单剂量匹罗卡品诱导小鼠癫痫及其海马神经损伤的研究

目的 探索不同剂量匹罗卡品单次腹腔注射对诱发小鼠癫痫及对海马损伤的影响,为癫痫建模提供参考。方法将100只健康的C57BL/6小鼠随机分为正常对照组、3个不同剂量匹罗卡品(250 mg/kg、290 mg/kg、350 mg/kg)腹腔注射组。比较3组的建模成功率、死亡率,通过免疫组化染色观察神经元和胶质细胞的数量变化,分析海马区组织的病变情况。结果 匹罗卡品250 mg/kg组小鼠的Racine评分4级以上发作率为12%,无死亡; 290 mg/kg组小鼠的4级以上发作率为70%,死亡率为32%; 350 mg/kg组小鼠的4级以上发作率为86%,死亡率为56%。免疫组化染色结果显示:与正常对照组相比,3个不同剂量匹罗卡品组小鼠诱发癫痫后,神经元数量均显著减少,小胶质细胞和星形胶质细胞数量均显著增加;但250 mg/kg组小鼠的活化小胶质细胞数量无显著变化,而其他两组显著增多。结论 匹罗卡品290 mg/kg单次注射诱发小鼠癫痫模型的成功率较高、死亡率较低,且海马区损伤明显,可以作为癫痫小鼠建模的参考方法。     

改良锂-匹罗卡品大鼠癫痫模型的制作及性别特点

目的 建立稳定可靠的颞叶癫痫动物模型，摸索并证实建模规律。方法 选用56只健康SD大鼠，雌雄近半，随机分配为试验组48只，对照组8只。氯化锂3mmol／kg腹腔注射预处理：18-20h后首剂注射匹罗卡品30mg／kg，此后每隔30分钟注射10mg／kg追加剂量，极量为60mg／kg，直至出现癫痫持续状态（SE）；SE1h后地西泮10mg／kg止痉，2h后注射生理盐水5mL／只，2／d．连续2d；SE后次日起灌胃鸡蛋牛奶糊7mL／只，2／d，连续6d；观察大鼠行为学改变。对照组大鼠除用等量生理盐水代替匹罗卡品外，其余处理措施均相同。结果大鼠成功点燃44只，病死10只．造模总成功只数为34。雌雄大鼠点燃只数相近．雄鼠病死只数低于雌鼠。结论中等剂量首剂．不超过3次小剂量累加腹腔注射匹罗卡品造模简易有效，可操作性强，效应匀齐。改良护理方法可明显降低病死率．雄鼠较雌鼠更适于造模。     

乌司他丁对氯化锂-匹罗卡品诱发癫痫大鼠脑保护作用及其机制研究

目的 研究乌司他丁对癫痫发作时大脑的保护作用及其机制。方法 通过对Wistar大鼠建立癫痫模型,建模成功后并对其进行4种分组处理,即A组注射生理盐水; B组注射卡马西平; C组注射卡马西平和少量乌司他丁(10 000 U/kg); D组注射卡马西平和大量乌司他丁(70 000 U/kg); 测量每组达到惊厥标准后24、48、72、96 h不同时间段的S100β蛋白和神经烯醇化酶(NSE)水及96 h后肿瘤坏死因子-α(TNF-α)与IL-1β水平; 通过免疫组化检测caspase-3、bax与bcl-2阳性细胞数; 观察神经元凋亡情况; 用水迷宫检测大鼠的认知功能,通过模型组与空白对照组和模型组间对比分析乌司他丁对癫痫大鼠的影响; 空白对照组全程注射等量生理盐水。结果 注射乌司他丁能够降低S100β和NSE水平,并且大剂量乌司他丁效果更显著(P<0.05); 乌司他丁对TNF-α与IL-1β表达进行抑制,且大剂量乌司他丁效果更显著(P<0.05); 注射乌司他丁后caspase-3与bax的阳性细胞减少而bcl-2的阳性细胞增加,且大剂量乌司他丁效果更显著(P<0.05); 注射乌司他丁能够减少海马CA3区神经元凋亡,且大剂量乌司他丁效果更显著(P<0.05); 注射乌司他丁能够改善癫痫大鼠的认知功能,且大剂量乌司他丁效果更显著(P<0.05)。结论 乌司他丁可能是通过抑制炎症因子的表达和caspase-3与bax的激活,促进bcl-2蛋白激活,并减少神经元凋亡来对癫痫大鼠的大脑进行保护。     

