摘要
研究背景和目的
每年全世界约有2200万的新发脑卒中患者,2012年最新发表的国际卒中试验3(The third international stroke trial, IST-3)研究表明,全球范围内在60岁以上人群中脑卒中为第二大死亡原因,而在15-59岁人群死亡原因中居第五位。中国人群最主要的类型是缺血性脑卒中,约占43%～73%。逐年增长的发病率与其高致死率及高致残率使脑卒中成为中国亟待解决的公众健康难题,促进神经功能恢复是脑梗死治疗的基本途径。神经功能恢复的结构基础在于两方面：一是脑梗死后早期神经结构最大限度的保留；二是脑梗死恢复期神经可塑性的调节。神经可塑性在脑梗死后的相当长时间内都可受到调节,后者是我们治疗的主要方向。作为局部脑缺血的一种反应,在急性期过后的一段时间里,脑组织的自身修复过程被激活,然而这些内源性中枢神经系统重塑不足以使神经功能恢复。如何刺激、放大其内源性修复机制是我们面临的重要问题,这个问题在近年来越来越受到重视。
目前认为神经可塑性在细胞结构水平表现为：树突分支、轴突出芽、树突棘密度、突触数目以及受体密度的改变。脑梗死灶同侧及对侧的轴突重塑、树突棘密度调节是脑梗死后神经结构修复以及自发神经功能恢复的关键因素。般来说,缺血性脑卒中后诱发锥体神经元轴突发生Waller变性,其程度取决于缺血病灶的严重程度,进而轴突纤维沿着梗死灶边缘发生再生重塑,存活的远端锥体束轴突出芽,其实在病灶同侧和对侧均可见轴突末梢出芽的增强。不仅如此,跨胼胝体的联合纤维也发生了重塑以及轴突出芽。近期研究的数据表明：脑梗死灶周围树突棘密度的变化与神经功能的恢复密切相关。
绝大多数树突表面分布有树突棘,每个树突棘至少含有一个兴奋性突触,因而树突棘对于脑神经元细胞之间的信号传递具有重要作用。树突棘在生长发育过程中有三种结局：绝大多数参与突触后成分的构成,一部分被修剪或沉默,另有小部分树突棘进一步增长发育成3级或4级树突。树突棘的结构具有异质性,表现为不同类型,如最常见的呈蘑菇状,以及蚓状和短粗状。对于成年大脑,大多数树突棘是处于一个稳定状态,但新近证据表明,在某些情况下树突棘的结构可以发生显著改变,突触信号传导的重塑可能因此而激活,树突棘更新率可以在以下条件下得到增强：外界感觉刺激、控制神经兴奋性包括长时程增强或长时程抑制、以及在某些神经疾病病理条件下。进一步的研究表明兴奋性受体介导的电流强度、兴奋性突触后电位传导、树突棘头端至母体树突的细胞内钙离子弥散动力学改变等,均与树突棘形态学改变有关。因而树突棘可塑性是中枢神经系统突触功能可塑性的一个重要方面。由于树突棘具有受环境影响而表现出较强可塑性的特点,提示树突棘对于脑可塑性的调节起到关键作用,那么在脑缺血病理情况下又起到哪些作用?
