Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

Purpose: Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE. Methods: Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key Findings: In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up‐regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3–4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin‐1β and lipopolysaccharide). Significance: These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte‐based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated.     

Vasoactive intestinal polypeptide and its receptor changes in human temporal lobe epilepsy

The distribution of the VIP receptor in the human hippocampus was studied by receptor autoradiography using [3-iodotyrosyl-¹²⁵I]Vasoactive Intestinal Peptide (VIP) as a ligand, and the relationship of receptor distribution to the distribution of the peptide (visualized by immunocytochemistry) was examined in hippocampi surgically removed from patients with medically intractable temporal lobe epilepsy (TLE) and hippocampi obtained at autopsy from neurologically normal subjects. In the autopsy hippocampi and hippocampi from TLE patients with extrahippocampal temporal lobe lesions [¹²⁵I]VIP binding was highest in the dentate molecular layer, with lower levels in the fields of Ammon's Horn (CA fields) and the subiculum. In hippocampi from patients with no temporal lobe lesions but considerable hippocampal neuronal loss there were significant elevations in the levels of ligand binding in all CA fields and the subiculum. Ligand binding densities in all CA fields of the patient hippocampi were strongly negatively correlated with neuronal numbers. Immunocytochemical localization of VIP shows no obvious change in the distribution patterns of VIP immunoreactivity in the patient groups. This is the first demonstration of VIP and its receptor distribution in the human hippocampus. It is suggested that the elevated levels of receptor binding in the hippocampal seizure focus may indicate a mechanism for greater excitability of neurons and/or for their survivability in the face of the increased excitation and potential for injury in a seizure focus.     

Aktl在颞叶癫痫大鼠海马中的表达变化

目的研究颞叶癫痼模型海马区神经元Aktl表达变化,探讨其在癫痫发生发展中的作用。方法采用氯化锂．匹罗卡品方法制备颞叶癫痴大鼠模型,Westernblotting检测海马区总蛋白、Quantitvone软件行灰度值分析;免疫组织化学染色观察海马各区Aktl蛋白表达变化,计数不同处理组阳性神经元数目。结果Westernblotting检测结果显示,与正常对照组相比,癫痫模型组大鼠于癫痫持续状态发作即刻海马区Aktl蛋白表达升高（t=2．445,P=0．034）,并于第30天时达峰值水平（}=1．214,P=0．002）,发作后24h表达水平迅速降低,并低于正常值范围（t=4．294,P=0．000）,其余各测量时间点表达无明显改变;与氯化锂组相比,癫痼模型组大鼠于癫痫持续状态后1h海马区Aktl蛋白表达开始降低,24h降至最低水平（t：4．134,P=0．000）,至发作48h后开始逐渐升高（t=2．481,P=0．002）,并于发作第7天时升至氯化锂组水平。免疫组织化学染色显示,癫痫持续状态发作后海马CA3区Aktl蛋白表达阳性神经元数目立即增加,12h达高峰（t=16．586,P：0．000）,48h减少并降至正常值水平（t=0．357,P=0．089）,发作后第10天再次增加（t=3．123,P=0．000）,于第30天时阳性神经元数目再次达峰值水平（t=18．339,P=0．000）,第50天开始恢复至正常值水平（t=3．219,P=0．000）;氯化锂组仅海马CA3区Aktl蛋白表达于实验初始（0h）升高并高于正常对照组（P〈0．05）,海马CA1和CA2区Aktl蛋白表达变化组间差异均无统计学意义（P〉O．05）。结论海马及海马CA3区Aktl蛋白表达均呈现癫疝持续状态后升高、降低、再升高的动态过程,提示可能存在神经元保护作用,对抗细胞凋亡、促进细胞存活。     

颞叶癫痫脑电图分析及病灶超微结构观察

目的 研究影像学检查无异常的颞叶癫痫患者,电生理异常与皮层棘波灶及海马超微病变的关系.方法 选择经CT或MRI检查未见异常的颞叶癫痫患者7例,术前做脑电图或24h视频脑电监测,术中在脑电监测下取颞叶大脑皮质棘波灶和海马组织,做电镜观察.结果 7例患者电生理检查均可见典型痫样放电.颞叶皮质痫灶和海马可见不同程度的神经元固缩,胶质细胞变性,胶质增生,突触数量及突触结构改变,血脑屏障破坏等改变.结论 影像学无异常的颞叶癫痫患者颞叶皮层痫灶和海马超微结构病理变化明显,特别是突触的变化,是导致癫痫患者脑电生理机能异常及癫痫反复自发性发作的形态学基础.     

