Evaluation Of Novel Epilepsy Treatment Approaches

该项目的总体目标是通过动物模型的药理学研究和人类受试者的临床研究来探索药物治疗癫痫的策略。研究继续评估神经活性类固醇在癫痫中的作用及其在癫痫治疗中的可能用途。神经活性类固醇是内源性类固醇激素（及其合成类似物），通过直接作用于膜离子通道，包括GABA-A和NMDA受体，迅速改变神经元的兴奋性。在之前的报道中，我们证实了生殖激素黄体酮具有强大的抗惊厥活性，我们证明了这是由于它转化为神经活性类固醇异孕酮。我们提出，在月经期（孕酮水平下降时），许多女性癫痫患者发作频率的增加可能与异孕酮的停药有关。目前，没有专门的治疗癫痫的方法。然而，我们对兔兔癫痫动物模型的研究表明，神经类固醇替代可能是有用的。我们已经启动了临床研究来验证双侧癫痫的神经类固醇停药假说，我们计划进行一项临床试验来评估神经类固醇替代的效用。在本报告期间，我们完成了一项研究，检查神经类固醇在应激诱导的癫痫易感性改变中的作用，特别关注四氢脱氧皮质酮（DOC），一种肾上腺类固醇，其合成在应激时增强。DOC通常被认为是一种矿物皮质激素前体；然而，我们获得的证据表明，它也是应激效应对癫痫发作的中介。DOC经过5 α -还原酶和3 α -羟基类固醇氧化还原酶的顺序代谢还原，形成二氢脱氧皮质酮（DHDOC）和THDOC，这是一种GABA-A受体调节的神经类固醇，具有抗惊厥特性。急性游泳应激使大鼠血浆THDOC浓度显著升高，PTZ发作阈值升高。全身小剂量的DOC可使血浆THDOC水平和PTZ发作阈值增加。阻断DOC向DHDOC转化的非那雄胺预处理逆转了应激的抗癫痫作用。DOC对小鼠的PTZ、DMCM（甲基-6,7-二甲氧基-4-乙基- β -羰基-3-羧酸酯）、微螺毒素和杏仁核点燃性癫痫也有保护作用。非那雄胺逆转了DOC的抗癫痫活性；吲哚美辛（一种3 α -羟基类固醇氧化还原酶抑制剂）也具有部分拮抗作用。非那雄胺对DHDOC和THDOC无保护作用，而吲哚美辛对DHDOC有部分逆转作用，但对THDOC无逆转作用。与非那雄胺和吲哚美辛的实验结果表明THDOC可能介导DOC的抗惊厥活性，DHDOC也可能介导DOC的抗惊厥活性。在培养海马神经元的全细胞电压钳记录中，我们发现DHDOC与THDOC一样，增强了gaba激活的氯离子电流和直接激活GABA-A受体电流，这与DHDOC在DOC抗癫痫活性中的作用相一致。我们的研究结果表明，DOC是急性应激生理效应的中介，可能通过其转化为神经类固醇，对GABA-A受体（包括THDOC和DHDOC）具有调节作用，从而促进应激诱导的癫痫易感性变化。我们的研究结果进一步表明，神经活性类固醇作为一种治疗压力相关癫痫发作的方法。在本报告期间，我们还开展了研究，检查孕酮受体（PR），一种核转录因子，在介导孕酮抗惊厥作用中的作用。虽然我们之前的研究表明，黄体酮对癫痫易感性的调节是通过其神经类固醇代谢物异孕酮间接发生的，但由于PR是黄体酮在多种组织中作用的主要靶点，我们试图排除黄体酮对癫痫发作的影响部分是通过PR介导的转录效应发生的可能性。我们使用PR“敲除”（PRKO）小鼠，其中PR基因被基因靶向删除（导致PR的A和B形式缺失）。黄体酮对PRKO小鼠抗戊四唑（PTZ）和杏仁核点燃性癫痫有保护作用，非那雄胺预处理后黄体酮的抗癫痫活性逆转。内源性神经类固醇异孕醇酮和THDOC以及合成神经活性类固醇加那洛酮在PRKO小鼠中也表现出有效的抗惊厥活性。然而，我们意外地发现，与野生型对照相比，PRKO小鼠中黄体酮的抗癫痫效力增加了2倍。此外，我们观察到PRKO小鼠对PTZ发作的总体易感性降低。为了开始了解这些癫痫易感性变化的分子基础，我们使用包含12,000个序列的微阵列进行了基因表达分析，其中包括小鼠UniGene数据库中的所有6,000个功能特征序列。我们假设PR缺失的影响可能是由于GABA-A受体亚基基因表达的改变。然而，对所有已知亚基的综合分析显示，PRKO与野生型动物之间没有显著差异。我们还调查了所有已知的嗜离子性谷氨酸受体基因。我们发现，只有KA2（与GluR5和GluR6形成异质通道的高亲和盐酸盐受体亚基）和NMDAR1 （NMDA受体的强制性亚基）表现出显著的差异表达，在PRKO动物中，这两种情况下都增加了约两倍。此外，微阵列分析显示，其他20个基因表达增加，2个表达减少。KA2亚基表达的变化是特别有趣的，因为与同质受体相比，异源盐酸盐受体表现出更快的脱敏，并且含有KA2的盐酸盐受体丰度的增加可以解释癫痫发作倾向的降低。

The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity and we demonstrated that this results from its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may, in part, be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. We have initiated clinical studies to validate the neurosteroid withdrawal hypothesis of catamenial epilepsy and we plan a clinical trial to evaluate the utility of neurosteroid replacement. In the present reporting period we completed a study examining the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on tetrahydrodeoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. DOC is conventionally considered a mineralocorticoid precursor; however, we obtained evidence indicating that it is also a mediator of stress effects on seizures. DOC undergoes sequential metabolic reduction by 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase to form dihydrodeoxycorticosterone (DHDOC) and THDOC, which is a GABA-A receptor modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the PTZ seizure threshold. Small systemic doses of DOC produced comparable increases in plasma THDOC levels and PTZ seizure threshold. Pretreatment with finasteride, which blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also protected mice against PTZ, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-caboxylate), picrotoxin and amygdala kindled seizures in mice. Finasteride reversed the antiseizure activity of DOC; partial antagonism was also obtained with indomethacin, an inhibitor of 3alpha-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC whereas indomethacin partially reversed DHDOC but not THDOC. The results with finasteride and indomethacin indicated that THDOC and possibly DHDOC could mediate the anticonvulsant activity of DOC. In whole cell voltage clamp recordings from cultured hippocampal neurons, we found that DHDOC, like THDOC, potentiated GABA-activated chloride currents and directly activated GABA-A receptor currents compatible with a role for DHDOC in the antiseizure activity of DOC. Our results demonstrate that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including THDOC and possibly also DHDOC. Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period we also carried out studies examining the role of the progesterone receptor (PR), a nuclear transcription factor, in mediating the anticonvulsant effects of progesterone. Although our prior research demonstrated that progesterone modulation of seizure susceptibility occurs indirectly through its neurosteroid metabolite allopregnanolone, since the PR is the primary target of progesterone action in diverse tissues, we sought to exclude the possibility that progesterone's effects on seizures in part occur through transcriptional effects mediated by the PR. In these studies, we used PR "knockout" (PRKO) mice in which the PR gene had been deleted by gene targeting (resulting in the absence of both the A and B forms of the PR). Progesterone protected PRKO mice against pentylenetetrazol (PTZ) and amygdala-kindled seizures, and the antiseizure activity of progesterone was reversed by pretreatment with finasteride. The endogenous neurosteroids allopregnanolone and THDOC, and the synthetic neuroactive steroid ganaxolone also exhibited potent anticonvulsant activity in PRKO mice. However, we unexpectedly found a 2-fold increase in the antiseizure potency of progesterone in PRKO mice compared to wild type controls. In addition, we observed that PRKO mice have an overall reduced susceptibility to PTZ seizures. To begin to understand the molecular basis for these alterations in seizure susceptibility, we have carried out gene expression analysis using a microarray representing 12,000 sequences including all 6,000 functionally characterized sequences in the mouse UniGene database. We hypothesized that the effects of deletion of the PR could be due to alterations in the expression of GABA-A receptor subunit genes. However, a comprehensive analysis of all known subunits revealed no significant differences between PRKO and wild-type animals. We also surveyed all known ionotropic glutamate receptor genes. We found that only KA2 (high affinity kainate receptor subunit that forms heteromeric channels with GluR5 and GluR6) and NMDAR1 (obligatory subunit of NMDA receptors) showed significant differential expression, with an increase in PRKO animals of about two-fold in both cases. In addition, the microarray analysis showed twenty other genes whose expression was increased and two that were decreased. The change in KA2 subunit expression was of particular interest inasmuch as heteromeric kainate receptors show more rapid desensitization as compared with homomeric receptors and increased abundance of KA2-containing kainate receptors could account for the reduced propensity for seizures. As a complement to our prior studies on drug and hormonal treatment of epilepsy, we have begun a new research direction to investigate alternative epilepsy treatment approaches. Our current emphasis is on the use of convection-enhanced brain infusion for the suppression or ablation of epileptic foci. We completed a study in collaboration with the Surgical Neurology Branch examining the long-term antiseizure effects of infusion of the excitotoxin ibotenate in a rat model of temporal lobe epilepsy. Kainate injection into the left amygdala of rats elicited chronic spontaneous limbic seizure activity. Two weeks after the injection, ibotenate, a nonepileptogenic excitatory amino acid that is an axon-sparing neuronal cell toxin, was infused into the left amygdala and piriform lobe. The infusion eliminated the electrical and behavioral seizure activity. We conclude that lesioning of an epileptic focus by convective distribution of ibotenate can produce an enduring suppression of seizure activity. These studies demonstrate a potential chemical neurosurgical approach to epilepsy therapy that could represent an alternative to conventional ablative neurosurgical procedures in the management of partial epilepsies.