rhEPO对大鼠颅脑损伤早期脑微血管内皮细胞紧密连接的保护作用

目的探讨重组人促红细胞生成素(rhEPO)对颅脑损伤早期脑微血管内皮细胞的保护作用及机制。方法将81只SD大鼠按随机数字表法随机分为假手术组(n=9)、颅脑损伤(TBI)组(n=36)和rhEPO治疗组(n=36)。用Marmarou等人的方法制作大鼠颅脑损伤模型,分光光度仪测定各组大鼠脑组织伊文氏蓝(EB)含量,实时荧光定量PCR和免疫组织化学法检测闭合蛋白-5(claudin-5)、咬合蛋白(occludin)和胞质紧密粘连蛋白-1(ZO-1)的表达。结果伤后3h、24h、3d,与假手术组对比,TBI组大鼠脑组织EB含量明显升高(P<0.05),claudin-5、occludin和ZO-1mRNA及其蛋白的表达显著降低(P<0.05);与TBI组对比,rhEPO治疗组大鼠脑组织EB含量显著降低(P<0.05),claudin-5、occludin和ZO-1mRNA及其蛋白的表达显著升高(P<0.05)。结论颅脑损伤早期应用rhEPO,可能通过增加紧密连接相关蛋白claudin-5、occludin和ZO-1的表达而保护脑微血管内皮细胞。     

实验性脑出血后紧密连接蛋白JAM-1的分布与表达变化

目的 探讨紧密连接蛋白JAM-1在脑出血后的分布与表达变化及其重要意义.方法 128只雄性SD大鼠按随机数字表法分为正F常对照组(16 只)及脑出血组(112只,立体定向注射75 μL自体血到右侧尾状核制作脑出血模型),并选择脑出血后6h、12h、24 h、48h,3 d、7d、14d 为观察时间点(每个时间点16只).静脉注射伊文思蓝检测大鼠血脑屏障通透性;采用免疫荧光染色及实时荧光定量PCR分析血肿周围脑组织紧密连接蛋白JAM-1的分布及表达情况.结果 脑组织中伊文思蓝含量在脑出血后24 h、48 h、3d、7d时明显增高,与正常对照组比较差异有统计学意义(P＜0.05).免疫荧光染色检测到脑出血后12h、24 h,48 h时血管上JAM-1表达减弱;3d时呈不连续表达,同时在粘附于血管腔表面的白细胞上表达;7d时可见血肿周围大量JAM-1阳性细胞;荧光双染进一步发现JAM-1表达在ED-1阳性的巨噬细胞上.荧光定量PCR结果显示,脑出血后12h、24h、48h JAM-1 mRNA表达明显降低,与正常对照组比较差异有统计学意义(P＜0.05);7d时明显增高,与正常对照组比较差异有统计学意义(P＜0.05).结论 脑出血后紧密连接蛋白JAM-l的表达变化不仅参与血脑屏障的破坏,同时也参与了脑出血后炎症反应.     

急性脑出血血肿周围组织水通道蛋白-4表达与血脑屏障损伤的关系

目的研究急性脑出血血肿周围组织水通道蛋白-4表达与血脑屏障损伤之间的关系。方法采用立体定向注射无肝素自体动脉血制作大鼠脑出血模型,对脑含水量、血脑屏障损害进行观察,RT-PCR和免疫印迹检测水通道蛋白-4 mRNA和蛋白表达,同时观察参与紧密连接的咬合蛋白(occ lud in)和带状闭合蛋白-1(zona occ ludens-1,ZO-1)表达和水通道蛋白-4表达的相关关系。电镜下观察不同时间点血脑屏障和神经星形胶质细胞终足改变。结果电镜提示出血后6 h血脑屏障紧密连接未破坏,但神经胶质细胞终足已经肿胀。与假手术组相比,此时的脑出血血肿周围组织大鼠水通道蛋白-4表达增高(P<0.05):脑出血3 h水通道蛋白-4 mRNA开始增强(吸光度比值1.05±0.13),水通道蛋白-4蛋白6 h开始增强(0.62±0.05),第3天达峰值(1.46±0.02)。电镜和伊文蓝显示血脑屏障从12 h才开始明显损害[12 h脑组织伊文蓝含量为(12.91±0.64)μg/g],同时ZO-1和occ lud in蛋白表达减弱,分别为0.71±0.05和0.72±0.04。与水通道蛋白-4蛋白表达呈负相关。结论脑出血后水通道蛋白-4增高和血脑屏障损伤之间存在相关关系;水通道蛋白-4增高和星形胶质细胞肿胀可能是血脑屏障损伤的因素之一。     

