Intermittent Photic Stimulation in Rabbit Model: A Technique to Induce Epileptic Seizures and Simultaneously Record Multisystem Parameters

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Photic stimulation experiment

1. Place a light source with a circular reflector 30 cm in front of the rabbit at eye level, with the flash intensity set to the maximum (16 candela). The light source is indicated by a white dot in Figure 1E.
   NOTE: A dimly lit room should be used to elicit the photosensitive response.
2.  As the rabbit's eyes are on the side of the head instead of the front of its head (as in humans), place 2 mirrors on either side of the rabbit, and 1 behind the rabbit so that the light enters the rabbit's eyes.
   NOTE: A flat mirror that is ≥ 20 cm tall, by ≥ 120 cm long creates a triangular enclosure around the rabbit to ensure that the flashing light enters the rabbit's eyes, as seen in Figure 1E.
3. Connect the light source to a controller that has an adjustable rate, intensity, and duration.
4. Record video using a camera with a red light and infrared recording capabilities.
5. Expose the rabbits to each frequency for 30 s with their eyes open and then another 30 s with a surgical mask covering their face to simulate or cause eye closure at each frequency.
   NOTE: Previous studies have shown that eye closure is the most provocative maneuver for eliciting photosensitivity to seizure. In addition, 10% of photosensitive patients only exhibit electroencephalographic signs while their eyes are closed. A seizure can be identified clinically by observing the presence of head and whole-body myoclonic jerks, clonus, or a tonic state. The EEG recording is more thoroughly analyzed for electroencephalographic correlation (e.g., spikes, poly-spikes, and rhythmic discharges) with motor manifestations for a definitive diagnosis of seizure activity. Movements in which the EEG is obscured by muscle artifact or waves of indeterminant epileptogenicity should be reviewed by an epileptologist for confirmation.
6. Increase the photic stimulator frequency from 1 Hz to 25 Hz in 2 Hz increments. Then perform the same photo-stimulation protocol, but this time decrease the frequency from 60 Hz to 25 Hz in 5 Hz increments.
   NOTE: If a rabbit has a seizure, the experiment should be stopped. Continue to monitor the rabbit for 30 min. Then return the rabbit to the housing room and monitor every 1 h for 3 h for full recovery. However, if the photic stimulation induces a photoparoxysmal response, then the remainder of ascending frequencies are skipped, and the series is started again by descending from 60 Hz until another photoparoxysmal response occurs. This will allow for the determination of the upper and lower photic stimulation thresholds. No delay is necessary as the photoparoxysmal response will cease after the photic stimulation is discontinued. If it is unclear whether a photoparoxysmal response has occurred, the frequency is repeated after a 10 s delay.
7. After the experiment is completed, remove EEG and ECG leads from the rabbit and return it to its home cage for routine care by husbandry staff. 

Resultados

Figure 1: Rabbit connected to equipment. (A) Location of ECG electrodes, Left Arm is indicated by a yellow dot. Right Arm is indicated by a white dot. Left Leg is indicated by a red dot. Ground anterior to the right leg is indicated by a green dot. (B) Rabbit in restrainer with ECG and EEG electrodes attached. (C) Juvenile rabbit in a restrainer ...

Materiais

Name	Company	Catalog Number	Comments
Computer	Dell	Optiplex 5040	Acquisition computer
ECG Electrode	RhythmLink	RLSND116-2.5	13mm 35-degree bent (0.4 mm diameter) subdermal pin electrodes
EEG Electrode	RhythmLink	RLSP513	5-twist 13mm straight (0.4mm diameter) subdermal pin electrodes
EEGLAB (2020)	Swartz Center for Computational Neuroscience	Open Access	Can perform spectral analysis of EEG
Ethernet-to-ethernet adapter	Linksys	USB3G16	Adapter for connecting the camera to the computer
Foam padding	Generic	N/A	Reduces pressure applied to the neck of small rabbits by the restrainer in order to prevent the adverse cardiorespiratory effects of neck compression
IR Light	Bosch	EX12LED-3BD-8W	Facilitates recordings in the dark
LabChart Pro (2019, Version 8.1.16)	ADInstruments	N/A	ECG Analysis
MATLAB (R2019b, Update 5)	MathWorks	N/A	Required to run EEGLAB
Microphone	Sony Stereo	ECM-D570P	Recording of audible manifestions of seizures
Micropore Medical Tape, Paper, White	3M	1530-1	Used to secure wires and create ear splint
Natus NeuroWorks	Natus	LC101-8	Acquisition and review software
Photic Stimulator	Grass	PS22	Stimulator to control frequency, delay, duration, intensity of the light pulses
Plastic wire organizer / bundler	12Vwire.com	LM-12-100-BLK	Bundle wires to cut down on noise
PS 22 Photic Stimulator	Grass Instruments	BZA641035	Strobe light with adjustable flash frequency, delay, and intensity
PVC pipe	Generic	N/A	Prevents small rabbits from kicking their hind legs and causing spinal injury
Quantum Amplifier	Natus	13926	Amplifier / digitizer
Quantum HeadBox Amplifier	Natus	22134	64-pin breakout box
Rabbit Restrainer	Plas-Labs	501-TC	Various size rabbit restrainers are available. 6" x 18" x 6" in this study.
Rubber pad (booster)	Generic	N/A	Raises small rabbits up in the restrainer to prevent neck compression
SpO2 ear clip	NONIN	61000	PureSAT/SpO2
SpO2 sensor adapter	NONIN	13931	XPOD PureSAT/SpO2
SRG-X120 1080p PTZ Camera with HDMI, IP & 3G-SDI Output	Sony	SRG-X120	Impela Camera