MDR-1和GFAP蛋白在难治性癫痫脑组织的表达

目的：观察不同病因的难治性癫痫手术切除脑组织中多药耐药基因蛋白（MDR-1）和胶质纤维酸性蛋白（GFAP）的表达。方法：在对22例难治性癫痫临床病理资料分析的基础上，应用免疫组化和免疫组化双标技术观察脑组织中MDR-1和GFAP蛋白的表达情况。结果：反应性胶质细胞增生是难治性癫痫共同的病理学特征。MDR-1蛋白的表达主要在一些增生性星形胶质细胞和毛细血管壁周围结构，而寡突胶质细胞、小胶质细胞及正常的神经元内无MDR-1蛋白的表达；增生性星形胶质细胞内MDR-1与GFAP具有共存现象。结论：在难治性癫痫的反应必脑胶质细胞内同时具有GFAP和MDR-1蛋白的高表达和共存。     

药物难治性癫痫患者脑内多药耐药相关蛋白1表达的研究

目的 研究药物难治性癫痫患者脑内皮层多药耐药相关蛋白1（multidrug resistant-associated protein 1，MRP1）表达的情况。方法 选择12例药物难治性癫痫患者癫痫切除灶与12例正常对照脑组织标本．用逆转录聚合酶链反应（RT-PCR）、免疫组化及免疫蛋白印记（Western blot）方法，分析比较MRP1基因在各组的表达。结果 药物难治性癫痫患者组脑内MRP1的表达显著高于正常对照组（P〈0．01）。在癫痫病灶内广泛分布的MRP1免疫阳性细胞主要为毛细血管内皮细胞和星形胶质细胞。结论 脑内高表达的MRP1参与了难治性癫痫的耐药机制。     

难治性癫痫大鼠脑内多药耐药相关蛋白1表达的研究

目的 研究杏仁核电刺激点燃的难治性癫痫大鼠脑内多药耐药相关蛋白1（multidrug resistant-associated protein 1 MRP1）表达的情况.方法 建立杏仁核电刺激点燃的难治性癫痫大鼠模型,用免疫组织化学及免疫蛋白印记（western blot）的方法,分析比较MRP1蛋白在癫痫模型组与正常对照组的表达.结果 药物难治性癫痫大鼠组脑内多药耐药相关蛋白的表达显著高于正常对照组（P〈0.01）.在癫痫大鼠脑内广泛分布的MRP1免疫阳性细胞主要为毛细血管内皮细胞和星形胶质细胞.结论 癫痫大鼠脑内高表达的MRP1参与了难治性癫痫的耐药机制.     

多药耐药相关蛋白-1在难治性颞叶癫痫患者脑组织中的表达及丙磺舒的干预作用

目的 观察难治性颞叶癫痫患者脑组织中多药耐药相关蛋白1( MRPl)及应用MRP1拮抗剂丙磺舒干预后的表达,探讨MRP1与难治性颞叶癫痫多药耐药的关系.方法 应用免疫组化检测难治性颞叶癫痫患者脑组织实验组和对照组MRP1的表达情况,同时应用免疫蛋白印记(Western blot)方法检测实验组、丙磺舒干预组和对照组MRP1的表达.结果 免疫组化结果显示MRP1在难治性颞叶癫痫患者脑组织中表达增强,与对照组比较差异有显著性(P＜0.05).Western blot结果显示丙磺舒干预组MRP1蛋白水平较实验组明显较少,差异有显著性(P＜0.05).结论 脑内高表达的MRP1参与难治性颞叶癫痫的耐药机制,丙磺舒可以降低脑组织内MRP1的表达.     