匹罗卡品致疒间大鼠海马GAP-43与TrkB基因表达及其意义

目的探讨生长相关蛋白(GAP-43)和脑源性神经营养因子(BDNF)受体TrkB基因在匹罗卡品致疒间大鼠海马的表达及其意义.方法应用原位杂交组织化学方法研究匹罗卡品(PILO)致疒间大鼠海马GAP-43及TrkB mRNA表达的变化.结果匹罗卡品致癫疒间持续状态后3～6小时,海马齿状回颗粒细胞、CA3区及CA1区锥体细胞层TrkB mRNA表达显著高于对照组(P<0.01或0.05);在慢性期第7～30天,呈现第2次表达增强.致疒间后6～12小时,正常状态下并不表达GAP-43的大鼠海马颗粒细胞其GAP-43 mRNA表达较对照组显著增高(P<0.01),24小时～7天表达减少,在癫疒间慢性期表达再次高于对照组.结论 GAP-43及TrkB是颞叶癫疒间病理基础--海马苔藓纤维出芽的重要分子机制;BDNF对苔藓纤维的作用部分是通过GAP-43实现的.     

匹罗卡品癫痫大鼠海马TrkB受体表达与苔藓纤维突触重建的关系

目的 探讨匹罗卡品诱导慢性癫痫大鼠海马齿状颗粒细胞苔藓纤维突触重建与神经营养素受体TrkB表达的关系。方法 取匹罗卡品诱导大鼠急性癫痫持续状态及慢性自发性颞叶癫痫发作期大鼠脑片，用免疫组织化学方法检测TrkB及突触体素(P38，一种突触形成标志物)在大鼠海马的表达。结果 急性癫痫持续状态诱导颗粒细胞表达TrkB一过性增高，第2次表达高峰呈现在7～30d；P38免疫反应性在齿状回内分子层则呈进行性增加，与neo-Timm显示的异常苔藓纤维出芽相一致。结论 TrkB受体激活有助于海马齿状回苔藓纤维轴突生长及突触形成从而有利于颞叶癫痫的发生。     

Fluoro-Jade C揭示匹罗卡品模型中梨状皮质神经元变性

目的应用Fluoro-Jade C(FJC)染色方法在小鼠匹罗卡品癫痫模型中检测梨状皮质结构中神经元的变性情况,以了解梨状皮质结构在慢性颞叶癫痫发生中的病理变化和癫痫反复发作的神经基础。方法雄性昆明小鼠10只(对照组5只,匹罗卡品处理组5只)。处理组在癫痫持续状态后3d处死处理组小鼠。在梨状皮质水平切制冠状切片,行FJC染色,在荧光显微镜下观察FJC阳性细胞的形态和在梨状皮质中的整体分布情况。结果在处理组,FJC染色的脑切片上可以很清楚地看到呈亮黄绿色荧光的FJC阳性细胞,呈神经元形态,胞体和突起均清晰显示。在梨状皮质和梨状内核内出现大量FJC阳性细胞,而对照组未见。结论在小鼠匹罗卡品癫痫模型中运用FJC染色技术显示梨状皮质内发生了大量神经元变性,此研究有利于更好地理解颞叶癫痫中中枢神经系统所发生的长期病理变化和自发反复发作的癫痫机制。     

