Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Summary

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Abstract

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Introduction

Electrocardiography (ECG) is routinely used in the clinical setting to assess the dynamics of cardiac electrical conduction and the electrical activation-recovery process. ECG is important for detecting, localizing, and assessing the risk of arrhythmias, ischemia, and infarctions. Typically, electrodes are affixed to the patient's chest, arms, and legs in order to provide a three-dimensional view of the heart. A positive deflection is produced when the direction of myocardial depolarization is toward the electrode and a negative deflection is produced when the direction of myocardial depolarization is away from the electrode. Electrographic components of the cardiac cycle include atrial depolarization (P wave), atrial-ventricular conduction (P-R interval), ventricular excitation (QRS complex), and ventricular repolarization (T wave). There are great similarities in ECG and action potential measures across many mammals including humans, rabbits, dogs, guinea pigs, pigs, goats, and horses^1,2,3.

Rabbits are an ideal model for cardiac translational research. The rabbit heart is similar to the human heart in terms of ion channel composition, and action potential properties^2,4,5. Rabbits have been used for the generation of genetic, acquired, and drug-induced models of heart disease^2,4,6,7,8. There are great similarities in the cardiac ECG and action potential response to drugs in humans and rabbits^7,10,11.

The heart rate and cardiac electrical activation-recovery process is very different in rodents, as compared to rabbits, humans, and other larger mammals^12,13,14. The rodent heart beats ~10 times as fast as humans. In contrast, to the iso-electric ST segment in human and rabbit ECGs, there is no ST segment in rodents^14,15,16. Also, rodents have a QRS-r' waveform with an inverted T wave^14,15,16. Measurements of the QT interval are very different in rodents vs. humans and rabbits^14,15,16. Furthermore, normal ECG values are very different in humans vs. rodents^12,15,16. These differences in the ECG waveforms can be attributed to differences in the action potential morphology and the ion channels that drive cardiac repolarization^9,14. While the transient outward potassium current is the major repolarizing current in the short (non-dome) cardiac action potential morphology in rodents, in humans and rabbits there is a large phase-2 dome on the action potential, and the delayed rectifier potassium currents (I_Kr and I_Ks) are the major repolarizing currents in humans and rabbits^4,9,13,17. Importantly, the expression of I_Kr and I_Ks is absent/minimal in rodents, and due to the temporal activation kinetics of I_Kr and I_Ks it does not have a role in the cardiac action potential morphology^9,13. Thus, rabbits provide a more translational model for assessing the mechanisms for drug-induced, acquired, and inherited ECG abnormalities and arrhythmias^4,7,13. Next, as numerous studies have shown the presence of both neuronal and cardiac electrical abnormalities in primary cardiac (Long QT Syndrome^18,19,20) or neuronal diseases (epilepsy^21,22,23,24), it is important to study the underlying mechanisms in an animal model that closely reproduces human physiology. While rodents may be sufficient to model the human brain, rodents are not an ideal model of human cardiac physiology⁷.

Electroencephalography (EEG) uses electrodes, usually placed on the scalp or intracranially, to record cortical electrical function. These electrodes can detect changes in the firing rate and synchronicity of groups of nearby pyramidal neurons in the cerebral cortex²⁵. This information can be used to assess cerebral function and awake/sleep state. Also, EEGs are useful to localize epileptiform activity, and distinguish epileptic seizures from non-epileptic events (e.g., psychogenic non-epileptiform activity and cardiogenic events). In order to diagnose epilepsy type, provoking factors, and origin of the seizure, epilepsy patients are subjected to various maneuvers which may bring on a seizure. Various methods include hyperventilation, photic stimulation, and sleep deprivation. This protocol demonstrates the use of photic stimulation to induce EEG aberrations and seizures in rabbits^26,27,28,29.