颞叶癫(癎)大鼠海马中miRNA表达谱的检测和分析

目的 探讨颞叶癫(癎)大鼠海马组织中miRNA分子表达谱的差异,为进一步研究相关miRNA在颞叶癫(癎)发病机制中的作用打下基础.方法 对同一父系和母系的子代大鼠,利用氯化锂-匹罗卡品化学诱导方法制备慢性颞叶癫(癎)大鼠模型.分别提取1只正常和3只颞叶癫(癎)大鼠海马组织的miRNA,采用高通量的miRNA微阵列芯片杂交,筛选颞叶癫(癎)海马组织中差异表达的内源性miRNA.结果 在大鼠海马组织中共检测到125个miRNA基因.与正常大鼠相比,颞叶癫(癎)大鼠海马组织中差异表达的miRNA有23个,其中有5个miRNA下调,18个miRNA上调.结论 与正常大鼠相比,颞叶癫(癎)大鼠海马组织中存在差异表达的miRNA分子,差异表达的miRNA分子可能参与癫(癎)的发病过程,具有潜在的研究价值.     

Relationship of seizure frequency to hippocampus volume and metabolism in temporal lobe epilepsy

PURPOSE: To examine the relationship between frequency of complex partial (CPS) and secondarily generalized tonic-clonic seizures (sGTCS) on hippocampal volume (HV) and temporal lobe metabolism. METHODS: We performed volumetric magnetic resonance imaging (MRI) and positron emission tomography with 18fluorodeoxyglucose (18FDG-PET) in 32 patients with epilepsy. Temporal lobe foci were localized by ictal video-EEG. RESULTS: We did not find any association between CPS frequency or lifetime number of sGTCS and HV or metabolism ipsilateral to electroencephalographic focus. CONCLUSION: The progress of metabolic or pathologic abnormalities of temporal lobe epilepsy may not be altered by adequate seizure control. The presence of an epileptic focus might be associated with progressive neuronal injury even in clinically well-controlled patients.     

Hippocampal Navβ3 expression in patients with temporal lobe epilepsy

Voltage-dependent sodium channels consist of a pore-forming alpha-subunit and regulatory beta-subunits. Alterations in these channels have been implicated in temporal lobe epilepsy (TLE) and several genetic epilepsy syndromes. Recently we identified Na_vβ3 as a TLE-regulated gene. Here we performed a detailed analysis of the hippocampal expression of Na_vβ3 in TLE patients with hippocampal sclerosis (HS) and without HS (non-HS) and compared expression with autopsy controls (ACs). Immunoblot analysis showed that Na_vβ3 levels were dramatically reduced in the hippocampus, but not in the cortex of non-HS patients when compared to HS patients. This was confirmed by immunohistochemistry showing reduced Na_vβ3 expression in all principal neurons of the hippocampus proper. Sequence analysis revealed no Na_vβ3 mutations. The functional consequences of the reduced Na_vβ3 expression in non-HS patients are unknown. Altered Na_vβ3 expression might influence microcircuitry in the hippocampus, affecting excitability and contributing to epileptogenesis in non-HS patients. Further experiments are required to elucidate these functional possibilities.     

颞叶癫痫:肿瘤性病变与海马硬化的手术治疗

目的：探讨由肿瘤及海马硬化所致的颞叶癫痫的手术治疗。方法：40例顽固性癫痫中，经MRI或CT诊断有19例内轴心性肿瘤性病变，有21例海马硬化，均行手术治疗，切除的标本进行组织学观察。结果：肿瘤性病变19例均作了肿瘤全切除加海马、杏仁核切除，海马硬化21例均作选择性海马、杏仁核切除术。40例中35例随访，随访时间为术后3个月至1年，5例失去联系。癫痫完全解除的有30例，5例仍需依赖药物治疗。结论：海马硬化是颞叶癫痫的主要原因，手术切除疗效满意。     

Increased PLA2 activity in the hippocampus of patients with temporal lobe epilepsy and psychosis

Objective

The aim of this work was to investigate whether increased activity of the enzyme phospholipase A₂ (PLA₂) in the brain, as frequently reported in schizophrenia, is also related to psychosis in epilepsy. Our working hypothesis was based on the increased prevalence of schizophrenia-like psychosis in patients with temporal lobe epilepsy (TLE) secondary to mesial temporal sclerosis (MTS), as compared to patients with other forms of epilepsy.

