促红细胞生成素对新生大鼠皮质神经元体外缺氧缺糖损伤的保护作用研究

目的研究rhEPO对新生大鼠皮质神经元氧糖剥夺(OGD)损伤的保护作用及rhEPO的作用剂量及作用时间。方法新生Wistar大鼠皮质神经元原代培养7 d~11 d,随机分为正常对照组、OGD损伤组和rhEPO处理组。(1)建立皮质神经元的OGD模型,给予不同剂量的rhEPO(0.1、1、10、100 U/ml);(2)在不同时间(OGD损伤后0 h、24 h、48 h)给予10 U/ml rhEPO。观察各组细胞形态学改变,MTT法检测细胞存活率。结果与OGD损伤组比较,rh EPO(0.1、1、10 U/ml)处理可增加OGD损伤的皮质神经元的存活率(P0.05),且浓度为10 U/ml时rhEPO保护作用最强(P0.05)。皮质神经元OGD损伤后立即加入rhEPO(10 U/ml)保护作用最强(P0.05),损伤后24 h及48 h给药没有增加皮质神经元存活率。结论 rhEPO对体外缺氧缺糖损伤的大鼠皮质神经元具有保护作用,rhEPO有效浓度为0.1~10 U/ml,且其浓度为10 U/ml时保护作用最强;rhEPO在神经元损伤后立即给药保护作用最强。     

促红细胞生成素预处理在脑缺血性损伤中神经保护作用的体外研究

目的在体外研究重组人促红细胞生成素(rh EPO)预处理在脑缺血性损伤中的神经保护作用及其合适的剂量范围、时间窗。方法取处于生长稳定期的高纯度原代皮质神经元,分为正常对照组、糖氧夺获(OGD)损伤组、rh EPO预处理组。rh EPO预处理组是在不同时间(OGD损伤前24 h、48 h、72 h)给予不同浓度(0.01 U/ml、0.1 U/ml、1 U/ml、10 U/ml、100 U/ml)的rh EPO,最后以MTT比色法检测存活率,光镜下观察细胞形态变化。结果rh EPO(0.01 U/ml、0.1 U/ml、1 U/ml、10 U/ml)预处理可明显增加OGD损伤的原代皮质神经元的存活率(P0.05),其中当浓度为1 U/ml时保护作用最强(P0.05);rh EPO(1 U/ml)预处理24 h、48 h可明显增加OGD损伤的原代皮质神经元的存活率(P0.05),其中rh EPO预处理48 h的保护作用强于24 h。在光学显微镜下,观察到的细胞形态和数量的变化与以上结果相符。结论 rh EPO预处理对OGD损伤的皮质神经元具有神经保护作用,其有效浓度为0.01~10 U/ml,其中最佳剂量为1 U/ml,其有效时间窗为OGD前48 h或24 h,最佳干预时间窗为OGD前48 h。     

刺五加皂甙对神经元谷氨酸毒性损伤的保护作用

目的:观察不同谷氨酸浓度和谷氨酸作用不同时间对神经元活性的影响,探讨细胞损伤后刺五加皂甙(ASS)的有效保护浓度。方法:取孕13～15dICR小鼠,无菌条件下对胎鼠大脑皮层神经元进行原代分离培养,建立谷氨酸诱导的皮层神经元损伤模型。用MTT、LDH测定神经元活性,用硝酸还原酶法测定细胞培养上清液中NO的含量,用流式细胞仪检测细胞凋亡率,并在电镜下观察细胞形态学变化。结果:①经谷氨酸处理的神经元,其细胞存活率呈剂量和时间依赖下降、ASS能不同程度提高细胞存活率。②谷氨酸处理组的神经元凋亡率、LDH释放量和NO含量均升高,与正常对照组及ASS组比较有明显差异(P<0.01)。结论:一定浓度的ASS对谷氨酸引起的神经元损伤有保护作用;ASS可能是通过抑制NO的释放和稳定细胞膜,拮抗细胞元损伤。     

