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.
Ethics statement: All experiments are performed following the policies established by the Institutional Animal Care and Use Committee (IACUC) at Florida International University (IACUC 13-004).
1. EEG Recordings
Figure 1. A picture of the EEG mini-cap placed on a particular rat.
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2. Brain Source Imaging
Once all procedures are properly completed, estimated sources can be visualized in the brain surface of the pre-clinical model. Figure 5 shows the estimated sources from one particular sub-type of spikes (top) and sharp-waves (bottom) from IEDs. In addition, Figure 6 displays how the source distribution changes in sequential time frames during a seizure establishment. These results support the capability of the proposed methodologies to record high-resolution EEG on rats with focal epilepsy and to conduct source analysis using the recorded EEG.
Figure 5. Estimated brain source locations of IEDs with respect to different clusters in spikes (top) and sharp-waves (bottom). (A) Time series, (B) EEG topography, and (C) cortical current sources. The evaluation is performed at a specific time marked with a red vertical line in (A).
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Figure 6. Estimated brain sources during seizure. The time instants are marked as red vertical lines.
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A new methodology to non-invasively record multichannel EEG in a particular preclinical model of focal epilepsy is described. The particulars for the recording and analysis procedures, with specific experimental tips, are provided. There were key factors to consider achieving successful results. First, for EEG recordings, obtaining high quality signals is essential. Proper viscosity of the EEG paste should be applied to each electrode during the mini-cap preparation, and the rat’s head and ear hair should be completely removed during shaving. Impedance check is the most important step to confirm the quality of EEG recordings. Second, for brain source imaging, generating proper volume conductor model is crucial. Each surface should be co-registered. Also, the generated electrode positions should have minimum distance error from the actual electrode locations on the rat’s scalp.
Even though this manuscript introduces source analysis procedures using Brainstorm12, they can be conducted using other open softwares16,17 and commercial products18,19. Also, besides sLORETA13, other inverse solutions such as multiple dipole models and Beamformer can be applied4.
One limitation of this approach is that behavior analysis cannot be conducted since the EEG recording is carried out under sedation. However, compared to the other methods for EEG recording in rats5,6, this approach is noninvasive.
Our preliminary results support the importance for a precise classification of IED markers from EEG recordings to determine the irritative zones in a rat with focal epilepsy, as well as to evaluate their relationship with the underlying mechanisms for seizure initiation11. In addition, it has been shown that EEG source localization for such specific IEDs showed a good correspondence with the respective BOLD activation and deactivation regions20.
Our study will stimulate the use of preclinical models to evaluate bed-bench-bed strategies developed by biomedical engineers. For example, IED extraction is nowadays performed in hospitals manually, which required a considerable human effort. The methodology proposed in this study does it automatically. We hypothesize that the use of this methodology will produce similar results when applied to patients with FCD. We are preparing IRB protocols for the evaluation of this and other aspects of the methodology in human dataset.
Moreover, the use of preclinical models will help us understand the capabilities and limitations of EEG source localization in epilepsy21. Accurate estimation of brain sources underling epileptogenesis is crucial for therapeutic strategies and surgical planning. Also, having a standard platform for EEG recording in rats will be useful for the evaluation of the effectiveness of several anti-epileptic drugs in preclinical trials. This is the first study in which epileptic rats are recorded non-invasively under sedation, which will open new doors for the evaluation of EEG biomarkers for epilepsy. However, the entire methodology presented in this study is extendable to other experimental conditions and brain disorders. The EEG mini-cap can be also used in other rodent’s types.
In the past, a forepaw stimulation paradigm in Wistar rats has been used to evaluate the quality and reproducibility of data recorded with the EEG mini-cap2. Moreover, validations for the brain source reconstruction have been performed from high-resolution skull EEG concurrently recorded with laminar local field potentials from Wistar rats under a whisker stimulation paradigm22. This methodology has been developed for Wistar rats because of the existence of an MRI atlas for this particular rat strain. However, it can be applied to other rodent types with their standard format of atlas including mouse23, Sprague-Dawley rats24, and Paxinos and Watson rats25. In addition, the fundamental procedures of our proposed methodology could be used in any rodent preclinical models for which EEG is an important modality. However, many aspects of this methodology are particularly for epilepsy, especially those related to EEG preprocessing (IED detection and classification). Also, researchers must be aware of proper drugs used for sedation in different cases. The use of isoflurane and dexdomitor in our study has been carefully considered due to the reduced impact on IEDs. Regarding EEG recordings, in the case of mouse, the relatively small scalp surface area would reduce the number of channels considerably.
The authors have nothing to disclose.
The authors would like to thank Pedro A. Valdes Hernandez, Francois Tadel, and Lloyd Smith for their valuable advice and fruitful discussion. We also thank Rafael Torres for the proof reading.
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 |