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

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Research

JoVE Journal

Medicine

19.5K 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

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Behavior

Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, considerable variability has been reported for the specific parameters used in different studies that have employed this model, making it difficult to harmonize and interpret the results between laboratories. For example, variability has been reported regarding the size and location of the craniectomy, how the Luer lock hub is placed relative to the craniectomy, the atmospheric pressure applied to the dura and the duration of the pressure pulse. Each of these parameters can impact injury severity, which directly correlates with the incidence of PTE. This has been manifested as a wide range of mortality rates, righting reflex times and incidence of convulsive seizures reported. Here we provide a detailed protocol for the method we have used to help facilitate harmonization between studies. We used FPI in combination with a wireless EEG telemetry system to continuously monitor for electrographic changes and detect seizure activity.&#160; FPI is induced by creating a 5 mm craniectomy over the left hemisphere, between the Bregma and Lambda and adjacent to the lateral ridge. A Luer lock hub is secured onto the skull over the craniectomy. This hub is connected to the FPI device, and a 20-millisecond pressure pulse is delivered directly to the intact dura through pressure tubing connected to the hub via a twist lock connector. Following recovery, rats are re-anesthetized to remove the hub. Five 0.5 mm, stainless steel EEG electrode screws are placed in contact with the dura through the skull and serve as four recording electrodes and one reference electrode. The electrode wires are collected into a pedestal connector which is secured into place with bone cement. Continuous video/EEG recordings are collected for up to 4 weeks post TBI.

Here we present a protocol to induce severe TBI with the lateral fluid percussion injury (FPI) model in adult, male Wistar rats. We also demonstrate the use of a wireless telemetry system to collect continuous video-EEG recordings and monitor for epileptiform discharges consistent with post-traumatic epileptogenesis.&#160;

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, ...

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

Neuroscience

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

Source: Kim, J. E. and Cho, K. O. <a href="https://www.jove.com/video/56831/the-pilocarpine-model-temporal-lobe-epilepsy-eeg-monitoring-using">The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice</a>. J. Vis. Exp. (2018).
This video presents the procedure for inducing status epilepticus (SE) in mice by systemic administration of pilocarpine to model temporal lobe epilepsy.

Leczenie układowe pilokarpiny: mysi model padaczki skroniowej

Source: Kim, J. E. and Cho, K. O. The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice. J. Vis. Exp. ...