多药耐药相关蛋白与难治性癫痫

难治性癫痫的发病机制尚不完全清楚,近年来发现在难治性癫痫患者病灶脑组织可检测出多药耐药相关蛋白(MRP)的过度表达,表明MRP与难治性癫痫耐药关系密切。MRP的过度表达受多种因素影响,其可能通过降低抗癫痫药物在血脑屏障的通透性,减少药物的有效浓度,从而引起抗癫痫药物耐药发生。鉴于MRP在难治性癫痫多药耐药中可能的重要作用,筛选非MRP底物的抗癫痫药物和选用适合长期服用的特异性强﹑安全性高﹑毒副作用性小的MRP抑制剂,可能会为今后临床治疗难治性癫痫翻开新的一页。     

GFAP和Fos蛋白在戊四氮致痫大鼠前脑中的表达变化

目的 研究大鼠在戊四氮导致癫痫发作时前脑内星形胶质细胞和神经元的形态学反应及其相互关系。方法 应用免疫组织化学单标记法分别显示前脑内GFAP和Fos蛋白表达的时间规律，并用免疫组织化学双重标记显示GFAP和Fos蛋白表达的相互关系。结果 在戊四氮导致大鼠癫痫发作早期，前脑的星形胶质细胞被激活，细胞体积增大，突起粗大，GFAP表达阳性，随着存活时间的变化，星形胶质细胞的反应经历先逐渐升高后降低的过程。被激活的星形胶质细胞和神经元表达Fos蛋白阳性，也呈现逐渐升高又降低的变化；另外，GFAP阳性星形胶质细胞和Fos阳性神经元在前脑主要分布在大脑皮层、海马、杏仁核等部位，二者的分布特征基本一致。结论 星形胶质细胞可能和神经元一起参与了戊四氮所致癫痫发作的变化。     

难治性癫痫患者脑组织穹隆体主蛋白表达的研究

目的探讨难治性癫痫患者脑组织穹隆体主蛋白(MVP)表达的改变。方法应用免疫组化、免疫荧光双标染色及Western blot方法检测38例难治性癫痫患者(癫痫组)手术切除的额叶致痫灶皮质及15例重型颅脑损伤患者(对照组)开颅减压术切除的额叶皮质中MVP的表达水平,并进行分析比较。结果对照组额叶皮质仅有MVP微量表达。癫痫组额叶皮质神经元MVP表达(主要在胞浆中和核膜上)均阳性,其MVP表达水平显著高于对照组(P0.05);同部位神经胶质细胞无MVP表达。结论药物难治性癫痫患者脑组织神经元的MVP表达增高,这可能是其对抗癫痫药物产生耐药的机制之一。     

红藻氨酸致痫大鼠海马Fos和GFAP的共同表达

目的 研究红藻氨酸（kainic acid,KA)诱导大鼠癫痫发作后海马（hippocampus,HI)内神经元和星形胶质细胞的时空效应性反应变化。方法 大鼠侧脑室内注射KA，用抗即刻早期基因Fos蛋白和抗胶质原纤维酸性蛋白（GFAP）的双重免疫荧光组织化学方法结合激光共聚焦显微镜技术，显示痫性发作后HI同一部位内反应性神经元与星形胶质细胞的分布。结果 KA诱导大鼠癫痫发作，HI内的Fos阳性神经元和GFAP阳性星形胶质细胞明显增多。两分布范围基本一致，且癫痫诱发30min后GFAP开始增多，1h达高峰；1h后Fos阳性产物开始增多；2h达高峰；部分Fos阳性神经元周围有GFAP免疫反应产物包绕，显示反应性神经元（Fos阳性）与反应性星形胶质细胞（GFAP阳性）之间关系密切。结论 HI内的神经元和星形胶质细胞与癫痫发作直接相关且存在相互关系。可能共同参与癫痫的发生及其调节。     

红藻氨酸诱发大鼠复杂部分性癫痫发作的海马神经元和星形胶质细胞反应及相互关系

目的：观察大鼠癫痫发作后海鸟内神经元与星形胶质细胞反应变化的时空效应及相互关系。方法：以红藻氨酸诱发的大鼠复杂部分性癫痂发作为模型，利用免疫组织化学法，在原位显示癫痫发作后15、30、60、90、120、180min6个时间点海马神经元Fos蛋白及星形胶质细胞内胶质原纤维酸性蛋白（GFAP）的表达变化、相互关系及分布规律。结果：致痫后15min海马内GFAP表达开始增多，60min达高峰。Fos阳性神经元在癞痴诱发后30min开始出现，120min达高峰。海马内GFAP阳性细胞与Fos阳性神经元分布规律基本一致。结论：在癫痫病理状态下，海马内星形胶质细胞的反应略早于神经元，两者之间分布呈平行关系，它们之间可能存在着复杂的信息通讯，以复合体的形式其同对各种病理生理刺激作出反应。     

