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 m...

<ol>
	<li>Chang, B. S., Lowenstein, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy.">Epilepsy.</a> N Engl J Med. 349 (13), 1257-1266 (2003).</li><li>Scharfman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+epilepsy.">The neurobiology of epilepsy.</a> Curr Neurol Neurosci Rep. 7 (4), 348-354 (2007).</li><li>Rakhade, S. N., Jensen, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptogenesis+in+the+immature+brain:+emerging+mechanisms.">Epileptogenesis in the immature brain: emerging mechanisms.</a> Nat Rev Neurol. 5 (7), 380-391 (2009).</li><li>Cavalheiro, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pilocarpine+model+of+epilepsy.">The pilocarpine model of epilepsy.</a> Ital J Neurol Sci. 16 (1-2), 33-37 (1995).</li><li>Curia, G., Longo, D., Biagini, G., Jones, R. S., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pilocarpine+model+of+temporal+lobe+epilepsy.">The pilocarpine model of temporal lobe epilepsy.</a> J Neurosci Methods. 172 (2), 143-157 (2008).</li><li>Morimoto, K., Fahnestock, M., Racine, R. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pilocarpine+model+of+epilepsy+in+mice.">The pilocarpine model of epilepsy in mice.</a> Epilepsia. 37 (10), 1015-1019 (1996).</li><li>Racine, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+seizure+activity+by+electrical+stimulation.+II.+Motor+seizure.">Modification of seizure activity by electrical stimulation. II. Motor seizure.</a> Electroencephalogr Clin Neurophysiol. 32 (3), 281-294 (1972).</li><li>Hester, M. S., Danzer, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+abnormal+adult-generated+hippocampal+granule+cells+predicts+seizure+frequency+and+severity.">Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity.</a> J Neurosci. 33 (21), 8926-8936 (2013).</li><li>Shibley, H., Smith, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilocarpine-induced+status+epilepticus+results+in+mossy+fiber+sprouting+and+spontaneous+seizures+in+C57BL/6+and+CD-1+mice.">Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice.</a> Epilepsy Research. 49 (2), 109-120 (2002).</li><li>Borges, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+pathological+changes+during+epileptogenesis+in+the+mouse+pilocarpine+model.">Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model.</a> Exp Neurol. 182 (1), 21-34 (2003).</li><li>Pavlova, M. K., Shea, S. A., Bromfield, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Day/night+patterns+of+focal+seizures.">Day/night patterns of focal seizures.</a> Epilepsy Behav. 5 (1), 44-49 (2004).</li></ol>

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.

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		<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>
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		<tr height="40">
			<td height="40" style="height:40px;">Ketamine hydrochloride</td>
			<td>Yuhan corporation</td>
			<td></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Xylazine hydrochloride</td>
			<td>Bayer Korea</td>
			<td></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Diazepam</td>
			<td>SAMJIN</td>
			<td></td>
			<td></td>
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		<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>
		</tr>
		<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>
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		<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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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 ...

