This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2014R1A1A3049456) and a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI15C2854).

All experimental procedures were approved by the Ethics Committee of the Catholic University of Korea and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23).
1. SE Induction
<ol>
	<li>Purchase 8-week-old male C57BL/6NHsd mice and weigh each mouse. Then, use a marker pen to mark the tails of all mice for their easy identification during SE induction.</li>
	<li>Calculate the amount of scopolamine methyl bromide (scopolamine; 2 mg/kg), terbutaline hemisulfate (terbutaline; 2 mg/kg), and pilocarpine hydrochloride (pilocarpine; 280 mg/kg) based on the mouse weight and add saline (0.9% NaCl, 10 mL/kg) to make solutions. 
	Note: For example, if the weight of the mouse is 25 g, the following amounts are applied: Scopolamine and terbutaline: 2 mg/kg * (25 g/1,000 g) = 0.05 mg, Saline: 10 mL/kg * (25 g/1,000 g) = 0.25 mL, Pilocarpine: 280 mg/kg * (25 g/1,000 g) = 7 mg, Saline: 10 mL/kg * (25 g/1,000 g) = 0.25 mL.</li>
	<li>Load the scopolamine and terbutaline mixture in a 1 mL syringe with a 30 G needle. Inject the solution intraperitoneally into each mouse, and then return the mice to their cages. At 30 min after scopolamine and terbutaline administration, inject pilocarpine solution into each mouse (i.p.; 1 mL syringe; 30 G needle). Immediately after pilocarpine injection, place the mice in an incubator (28-30&#176;C) for observation.</li>
	<li>Carefully monitor the behavior of the mice until SE is induced. If limbic motor seizures that correspond to stage 3 or higher according to Racine&#39;s scale are detected, record the time and monitor the mice to determine whether the seizures occur more frequently. Once continuous motor seizures last more than 2 min, place the mouse in a new cage at room temperature and keep monitoring for 3 h to determine whether their convulsive seizures continue and SE is induced. Euthanize the mice that failed to enter SE at about 2 h after pilocarpine injection. 
	Note: Racine&#39;s scale; stage 1, mouth and facial movement; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing with forelimb clonus; stage 5, rearing and falling with forelimb clonus16. If the mouse is not transferred to the cage at room temperature immediately after SE induction, the mouse can die due to hyperthermia.</li>
	<li>Terminate behavioral acute seizures at 3 h after SE onset by injecting diazepam solution (i.p.; 10 mg/kg; 1 mL syringe; 30 G needle). Make the diazepam solution by diluting 5 mg/mL of diazepam in 10% polyoxyl 35 hydrogenated castor oil by adding saline (i.p.; 10 mL/kg; 1 mL syringe; 30 G needle).&#160; 
	Note: 10% polyoxyl 35 hydrogenated castor oil solution: 1 mL polyoxyl 35 hydrogenated castor oil solution + 9 mL saline. Store the solution at room temperature. For example, if the mouse body weight is 25 g, inject 250 &#181;L of diazepam solution: 50 &#181;L 5 mg/mL diazepam + 200 &#181;L 10% polyoxyl 35 hydrogenated castor oil solution = 250 &#181;L 1 mg/mL diazepam.</li>
	<li>For sham mice, perform an i.p. injection of the scopolamine methyl bromide and terbutaline hemisulfate mixture (i.p.; both 2 mg/kg, 10 mL/kg; 1 mL syringe; 30 G needle). 30 min later, inject the saline intraperitoneally (i.p.; 10 mL/kg; 1 mL syringe; 30 G needle). 
	Note: If the mouse body weight is 25 g, inject 250 &#181;L of saline instead of pilocarpine.</li>
	<li>After diazepam injection (i.p.; 1 mL syringe; 30 G needle), administer 1 mL of 5% dextrose per individual mouse (i.p.; 1 mL syringe; 26 G needle). 
	Note: 1 mL of 5% dextrose injection provides energy source and hydration that can increase the survival rate.</li>
	<li>During post-care in the incubator (28-30 &#176;C), wipe off excessive secretions such as saliva, tears, and feces.</li>
	<li>At day 1 after SE induction, weigh the mice and keep them in the incubator (28-30 &#176;C) for one extra day. At day 2, after SE induction, weigh the mice and return them to their home cage. Provide moist chow to facilitate their recovery. 
	Note: In this experiment, the mortality rate for C57BL/6NHsd after SE termination was 8.57% on average (3 out of 35 mice tested).</li>
	<li>Measure daily body weight of the animals until 7 days post-pilocarpine and inject the mice with 5% dextrose(i.p.; 1 mL syringe; 26 G needle) when the body weight has not increased. Stop body weight monitoring when the mice start to regain body weight and consume moist chow without difficulty at 2 days after pilocarpine injection. 
	Note: If the body weight of a mouse did not increase until 7 days post SE, exclude the mouse from the experiment and euthanize by carbon dioxide. Depending on the background of the mouse, death can occasionally occur; however, in the case of C57BL/6NHsd, less than 1% of mice died in these experiments. With survival of more than 7 days after SE induction, the mice rarely die during the latent period.</li>
</ol>
2. Implantation Surgery for Video-EEG Monitoring
<ol>
	<li>Prepare 12-week-old mice (4 weeks after SE induction) to perform telemetry implantation for EEG monitoring. 
	Note: Timing of implantation can be modified depending on the experimental purposes and the mouse strains.</li>
	<li>Before surgery, anesthetize the mouse with a mixture (4:0.5) of ketamine (50 mg/mL) and xylazine (23.3 mg/mL) solution dissolved in saline at a dose of 2.5 &#181;L/g body weight (i.p.; 1 mL syringe; 26 G needle). Monitor respiratory rate and respiratory effort of the mouse at regular intervals (15 min interval maximum) and assess the depth of anesthesia by the pedal withdrawal reflex.</li>
	<li>Place the mouse in a stereotaxic frame with ear bars and a bite plate and apply vet ointment on both eyes to avoid blindness.</li>
	<li>To maintain sterile conditions during the surgery, shave surgical sites using a razor blade. Be careful to prevent fur contamination of the surgical field. After shaving, disinfect the skin with 70% ethanol and iodine solution. 
	Note: Surgery should be performed in an aseptic manner wearing sterile gloves and masks, using sterilized equipment and aseptic techniques.</li>
	<li>After confirming the depth of anesthesia, make a midline incision of the skin to expose the skull. Make two burr holes with a drill attached to the stereotaxic apparatus at the coordinates from the Bregma in the anteroposterior axis (AP): +0.1 mm, mediolateral axis (ML): +0.1 mm (Reference), and AP: &#8722;0.2 mm, ML: +0.22 mm (Left parietal cortex)13.
	<ol>
		<li>Wipe the skin with PBS-soaked cotton swaps, followed by 70% ethanol and iodine solution. Make a longitudinal incision in the middle of the head with scissors to expose the Lambda (the point where the sagittal and the lambdoid suture meet), the Bregma (the point of intersection of the coronal suture by the sagittal suture), and the target location.</li>
