体外血脑屏障模型的建立及功能测定

目的利用大鼠原代微血管内皮细胞及星形胶质细胞建立体外血脑屏障模型,并通过跨内皮细胞电阻(Trans-epithelium electrical resistant,TEER)方法对血脑屏障模型进行功能测定。方法原代分离纯化SD大鼠脑微血管内皮细胞和星形胶质细胞,用免疫荧光检测内皮细胞标志物VWF,紧密连接蛋白ZO-1,星形胶质细胞标志物GFAP;用微血管内皮细胞和星形胶质细胞在Transwell小室上建立体外血脑屏障模型,观察TEER值的动态变化。结果原代的微血管内皮细胞培养至融合后具有典型的梭形"铺路石"样外观,VWF鉴定细胞纯度达到95%以上,ZO-1免疫荧光鉴定证实细胞间形成紧密连接;原代培养的星形胶质细胞呈现具有多个突起的典型形态,GFAP鉴定细胞纯度达到95%以上;在第10 d,单独微血管内皮细胞血脑屏障模型的TEER值为(42±1.41)Ωcm2,内皮细胞和星形胶质细胞共培养血脑屏障模型的TEER值为(65±1.42)Ωcm2。结论建立了体外血脑屏障模型,通过TEER值测定证明共培养使模型更加完整,更加接近在体血脑屏障模型的特性。     

人脑出血后血肿周围组织HO-1、GFAP和cyclinD1的表达研究

目的探讨脑出血后血肿周围组织血红素氧合酶-1(HO-1)、胶质纤维酸性蛋白(GFAP)和细胞周期蛋白D1(cvclinD1)表达规律,及其与神经修复之间的关系。方法 HE染色观察脑出血后神经元和星形胶质细胞形态变化,免疫组织化学染色检测脑出血后不同时间点血肿周围组织H0-1、GFAP和cycIinD1表达水平。结果脑出血后2h星形胶质细胞胞质内即开始表达HO-1[(5.30±1.00)个/高倍视野]、GFAP[(22.60±1.40)个/高倍视野]和cvclinD1[(11.50±1.20)个/高倍视野],达峰值水平后逐渐下降,脑出血后不同时间点表达水平均高于健侧正常脑组织,且差异具有统计学意义(均P=0.000)。结论人脑出血后血肿周围组织HO-1、GFAP和cvclinD1表达变化呈"抛物线"样,HO-1和cvclinD1共同参与了脑出血后星形胶质细胞的增生与活化,以及脑出血后的继发性损伤和修复。     

脑出血后血红蛋白对血脑屏障的影响

目的 探讨脑出血后血红蛋白(Hb)对血脑屏障结构和功能的影响. 方法 108只雄性SD大鼠按随机数字表法分为正常对照组(12只)、Hb组(48只)和假手术组(48只);再进一步依据模型制作完成后观察时间点的不同,将Hb组和假手术组分为6h、24 h、3d、7d共4个亚组(每个亚组12只).应用HE染色、铁染色分别观察Hb注入大鼠脑内后组织学变化和铁离子释放规律.利用伊文氏蓝检测血脑屏障的通透性.免疫荧光和荧光定量PCR检测血脑屏障紧密连接蛋白claudin-5和ZO-1表达的变化. 结果 Hb注入后大鼠脑组织中可见明显的水肿坏死,3d和7d时可见铁离子的释放和蓄积.Hb组的伊文氏蓝渗出量明显高于假手术组,差异有统计学意义(P＜0.05).免疫荧光结果显示Hb注入大鼠脑内后可见血管内皮细胞间紧密连接蛋白claudin-5和ZO-1表达不连续,表达水平下降.在mRNA水平,Hb组claudin-5和ZO-1表达水平明显低于假手术组,差异有统计学意义(P＜0.05). 结论 脑出血发生后,Hb可能直接引起血脑屏障结构和功能的破坏,进而参与脑水肿的发生、发展过程.     

脑出血大鼠血肿周围组织水孔蛋白-4表达分析

目的研究大鼠脑出血(ICH)后皮质、CPu血肿周围组织水孔蛋白-4(AQP4)表达的变化,以探讨其在脑水肿形成过程中的作用。方法采用立体定向技术,经尾动脉采血制作大鼠CPu出血模型。采用免疫组化技术对皮质及血肿周围组织AQP4蛋白进行动态检测。结果ICH6 h后CPu血肿周围组织邻近毛细血管的AQP4蛋白表达开始增高,于出血第1~3天达高峰期,之后逐渐下降,至第7天仍高于正常水平。在出血侧皮质AQP4蛋白表达亦相应增加,但不如血肿周围组织明显。结论ICH时血肿成分可诱导AQP4蛋白表达增加,脑毛细血管周围星形胶质细胞终足表达增强的AQP4蛋白可能促进水通过血-脑脊液屏障向脑实质流动,直接参与ICH后血管源性脑水肿的形成。     

