胚胎大鼠神经干细胞电生理特性检测

目的 探讨胚胎大鼠神经干细胞体外培养分化的子代细胞电生理特性。方法 传代细胞单层贴壁培养,细胞内电流钳记录静息膜电位、动作电位、膜阻抗等电生理指标。结果 神经干细胞在现有培养条件下可初步分化为神经元和胶质细胞,并表现出不同的电生理特性。结论 神经干细胞表现出多向分化潜能,但现有培养条件尚不能使子代细胞电生理功能完全成熟。     

大鼠海马干细胞分化的神经元内钙离子浓度的测定

目的　采用激光扫描共聚焦技术 ,检测大鼠海马干细胞分化的神经元内钙离子浓度 ,以期为神经干细胞进一步应用于细胞替代治疗提供理论依据。方法　分离培养新生鼠的海马干细胞 ,采用激光扫描共聚焦技术测定其分化的神经元内钙离子浓度 ,并与原代培养的海马神经元内的钙离子浓度进行比较。结果　大鼠海马干细胞分化的神经元平均荧光强度为 94 3.6 5± 89.33,相同条件下原代培养的大鼠海马神经元平均荧光强度为 713.94± 5 5 .5 9(t=1.5 5 ,P >0 .10 )。结论　大鼠海马干细胞分化的神经元已具有神经元的一些特征 ,其细胞内钙离子的分布与原代培养的海马神经元相似。     

星形胶质细胞诱导成年大鼠骨髓基质细胞分化前后的电生理功能鉴定

目的 探讨骨髓基质细胞向神经元方向分化后是否具备神经元的电生理功能。方法 应用膜片钳技术,釆用全细胞记录方式.对由星形胶质细胞诱导的分化前、分化3d、分化7d的成年大鼠骨髓基质细胞进行电生理功能测定。结果分化前后的骨髓基质细胞未记录到内向Na~+电流,但成功记录到了静息膜电位,分別为:(-12.71±3.25)mV(n=7)、(-26.29±10.34)mV(n=7)、(-45.0±8.46)mV(n=6);刺激脉冲为40 mV时三组的外向延迟整流K~+电流分別为:(0.31±0.17)nA(n=7)、(1.34±0.59)nA(n=8)、(1.99±0.97)nA(n=7);刺激脉冲为-40 mV时的内向整流K~+电流为:(79±58)pA(n=7)、(260±150)pA(n=8)、(420±120)pA(n=7),伴随着分化时间的逐渐延长,两种K~+电流及膜电位逐渐增高。结论(1)骨髓基质细胞表达弱的外向延迟整流K~+电流及内向整流K~+电流可能是其特征性的电生理指标。(2)骨髓基质细胞经过星形胶质细胞的诱导能够向功能性神经元方向分化。     

NSCs在OECs诱导下定向分化及神经元电生理特性研究

目的探索大鼠嗅鞘细胞（OECs）对神经干细胞（NSCs）分化的影响,并记录分化后神经元电生理特性。方法在无血清改良Eagle培养基（DMEM／F12）培养的NSCs中,加入OECs条件培养基,观察分化情况,巢蛋白（nestin）鉴定NSCs、神经丝蛋白200（NF200）鉴定神经元、膜片钳检测神经元电生理特性。结果OECs能促进NSCs分化为神经元,分化后的神经元记录到快速激活快速失活、能被河豚毒素（TTX）特异阻断的钠电流及慢激活慢失活、能被四乙基氯化铵（TEA）特异阻断的延迟整流性钾电流。结论OECs能诱导NSCs分化,分化后的神经元具有活跃的电生理特性,具有替代凋亡、坏死神经元的潜能。     

大鼠伏隔核神经元膜特性及乙醇对其影响

目的 :观察伏隔核神经元的膜特性及致醉剂量的乙醇 (44mmol L)对其影响。 方法 :在大鼠伏隔核脑片上 ,采用细胞内电流钳记录技术。结果 :所观察到的神经元包括五种类型 ,神经元的静息膜电位 (mV)为 - 79.1± 2 .4 ,输入阻抗 (MΩ)为 4 8.4± 2 .3,锋电位的幅度 (mV)平均为 98.5± 4 .1;致醉剂量的乙醇 (44mmol L)对大部分伏隔核神经元的膜电位、输入阻抗及I V曲线无明显影响。 结论 :在伏隔核内 ,存在多种膜特性类型的神经元 ;致醉剂量的乙醇对大鼠伏隔核神经元的膜特性无明显影响 ,说明乙醇对伏隔核神经元的抑制作用主要并不是通过影响膜特性来实现的。     