		<li>Identify anatomical landmarks such as Lambda and Bregma. Position the drill bit at Bregma and note the X, Y coordinates for this point.</li>
		<li>Use a brain atlas books or published references to determine the appropriate coordinates of the brain regions for EEG recording. Then, calculate the coordinates for the screws (reference and recording) using the Bregma coordinates measured at step 2.5.2.</li>
		<li>Slowly lower the drill bit to make a burr hole being careful to avoid inserting the drill bit too far at the point where the skull becomes thinner.</li>
	</ol>
	</li>
	<li>Insert the body of the single channel wireless EEG transmitter behind the nape of the neck subcutaneously and place flexible leads near the skull.</li>
	<li>Place a stainless-steel screw (2 mm length) into each burr hole with tweezers and tighten it using a screw driver by clockwise rotation. Make sure the screw is not inserted too far deep by adjusting the degree of screw rotation in order to prevent damage to the dura or the brain tissues.&#160;</li>
	<li>Strip the insulation coat 2 mm from the tip of the leads and stretch the wire long enough to round the screw shaft for a good connection between the wire and the screw. Connect the reference lead to the front screw and the recording lead to the screw in the parietal cortex. Then, apply dental cement to secure the entire assembly in place without exposing the metal parts.</li>
	<li>After verifying that the dental cement has completely dried by visual inspection, suture the skin and apply topical mupirocin ointment. Place the mouse in a 30 &#176;C incubator alone until it recovers from the surgery (about 30 min). Then, return the mouse to the home cage until continuous video-EEG monitoring begins. 
	Note: After surgery, each mouse is injected (i.p.; 1 mL syringe; 30 G needle) with 5 mg/kg of gentamicin (antibiotics) and 5 mg/kg of ketoprofen (analgesics). Animals should be monitored until they become fully ambulant. Transmitter-implanted mice have a recovery period of approximately 1 week.</li>
</ol>
3. Video and EEG Monitoring and Analysis
<ol>
	<li>Place a mouse in an individual cage and place the cage on top of the wireless receiver plates where the Faraday cage is installed to avoid interruption of electrical signals from any sources including a computer, AC lines, and adjacent receivers. 
	Note: Refer to the manual provided by the manufacturer. Care must be taken to avoid blind spots.</li>
	<li>Record the electrical activity using the wireless video-EEG telemetry system.
	<ol>
		<li>Open the software for video-EEG recording. Click &#34;Hardware&#34; and select &#34;Edit configuration&#34;. Match the receiver and the transmitter implanted into each mouse. Click &#34;Channel configuration&#34; and confirm all the channels are active. Click &#34;Video configuration&#34; to synchronize the video with the EEG data (resolution of 40 x 480 and frame rate of 30.00). 
		Note: Install an IP camera with high sensitivity if it is critical to collect good image qualities that can identify subtle behavioral changes of the mice at night.</li>
		<li>Click &#34;Setup&#34; and &#34;P3 setup&#34; to input individual animal information, matched camera, and graph setting.</li>
		<li>Click &#34;Acquisition&#34; and &#34;A/D sampling rate&#34; to setup sampling rates. Click &#34;Data set name&#34; to designate each experiment. 
		Note: Sampling rate for EEG needs to be higher than 500 Hz. Due to the large video and EEG data size, it is recommended that only 24 h of data are handled per session rather than using a continuous 2-week-recording as one file. Data can be separately saved at 8 h or 12 h intervals in case of RAM memory shortage.</li>
		<li>Activate the transmitter with a magnetic bar and click &#34;Start acquisition&#34; to collect the video-EEG recording. Continuously monitor the mouse by video-EEG system for at least 2 weeks. 
		Note: For C57BL/6 mice, SRS tends to occur in clusters that last about 5.2 days and the duration of the seizure-free period is about 6.7 days17.</li>
	</ol>
	</li>
	<li>Determine the seizure frequency and duration by manual inspection using the analysis software.
	<ol>
		<li>Mark the area showing SRS and export the data for statistical analysis. Be cautious to exclude EEG artifacts that can be generated by scratching the head or chewing; these can be removed by checking the video data. 
		Note: SRS is defined as a sudden onset of repetitive epileptiform spiking activity (&#8805; 3 Hz) that persists for more than 10 s. Convulsive behaviors can be frequently accompanied with a burst of spiking activity. Non-convulsive seizures can also demonstrate electrographic spiking activities without convulsive behaviors. Seizure termination is defined as occurring when the firing spikes return to baseline activity.</li>
		<li>For analysis of seizure frequency, divide the total number of SRS cases detected during the 2 weeks by the total number of recording days for each individual animal.</li>
		<li>For analysis of seizure duration, measure the time from the onset to the end of the epileptiform spiking activities.</li>
	</ol>
	</li>
	<li>After completion of the video-EEG recording, fix the brain with 4% paraformaldehyde by transcardial perfusion.&#160;To anesthetize the mouse, inject a solution (4:0.5) of ketamine (50 mg/mL) and xylazine (23.3 mg/mL) dissolved in saline at a dose of 10 &#956;L/g body weight (i.p.; 1 mL syringe; 26 G needle). Once the mouse is confirmed to be deeply anesthetized, perform transcardial perfusion.</li>
	<li>Before isolating the mouse brain, retrieve the transmitter from the mouse for reuse after cleansing.
	<ol>
		<li>Remove the dental cement around the leads using forceps.</li>
		<li>Soak the tip of the leads with dental cement in 100% acetone until the dental cement is dissolved.</li>
		<li>Remove tissue contaminants around the transmitter by rinsing it with distilled water.</li>
		<li>Rinse the transmitter with 70% ethanol for 3 h and air dry it for storage. Immediately prior to the next use, rinse the screw and transmitter with 70% ethanol, followed by distilled water, and place them in saline. 
		Note: Care must be taken to avoid cutting the flexible wires when decapitating the mouse since, if the flexible leads are too short, the noise in the next implantation and video-EEG recording may increase.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This work describes the experimental procedures for the SE induction and the evaluation of chronic seizures.
Several factors can affect successful SE induction. Accurate behavioral monitoring according to the Racine scale is critical for the development of SRS. Head nodding, forelimb clonus, rearing, and falling are the behavioral hallmarks of acute seizures developing into SE phase4,16. Once the first motor seizure is detected, the le...