人脑出血后血肿周围组织HO-1、GFAP和cyclinD1的表达研究

目的探讨脑出血后血肿周围组织血红素氧合酶-1（HO-1）、胶质纤维酸性蛋白（GFAP）和细胞周期蛋白D1（cvclinD1）表达规律,及其与神经修复之间的关系。方法HE染色观察脑出血后神经元和星形胶质细胞形态变化,免疫组织化学染色检测脑出血后不同时间点血肿周固组织HO-1、GFAP和cvclinD1表达水平。结果脑出血后2h星形胶质细胞胞质内即开始表达HO-1[（5．30±1．00、）个,高倍视野]、GFAP[（22．60±1．40）个／高倍视野]和cyclinD1[（11．50±1．20）个,高倍视野],达峰值水平后逐渐下降,脑出血后不同时间点表达水平均高于健侧正常脑组织．且差异具有统计学意义（均P=0.000）。结论人脑出血后血肿周围组织HO-1、GFAP和cvclinD1表达变化呈“抛物线”样,HO-1和evclinD1共同参与了脑出血后星形胶质细胞的增生与活化,以及脑出血后的继发性损伤和修复。     

头孢曲松钠减轻水通道蛋白4抗体对星形胶质细胞的毒性作用

目的探讨头孢曲松钠在水通道蛋白4(AQP4)抗体诱导的星形胶质细胞损伤中的作用以及机制。方法常规体外培养新生SD大鼠大脑皮质细胞,将培养的细胞分为4组,分别加入健康人血清(对照组)、AQP4抗体阳性患者血清、头孢曲松钠+AQP4抗体阳性血清以及单纯头孢曲松钠。细胞培养24h后采用免疫组织化荧光染色观察不同组星形胶质细胞数目的变化,采用比色法测定上清液谷氨酸浓度以及免疫印迹分析谷氨酸转运体-1(GLT-1)蛋白表达水平。结果和对照组比较,AQP4抗体阳性血清组星形胶质细胞数目和谷氨酸转运体-1(GLT-1)蛋白表达明显减少,上清液谷氨酸浓度明显增高(均P0.01),而单纯头孢曲松钠组仅显著增加GLT-1蛋白表达(P0.01),并不影响星形胶质细胞数目和谷氨酸水平(P0.05);和AQP4抗体阳性组比较,头孢曲松钠+AQP4抗体阳性血清组星形胶质细胞数目和GLT-1蛋白表达增加,上清液谷氨酸浓度明显下降(P0.01)。结论头孢曲松钠可能通过上调星形胶质细胞GLT-1表达、减少细胞外液谷氨酸水平发挥减轻AQP4抗体对体外培养的大鼠脑皮质星形胶质细胞的毒性作用。     

水通道蛋白-4在脑出血大鼠脑组织的分布

目的 观察脑出血大鼠水通道蛋白-4(AQP4)在脑组织的分布变化及其在出血性脑水肿中的作用。方法 采用定量胶原酶注人大鼠尾状核建立脑出血模型，应用免疫组化法观察脑出血后不同时间大鼠脑组织AQP4的表达。结果 AQP4阳性细胞主要分布在脑出血血肿周围区和大脑皮质的星形胶质细胞、脑室周围、视上核和室旁核。脑出血后6h起，AQP4表达增强，出血后72h达高峰，出血1周后仍高于正常水平。结论 脑出血后，AQP4在与水代谢密切相关的部位表达明显增强，提示AQP4在出血性脑水肿的形成过程中起重要作用。     

高血压脑出血血肿周围组织血-脑屏障变化的动态观察

目的动态观察高血压脑出血血肿周围组织血-脑屏障的变化.方法对6例高血压脑出血患者实施立体定向血肿碎吸术,术中取血肿周围少许脑组织进行超微病理观察.结果血-脑屏障的损伤与出血时间的长短呈正比.脑出血后24 h,可见星形胶质细胞肿胀,部分细胞崩解、坏死.毛细血管周围细胞足突肿胀,血-脑屏障损坏.72 h,部分星形胶质细胞高度肿胀,细胞器溶解.毛细血管内皮细胞胞核增大,胞质突入管腔,内皮细胞间紧密连接消失.4～7 d,星形细胞高度肿胀,胞质内充满水肿液,细胞器消失,细胞变性.毛细血管星形细胞足突明显肿胀,血管周围可见微小出血灶.结论血-脑屏障的变化与患者的预后有密切关系.     

高血压脑出血血肿周围组织血-脑屏障变化的动态观察

目的动态观察高血压脑出血血肿周围组织血-脑屏障的变化.方法对6例高血压脑出血患者实施立体定向血肿碎吸术,术中取血肿周围少许脑组织进行超微病理观察.结果血-脑屏障的损伤与出血时间的长短呈正比.脑出血后24h,可见星形胶质细胞肿胀,部分细胞崩解、坏死.毛细血管周围细胞足突肿胀,血-脑屏障损坏.72h,部分星形胶质细胞高度肿胀,细胞器溶解.毛细血管内皮细胞胞核增大,胞质突入管腔,内皮细胞间紧密连接消失.4～7d,星形细胞高度肿胀,胞质内充满水肿液,细胞器消失,细胞变性.毛细血管星形细胞足突明显肿胀,血管周围可见微小出血灶.结论血-脑屏障的变化与患者的预后有密切关系.     