氯化锂-匹罗卡品致痫大鼠海马EphA5及ephrinA3的表达及意义

目的探讨颞叶癫痫发作后海马EphA5及ephrinA3基因的表达变化和轴突出芽的关系。方法建立氯化锂-匹罗卡品颞叶癫痫大鼠模型,利用原位杂交方法检测致痫后12h、24h、7d、15d、30d、60d海马CA3区、CA1区EphA5及ephrinA3 mRNA的表达,快速Golgi染色观察CA1区的轴突出芽。结果致痫后,EphA5 mRNA在CA3区表达下调,ephrinA3 mRNA在CA1区表达下调,均在7d降至最低点,与对照组相比差异有显著意义(P<0.01),此后逐渐回升,但15d时仍低于对照组(P<0.05),在30d和60d与对照组相比差异无统计学意义(P>0.05)。快速Golgi染色显示,对照组大鼠CA1区轴突走行正常,匹罗卡品致大鼠SE后7dCA1区锥体细胞层出现显著增多的轴突染色。结论CA3区的EphA5和CA1区的ephrinA3的表达下调可能与CA1区的轴突出芽、突触重建有关。     

建立杏仁核电刺激慢点燃和匹罗卡品化学点燃耐药性颞叶癫痫模型并对比癫痫发作和海马超微结构的变化

目的用两种方法建立颞叶耐药癫痫模型,探讨哪种方法更适合建立多药耐药颞叶癫痫模型。方法选用SD大鼠130只,10只作为正常组,120只分别制作杏仁核和匹罗卡品模型,模型成功后用抗癫痫药苯巴比妥和苯妥英钠或卡马西平进行筛选,分别选择10只杏仁核模型和匹罗卡品模型比较两种方法建立的模型癫痫发作持续时间、发作频率、发作级别、脑电图及电镜下超微结构的变化。结果成功制作杏仁核模型31只,匹罗卡品模型29只,匹罗卡品模型癫痫发作频率(2. 09±0. 044)高于杏仁核组(1. 01±0. 037),持续时间(61. 37±4. 22)长于杏仁核组(43. 16±5. 91),而癫痫发作的级别无明显差异;经过耐药性癫痫模型的筛选,选出杏仁核耐药模型8只,匹罗卡品耐药模型12只。匹罗卡品模型与杏仁核模型相比脑电频率更高,波幅增宽,超微结构的损伤更严重。结论匹罗卡品模型自发性率高,耐药率高,脑电变化明显,超微结构损伤重,更适合耐药性癫痫机制的研究。     

银杏叶提取物对锂-匹罗卡品癫痫大鼠海马结构c-Fos蛋白表达的影响

目的:研究银杏叶提取物(GBE)对癫痫的干预作用及其对癫痫大鼠海马结构c-Fos蛋白表达的影响。方 法:采用锂-匹罗卡品癫痫(LPS)大鼠模型。观察动物行为改变,应用免疫组化方法观察海马结构c-Fos蛋白的表达。结 果:GBE对该模型癫痫发作的预防有效率和治疗有效率分别为94．44％和35．48％。与LPS组相比,GBE预处理组的4 级以上发作率和发作分级明显下降;站立发作潜伏期延长。注射匹罗卡品后3小时,生理盐水对照组、GBE单独给药组 和GBE预处理组大鼠海马结构c-Fos蛋白表达阴性。LPS组和GBE急性给药组大鼠海马结构c-Fos蛋白表达阳性。 结论:GBE预处理和急性给药两种给药模式均有一定的抗LPS作用,前者明显优于后者。GBE抗LPS作用的机制可能 与GBE能抑制海马结构c-Fos蛋白表达有关。     