TLE is one of the most common acquired epilepsies1. People with epilepsy experience recurrent seizures as a result of abnormal neuronal activities in the brain2,3. Given that TLE is often intractable, it is crucial to understand the basic mechanisms underlying the development of epilepsy.
Animal models that can recapitulate the key characteristics of human TLE can offer better appreciation of TLE pathophysiology, allowing us to readily monitor and manipulate critical factors in epileptogenesis. Among them, chemoconvulsants-induced SE has been widely used4,5. Unlike other epilepsy models, such as electrical stimulation which shows no hippocampal sclerosis and robust SRS6,7,8, the systemic injection of chemoconvulsants can mimic clinical pathogenesis of human TLE, i.e., initial brain injury, a latent period, and a chronic epileptic stage manifesting SRS5,9,10. Therefore, this technique can be utilized in various studies explaining the mechanisms of acute brain damage, epileptogenesis, or seizure suppression. Moreover, histopathological alterations induced by chemoconvulsants are similar to those seen in human TLE, providing an additional rationale for use of TLE rodent models10,11,12. Notably, structural damages involving the hippocampus have been consistently reproduced in both kainic acid- and pilocarpine-induced SE models. However, compared to kainic acid injection, the pilocarpine model can produce more robust SRS in mice, which can offer sizable advantages for studying chronic epilepsy when considering the wide availability of transgenic mouse lines5,13,14,15. Moreover, seizure progression after pilocarpine injection is generally faster than in the kainic acid model, providing additional evidence for the effective use of a pilocarpine model of epilepsy.
Here, we demonstrate a method of inducing SE by the i.p. injection of pilocarpine and by performing video and EEG monitoring in chronic epilepsy.

