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

Summary

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.

Abstract

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.

Introduction

TLE is one of the most common acquired epilepsies¹. People with epilepsy experience recurrent seizures as a result of abnormal neuronal activities in the brain^2,3. 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 used^4,5. Unlike other epilepsy models, such as electrical stimulation which shows no hippocampal sclerosis and robust SRS^6,7,8, 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 SRS^5,9,10. 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 models^10,11,12. 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 lines^5,13,14,15. 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.

Protocol

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

1. 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.
2. 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.
3. 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°C) for observation.
4. 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'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'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.
5. 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). 
   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 µL of diazepam solution: 50 µL 5 mg/mL diazepam + 200 µL 10% polyoxyl 35 hydrogenated castor oil solution = 250 µL 1 mg/mL diazepam.
6. 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 µL of saline instead of pilocarpine.
7. 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.
8. During post-care in the incubator (28-30 °C), wipe off excessive secretions such as saliva, tears, and feces.
9. At day 1 after SE induction, weigh the mice and keep them in the incubator (28-30 °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).
10. 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.

2. Implantation Surgery for Video-EEG Monitoring

1. 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.
2. 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 µL/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.
3. Place the mouse in a stereotaxic frame with ear bars and a bite plate and apply vet ointment on both eyes to avoid blindness.
4. 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.
5. 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: −0.2 mm, ML: +0.22 mm (Left parietal cortex)¹³.
   1. 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.
   2. Identify anatomical landmarks such as Lambda and Bregma. Position the drill bit at Bregma and note the X, Y coordinates for this point.
   3. 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.
   4. 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.
6. Insert the body of the single channel wireless EEG transmitter behind the nape of the neck subcutaneously and place flexible leads near the skull.
7. 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. 
8. 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.
9. 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 °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.

3. Video and EEG Monitoring and Analysis

1. 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.
2. Record the electrical activity using the wireless video-EEG telemetry system.
   1. Open the software for video-EEG recording. Click "Hardware" and select "Edit configuration". Match the receiver and the transmitter implanted into each mouse. Click "Channel configuration" and confirm all the channels are active. Click "Video configuration" 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.
   2. Click "Setup" and "P3 setup" to input individual animal information, matched camera, and graph setting.
   3. Click "Acquisition" and "A/D sampling rate" to setup sampling rates. Click "Data set name" 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.
   4. Activate the transmitter with a magnetic bar and click "Start acquisition" 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.
3. Determine the seizure frequency and duration by manual inspection using the analysis software.
   1. 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 (≥ 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.
   2. 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.
   3. For analysis of seizure duration, measure the time from the onset to the end of the epileptiform spiking activities.
4. After completion of the video-EEG recording, fix the brain with 4% paraformaldehyde by transcardial perfusion. 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 μL/g body weight (i.p.; 1 mL syringe; 26 G needle). Once the mouse is confirmed to be deeply anesthetized, perform transcardial perfusion.
5. Before isolating the mouse brain, retrieve the transmitter from the mouse for reuse after cleansing.
   1. Remove the dental cement around the leads using forceps.
   2. Soak the tip of the leads with dental cement in 100% acetone until the dental cement is dissolved.
   3. Remove tissue contaminants around the transmitter by rinsing it with distilled water.
   4. 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.

Results

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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Discussion

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 phase^4,16. Once the first motor seizure is detected, the le...

Materials

List of materials used in this article

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