【作者】 杨帆；

【导师】 林卫红；

【作者基本信息】 吉林大学 ， 神经病学， 2021， 博士

【摘要】 研究背景:手术是治疗难治性癫痫的有效方法,但如何精确定位致痫灶的位置,并且对致痫灶进行空间成像仍是一个挑战,尤其是在没有发现解剖异常的情况下。神经血管耦联(neurovascular coupling,NVC)描述了神经元活动、代谢变化和脑血流量之间的亲密关系。神经元激活、代谢增加可以导致脑血流量增加,同时脑血管的功能性充血反应,也增加了血液中的氧气含量,可以进一步增强神经元活动。基于神经血管耦联机制的脑成像技术已在临床工作中得到广泛应用,但依然对于癫痫发作期的神经血管耦联机制了解很少。目前,对癫痫发作期神经血管耦联机制的研究主要应用麻醉动物,并且只在致痫灶所处有限区域内进行采样。研究目的:本论文将探究癫痫发作期,清醒和异氟烷麻醉两种状态下,双侧大脑半球神经元活动与血流动力学反应之间的关系,探索造成两种状态间差异的作用机制;并探讨基于神经血管耦联的脑成像中可描绘癫痫发作时空分布的血流动力学信号源。研究方法:我们使用Thy1-GCa MP6f转基因小鼠,在大脑新皮层区构建4-氨基吡啶(4-Aminopyridine,4-AP)诱导的急性癫痫发作模型。之后,在清醒和麻醉两种状态下,应用玻璃电极记录癫痫发作起始处局部场电位(local field potential,LFP)的同时,利用广视野钙荧光信号成像和内源性信号光成像来描绘癫痫发作期的神经元活动和血流动力学变化的时空分布。最后向清醒小鼠体内静脉注射葡聚糖共轭异硫氰酸荧光素(dextran-conjugated fluorescein isothiocyanate,FITC-dx)破坏内皮细胞模拟麻醉作用,记录血流动力学变化并分析相关机制。研究结果:我们发现清醒状态下,小鼠癫痫发作有三种传播模式:第一种是癫痫样放电只局限于致痫灶周围;第二种癫痫发作呈对称性跳跃至对侧大脑半球同源区;第三种则可见癫痫样放电以致痫灶为中心呈同心圆状传播并平稳跨过中线传至对侧。同时,麻醉不仅影响癫痫发作自身的放电频率,致使局部场电位中高频脑电的比重增加、钙荧光信号的准确性降低,同时限制了癫痫发作向对侧大脑半球的扩散。另外,麻醉还减弱了血流动力学反应,引起脱氧血红蛋白(deoxygenated hemoglobin,Hbr)浓度升高的面积显著增加,大脑新皮层癫痫发作期组织缺氧面积增加。FITC-dx破坏清醒小鼠的内皮细胞,同样显示Hbr面积增加,而氧合血红蛋白(oxygenated hemoglobin,Hb O)浓度增幅降低播散面积缩小。总血红蛋白(total hemoglobin,Hb T)是脑血容量(cerebral blood volume,CBV)的反映,在清醒和麻醉动物中,是辨别新皮层癫痫发作起始和播散位置最可靠的信号源。研究结论:(1)清醒小鼠癫痫发作有多种传播模式,且传导机制多变,但异氟烷麻醉会限制癫痫的传播。麻醉下进行脑功能成像可能不能完全揭示致痫灶的位置。(2)异氟烷麻醉状态癫痫发作期的神经血管耦联机制不同于清醒状态,表现为癫痫发作期的神经元放电频率增加,血流动力学反应减弱。(3)麻醉剂异氟烷可能通过影响内皮细胞减弱血管功能性充血反应,对癫痫发作期神经血管耦联产生影响。(4)基于脑血容量或者Hb T的成像技术,比基于Hb O信号的血氧水平依赖的(blood oxygen level dependent,BOLD)功能磁共振成像(functional magnetic resonance imaging,f MRI),更能准确描绘癫痫发作的时空分布状态。更多还原

【Abstract】 Background:Unambiguously identifying an epileptic focus with high spatial resolution is a challenge,especially in cases where no anatomic abnormality can be detected.Neurovascular coupling describes the intimate relationship between neuronal activation,increased metabolism,and the resulting rise in cerebral blood flow,or functional hyperemia required to adequately oxygenate active neurons.Neurovascular couplingbased brain mapping techniques are often applied in clinic despite the unclear ictal neurovascular coupling mechanisms,which have been derived primarily from recordings in anesthetized animals with a limited spatial sampling of the ictal core.Objectives:We were going to investigate the relationship between neuronal activities and hemodynamic signals in ictal event in awake and anesthetized mice,and describe the mechanism of the difference between the two states,and explore a better signal for seizure mapping based on neurovascular coupling brain imaging.Method:Focal injection of 4-Aminopyridine(4-AP)was employed to create an acute neocortical seizure model in both anesthetized and awake Thy1-GCa MP6 f transgenic mice.The local field potential(LFP)was recorded through the 4-AP electrode.Meanwhile,a “temporal separation” technique was employed to simultaneously image wide-field calcium and a multispectral intrinsic optical signal.Finally,dextranconjugated fluorescein isothiocyanate(FITC-dx)was injected intravenously to investigate the role of vasodilation in our imaging results.Result:In awake mice,three different calcium propagation patterns were observed.In 9/17 seizures,the calcium signal remained ipsilateral.In 6/17 seizures,the seizure activity first propagated throughout the ipsilateral cortex and then jumped to the homotopic location to the 4-AP injection site in the contralateral hemisphere.In the remaining 2events,the seizure first propagated throughout the ipsilateral cortex and then contiguously propagated to the contralateral cortex.Isoflurane increases highfrequency activity in the LFP,reduces the accuracy of the calcium signal,and restricts contralateral electrographic spread.Anesthesia likewise blunts the hemodynamic response leading to a profound increase in the area of the rise in deoxygenated hemoglobin(Hbr)and rendering the “epileptic dip”.The destruction of endothelial cells by FITC-dx caused a similar decrease in oxygenated hemoglobin(Hb O)and an increase in Hbr.For awake and anesthetized mapping of ictal event,the signal derived from cerebral blood volume(CBV),or total hemoglobin(Hb T),provides the most reliable signal for mapping both the ictal onset and spread.Conclusion:(1)Ictal event has a variety of propagation patterns and mechanisms,but isoflurane-anesthesia will limit the propagation.Brain functional imaging under anesthesia may not unambiguously identify epileptogenic foci.(2)Isoflurane-anesthesia can affect the neuronal activities and blunt the hemodynamic response during seizures compared with unanesthesia.The mechanism of neurovascular coupling during ictal events in isoflurane-anesthetized mice is different from that in awake mice.(3)Isoflurane may reduce vascular functional hyperemia by interfering with endothelial cells and affect hemodynamics during seizures.(4)The CBV-based imaging techniques should be more accurate than blood oxygen level dependent(BOLD)-based imaging techniques for seizure mapping in awake and anesthetized patients and animals.更多还原