Methods

We determined PLA₂ activity in hippocampal tissue from 7 patients with TLE-MTS and psychosis, as compared to 9 TLE-MTS patients without psychosis. Hippocampal tissue was obtained from patients who underwent an anterior temporal lobectomy due to therapy-resistant epilepsy.

Results

We found that patients with TLE-MTS and psychosis had a significantly increased calcium-independent PLA₂ activity as compared to patients without psychosis (p = 0.016).

Conclusion

Our finding suggest that an increment in brain PLA₂ activity is not specific for schizophrenia, but rather may be associated to the manifestation of schizophrenia-like psychotic symptoms in general.     

Increased persistent sodium currents in rat entorhinal cortex layer V neurons in a post-status epilepticus model of temporal lobe epilepsy

PURPOSE: Spontaneous seizures in rats emerge several weeks after induction of status epilepticus with pharmacologic treatment or electrical stimulation, providing an animal model for human temporal lobe epilepsy. In this study, we investigated whether status epilepticus caused changes in the function of voltage-gated sodium channels in entorhinal cortex layer V neurons, a cellular group important for the genesis of limbic seizures. METHODS: We induced status epilepticus in rats, by using lithium-pilocarpine, and then 2-12 weeks later, used whole-cell voltage-clamp to examine voltage-activated sodium currents of acutely dissociated layer V neurons. RESULTS: Transient sodium currents of entorhinal cortex layer V neurons isolated from 9- to 12-week post-status epilepticus rats were similar to currents in age-matched controls; however, low-threshold persistent sodium currents were significantly larger. This increase in persistent activity was not seen 2-3 weeks after pilocarpine treatment; thus it occurred after a delay comparable to the delay in the appearance of spontaneous seizures. CONCLUSIONS: Increased persistent currents are expected to accentuate neuronal excitability and thus may contribute to the genesis of spontaneous seizures after status epilepticus.     

Ontogeny of altered synaptic function in a rat model of chronic temporal lobe epilepsy

In the limbic status model of chronic temporal lobe epilepsy, hippocampal stimulation induces acute status epilepticus in rats; recurrent, spontaneous seizures develop following an asymptomatic silent period lasting several weeks. Previous work has shown increased excitability and decreased inhibition in CA1 pyramidal neurons in chronically epileptic animals. To determine the relationship of altered cellular responses to seizure onset, in vitro intracellular recording was used to follow the evolution of changes in synaptic physiology occurring during the seizure-free silent period. Pyramidal cells displayed increasing epileptiform activity throughout the period investigated, 3–14 days following status; the mean number of evoked action potentials from 1.1±0.05 in control cells to 2.4±0.4 early (3 days after status) and 4.3±0.7 late (14 days) in the silent period. Monosynaptic inhibitory postsynaptic potentials mediated by γ-aminobutyric acid-A receptors in silent period cells differed markedly from controls. Area, rise time, and duration of these potentials decreased by 40–60% within 3 days following status and to values commensurate with chronically epileptic animals in 7 to 10 days. γ-Aminobutyric acid-B receptor-mediated IPSPs diminished more gradually in the silent period, reaching a minimum at day 14. In contrast, presynaptic γ-aminobutyric acid-B receptor function showed maximum impairment 3 days after status. The benzodiazepine type 1 receptor agonist zolpidem reduced hyperexcitability in both silent period and chronically epileptic cells, but was more effective at unmasking the underlying IPSP in silent period neurons. The results indicate that changes in different components of pyramidal cell inhibitory synaptic physiology associated with chronic epilepsy in this model evolve individually at different rates, but are all complete before seizure onset. Although the results do not imply causality, they do suggest that the development of physiological changes in CA1 pyramidal cells may contribute to the lag period preceding the onset of chronic seizures.     