难治性癫癎患者脑组织中5种耐药基因产物的同步表达及临床意义

目的　观察难治性癫患者脑组织中 5种耐药基因产物的同步表达 ,探索其临床意义。方法　 17例难治性癫患者分为长 (≥ 10年 )、短病程 (<10年 )两组 ,测定两组患者术后脑组织中P 糖蛋白 (Pgp)、多药耐药相关蛋白 (MRP)、谷胱甘肽硫转移酶 π(GST π)、肺耐药相关蛋白 (LRP)及拓扑异构酶Ⅱ (Topo Ⅱ )的表达 ,并与对照组比较。结果　短病程组患者胶质细胞Pgp表达 (M =4 0 )较长病程组 (M =0 0 )、对照组 (M =0 0 )增强 ,长、短病程组神经元Pgp表达 (M值均为 3 0 )较对照组 (M =0 0 )增强 ;神经元MRP、LRP表达在长病程组较短病程组增多 ,短病程组较对照组增多 ;GST π在神经元的表达长病程组 (M =6 5 )较短病程组 (M =2 0 )、对照组 (M =0 0 )增强 ;长、短病程组神经元Topo Ⅱ表达 (M值分别为 5 0、4 0 )较对照组 (M =0 0 )增强 ,差异均具有显著性意义 (P<0 0 1)。结论　Pgp、MRP、GST π、LRP及Topo Ⅱ均参与难治性癫耐药的形成。在病程早期 ,耐药机制以Pgp为主 ,并有LRP、MRP参加 ;在后期 ,Pgp作用减弱 ,LRP、MRP的作用进一步增强 ,同时GST π参与后期耐药的形成 ;TopoⅡ可能与难治性癫形成有关 ,具体机制尚待进一步研究。     

Expression and cellular distribution of multidrug resistance-related proteins in patients with focal cortical dysplasia.

Recent arouse of interest indicated that drug resistant proteins are markedly over-expressed in the epileptogenic tissue and they may be responsible for the one-third of the epileptic patients who were refractory to anti-epileptic drugs (AEDs). Since several AEDs may act as substrates for these drug resistant proteins, the enhanced function of such proteins may increase drug extrusion, resulting in inadequate response to drug therapy in patients with epilepsy. We studied expression of the multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) in the epileptic tissues resected surgically in 28 patients with focal cortical dysplasia (FCD) by immunohistochemistry. The results were compared with 10 normal necropsy brain tissues. Normal brain showed no MDR1 expression in neurons and astrocytes, while MRP1 expression was very weak, which were encountered in a few samples. MDR1 expression was mainly localized on the vascular endothelial cells. In contrast to normal brain, we found intense MDR1 and MRP1 expression in both neurons and reactive astrocytes in the vast majority of dysplastic tissues. The majority of the dysplastic neurons demonstrated moderate to strong MRP1 immunoreactivity. Endothelial cells showed both MDR1 and MRP1 expression in the majority of the specimens studied. Multidrug transporters are over-expressed in the epileptogenic zone in patients with FCD. These results are concordant with previous studies, in which over-expression of multidrug proteins were shown in epileptogenic brain tissue in patients with FCD, that the over-expression of drug transport proteins in tissue from patients with refractory epilepsy may explain one possible mechanism for drug resistant in these pathologies.     

