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。     

脑发育异常性癫痫脑组织的MDR-1、MRP和GFAP表达一例并文献复习

目的：观察灰质异位性难治性癫痫中多药耐药基因蛋白（MDR-1）、耐药基因相关蛋白（MRP）及胶质纤维酸性蛋白（GFAP）在发育异常性脑组织中的表达情况。方法：对确诊为脑灰质异位性症状性癫痫患者手术切除下病灶组织行免疫组化染色，观察脑组织中MDR-1、MRP及GFAP的表达情况。结果：在手术切除的病灶组织中，除一些增生性的星形胶质细胞同时具上述三种蛋白的阳性标记外，在一些异形的神经元内同时还存在有MDR-1和MRP的阳性表达。结论：灰质异位性难治性癫痫中MDR-1、MRP及GFAP不仅可在一些反应性的星形胶质细胞中表达，而且还可在一些发育异常的异形神经元中表达。     

难治性癫痫患者致痫灶细胞凋亡现象的研究

目的观察难治性癫痫患者手术切除癫痫灶中细胞凋亡现象,探讨神经元凋亡在癫痫发病机制中的意义,为临床治疗提供新的理论依据.方法收集16例经手术治疗的难治性癫痫患者致痫灶标本,在对临床资料全面分析的基础上,应用光镜、电镜及凋亡细胞DNA原位末端标记法（TUNEL）观察标本中存在的细胞凋亡现象.结果致痫灶周围神经元普遍固缩、减少,反应性胶质细胞增生.TUNEL阳性细胞数量较对照组显著增高（P＜0.01）,且绝大部分为神经元.结论难治性癫痫患者致痫灶周围存在细胞凋亡现象,神经元凋亡可能参与癫痫的发病过程.     

GFAPδ immunostaining improves visualization of normal and pathologic astrocytic heterogeneity

Neuropathological analysis of cellular mechanisms underlying gliosis and brain tumors is slowed by the lack of markers allowing to distinguish glial subpopulations in normal or pathological human brains. We therefore evaluated GFAPδ immunostaining in a wide panel of astrogliosis and gliomas, and compared these with GFAP and vimentin. In normal tissue, gliosis and gliomas, GFAPδ immunostaining was observed in astrocytes with relatively high GFAP levels. GFAPδ immunostaining was most conspicuous in glia limitans astrocytes. In Chaslin's gliosis accompanying chronic epilepsy, GFAPδ immunostaining evidenced the glia limitans reactive astrocytes as the source of the dense fibrillary meshwork typical of Chaslin's gliosis. Interestingly GFAPδ and vimentin immunostainings coincided in normal tissues and gliosis, but not in gliomas. Altogether these results show that combined GFAP, GFAPδ and vimentin labelling reveals fine gliofilament regulation in normal and pathological brain.     

Expression of multidrug resistance 1 gene and C3435T genetic polymorphism in peripheral blood of patients with intractable epilepsy

BACKGROUND:Increased expression of multidrug resistance 1 (MDR1) mRNA in peripheral blood of pa-tients with intractable epilepsy is not due to epilepsy drugs,but epilepsy behavior. Monitoring MDR1 ex-pression in peripheral blood is a target for MDR1 gene evaluation. OBJECTIVE: To investigate the influence of antiepileptic drugs and seizures on MDR expression in intrac-table epilepsy,and to analyze the genetic polymorphisms of C3435T in the MDR1 gene. DESIGN,TIME AND SETTING: Factorial designs and comparativ...     

Outcome of surgical treatment in non-lesional intractable childhood epilepsy

PurposeThe aim of this study is to investigate seizure outcomes following epilepsy surgery in non-lesional neocortical pediatric epilepsy.MethodsWe performed a retrospective study on 27 patients with intractable childhood epilepsy who received epilepsy surgery between 1999 and 2006 at Sang-gye Paik Hospital and Severance Children's Hospital. None of the patients had any detectable lesions on MRI. Surgical outcome was assessed at least 2 years postoperatively; clinical characteristics, surgical outcome, pathologic findings, types of surgery, localizing features on SPECT, FDG-PET, and long-term video-EEG were reviewed.ResultsEighteen patients (67%) demonstrated an Engel class I outcome postoperatively. The mean follow-up duration was 4.3 years (range, 2.2–9 years). Eighteen out of 27 cases (67%) showed focal localizing features on ictal SPECT, and 21 of 27 cases (78%) showed abnormal findings on PET. Single lobectomy was the most common procedure, and was performed in 20 patients (74%). Multilobar resection was performed in seven patients (26%). Cortical dysplasia was the most common finding during pathological examination, and was observed in 15 (56%) cases. In addition, gliosis was found in two patients (7%) and non-specific pathological findings were described in 10 patients (37%).ConclusionsIn children with intractable epilepsy and a MRI demonstrating no abnormal lesions, epileptic surgery should be strongly considered when cortical pathology can be identified from other studies.     