Scn2a sodium channel mutation results in hyperexcitability in the hippocampus in vitro

PURPOSE: To investigate in vitro, the cellular network activity of the hippocampus in Q54 mice that display spontaneous seizures because of a gain-of-function mutation of the Scn2a sodium channel gene. METHODS: Extacellular recordings were obtained from CA1 and CA3 pyramidal neurons in hippocampal slices prepared from Q54 transgenic and nontransgenic littermates (WT) under physiologic conditions as well as during periods of orthodromic stimulation of the Schaffer collaterals. Cerebral spinal fluid samples were analyzed and cresyl violet histology of the hippocampus was conducted. RESULTS: Increased spontaneous extracellular activity was found in both CA1 and CA3 regions of Q54 hippocampal slices. Q54 slices also demonstrated significantly greater spontaneous and afterdischarge activity as well as population spike amplitude and duration following tetanic stimulus in comparison to WT slices. Frequency analysis of tetanically stimulated recordings indicated high-frequency components (100 and 200 Hz) unique to Q45 slices. Analysis of cresyl violet histology supports healthy Q54 slices up to 10 weeks, while Q54 cerebral spinal fluid shows elevated osmolarity. CONCLUSION: Evidence for hyperexcitability and increased synaptic efficacy in Q54 mice was found by observing spontaneous activity as well as evoked activity. Response to tetanic stimulation included unique high-frequency oscillations, and resulted in an increased population spike amplitude and duration. Histological assessment shows equivalent neuronal development in both experimental groups. The data support the hypothesis that modified Scn2a channels in Q54 mice result in network hyperexcitability of the hippocampus necessary for the development and maintenance of temporal lobe seizures.     

Pro-convulsant actions of theophylline and caffeine in the hippocampus: implications for the management of temporal lobe epilepsy

The pro-convulsant actions of theophylline and caffeine have been investigated using the hippocampal slice preparation and rats administered kainic acid or Metrazol. Both theophylline and caffeine induced the generation of epileptiform activity in the CA3 region of the hippocampal slice with convulsive dose50 (CD50) values of 3 microM respectively. Kainic acid-induced bursting in hippocampal slices was enhanced by theophylline (0.3-30 microM) and caffeine (1-100 microM). Theophylline induced burst firing in response to electrical stimulation in hippocampal area CA3 but not area CA1. Theophylline (50 mg/kg) strongly potentiated the effect of the limbic convulsant kainic acid in vivo whilst a dose of 200 mg/kg was necessary to significantly lower the threshold dose of Metrazol required to induce generalized convulsions. We conclude that alkylxanthines, probably by antagonizing the effect of endogenous adenosine, exert a pro-convulsant action in the hippocampus which preferentially promotes limbic seizures.     

难治性颞叶癫癎患者脑内KCNJ4基因表达的研究

目的观察内向整流钾离子通道亚单位基因KCNJ4 mRNA及其编码的蛋白产物Kir2.3在难治性颞叶癫癎患者和急性脑外伤患者脑内的表达差异,从分子水平探讨难治性颞叶癫癎可能的发病机制.方法 12例难治性颞叶癫癎患者手术切除的颞叶组织,10例急性脑外伤患者相应部位颞叶组织,利用逆转录-聚合酶链反应(RT-PCR)与蛋白印迹检测(Western-blot)方法检测两组间KCNJ4 mRNA(KCNJ4 mRNA/β-actin)与Kir2.3通道蛋白(Kir2.3/β-actin)的表达.结果难治性颞叶癫癎患者与脑外伤患者比较,颞叶组织KCNJ4 mRNA(0.438±0.178)及Kir2.3通道蛋白(0.063)的表达水平均降低,差异有统计学意义(P＜0.05).结论 KCNJ4 mRNA表达水平的下调及其编码产物Kir2.3通道蛋白表达水平的下降,可能是难治性颞叶癫癎发生发展的重要的分子学基础之一.     