Expression of multidrug resistance type 1 gene (MDR1) P-glycoprotein in intractable epilepsy with different aetiologies: a double-labelling and electron microscopy study

Abstract The objective of this study was to analyse the clinical characteristics, pathological features and expression patterns of multiple drug resistance type 1 (MDR1) and glial fibrillary acidic protein (GFAP) in intractable epilepsy patients with variable aetiologies and to analyse the relationships between the clinical and pathological findings. Twenty-six patients (15 males, 11 females, age range 4–25 years, mean age 22.92 years, SD 11.19 years) with intractable epilepsy were included in this study; the clinical characteristics were considered, and the pathological changes as well as expression of MDR1 and GFAP in surgically removed brain tissues of each subject were examined under light and electron microscopy. All patients presented a long-lasting, refractory epilepsy, mostly of the partial type, due to different causes, such as trauma, vascular injuries, encephalitis, cortical dysplasia, cavernous angioma and Sturge-Weber disease. Neuronal degenerative damage, reactive proliferation of astrocytes, as well as overexpression of GFAP and MDR1, appeared as common pathological features in all cases. The detection of MDR1 by electron microscopy allowed us to precisely define its cellular location in reactive astrocytes and to exclude the presence of the antigen in other cellular types. In all cases, pathological features, at both light and electron microscopy, were similar, independent of the different clinical presentation and aetiology.     

Distribution and functional activity of P-glycoprotein and multidrug resistance-associated proteins in human brain microvascular endothelial cells in hippocampal sclerosis

Multidrug resistance protein, also referred as P-glycoprotein (P-gp, MDR1; ABCB1) and multidrug resistance-associated protein (MRP) 1 (ABCC1) and 2 (ABCC2) are, thus far, candidates to cause antiepileptic drug (AED) resistance epilepsy. In this study, we investigated P-gp, MRP1 and MRP2 expression, localization and functional activity on cryosections and isolated human brain-derived microvascular endothelial cells (HBMEC) from epileptic patients (HBMEC-EPI) with hippocampal sclerosis (HS), as compared with HBMEC isolated from normal brain cortex (HBMEC-CTR). We examined the expression and distribution of three transporters, P-gp, MRP1 and MRP2 on two major parts of the resected tissue, the hippocampus and the parahippocampal gyrus (Gph). P-gp showed diffuse expression not only in endothelium but also by parenchymal cells in both the hippocampus and the Gph. MRP1 labeling was observed in parenchymal cells in the Gph. By contrast, MRP2 was mainly found in endothelium of the hippocampus. P-gp and MRP1 expression in the Gph was relatively high in the patient with long-term seizure history. Quantitative RT-PCR analysis of HBMEC revealed that MDR1, MRP1 as well as MRP5 (ABCC5) and MRP6 (ABCC6) were overexpressed in HBMEC-EPI at the mRNA level. HBMEC from both normal and epilepsy groups displayed protein expression of P-gp, whereas MRP1 and MRP2 were seen only in HBMEC-EPI. Accordingly, it is of particular interest that MRP functional activities were observed in HBMEC-EPI, but not in HBMEC-CTR. Our results suggest that complex MDR expression changes not only in the hippocampus but in the Gph may play a role in AED pharmacoresistance in intractable epilepsy patients with mesial temporal lobe epilepsy (MTLE) by altering the permeability of AEDs across the blood-brain barrier (BBB).     