P2X3受体对中脑导水管周围灰质神经元放电的促进作用

目的:应用脑片膜片钳记录技术,探讨腺嘌呤核苷酸离子通道型3(P2X3)受体活化对大鼠中脑导水管周围灰质(PAG)外侧区(LPAG)神经元膜电位及动作电位发放频率的影响.方法:制备大鼠PAG脑片,在膜片钳全细胞记录模式下,观察LPAG神经元膜电位及动作电位发放频率以及给予P2X3受体激动剂α、β-me三磷酸腺苷(ATP)后对LPAG神经元兴奋性的影响.结果:记录 32个LPAG神经元中有29个神经元观察到膜电位升高以及动作电位频率的增加(P<0.05).结论:P2X3受体活化具有促进LPAG神经元膜电位升高以及动作电位发放频率增加的作用.     

骨髓间充质干细胞分化为神经元样细胞后电生理特性的改变

背景：目前体外实验对骨髓间充质干细胞来源的神经元样细胞的研究多集中于形态学层面和神经标志物方面,对分化后的电生理功能研究较少。 目的：观察脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞后电生理特性的变化。 设计、时间及地点：细胞学体外培养,对比观察,于2005-06/2007-10在天津市环湖医院细胞室和南开大学生命科学院完成。 材料：6周龄雄性Wistar大鼠3只,体质量160 g左右。 方法：贴壁培养法体外分离纯化间充质干细胞,用脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸联合诱导间充质干细胞向神经元样细胞分化。诱导前和诱导3 d后分别用膜片钳技术检测细胞膜电流。 主要观察指标：流式细胞仪检测间充质干细胞表型;倒置显微镜观察诱导分化前后细胞形态变化;免疫细胞化学鉴定神经元特异性烯醇化酶的表达,以及全细胞电流测定结果。 结果：①流式细胞仪检测结果显示,CD90阳性率(99±3)%,CD31阳性率(3.4±0.8)%,CD34阳性率(0.3±0.1)%。说明这一细胞群大部分处于未分化的干细胞状态,其纯度可达95%。②光镜下可见未经诱导的间充质干细胞多为扁平形带突起的细胞,似纤维样细胞,诱导3 d后出现神经元样细胞。③免疫细胞化学结果显示,诱导前间充质干细胞的神经元特异性烯醇化酶呈弱阳性,诱导后呈强阳性。诱导72 h时分化率为(24.01±3.76)%。④诱导组神经元样细胞外向电流峰值及最大外向电流密度高于对照组(P < 0.05),但未发现内向钠电流。 结论：脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导方法可以诱导间充质干细胞向神经元方向分化,虽未发现具有成熟神经元电生理功能,但有向成熟神经元分化的趋势。     

4000/人胚胎干细胞源TH 阳性细胞电压门控性通道的初步研究

目的：检测人胚胎干细胞源TH阳性细胞的神经元性电生理特性。方法：采用我们实验室改良后的“无血清四步法经拟胚体培养体系”的方法,体外诱导人胚胎干细胞源性TH阳性细胞,在对其细胞核型及特异性标志物进行检测的基础上,运用全细胞膜片钳记录的方法,检测其细胞膜上电压门控性离子通道的电生理特性。结果：分化前后细胞核型保持正常;诱导得到的细胞形态一致,大多数细胞（>90%）表达多巴胺能神经元的标志β-tubulion和TH,并仍表达神经前体细胞的标志nestin;膜片钳检测显示诱导分化的TH阳性细胞具有神经元性电压门控钠、钾离子通道。结论：人胚胎干细胞经体外定向诱导分化为TH阳性细胞后具有一定的DA能神经元特性,特别是神经元性电生理特性。     

