This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.
Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.
It has been shown that interictal epileptiform discharges (IEDs) observed from EEG constitute useful markers of epileptogenesis in patients with focal epilepsy3. The regions inside the brain from which these IEDs originate, named irritative zones, can in practice be localized based on EEG recordings4. Preclinical models are essential to find correlates between these irritative zones and the actual regions underlying seizure initiation. However, recording EEG from small animals is challenging because of the small surface area of the head compared to the human scalp. Although invasive methods for chronic recording in rats can be used5, 6, techniques are not available at this moment to acquire traditional EEG recordings on rodents under acute conditions without the need of anesthesia.
To solve this problem, we apply a patented EEG mini-cap2, which allows us to record 32-channel EEG data from rodents noninvasively. In this study, we also provide evidence about the need of an analgesic to preserve IED frequency. Therefore, although fixation of EEG mini-cap was performed under isoflurane, EEG recordings were obtained with rats only under sedation (dexdomitor)7. The method proposed in this study can be used in any preclinical rat model of focal epilepsy. To illustrate the capabilities of this methodology, we apply it to understand the correlates between irritative and seizure-onset zones in focal cortical dysplasia (FCD). To that end, we use a “double-hit” model of FCD8 in Wistar rats.
To perform brain source analysis, it is required to: a) accurately extract IEDs from EEG raw data and b) obtain a volume conductor model for the individual animal head. To generate a practical volume conductor model, we use an in vivo rat MRI atlas, comprising average images of intensity/shape and obtained via non-linear registration of T2 images of 31 Wistar rats9. The forward model for the generated volume conductor was computed by boundary element method (BEM)10. As in the case of humans, two typical patterns of IEDs (sharp-waves and spikes) were detected and sub-classified into different clusters through an intelligent feature extraction, feature selection, and classification process11. These sub-classified signals are used to estimate the brain source localizations associated with different types of irritative zones. We present the source analysis steps using a well-known public software called Brainstorm12. The EEG source localizations for each IED sub-type and the seizure onset time frames were performed using standardized low-resolution brain electromagnetic tomography (sLORETA)13, which is available in Brainstorm.
一种新的方法,以非侵入性地记录多信道脑电图局灶性癫痫的特定临床前模型中进行说明。用于记录和分析程序的详情,与具体的实验提示,提供。有考虑取得成功结果的关键因素。首先,脑电图记录,获得高品质的信号是必不可少的。脑电图糊的适当粘度应该迷你帽制备过程中施加到每个电极,以及鼠的头部和耳朵发应在剃刮期间被完全除去。阻抗检查是为了确认脑电图记录质量的最重要的一步。其次,脑成像来源,产生量适当导体模式是至关重要的。每个表面应共登记。此外,所产生的电极的位置应具有对大鼠的头皮的实际电极的位置的最小距离误差。
尽管这个手稿源介绍采用头脑风暴12分析程序,他们可以使用其他软件开放16,17和商业产品18,19进行。此外,除了sLORETA 13,其他逆解决方案,如多偶极模型和波束形成器可用于4。
这种方法的一个限制是,行为分析,不能进行自脑电图记录镇静下进行。然而,相对于其它方法的脑电图记录大鼠5,6,这种方法是非侵入性的。
我们的初步结果支持为从脑电图记录IED标记,以确定在局灶性癫痫大鼠的刺激性区,以及评估其与癫痫发作起始11的基本机制关系的精确分类的重要性。此外,已经表明,脑电图源定位为这样的特定的IED表现出良好的对应关系与RESPective BOLD激活和去激活区20。
我们的研究将刺激使用临床前模型来评价生物医学工程师研制床工作台床的策略。例如,时下是手动的医院,这需要大量的人力精力进行IED提取。在这项研究中提出的方法自动完成。我们推测,当施加到患者FCD使用这种方法会产生类似的结果。我们正在准备的IRB协议的这个评价和对人的数据集的方法的其他方面。
此外,使用临床前模型将帮助我们理解在癫痫脑电图21源定位的能力和局限。脑源下属的癫痫准确的估计是治疗策略和手术规划至关重要。此外,具有大鼠脑电记录一个标准的平台将是有益的几种抗癫痫药物在临床前试验中的效力的评估。这是第一个研究中,癫痫的大鼠都记录非侵入镇静下,这将打开新的大门脑电图生物标志物用于癫痫的评价。然而,在该研究中提出的整个方法可延伸到其他的实验条件和脑部疾病。脑电图迷你帽,也可以使用在其他啮齿动物的类型。
在过去,在Wistar大鼠中一个前爪刺激范例已被用于评估记录的脑电图迷你盖2的数据的质量和再现性。此外,验证为脑源重建已执行从高分辨率颅骨脑电图同时录制与来自Wistar大鼠层局部场电位下一个晶须刺激范例22。这种方法已经开发Wistar大鼠,因为核磁共振图谱的存在对于这个特殊的鼠s火车。但是,它可以被应用到其它啮齿动物类型与图谱包括鼠标23,Sprague-Dawley大鼠24,以及Paxinos和Watson大鼠25的自己的标准格式。此外,我们所提出的方法的基本程序可在任何啮齿类动物临床前模型的量脑电图是一个重要的方式被使用。然而,这种方法的许多方面都特别为癫痫,特别是那些与脑电图预处理(IED检测和分类)。此外,研究人员必须知道在不同情况下使用镇静药物的正确。在我们的研究中使用异氟醚和dexdomitor已精心由于对简易爆炸装置的影响减少考虑。关于脑电图记录,在小鼠的情况下,相对小的头皮表面面积将大大减少信道数。
The authors have nothing to disclose.
作者要感谢佩德罗A.巴尔德斯埃尔南德斯,弗朗索瓦Tadel和劳埃德·史密斯提出宝贵的意见和富有成果的讨论。我们也感谢拉斐尔托雷斯的校对。
Data Qcquisition Computer | Hewlett-Packard | Z210 Workstation | |
Dexdomitor | Orion Pharma | 6295000 | Dexmedetomidine hydrochloride |
EEG Analysis Software | The Mathworks Inc. | MATLAB R2011b | |
Brainstorm | Sylvain et al. 2001 | ||
OpenMEEG | Bramfort et al. 2010 | ||
EEG Data Streamer | Tucker-Davis Technologies | RS4 Data Streamer | |
EEG Electrode Paste | Biotach | YGB 103 | |
EEG Preamplifier | BioSemi | Active Two | |
Brain Products | BrainAmp | ||
Tucker-Davis Technologies | PZ3 Low Impedance Amplifier | ||
EEG Processor | Tucker-Davis Technologies | RZ2 BioAmp Processor | |
EEG Recording Software | Tucker-Davis Technologies | OpenEx – OpenDeveloper | |
EEG SCSI Connector | BioSemi | Active Two SCSI Connector | |
Brain Products | D-sub Connector | ||
Tucker-Davis Technologies | Zif-Clif Digital Headstage | ||
High Resolution EEG Mini-cap | Cortech Solutions | DA-AR-ELRCS32 | US patent Application No. 13/641,834 |
Isoflurane, USP | VedcoPiramal Healthcare | NDC 66794-013-25 | |
Isopropyl Alcohol | Aqua Solutions | 3112213 | 90% v/v solution |
Lubricant Ophthalmic Ointment | Rugby | NDC 0536-6550-91 | Sterile |
NaCl | Abbott | 2B8203 | Vaterinary 0.9% Sodium Chroride Injection USP |
Physiology Recording Software | ADInstruments | LabChart 7.0 | |
Physiology Recording System | ADInstruments | PowerLab 8/35 | |
Syringe | Monoject | 200555 | 12cc |