Simultaneous video-EEG-ECG recordings have been extensively used in humans and rodents to assess behavioral, neuronal, and cardiac activity during the pre-ictal, ictal, and post-ictal states³⁰. While several studies have conducted EEG and ECG recordings separately in rabbits^4,31,32,33, a system for acquiring and analyzing simultaneous video-EEG-ECG in the conscious restrained rabbit is not well established³⁴. This paper describes the design and implementation of a protocol that can record simultaneous video-EEG-ECG -capnography-oximetry data in conscious rabbits in order to assess neuro-cardiac electrical and respiratory function. Results gathered from this method can indicate the susceptibility, triggers, dynamics and concordance between arrhythmias, seizures, respiratory disturbances, and physical manifestations. An advantage of our experimental system is that we acquire conscious recordings without the need of a sedative. The rabbits remain in the restrainers for ≥5 h, with minimal movement. As anesthetics perturb neuronal, cardiac, respiratory, and autonomic function, recordings during the conscious state provide the most physiological data.

This recording system may ultimately provide detailed insights to advance the understanding of the neurologic, cardiac and respiratory mechanisms for sudden unexpected death in epilepsy (SUDEP). In addition to neurologic and cardiac monitoring above, recent evidence has also supported the role of respiratory failure as a potential contribution to sudden death after a seizure^35,36. To monitor the respiratory status of the rabbits, oximetry and capnography were implemented to evaluate the status of the respiratory system before, during and after a seizure. The protocol presented here was designed with the purpose of assessing the threshold for pharmacologically and photic-stimuli induced rabbit seizures. This protocol can detect subtle EEG and ECG abnormalities that may not result in physical manifestations. In addition, this method can be used for cardiac safety and anti-arrhythmic efficacy testing of novel drugs and devices. 

Protocol

All experiments were carried out in accordance with the National Institutes of Health (NIH) guidelines and Upstate Medical University Institutional Animal Care and Use Committee (IACUC). In addition, an outline of this protocol is provided in Figure 1.

1. Preparing recording equipment

1. Connect the computer to an amplifier with a 64-pin headbox.
   NOTE: Each animal has four straight subdermal scalp pin electrodes (7 or 13 mm) for EEGs from the 4 quadrants of the head, 3 bent subdermal chest pin electrodes (13 mm, 35˚ angle) for ECG (Einthoven’s triangle), 1 bent subdermal pin ground electrode on the right leg, and 1 straight subdermal scalp pin electrode on the center of the head serves as the reference.
2. To make every 8^th pin on the headbox a reference, update the acquisition software settings, acquisition tab, so the Reference Electrode is “Independent” (i.e., research mode).
   NOTE: This enables recordings from up to 7 animals simultaneously, each with 7 electrodes plus a dedicated reference electrode and a ground electrode, all through one amplifier, digitizer, and computer. All electrodes are acquired as unipolar channels and compared to the reference (center of the head.) Additional bipolar and augmented lead configurations/montages can be setup during or after the recording. As the setup has the capability to record from multiple animals simultaneously, a ground electrode from each animal is connected in parallel to the ground input on the amplifier (Figure 2).
3. Remove rabbits from their cage and weigh them to calculate the appropriate drug dose for each animal. Place the rabbits in a transport carrier and bring them to a separate room in order to minimize stress to non-experimental animals. In this study, male and female New Zealand White rabbits, and their subsequent offspring were used. Experiments were performed on rabbits > 1 month of age. At the time of the experiment, these rabbits weighed between 0.47 – 5.00 kg.
   NOTE: Since the rabbits need to be in the same room and in view of the camera, do not completely isolate the rabbits. There is the potential for visual and auditory manifestations from one rabbit stressing another rabbit. Therefore, it is ideal to have one rabbit in the room at a time, which is done for the photic stimulation experiments. For all other experiments, the rabbits are spaced out as much as possible, while keeping all of them within the view of the video camera. Ideally, barriers are used or only one animal is studied at a time. This was not a major confounder as the rabbits’ heart rates remained fairly stable during the experiments and there was the frequent presence of sleep spindles. Recordings from multiple animals simultaneously assures that both control and test animal data are acquired under the same environmental conditions.