MicroRNA-132拮抗剂两种不同注射方式在匹罗卡品诱导的幼年大鼠癫痫急性期的差异

目的探讨microRNA-132拮抗剂对氯化锂-匹罗卡品诱导的幼年SD大鼠内侧颞叶癫痫(mesial temporal lobe epilepsy,MTLE)模型急性期的影响。方法实验分为4组:microRNA-132拮抗剂侧脑室置管组;microRNA-132拮抗剂阴性对照置管组;microRNA-132拮抗剂直接注射组;microRNA-132拮抗剂阴性对照直接侧脑室注射组;实验各组药物干预在造模前24 h进行。利用氯化锂-匹罗卡品建立SD大鼠MTLE模型,通过行为学观察各组大鼠癫痫持续状态发作潜伏时长,Racine评分观察各组大鼠抽搐发作的严重程度,脑电图监测癫痫样放电的频率及波幅,并统计实验各组大鼠诱导SE的成功率及24 h后的死亡率。结果在实验各组中,建模成功率无显著性差异,microRNA-132拮抗剂直接注射组幼年SD大鼠造模以后达到SE的潜伏时较其阴性对照组明显延长、癫痫发作的Racine评分明显下降、脑电图结果显示痫样放电的幅度及频率显著下降,死亡率降低,结果有统计学意义(P0.05)。MicroRNA-132拮抗剂侧脑室置管注射组与microRNA-132拮抗剂侧脑室直接注射组结果无统计学差异(P0.05)。结论 microRNA-132拮抗剂预处理SD大鼠能明显延长氯化锂-匹罗卡品诱导的SD幼年大鼠癫痫发作的潜伏期,减轻急性期抽搐严重程度及大脑样痫放电,降低大鼠癫痫持续状态后的死亡率。提示microRNA-132拮抗剂对氯化锂-匹罗卡品诱导的幼年SD大鼠MTLE的发生具有抑制作用,抑制microRNA-132有可能成为癫痫持续状态药物治疗的潜在靶点和新方向。     

Surgical outcome and predictive factors in adult patients with intractable epilepsy and focal cortical dysplasia

OBJECTIVES: To determine the surgical outcome and prognostic factors in adult patients with intractable epilepsy and focal cortical dysplasia (FCD). MATERIALS AND METHODS: We retrospectively studied the operative outcome in 21 consecutive adult patients with FCD who underwent surgical treatment for intractable partial epilepsy. RESULTS: The mean age at surgery was 32.7 years (range, 18-58 years). The median post-operative follow-up was 2.5 years. The FCD was extratemporal in 11 patients, involved the temporal lobe in 10 patients, and was multilobar in eight patients. Eleven patients (52%) were rendered seizure-free, four patients (19%) had >95% reduction in seizures, and two patients (10%) had an 80-94% reduction in seizures. A seizure-free outcome was associated with shorter duration of epilepsy (P = 0.02). CONCLUSION: Adult patients with FCD may be candidates for surgical treatment of intractable partial epilepsy. Most individuals have neocortical, extrahippocampal seizures and approximately 50% of patients are rendered seizure-free.     

海马神经元激活物致疒间大鼠大脑皮质和海马γ-氨基丁酸受体的表达

目的 观察大鼠海马神经元在激活物作用下γ-氨基丁酸（GABA。）受体的表达,进一步探讨癫痫发病机制。方法 将大鼠海马神经尢被戊四氮（PTZ）作用后的激活物（实验组）及无血清培养基（对照组）注入大鼠侧脑室后观察其行为、脑电图（EEG）及脑组织GABA,受体表达的变化。结果 实验组大鼠注射后15～30min出现Ⅱ～Ⅲ级惊厥反应,EEG呈短程中高幅尖波、尖慢复合波;对照组的行为及EEG未见异常;各时点实验组大鼠脑组织GABA、免疫反应阳性细胞表达明显低于对照组（均P〈0．05）,对照组GABA,免疫反应阳性细胞广泛分布于大脑皮质、海马回、齿状回。结论 海马神经元激活物具有明显致痫作用,其致痫效应与GABAA受体含量下降有关。     

Calcified cysticercotic lesions and intractable epilepsy: a cross sectional study of 512 patients

Background

Neurocysticercosis is a major cause of epilepsy in developing countries and is endemic in Brazil. To test the hypothesis that the aetiological profile of patients with intractable epilepsy in Brazil includes neurocysticercosis, we conducted a cross sectional study investigating the aetiology of intractable epilepsy.

