The Effects Of TDCS On Pannexin1Channel And Neural Plasticity After Cerebral Infarction In MCAO Rat

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 materials1Animals and experimental groupingOne 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 therapyTo 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 assessmentMotor 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 stainingOn 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 immunohisochemistryThe 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 PCRThe 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 analysisThe 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 infarctionOn 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 infarctionIn 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 groupPannexin1was 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).ConclusionIn 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.