术后7天,取出手术侧L4-L5的DRG。细胞培养方法同第一部分。
4.细胞活性检测
成年大鼠DRG神经元在细胞松弛素B或鬼笔环肽培养基中培养2小时后,用于检测DRG神经元的活性,检测方法同第一部分。
5.全细胞膜片钳记录TRPV4通道的电流
同第一部分。
6.免疫细胞化学
同第一部分。
结果
1.微丝对CCD大鼠痛觉过敏的影响
所有实验大鼠在手术前后及给药处理前后步态均正正常,足无畸形,评分均为1分,损伤前、后各组间无显著性差异。
微丝解聚剂细胞松弛素B和细胞松弛素D皆可缓解CCD大鼠的机械痛敏和热痛敏(n=9,P＜0.01)。细胞松弛素B(200μg-300μg)可部分抑制CCD大鼠的痛敏,且此抑制作用呈剂量依赖性表现,然而,100μg细胞松弛素B无此抑制作用(P＞0.05,n=9)。CCD大鼠痛敏的缓解在注射细胞松弛素B后1小时出现,2小时达到高峰,持续少于24小时。
微丝聚合剂鬼笔环肽单独注射对CCD大鼠的机械和热痛敏的影响无统计学意义(P＞0.05,n=9)。细胞松弛素B注射前1小时注射鬼笔环肽可以抑制细胞松弛素B对CCD大鼠痛敏的缓解作用(n=9)。
2.微丝对CCD大鼠DRG神经元TRPV4通道功能的影响
CCD组TRPV4的电流峰值远高于假手术组(CCD组n=12,假手术组n=10,P＜0.05)。
细胞松弛素B和细胞松弛素D均可显著抑制(?)TRPV4的电流峰值(CCD组为310.19±9.17pA,细胞松弛素B组为245.48±12.12pA,细胞松弛素D组为237.81±10.17pA,各组中n=12,P＜0.05)。这与二者对CCD大鼠痛敏的影响一致。经微丝解聚剂孵育后,DRG神经元的I/V曲线表现出典型的TRPV4外向整流的特性,然而,逆转电位更倾向于正相电位,这与微丝对TRPV4(?)降值电流的抑制相一致。
CCD大鼠DRG神经元的峰值时间延迟(CCD组为243.60±20.00s,假手术组为200.37±15.40s,P＜0.05)。经微丝解聚剂孵育后,CCD大鼠DRG神经元TRPV4电流峰值时间提前(细胞松弛素B组为123.44±10.10s,细胞松弛素D组为193.76±12.40s,P＜0.05)。
微丝稳定剂鬼笔环肽(10-SM)可导致TRPV4电流的峰值降低和延迟(80.87±5.42pA,311.72±14.56s,n=12,P＜0.01),这与鬼笔环肽对CCD大鼠痛敏的影响不一致。与鬼笔环肽对TRPV4电流影响一致的是,DRG神经元I/V曲线更倾向于正相电位。MTT实验结果表明,微丝的解聚剂和稳定剂对于DRG神经元的活性无影响(P＞0.05)。
3.微丝对CCD大鼠DRG神经元TRPV4定位的影响
CCD大鼠DRG神经元细胞膜上表达的TRPV4多于细胞浆中表达的TRPV4。经细胞松弛素B(10-4M)孵育2小时后,DRG神经元细胞膜上TRPV4的表达量明显降低(CCD组为83.62±2.14%,细胞松弛素B组为69.63±3.41%,P＜0.05)。此外,鬼笔环肽(10-SM)也导致DRG神经元细胞膜上TRPV4的表达量降低(54.24±2.44%,P＜0.05)
结论
1.微丝解聚缓解CCD大鼠的痛觉过敏,微丝稳定剂可消除微丝解聚剂的镇痛作用。
2.微丝解聚抑伟TRPV4通道的电流峰值,降低神经元细胞膜上TRPV4的表达,因此,微丝通过TRPV4通道调节CCD大鼠的痛觉过敏。
Neuropathic pain is one kind of pain irritated by primary damage and malfunction of nervous systems. Chronic compression of dorsal root ganglion (DRG)(CCD) is a typical model of neuropathic pain. It presents spontaneous pain, mechanical allodynia, and thermal hyperalgesia. However, the mechanism of the allodynia is not clear.
Microtubules and microfilaments are important components of cytoskeleton. Microtubules are made up of tubulin, which includes a-tubulin (TUBA),(3-tubulin (TUBB) and y-tubulin (TUBG). Taxol is a microtubule stabilizer. Colchicine binds to the subunit of tubulin heterodimers to form a tubulin-colchicine complex that subsequently inhibits the polymerization of microtubule. Microfilament cytoskeleton structure is the thinnest fiber, which is about7nm in diameter. Microfilaments are mainly composed of actin, which contains free globular actin (G-actin) and fibrous actin polymerization (F-actin); however; only the latter is functional. Cytochalasin is the depolymerizing agent, inhibits the polymerization of microfilaments. Phalloidin is the stabilizer of microfilament, inhibits the depolymerization of microfilaments. Microtubules and microfilaments are in the state of dynamic balance, which is necessary for completing the physiological function.
Several findings suggest that cytoskeletal elements are involved in the mechanical transduction in sensory neurons and cytoskeletal elements may play a role in the development of chronic pain. Recent evidence shows that colchicine could relieve the hyperalgesia in rats with loose ligation of the sciatic nerve (CCI). In addition, intact actin filaments is reported to be necessary for inflammatory pain, and disruptors of the microfilaments markedly attenuate the hyperalgesia in rat paws caused by injection of epinephrine or its downstream mediators. However, the effects of microtubules and microfilaments dynamics on CCD-induced allodynia have not been clearly determined.
In CCD rats, several types of ion channels are reported to contribute to the allodynia, such as voltage-gated Na+and K+, hyperpolarization-activated cation current and transient receptor potential (TRP). As one of the TRP families, transient receptor potential vanilloid4(TRPV4) is a Ca2+-permeable polymodal receptor. TRPV4can be activated by various stimuli, including osmo-stimuli, mechano-stimuli, moderate heat, endogenous substances, and4a-phorbol12,13-didecanoate (4a-PDD). TRPV4is verified to contribute to the mechanical and thermal allodynia induced by CCD surgery in our previous study.
Microtubules and microfilaments interact with TRPV4and regulate the TRPV4channel activity. Disruption of the actin cytoskeleton impairs the TRPV4-mediated currents and Ca2+-influx in TRPV4expressing cells in response to hypotonic shock. Therefore, we hypothesized that TRPV4may be involved in the effects of microtubules and microfilaments on CCD-induced allodynia.
In order to test the hypothesis, we investigated the effects of microtubules and microfilaments on TRPV4activity and distribution in the DRG neurons in CCD rats, so to supply a possible mechanism for the effects of microtubules and microfilaments on CCD-induced allodynia.
Objective
To investigate the role of microtubules in allodynia induced by CCD surgery. In addition, the effects of microtubules dynamics on TRPV4were also tested.
Materials and methods
1. Chronic compression of dorsal root ganglion
After anesthetized by sodium pentobarbital (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5of CCD rats were exposed unilaterally. Two stainless steel L-shaped rods (0.63mm in diameter and4mm in length) were inserted into the L4and L5foramina respectively, to compress the DRGs. The muscle and skin layers were then sutured.
2. Behavioral testing
The behavioral testing was carried out in the ipsilateral hind paw of the animals prior to surgery and on postoperative days4,6,7,14and28. The effect of colchicine on CCD-induced allodynia was tested0.5-8h post injection.
Paw withdrawal mechanical threshold (PWMT) was evaluated with calibrated von Frey fibers. Starting with the lowest filament force, von Frey fibers were pressed against the lateral plantar surface of hindpaws in ascending order with sufficient force to cause slight bending and held for5s. A positive response was noted if the paw was immediately withdrawn. The stimulation of the same force was repeated five times at intervals of5s. If there were no less than three withdrawal responses to any of the five applications, the next lower force fibers was repeated.