脑缺血引起兴奋性氨基酸毒性作用、钙离子超载、自由基损伤、NO毒性作用、凋亡激活以及炎症反应等一系列病理生理变化,神经元树突继之出现变性,其突出的形态学改变特点是沿着树突茎出现曲张样膨大,最初表现轻微,但随着缺血状态加重而逐渐加剧,最后常常表现为串珠样。离体研究和在体研究均可观察到树突的这种病理改变与缺血程度和病灶体积大小有关。正常情况下皮质树突与毛细血管的间距平均为13μ m,而缺血后最初几个小时在血管周围80μ仍可存在完整树突。有研究认为体外培养皮质神经元经氧-糖剥夺或给与谷氨酸受体激动剂,将导致严重的树突膨胀和形态学改变,但在缺血缺氧不超过2小时就能在结构上完全康复。在光化学法诱导的脑梗死模型上同样可以观察到受累树突的康复,同时观察到大多数再生的树突棘重新分布定位于梗死灶周围它们破坏、消失的位点,这可能是因为突触前成分在脑卒中早期处于相对较稳定的状态。有研究证明在海马脑片上这些再生的树突棘能诱发出来突触反应,这证明再生的树突棘是具有生理功能的。有研究表明脑卒中诱发激活树突分支增多、树突棘密度增加,这提示树突和树突棘的重塑是脑可塑性的基础。
根据大脑中动脉梗死后损伤情况,梗死的脑组织分为三个区域：永久缺血坏死区域,临近的再灌注损伤、胶质瘢痕及细胞凋亡区域,外周的正常区域,缺血性脑梗死引发的神经修复再生过程主要发生在后两个区域。梗死灶周围区域(peri-infarct region)处于梗死灶坏死核心的周围,处于脑组织的低灌注区域,但目前就peri-infarct region的形成机制以及范围大小仍有争议。有研究将其界定为梗死灶坏死核心的周围存活的组织区域,对于啮齿类动物而言可能不到lmm,实际上此区域界定的模糊与再灌注时间、代谢以及细胞凋亡有关,与临床“半暗带”概念有相似之处。众多研究表明该区域是神经再生发生的主要区域,因为有很多神经生长促进和抑制因子表达在此发生重要的变化。另有电生理、影像学研究认为该区域在受到外界环境影响下更具有活性,肢体功能的早期康复与梗死周围区域皮质功能重组有关。实际上脑组织的缺血性损害主要发生于梗死后的24小时内,在荧光标记的转基因动物中观察到缺血后树突完整性破坏稳定于梗死后的6小时之内,进一步离体研究表明受累树突分支的数十微米内结构处于一个损害-修复的动态平衡,并且具有较为清楚的界限,然而在此界限边缘看似未损伤的树突其实在树突棘水平发生着细微的变化,主要表现为数目的进行性减少或是密度的下降。有体外研究认为缺血后树突棘可有增长,提示增长的树突棘可以限制破坏性离子电信号从头端传向树突。越来越多研究表明在脑梗死后的数天至数周内,脑梗死周围区域是神经可塑性的热点区域,对神经功能康复具有重要意义。
经颅直流电刺激(Transcranial direct current stimulation, TDCS)是近年来日益受到国内外学者的重视的康复技术,TDCS作为无创性脑刺激技术之一具有其独特的优势,可以和物理治疗同步进行,而且安全性高。不少临床研究观察到TDCS对于肢体运动功能提高、记忆改善、镇痛等方面具有肯定的疗效,不仅如此,对于语言、吞咽的恢复也有帮助。但其作用机理目前尚不清楚,且未见系统的研究报道TDCS可以通过电极定向、定位调节相应区域的神经网络兴奋性来促进神经功能恢复,是用来研究大脑活动性、促进神经可塑性的理想方法,而且近年来的研究表明TDCS能够通过调节大脑活动影响神经可塑性,比rTMS具有更少的副作用。如前所述,脑卒中诱发激活树突分支增多、树突棘密度增加,树突和树突棘的重塑是脑可塑性的基础,且脑梗死病理生理过程本身就可以诱导病灶周围的神经再生,特别是脑梗死周围区域1-12周神经兴奋性的增加对于肢体功能恢复至关重要。
目前认为TDCS可以将足够的电流引入大脑皮质,特点是不诱发动作电位,而只调节神经元的膜静息电位,从而仅调节已经处于活动状态的神经元兴奋性。TDCS既能够增强神经元的活动,也能抑制它们的活动,这要看电流的方向和神经元的排列方式。大脑皮层神经元树突指向头皮方向,当带正电荷的经颅直流电刺激电极(正极)靠近树突时,电流就导致处于活动状态的神经元兴奋性增加,而负极的作用正好相反。有临床研究观察到兴奋性氨基酸受体NMDAR阻滞剂右美沙芬可以阻断TDCS作用于神经细胞的正极、负极效应,从而推测NMDA受体参与TDCS改变神经可塑性。另有研究观察到卡马西平选择性消除了TDCS的正极效应而未影响负极效应,推测TDCS正极效应需要离子通道的参与,膜电位去极化及细胞间相互作用可能是其主要机制之一。虽然TDCS作用于神经可塑性的机制目前仍不明确,需要进一步的动物实验研究去探讨,但上述研究为我们提供了研究切入点：神经细胞联系通道——缝隙连接通道。