海马硬化与颞叶癫痫术前定位探讨(附12例报告)

目的　探讨对顽固性颞叶癫痫的术前定位方法。方法　将 Ｍ Ｒ 对海马硬化的诊断作为一重要参考指标与 Ｅ Ｅ Ｇ 检查相结合,对１２ 例顽固性颞叶癫痫进行术前定位诊断,并与术中皮层 Ｅ Ｅ Ｇ 检查结果和术后随访结果比较。结果　 Ｍ Ｒ 检查发现９ 例海马硬化,皮层 Ｅ Ｅ Ｇ 证实有同侧颞叶内侧放电,行选择性海马杏仁核切除术后病理证实海马硬化;未发现海马硬化改变３ 例行前颞叶切除术。随访０５ ～２ 年,疗效满意。结论　 Ｅ Ｅ Ｇ 是诊断颞叶癫痫的最重要手段, Ｍ Ｒ 发现海马硬化对致痫灶定位有重要意义。     

发育期大鼠内侧颞叶癫痫模型丙戊酸治疗前后的蛋白质组学研究

目的 研究丙戊酸(VPA)对发育期大鼠内侧颞叶癫痫(MTLE)模型海马蛋白质表达谱的影响,寻求与VPA抗癫痫作用相关的蛋白质以及探讨VPA在MTLE发病早期发挥抗癫痫作用的机制.方法 建立氯化锂-匹罗卡品诱导的发育期大鼠MTLE模型,采用二维凝胶电泳(2-DE)技术分离海马总蛋白,PDQuest软件识别差异表达蛋白质,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)进行鉴定.结果 建立了VPA治疗前后的MTLE模型大鼠海马蛋白质的2-DE图谱.质谱鉴定出9个差异表达的蛋白质,其中4个在VPA治疗后表达上调;5个下调.结论 本实验成功建立了发育期大鼠MTLE模型VPA处理前后的海马蛋白质2-DE图谱;β-synuclein、Neurogranin等差异表达蛋白可能与VPA在MTLE发病早期发挥抗癫痫作用的机制相关.     

脑立体定向植入海马电极监测颞叶内侧癫痫的临床研究

目的 观察应用脑立体定向微创穿刺技术植入海马电极监测颞叶内侧癫痫的效果.方法 13例耐药性颞叶内侧癫痫患者,主要表现为复杂部分性癫痫发作及继发性全身强直阵挛性发作.根据临床症状、MRI等资料初步确定癫痫灶位于海马区域,在脑立体定向仪引导下于双侧海马植入8-触点深部电极,监测24 ～ 72 h,从而确认癫痫灶是否位于海马区域.结果 13例患者经过72 h监测,共监测到7例有29次临床发作,发作期脑电变化表现为在背景波形基础上出现阵发性高幅慢波或棘尖慢复合波,从某个电极点开始,迅速扩展到同侧其他电极点甚至对侧电极;头皮脑电在延迟1～2s后出现3～4 Hz的高幅δ节律.6例未监测到临床发作的患者,海马电极监测到发作性局灶性高幅慢波或尖慢综合波,而头皮电极未监测到明显异常.13例患者中6例接受选择性海马杏仁核切除或立体定向病灶损毁术,随访3～8个月,效果满意.结论 脑立体定向植入海马电极监测颞叶内侧癫痫是一种安全可靠的方法,可以判断癫痫病灶的起源,为外科进行选择性海马杏仁核切除提供有力依据,对于视频脑电图或其他手段难以记录到癫痫样波形或难以判断癫痫样放电起源的患者可进行脑立体定向深部电极脑电图监测.     

Voltage-gated sodium channel Nav1.1, Nav1.3 and beta1 subunit were up-regulated in the hippocampus of spontaneously epileptic rat

The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. Since the first point mutation in the voltage-gated sodium channel (VGSC) beta(1) subunit in human generalized epilepsy with febrile seizures plus (GEFS+) was identified, more and more types of genetic epilepsy have been causally suggested to be related to gene changes in VGSC. However, there are no reports that can elucidate the effects of VGSC in SER. The present study was undertaken to detect sodium channel I alpha-isoform (Na(v)1.1), sodium channel III alpha-isoform (Na(v)1.3) and beta(1) subunit from both the level of mRNA and protein in SERs hippocampus compared with control Wistar rats. In this study, the mRNA expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were significantly higher than those in control rats hippocampus by real-time RT-PCR; The protein distributions and expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were detected by immunofluorescence, immunohistochemistry and western blot, and the protein expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit were significantly increased. In conclusion, our study suggested for the first time that sodium channel Na(v)1.1, Na(v)1.3 and beta(1) subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER. The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.