Thermal allodynia was assessed using the paw withdrawal latencies (PWLs) of the animals in response to radiant heat (BME-410C) in a quiet room. Five values of the PWL were obtained for each rat at5-minute intervals. The intensity of the heat source was pre-calibrated to result in a baseline latency of approximately10s, and the cutoff time was set to20s.
3. DRG neuron culture
The L4and L5ganglia were harvested and incubated with collagenase and trypsin (Sigma-Aldrich, St. Louis, MO), followed by mechanically dissociated into single cells. Cells were plated onto coverslips coated with poly-L-lysine and cultured in Neurobasal medium (Invitrogen, Carlsbad, CA) that had been supplemented with N2, NGF and glutamine (Gibco Invitrogen, Grand Island, NY). The neurons were cultured for4days prior to use.
4. HEK293cells culture and transfection
HEK293cells were transfected with TRPV4-GFP plasmids (kindly supported by Professor Yu Xiao from the physiology department of Shandong University) using Lipofectamine2000reagent and following the manufacturer's protocol (Invitrogen, Carlsbad, CA). The fluorescent cells were used for both recording currents and measuring the levels of TRPV4gene and protein expression.
5. Cell viability assay
Cell viability was measured via3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (the MTT test). Cells were cultured on96-well plates, and8wells were used for each condition. The mean value from the8wells for a given condition was calculated and was taken as one sample. Six samples were taken for each condition. The cell viability of the group that was incubated in the normal medium was taken to represent100%viability.
6. Whole-cell current recording
Currents were digitized at a sampling rate of5kHz and filtered at1kHz for analysis (Axon200B amplifier with pCLAMP software; Molecular Devices, Foster City, CA). The bath solution contained the following compounds (in mM):124NaCl,5KC1,1.2KH2PO4,1.3MgCl2,2.4CaCl2, and26NaHCO3(310mOsm), pH adjusted to7.35with NaOH. The pipette solution contained the following compounds (in mM):140CsCl,2NaCl,3MgCl2,10Hepes, and5EGTA (290mOsm), pH adjusted to7.25with CsOH.
7. Real-time quantitative RT-PCR
7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. Fragments of TRPV4or β-actin were then amplified from the cDNA via PCR The2-△△CT method was used to analyze the data according to the relative gene expression levels.
8. Western blot analysis
7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. The protein levels were expressed as a ratio of the density of the detected band relative to the density of the β-actin (1:4000, CST, USA).
9. Immunofluorescence microscopy
DRG neurons were used for immunofluorescence studies. After blocked in0.1%Triton-100and5%(w/v) heatinactivated goat serum in PBS, DRG neurons were incubated with rabbit anti-TRPV4(1:200, Abcam, Cambridge, UK) antibodies overnight at4℃. Unbound antibodies were removed by three PBS washes for15min each at room temperature. Then DRG neurons were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, Zhongshan Goldenbridge, Beijing, China) for1h at room temperature. Cell nuclei were counterstained with DAPI prior to analysis under a confocal laser scanning microscope (LSM710, Zeiss, Jena, Germany).
Results
1. Effects of microtubules depolymerization by colchicine on CCD-induced allodynia
After surgery, CCD rats expressed a significant decrease in PWLs (in response to temperature and mechanical stimulus) of the ipsilateral hind paws (P<0.05). The decrease in PWLs peaked at7days post-CCD (P<0.01); we thus performed subsequent experiments at7days post-operation.
When compared with intrathecal administration of saline in CCD rats, administration of colchicine (625μg/kg and1250μg/kg) produced a dose-dependent and partial reduction of allodynia in CCD rats (P<0.05); however, the similar result could not be observed in CCD rats injected with colchicine in the dose of312.5μg/kg (all P>0.05). The significant reduction in allodynia was observed at0.5h, peaked at2h, and lasted for about8h post-injection.
2. Effects of colchicine on the viability of DRG neurons and HEK293-TRPV4cells
MTT results showed that, the viability of DRG neurons and HEK293-TRPV4cells were not influenced by the application of colchicine.
3. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neurons
At a holding potential of-60mV. the peak value of the inward current in DRG neurons was recorded as230.55±17.23pA. The peak current values in neurons that were incubated with colchicine at concentrations of0.01μg/ml,0.05μg/ml and0.1μg/ml were201.62±4.72pA,165.25±7.44pA, and128.41±3.75pA, respectively; these values represent colchicine-induced inhibition of12.6%,28.3%and44.3%, respectively. In general, incubation with a solution that contained colchicine suppressed the inward current, and exposure to a0.1μg/ml concentration of colchicine induced a minimal peak current value and a near maximum inhibition of the TRPV4channels (P<0.05). Thus,0.1μg/ml of colchicine was subsequently used as the preferred concentration for the inhibition of the TRPV4channels.
4. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neurons in CCD rats
The peak values of the4aPDD-induced TRPV4currents in CCD group were also suppressed by colchicine. The4aPDD-induced peak current had a value of310.19±9.17pA, and this current was depressed by26%(to230.10±12.10pA) when the neurons were exposed to colchicine. The currents of the neurons in the presence of colchicine were activated at55.7s, reached a peak at only199.1s after activation (243.6s after activation in the absence of colchicine, P<0.05), and relaxed to a pseudosteady state for the remainder of the recording.
5. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in HEK293-TRPV4cells
Colchicine administration also resulted in a reduction and advance of the peak current in HEK293-TRPV4cells (412.50±27.15pA and131.6s after activation for HEK293-TRPV4cells;219.53±5.24pA and42.7s after activation for HEK293-TRPV4cells in the presence of colchicine, P<0.05). HEK293-TRPV4cells incubated with colchicine expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages.
6. Effects of microtubules depolymerization by colchicine on the expression of TRPV4
The level of TRPV4protein expression was diminished when animals were injected with colchicine in the doses of625ug/kg and1250μg/kg, and significant decreases were observed at2h and4h post-injections (P<0.05). Pretreatment with colchicine significantly and dose-dependently depressed the expression of TRPV4mRNA with the minimum expression level occurred at2h after exposure to colchicine in the dose of1250μg/kg (P<0.05). The inhibition of colchicine on TRPV4expression was associated with colchicine-induced relief of allodynia in CCD rats.
7. Effects of microtubules depolymerization by colchicine on the subcellular localization of TRPV4
Intrathecal administration of colchicine failed to alter the ratio of neurons with pronounced localization of TRPV4. TRPV4immunofluoresecnce was observed more in the plasma membrane than that in the cytoplasm, and this distribution was unaffected by colchicine. The subcellular localization of TRPV4observed in the current study was in agreement with previous findings.
Conclusion
Depolymerization of microtubules by colchicine could attenuate the allodynia in CCD rats. TRPV4contributes to the relief of allodynia by the depolymerization of microtubules.
Objective
To investigate the role of microfilaments in allodynia induced by chronic compression of the dorsal root ganglion; to investigate the effects of microfilaments on TRPV4.
Materials and methods
1.CCD surgery
Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5vertebrae were exposed unilaterally in accordance with a previously described procedure. Two hollow, stainless steel, L-shaped rods (0.66-mm diameter and4-mm length), connected to silicon tubing (0.51mm ID,0.94OD,30-40mm length) filled with heparin, were inserted into L4and L5foramen respectively, to compress the DRG. The other end of the tubing was sealed, except when injecting drugs. The muscle and skin layers were then sutured. Sham surgery involved identical surgical procedures to those described but without insertion of the rods.
2. Behavioral testing
All behavioral tests were carried out in a quiet room and conducted under blind conditions. Behavioral tests were conducted on the7th day post-surgery and1,2,4,6,8, and24h after injection of chemicals or saline. Chemicals and saline were all injected by the silicon tubing.