目前缝隙连接通道在脑缺血中的作用日益受到关注。pannexins家族是近年来确定的组成通道的蛋白家族。目前研究认为缺血诱导pannexin1通道的开放,继而膜通透性增加,同时出现阳离子电流的调节异常,此过程虽也有ASIC1a通道、电压依赖Na+通道、NMDA受体以及TRP通道的参与,但通道的电流震荡是关键的环节。Pannexin1通道开放导致的电流震荡使神经元持续处在膜静息电位水平(-60mV),这提示通道电流是导致缺氧去极化的主要成因,同时pannexin1通道的开放导致葡萄糖和ATP的外渗,以上从细胞水平说明pannexin1通道开放是导致缺血后神经电兴奋性改变、神经细胞间通信的关键环节。有意思的是Pannexin1被发现和PSD-95(postsynaptic density protein95)共同存在于突触后膜,参与影响突触可塑性。可以预见到脑梗死后双侧大脑半球的神经兴奋性以及pannexin1通道诱导的电流震荡是不平衡的,早期应用TDCS干预这种不平衡对于神经功能的康复有着重要意义。
研究目的
脑梗死后TDCS干预有助于神经功能恢复,那TDCS对皮层神经元pannexin1通道、神经可塑性有何影响?我们预期通过本课题明确pannexin1通道在脑梗死后的改变以及TDCS对其的影响,并初步探讨TDCS的干预时机及其作用于神经功能重组的机制,为脑梗死后治疗提供新的靶点,并为TDCS的应用提供理论依据。本课题采用大脑中动脉闭塞模型(MCAO),对peri-infarct region进行如下观察研究：
1.观察成年大鼠脑梗死后TDCS对脑梗死灶周围区树突棘形态学的影响；
2.观察成年大鼠脑梗死后TDCS对脑梗死灶周围区pannexin1表达的影响；
3.结合TDCS对脑梗死大鼠行为学影响的观察,探讨TDCS促进神经可塑性的机制。
方法
1、选用9～10周龄雄鼠,体重300-350g,共120只。从成功复制的MCAO模型大鼠中用随机数字表随机选取80只,其中40只作为单侧大脑中动脉闭塞组(MCAO组)即脑梗死对照组,40只作为TDCS治疗组,其余MCAO模型大鼠备用。主要观察时间点设为3天、7天、14天。
具体分组如下：
1) MCAO组(n=40)：线栓法阻断MCAO。
其中36只用于研究TDCS对树突棘密度、对Pannexin1mRNA表达、Neurocan mRNA表达的影响,分3个时间点(3天、7天、14天),每组12只。第3组(14天组)的大鼠中取8只,采用走平衡木实验在第3天第7天、第14天进行神经功能评分。
其余4只,其中1只用于TTC染色,1只用于Nissl染色,2只用于观察Pannexin1和Neurocan在脑内的分布。
2) TDCS治疗组(n=40)：线栓法阻断MCA+TDCS治疗。具体实验动物分组方法与MCAO组相同。
3)假手术组(n=40)：仅暴露颈部动脉。具体实验动物分组方法与MCAO组相同。
2、大鼠肢体运动功能评定：采用走平衡木实验(beam walking test)对运动协调功能进行神经功能评分。重复测量三次取平均值。
具体评分标准如下分数标准0分能在横木上保持静态平衡,行走不会跌倒1分双上肢抱平衡木可在横木上停留,行走时跌倒机率<50%2分抱平衡木可在横木上停留,且有一肢体掉落,行走时跌倒机率>50%3分在平衡木上不能行走,且两肢体均掉落,在横木上停留>60s4分可在平衡木上保持平衡>40s5分可在平衡木上保持平衡>20s6分将大鼠放在平衡木上坠地<20s
3、TDCS刺激干预方法
SD大鼠MCAO后次日即给予直流电TDCS治疗,采用G6805-2B型刺激仪(上海医用电子仪器),参数设置为频率10Hz,强度O.1mA,每天给予30分钟。刺激电极位置：正极(上调兴奋性)在耳间线后约2mm、中线偏右2-3mm,负极(下调兴奋性)在耳间线前5mm、中线偏左2-3mm。在刺激的第3天、7天、14天分别选取相应大鼠处死进行下一步实验。
4、Golgi染色在光学显微镜下观察皮质锥体神经元从二级树突基部开始每20μm长度范围内的树突棘个数,代表树突棘密度。
5、免疫组化检测Pannexin1、Neurocan的表达分布,以及分别与MAP-2和GFAP的关系。
6、荧光实时定量PCR检测Pannexin1mRNA、Neurocan mRNA的表达。统计学处理
全部数据均采用SPSS17.0统计软件分析比较。
研究TDCS对行为学的影响采用重复测量方差分析,组内两两比较采用LSD方法。
研究TDCS对树突棘密度、对Pannexin1mRNA表达、Neurocan mRNA表达的影响采用析因设计方差分析,组内两两比较采用LSD方法。
P<0.05表示差异具有统计学意义。
结果
1、TDCS对脑梗死后神经功能的影响