LKB1表达下调对培养海马神经元突触mEPSC的影响

目的研究离体培养的海马神经元LKB1表达下调对于神经元微小兴奋性突触后电流(mEP-SC)的影响。方法选用17d的胚胎大鼠培养海马神经元,分别用电穿孔的方法转染CAG-RE质粒和LKB1RNAi质粒,培养10~12d后进行电生理记录,选用全细胞膜片钳方式及自由记录模式,细胞外液加TTX阻断动作电位,加Bicucullin抑制GABA电流,记录神经元的mEPSC,比较2组神经元的mEPSC频率和幅度的差别。结果转染CAG-RE的神经元mEPSC幅度平均为25.6pA,频率平均为(5.21±0.25)Hz,是基线的99.8%;转染LKB1RNAi的神经元mEPSC幅度平均为35.1pA,频率平均为(5.79±0.27)Hz,是基线的127.1%;比较2组间频率、幅度变化,差别有显著性意义(P<0.05)。结论LKB1基因表达下调增强了培养海马神经元突触传递的效率。     

骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究

目的探索骨髓基质细胞(BMSCs)诱导分化为神经干细胞(NSCs),比较血清、维甲酸(RA)、胶质细胞源神经营养因子(GDNF)、脑源性神经营养因子(BDNF)及2-巯基乙醇(2-ME)等不同浓度诱导条件下BMSCs分化情况,以及分化细胞的电生理特性。方法以恒河猴骨髓中分离出的BMSCs为实验对象,利用神经干细胞培养基和RA、GDNF、BDNF、2-ME等生长因子在不同血清浓度下进行培养增殖和诱导分化。Nestin、CD133抗体免疫细胞化学染色鉴定NSCs,NSE、β-tublin鉴定神经元、GFAP鉴定神经胶质细胞。膜片钳检测细胞的电生理特性。结果低浓度血清(2.5%)+RA(0.3mg/L)+GDNF(20μg/L)诱导分化效果较好,且分化的神经元样细胞较未分化细胞的膜特性[静息膜电位(RMP)、膜电容(Cm)、串联电阻值(Rs)]有了显著改变(P﹤0.01)。部分形态成熟的神经元样细胞表现出TTX敏感的快速激活、快速失活的电压依赖性的Na+通道,而未分化细胞却未记录到内向电流;两类细胞均可记录到外向的K+电流,但神经元样细胞的电流峰值强度要显著高于未分化细胞,并且包括两种电流成分:瞬时外向K+电流和延迟整流型的K+电流。结论RA+GDNF及配合使用低浓度血清能够有效诱导骨髓源神经干细胞向成熟神经系细胞分化,且分化的神经元样细胞具有快速激活、快速失活的电压依赖性Na+通道,类似神经细胞的电生理特性。     

Diagnostic Difficulties and Treatment Implications

Summary: Differentiation between types of epileptic seizures has been aided in recent years by the introduction of intensive neurodiagnostic techniques and the development of increasingly detailed classification systems. Paradoxically, these developments have not simplified the task of matching the appropriate antiepileptic drug to a particular seizure type. It is reasonable to assume that anticonvulsant drugs will have different effects on different types of seizures, but faulty, circular reasoning can enter the picture if one also assumes that responses of seizures to different drugs signify different seizure types. There are several examples of differential diagnoses that can fall prey to this problem, including the diagnosis between partial seizures with secondary generalization and generalized tonic-clonic seizures, and the diagnosis between complex partial seizures and absence seizures with automatisms, among others. Considerations of etiology in future classification systems can further complicate the problem: should one then choose an anticonvulsant drug on the basis of individual seizure type or on the basis of the type of epilepsy? Ramifications of this issue extend even to the drug approval process. Official sanction is not given for use of a drug for a seizure type not included in the original efficacy studies, even if later scientific evidence shows that seizure type to be related to a type that is included. New trials must be undertaken. These problems arise from how we choose to classify seizures.     

Cognitive Dysfunction Associated with Antiepileptic Drug Therapy

Summary: Epilepsy is frequently associated with cognitive dysfunction. However, the reasons for this correlation are unclear. Possible influential factors include patient age; duration, frequency, etiology, and type of seizures; hereditary factors; psychosocial issues; and antiepileptic drug (AED) therapy. Whereas many of these factors are beyond the physician's control, AED therapy is one element that can be addressed in treatment decisions by recognizing the potential cognitive effects of particular AEDs. For example, phenobarbital impairs memory and concentration; phenytoin affects attention, problem solving ability, and performance of visuomotor tasks. In contrast, carbamazepine may affect concentration, while valproate would appear to have minimal effects on cognition. Moreover, cognitive effects of AEDs are amplified with coadministration of multiple anticonvulsants (polytherapy). A review of studies on the cognitive effects of monotherapy with AEDs, as opposed to those of polytherapy, provides evidence that drug-related cognitive dysfunction can be reversed if patients are switched to a simpler therapeutic regimen. Future research should be directed toward developing reliable measures for assessing and monitoring cognition, and understanding the particular cognitive side effects of each AED. Physicians also need to revise their opinions about which side effects are "tolerable" for epileptic patients.     