3. DRG neurons culture
The L4and L5ganglia were removed from the operated side of each animal on7days after surgery. The neurons were cultured according to the methods in chapter I.
4. Cell viability assay
The viability of DRG neurons were tested according to the method in chapter I.
5. Whole-cell current recording
The whole-cell current of TRPV4were tested according to the method in chapter I.
6. Immunofluorescence microscopy
The localization of TRPV4is tested according to the method in chapter I.
Results
1. Effects of microfilaments dynamics on the allodynia in CCD rats
All rats walked normally after the CCD surgery, indicating that CCD surgery did not injury the motor behavior.
Both of two disrupting agents (CB and CD) resulted in attenuation of CCD-induced mechanical and thermal allodynia (n=9in each group, both P<0.01). Compared with the administration of saline in CCD rats, administration of cytochalasin B (200μg and300μg) produced a dose-dependent and partial reduction of allodynia in CCD rats. However, similar result could not be observed in CCD rats injected with cytochalasin B in the dose of100ug (P>0.05, n=9in each group). The significant reduction in allodynia was observed at1h, peaked at2h, and lasted less than24h post-injection.
Phalloidin didn't affect the mechanical and thermal allodynia induced by CCD surgery (all P>0.05, n=9in each group). Phalloidin was injected to investigate whether it could prevent cytochalasin B-induced loss of allodynia in CCD rats, confirming that the inhibition of CCD-induced allodynia by anti-microfilaments agents was due to microfilaments disruption. Injection of phalloidin1h preceding administration of cytochalasin B significantly inhibited the cytochalasin B-induced attenuation of allodynia in CCD rats (n=9).
2. Effects of microfilaments dynamics on TRPV4currents in DRG neurons in CCD rats
Similar to CCD-induced allodynia in vivo, whether the potentiation of TRPV4in CCD rats was dependent on intact actin filaments was tested. The peak inward current was significantly higher in CCD group than in sham group (n=12in CCD group, n=10in sham group, P<0.05). The effects of dynamic changes of microfilaments on TRPV4-mediated channel activity were tested next.
Exposure of cultured compressed DRG neurons to disruptors (cytochalasin B and cytochalasin D, both10-4M) for2h prior to the patch clamp significantly inhibited the potentiation of TRPV4(the peak value is310.19±9.17pA in CCD group,245.48±12.12pA in DRG neurons exposure to cytochalasin B, and237.81±10.17pA in DRG neurons exposure to cytochalasin D, n=12in each group, P<0.05), which are consistent with the attenuation of the effects of disruptors on CCD-induced allodynia. DRG neurons incubated with disruptors expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages, which are in accordance with the inhibition of TRPV4currents by disruption of microfilaments. The time course was analyzed to further confirm the effect of disruption of microfilaments on TRPV4channel opening. A delayed peak time was observed in DRG neurons from CCD rats (243.60±20.00s for CCD group and200.37±15.40s for sham group, P<0.05). Disruption of microfilaments induced an advance of the peak current in DRG neurons of CCD rats (123.44±10.10s for DRG neurons exposure to cytochalasin B, and193.76±12.40s in DRG neurons exposure to cytochalasin D, P<0.05).
Phalloidin (10-5M) resulted in a reduction and delay of TRPV4current (80.87±5.42pA and311.72±14.56s in DRG neuron exposure to phalloidin, n=12, P<0.01), which is not consistent with the effect of phalloidin on CCD-induced allodynia. In association with the inhibition of TRPV4currents, the reversal potentials shifted toward more positive voltages. MTT tests were carried out to exclude the effects of chemicals (cytochalasin B, cytochalasin D and phalloidin) on the cell viabilities of DRG neurons. The results showed that both disruptors and stabilizer of microfilaments had no significant influence on the cell viabilities of DRG neurons (P>0.05).
3. Effects of microfilaments dynamics on the subcellular localization of TRPV4in DRG neurons in CCD rats