脑出血后血脑屏障紧密连接蛋白occludin的表达变化

目的 探讨脑出血后血脑屏障微血管内皮细胞间紧密连接蛋白occludin的表达变化. 方法 将SD雄性大鼠按随机数字表法分为正常对照组和脑出血组,再按时间因素将脑出血组分为出血后6h、24h、48h、72h、7d、14d6个亚组.采用脑内注入自体血法制作脑出血模型.HE染色观察脑出血后血肿周围脑组织的形态学改变;透射电镜观察脑出血后血肿周围紧密连接的超微结构改变;免疫荧光染色检测脑出血后血肿周围紧密连接蛋白occludin的表达分布状况;定量RT-PCR检测脑出血后血肿周围脑组织中occludin mRNA的表达状况. 结果 与正常对照组相比,脑出血组血肿周围脑组织出现水肿,在48 h左右尤为明显,局部可见明显脑细胞坏死及炎细胞浸润.脑出血后血肿周围紧密连接发生明显破坏,内皮细胞间出现裂隙.免疫荧光染色结果显示:正常对照组紧密连接蛋白occludin呈强阳性表达.脑出血后6hoccludin的表达即开始下降,呈阳性表达;脑出血后24～72h occludin的表达维持在较低水平,呈弱阳性表达.定量RT-PCR结果显示:脑出血后血肿周围脑组织中occludin mRNA相对含量明显降低,在6～72 h持续维持在较低水平,与正常对照组比较差异均有统计学意义(P＜0.05). 结论 脑出血发生后,紧密连接蛋白occludin 的表达下降,这可能是脑出血发生后血脑屏障破坏及脑水肿发生发展的重要分子基础之一.     

黄体酮对脑缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的影响

目的探讨黄体酮对大鼠局灶性脑缺血再灌注后血脑屏障紧密连接蛋白ZO-1、occludin表达及血脑屏障通透性的影响。 方法将42只健康雄性SD大鼠按随机数字表法分为假手术组（6只）和缺血再灌注组,后者再按再灌注时间分为缺血2h再灌注3h、6h、12h、24 h、48 h及72h组（各6只）。缺血再灌注组用线栓法制备成大鼠大脑中动脉缺血再灌注模型。采用荧光分光光度法测定缺血侧脑组织中伊文氏蓝(EB)含量来评价血脑屏障的通透性,Western blotting法检测脑组织ZO-1和occludin的表达。取EB漏出最多组的时间点,增设黄体酮干预组和溶剂对照组（各6只）,与相同时间点的缺血再灌注组比较,观察黄体酮对ZO-1、occludin表达及血脑屏障通透性的影响。 结果 缺血2h再灌注3h时脑组织EB含量开始增加,再灌注24 h时达高峰;ZO-1、occludin的表达在缺血2h再灌注3h时开始下降,再灌注24 h时达最低。黄体酮干预组EB含量明显低于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。黄体酮干预组ZO-1和occludin的表达水平均明显高于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。 结论 黄体酮町抑制缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的降低,从而起到保护血脑屏障的作用。     

黄芪甲苷对脑缺血再灌注后血脑屏障的保护作用及occludin蛋白表达的影响

目的探讨黄芪甲苷对大鼠脑缺血再灌注后血脑屏障的保护作用及occludin蛋白表达的影响。方法SD大鼠72只,随机等分为4组,每组18只。A组为假手术组;B组为生理盐水对照组;C组为小剂量黄芪甲苷治疗组,D组为大剂量黄芪甲苷治疗组。采用干湿重法、分光光度计法及免疫组化法分别检测各组大鼠脑组织含水量、伊文氏蓝含量及occludin蛋白的表达水平。结果与A组相比,B组大鼠脑组织含水量、伊文氏蓝含量明显增多,occludin蛋白的表达明显减少(P0.01);与B组相比,C组和D组大鼠脑组织含水量、伊文氏蓝含量显著减少,occludin蛋白的表达显著增加(P0.05;C组与D组相比,大鼠脑组织含水量、伊文氏蓝含量及occludin蛋白的表达无显著差异(P0.05)。结论黄芪甲苷对脑缺血再灌注后血脑屏障具有保护作用,这可能与黄芪甲苷上调occludin蛋白的表达有关。     

黄芪甲苷对脑缺血再灌注后血脑屏障的保护作用及IL-1β含量、MMP-9蛋白表达的影响

目的 探讨黄芪甲苷对大鼠脑缺血再灌注后血脑屏障的保护作用及其机制。方法 将SD大鼠72只随机等分为4组:假手术组、生理盐水对照组、小剂量黄芪甲苷治疗组(10 mg/kg)和大剂量黄芪甲苷治疗组(20 mg/kg),采用分光光度计法、酶联免疫吸附法及免疫组化法分别检测各组大鼠脑组织伊文氏蓝、IL-1β含量及MMP-9蛋白的表达水平。结果 与假手术组相比,生理盐水对照组脑组织伊文氏蓝含量明显增多、IL-1β含量显著增高、MMP-9蛋白的表达明显增强(P<0.01); 与生理盐水对照组相比,小剂量黄芪甲苷治疗组及大剂量黄芪甲苷治疗组脑组织伊文氏蓝含量均显著减少、IL-1β含量明显降低、MMP-9蛋白表达明显减弱(P<0.05); 小剂量黄芪甲苷治疗组与大剂量黄芪甲苷治疗组相比,伊文氏蓝、IL-1β含量及MMP-9蛋白表达无显著差异(P>0.05)。结论 黄芪甲苷对脑缺血再灌注后血脑屏障具有保护作用,这可能与其下调IL-1β含量、抑制MMP-9蛋白的表达有关。     