脑梗死后不同时间点之间(F=27.941,P<0.001)、TDCS治疗组和MCAO组之间(F=39.443,P<0.001)的评分差异有统计学意义,脑梗死后不同时间点与不同组别间无交互效应(F=2.294,P=0.119)。MCAO组在不同时间之间的评分差异有统计学意义(F=7.824,P=0.005),TDCS治疗组在不同时间之间的评分差异有统计学意义(F=24.843,P<0.001)。TDCS治疗组与MCAO组之间第3天(t=2.688,P=0.018)、第7天(t=5.789,P<0.001)、第14天(t=5.017,P<0.001)的BWT评分差异有统计学意义,TDCS治疗组的评分均低于MCAO组,提示TDCS治疗组肢体功能较MCAO组好。MCAO组内BWT评分在第7天与第3天之间、第14天与第7天差异均无统计学意义(P值分别为0.170、0.20)；第14天BWT评分低于第3天,差异有统计学意义(P=0.011)。TDCS组内第7天BWT评分低于第3天,差异有统计学意义(P=0.040)；BWT评分在第14天与第7天之间差异有统计学意义(P=0.033)：第14天BWT评分低于第3天,差异有统计学意义(P<0.001)。
2、TDCS对各组树突棘的影响
Golgi染色后通过光镜观察发现,SO组、MCAO组以及TDCS组的神经树突棘大多呈蘑菇状。MCAO组的树突棘表现出缺失、或稀少的树突棘。析因设计方差分析显示不同分组问树突棘密度差异有统计学意义(F=384.352,P<0.001);而不同时间树突棘密度差异有统计学差异(F=13.402,P<0.001);干预因素(TDCS治疗、MCAO手术)和时间之间有交互效应(F=4.509,P=0.002)。TDCS治疗组、MCAO组以及SO组术后第3天(F=195.773,P<0.001)、7天(F=112.039,P<0.001)、14天(F=100.864,P<0.001)的树突棘密度差异均有统计学意义。在术后第3天、7天、14天时MCAO组、SO组之间神经树突棘密度差异有统计学意义(P值均小于0.001),MCAO组神经树突棘密度均较SO组降低。在术后第3天、7天、14天时TDCS治疗组与MCAO组之间神经树突棘密度差异有统计学意义(P值均小于0.001),TDCS治疗组神经树突棘密度均较MCAO组增高。在术后第3天、7天、14天时TDCS组与SO组之间神经树突棘密度差异有统计学意义(P值均小于0.05)。
3、Pannexin1在脑内的分布
Pannexin1在全脑均有表达,梗死灶侧阳性信号较非梗死灶侧强。大鼠大脑皮层的V2MM第二视皮质内侧内区、V2ML第二视皮质内侧外区、Aul第一听皮质、AuV第二听皮质腹侧区、海马CA1/CA2/CA3/齿状回(DG)、S1BF感觉皮质桶状区、PMCo皮质后内侧杏仁核、PLCo皮质后外侧杏仁核、Pir梨形皮质、PtA顶叶联络皮质表达较强；其主要分布在皮层6层结构中的Ⅱ(外颗粒)层、Ⅲ(锥体细胞)层、Ⅳ(内颗粒)层及Ⅴ(外锥体)层中。
双重免疫荧光标记结果显示：Pannexin1主要分布在皮层的锥体细胞、颗粒细胞中。Pannexin1与MAP-2共定位后发现：Pannexin1与MAP2均为阳性的细胞约占MAP2阳性细胞总数的80%-95%,可见Pannexin1在胞体及近端树突中均有较强阳性信号表达,提示Pannexin1主要分布于神经元胞膜,部分表达于胞浆。
4、TDCS对Pannexin1表达的影响
RT-PCR检测脑梗死周围区域Pannexin1mRNA相对表达量的变化。析因设计方差分析显示不同分组(F=237.734,P<0.001)、不同时间(F=34.868,P<0.001)差异均有统计学意义,干预因素(TDCS治疗、MCAO手术)和时间之间有交互效应(F=45.483,P<0.001)。TDCS治疗组、MCAO组以及SO组术后第3天(F=77.498,P<0.001)、7天(F=194.79,P<0.001)、14天(F=70.962,P<0.001)的Pannexin1mRNA相对表达量差异均有统计学意义。术后第3天、7天、14天时MCAO组与SO组Pannexin1mRNA相对表达量差异均有统计学意义(P<0.001),MCAO组Pannexin1mRNA相对表达量均高于假手术组。术后第3天TDCS治疗组Pannexin1mRNA表达量与MCAO组间差异无统计学意义(P=0.264),术后第7天、14天时TDCS治疗组Pannexin1mRNA相对表达量与MCAO组差异有统计学意义(P<0.001),术后第7天、14天时TDCS治疗组Pannexin1mRNA表达量较MCAO组降低。在术后第3天、7天、14天时TDCS组与SO组Pannexin1mRNA相对表达量差异均有统计学意义(P<0.05)。