Results demonstrated that more TRPV4immunofluoresecnce was observed in the plasma membrane than the cytoplasm in all groups. Cytochalasin B (10-4M) resulted in an apparent decrease of the plasma membrane association of TRPV4(83.62±2.14%in CCD group and69.63±3.41%in cytochalasin B group, P<0.05). Meanwhile, phalloidin (10-5M) also leaded to a decrease of plasma membrane association of TRPV4(54.24±2.44%, P<0.05).
Conclusions
This study demonstrates that microfilaments play an important role in allodynia induced by chronic compression of the dorsal root ganglion and TRPV4contributes to the attenuation of allodynia by depolymerization of microfilaments.
微管与微丝是细胞骨架的主要组成部分,参与维持细胞形态、保持细胞内部结构的有序性、转运细胞内物质等生理功能。微管是细胞骨架纤维中最粗的纤维,是由微管蛋白组成的直径约为25nm的中空圆柱状纤维。微管蛋白主要包括α-微管蛋白(a-tubulin, TUBA)、β-微管蛋白(p-tubulin, TUBB)和γ-微管蛋白(γ-tubulin,TUBG)。TUBA和TUBB以非共价键的形式连接形成异二聚体,异二聚体首尾相连则形成微管蛋白原纤维。哺乳动物中,13条微管蛋白原纤维构成1个中空的微管。紫杉醇是微管的稳定剂,稳定微管的聚合状态,抑制微管解聚。秋水仙碱是一种微管解聚剂,抑制微管聚合并促进微管解聚。微丝是细胞骨架结构中最细的纤维,直径7nm左右,主要由肌动蛋白螺旋状聚合形成,包含游离球状肌动蛋白(G-actin)和聚合纤维状肌动蛋白(F-actin)两种形式,只有后者具有生物学作用。细胞松弛素是微丝的解聚剂,可抑制微丝聚合并促进微丝解聚。鬼笔环肽是一种微丝稳定剂,具有稳定聚合微丝、抑制微丝解聚的作用。微管与微丝处于不断的解聚与聚合的动态平衡状态,这种动态平衡是完成生理功能的必要条件。
研究证实,微管和微丝的解聚可降低炎性疼痛。炎性疼痛中,炎症细胞和炎症介质在趋化因子的作用下迁移到病变处发挥作用,其迁移是在微管的作用下完成的,低浓度(10-8mol/L)短时间(30-120分钟)应用微管解聚剂秋水仙碱可抑制炎性细胞的迁移,控制炎症。肾上腺素(epinephrine, EPI)及其下游产物可提高感觉神经元对疼痛的敏感性,而单独应用微管解聚剂或微丝解聚剂均可抑制肾上腺素诱导的痛觉过敏。微管也能够介导神经性疼痛。坐骨神经慢性压迫(sciatic chronic constriction injury, CCI)后,大鼠出现后肢的热痛觉过敏,注射秋水仙碱可缓解CCI所致的热痛觉过敏。因此,我们推测微管、微丝可能介导CCD大鼠机械痛觉过敏和热痛觉过敏,因此,本课题拟利用微管和微丝的解聚剂及聚合剂研究二者在CCD大鼠机械痛敏和热痛敏中的作用。
持续受压后,大鼠的痛觉过敏和DRG神经元的高兴奋性与多种离子通道有关,如电压门控性Na+通道和K+通道、超极化激活性阳离子通道和瞬时感受器电位离子通道(transient receptor potential, TRP)通道等。瞬时感受器电位离子通道香草素受体亚家族4(transient receptor potential vanilloid subtype4, TRPV4)是TRP超家族成员之一,是一种Ca2+通透性较高的阳离子通道。DRG神经元的细胞膜、细胞浆和细胞核上均可检测到TRPV4,细胞膜上表达的TRPV4通道是介导痛觉过敏的主要部分。实验证据支持DRG中表达的TRPV4参与介导多种神经性疼痛和炎性疼痛。前列腺素E2(prostaglandin E2, PGE2)可增强低渗或中度高渗溶液刺激引起的异常疼痛和痛觉过敏,TRPV4反义寡核苷酸干扰和基因敲除后,大鼠和小鼠的异常疼痛和痛觉过敏缓解。TRPV4反义寡核苷酸干扰后,微管稳定剂紫杉醇引起的机械痛觉过敏降低。本课题组的前期研究证实,TRPV4通道参与介导CCD大鼠的机械痛觉过敏和热痛觉过敏。
微管、微丝与TRPV4直接相连并可调节TRPV4通道的功能。如微丝解聚后,TRPV4通道介导的内向电流和低渗休克导致的TRPV4通道的Ca2+内流受到抑制,TRPV4与肌动蛋白的共表达减少。结合TRPV4在CCD大鼠痛敏中的作用,我们推测,TRPV4可能参与微管、微丝对CCD大鼠痛敏的调节过程。
因此,本课题中,我们拟观察微管、微丝对CCD大鼠痛敏的影响,并研究微管、微丝对CCD大鼠DRG神经元TRPV4通道功能、表达和定位的影响,为微管、微丝对CCD大鼠痛敏的调节提供可能的作用机制。
目的
1.研究微管解聚剂秋水仙碱对CCD大鼠机械痛觉过敏和热痛觉过敏的影响。
2.研究秋水仙碱对TRPV4通道功能和表达的影响,探讨微管解聚影响CCD大鼠痛觉过敏的机制。
材料与方法
1.CCD模型的制备
应用戊巴比妥钠(50mg/kg)行腹腔注射麻醉后,沿L4-S1棘突作后正中偏右切口,暴露横突的根部及L4-L5椎间外孔后,将每侧长4mm,直径0.63mm的“L”形不锈钢棒沿L4-L5椎间外孔的前壁上方水平插入椎管内,另一端位于椎管壁外。通过控制“L”形棒椎管壁外侧端使其椎管内端沿椎骨内壁缓慢下滑至椎管中部,恒定地压迫DRG及邻近神经根。术后用生理盐水冲洗切口,依次缝合肌肉、腰背筋膜和皮肤,腹腔注射青霉素40万单位预防感染,术后密切观察大鼠生命体征变化。
2.神经行为学的测定
对CCD大鼠的手术侧及正常大鼠的同侧后肢进行行为学检测,时间为：术前,术后第4、6、7、14、28天；药物对行为学影响检测时间为：药物处理前测量,排除痛觉表现不明显者(<5%)；药物处理后0.5、1、2、4、8小时分别测量。
大鼠的机械痛觉过敏采用机械缩足反射阈值(Mechanical withdrawal threshold, MWT)测量,使用BME-403型、Von Frey Fibers机械痛刺激仪进行。将不同强度的Von Frey纤维由低到高依次地刺激CCD大鼠手术侧或正常大鼠同侧后肢足底中部并维持5秒,阳性反应为出现快速缩足或舔足现象。每个强度的纤维刺激5次,机械缩足反射阈值为至少能够引发3次缩足反应的纤维强度。
大鼠热痛觉过敏通过热辐射刺激缩爪反应潜伏期(paw withdrawal latency,PWL)来确定,采用BME-410C型热痛刺激仪测量。大鼠安静后,将光源焦距对准CCD大鼠手术侧或正常大鼠同侧后肢足底中心。从开始照射到引起后肢回缩反应所需的时间,即为当次热辐射刺激缩爪反应潜伏期。每肢照射5次,测量结果的均值即为此大鼠的热辐射刺激缩爪反应潜伏期。
3.成年大鼠DRG神经元的培养
取出CCD术后第7天手术侧L4-L5的DRG和正常大鼠同侧L4-L5的DRG,Ⅰ型胶原酶和胰蛋白酶分别消化1小时和30分钟后,应用Neurobasal+1%N2+20ng/mlNGF+1%谷氨酰胺+100U/ml青链霉素培养,培养第4天的DRG神经元用于全细胞膜片钳技术、细胞活性测定及免疫细胞化学检测等后续实验。
4.HEK293细胞的培养和转染
HEK293细胞培养于10%胎牛血清和1%双抗的DMEM培养基中,3-4天传代一次,传代后24小时转染。Lipofectamine2000脂质体和(?)TRPV4-GFP质粒先各自加入一定量的缓冲液OPTI-MEM中,混匀后静置5分钟。将两者再混匀,室温放置20-30分钟。将质粒脂质体混合物加入HEK293细胞培养液中孵育4-6小时,然后将培养液更换为包含血清和抗生素的DMEM培养液。转染后24-48小时进行电生理实验或细胞活性检测。
5.细胞活性检测
将成年大鼠DRG神经元和HEK293-TRPV4细胞以1×104／孔的密度接种于96孔培养板,在不同浓度秋水仙碱培养基中培养2小时后,吸去原培养液,每孔加入80μl培养液和20μl5mg/ml的四甲基偶氮唑(3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide, MTT)。经37℃孵箱孵育4小时后,将上清液吸去后应用PBS冲洗后,每孔加入150μl甲基亚砜(Dimethyl sulphoxide, DMSO)溶剂。将孔板放置在摇床上,通过10分钟的低速震荡即可充分溶解结晶。应用酶标检测仪测定波长490nm处的吸光度OD值,各组均以正正常培养基培养的成年大鼠DRG神经元的吸光度作为100%,其他情况下的吸光度数值需与之相比较。
6.全细胞膜片钳记录TRPV4通道的电流
采用常规高阻抗封接的全细胞记录方式,检测DRG神经元及HEK293-TRPV4细胞中TRPV4通道介导的内向电流。电极内液(mmol/L):140CsCl,2NaCl,3MgCl2,10Hepes,5EGTA (290mOsm),用CsOH调节pH至7.25,用针头式滤器(孔径0.22μm)过滤后分装,-20℃保存。电极入水电阻为3-6MΩ。电极外液(mmol/L):124NaCl,5KC1,1.2KH2PO4,1.3MgCl2,2.4CaCl2,26NaHCO3(310mOsm),用NaOH调节pH至7.35,4℃保存。
7.实时定量RT-PCR检测
CCD术后第7天,鞘内注射不同浓度秋水仙碱0.5-8小时后,从各组大鼠手术侧L4-L5DRG中提取RNA,实时定量RT-PCR检测TRPV4mRNA表达的变化。
8. Western blotting检测