Nestin expression in ganglioglioma

It has been suggested that gangliogliomas represent a neoplastic transformation of a dysplastic focus or heterotopia. Other theories propose that gangliogliomas arise from multipotent stem cells with the ability to differentiate along glial and neuronal cell lines. Our goal was to characterize the expression of nestin, a neuroepithelial precursor/stem cell antigen, in gangliogliomas along with other pathological and clinical features of this entity. The clinical and operative features of 18 recent cases meeting the histological criteria for ganglioglioma were reviewed. The expression of nestin, microtubule-associated protein 2 (MAP2), neurofilament, and glial fibrillary acidic protein (GFAP) was assessed by immunohistochemistry and confocal scanning laser microscopy. Abundant MAP2- and nestin-positive neuronal cells were found by immunohistochemistry in all 18 gangliogliomas. GFAP staining was found in reactive and lesional astrocytes but not in cells of neuronal morphology. Confocal microscopy demonstrated colocalization of nestin and MAP2 in select neuronal cells. The true lineage of gangliogliomas remains controversial. Our findings confirm the presence of cells within these lesions that harbor a persistent stem cell cytoskeletal protein (nestin). Further insight into the cytoskeletal derangement of nestin-positive neuronal cells may shed further light on the pathogenesis of gangliogliomas and its associated epilepsy.     

药物难治性癫痫患者脑内多种耐药基因表达的初步研究

目的研究药物难治性癫痫患者与正常对照组脑内多种药物耐药基因（ＭＤＲ１）表达的情况。方法　选择１５例药物难治性癫痫患者和５例对照组患者的脑组织标本,用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）技术,以Ｂ－ａｃｔｉｎ为内参照对ＭＤＲ１基因进行扩增,经琼脂糖凝胶电泳分别得到２２６ｂｐ和５４８ｂｐ的条带,通过照相、扫描、计算ＭＤＲ１／ｂ－ａｃｔｉｎ比值进行半定量分析。结果统计学处理提示,药物难治性癫痫患者脑内ＭＤＲ１的表达明显高于对照组（Ｐ＜０．０１）。结论　Ｐ－糖蛋白为ＭＤＲ１的产物,能将药物从脑内排入血循环而降低脑内药物浓度。药物难治性癫痫患者因脑内ＭＤＲ１的高表达,导致脑内抗癫痫药物（ＡＥＤ）浓度较低而耐药。故应用ＭＤＲ１抑制剂也许可以提高ＡＥＤ在脑内的药物浓度,使癫痫的药物治疗效果更令人满意。     

Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commerically available liposome complexed with 3′ or 5′ antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo. © 1993 Wiley-Liss, Inc.     

DJ‐1 immunoreactivity in human brain astrocytes is dependent on infarct presence and infarct age

DJ‐1 is a protein with anti‐oxidative stress and anti‐apoptotic properties that is abundantly expressed in reactive CNS astrocytes in chronic neurodegenerative disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and Pick's disease. Genetic mutations which eliminate DJ‐1 expression in humans are sufficient to produce an early‐onset form of familial PD, PARK7, suggesting that DJ‐1 is a critical component of the neuroprotective arsenal of the brain. Previous studies in parkinsonism/dementia brain tissues have revealed that reactive astrocytes within and surrounding incidentally identified infarcts were often robustly immunoreactive for DJ‐1, especially if the infarcts showed histological features consistent with older age. Given this, we sought to evaluate astrocytic DJ‐1 expression in human stroke more extensively, and with a particular emphasis on determining whether immunohistochemical DJ‐1 expression in astrocytes correlates with histological infarct age. The studies presented here show that DJ‐1 is abundantly expressed in reactive infarct region astrocytes in both gray and white matter, that subacute and chronic infarct region astrocytes are much more robustly DJ‐1+ than are acute infarct and non‐infarct region astrocytes, and that DJ‐1 staining intensity in astrocytes generally correlates with that of the reactive astrocyte marker GFAP. Confocal imaging of DJ‐1 and GFAP dual‐labelled human brain sections were used to confirm the localization to and expression of DJ‐1 in astrocytes. Neuronal DJ‐1 staining was minimal under all infarct and non‐infarct conditions. Our data support the conclusion that the major cellular DJ‐1 response to stroke in the human brain is astrocytic, and that there is a temporal correlation between DJ‐1 expression in these cells and advanced infarct age.     

Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy

PURPOSE: This study investigated the cellular distribution of different multidrug resistance (MDR)-related proteins such as P-glycoprotein (P-gp), the multidrug resistance-associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE). METHODS: Single- and double-label immunocytochemistry was used on brain sections of control hippocampus and of hippocampus of refractory MTLE patients. RESULTS: In TLE cases with hippocampal sclerosis (HS), all four MDR proteins examined that had low or no expression in control tissue were upregulated, albeit with different cellular distribution patterns. P-gp immunoreactivity (IR) was observed in astrocytes in regions with diffuse reactive gliosis. In 75% of HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling. Reactive astrocytes displayed low MRP1 IR. However, glial MRP1 expression was noted in glial endfoot processes around blood vessels. Neuronal MRP1 expression was observed in hypertrophic hilar neurons and in a few residual neurons of the CA1 region. Hippocampal MRP2 expression was observed in the large majority of HS cases in blood vessels. Hypertrophic hilar neurons and blood vessels within the sclerotic hippocampus expressed major vault protein (MVP). CONCLUSIONS: These findings indicate that MDR proteins are upregulated in concert in the hippocampus of patients with refractory MTLE, supporting their role in the mechanisms underlying drug resistance. The specific cell-distribution patterns within the sclerotic hippocampus suggest different cellular functions, not necessarily linked only to clinical drug resistance.     

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.     

Sodium channel beta1-subunit expression is increased in reactive astrocytes in a rat model for mesial temporal lobe epilepsy

As several epilepsy syndromes are associated with changes in sodium channel subunits we investigated the expression of beta1 sodium channel protein in a rat epilepsy model. In this model a chronic epileptic syndrome develops after electrically induced status epilepticus (SE). Many neuropathological characteristics of mesial temporal lobe epilepsy can be reproduced (cell loss, gliosis and synaptic reorganization). In control hippocampus beta1 subunit protein was moderately expressed in neurons and weakly expressed in resting astrocytes. beta1 sodium channel immunoreactivity increased markedly within 1 week after SE mainly in astrocytes that were colocalized with vimentin (marker for reactive astrocytes). This up-regulation was still present in reactive astrocytes of chronic epileptic rats (> 3 months after SE). Considering the fact that the beta1 subunits may function as cell adhesion molecules interacting with extracellular matrix, the observed increase in reactive astrocytes might subserve a function in cellular and synaptic reorganization during epileptogenesis.     

Regressive changes of astroglia in white matter lesions in cerebrovascular disease and Alzheimer’s disease patients

The pathogenesis of white matter lesions, which are frequently found in ischemic cerebrovascular disease and Alzheimer’s disease, remains unclear. Using light and electron microscopic immunohistochemistry for glial fibrillary acidic protein (GFAP) as a marker, the present study focused on the role of astroglia which show characteristic morphological alterations. Of 29 brains of patients with cerebrovascular disease and Alzheimer’s disease, 4 brains showed extensive swelling and vacuolation of white matter astroglia with their processes disintegrated and beaded (termed clasmatodendrosis). No such cells were observed in 6 control patients. Clasmatodendritic astroglia were not intensely eosinophilic using hematoxylin and eosin staining and included large lipophilic granules in their perikarya. These astroglia were immunoreactive for serum proteins such as immunoglobulins, fibrinogen and complement C3, C1q and C3d, as well as for proteins which are known to increase in reactive astroglia, such as vimentin, α-B crystallin, apolipoprotein-E and laminin. Double labeling for GFAP and microglial cell markers indicated that these cells were of astroglial lineage. Immunoelectron microscopy for GFAP revealed that clasmatodendritic astroglia had condensed chromatin, lysosomes and large membrane-bound osmiophilic cytoplasmic inclusions, which corresponded to the lipophilic granules observed with light microscopy. These cytochemical features collectively suggest that clasmatodendritic astroglia incorporate edema fluid and phagocytose cellular debris, and eventually degenerate as a result of cerebral edema. Received: 3 December 1996 / Revised, accepted: 24 January 1997     

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down‐regulated proportionally to neuronal loss in the human epileptic hippocampus

Human mesial temporal lobe epilepsy (MTLE) features subregion‐specific hippocampal neurodegeneration and reactive astrogliosis, including up‐regulation of the glial fibrillary acidic protein (GFAP) and down‐regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE‐associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high‐resolution image analysis for digital pathology and whole‐slide imaging. In the cortex, GS‐positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP‐positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1–5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up‐regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS‐pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down‐regulated proportionally to the degree of neuronal loss.     

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。     

HIV-1 Nef co-localizes with the astrocyte-specific cytoskeleton protein GFAP in persistently nef-expressing human astrocytes

In T-cells HIV-1 Nef exerts various functions and interacts with actin. In astrocytes interaction of Nef with cellular proteins is poorly understood. Therefore, human astrocytic cell clones stably transfected with nef-genes derived from HIV-1 Bru and its myristoylation-defective TH-variant were investigated by confocal laser scanning microscopy for expression of Nef and cytoskeleton proteins actin and GFAP, a marker for activated astrocytes. Myristoylated Nef was detected in cytoplasm, Golgi and plasmamembrane, while non-myristoylated Nef was exclusively cytoplasmic. Nef co-localised with GFAP in the perinuclear region of astrocytes. In contrast, Nef did not interact with actin filaments in human astrocytes. Nef/GFAP interaction could contribute to changes in morphology and activation state of astrocytes shown previously which are both critical for development of astrogliosis in HIV-1 infected brain.     

Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes

PURPOSE: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. METHODS: Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. RESULTS: Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. CONCLUSIONS: Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.