5、Neurocan在脑内分布
Neurocan阳性信号主要分布于梗死灶周围区域oGFAP阳性区域显示增生的胶质细胞,Neurocan阳性区域呈不规则片状分布,Neurocan和GFAP的双标结果提示Neurocan阳性区域要大于GFAP阳性区域,Neurocan阳性染色主要分布于胶质细胞及细胞间隙。
6、TDCS对Neurocan表达的影响
析因设计方差分析显示不同分组(F=1986.124,P<0.001)、不同时间(F=307.183,P<0.001)梗死灶周围Neurocan mRNA相对表达量差异均有统计学意义,干预因素(TDCS治疗、MCAO手术)和时间之间有交互效应(F=118.138,P<0.001)。TDCS治疗组、MCAO组以及SO组术后第3天(F=594.946,P<0.001)、7天(F=2484.108,P<0.001)、14天的Pannexin1mRNA(F=204.715,P<0.001)相对表达量差异均有统计学意义。在术后第3天、7天、14天时MCAO组与SO组Neurocan mRNA相对表达量差异均有统计学意义(P<0.001),MCAO组Neurocan mRNA相对表达量均较SO组增加。在术后第3天、7天、14天时TDCS治疗组与MCAO组Neurocan mRNA相对表达量差异均有统计学意义(P<0.001),TDCS治疗组Neurocan mRNA相对表达量均较MCAO组降低。在术后3天、7天时TDCS治疗组与SO组Neurocan mRNA相对表达量差异有统计学意义(P<0.001),但在14天时TDCS治疗组与SO组Neurocan mRNA相对表达量差异无统计学意义(P=0.741)。
结论
TDCS促进脑梗死早期肢体功能康复的机制之一可能是增加了脑梗死后梗死灶周围的树突棘密度,表明其改善神经功能的重要机制之一是促进了神经可塑性。在脑梗死后的早期应用TDCS会有更好的运动功能恢复,而且能够下调pannexin1mRNA的表达。同时我们观察到早期应用TDCS可以抑制Neurocan mRNA的表达。
1.脑梗死周围区域树突棘密度在脑梗死后呈现逐渐下降的趋势,TDCS则促进脑梗死后的树突棘再生,使树突棘密度增加;
2. Pannexin1广泛分布于大脑皮层、海马,定位于胞膜,部分表达于胞浆,脑梗死周围区域pannexin1表达在梗死后增加,TDCS治疗抑制其表达；
3. Neurocan主要表达于脑梗死周围局部区域,定位于增生的胶质细胞及间隙,脑梗死周围区域Neurocan表达在梗死后增加,TDCS治疗抑制其表达。
Background and Objection:
According to the World Health Report2002,15million people suffer stroke worldwide each year. One third of them die and another one third are permanently disabled (WHO,2002). Over the past four decades, there was more than a40%decrease in stroke incidence in developed countries and greater than a100%increase in stroke incidence in developing countries. The overall stroke incidence rates in developing countries exceeded those in developed countries by20%from2000to2008(Feigin et al.,2009). Although in developed countries, the incidence of stroke is declining due to better control of blood pressure and a reduced smoking population, the overall rate of stroke remains high due to the aging of the population (WHO,2002). Stroke is the fourth leading cause of death in America and a leading cause of adult disability (National Center for Health Statistics, accessed March30,2012).
