Prepping Conscious Rabbit Model for Simultaneous Video-EEG-ECG-Oximetry-Capnography for Experimental Interventions: A Procedure to Implant EEE-ECG Electrodes and Attach Respiratory Probes in Rabbit Model

Overview

This video demonstrates the procedure to implant EEG-ECG electrodes and attach respiratory probes in a rabbit model for acquiring and analyzing records of its bio signals.

Protocol

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

1. Implanting EEG-ECG electrodes and attaching respiratory monitors

1. Remove one rabbit from the transport carrier and place in the lap of a seated investigator.
2. Hold the rabbit vertically and keep it close to the investigator's body.
3. Lower the rabbit into a supine position, with the rabbit's head at the investigator's knees, and the rabbit's head lower than the rest of its body. 
   NOTE: This maneuver relaxes the animal and minimizes the likelihood of it trying to move or escape while placing the electrodes.
4. Now that the rabbit is secured in a supine position, ask a second investigator to spread the fur until the skin can be identified and isolated from the underlying tissue.
5. Insert 35˚ bent electrodes subdermally in each axilla (Figure 1A).
   NOTE: The electrodes should be pushed through so that they are securely hooked into the skin, but do not penetrate deeper structures. Having the electrode enter and then exit out of the skin (through-and-through) reduces the chance of the leads becoming dislodged when placing the rabbit in the restrainer or if it moves during the experiment (Figure 1B). All electrodes are sterilizaed with 70% ethanol prior to placement.
6. Place leads on the chest posterior to the right and left forelimbs and on the abdomen anterior to the left hind limb. Place a ground pin-electrode anterior to the right hind limb on the abdomen (Figure 2A).
7. Once all of the ECG leads are properly placed, return the rabbit to a prone position, with the leads running up one side of the rabbit abdomen, and transfer the rabbit into an appropriately sized restrainer (e.g., 6" x 18" x 6").  When placing the rabbit in the restrainer, pull the loose wire upward to minimize the rabbit from pulling out the electrodes with its legs. Tape the wires to the side of the restrainer so that they do not get caught under the rabbit during the experiment (Figure 2B).
8. Secure the rabbit in the restrainer by lowering the restraint around the neck and locking it into place. Additionally, move the hind limbs up underneath the animal and secure the rear restraint.  
   NOTE: One should be able to fit 1-2 fingers within the space under the neck to assure it is not too tight. Particularly during experiments where there may be motor movement, it is important to tighten down the restraint to minimize movement, potential spinal injuries, limb dislocation, and the ability to kick out the rear restraint (Figure 2B). Rabbits have been maintained in the restrainer for ~5 h without any issues related to increased movement or signs of dehydration.
   1. For small rabbits (e.g., less than 2 months) place a rubber booster pad under the animal to raise the rabbit up, which prevents the rabbit from resting its neck on the bottom of the head restraint (Figure 2C).
      NOTE: A sudden drop in respiratory and heart rate may be secondary to neck impingement. If this occurs, loosen the neck restrainer and lift the rabbit's head to relieve any neck compression.
   2. When the rear restraint does not closely trace the back/spine of the rabbit, place a PVC spacer to prevent any movement that could cause spinal injuries.
      NOTE: For example, ~14 cm long x 4" inner diameter PVC pipe, with the lower 25-33% removed can be placed over the rabbit with foam to provide appropriate restraint (Figure 2C).
9. Now that the rabbit is securely placed into the restrainer, insert the 7-13 mm subdermal straight pin-electrodes into the scalp (Figure 1A). Using a 45˚ angle approach of entry, run the wires up between the ears, and loosely tether the wires to the restrainer behind the head to maintain lead placement. Place 5 EEG leads in the following positions: right anterior, left anterior, right occipital, left occipital and a central reference (Cz) lead at the point between the other 4 leads (Figure 2D).
   NOTE: Electrodes are properly placed when they are positioned into subcutaneous tissue against the skull. This placement minimizes artifact from the nose, ears, and other surrounding muscles. Some artifact from rhythmic nose movement is unavoidable. The anterior EEG leads should be placed medial to the rabbit's eyes and point anteriorly. The occipital leads should be placed anterior to the ears and will point in the medial direction. Cz is placed in the center of the top of the head at a point that is between all 4 electrodes (half-way between Lambda and Bregma, along the suture line). The pin of the Cz electrode points anteriorly.
   1. Pass the EEG wires up between the ears, to avoid the rabbit trying to bite the wires.
10. Attach the pulse oximeter plethysmograph to the rabbit's ear over the marginal ear vein.
    NOTE: It may be necessary to shave excess hair from the ear to improve signal or use some gauze to keep the sensor in place.
    1. Ensure that the heart rate on the plethysmography correlates with the heart rate from the ECG and that the oxygen saturation is displayed (Figure 3C).
11. Gently place the facemask with capnography tubing over the rabbit's mouth and nose (Figure 2H). Secure the facemask with string wrapped around the mask and attach both ends of the string to the restrainer. Attach the other end of the capnography tubing to the vital signs monitor.
    NOTE: It is important to prevent the string from laying over the rabbit's eyes during the experiment. To do this, tape the string to the middle of the restrainer between the rabbit's ears. In order to improve the capnography signal, create a one-way valve using tape and a thin piece of nitrile that will allow oxygen to enter the T-piece, and will direct exhaled CO2 into the capnography tubing (Figure 2I).