2. 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 3A).
   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 3B). 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 4A).
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 4B).
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 4B). 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 4C).
      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 4C).
9. Now that the rabbit is securely placed into the restrainer, insert the 7-13 mm subdermal straight pin-electrodes into the scalp (Figure 3A). 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 4D).
   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 5C).
11. Gently place the facemask with capnography tubing over the rabbit’s mouth and nose (Figure 4H). 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 4I).

3. 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 of 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 5A).
   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.

4. Experimental protocols

NOTE: Each of the following experiments are performed on separate days if they are performed on the same animal. There is a 2-week delay between the oral tests compound drug studies, and the acute terminal pro-convulsant drug study. When necessary, the photic-stimulation experiment is performed, followed by a 30 min wait, and then the PTZ drug study.

1. To enable the rabbits to acclimate in the restrainers and for the investigator to objectively confirm stabilization of cardiorespiratory rates, instrument all rabbits with the cardiorespiratory and neuronal sensors and perform continuous video monitoring for > 1 hour, 1 - 3 times per animal. 
2. Photic stimulation experiment
   1. In addition to the method described above, 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)²⁹. The light source is indicated by a white dot in Figure 4E.
      ​NOTE: A dimly lit room should be used to elicit the photosensitive response³⁷.
   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 4E.
   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 seizure²⁹. In addition, 10% of photosensitive patients only exhibit electroencephalographic signs while their eyes are closed²⁹. 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 delay³⁸.
   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.
3. Oral administration of medications
   1. As many drugs are taken orally, prepare oral compounds by mixing with food-grade apple sauce. Mix 0.3 mg/kg of E-4031 in 3 mL of apple sauce and load into a 3 mL oral/irrigation syringe without a needle.
      NOTE: Several medications can be administered in this fashion including, test compounds, drugs that are known to alter the QT duration (moxifloxacin or E-4031), and a negative control or vehicle. Some drugs are not available in an intravenous formulation. In addition, many medications are prescribed in an oral formulation and therefore an intravenous administration may have less clinical relevance.
   2. Lift the upper lips and slide the tip of the oral syringe into the side of the rabbit’s mouth, which is unobstructed by the rabbit’s teeth, and inject all of the medication and apple sauce into the rabbit’s mouth.
   3. Continue the video-EEG-ECG recording for 2 h and then return the animal to its home cage for routine care.
   4. On the experimental day 2 and 3, connect the rabbit to the video-EEG-ECG, record 10-20 min of baseline, then inject the same medication and record for 2 h.
   5. After 1 week of washout, perform 10-20 min of baseline, and then give each rabbit a single dose of placebo for 3 consecutive days and record for 2 h.
      NOTE: Administration of oral medications may be designed as a crossover study, in which the placebo is given during week 1 and the medication in week 2.
4. Intravenous medication experiment (Pentylenetetrazol, PTZ)
   1. In order to visualize the marginal ear vein, shave the posterior surface of the rabbit’s ear. Use a 70% ethanol wipe to disinfect the site and dilate the marginal ear vein. This is indicated by the black dashed oval in Figure 4F.
   2. At this point, have one experimenter cover the rabbit’s face with their hand in order to decrease the stress of the procedure to the rabbit. A second experimenter carefully cannulates the marginal ear vein with a  sterile 25-G angiocatheter.
   3. Once the catheter is in the vein, place a sterile injection plug at the end of the catheter so that a needle can introduce medication intravenously. The location of the injection plug is indicated by a blue circle in Figure 4G.
   4. Make a splint by wrapping 4 x 4 inch gauze with tape so that it forms a tube shape and placing it inside the rabbit’s ear. Then tape the splint to the ear so that the catheter is secured in place and remains upright, similar to the non-catheterized ear.
   5. Inject 1 mL of 10 USP units per mL of heparinized saline.
      NOTE: The catheter and vessel should be visibly cleared of air and remain patent. If the catheter is not in the vessel, the syringe will not push easily and there will be accumulation of saline in the subcutaneous tissue.
   6. Give rabbits incremental doses of PTZ intravenously from 1 mg/kg to 10 mg/kg in 1 mg/kg increments every 10 min. Make a note at the start of each dose to indicate which animal is being injected and the concentration of the medication.
      NOTE: This enables assessments of the acute and additive effects of PTZ administration. Alternatively, to further assess the chronic effects of low dose PTZ, the rabbit is given repeated doses at each low dose concentration, 7 doses at 2 mg/kg, 3 doses at 5 mg/kg, then 3 doses at 10 mg/kg, each dose is separated by 10 min.
   7. After each dose, carefully monitor the video-EEG-ECG-capnography-oximetry for any neuro-cardiac electrical and respiratory abnormalities and visual evidence of epileptiform activity. Note these changes in real-time and during post-analysis.
      NOTE: Seizure activity often begins within 60 s of PTZ administration.

