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 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.
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.
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
2. Implantation Surgery for Video-EEG Monitoring
3. Video and EEG Monitoring and Analysis
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.
Figure 1 shows cell deaths in the hippocampus assessed by cresyl violet staining. At 7 days after SE, pyknotic cells were detected in the hilar regions (Figure 1Ad) of the dentate gyrus and the pyramidal cell layer of the CA3 subfield of the hippocampus (Figure 1Af), whereas intact hippocampus was observed in the sham animals injected with saline instead of pilocarpine (Figure 1Aa-c). Interestingly, animals who underwent multiple seizures, but failed to enter SE, showed no cell death in either the hilar region or the pyramidal cell layer (Figure 1Ag-i). At 6 weeks after SE, a marked reduction was observed in the number of hilar cells (Figure 1Bm). In addition, neuronal loss was observed in the CA1 (Figure 1Bn) and CA3 (Figure 1Bo) regions.
Figure 2 shows the representative EEG traces in the sham and SE groups (Figure 2A). Compared to the sham group showing physiologic electrical activity, the SE group occasionally showed epileptiform discharges accompanied with convulsive behaviors. The locations of the epidural electrodes are illustrated in Figure 2C. Figure 2B demonstrates an example of the electrical noises from an adjacent monitor and the failure of signal collection. We also demonstrated EEG traces of sham-manipulated animals who showed spontaneous seizures during the 2 weeks of video-EEG recording (Figure 2E) and found cortical injuries at perfusion, possibly derived from the excessively deep placement of the epidural screws (Figure 2D).
Figure 1: Neuronal death in the hippocampus after pilocarpine-induced SE. Representative images from the groups of (A) 7 days and (B) 6 weeks after pilocarpine or saline injection. Magnified images show the hilus (a, j), CA1 subfield (b, k), and CA3 subfield (c, l) of saline-treated sham group, the hilus (d, m), CA1 subfield (e, n), and the CA3 subfield (f, o) of the SE group, and the hilus (g), CA1 subfield (h), and CA3 subfield (i) of the mice that do not develop SE. Black arrows in a, g, and j indicate some of the representative hilar cells, and arrowheads in d and m indicate pyknotic cells. Scale bar in the bottom left side = 200 µm, valid for the entire left most column, scale bar in a and c = 20 µm, valid for the entire middle column and right column. Abbreviations in this figure: CA1, CA3: Cornu Ammonis hippocampal subfields; DG: Dentate gyrus. Please click here to view a larger version of this figure.
Figure 2: Video and EEG recording shows spontaneous recurrent seizures in the epileptic mouse. (A) Representative EEG traces from sham- (top) and SE-manipulated animals (bottom). Note that SE-subjected animals show epileptiform spiking activity (≥ 3 Hz), not exhibited by sham animals. (B) A representative image of electrical noises and failure of the signal collection. Note the high amplitude unipolar spikes and discontinuation of electrical signals. (C) Schematic drawing of the mouse brain with locations of electrodes. (D) Two images showing the intact and injured brain in the cortex, possibly due to excessively deep insertion of epidural screws. The black arrow in the right image indicates the site of cortical damage. Scale bar = 250 µm. (E) Representative EEG traces of sham-manipulated animals with or without brain injury. Note that sham-manipulated animals with cortical injury generated spiking activity and convulsive SRS. Please click here to view a larger version of this figure.
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 length of the interval between the later convulsive seizures decreases before entering SE. Moreover, SE should be sustained for at least 6 h unless behavioral seizures are terminated by diazepam5. It is worth noting that researchers should be aware of the possibility that sustained seizure activities can be detected via electrocorticograms, even though behavioral seizures seem to subside with diazepam injection. Increasing ambient temperature after pilocarpine injection until SE induction can greatly improve the number of animals with SE. It is also important to provide intensive post-seizure care, including sufficient hydration, nutritional support, and weight monitoring for higher survival rates than about 70-80% on average5,18. Depending on the animal’s genetic background, susceptibility to pilocarpine may differ. If animals display severe acute seizures with high mortality, the reduction of pilocarpine dosage, SE duration, or increased scopolamine and terbutaline can be helpful. While severe SE is known to be related to higher incidence of SRS, with higher mortality, it is crucial to consider these factors. Hippocampal neuronal deaths are consistently observed after SE. At 6 h post-SE, extensive loss of hilar interneurons has been reported19. At 7 days after SE, pyramidal neurons in the CA3 subfield typically die, and are continuously observed at 6 weeks after SE induction, although the extent of neuronal damage in the pyramidal cell layer is variable in mice. Neuronal loss in the CA1 subfield of the hippocampus can also be frequently observed. Interestingly, animals with multiple seizures who do not enter SE showed almost intact hippocampus similar to the sham group, indicating histologic cell death can be a good marker of sufficiently severe acute seizures.
At approximately 4 weeks after SE, all pilocarpine-treated C57BL/6NHsd mice became epileptic and showed SRS, demonstrating one of the major merits using this model. Since chronic seizures are clustered, the video-EEG should be recorded continuously for at least 2 weeks rather than intermittently, even though the recording period can be modified depending on the mouse strains or experimental purposes17. Moreover, spontaneous seizures in TLE have diurnal variations demonstrating high SRS frequency in the late afternoon20. Practically, for accurate video-EEG recording, any sources including AC lines, computers, cameras, or transmitters implanted in the mouse in the adjacent receiver need to be blocked. The installation of the Faraday cage and cameras with high sensitivity during the day and night can diminish electrical noise and help to identify generalized chronic seizures, respectively. Finally, it is extremely important to avoid brain damage when the screws are placed on the surface of the dura mater as it can generate misleading EEG data. Although the SRS frequency was not high, any mechanical cortical injuries by forced screw insertion could generate convulsive seizures in only one week.
In conclusion, despite the time constraints for modeling and the requirement of in-depth training to perform implant surgeries and EEG analysis, the pilocarpine-induced SE is one of the best models for studying chronic epilepsy among currently available animal models of TLE. Here, we describe a method of inducing SE after pilocarpine injection and assessing chronic seizures using a wireless telemetry video-EEG system. We believe this protocol can provide detailed information for suitable animal models for chronic epilepsy and epileptogenesis.
The authors have nothing to disclose.
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&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2854).
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 | |
Intensive care unit | Daejong instrument industry Co., Ltd. | 28~30℃ | |
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 |