2. Recording of video-EEG-ECG

1. Perform video-EEG-ECG recording using a commercially available EEG software.
   NOTE: The biopotential leads and video are time locked to later correlate the electrical and video signals (e.g., EEG spike with a myoclonic jerk).
2. Confirm optimal connectivity, with no baseline drift, no 60 Hz electrical noise, and high signal-to-noise ratio. Specifically, ensure that each phase of the cardiac waveform can be visualized on the ECG and that the delta, theta, and alpha waves are not visually obscured by high frequency noise on the EEG.
   1. If all the electrodes are producing excessive amounts of noise, then adjust the central reference lead. If only one electrode is excessively noisy, then push that electrode deeper into the skin or reposition it until there is no metal exposed.
3. Adjust the video so that all rabbits can be seen simultaneously, which allows for the correlation of motor activity with EEG findings (Figure 3A).
   NOTE: The system accommodates simultaneous EEG/ECG/oximetry/capnography recordings from up to 7 rabbits.
4. Start the baseline recording from each animal for a minimum of 10-20 min or until the heart rate stabilizes to a calm relaxed state (200-250 bpm) and the rabbits do not exhibit large movements for at least 5 min. Acquire full bandwidth electrographic data without any filters. In order to better visualize data set the low frequency filter (=high pass filter) at 1 Hz and the high frequency filter (=low pass filter) at 59 Hz.
   NOTE: Another sign that the rabbit is relaxed is the onset of EEG sleep spindles (discussed later).
5. Add time-locked notes during the experiment in real-time to indicate the timing of interventions (e.g., drug delivery) and neuro-cardiac events (e.g., EEG spike, motor seizures, ectopic beat, and arrhythmias), and motor/investigator artifacts.
   NOTE: Due to the frequency that the investigator needs to apply an intervention (e.g., photic-stimulation, drug delivery), to minimize the stress of an investigator entering and exiting the room and opening/closing the door, the investigator remains on the opposite side of the room throughout the experiment. The investigator sits as far from the animal as possible, and remains still and quiet to minimize potentially disturbing the animals.

Representative Results

Figure 1: Picture of EEG and ECG electrodes. (A) Bent ECG electrodes and straight EEG electrodes. (B) How to hook the ECG electrode in the subcutaneous tissue of the rabbit, so that it is through and through. Abbreviations (LL: Left limb, RA: Right arm, RL: Right limb, LA: Left arm, RF: Right frontal, LF: Left frontal, Cz: Center, RO: Right occipital, LO: Left Occipital).

Figure 2: 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 with appropriate modifications to accommodate a smaller rabbit, including a booster beneath the rabbit, neck foam and cut PVC pipe. (D) Rabbit in restrainer with location of EEG electrodes. Right Frontal is indicated by an orange dot. Left Frontal is indicated by a red dot. Right Occipital is indicated by a yellow dot. Left Occipital is indicated by a blue dot. The reference is indicated by a black dot. (E) Rabbit in restrainer with photic stimulator and mirror booth setup. Light source is indicated by a white dot. (F) Marginal ear vein after rabbit's ear has been shaven and wiped with alcohol. (G) Rabbit with angiocatheter securely taped in the left marginal ear vein. Site of injection plug is indicated with a blue dot. (H) Rabbit with facemask attached to the capnography tubing by a T-piece that contains a one-way valve. (I) Diagram of the facemask and T-piece connected to the capnography tubing. During inspiration, room air is able to enter the T-piece through a one-way valve (green arrow). During expiration, CO₂ leaves the T-piece by entering the capnography tubing (yellow arrow.) Because of the small amount of dead space, very little CO₂ is retained in the T-piece and is generally less than 5 mmHg.

Figure 3: Simultaneous Rabbit Video-EEG-ECG-Capnography-Oximetry. (A) Simultaneous video-EEG-ECG recording of 3 rabbits. (B) Zoomed in view of simultaneous video-EEG-ECG recording from Rabbit #2. (LL: Left limb, RA: Right arm, LA: Left arm) (C) Simultaneous recording of capnography (yellow) and plethysmography (blue). Measurements showing inspired CO₂, end tidal CO₂, respiratory rate, pulse rate and pulse oximetry are included in the figure.

Materials

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
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
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
Webcol Alcohol Prep, Sterile, Large, 2-ply	Covidien	5110	To prepare ear vein before catheterization
LabChart Pro (2019, Version 8.1.16)	ADInstruments	N/A	ECG Analysis
Quantum Amplifier	Natus	13926	Amplifier / digitizer
Quantum HeadBox Amplifier	Natus	22134	64-pin breakout box
SRG-X120 1080p PTZ Camera with HDMI, IP & 3G-SDI Output	Sony	SRG-X120	Impela Camera