体外培养大鼠皮质神经元机械性损伤后代谢型谷氨酸受体1a表达规律及其竞争性拮抗剂1-氨基茚满1，5-二羧酸的保护作用

体外培养大鼠皮质神经元机械性损伤模型,伤后30min,损伤神经元存活率较正常神经元明显下降,且随损伤程度加重神经元存活率随之降低。RT-PCR结果,显示神经元重度损伤1h后代谢型谷氨酸受体1a mRNA的表达明显升高。免疫组化染色发现神经元机械性损伤后30min,代谢型谷氨酸受体1a阳性细胞较正常神经元明显增多。伤后12h,1-氨基茚满1,5-二羧酸处理的损伤神经元乳酸脱氢酶活性较单纯损伤的神经元明显降低。伤后1h后1-氨基茚满1,5-二羧酸处理的损伤神经元较单纯损伤神经元细胞内Ca2 +浓度明显降低。提示皮质神经元机械性损伤后代谢型谷氨酸受体1a表达明显增强,创伤引起的神经细胞内游离Ca2 +浓度升高可被代谢型谷氨酸受体1a 拮抗剂1-氨基茚满1,5-二羧酸阻断,说明1-氨基茚满1,5-二羧酸有显著的神经保护作用。     

一氧化氮在缺氧复氧性神经元凋亡中的作用

目的观察神经元缺氧复氧损伤时NO的动态变化及其与神经元凋亡的关系。方法取孕13~15d ICR小鼠,无菌条件下对胎鼠大脑皮质神经元进行原代分离培养,建立缺氧复氧诱导的皮质神经元凋亡模型。用流式细胞仪检测细胞凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT测定细胞活性,并在电镜下观察细胞形态学变化。结果经缺氧复氧处理的神经元,随着缺氧时间延长细胞存活率逐渐下降,神经元凋亡率呈时间依赖性升高,NO含量逐渐升高,至缺氧8h复氧24h达到高峰,与正常对照组比较有显著性差异(P<0.01)。细胞超微结构呈现凋亡样改变。结论NO介导了缺氧复氧性神经元凋亡过程。     

海马神经元兴奋性毒性模型的建立及其意义

目的探讨不同浓度谷氨酸对海马神经元的损伤作用,流式细胞仪在建立理想的兴奋性毒性模型中的应用。方法在体外原代培养10d的海马神经元中分别加入不同浓度的谷氨酸(100、200、400、600、800μmol/L),24h后相差显微镜下观察细胞形态变化,用MTT法检测存活率和流式细胞仪检测凋亡率以评定谷氨酸对海马神经元的损伤程度。结果不同浓度的谷氨酸组与对照组的细胞存活率差异有显著性(P<0.01),并呈浓度依赖性,随着谷氨酸浓度的升高,神经元的存活率降低;谷氨酸终浓度(100、200、400μmol/L)组的细胞凋亡率与对照组比较差异有显著性(P<0.01);600和800μmol/L谷氨酸组的细胞凋亡率与对照组比较差异无显著性(P>0.05),但细胞凋亡率随着谷氨酸浓度的增加而降低。结论过高浓度的谷氨酸导致细胞急性坏死而非迟发性凋亡,运用流式细胞仪检测体外培养海马神经元凋亡率是一种特异性高的检测方法,值得推广。     

刺五加皂甙对皮质神经元缺血缺氧性损伤的保护作用

目的探讨刺五加皂甙(ASS)对皮质神经元缺血缺氧性损伤的保护作用。方法胎鼠皮质神经元经缺血缺氧和谷氨酸(Glu)作用模拟缺血性皮质神经元损伤模型。随机分成缺血缺氧组(HI组)、Glu组、ASS组和对照组;ASS组在实验前、中均加入终浓度为50mg/L的ASS。用流式细胞仪检测神经元凋亡率,用硝酸还原酶法测定细胞培养上清液中一氧化氮(NO)的含量,用3[4,5二甲基磺胺噻唑]2,5二苯基四唑溴化物(MTT)比色法、乳酸脱氢酶(LDH)释放量测定神经元活性,电镜下观察细胞形态学变化。结果(1)随着缺血缺氧时间延长神经元存活率逐渐下降,至缺血缺氧8h达到高峰;Glu作用的神经元,其存活率随作用时间延长而下降。(2)HI组及Glu组皮质神经元存活率显著低于正常对照组,LDH释放量、NO含量及凋亡率均显著高于对照组(均P<0.05);与HI组和Glu组比较,HI ASS组及Glu ASS组皮质神经元的存活率显著升高,而LDH释放量、NO含量及凋亡率均显著降低(均P<0.05)。结论ASS能提高缺血神经元存活率、降低凋亡率,抑制NO和LDH释放,从而起到神经保护作用。     