<table border="0" cellpadding="0" cellspacing="0" style="width:829px;" width="829">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:189px;">C57BL/6</td>
			<td style="width:233px;">Envigo</td>
			<td style="width:175px;">C57BL/6NHsd</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scopolamine methyl nitrate</td>
			<td>Sigma</td>
			<td>S2250</td>
			<td>Make 10X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Terbutaline hemisulfate salt</td>
			<td>Sigma</td>
			<td>T2528</td>
			<td>Make 10X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pilocarpine hydrochloride</td>
			<td>Sigma</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Intensive care unit</td>
			<td>Daejong instrument industry Co., Ltd.</td>
			<td></td>
			<td>28~30&#8451;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine hydrochloride</td>
			<td>Yuhan corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylazine hydrochloride</td>
			<td>Bayer Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Diazepam</td>
			<td>SAMJIN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Castor oil (Kolliphor EL)</td>
			<td>Sigma</td>
			<td>C5135</td>
			<td>Polyoxyl 35 hydrogenated castor oil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Saline</td>
			<td>Daihan pharm. Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5% Dextrose</td>
			<td>Daihan pharm. Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Iodine solution (Povidin)</td>
			<td>Firson</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">vet ointment (Terramycin)</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue Nylon</td>
			<td>AILEE</td>
			<td>NB617</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mupirocin (Bearoban)</td>
			<td>Daewoong Pharmaceutical Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketoprofen</td>
			<td>Samchundang Pharm. Co., Ltd</td>
			<td></td>
			<td>5 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gentamicin</td>
			<td>Huons, Ltd.</td>
			<td></td>
			<td>5 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1 mL syringe</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">26 guage needle</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td>26 G * 13 mm (1/2&#34;)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">30 guage needle</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td>30 G * 13 mm (1/2&#34;)</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Razor blade</td>
			<td>Dorco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Drill</td>
			<td>Saeshin precision Co., Ltd.</td>
			<td></td>
			<td>207A, 35K (speed)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Telemetry video/EEG system</td>
			<td>Data sciences International. Inc.</td>
			<td>Version 5.20-SP6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Implantable transmitter</td>
			<td>Data sciences International. Inc.</td>
			<td>ETA-F10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Screw</td>
			<td>Sungho Steel</td>
			<td></td>
			<td>M1.4, 2 mm length stainless steel</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vertex dental material&#160;</td>
			<td>Dentimex</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetone</td>
			<td>Duksan pure chemicals Co., Ltd.</td>
			<td></td>
			<td>CAS 67-64-1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde (PFA)</td>
			<td>millipore</td>
			<td>1.04005.1000</td>
			<td>4 %&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>30 % solution in 0.01 M PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cresyl violet acetate</td>
			<td>Sigma</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>EMD Millipore Co.</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">xylene</td>
			<td>Duksan pure chemicals Co., Ltd.</td>
			<td>UN1307</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetic acid glacial</td>
			<td>Junsei chemical</td>
			<td>31010-0350</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FSC33 Clear&#160;</td>
			<td>Leica biosystems</td>
			<td></td>
			<td>OCT compound for tissue freezing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPX Mounting for histology</td>
			<td>Sigma</td>
			<td>6522</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps</td>
			<td>Fine science tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scissors</td>
			<td>Solco biomedical</td>
			<td>02-2445</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Stereotaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>E51070012</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Successful SE can induce hippocampal cell death and SRS (Figure 1 and Figure 2). We terminated behavioral acute seizures by diazepam injection at 3 h after SE onset and sacrificed the mice 7 days or 6 weeks later.
For video-EEG monitoring, the mice received implant surgery at 4 weeks after SE, and SRS cases were evaluated for 2 weeks from 5-7 weeks after SE onset.
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Watch this Scientific Journal Video about The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice at JoVE.com