The overall goal of this procedure is to induce status epilepticus using pilocarpine and to monitor spontaneous recurrent seizures using a radiotelemetry system in mice. This protocol can be utilized for studying the pathophysiology mechanisms of acute seizures, epileptogenesis, and chronic epilepsy. The main advantage of this technique is to provide a mouse model of chronic epilepsy showing spontaneous recurrent seizures with high survivor rate after acute seizures.Demonstrating the procedure will be Dr.Ji-Eun Kim, a postdoc in my laboratory. Begin by weighing each eight-week-old, male mouse. Make two milligrams per kilogram of scopolamine and terbutaline, as well as 280 milligrams per kilogram of pilocarpine solution.Then, inject the solution intraperitoneally into each mouse, and return the mice to their cages. 30 minutes after the scopolamine and terbutaline administration, inject pilocarpine solution into each mouse. Immediately after pilocarpine injection, place the mice in an incubator with 28 to 30 degrees Celsius for observation.Next, monitor the behavior of the mice until status epilepticus, or SE, is induced. If limbic motor seizures that correspond to stage three or higher according to Racine's scale are detected, record the time and monitor the mice. Once continuous motor seizures last more than two minutes, place the mouse in a new cage at room temperature and keep monitoring for three hours to determine whether their convulsive seizures continue and SE is induced.Then, terminate behavioral acute seizures at three hours after SE onset by injecting 10 milligrams per kilogram of diazepam solution. After diazepam injection, administer one milliliter of 5%dextrose per individual mouse to help with recovery. At day one after SE induction, weigh the mice and keep them in the incubator for one extra day.At day two after SE induction, weigh the mice and return them to their home cage. Provide moist food to facilitate recovery. Finally, measure daily body weight of the animals until seven days post-pilocarpine.And if the body weight has not increased, inject the mice with 5%dextrose. Prepare 12-week-old mice, four weeks after SE induction, to perform telemetry implantation for EEG monitoring. Place the mouse in a stereotaxic frame with ear bars and a bite plate, and apply vet ointment on both eyes to avoid blindness.Then, to maintain sterile conditions during the surgery, shave surgical sites using a razor blade, and be careful to prevent fur contamination of the surgical field. After shaving, disinfect the skin with 70%ethanol and iodine solution. Next, wipe the skin with PBS-soaked cotton swabs, followed by 70%ethanol and iodine solution.Make a longitudinal incision in the middle of the head with scissors to expose the lambda, the bregma, and the target location. Then, position the drill bit at bregma, and note the X, Y coordinates for this point to calculate the coordinates for the screws. Next, 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.Insert the body of the single-channel wireless EEG transmitter behind the nape of the neck subcutaneously, and place flexible leads near the skull. Next, place a stainless-steel screw into each burr hole with tweezers, and tighten it using a screwdriver by clockwise rotation. Ensure the screw is not inserted too far deep to prevent damage to the dura or the brain tissues.Strip the insulation coat two millimeters 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.Finally, after the dental cement has dried, suture the skin and apply topical mupirocin ointment. Place the mouse in a 30 degree Celsius incubator alone until it recovers from the surgery. Then, return the mouse to the home cage.Begin by placing a mouse in an individual cage and placing 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. Then, record the electrical activity using the wireless video-EEG telemetry system. Determine seizure frequency by dividing the total number of SRS cases detected during the two weeks by the total number of recording days for each individual animal.Then, to analyze seizure duration, measure the time from the onset to the end of the epileptiform spiking activities. After completion of the video-EEG recording and the fixation of the brain, retrieve the transmitter from the mouse for reuse after cleansing. To do this, remove the dental cement around the leads using forceps.Then, soak the tip of the leads with dental cement in 100%acetone until the dental cement is dissolved. Next, remove tissue contaminants around the transmitter by rinsing it with distilled water. Finally, rinse the transmitter with 70%ethanol for three hours and air dry it for storage.At seven days after SE, pyknotic cells were detected in the hilar regions of the dentate gyrus and the pyramidal cell layer of the CA3 subfield of the hippocampus. At six weeks after SE, a marked reduction was observed in the number of hilar cells. In addition, neuronal loss was observed in the CA1 and CA3 regions.Additionally, compared to the sham group showing physiologic electrical activity, the SE group occasionally showed epileptiform discharges accompanied with convulsive behaviors. Excessively deep placement of the epidural screws during implantation surgery can induce cortical injuries, which can result in spontaneous seizures even in sham-manipulated animals. Once mastered, induction of status epilepticus and screw implantation can be done in an hour if it is performed properly.While attempting this procedure, it is important to remember the behavioral manifestation after pilocarpine injection and not to damage brain tissues during implantation surgery. Following this procedure, other methods, like behavior test for memory and motor problems in chronic epilepsy, can be performed in order to answer additional questions, like epilepsy-associated circuitry issues. After watching this video, you should have a good understanding of how to induce status epilepticus and monitor chronic seizures in mice.

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Research

JoVE Journal

Medicine

19.2K visualizações.  The Catholic University of Korea. The overall goal of this procedure is to induce status epilepticus using pilocarpine and to monitor spontaneous recurrent seizures using a radiotelemetry system in mice. This protocol can be utilized for studying the pathophysiology mechanisms of acute seizures, epileptogenesis, and chronic epilepsy. The main advantage of this technique is to provide a mouse model of chronic epilepsy showing spontaneous recurrent seizures with high survivor rate after acute seizures.Demonstrating the p...