Name: brain source imaging in preclinical rat models of focal epilepsy using high-resolution eeg recordings
Uploaded: 2015-06-06T14:00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings at JoVE.com

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.

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
<ol>
	<li>Preparation of the EEG mini-cap
	<ol>
		<li>Immerse the electrode tips of the EEG mini-cap at least 12 hrs in distilled water with 0.2% chloride. Rinse the EEG mini-cap gently in distilled water. Dry the cap and the electrodes in the air.</li>
		<li>Mix EEG electrode paste with 0.9% NaCl solution in the volume proportion of 2 : 1. Add a drop of methylene blue, which will help to visualize the electrode paste inside the electrodes and on the skin. Take the mixed paste in a syringe. Make sure there are no air bubbles in the syringe. Inject the gel into each of the 32 electrodes, filling them without introducing any air bubbles. It is recommended to inject from the bottom rather than the top. This provides better access to each electrode and reduces the possibility of the gel spilling over.</li>
		<li>Turn on EEG and physiological recording system, and open the corresponding recording software on the computer in use.</li>
	</ol>
	</li>
	<li>Animal preparation and anesthesia 
	NOTE: Chronic epilepsy was created using a protocol for FCD8 in Wistar rats. The EEG recording were conducted in adult Wistar rats (8 weeks old, 300 - 400 g).
	<ol>
		<li>Record the rat&#8217;s weight in an experiment sheet. Use this information to calculate sedative dose (dexdomitor 0.25 mg/kg). Induce anesthesia in the rat with 5% isoflurane and 100% oxygen (1 L/min at 14.7 psi).</li>
		<li>After trim the rat&#8217;s head, reduce isoflurane to 2% and maintain it during the entire setting of the EEG mini-cap. Check rat reflexes are absent (toe-pinch).&#160;Place the rat on a heating pad in the stereotaxic apparatus by fixing the ear canals using ear bars. Make sure anesthesia nose cone is secure.</li>
		<li>Apply lubricant ophthalmic ointment to each eye.</li>
		<li>Shave the extra hair on the rat head and ears using a razor. Avoid any bleeding during shaving. 
		NOTE: Any hair left on the skin will produce noise in the EEG recordings. Rub the rat&#8217;s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin.</li>
		<li>Place a saline swab on the scalp and cover it completely to keep good skin conductance until the EEG mini-cap is ready to be placed.</li>
		<li>Connect temperature, respiration, and three lead electrocardiogram probes. Note that the temperature is measured by a rectal probe. Continuously monitor the physiology of the rat during the recording procedures. Ensure that the normal temperature is 37 &#176;C, respiration range is 30 - 60 breaths per minute, and heart rate is around 350 - 450 beats per minute .</li>
	</ol>
	</li>
	<li>Recording procedures
	<ol>
		<li>Remove the saline swab on the rat&#8217;s scalp and place the prepared EEG mini-cap on its skin. Fix the mini-cap with rubber bands. Place one rubber band on the front side of the scalp, usually in front of the eyes, and another band at the back of the scalp between the ears and neck. Use a plastic protector under the neck to facilitate normal respiration.</li>
		<li>Put a layer of high conductance electrode paste on both the ground and reference electrodes. Place them on the respective ear. 
		NOTE: The reference electrode can be possibly placed in other locations.</li>
		<li>Connect the EEG mini-cap to the amplifiers and observe a preview of the workbench for electrode impedance. Check the performance of all electrodes. For a high-quality recording, ensure that the impedance value is in the range of 5 - 30 k&#937;. If there are any noisy electrodes, provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode.</li>
		<li>Administer dexdomitor (0.25 mg/kg) intraperitoneally and immediately reduce isoflurane rate to 0%. If the respiration rate is not within 30 - 60 breaths per minute range, start increasing the isoflurane rate gently. Do not exceed the value of 1% isoflurane. Monitor this step carefully because the mixture of isoflurane and dexdomitor could prompt the animals to a critical condition. 
		NOTE: On the preclinical model of focal epilepsy, isoflurane affects IEDs, whereas dexdomitor does not. Subjects under isoflurane have weaker epileptogenic property, i.e., relatively fewer IEDs can be detected compared to other conditions7,14. The dexdomitor dose is effective for about 2 hrs. Thus, to save the time for its effect, the preparation was carried out under isoflurane.</li>
		<li>Conduct EEG recordings. After the recording, mark the positions of the three jutting circles of the EEG mini-cap on top of the skin by inserting a color pen inside them before the EEG mini-cap is removed. Use them as landmarks for MRI co-registration. Take a picture of the rat head with the landmarks. Place the rat back inside the cage and monitor it until complete recovery from dexdomitor&#8217;s effect. 
		NOTE: In this experiment, red color (opponent color to green) was used to distinguish from the electrode positions (green). However, it is recommended to use other colors (purple/green) if small bleeding spots are observed in the skin.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52700/52700fig1.jpg" /> 
Figure 1. A picture of the EEG mini-cap placed on a particular rat.
<a href="https://www.jove.com/files/ftp_upload/52700/52700fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Brain Source Imaging
<ol>
	<li>IED classification 
	NOTE: The IED detection and classification is performed using self-developed codes in MATLAB based on the previous study15. This software will be available by request.
	<ol>
		<li>Discard noisy channels by visually inspecting the EEG tracers. Remove EKG artifacts using an automatic method for periodic waveform subtraction, which is based on a template and a correlation analysis. 
		NOTE: Usually, the experimenter who recorded the EEG shares the written experimental sheet for the observed bad channel information based on the impedance values. Software to remove EKG artifacts will be also available by request. 
		<img alt="Figure 2" src="/files/ftp_upload/52700/52700fig2.jpg" /> 
		Figure 2. An example of the EEG trace showing different types of IEDs. The red box indicates one type of IEDs. 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
		&#160;</li>
		<li>Apply a band-pass filter with cutoff frequencies of 3 - 150 Hz and a notch filter to remove the line frequency (60 Hz in general and 50 Hz in some countries) component offline.</li>