Methods

A total of 512 patients evaluated at the outpatient clinic for intractable epilepsy at the Ribeirão Preto School of Medicine were included in the survey. Medical intractability was determined on the basis of seizure incidence and severity, and response to appropriate epilepsy management. Neuroimaging included brain CT with non‐contrasted and contrasted phases and high resolution MRI. Patients were divided into neurocysticercosis and non‐neurocysticercosis groups according to previous diagnostic criteria.

Results

The most common epileptogenic lesions were mesial temporal sclerosis (MTS; 56.0%), malformations of cortical development (12.1%), and brain tumours (9.9%). Neuroimaging was normal in 8.7% of patients. Calcifications were found in 27% of patients and were significantly more common in patients with MTS than in those without MTS (p<0.001). Isolated neurocysticercosis was found in only eight patients (1.56%).

Conclusions

These data suggest that neurocysticercosis is an uncommon cause of intractable epilepsy, even in an endemic region such as Brazil, and that it may only represent a coexistent pathology. However, an analysis of our findings reveals that neurocysticercosis was more common in patients with MTS. This finding could suggest either that there is a cause‐effect relationship between MTS and neurocysticercosis, or that MTS and neurocysticercosis co‐vary with a missing variable, such as socio‐economic status.     

难治性癫痫的致痫灶定位及手术治疗研究

目的评价难治性癫痫的致痫灶定位方法和皮层电极监测下致痫灶切除,加行多处软脑膜下横纤维切断术(MST)治疗癫痫的疗效。方法对47例难治性癫痫病人的致痫灶,采用CT MRI EEG 单光子发射计算机体层摄影(SPECT) 皮层脑电脑(ECoG)联合检测定位。对检出的阳性病灶在皮层电极监测显微镜下行致痫灶切除,切除后监测仍有癫痫波者加行MST;致痫灶位于重要功能区者单行MST。结果致痫灶阳性检出率86%。皮层电极检测显微镜下致痫灶切除加MST,术后91%的病人癫痫发作停止,半年后约15%的病人复发,但症状较术前减轻,持续时间较术前短。结论CT MRI EEG SPECT ECoG联合检测,对手术定位具有较高价值。皮层电极监测下致痫灶切除术及MST创伤轻微、效果比较可靠、治愈率高、并发症少、复发率低。病灶及致痫灶的不完全切除和形成皮层软化及疤痕,可能是导致癫痫复发的重要原因。     

Influence of mTOR signaling pathway on ketamine-induced injuries in the hippocampal neurons of rats

Objective: To explore the influences of mammalian target of rapamycin (mTOR) signaling pathway on ketamine-induced apoptosis, oxidative stress and Ca²⁺ concentration in the hippocampal neurons of rats.

Methods: The primary hippocampal neurons isolated from fetal Sprague Dawley rats were treated with ketamine (0, 50, 100 and 500 μM) for 4 days to observe its effect on mTOR signaling pathway and apoptosis of rat hippocampal neurons. Then, the hippocampal neurons were divided into C (Control), R (Rapamycin, an inhibitor of mTOR signaling pathway), K (Ketamine) and R + K (Rapamycin + Ketamine) groups to detect the apoptosis, reactive oxygen species (ROS) production, and Ca²⁺ concentration via the terminal transferase uridyl nick end labelling (TUNEL) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) method and Fluo-3 acetoxymethyl ester (Fluo-3AM) staining, respectively. The expressions of mTOR signaling pathway and apoptosis-related proteins in hippocampal neurons were examined by qRT-PCR and Western blot.