Hepatic Considerations in the Use of Antiepileptic Drugs

Summary: Virtually all of the major antiepileptic drugs (AEDs) can cause hepatotoxicity, although fatal hepatic reactions are rare. The mechanisms, incidences, and risk profiles for such reactions differ from drug to drug. With carbamazepine and phenytoin, hepatotoxicity may be due to drug hypersensitivity. Although the profiles of patients at risk have not been well-defined for these two antiepileptic drugs, it would appear from reports in the literature that older adolescents and adults are at higher risk than children of developing serious or fatal hepatotoxicity. Once hepatotoxicity develops, mortality rates are 10–38% with phenytoin and 25% for carbamazepine. The risk profile for valproate fatal hepatotoxicity has been more clearly defined. Those at primary risk of fatal hepatic dysfunction are children under the age of 2 years who are receiving multiple anticonvulsants and also have significant medical problems in addition to severe epilepsy. The risk is considerably lower for patients over the age of 2 years on valproate monotherapy. In contrast to the risk profile with other AEDs, adults receiving valproate as monotherapy have the lowest risk of hepatotoxicity. Fatal hepatic dysfunction coincident with valproate may be the result of aberrant drug metabolism. Concomitant use of AEDs that induce microsomal P450 enzymes (e.g., phenytoin and phenobarbital) may enhance the production of a toxic metabolite, and hence the greater risk of hepatotoxicity with polypharmacy.     

Vascular Malformations and Epilepsy: Clinical Considerations and Basic Mechanisms

Summary: Vascular malformations (VMs) are associated with epilepsy. The natural history of the various VMs, clinical presentation, and tendency to provoke epilepsy determine treatment strategies. Investigations have probed the mechanisms of epileptogenesis associated with these lesions. Electrophysiologic changes are associated with epileptogenic cortex adjacent to VMs. Putative pathophysiologic mechanisms of epileptogenesis include neuronal cell loss, glial proliferation and abnormal glial physiology, altered neurotransmitter levels, free radical formation, and aberrant second messenger physiology.     

Carbamazepine Efficacy in Adults With Partial and Generalized Tonic-Clonic Seizures

Summary: Carbamazepine and phenytoin are drugs of choice in initial monotherapy for adult partial and secondarily generalized tonic-clonic seizures. These designations reflect the results of the Veterans Administration Epilepsy Cooperative Study Group of 1985. An earlier comparative study of carbamazepine and phenytoin by Ramsay and associates found both drugs equally effective in controlling new-onset seizures. Among the advantages of carbamazepine is that it causes relatively few cognitive and dysmorphic side effects. Its disadvantages are its unavailability in parenteral formulation and its metabolic autoinduction. The latter must be compensated for by planned dosage increases to maintain therapeutic plasma steady-state levels during the first 2 or 3 months of treatment. Carbamazepine is judged a drug of choice in the treatment of these secondarily generalized tonic-clonic seizures, and the drug of choice in children, adolescents, and women susceptible to the dysmorphic side effects associated with other anticonvulsant agents.     

Etiologic and Preventive Aspects of Epilepsy in the Child–Bridging the Gap Between Laboratory and Clinic

Summary: Four broad categories of basic phenomena are pertinent to developing ways to prevent epilepsy. These include mechanisms of epileptogenesis, ictal initiation and temporary entrainment by the seizure discharge of normally functioning brain, seizure propagation, and control mechanisms that function both to restrain the cascade of epileptic events culminating in a seizure and to arrest the epileptic event and restore the interictal state. In newborns and children, hypoxia-ischemia is a major factor leading to epileptogenesis, and several schemes are proposed to classify, quantify, and prevent hypoxic-ischemic encephalopathy. Control mechanisms must be better understood in order to develop prophylactic recommendations for epilepsy, and an experimental model of "kindling antagonism" may increase our understanding of these. Programs of prevention of seizures in children will evolve only if basic researchers and clinicians work productively together to develop an adequate understanding of factors important in epileptogenesis and antiepileptogenic control mechanisms.     