The basic goal of cerebral infarction treatment is to promote the recovery of neurological function. The recovery of neural function after cerebral infarction relies on neural plasticity and regional neural functional reorganization (i.e., integration of the neurological function of the damaged areas to the surrounding undamaged areas or the contralateral cerebral hemisphere)(Pascual-Leone et al.,2005). From the electrophysiological point of view, enhancement of ipsilateral cortical excitability and reduction of excitability of the contralateral cortex is the basic starting point for the neurological function recovery (Talelli and Rothwell,2006). Transcranial direct current stimulation (TDCS) is a noninvasive, safe, and inexpensive technique that has been studied as a therapeutic approach for different neurologic disorders (Arul-Anandam et al.,2009; Williams et al.,2009). In stroke patients, the contralesional motor region exerts an undue inhibitory influence on the lesional motor region, which might hinder recovery. Simultaneous anodal TDCS of the affected hemisphere and cathodal TDCS of the unaffected hemisphere may increase the cortical excitability of one hemisphere while causing decrease of cortical excitability in the contralateral hemisphere, making TDCS an especially useful tool for the rehabilitation of patients with stroke (Boggio et al.,2007). Transcranial direct current stimulation may be used alone or combined with standard physical therapies to induce changes in cortical excitability and improve motor function in stroke patients (Boggio et al.,2007; Bolognini et al.,2009). These effects may be affected by polarity, duration of therapy and adopted current intensity (Bolognini et al.,2009).
However, the mechanism underlying such neuroplastic changes after TDCS still remains unclear (Venkatakrishnan and Sandrini,2011). Currently, it is believed that TDCS can introduce enough current to the cerebral cortex without inducing action potentials. It only regulates the membrane resting potential of neurons, which can reduce the spontaneous discharge rate (Liebetanz et al.,2002). Therefore, it only regulates the excitability of neurons in the active state, and will not cause spontaneous discharge of dormant neurons (Wagner et al.,2007). Additionally, TDCS is associated with augmentation or weakening of N-Methyl-D-aspartate (NMDA) receptor activity (Kim et al.,2010). A clinical study observed that the NMDA receptor antagonist dextromethorphan can block the effects of anodal and cathodal TDCS on nerve cells (Liebetanz et al.,2002) and it was speculated that NMDA receptors are involved in TDCS-induced modulation of neural plasticity. Activation of NMDA receptors results in the opening of nonselective ion channels. Calcium flux through NMDA receptors is thought be critical in synaptic plasticity, a cellular mechanism for learning and memory. These findings suggest a relationship between ion channels and TDCS. Another study observed that carbamazepine selectively eliminated the effects of anodal TDCS without affecting the effects of cathodal TDCS (Nitsche et al.,2004). Carbamazepine stabilizes the inactivated state of sodium channels, suggesting that the effects of anodal TDCS require the participation of ion channels; membrane potential depolarization and cell-cell interactions may be one of its main mechanisms. Previously, many experiments proved that peripheral electroacupuncture therapy (ET) had neural protective function after cerebral ischemia (Huo et al.,2004). Electroacupuncture therapy is a kind of therapy that delivers an electrical current pulse into body through a milli-needle or skin electrode. Our research group carried out a series of studies to explore the mechanisms related to rehabilitation after cerebral ischemia. In a previous study, we found that ET (frequency10Hz; intensity1mA;30min per day) at four acupuncture points 'NEIGUAN'(PC6),'WAIGUAN'(SJ5),'SANYINJIAO'(SP6), and'ZUSANLI'(ST36) significantly suppressed upregulated Na(v)1.1and Na(v)1.6expression after cerebral ischemia (Ren et al.,2010).
The pannexins family is a recently identified protein family that forms large-pore nonselective channels in the plasma membrane of cells. Studies suggest that ischemia may induce opening of the pannexin1, resulting in increased membrane permeability and ionic dysregulation (Bargiotas et al.,2009). Although ASIC1a channels, voltage-dependent Na+channels, NMDA receptors, and transient receptor potential channels participate in this process, the channels are thought to play a key role (Bruzzone et al.,2003). Current oscillation caused by the opening of pannexin1keeps the neurons at the membrane resting potential (-60mV), suggesting that the channel current is the main cause of hypoxic depolarization. At that same time, the opening of pannexin1leads to exosmosis of glucose and ATP, indicating that the opening of pannexin1is a key link leading to changes in neuron excitability and intercellular communication after ischemic injury (Thompson et al.,2006). An interesting finding is that pannexin1and postsynaptic density protein95are present in the postsynaptic membrane, and participate in modulation of synaptic plasticity (Zoidl et al.,2007). In the current study, we aimed to investigate the effects of TDCS on pannexin1in cortical neurons and neural plasticity in the early stage of cerebral ischemia, and explore the optimal time window of TDCS therapy after cerebral infarction.