TLR3在poly（I：C）-LMW预处理后缺糖缺氧诱导的原代皮质神经元的表达意义

目的观察TRL3激动剂poly（I：C）-LMW预处理对原代皮质神经元Toll样受体3（TLR3）表达的影响,探讨TLR3在缺糖缺氧诱导的原代皮质神经元损伤中的作用。方法取体外培养7d的大鼠原代皮质神经元细胞,对细胞分别予以正常条件下培养（空白对照组）;缺糖缺氧2h,复糖复氧24h（OGD组）;TRL3激动剂预处理12h（激动剂组）;TRL3激动剂预处理12h后缺糖缺氧2h,复糖复氧24h（激动剂＋OGD组）。免疫荧光法观察各组细胞生长状态,分别以Western blot法、RT-PCR法测定TLR3蛋白及TLR3mRNA的表达情况。结果 与空白对照组相比,激动剂及缺氧复氧处理方法均诱导原代皮质神经元TLR3、TLR3mRNA表达增强（P〈0.05）;激动剂预处理后,与OGD组相比,激动剂＋OGD组神经元细胞状态较好,数目较多（P〈0.05）。结论 TLR3参与缺糖缺氧诱导的原代皮质神经元的损伤过程,激动剂可减轻神经元缺糖缺氧后损伤。     

RIP1/3对创伤性脑损伤后神经元的作用及其机制研究

目的 探讨RIP1/3对创伤性脑损伤(TBI)后神经元的影响及其作用机制。方法 将体外培养的皮层神经元分为4组:siRIP组、Nec-1预处理组、阴性质粒转染组和对照组。通过慢病毒转染法分别干涉RIP1、RIP3表达,建立离体TBI损伤模型,通过流式细胞学检测划伤后神经元存活情况。对体外培养的皮层神经元敲除RIP1/3,建立谷氨酸诱导神经元损伤模型,通过流式细胞学检测谷氨酸刺激后神经元存活情况。结果 siRIP组及Nec-1预处理组机械性损伤后神经元存活率高于阴性质粒转染组和对照组。Nec-1预处理组谷氨酸损伤后神经元存活率高于对照组,而siRIP组存活率与对照组和阴性转染组比较未见显著差异。结论 RIP1和RIP3可能对TBI诱导后神经元死亡有作用,而RIP1抑制剂Nec-1可能对TBI具有脑保护作用。RIP1/3与谷氨酸兴奋毒性诱导细胞死亡无关,而Nec-1对于谷氨酸损伤具有潜在保护作用,并可能存在特异性靶点。     

神经源性丝氨酸蛋白酶抑制剂在神经元缺氧复氧损伤中的保护作用

目的：观察组织型纤溶酶原激活物（tPA）对体外不同条件培养的神经元的损伤作用，探讨神经源性丝氨酸蛋白酶抑制剂（NSP）是否可以减轻tPA对神经元的损伤。方法：体外原代培养大鼠皮质神经元，建立缺氧复氧（H／R）模型。在不同的培养条件下使用不同的试剂干预方法。采用倒置相差显微镜观察免疫荧光染色后的细胞形态学变化、活细胞计数试剂盒测定神经元存活率、LDH释放实验测定细胞毒性和Tunel试剂盒测定细胞凋亡率。结果：tPA对正常神经元具有毒性损伤作用。经tPA干硕的H／R组与单纯H／R组相比，前者神经元损伤加重（P〈0．05）。NSP＋tPA联合干预的H／R组细胞损伤均轻于tPA干预的H／R组（P〈0．05）。结论：NSP可减轻tPA对神经元的毒性损伤而起到保护神经元的作用。     

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.     

Special Pharmacokinetic Considerations in Children

Summary: Pediatric patients have greater degrees of pharmacokinetic variability and unpredictability than adults. This variability results from the effects of pharmacogenetics, age and growth, prior and current comedication, and disease. Newborns with seizures have the least predictable dosage requirements, and their needs change as drug-eliminating mechanisms mature in the neonatal period. Infants have the highest relative capacities to eliminate antiepileptics of any age group and require the largest relative doses. In addition to age-related trends, children demonstrate the same drug-specific, pharmacokinetic phenomena that adults do, including nonlinear phenytoin elimination, nonlinear valproate binding, and autoinduction of carbamazepine. Intercurrent illness and drug interactions further modify the age-related pharmacokinetic patterns in children and make dosage requirements even more unpredictable. Recent studies have shown that febrile illness can affect drug elimination, sometimes decreasing drug levels by 50% or more. Intermittent treatment with benzodiazepines administered either orally or rectally can be an important adjunct and help minimize this type of problem for children with marginally controlled epilepsy. Intermittent benzodiazepines are also helpful for children who have febrile seizures and who need only occasional antiepileptic protection.