		<li>Detect two types of IEDs (spikes and sharp-waves). Spikes and sharp-waves constitute large electrical events of 20 - 70 ms and 70 - 200 ms in duration respectively. Therefore, after applying a respective band-pass filter (cutoff frequencies of 15 - 50 Hz for spikes and 5 - 15 Hz for sharp-waves), the IEDs are detected based on amplitude thresholds15. 
		NOTE: The thresholds are automatically set to 4&#963; as suggested in the previous study for multiunit activity15. Here, &#963; is an estimated standard deviation of the bandpass-filtered signal, &#963; = median{|filtered signal| &#8260; 0.6745}.</li>
		<li>Sub-classify spikes and sharp-waves into different clusters. The distinctive features of different spikes and sharp-waves are extracted using wavelet transform15. They are sub-classified into multiple clusters using k-means, and the optimal cluster number k is determined using silhouette.</li>
		<li>Average the sub-classified signals within the same cluster. The average EEG signals for each IED sub-type will be used for brain source analysis.</li>
	</ol>
	</li>
	<li>Volume conductor model 
	NOTE: For the following sections, the open source software, Brainstorm12, will be used with the MRI atlas for Wistar rats9. However, individual rat&#8217;s MRI can also be used to generate the volume conductor model if available. The MRI atlas9 can be downloaded at 
	<a href="http://www.idac.tohoku.ac.jp/bir/en/db/rb/101028.html">http://www.idac.tohoku.ac.jp/bir/en/</a>. This website provides the atlas as NIFTI format under &#8220;Wistar Rat MRI Atlas&#8221; section, and it can be accessible after registration. The software needed for pre-processing can be also found in this website.
	<ol>
		<li>Input MRI and brain surface to the software12. 
		<img alt="Visual 1" src="/files/ftp_upload/52700/52700vis1.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Generate head surface with default setting. 
		<img alt="Visual 2" src="/files/ftp_upload/52700/52700vis2.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Generate scalp and inner/outer skull surfaces based on MRI for lead field computation12. 
		NOTE: The resolution of the vertices influences the accuracy of the estimated source, but large number of vertices results in high computational complexity. Recommended number of vertices of each layer is 642 for acceptable accuracy with fair computational complexity. The thickness of the skull can be checked from the MRI, and in the case of the MRI atlas, it is approximately 1 mm. After inserting above values in the software, corresponding triangle face-vertex meshes for each surface will be created. 
		<img alt="Visual 3" src="/files/ftp_upload/52700/52700vis3.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Check the orientation and location of each surface with respect to the MRI using the visualization option. Modify accordingly, if any surfaces are not co-registered12. 
		<img alt="Visual 4" height="351" src="/files/ftp_upload/52700/52700vis4.jpg" width="321" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Using the rat head picture acquired in 1.3.5. co-register the positions of the 3 landmarks (R1, R2, and R3) in the MRI. Use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold (Figure 3B). 
		<img alt="Figure 3" src="/files/ftp_upload/52700/52700fig3.jpg" /> 
		Figure 3. (A) Rat head picture used to obtain electrode positions and (b) the EEG mini-cap diagram with the coordinate system. Red dots in (A) indicate the landmarks mentioned in 1.3.5. which correspond to the red numbers in (B). Also, the green marks in (A) depict the 32 electrode positions, and they correspond to the blue numbers in (B). 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
		&#160;</li>
		<li>Generate N &#215; 3 electrode position matrix based on the 3 landmarks. Here, N is the number of channels (N = 32) and the column represents the corresponding x, y, and z coordinate values. 
		NOTE: The EEG mini-cap is a rigid scaffold. Therefore, once the 3 reference grids (R1, R2, and R3) are obtained, the position of the electrodes are automatically set. The user will only need to redefine the Z-values on a way that the mini-cap is appropriately projected on the scalp. The N point grids can be numbered sequentially as depicted in Figure 3B blue numbers. The standard scaffold for the EEG mini-cap is commercially available (Table of Materials). The software for co-registration is also available to the community.</li>
		<li>Input the generated channel file. 
		<img alt="Visual 5" src="/files/ftp_upload/52700/52700vis5.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Display and confirm the location of all electrodes. Modify any misplaced electrodes12. The final coordinate system for the electrode positions should coincide with the coordinate system used for the above-mentioned surfaces. 
		NOTE: The created surfaces can be visually inspected on an MRI by using the visualization option, &#8220;MRI registration\Check MRI/surface registration.&#8221; And then, a selected surface will be displayed as yellow line on the MRI. In addition, the 3 landmarks and the 32 electrode positions can be displayed on the MRI by selecting the toolbox&#8217;s option, &#8220;Display Sensors\MRI Viewer.&#8221; The locations can be visually inspected by comparing the distributions based on the rat&#8217;s eye and ear locations (Figure 4). 
		<img alt="Figure 4" src="/files/ftp_upload/52700/52700fig4.jpg" /> 
		Figure 4. (A) MRI atlas with co-registered brain surface (yellow line), (B) the created volume conductor model with the aligned 32 electrodes and 3 landmarks (red dots), and (C) MRI atlas with co-registered reference grid R1. 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
	</ol>
	</li>
	<li>Brain Source Imaging
	<ol>
		<li>Compute lead field matrix13. Input the conductivity values which satisfy the ratio of skin, skull, and brain as 1 : 1/80 : 1. Obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2. 
		NOTE: The toolbox12 offers the interface with the other software to compute BEM10. Therefore, only the conductivity values are required as input. 
		<img alt="Visual 6" src="/files/ftp_upload/52700/52700vis6.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Input the average EEG signals for each IED sub-type stored in 2.1.4. 
		<img alt="Visual 7" src="/files/ftp_upload/52700/52700vis7.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Obtain sLORETA solution13 based on the computed lead field matrix and the input EEG signals. By selecting the source estimation method option, the inverse solution can be obtained12. 
		<img alt="Visual 8" src="/files/ftp_upload/52700/52700vis8.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/52700/52700vis8highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
		<li>Plot the estimated sources.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