CCD术后第7天,鞘内注射不同浓度秋水仙碱0.5-8小时后,从各组大鼠手术侧L4-L5DRG中提取蛋白质,Western blotting检测TRPV4蛋白表达的变化。
9.组织免疫荧光及免疫细胞化学检测TRPV4的定位
将DRG组织切片及培养的DRG神经元用冷丙酮固定10分钟后(-20℃),加入2%BSA室温封闭1小时,然后加入TRPV4一抗4℃孵育过夜。PBS冲洗3次后,加入带有绿色荧光标记的TRPV4二抗室温孵育1小时。使用DAPI细胞核染色后,封片,荧光显微镜下观察TRPV4在DRG神经元的定位区域。
结果
1.鞘内注射微管解聚剂秋水仙碱可缓解CCD大鼠的痛觉过敏
CCD术后,大鼠手术侧机械和热痛觉阈值降低(P＜0.05)。术后7天时,手术侧机械痛觉阈值和热辐射刺激缩爪反应潜伏期降至最低(P＜0.01),此后逐渐升高。
625μg/kg-1250μg/kg的秋水仙碱可部分抑制CCD后大鼠的机械痛敏和热痛敏,此抑制作用(P＜0.05)呈剂量依赖性升高。此抑制作用于鞘注秋水仙碱后30分钟可测得,2小时作用达到最大,此抑制作用可持续至8小时,而312.5μg/kg秋水仙碱则无抑制作用(P＞0.05)。
2.微管解聚剂秋水仙碱对DRG神经元以及HEK293-TRPV4细胞活性的影响
0.1μg/ml秋水仙碱对DRG神经元和HEK293-TRPV4细胞的活性无影响。其中,0.1μg/ml秋水仙碱分别孵育DRG神经元和HEK293-TRPV4细胞0.5小时和2小时,细胞活性均未受影响(P＞0.05)
3.微管解聚剂秋水仙碱对正常大鼠DRG神经元TRPV4通道功能的影响
秋水仙碱孵育后,正常大鼠DRG神经元TRPV4的电流降低。秋水仙碱0.01μ/ml,0.05μg/ml和O.1μg/ml孵育DRG神经元2小时后,TRPV4的内向电流分别为201.62±4.72pA、165.25±7.44pA和128.41±±3.75pA。因此,秋水仙碱对DRG神经元TRPV4电流的抑制呈剂量依赖性,且0.1μg/ml秋水仙碱对TRPV4电流的影响最大(P<0.05)。因此,在本课题中,将0.1μg/ml秋水仙碱作为最适浓度。
4.微管解聚剂秋水仙碱对CCD大鼠DRG神经元TRPV4通道功能的影响
与正常大鼠DRG神经元TRPV4电流相比,CCD大鼠DRG神经元TRPV4通道介导的内向电流峰值增加(CCD大鼠电流峰值为310.19±9.17pA,正常大鼠为230.55±17.23pA,P<0.05)。CCD大鼠DRG神经元TRPV4通道的电流峰值延迟(CCD大鼠为243.6s,正常大鼠为172.4s,P＜0.05)。0.1μg/ml秋水仙碱孵育2小时后,CCD大鼠DRG神经元TRPV4通道电流峰值降低,且峰值时间延迟(230.10±12.10pA,199.1s,P＜0.05)
5.微管解聚剂秋水仙碱对HEK293-TRPV4细胞TRPV4通道功能的影响
0.1μg/ml秋水仙碱孵育2小时后,HEK293-TRPV4细胞的TRPV4通道电流峰值降低,且峰值时间延迟(正常HEK293-TRPV4细胞为412.50±27.15pA,131.6s;秋水仙碱组为219.53±5.24pA和42.7s,P＜0.05)。孵育秋水仙碱后,HEK293-TRPV4细胞的内向电流表现出典型的TRPV4通道的外向整流特性,但逆转电位更倾向于正相电位。
6.微管解聚剂秋水仙碱对TRPV4通道表达的影响
鞘内注射625μg/kg-1250μg/kg秋水仙碱时,TRPV4通道的基因表达明显降低,其中,鞘内注射1250μg/kg秋水仙碱2小时,TRPV4通道的基因表达下降最明显(P＜0.05)。当鞘内注射625μg/kg-1250μg/kg秋水仙碱时,TRPV4通道的蛋白表达显著降低,其中,鞘内注射1250μg/kg秋水仙碱2小时和4小时,TRPV4通道的蛋白表达下降最明显(P＜0.05)
7.微管解聚剂秋水仙碱对TRPV4定位的影响
组织免疫荧光的结果提示,鞘内注射1250μg/kg秋水仙碱未影响CCD大鼠背根神经节TRPV4的定位。DRG神经元的免疫细胞化学结果示,DRG神经元上胞膜表达的TRPV4多于胞浆内表达的TRPV4,并且秋水仙碱并未影响TRPV4的定位。
结论
1.微管解聚剂秋水仙碱缓解CCD大鼠的机械痛敏和热痛敏。
2.秋水仙碱可抑带(?)TRPV4的表达,降低TRPV4通道的电流峰值,因此,TRPV4介导了秋水仙碱对CCD大鼠痛敏的缓解作用。
目的
1.研究微丝在CCD大鼠痛觉过敏中的作用。
2.研究微丝对CCD大鼠DRG神经元TRPV4通道功能和定位的影响,探讨微丝调控CCD大鼠痛觉过敏的机制。
方法
1.CCD模型的制备
大鼠被随机分为CCD组和假手术组。戊巴比妥钠(50mg/kg)麻醉后,沿L4-S1棘突作后正中偏右切口,暴露右侧L4-L5椎间外孔后,将每侧长4mm,外径0.63mm的中空的“L”形不锈钢棒沿L4-L5椎间外孔的前壁上方水平插入椎管内,恒定地压迫DRG及其邻近的神经根。不锈钢棒外套有内径0.51mm,30-40mm长的硅胶管,硅胶管的外端置于椎管壁外,内端随中空钢棒一起插入椎管内,硅胶管内充满肝素。硅胶管的外端是封闭的,只有注射药物时打开。术后用生理盐水冲洗切口,依次用4-0丝线缝合肌肉、筋膜和皮肤,并注射青霉素40万单位以预防感染。假手术组除不插钢棒压迫神经节外,其余操作同手术组。术后大鼠没有发生伤侧肢体自噬现象。
2.神经行为学的测定
分别于手术后7天注射化学药物前及注射药物后1、2、4、6、8、24小时测量,排除痛觉表现不明显者(<5%)；所有的测量在一个安静的环境内进行,室温保持在26±0.5℃,且均使用单盲法。药物通过与压迫DRG的中空不锈钢管相连的硅胶管直接注射。
大鼠机械缩足反射阈值和热辐射刺激缩爪反应潜伏期的测量方法同第一部分。
3.成年大鼠DRG神经元的培养
CCD术后7天,取出手术侧L4-L5的DRG。细胞培养方法同第一部分。
4.细胞活性检测
成年大鼠DRG神经元在细胞松弛素B或鬼笔环肽培养基中培养2小时后,用于检测DRG神经元的活性,检测方法同第一部分。
5.全细胞膜片钳记录TRPV4通道的电流
同第一部分。
6.免疫细胞化学
同第一部分。
结果
1.微丝对CCD大鼠痛觉过敏的影响
所有实验大鼠在手术前后及给药处理前后步态均正正常,足无畸形,评分均为1分,损伤前、后各组间无显著性差异。
微丝解聚剂细胞松弛素B和细胞松弛素D皆可缓解CCD大鼠的机械痛敏和热痛敏(n=9,P＜0.01)。细胞松弛素B(200μg-300μg)可部分抑制CCD大鼠的痛敏,且此抑制作用呈剂量依赖性表现,然而,100μg细胞松弛素B无此抑制作用(P＞0.05,n=9)。CCD大鼠痛敏的缓解在注射细胞松弛素B后1小时出现,2小时达到高峰,持续少于24小时。
微丝聚合剂鬼笔环肽单独注射对CCD大鼠的机械和热痛敏的影响无统计学意义(P＞0.05,n=9)。细胞松弛素B注射前1小时注射鬼笔环肽可以抑制细胞松弛素B对CCD大鼠痛敏的缓解作用(n=9)。
2.微丝对CCD大鼠DRG神经元TRPV4通道功能的影响
CCD组TRPV4的电流峰值远高于假手术组(CCD组n=12,假手术组n=10,P＜0.05)。
细胞松弛素B和细胞松弛素D均可显著抑制(?)TRPV4的电流峰值(CCD组为310.19±9.17pA,细胞松弛素B组为245.48±12.12pA,细胞松弛素D组为237.81±10.17pA,各组中n=12,P＜0.05)。这与二者对CCD大鼠痛敏的影响一致。经微丝解聚剂孵育后,DRG神经元的I/V曲线表现出典型的TRPV4外向整流的特性,然而,逆转电位更倾向于正相电位,这与微丝对TRPV4(?)降值电流的抑制相一致。
CCD大鼠DRG神经元的峰值时间延迟(CCD组为243.60±20.00s,假手术组为200.37±15.40s,P＜0.05)。经微丝解聚剂孵育后,CCD大鼠DRG神经元TRPV4电流峰值时间提前(细胞松弛素B组为123.44±10.10s,细胞松弛素D组为193.76±12.40s,P＜0.05)。
微丝稳定剂鬼笔环肽(10-SM)可导致TRPV4电流的峰值降低和延迟(80.87±5.42pA,311.72±14.56s,n=12,P＜0.01),这与鬼笔环肽对CCD大鼠痛敏的影响不一致。与鬼笔环肽对TRPV4电流影响一致的是,DRG神经元I/V曲线更倾向于正相电位。MTT实验结果表明,微丝的解聚剂和稳定剂对于DRG神经元的活性无影响(P＞0.05)。
3.微丝对CCD大鼠DRG神经元TRPV4定位的影响
CCD大鼠DRG神经元细胞膜上表达的TRPV4多于细胞浆中表达的TRPV4。经细胞松弛素B(10-4M)孵育2小时后,DRG神经元细胞膜上TRPV4的表达量明显降低(CCD组为83.62±2.14%,细胞松弛素B组为69.63±3.41%,P＜0.05)。此外,鬼笔环肽(10-SM)也导致DRG神经元细胞膜上TRPV4的表达量降低(54.24±2.44%,P＜0.05)
结论
1.微丝解聚缓解CCD大鼠的痛觉过敏,微丝稳定剂可消除微丝解聚剂的镇痛作用。
2.微丝解聚抑伟TRPV4通道的电流峰值,降低神经元细胞膜上TRPV4的表达,因此,微丝通过TRPV4通道调节CCD大鼠的痛觉过敏。
Neuropathic pain is one kind of pain irritated by primary damage and malfunction of nervous systems. Chronic compression of dorsal root ganglion (DRG)(CCD) is a typical model of neuropathic pain. It presents spontaneous pain, mechanical allodynia, and thermal hyperalgesia. However, the mechanism of the allodynia is not clear.