难治性癫(癎)相关耐药蛋白在局灶性皮质发育不良脑组织中的表达

目的 通过对P-糖蛋白、多药耐药相关蛋白和肺耐药相关蛋白在难治性癫痫相关局灶性皮质发育不良脑组织中表达部位的初步研究,以及对其在不同程度病变脑组织中表达量的比较,进一步阐明难治性癫疴的耐药机制,为癫(癎)患者的临床合理用药提供理论依据.方法 选取16例难治性癫(癎)患者手术切除脑组织标本作为患者组(局灶性皮质发育不良Ⅰ型和Ⅱ型患者各8例),5例无癫(癎)发作病史的胶质瘤患者手术切除脑组织标本的非病灶区域作为对照组.应用Envision二步法进行免疫组织化学标记,观察3种耐药蛋白在脑组织中的表达部位和表达强度;应用Western blot法进行SDS-聚丙烯酰胺凝胶电泳,对3种耐药蛋白在脑组织中的表达进行定量分析.结果 P-糖蛋白主要表达于毛细血管内皮细胞,多药耐药相关蛋白主要表达于脑组织内的神经元成分,肺耐药相关蛋白的表达则涌盖了毛细血管内皮细胞、气球细胞及病灶区域部分基质.3种耐药蛋白在局灶性皮质发育不良脑组织中的表达均显著高于对照组脑组织(P-糖蛋白:0.520±0.121,多药耐药蛋白:0.132±0.018,肺耐药相关蛋白:0.092 4-0.018,U=0.000,P<0.01),其中P-糖蛋白和肺耐药相关蛋白在局灶性皮质发育不良Ⅱ型患者的病灶区域(3.809±0.842、0.655±0.303)表达高于病灶周围区域(2.636 4±0.622、0.290±0.096,U=6.000、4.500,P<0.01).结论 P-糖蛋白、多药耐药相关蛋白和肺耐药相关蛋白在不同程度的局灶性皮质发育不良脑组织中具有不同的表达部位和表达量,提示其作用机制和作用强度有所差异.     

Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional pathologies

Aims: Glial fibrillary acidic protein (GFAP)‐δ is a novel isoform that differs in its C‐terminal sequence from other GFAP isoforms. Previous studies suggest restriction of expression to the subpial layer, subventricular zone and the subgranular zone astrocytes, with an absence in pathological conditions causing reactive gliosis. GFAP‐δ is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP‐δ‐positive glia may be multipotent stem cells. The aim of this study was to investigate its expression in common causes of lesion‐related refractory epilepsy. Methods: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD) type IIB, cortical tuberous sclerosis (TSC) lesions, gangliogliomas, grey matter heterotopias and hemimegalencephaly from a wide age range of patients using both surgical and post mortem tissue specimens were studied. Results: GFAP‐δ expression was observed in CA4 and CA1 astrocytes in HS with less frequent labelling in the granule cell layer, even where granule cell dispersion was present. No significant labelling was noted in the subiculum in HS cases or in any subfields in non‐HS epilepsy cases. Balloon cells in FCDIIB and hemimegalencephaly, giant cells in TSC and the astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP‐δ was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP‐δ‐positive balloon cells than conventional GFAP. There was no GFAP‐δ expression within nodular heterotopia. Conclusions: GFAP‐δ expression patterns in HS overall appears to mirror regional reactive gliosis. It is a useful marker for the demonstration of balloon cells in FCD and TSC, which may be relevant to their abnormal size and localization. The lack of GFAP‐δ within heterotopia supports their composition from cells destined for deeper cortical layers.     

Enhanced expression of microtubule-associated protein 2 in large neurons of cortical dysplasia

To evaluate neuronal cytoarchitectural changes in cortical dysplasia, we examined microtubule-associated protein 2 (MAP2) expression in surgically resected specimens obtained from 20 patients (age range, 3 months to 10 years) treated for intractable epilepsy. Large neurons were investigated in the specimens from all patients and showed significantly strong immunoreactivity with antibodies against MAP2 in the perikaryon and proximal portion of their processes. In situ hybridization with MAP2 antitense riboprobe showed increased hybridization signal intensities in the large neurons, which correlated with the pattern of immunoreactivity for MAP2. We conclude that MAP2 is strongly expressed in the large neurons in cortical dysplasia. The results of preliminary immunoblotting in 1 patient with focal cortical dysplasia showed that the low-molecular-weight form of MAP2 (MAP2c) was strongly expressed in the dysplastic cortex, suggesting that MAP2c may be a major component contributing to the increased expression of MAP2 in the large neurons of cortical dysplasia. Since it has been suggested that MAP2 plays a crucial role in the branching and remodeling of neuronal processes, increased expression of MAP2 may reflect activated plasticity of the large neurons in cortical dyspasia.