Methods and materials
1Animals and experimental grouping
One hundred and twenty adult male Sprague-Dawley rats aged4-5months were included in this study. The rats were randomly assigned to the following three groups: sham operation (SO) group, middle cerebral artery occlusion (MCAO) group and TDCS group. In the MCAO and TDCS groups, the cerebral infarction model was constructed with unilateral middle cerebral artery electrocoagulation contralateral to the reaching forelimb (Bederson et al.1986). In the SO group, the middle cerebral arteries of the rats were not coagulated, but the remaining operations were the same at that in the cerebral infarction model Postoperative benzylpenicillin(100,000unit/kg) was used to prevent infection. Bilateral pericranium electrode implantation was performed in each group, but only the TDCS group received TDCS therapy. This study was approved by the Institutional Animal Care and Use Committee of our hospital and was carried out in accordance with the Declaration of Helsinki and with the Institute of Laboratory Animal Resources (1996).
2TDCS therapy
To use anodal TDCS to upregulate excitability of the ipsilesional motor cortex and cathodal TDCS to downregulate excitability of the contralesional motor cortex, anodal and cathodal TDCS (Type G6805-2B; Medical Electronic Apparatus Company, Shanghai, China) was given for30min each day starting on day1after surgery. Rats received TDCS daily until sacrifice. The TDCS parameters were set as follows: frequency,10Hz; intensity,0.1mA.(Kim et al.,2010). The active electrode was positioned5mm to the left and2mm in front of the interaural line. Rats were killed on the3rd,7th and14th day after TDCS.
3Motor function assessment
Motor function was assessed using the beam walking test (BWT) developed by Feeney et al.(1982). Assessments were performed on days3,7, and14after TDCS, and the recovery of the fine motor function of rats was classified into seven grades (0=normal and7=severe disorder). A higher score indicates poorer fine motor function.
4Observation of dendritic spines in brain slices using Golgi staining
On the3rd,7th and14th day, rats in each group were anesthetized with a lethal dose of chloral hydrate and perfused intracardially with0.1M phosphate buffer followed by4%paraformaldehyde in the same buffer. The brains were cut into coronal slices50mm thick. After being post-fixed in1%osmic acid for30min and poached in3.5%kalium bichromicum for1-3h, the slices were put in1%sliver nitrate cream for6-24h, dehydrated in gradient alcohol and made transparent with methyl salicylate. The amount of dendritic spines was observed under an optic microscope and the density and length of the dendritic spines were analyzed with an IBAS2.0imaging analysis system.
5Detection of pannexin1distribution in the brain using immunohisochemistry
The brains of rats in each group were collected as previously described and were cut using a freezing microtome into coronal sections30mm thick. After inactivation of endogenous peroxidase, the tissue sections were rinsed several times with0.01M phosphate buffered saline (PBS) and then placed in blocking solution (2%goat serum/0.3%, Triton X-100/0.1%, BSA in PBS) for1h. Following further rinses in PBS, the sections were incubated at4℃for24h in anti-pannexin1(1:100, Santa Cruz) and MAP-2(1:200, Chemicion). After incubation, the sections were again rinsed and incubated for1h in anti-Alexa, followed by further rinses in PBS and incubation for1h in anti-FITC at room temperature. After rinses in PBS, the sections were mounted by aquosity mounting and observed immediately under a confocal laser microscope.
6Detection of pannexin1mRNA expression in the surrounding areas of cerebral infarction using real-time PCR
The brain tissue approximately3-5mm from the cerebral infarction was collected as previously described for real-time polymerase chain reaction (PCR). Primer and probe were supplied by Zhongshan University Daan gene Company and designed by Primer Express2.0software and synthesized using an ABI3900high-flux DNA synthesizer. Total RNA was extracted from tissues using Trizol (Invitrogen). Real-time PCR was carried out in a30-ml reaction mixture. Primers for a housekeeping gene (GAPDH) were used as controls (30cycles). To ensure that PCR cycles ended before saturation, generally the cycle number for each primer was first tried at30cycles and then decreased or increased by1-4cyclcs,depending on the intensity of the initial PCR products. PCR was performed with the following conditions:93℃,3min;55℃,1min; and72℃2.5min, for30cycles. Primers used were the following:
Pannexin1forward primer:5'-TCTTCTGGCGCTTCTCTGC-3', reverse primer:5'-GGTCCAGGTCCGTCTCTTAGG-3'.