Microtubules and microfilaments are important components of cytoskeleton. Microtubules are made up of tubulin, which includes a-tubulin (TUBA),(3-tubulin (TUBB) and y-tubulin (TUBG). Taxol is a microtubule stabilizer. Colchicine binds to the subunit of tubulin heterodimers to form a tubulin-colchicine complex that subsequently inhibits the polymerization of microtubule. Microfilament cytoskeleton structure is the thinnest fiber, which is about7nm in diameter. Microfilaments are mainly composed of actin, which contains free globular actin (G-actin) and fibrous actin polymerization (F-actin); however; only the latter is functional. Cytochalasin is the depolymerizing agent, inhibits the polymerization of microfilaments. Phalloidin is the stabilizer of microfilament, inhibits the depolymerization of microfilaments. Microtubules and microfilaments are in the state of dynamic balance, which is necessary for completing the physiological function.
Several findings suggest that cytoskeletal elements are involved in the mechanical transduction in sensory neurons and cytoskeletal elements may play a role in the development of chronic pain. Recent evidence shows that colchicine could relieve the hyperalgesia in rats with loose ligation of the sciatic nerve (CCI). In addition, intact actin filaments is reported to be necessary for inflammatory pain, and disruptors of the microfilaments markedly attenuate the hyperalgesia in rat paws caused by injection of epinephrine or its downstream mediators. However, the effects of microtubules and microfilaments dynamics on CCD-induced allodynia have not been clearly determined.
In CCD rats, several types of ion channels are reported to contribute to the allodynia, such as voltage-gated Na+and K+, hyperpolarization-activated cation current and transient receptor potential (TRP). As one of the TRP families, transient receptor potential vanilloid4(TRPV4) is a Ca2+-permeable polymodal receptor. TRPV4can be activated by various stimuli, including osmo-stimuli, mechano-stimuli, moderate heat, endogenous substances, and4a-phorbol12,13-didecanoate (4a-PDD). TRPV4is verified to contribute to the mechanical and thermal allodynia induced by CCD surgery in our previous study.
Microtubules and microfilaments interact with TRPV4and regulate the TRPV4channel activity. Disruption of the actin cytoskeleton impairs the TRPV4-mediated currents and Ca2+-influx in TRPV4expressing cells in response to hypotonic shock. Therefore, we hypothesized that TRPV4may be involved in the effects of microtubules and microfilaments on CCD-induced allodynia.
In order to test the hypothesis, we investigated the effects of microtubules and microfilaments on TRPV4activity and distribution in the DRG neurons in CCD rats, so to supply a possible mechanism for the effects of microtubules and microfilaments on CCD-induced allodynia.
Objective
To investigate the role of microtubules in allodynia induced by CCD surgery. In addition, the effects of microtubules dynamics on TRPV4were also tested.
Materials and methods
1. Chronic compression of dorsal root ganglion
After anesthetized by sodium pentobarbital (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5of CCD rats were exposed unilaterally. Two stainless steel L-shaped rods (0.63mm in diameter and4mm in length) were inserted into the L4and L5foramina respectively, to compress the DRGs. The muscle and skin layers were then sutured.
2. Behavioral testing
The behavioral testing was carried out in the ipsilateral hind paw of the animals prior to surgery and on postoperative days4,6,7,14and28. The effect of colchicine on CCD-induced allodynia was tested0.5-8h post injection.
Paw withdrawal mechanical threshold (PWMT) was evaluated with calibrated von Frey fibers. Starting with the lowest filament force, von Frey fibers were pressed against the lateral plantar surface of hindpaws in ascending order with sufficient force to cause slight bending and held for5s. A positive response was noted if the paw was immediately withdrawn. The stimulation of the same force was repeated five times at intervals of5s. If there were no less than three withdrawal responses to any of the five applications, the next lower force fibers was repeated.
Thermal allodynia was assessed using the paw withdrawal latencies (PWLs) of the animals in response to radiant heat (BME-410C) in a quiet room. Five values of the PWL were obtained for each rat at5-minute intervals. The intensity of the heat source was pre-calibrated to result in a baseline latency of approximately10s, and the cutoff time was set to20s.
3. DRG neuron culture
The L4and L5ganglia were harvested and incubated with collagenase and trypsin (Sigma-Aldrich, St. Louis, MO), followed by mechanically dissociated into single cells. Cells were plated onto coverslips coated with poly-L-lysine and cultured in Neurobasal medium (Invitrogen, Carlsbad, CA) that had been supplemented with N2, NGF and glutamine (Gibco Invitrogen, Grand Island, NY). The neurons were cultured for4days prior to use.
4. HEK293cells culture and transfection
HEK293cells were transfected with TRPV4-GFP plasmids (kindly supported by Professor Yu Xiao from the physiology department of Shandong University) using Lipofectamine2000reagent and following the manufacturer's protocol (Invitrogen, Carlsbad, CA). The fluorescent cells were used for both recording currents and measuring the levels of TRPV4gene and protein expression.
5. Cell viability assay
Cell viability was measured via3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (the MTT test). Cells were cultured on96-well plates, and8wells were used for each condition. The mean value from the8wells for a given condition was calculated and was taken as one sample. Six samples were taken for each condition. The cell viability of the group that was incubated in the normal medium was taken to represent100%viability.
6. Whole-cell current recording
Currents were digitized at a sampling rate of5kHz and filtered at1kHz for analysis (Axon200B amplifier with pCLAMP software; Molecular Devices, Foster City, CA). The bath solution contained the following compounds (in mM):124NaCl,5KC1,1.2KH2PO4,1.3MgCl2,2.4CaCl2, and26NaHCO3(310mOsm), pH adjusted to7.35with NaOH. The pipette solution contained the following compounds (in mM):140CsCl,2NaCl,3MgCl2,10Hepes, and5EGTA (290mOsm), pH adjusted to7.25with CsOH.