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&#8217;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&#8217;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&#8217;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.

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 &#8220;double-hit&#8221; 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Data Qcquisition Computer</td>
			<td>Hewlett-Packard</td>
			<td>Z210 Workstation</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexdomitor</td>
			<td>Orion Pharma</td>
			<td>6295000</td>
			<td>Dexmedetomidine hydrochloride</td>
		</tr>
		<tr>
			<td>EEG Analysis Software</td>
			<td>The Mathworks Inc.</td>
			<td>MATLAB R2011b</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Brainstorm</td>
			<td>Sylvain et al. 2001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>OpenMEEG</td>
			<td>Bramfort et al. 2010</td>
		</tr>
		<tr>
			<td>EEG Data Streamer</td>
			<td>Tucker-Davis Technologies</td>
			<td>RS4 Data Streamer</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG Electrode Paste</td>
			<td>Biotach</td>
			<td>YGB 103</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG Preamplifier</td>
			<td>BioSemi</td>
			<td>Active Two</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Brain Products</td>
			<td>BrainAmp</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Tucker-Davis Technologies</td>
			<td>PZ3 Low Impedance Amplifier</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG Processor</td>
			<td>Tucker-Davis Technologies</td>
			<td>RZ2 BioAmp Processor</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG Recording Software</td>
			<td>Tucker-Davis Technologies</td>
			<td>OpenEx - OpenDeveloper</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG SCSI Connector</td>
			<td>BioSemi</td>
			<td>Active Two SCSI Connector</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Brain Products</td>
			<td>D-sub Connector</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Tucker-Davis Technologies</td>
			<td>Zif-Clif Digital Headstage</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution EEG Mini-cap</td>
			<td>Cortech Solutions</td>
			<td>DA-AR-ELRCS32</td>
			<td>US patent Application No. 13/641,834</td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>VedcoPiramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Aqua Solutions</td>
			<td>3112213</td>
			<td>90% v/v solution</td>
		</tr>
		<tr>
			<td>Lubricant Ophthalmic Ointment</td>
			<td>Rugby</td>
			<td>NDC 0536-6550-91</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Abbott</td>
			<td>2B8203</td>
			<td>Vaterinary 0.9% Sodium Chroride Injection USP</td>
		</tr>
		<tr>
			<td>Physiology Recording Software</td>
			<td>ADInstruments</td>
			<td>LabChart 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiology Recording System</td>
			<td>ADInstruments</td>
			<td>PowerLab 8/35</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Monoject</td>
			<td>200555</td>
			<td>12cc</td>
		</tr>
	</tbody>
</table>

brain source imaging in preclinical rat models of focal epilepsy using high-resolution eeg recordings

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.

&#160;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.
<img alt="Visual 6" src="/files/ftp_upload/52700/52700fig5.jpg" />
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). 
<a href="https://www.jove.com/files/ftp_upload/52700/52700fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
<img alt="Visual 6" src="/files/ftp_upload/52700/52700fig6.jpg" /> 
Figure 6. Estimated brain sources during seizure.&#160;The time instants are marked as red vertical lines. 
<a href="https://www.jove.com/files/ftp_upload/52700/52700fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings at JoVE.com

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

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 ...

brain-source-imaging-preclinical-rat-models-focal-epilepsy-using-high

Research

JoVE Journal

Medicine

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...