Results: Ketamine could dose-dependently promote the apoptosis of rat hippocampal neurons with upregulation of p-mTOR and its downstream regulators (p-4E-BP-1 and p-p70S6K). However, ketamine-induced apoptosis in hippocampal neurons was reversed significantly by the administration of rapamycin, as evident by the decrease in expressions of pro-apoptotic proteins (Bax and cleaved Caspase-3) and the increase in anti-apoptotic protein (Bcl-2). Meanwhile, the ROS generation and Ca²⁺ concentration was inhibited accompanied with reduced malonildialdehyde levels but elevated superoxide and glutathione peroxidase activities.

Conclusion: Inhibition of mTOR signaling pathway protected rat hippocampal neurons from ketamine-induced injuries via reducing apoptosis, oxidative stress, as well as Ca²⁺ concentration.

Abbreviations: mTOR: mammalian target of rapamycin; SD: Sprague-Dawley; SPF: Specific-pathogen free; ROS: reactive oxygen species; TUNEL: terminal transferase uridyl nick end labelling; DCFH-DA: Dichloro-dihydro-fluorescein diacetate; Fluo-3A: Fluo-3 acetoxymethyl ester; NMDAR: non-competitive N-methyl-D-aspartame glutamate receptor; 4E-BP1: 4E binding protein 1; p70S6K: p70 S6 Kinase; PCR: Polymerase chain reaction; MDA: malonildialdehyde; GSH-PX: glutathione peroxidase; ANOVA: One-way Analysis of Variance.     

Cerebral blood flow in children with intractable epilepsy

A good correlation of Doppler internal carotid blood velocity determinations with hemispheric blood flow measurements by the 133-xenon inhalation method was demonstrated in 14 epileptic patients without abnormal CT findings. The highest correlation was seen between the flow of gray matter (F1) and the internal carotid end-diastolic velocity (d) (left side r = 0.841, right side r = 0.817). As end-diastolic velocity (d) well correlated with the value obtained by the 133-xenon inhalation method, the d value was compared between 77 healthy children and 13 patients with intractable epilepsy. The mean d value of both internal carotid arteries in patients was 16.7 +/- 2.9 mm (mean +/- SD), and that of healthy children 20.9 +/- 4.3 mm, the difference being statistically significant. The low cerebral blood flow in patients might be due to multiple antiepileptic drugs administered and/or mental retardation and cerebral hypofunction related to seizures.     

无影像学病灶顽固性癫癎病人的手术治疗

目的探讨脑MRI与CT为阴性的顽固性癎癫病人的手术治疗及术前评估时应注意的问题。方法回顾性分析MRI及CT为阴性表现的22例癫癎手术病人的资料,按照Engel疗效分级标准,将其效果分为满意组(10例)与非满意组(12例),对两组的术前检查情况进行对比。结果手术后整体满意率为45%。发作间期头皮EEG癫癎波局限在单一脑叶者,满意组中多于非满意组;颅内电极发作期脑电图局限性起源者,满意组中多于非满意组;单光子发射断层显像(SPECT)报告结果与手术部位关系各指标比较,两组无显著性差异;单脑叶切除术者,满意组中多于非满意组。结论MRI为阴性的癫癎手术效果不如病灶性癫癎。术前头皮视频脑电图(VEEG)发作间期性波局限在单一脑叶者治疗效果优于非局限者。手术前评估除重视各项非侵袭性检查外,颅内电极检查往往是不可缺少的。发作间期SPECT目前还不能做为一项决定性的定位手段。多脑叶切除手术效果不优于单脑叶切除,手术的关键是致灶的彻底切除。     

癫痫的放射外科

立体定向放射外科治疗难治性癫痫早有报道，具有定位精确，对周围组织损伤小，疗效好，安全、无创等优点。但对致痫灶的定位，照射剂量的选择及机理的研究目前尚无定论。近年来，采用PET、MEG等综合定位，低剂量照射靶区的治疗方法得到肯定。