7. Real-time quantitative RT-PCR
7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. Fragments of TRPV4or β-actin were then amplified from the cDNA via PCR The2-△△CT method was used to analyze the data according to the relative gene expression levels.
8. Western blot analysis
7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. The protein levels were expressed as a ratio of the density of the detected band relative to the density of the β-actin (1:4000, CST, USA).
9. Immunofluorescence microscopy
DRG neurons were used for immunofluorescence studies. After blocked in0.1%Triton-100and5%(w/v) heatinactivated goat serum in PBS, DRG neurons were incubated with rabbit anti-TRPV4(1:200, Abcam, Cambridge, UK) antibodies overnight at4℃. Unbound antibodies were removed by three PBS washes for15min each at room temperature. Then DRG neurons were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, Zhongshan Goldenbridge, Beijing, China) for1h at room temperature. Cell nuclei were counterstained with DAPI prior to analysis under a confocal laser scanning microscope (LSM710, Zeiss, Jena, Germany).
Results
1. Effects of microtubules depolymerization by colchicine on CCD-induced allodynia
After surgery, CCD rats expressed a significant decrease in PWLs (in response to temperature and mechanical stimulus) of the ipsilateral hind paws (P<0.05). The decrease in PWLs peaked at7days post-CCD (P<0.01); we thus performed subsequent experiments at7days post-operation.
When compared with intrathecal administration of saline in CCD rats, administration of colchicine (625μg/kg and1250μg/kg) produced a dose-dependent and partial reduction of allodynia in CCD rats (P<0.05); however, the similar result could not be observed in CCD rats injected with colchicine in the dose of312.5μg/kg (all P>0.05). The significant reduction in allodynia was observed at0.5h, peaked at2h, and lasted for about8h post-injection.
2. Effects of colchicine on the viability of DRG neurons and HEK293-TRPV4cells
MTT results showed that, the viability of DRG neurons and HEK293-TRPV4cells were not influenced by the application of colchicine.
3. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neurons
At a holding potential of-60mV. the peak value of the inward current in DRG neurons was recorded as230.55±17.23pA. The peak current values in neurons that were incubated with colchicine at concentrations of0.01μg/ml,0.05μg/ml and0.1μg/ml were201.62±4.72pA,165.25±7.44pA, and128.41±3.75pA, respectively; these values represent colchicine-induced inhibition of12.6%,28.3%and44.3%, respectively. In general, incubation with a solution that contained colchicine suppressed the inward current, and exposure to a0.1μg/ml concentration of colchicine induced a minimal peak current value and a near maximum inhibition of the TRPV4channels (P<0.05). Thus,0.1μg/ml of colchicine was subsequently used as the preferred concentration for the inhibition of the TRPV4channels.
4. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neurons in CCD rats
The peak values of the4aPDD-induced TRPV4currents in CCD group were also suppressed by colchicine. The4aPDD-induced peak current had a value of310.19±9.17pA, and this current was depressed by26%(to230.10±12.10pA) when the neurons were exposed to colchicine. The currents of the neurons in the presence of colchicine were activated at55.7s, reached a peak at only199.1s after activation (243.6s after activation in the absence of colchicine, P<0.05), and relaxed to a pseudosteady state for the remainder of the recording.
5. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in HEK293-TRPV4cells
Colchicine administration also resulted in a reduction and advance of the peak current in HEK293-TRPV4cells (412.50±27.15pA and131.6s after activation for HEK293-TRPV4cells;219.53±5.24pA and42.7s after activation for HEK293-TRPV4cells in the presence of colchicine, P<0.05). HEK293-TRPV4cells incubated with colchicine expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages.
6. Effects of microtubules depolymerization by colchicine on the expression of TRPV4
The level of TRPV4protein expression was diminished when animals were injected with colchicine in the doses of625ug/kg and1250μg/kg, and significant decreases were observed at2h and4h post-injections (P<0.05). Pretreatment with colchicine significantly and dose-dependently depressed the expression of TRPV4mRNA with the minimum expression level occurred at2h after exposure to colchicine in the dose of1250μg/kg (P<0.05). The inhibition of colchicine on TRPV4expression was associated with colchicine-induced relief of allodynia in CCD rats.
7. Effects of microtubules depolymerization by colchicine on the subcellular localization of TRPV4
Intrathecal administration of colchicine failed to alter the ratio of neurons with pronounced localization of TRPV4. TRPV4immunofluoresecnce was observed more in the plasma membrane than that in the cytoplasm, and this distribution was unaffected by colchicine. The subcellular localization of TRPV4observed in the current study was in agreement with previous findings.
Conclusion
Depolymerization of microtubules by colchicine could attenuate the allodynia in CCD rats. TRPV4contributes to the relief of allodynia by the depolymerization of microtubules.
Objective
To investigate the role of microfilaments in allodynia induced by chronic compression of the dorsal root ganglion; to investigate the effects of microfilaments on TRPV4.
Materials and methods
1.CCD surgery
Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5vertebrae were exposed unilaterally in accordance with a previously described procedure. Two hollow, stainless steel, L-shaped rods (0.66-mm diameter and4-mm length), connected to silicon tubing (0.51mm ID,0.94OD,30-40mm length) filled with heparin, were inserted into L4and L5foramen respectively, to compress the DRG. The other end of the tubing was sealed, except when injecting drugs. The muscle and skin layers were then sutured. Sham surgery involved identical surgical procedures to those described but without insertion of the rods.
2. Behavioral testing
All behavioral tests were carried out in a quiet room and conducted under blind conditions. Behavioral tests were conducted on the7th day post-surgery and1,2,4,6,8, and24h after injection of chemicals or saline. Chemicals and saline were all injected by the silicon tubing.
3. DRG neurons culture
The L4and L5ganglia were removed from the operated side of each animal on7days after surgery. The neurons were cultured according to the methods in chapter I.
4. Cell viability assay
The viability of DRG neurons were tested according to the method in chapter I.
5. Whole-cell current recording
The whole-cell current of TRPV4were tested according to the method in chapter I.
6. Immunofluorescence microscopy
The localization of TRPV4is tested according to the method in chapter I.
Results
1. Effects of microfilaments dynamics on the allodynia in CCD rats
All rats walked normally after the CCD surgery, indicating that CCD surgery did not injury the motor behavior.
Both of two disrupting agents (CB and CD) resulted in attenuation of CCD-induced mechanical and thermal allodynia (n=9in each group, both P<0.01). Compared with the administration of saline in CCD rats, administration of cytochalasin B (200μg and300μg) produced a dose-dependent and partial reduction of allodynia in CCD rats. However, similar result could not be observed in CCD rats injected with cytochalasin B in the dose of100ug (P>0.05, n=9in each group). The significant reduction in allodynia was observed at1h, peaked at2h, and lasted less than24h post-injection.
Phalloidin didn't affect the mechanical and thermal allodynia induced by CCD surgery (all P>0.05, n=9in each group). Phalloidin was injected to investigate whether it could prevent cytochalasin B-induced loss of allodynia in CCD rats, confirming that the inhibition of CCD-induced allodynia by anti-microfilaments agents was due to microfilaments disruption. Injection of phalloidin1h preceding administration of cytochalasin B significantly inhibited the cytochalasin B-induced attenuation of allodynia in CCD rats (n=9).
2. Effects of microfilaments dynamics on TRPV4currents in DRG neurons in CCD rats
Similar to CCD-induced allodynia in vivo, whether the potentiation of TRPV4in CCD rats was dependent on intact actin filaments was tested. The peak inward current was significantly higher in CCD group than in sham group (n=12in CCD group, n=10in sham group, P<0.05). The effects of dynamic changes of microfilaments on TRPV4-mediated channel activity were tested next.