5.Conclusion of Non-Survival Experiments.

1. If the rabbit did not experience sudden death during the course of the PTZ experiment, administer 1mL of 390 mg/mL of sodium pentobarbital for every 4.54 kg of body weight (or 1.5 mL to all rabbits), followed by a 1 mL flush of normal saline. Monitor the ECG to ensure that the rabbit undergoes cardiac arrest.
2. Once the rabbit experiences cardiac arrest, quickly perform a necropsy to freshly isolate various organs, including the heart, lungs, liver, brain, skeletal muscle, and any other tissue necessary for subsequent molecular/biochemical analyses.
3.  Dispose of the rabbit according to institutional policies.

6. Analysis of ECG

1. Use commercially available ECG analysis software to visually inspect the ECG, and to identify periods of tachycardia, bradycardia, ectopic beats and other arrhythmias (Figure 6). To reduce the amount of data to review, create a tachogram, which will increase the ease with which periods of tachycardia, bradycardia, or irregularities of the RR interval can be identified. 
   NOTE: ECG abnormalities (e.g., QT_c prolongation) and arrhythmias are manually identified by reviewing the ECG for abnormalities in the rate (e.g., brady-/tachy-arrhythmias), rhythm (e.g., premature atrial/ventricular complexes), conduction (e.g., atrio-ventricular block), and waveform (e.g., non-sinus atrial/ventricular tachycardia and fibrillation.) Arrhythmias can be detected by reviewing the tachogram for irregularities in the RR interval. Tachycardia can be identified by sections of the tachogram in which the heart rate is above 300 beats per minute. Bradycardia is identified when the heart rate is less than 120 beats per minute on the tachogram.
2. Using commercially available ECG analysis software, perform standard ECG measurements (heart rate, cardiac cycle intervals) at baseline and upon provocation (e.g., investigator manipulating the animal, administration of test agents, and seizure induced ECG changes).