GAPDH:forward primer:5'-ATGTGTCCGTCGTGGATCTGA-3', reverse primer:5'-ATGCCTGCTTCACCACCTTCT-3'.
7. Statistical analysis
The statistical analysis was performed using SPSS17.0. Data were expressed as mean±SD. The repeated measure analysis of variance was used to detect any difference in the BWT scores, and the factory design analysis of variance was used to detect any difference in the density of dendritic spines and expression of pannexin1mRNA among groups, and when significant he was detected, LSD was used for between-group comparisons. The significance level P was set at0.05.
Results:
1. Effects of TDCS on motor function in rat model of cerebral infarction
On day3, there was a difference in the BWT scores between the MCAO groups and TDCS groups, and the rats in the MCAO group had significantly higher BWT scores on days7and14than the TDCS group (4.4±0.5vs.2.5±0.8and3.6±0.7vs.2.0±0.5, P<0.001), indicating a motor function improvement in the TDCS group. In the MCAO group, there was a significant reduction in BWT scores on day14and in the TDCS group, there was a significant reduction in BWT scores earlier on day7.
2. Effects of TDCS on the dendritic spines in the brain after cerebral infarction
In the SO, MCAO, and TDCS groups, the dendritic spines were mostly mushroom-shaped. The dendritic spines were sparse and missing in the MCAO group. Compared with the SO group, the MCAO group had lower spine density on days3,7, and14(P<0.001). The density of dendritic spines in the TDCS group showed a significant increase compared with that in the MCAO group on days3,7, and14(all P<0.001).
3. Distribution of pannexin1in the brain after cerebral ischemia and comparison of pannexin1mRNA expression by group
Pannexin1was mainly distributed in the hippocampus, cortex, and regions around the cerebral infarction. Co-localization between pannexin1and MAP2in the regions around the cerebral infarction showed strong positive expression of pannexin1in the neuron body and the cytoplasm of proximal dendritic spines. This suggested that pannexin1is distributed mainly in the neuron membrane, and is partially located in the cytoplasm. Pannexin1mRNA expression among the SO, MCAO, and TDCS groups was compared in the areas surrounding cerebral infarction. Compared with the SO group, the MCAO group had significantly increased pannexin1mRNA expression on days3,7, and14(P<0.001), and the peak pannexin1mRNA expression was observed on day7. Transcranial direct current stimulation did not decrease the elevated pannexin1mRNA expression after cerebral infarction on day3, but did reduce the significantly increased pannexin1mRNA expression after cerebral infarction on days7and14(P<0.001). The reduced pannexin1mRNA expression levels in the TDCS group on days3,7, and14were significantly different from that in the SO group (P<0.05)
4. The signal from Neurocan was detected in and around the GFAP-positive cells, however not in SO. This Indicate that some part of Neurocan was produced by the reactive astrocytes. Compared to SO, the expression of Neurocan mRNA significantly increased from the third day (P<0.001), and in the first week reached peak level (P<0.001), especially the second week kept higher expression level (P<0.001). After TDCS treatment the expression of Neurocan mRNA started to decrease from the third day (P<0.001), and kept downtrend to a lower amount in the second week (P<0.001). After TDCS treatment the expression of Neurocan started to decrease from the first week (P<0.001), and kept downtrend to a trace amount in the second week (P<0.001).
Conclusion
In summary, TDCS increases the dendritic spine density after cerebral infarction, indicating that it may promote neural plasticity after stroke. Early TDCS intervention from day3to day14after stroke demonstrates motor function improvement, and from day7to day14can down-regulate the elevated pannexin1mRNA expression after cerebral ischemia.
Neurocan is located in peri-infarct region, and mainly in proliferative glia cytoplasm. The expression of Neurocan in peri-infarct region kept increasing in the third day and the1st week, and decreased in the second week. The expression of Neurocan can be suppressed by TDCS.
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[21]Ambrosi C, Gassmann O, Pranskevich JN, Boassa D, Smock A, Wang J, Dahl G, Steinem C, Sosinsky GE. Pannexinl and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. J Biol Chem.2010.285(32): 24420-31.
[22]Bhalla-Gehi R, Penuela S, Churko JM, Shao Q, Laird DW. Pannexinl and pannexin3 delivery, cell surface dynamics, and cytoskeletal interactions. J Biol Chem.2010.285(12): 9147-60.
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