Name: multi-system monitoring for identification of seizures, arrhythmias and apnea in conscious restrained rabbits
Uploaded: 2021-03-27T13:00:00
Description: Watch this Scientific Journal Video about Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits at JoVE.com

Authors acknowledge this study was supported by grants from the American Heart Association, American Epilepsy Society, and SUNY Upstate Department of Pharmacology.

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
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	<li>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 EE...

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	<li>Kaese, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ECG+in+cardiovascular-relevant+animal+models+of+electrophysiology.">The ECG in cardiovascular-relevant animal models of electrophysiology.</a> Herzschrittmacherther Elektrophysiology. 24 (2), 84-91 (2013).</li><li>Pogwizd, S. M., Bers, D. 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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...

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&#39;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&#160;including humans, rabbits, dogs, guinea pigs, pigs, goats, and horses1,2,3.
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 properties2,4,5. Rabbits have been used for the generation of genetic, acquired, and drug-induced models of heart disease2,4,6,7,8. There are great similarities in the cardiac ECG and action potential response to drugs in humans and rabbits7,10,11.
The heart rate and cardiac electrical activation-recovery process is very different in rodents, as compared to rabbits, humans, and other larger mammals12,13,14.&#160;The rodent heart beats ~10 times as fast as humans. In contrast, to the iso-electric ST segment in human&#160;and rabbit ECGs, there is no ST segment in rodents14,15,16. Also, rodents have a QRS-r&#39; waveform with an inverted T wave14,15,16. Measurements of the QT interval are very different in rodents vs. humans and rabbits14,15,16. Furthermore, normal ECG values are very different in humans vs. rodents12,15,16. These differences in the ECG waveforms can be attributed to differences in the action potential morphology and the ion channels that drive cardiac repolarization9,14. 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 (IKr&#160;and IKs) are the major repolarizing currents in humans and rabbits4,9,13,17. Importantly, the expression of IKr and IKs is absent/minimal in rodents, and due to the temporal activation kinetics of IKr and IKs it does not have a role in the cardiac action potential morphology9,13. Thus, rabbits provide a more translational model for assessing the mechanisms for drug-induced, acquired, and inherited ECG abnormalities and arrhythmias4,7,13. Next, as numerous studies have shown the presence of both neuronal and cardiac electrical abnormalities in primary cardiac (Long QT Syndrome18,19,20) or neuronal diseases (epilepsy21,22,23,24), 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&#160;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 rabbits26,27,28,29.
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 rabbits4,31,32,33, 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&#160;-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&#160;and concordance between arrhythmias, seizures, respiratory disturbances,&#160;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 &#8805;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 seizure35,36. 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.&#160;