Carbamazepine Efficacy and Utilization in Children

Summary: Carbamazepine is effective for preventing partial and generalized tonic-clonic seizures in children. Although absence epilepsies are more common in children than adults, an estimated 80% of children with epilepsy have seizure types or epilepsies that are potentially responsive to carbamazepine. The differential diagnosis of ictal staring is an especially important issue in children because absence and atypical absence seizures are more prevalent in children than adults. Age-related pharmacokinetic differences and drug interactions are major considerations in children. On average, children have higher clearance rates of carbamazepine, shorter half-lives, and higher ratios of carbamazepine-10, 11-epoxide to carbamazepine than adults. In addition, children with severe epilepsy are more likely to require multiple-drug therapy, which can lead to complex drug interactions. When carbamazepine is administered along with valproate, drug protein binding interactions can cause intermittent side effects.     

Predisposing and Causative Factors in Childhood Epilepsy

Summary: We review information from large studies of defined populations, examining the role of known factors and especially of prenatal and perinatal factors in contributing to nonfebrile seizure disorders of early childhood. We depend especially, but not exclusively, on the recently completed analyses from the Collaborative Perinatal Project of the National Institute of Neurological and Communicative Disorders and Stroke, the NCPP. About 4% of children in the NCPP who had at least one non-febrile nonsymptomatic seizure by the age of 7 years had a previous seizure during acute neurologic illness, such as meningitis or during the acute illness after trauma. Many such seizures should potentially be preventable. Of children with seizures, 10% had had a neonatal seizure and 13% had had a febrile seizure. Among the hundreds of prenatal and perinatal factors explored as predictors of childhood seizure disorders, the principal predictors identified were congenital malformations of the fetus, cerebral and noncerebral; family history of certain neurologic disorders; and neonatal seizures. In agreement with the British National Child Development Study, labor and delivery factors in the NCPP appeared to contribute very little to childhood seizure disorders. Maldevelopment, rather than damage at birth to an initially intact nervous system, appeared to be the more common mechanism. Most seizure disorders of early childhood remained unexplained by the large set of prenatal and perinatal characteristics examined.     

Classification of methods in transcranial Electrical Stimulation (tES) and evolving strategy from historical approaches to contemporary innovations

Transcranial Electrical Stimulation (tES) encompasses all methods of non-invasive current application to the brain used in research and clinical practice. We present the first comprehensive and technical review, explaining the evolution of tES in both terminology and dosage over the past 100 years of research to present day. Current transcranial Pulsed Current Stimulation (tPCS) approaches such as Cranial Electrotherapy Stimulation (CES) descended from Electrosleep (ES) through Cranial Electro-stimulation Therapy (CET), Transcerebral Electrotherapy (TCET), and NeuroElectric Therapy (NET) while others like Transcutaneous Cranial Electrical Stimulation (TCES) descended from Electroanesthesia (EA) through Limoge, and Interferential Stimulation. Prior to a contemporary resurgence in interest, variations of transcranial Direct Current Stimulation were explored intermittently, including Polarizing current, Galvanic Vestibular Stimulation (GVS), and Transcranial Micropolarization. The development of these approaches alongside Electroconvulsive Therapy (ECT) and pharmacological developments are considered. Both the roots and unique features of contemporary approaches such as transcranial Alternating Current Stimulation (tACS) and transcranial Random Noise Stimulation (tRNS) are discussed. Trends and incremental developments in electrode montage and waveform spanning decades are presented leading to the present day. Commercial devices, seminal conferences, and regulatory decisions are noted. We conclude with six rules on how increasing medical and technological sophistication may now be leveraged for broader success and adoption of tES.     

Anticonvulsant Drugs and Cognitive Function: A Review of the Literature

Summary: Alterations of cognitive function are separate from disturbances of behavior seen in association with epilepsy. The nature of the cognitive disability may to a certain extent depend on the seizure type. Partial seizures, mainly derived from a temporal lobe focus, impair memory tasks, while generalized seizures seem to have more effect on attentional abilities. A number of studies, reviewed in this paper, suggest that anticonvulsant drugs further impair cognitive function. Maximal impairments are seen in patients receiving polytherapy: rationalization of polytherapy improves cognitive abilities. Studies in children and adults have allowed differentiation of the effects of various commonly used antiepileptic agents. Maximal cognitive deficits are seen with. phenytoin, while phenobarbital and sodium valproate induce moderate disturbances, and carbamazepine seems relatively free from such toxicity. Further research is needed on the interrelationship between types of seizure disorders, types of anticonvulsant medications, and cognitive function.