自体骨髓单核细胞移植对脑梗死的治疗作用

目的 观察自体骨髓单核细胞移植对大脑中动脉栓塞(MCAO)大鼠脑梗死体积、血脑屏障破坏程度及行为学评分等的影响,评价骨髓单核细胞移植对脑梗死的治疗价值. 方法 将72只成年雄性SD大鼠按完全随机数字表法分为假手术组、模型组、生理盐水组及骨髓单核细胞移植组,后三组采用线栓法制作MCAO大鼠模型.采用梯度离心法分离大鼠股骨骨髓腔中的单核细胞,并通过流式细胞术检测CD45+细胞比例.将分离得到的自体骨髓单核细胞经颈静脉途径移植到骨髓单核细胞移植组大鼠体内,生理盐水组大鼠给予等体积的生理盐水注射.造模后72 h时伊文氏蓝染色及脑组织含水量检测显示血脑屏障的破坏情况,造模后1d、3d、7d时通过Zea-Longer评分评价大鼠神经功能变化,最后一次Zea-Longer评分结束后TTC染色检测大鼠脑梗死体积. 结果 流式细胞术检测发现骨髓单核细胞中CD45+细胞的比例为91.2％.脑梗死体积、伊文氏蓝含量及脑组织含水量在模型组与生理盐水组中均较高,且相互比较差异无统计学意义(P＞0.05)；骨髓单核细胞移植组脑梗死体积、伊文氏蓝含量及脑组织含水量均明显下降,较模型组与生理盐水组差异均有统计学意义(P＜0.05).Zea-Longer评分显示,除假手术组外,其余大鼠均有不同程度的神经功能缺损；造模后7d时骨髓单核细胞移植组神经功能评分明显改善,与模型组及生理盐水组比较差异均有统计学意义(P＜0.05). 结论 骨髓单核细胞分离过程简单,利用其移植治疗脑梗死可以显著减轻脑损伤程度并促进神经功能恢复,对脑梗死具有很高的治疗价值.     

黄体酮对局灶性脑缺血再灌注大鼠水通道蛋白-4表达及血脑屏障通透性的影响

目的研究黄体酮对大鼠局灶性脑缺血再灌注后脑组织内水通道蛋白-4(AQP4)的表达及血脑屏障通透性的影响。方法健康雄性SD大鼠96只,随机分为4大组:假手术组、手术组、溶剂治疗组和黄体酮治疗组,建立大脑中动脉栓塞再灌注(MCAO/R)模型,分别在缺血2h再灌注6h、1d、3d、5d 4个时间点将大鼠麻醉后断头取脑,采用Western blot法、伊文氏蓝(EB)渗出量及干湿重法分别测定脑组织AQP4的表达、血脑屏障的通透性及脑含水量。结果手术组AQP4的表达、EB的含量及脑组织含水量明显高于假手术组;黄体酮组AQP4的表达、EB的含量及脑组织含水量较手术组及溶剂组明显降低,差异有统计学意义。结论黄体酮可以降低缺血再灌注大鼠脑组织AQP4的表达,从而降低血脑屏障的通透性,减轻脑水肿。     

缺血后处理对大鼠脑缺血再灌注后紧密连接的保护作用及ZO-1蛋白表达的影响

目的观察缺血后处理对脑缺血再灌注后紧密连接的保护作用及相关蛋白ZO-1表达的影响。方法 45只Wistar雄性大鼠随机分为假手术(Sham)组、缺血再灌注(I/R)组、缺血后处理(IP)组。采用线栓法建立大鼠大脑中动脉缺血模型,脑缺血2 h后,I/R组予再灌注,IP组给予缺血后处理之后予再灌注。于脑缺血再灌注后24 h行TTC染色观察脑梗死体积,应用电镜观察紧密连接蛋白结构改变,Western blot观察ZO-1蛋白表达的变化。结果 IP组脑梗死体积明显小于I/R组,与Sham组相比,I/R组血脑屏障紧密连接开放,ZO-1蛋白表达明显减少;与I/R组比较,IP组紧密连接开放程度减轻及ZO-1表达增加。结论缺血后处理减小脑梗死体积;缺血后处理能够减轻紧密连接破坏,其保护机制可能与ZO-1蛋白表达增加有关。     

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.