Exposure of cultured compressed DRG neurons to disruptors (cytochalasin B and cytochalasin D, both10-4M) for2h prior to the patch clamp significantly inhibited the potentiation of TRPV4(the peak value is310.19±9.17pA in CCD group,245.48±12.12pA in DRG neurons exposure to cytochalasin B, and237.81±10.17pA in DRG neurons exposure to cytochalasin D, n=12in each group, P<0.05), which are consistent with the attenuation of the effects of disruptors on CCD-induced allodynia. DRG neurons incubated with disruptors expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages, which are in accordance with the inhibition of TRPV4currents by disruption of microfilaments. The time course was analyzed to further confirm the effect of disruption of microfilaments on TRPV4channel opening. A delayed peak time was observed in DRG neurons from CCD rats (243.60±20.00s for CCD group and200.37±15.40s for sham group, P<0.05). Disruption of microfilaments induced an advance of the peak current in DRG neurons of CCD rats (123.44±10.10s for DRG neurons exposure to cytochalasin B, and193.76±12.40s in DRG neurons exposure to cytochalasin D, P<0.05).
Phalloidin (10-5M) resulted in a reduction and delay of TRPV4current (80.87±5.42pA and311.72±14.56s in DRG neuron exposure to phalloidin, n=12, P<0.01), which is not consistent with the effect of phalloidin on CCD-induced allodynia. In association with the inhibition of TRPV4currents, the reversal potentials shifted toward more positive voltages. MTT tests were carried out to exclude the effects of chemicals (cytochalasin B, cytochalasin D and phalloidin) on the cell viabilities of DRG neurons. The results showed that both disruptors and stabilizer of microfilaments had no significant influence on the cell viabilities of DRG neurons (P>0.05).
3. Effects of microfilaments dynamics on the subcellular localization of TRPV4in DRG neurons in CCD rats
Results demonstrated that more TRPV4immunofluoresecnce was observed in the plasma membrane than the cytoplasm in all groups. Cytochalasin B (10-4M) resulted in an apparent decrease of the plasma membrane association of TRPV4(83.62±2.14%in CCD group and69.63±3.41%in cytochalasin B group, P<0.05). Meanwhile, phalloidin (10-5M) also leaded to a decrease of plasma membrane association of TRPV4(54.24±2.44%, P<0.05).
Conclusions
This study demonstrates that microfilaments play an important role in allodynia induced by chronic compression of the dorsal root ganglion and TRPV4contributes to the attenuation of allodynia by depolymerization of microfilaments.
引文
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1. Hu SJ, Xing JL. An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain 1998; 77:15-23.
2. Song XJ, Hu SJ, Greenquist KW, Zhang JM, LaMotte RH. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia. J Neurophysiol 1999; 82:3347-3358.
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5. Song XJ, Zhang JM, Hu SJ, LaMotte RH. Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators. Pain 2003; 104:701-709.
6. Nogales E, Downing KH, Amos LA, et al. Tubulin and FtsZ form a distinct family of GTPases. Nat Struct Biol 1998;5:451-458.
7. Dina OA, McCarter GC, de Coupade C, Levine JD. Role of the sensory neuron cytoskeleton in second messenger signaling for inflammatory pain. Neuron 2003;39:613-624.
8. Zhang Y, Wang YH, Ge HY, Arendt-Nielsen L, Wang R, Yue SW. A transient receptor potential vanilloid 4 contributes to mechanical allodynia following chronic compression of dorsal root ganglion in rats. Neurosci Lett 2008;432:222-227.
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14. Alessandri-Haber N, Joseph E, Dina OA, Liedtke W, Levine JD. TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator. Pain 2005;118:70-79.
15. Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, et al. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 2003;39:497-511.
16. Todaka H, Taniguchi J, Satoh J, Mizuno A, Suzuki M. Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia. J Biol Chem 2004;279:35133-35138.
17. Alessandri-Haber N, Dina OA, Yeh JJ, Parada CA, Reichling DB, Levine JD. Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat. J Neurosci 2004;24:4444-4452.
18. Alessandri-Haber N, Dina OA, Joseph EK, Reichling DB, Levine JD. Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia. J Neurosci 2008;28:1046-1057.
19. Ding XL, Wang YH, Ning LP, Zhang Y, Ge HY, Jiang H, et al. Involvement of TRPV4-NO-cGMP-PKG pathways in the development of thermal hyperalgesia following chronic compression of the dorsal root ganglion in rats. Behav Brain Res 2009;208:194-201.
20. Goswami C, Kuhn J, Heppenstall PA, Hucho T. Importance of non-selective cation channel TRPV4 interaction with cytoskeleton and their reciprocal regulations in cultured cells. PLoS One 2010;5:e11654.
21. Suzuki M, Hirao A, Mizuno A. Microtubule-associated [corrected] protein 7 increases the membrane expression of transient receptor potential vanilloid 4 (TRPV4). J Biol Chem 2003;278:51448-51453.
22. Becker D, Bereiter-Hahn J, Jendrach M. Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation. Eur J Cell Biol 2009;88:141-152.
23. Shibasaki K, Suzuki M, Mizuno A, Tominaga M. Effects of body temperature on neural activity in the hippocampus:regulation of resting membrane potentials by transient receptor potential vanilloid 4. J Neurosci 2007;27:1566-1575.
24. Wei H, Zhang Y, Fan ZZ, Ge HY, Arendt-Nielsen L, Jiang H, Yao W, Yue SW. Effects of colchicine-induced microtubule depolymerization on TRPV4 in rats with chronic compression of the dorsal root ganglion. Neurosci Lett 2013;534:344-350.
25. Wieland T. Poisonous principles of mushrooms of the genus Amanita. Four-carbon amines acting on the central nervous system and cell-destroying cyclic peptides are produced. Science 1968; 159:946-952.
26. Bowe JE, Chander A, Liu B, Persaud SJ, Jones PM. The permissive effects of glucose on receptor-operated potentiation of insulin secretion from mouse islets:a role for ERK1/2 activation and cytoskeletal remodelling. Diabetologia 2013; DOI: 10.1007/s00125-012-2828-2 [Epub ahead of print]
27. Nury T, Samadi M, Varin A, Lopez T, Zarrouk A, Boumhras M, et al. Biological activities of the LXRalpha and beta agonist,4beta-hydroxycholesterol, and of its isomer,4alpha-hydroxycholesterol, on oligodendrocytes:Effects on cell growth and viability, oxidative and inflammatory status. Biochimie 2012;95:518-530.
28. Paiva-Lima P, Rezende RM, Leite R, Duarte ID, Bakhle Y, Francischi JN. Crucial involvement of actin filaments in celecoxib and morphine analgesia in a model of inflammatory pain. J Pain Res 2012;5:535-545.
29. Becker D, Muller M, Leuner K, Jendrach M. The C-terminal domain of TRPV4 is essential for plasma membrane localization. Mol Membr Biol 2008;25:139-151.
30. Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, Piomelli D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 1994;372:686-691.
31. Liedtke W. TRPV4 as osmosensor:a transgenic approach. Pflugers Arch 2005; 451:176-180.
32. Lowry C, Lightman S, Nutt D. That warm fuzzy feeling:brain serotonergic neurons and the regulation of emotion. J Psychopharmacol 2009;23:392-400.
33. Vennekens R, Hoenderop JG, Prenen J, Stuiver M, Willems PH, Droogmans G, Nilius B, Bindels RJ. Permeation and gating properties of the novel epithelial Ca2+ channel. J Biol Chem 2000;275:3963-3969.
34. Goswami C, Kuhn J, Dina OA, Fernandez-Ballester G, Levine JD, Ferrer-Montiel A, et al. Estrogen destabilizes microtubules through an ion-conductivity-independent TRPV1 pathway. J Neurochem 2011; 117:995-1008.
35. Aley KO, Reichling DB, Levine JD. Vincristine hyperalgesia in the rat:a model of painful vincristine neuropathy in humans. Neuroscience 1996;73:259-265.