高血压脑出血血肿周围组织血—脑屏障变化的动脉观察

目的 动态观察高血压脑出血血肿周围组织血-脑屏障的变化。方法 对6例高血压脑出血患者实施立体定向血肿碎吸术,术中取血肿周围少许脑组织进行超微病理观察。结果 血-脑屏障的损伤与出血时间的长短呈正比。脑出血后24h,可见星形胶质细胞肿胀,部分细胞崩解、坏死。毛细血管周围细胞足突肿胀,血-脑屏障损坏。72h,部分星形胶质细胞高度肿胀,细胞器溶解。毛细血管内皮细胞胞核增大,胞质突入管腔,内皮细胞间紧密连接消失。4-7d,星形细胞高度肿胀,胞质内充满水肿液,细胞器消失,细胞变性。毛细血管星形细胞足突明显肿胀,血管周围可见微小出血灶。结论 血-脑屏障的变化与患者的预后有密切关系。     

Epilepsy and anomalies of neuronal migration: MRI and clinical aspects

Neuronal migration disorders are the result of disturbed brain development. In such disorders, neurons are abnormally located. In diagnosing these conditions, magnetic resonance imaging is superior to any other imaging technique. This enables us to improve our knowledge of the clinical correlates of neuronal migration. With reference to migrational disorder, a retrospective study of all 303 patients with epileptic seizures referred for magnetic resonance imaging during a 3-year period was performed, 13 patients (aged 12-41, mean age 27) were identified. They represent 4.3% of the entire study group. Of the patients with known epilepsy, 6.7% and of the mentally retarded, 13.7% had migrational disorders. Four patients had schizencephaly as the dominant finding, one was classified as hemimegalencephaly, 2 had isolated heterotopias, and 6 had localized pachy- and/or poly-microgyria. The clinical pictures are complex. Ectopias of grey matter are recognised foci of epilepsy, but from an epileptological and a clinical viewpoint little attention has been given to these disorders. The present study shows that malmigration is not rare in epilepsy patients, especially not in the mentally retarded.     

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.     

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.     

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.     

Neonatal Seizures: Problems in Diagnosis and Classification

Summary: The clinical identification of neonatal seizures is critical for the recognition of brain dysfunction; however, diagnosis is often difficult because of the poorly organized and varied nature of these behaviors. Current classification systems are limited in their ability to communicate motor, autonomic, and electroencephalo-graphic features of seizures precisely and to provide a basis for uniform effective diagnosis, therapy, and determination of prognosis. Recent investigations of neonates, utilizing bedside electroencephalographic/polygraphic/ video monitoring techniques, have provided the basis for improved diagnosis and classification of seizures in the newborn. These studies have demonstrated that not all clinical phenomena currently considered to be seizures require electrocortical epileptiform activity for their initiation or elaboration. In addition, the specific clinical character of the phenomena considered to be seizures, the clinical state of the infant, and the character of the EEG indicate the probable pathophysiological mechanisms involved and suggest probable etiologies, prognosis, and therapy. Similarities between animal models that demonstrate reflex physiology and neonates with motor automatisms and tonic posturing suggest that these clinical behaviors may not be epileptic in origin but, rather, primitive movements of progression and posture mediated by brainstem mechanisms. Although not all clinical behaviors currently considered to be neonatal seizures may have similar pathophysiological mechanisms, they are clinically significant because they all indicate brain dysfunction.     

Valproate Monotherapy in the Management of Generalized and Partial Seizures

Summary: For decades, therapeutic tradition has promoted the concept of polypharmacy in the management of epilepsy. In recent years, however, studies have shown that, for most patients, monotherapy can provide comparable or better seizure control than administration of multiple anticonvulsants, while diminishing the potential for adverse reactions, drug interactions, and poor compliance. Valproate is an important monotherapeutic agent that is highly effective in the control of idiopathic primary and secondarily generalized epilepsies, and partial seizures that do not generalize. Comparative studies have found that valproate is at least as effective as phenytoin and carbamazepine in the treatment of generalized and partial seizures. Given the similar efficacy, other factors such as pharmacokinetics and side effects may therefore determine anticonvulsant selection for monotherapy.     

Un nouveau cadre conceptuel de travail pour la psychiatrie

In an attempt to place psychiatric thinking and the training of future psychiatrists more centrally into the context of modern biology, the author outlines the beginnings of a new intellectual framework for psychiatry that derives from current biological thinking about the relationship of mind to brain. The purpose of this framework is twofold. First, it is designed to emphasize that the professional requirements for future psychiatrists will demand a greater knowledge of the structure and functioning of the brain than is currently available in most training programs. Second, it is designed to illustrate that the unique domain which psychiatry occupies within academic medicine, the analysis of the interaction between social and biological determinants of behavior, can best be studied by also having a full understanding of the biological components of behavior.