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

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 ...

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JoVE Journal

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19.4K Views.  The Catholic University of Korea. 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.

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

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal fluctuations over time. Temporal Lobe Epilepsy (TLE) is the most common type of adult epilepsy and involves multiple brain networks. The default mode network (DMN) is involved in conscious, resting state cognition and is thought to be affected in TLE where seizures cause impairment of consciousness. The DMN in epilepsy was examined using seed based fcMRI. The anterior and posterior hubs of the DMN were used as seeds in this analysis. The results show a disconnection between the anterior and posterior hubs of the DMN in TLE during the basal state. In addition, increased DMN connectivity to other brain regions in left TLE along with decreased connectivity in right TLE is revealed. The analysis demonstrates how seed-based fcMRI can be used to probe cerebral networks in brain disorders such as TLE.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

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.

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 ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Assessment of Static Graviceptive Perception in the Roll-Plane using the Subjective Visual Vertical Paradigm

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow the examination of all semicircular canals in the clinical setting. Assessment methods of the otolithic system, which is responsible for the perception of linear acceleration and perception of gravity, are far less in clinical use. There are several experimental approaches for measuring the perception of gravity. The most frequently used method is the determination of the subjective visual vertical. This is usually measured with the head in an upright position. We present here an assessment method for testing otolith function in the roll plane. The subjective visual vertical is measured in the head upright position as well as with head inclination of &#177; 15&#176; and &#177; 30&#176; in the roll plane. This extended functional paradigm is an easy-to-perform clinical test of otolith function and ensures increased information content for the detection of impaired graviceptive perception.

The perception of gravity is commonly determined by the subjective visual vertical in the head upright position. The additional assessment at head tilts of &#177; 15&#176; and &#177; 30&#176; in the roll plane ensures increased information content for the detection of impaired graviceptive perception.

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...