Neonatal ischemic seizures: age and gender susceptibility to refractoriness

描述(申请人提供)：未成熟的大脑易受缺血后癫痫的影响。目前的临床实践包括将苯巴比妥(PB)作为确诊或疑似新生儿癫痫发作的一线经验性治疗。本研究将探讨CD1小鼠脑缺血后KCC2表达和调控的年龄和性别差异。在准备工作中，我们在P7、P10和P12仔鼠体内建立了KCC2表达的年龄依赖性增加和一线抗惊厥药物苯巴比妥对新生儿缺血性惊厥的年龄依赖性疗效。我们发现苯巴比妥在P7时抑制缺血性发作无效，在P10和P12时显着有效。我们的数据显示，在脑缺血后癫痫发作的严重程度和苯巴比妥在P7时抑制癫痫发作的疗效方面，与性别有关的生物学差异显著。以下假设将在CD1小鼠单侧颈总动脉结扎(即没有全球缺氧)诱导的新生儿卒中模型中得到验证：1.缺血损伤下调成人型电子中和氯化物协同转运体KCC2(氯离子挤出K-氯协同转运体)在P7和P10的急性和亚急性发育表达谱；2.早期阻断缺血后KCC2下调的表达可能是防止难治性新生儿癫痫发作的一种新策略。3.对P7和P10 CD1仔鼠急性卒中后事件的定量视频脑电以及它们对标准和新型抗惊厥药物如PB和BTN(BTN；有效的NKCC1拮抗剂)的反应将有助于预测在阻断新生儿卒中后KCC2下调后立即与早期联合治疗的疗效。新型抗惊厥药物BTN的额外无效；在我们先前资助的EFA(2011)研究中发现了一种有效的NKCC1拮抗剂，这突显了BTN在不同癫痫模型中不同疗效的重要性。特别令人感兴趣的是我们的初步发现，在这个模型中，KCC2在缺血后下调，而NKCC1的表达保持稳定，并有上调的趋势。因此，在未成熟脑中诱导急性癫痫发作的机制，以及该方案通过KCC2的磷酸化或降解而增强或恶化氯离子共转运体功能的特定效果，可能决定了抗惊厥药物的疗效依赖于氯离子梯度的抗癫痫作用。在缺血性癫痫发作发生后，将研究立即使用苯巴比妥和NKCC1拮抗剂布美他尼联合治疗预防KCC2下调的缺血后反应，并将其与早期治疗进行比较，并评估性二形性。

DESCRIPTION (provided by applicant): The immature brain is susceptible to post-ischemic seizures. Current clinical practice includes empiric treatments with phenobarbital (PB) as first line for confirmed or suspected seizures in the newborn. This proposal will investigate the age and gender dependent differences in KCC2 expression and modulation following ischemic neonatal brains of CD1 mice. In preparation we have established the age-dependent increase in KCC2 expression and the age-dependent efficacy of first line anticonvulsant drug, phenobarbital on neonatal ischemic seizures in-vivo in P7, P10 and P12 pups. We have found phenobarbital to be inefficacious in suppressing ischemic seizures at P7 and significantly efficacious at P10 and P12 using quantitative video-EEG. Our data show a significant biological sex related difference both in the severity of post-ischemic seizures and in the efficacy of phenobarbital to suppress seizures at P7. The following hypotheses will be tested in the CD1 mouse model of neonatal stroke induced by unilateral common carotid ligation only (i.e., no global hypoxia): 1.Ischemic insults downregulate the acute and delay the sub-acute developmental expression profile of the adult-form electroneutral chloride cotransporter KCC2 (chloride-extruding K-Cl cotransporter) at P7 and P10; 2. Early blocking of KCC2 downregulation following ischemia may be a novel strategy to prevent emergence of refractory neonatal seizures. 3. Quantitative video-EEG of acute post-stroke events from P7 and P10 CD1 pups and their response to standard and novel anticonvulsants like PB and bumetanide (BTN; potent NKCC1 antagonist) will help predict the efficacy of combination therapies immediate versus early after blocking KCC2 downregulation following neonatal stroke. The added inefficacy of novel anticonvulsant BTN; a potent NKCC1 antagonist detected in our previously funded study by the EFA (2011) has highlighted the importance of the differential efficacy of BTN in different seizure models. Especially of interest are our preliminary findings that KCC2 is downregulated after ischemia in this model whereas NKCC1 expression remains stable with trends towards upregulation. Therefore the mechanism by which acute seizures are induced in immature brains and the specific effect that protocol has on enhancing or worsening chloride cotransporter function by phosphorylation or degradation of KCC2 may dictate the efficacy of anticonvulsants that depend on the chloride gradient for their anti-seizure effects. Post-ischemic response of preventing KCC2 downregulation to immediate treatment with combination therapy of phenobarbital and NKCC1 antagonist bumetanide will be investigated and compared to early treatment after the occurrence of ischemic seizures and evaluated for sexual dimorphism.