7. Analysis of video-EEG

1. Visually scroll through the video and EEG tracing using commercially available software to identify the baseline signal (Figure 7) and the presence of expected EEG discharges such as sleep spindles (Figure 8) and vertex waves (Figure 9).
   NOTE: Although full bandwidth electrographic data is acquired without any filters, data should be displayed with the low frequency filter (i.e., high pass filter) set at 1 Hz, and based on Nyquist’s theorem, the high frequency filter (i.e., low pass filter) is set at 120 Hz to avoid missing any signal. The filters can be adjusted to allow for better visualization and noise reduction (e.g., 1-59 Hz) when reviewing lower frequency (<25 Hz) EEG activity.
2. In addition to capnography waveforms, use nose movement artifact on the EEG to determine the presence versus absence of breathing. This can also be correlated with nose movements seen on the video recording.
3. Visually scroll through the video and EEG tracing using commercially available software to distinguish epileptic vs. non-epileptic (e.g., conscious) movements for at least 1 min after each dose of PTZ (Figure 10). Scan for interictal epileptic discharges and for EEG changes before, during, and after seizures. A seizure can be identified clinically by observing the presence of head and whole-body myoclonic jerks, clonus, or a tonic state with an EEG correlate. The EEG changes may include EEG spikes, poly-spikes, and rhythmic discharges.
   NOTE: Movements in which the EEG is obscured by muscle artifact or waves of indeterminant epileptogenicity should be reviewed by a neurologist for confirmation. It may be advantageous to focus the video on one rabbit to view its behavior, as well as its EEG and ECG recordings, more closely (Figure 5B).
4. Score the video-EEG for seizures based on the type and severity of motor manifestations, which typically occur within 1 min after PTZ injection (Table 1).
5. After a photic stimulation experiment, analyze the occipital leads of the EEG for the presence and absence of the occipital driving rhythm by creating a spectral analysis plot in commercially available EEG analysis software. The occipital driving rhythm will create a peak in the spectral analysis that corresponds to the frequency of the photic stimulator (Figure 11).
   NOTE: Photic stimulation may produce harmonic frequency peaks in addition to the peak of the fundamental frequency.

7. Analysis of respiratory function

1. Review the output from the vital signs monitor (Figure 4I) and export the signal for further analysis.
2. Note the change in respiratory pattern during a seizure and after a seizure, especially the timepoint when apnea begins.

Results

The method described above is capable of detecting abnormalities in the electrical conduction system of the brain and the heart as well as respiratory disturbances. A data acquisition software is used to assess the ECG morphology and detect any abnormal heart rates, conduction disturbances, or ECG rhythms (atrial/ventricular ectopic beats, and brady-/tachy-arrhythmias) (Figure 6). In addition to visualizing the ECG morphology, the traces are analyzed to quant...

Discussion

This experimental setup facilitates detailed simultaneous video-EEG-ECG-oximetry-capnography recordings and analyses in rabbits, particularly in models of cardiac and/or neuronal diseases. The results of this article show that this method is capable of detecting seizures and arrhythmias and differentiating them from electrographic artifacts. Expected results were obtained when giving rabbits a proconvulsant, which induced seizures. The data obtained from the video-EEG recordings were able to be further analyzed to differ...

Materials

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Irrigation, USP - Flexible Container	PFIZER (HOSPIRA)	7983-09	Dilutant
10cc Luer Lock syringe with 20G x 1" Needle	Sur-Vet	SS-10L2025	Used as a flush after drug injection
4x4 gauze sponges	Fisher Scientific	22-415-469	Rolled in a tube to splint ear with angiocatheter
Apple Sauce	Kirkland	897971	Vehicle for oral medications
Computer	Dell	Optiplex 5040	Acquisition computer
E-4031	Tocris	1808	Agent known to prolong the QT interval
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
Euthanasia-III Solution	Med-Pharmex	ANADA 200-280	Contains pentobarbital sodium and phenytoin sodium, controlled substance
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
Heparin Lock Flush	Medline	EMZ50051240	To maintain patency of angiocatheter
IR Light	Bosch	EX12LED-3BD-8W	Facilitates recordings in the dark
LabChart Pro (2019, Version 8.1.16)	ADInstruments	N/A	ECG Analysis
JELCO PROTECTIV Safety I.V. Catheters, 25 gauge	Smiths Medical	3060	Used to catherize marginal ear vein
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
Pentylenetetrazol (1 - 10 mg/kg always in 1mL volume)	Sigma-Aldrich	88580	Dilutions prepared in saline
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
Terumo Sur-Vet Tuberculin Syringe 1cc 25G X 5/8" Regular Luer	Sur-Vet	13882	Used to inject intravenous medications
Veterinary Injection Plug Luer Lock	Sur-Vet	SRIP2V	Injection plug for inserting the needle for intravenous medication
Webcol Alcohol Prep, Sterile, Large, 2-ply	Covidien	5110	To prepare ear vein before catheterization