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			<td>Optiplex 5040</td>
			<td>Acquisition computer</td>
		</tr>
		<tr>
			<td>E-4031</td>
			<td>Tocris</td>
			<td>1808</td>
			<td>Agent known to prolong the QT interval</td>
		</tr>
		<tr>
			<td>ECG Electrode</td>
			<td>RhythmLink</td>
			<td>RLSND116-2.5</td>
			<td>13mm 35-degree bent (0.4 mm diameter) subdermal pin electrodes</td>
		</tr>
		<tr>
			<td>EEG Electrode</td>
			<td>RhythmLink</td>
			<td>RLSP513</td>
			<td>5-twist 13mm straight (0.4mm diameter) subdermal pin electrodes</td>
		</tr>
		<tr>
			<td>EEGLAB (2020)</td>
			<td>Swartz Center for Computational Neuroscience</td>
			<td>Open Access</td>
			<td>Can perform spectral analysis of EEG</td>
		</tr>
		<tr>
			<td>Ethernet-to-ethernet adapter</td>
			<td>Linksys</td>
			<td>USB3G16</td>
			<td>Adapter for connecting the camera to the computer</td>
		</tr>
		<tr>
			<td>Euthanasia-III Solution</td>
			<td>Med-Pharmex</td>
			<td>ANADA 200-280</td>
			<td>Contains pentobarbital sodium and phenytoin sodium, controlled substance</td>
		</tr>
		<tr>
			<td>Foam padding</td>
			<td>Generic</td>
			<td>N/A</td>
			<td>Reduces pressure applied to the neck of small rabbits by the restrainer in order to prevent the adverse cardiorespiratory effects of neck compression</td>
		</tr>
		<tr>
			<td>Heparin Lock Flush</td>
			<td>Medline</td>
			<td>EMZ50051240</td>
			<td>To maintain patency of angiocatheter</td>
		</tr>
		<tr>
			<td>IR Light</td>
			<td>Bosch</td>
			<td>EX12LED-3BD-8W</td>
			<td>Facilitates recordings in the dark</td>
		</tr>
		<tr>
			<td>LabChart Pro (2019, Version 8.1.16)</td>
			<td>ADInstruments</td>
			<td>N/A</td>
			<td>ECG Analysis</td>
		</tr>
		<tr>
			<td>JELCO PROTECTIV Safety I.V. Catheters, 25 gauge</td>
			<td>Smiths Medical</td>
			<td>3060</td>
			<td>Used to catherize marginal ear vein</td>
		</tr>
		<tr>
			<td>MATLAB (R2019b, Update 5)</td>
			<td>MathWorks</td>
			<td>N/A</td>
			<td>Required to run EEGLAB</td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>Sony Stereo</td>
			<td>ECM-D570P</td>
			<td>Recording of audible manifestions of seizures</td>
		</tr>
		<tr>
			<td>Micropore Medical Tape, Paper, White</td>
			<td>3M</td>
			<td>1530-1</td>
			<td>Used to secure wires and create ear splint</td>
		</tr>
		<tr>
			<td>Natus NeuroWorks</td>
			<td>Natus</td>
			<td>LC101-8</td>
			<td>Acquisition and review software</td>
		</tr>
		<tr>
			<td>Pentylenetetrazol (1 - 10 mg/kg always in 1mL volume)</td>
			<td>Sigma-Aldrich</td>
			<td>88580</td>
			<td>Dilutions prepared in saline</td>
		</tr>
		<tr>
			<td>Photic Stimulator</td>
			<td>Grass</td>
			<td>PS22</td>
			<td>Stimulator to control frequency, delay, duration, intensity of the light pulses</td>
		</tr>
		<tr>
			<td>Plastic wire organizer / bundler</td>
			<td>12Vwire.com</td>
			<td>LM-12-100-BLK</td>
			<td>Bundle wires to cut down on noise</td>
		</tr>
		<tr>
			<td>PS 22 Photic Stimulator</td>
			<td>Grass Instruments</td>
			<td>BZA641035</td>
			<td>Strobe light with adjustable flash frequency, delay, and intensity</td>
		</tr>
		<tr>
			<td>PVC pipe</td>
			<td>Generic</td>
			<td>N/A</td>
			<td>Prevents small rabbits from kicking their hind legs and causing spinal injury</td>
		</tr>
		<tr>
			<td>Quantum Amplifier</td>
			<td>Natus</td>
			<td>13926</td>
			<td>Amplifier / digitizer</td>
		</tr>
		<tr>
			<td>Quantum HeadBox Amplifier</td>
			<td>Natus</td>
			<td>22134</td>
			<td>64-pin breakout box</td>
		</tr>
		<tr>
			<td>Rabbit Restrainer</td>
			<td>Plas-Labs</td>
			<td>501-TC</td>
			<td>Various size rabbit restrainers are available. 6&#34; x 18&#34; x 6&#34; in this study.</td>
		</tr>
		<tr>
			<td>Rubber pad (booster)</td>
			<td>Generic</td>
			<td>N/A</td>
			<td>Raises small rabbits up in the restrainer to prevent neck compression</td>
		</tr>
		<tr>
			<td>SpO2 ear clip</td>
			<td>NONIN</td>
			<td>61000</td>
			<td>PureSAT/SpO2</td>
		</tr>
		<tr>
			<td>SpO2 sensor adapter</td>
			<td>NONIN</td>
			<td>13931</td>
			<td>XPOD PureSAT/SpO2</td>
		</tr>
		<tr>
			<td>SRG-X120 1080p PTZ Camera with HDMI, IP &#38; 3G-SDI Output</td>
			<td>Sony</td>
			<td>SRG-X120</td>
			<td>Impela Camera</td>
		</tr>
		<tr>
			<td>Terumo Sur-Vet Tuberculin Syringe 1cc 25G X 5/8&#34; Regular Luer</td>
			<td>Sur-Vet</td>
			<td>13882</td>
			<td>Used to inject intravenous medications</td>
		</tr>
		<tr>
			<td>Veterinary Injection Plug Luer Lock</td>
			<td>Sur-Vet</td>
			<td>SRIP2V</td>
			<td>Injection plug for inserting the needle for intravenous medication</td>
		</tr>
		<tr>
			<td>Webcol Alcohol Prep, Sterile, Large, 2-ply</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>To prepare ear vein before catheterization</td>
		</tr>
	</tbody>
</table>

multi-system monitoring for identification of seizures, arrhythmias and apnea in conscious restrained rabbits

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.

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...

Watch this Scientific Journal Video about Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits at JoVE.com

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

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.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Cardiac and respiratory dysfunction contributes to sudden death after a seizure. Therefore, to study this phenomenon, a multi-system approach was applied to a rabbit model. This technique enables simultaneous monitoring and later analysis of the neurologic, cardiac, and respiratory function of multiple rabbits surrounding seizures, arrhythmias, and preceding death.While a disease may be primarily neuronal or cardiac, it may include electrical disturbances in both the brain and the heart, such as seizures and arrhythmias, as well as respiratory dysfunction. It can be difficult to work with rabbits because of their hind leg strength. Take care to keep the rabbits calm when moving them, and when attaching the monitors.Begin by connecting the computer to an amplifier with a 64-pin headbox. To make every eighth pin on the headbox a reference, set the reference electrode to independent. For implantation of the ECG electrodes, secure a rabbit in the supine position with the head at the investigator's knees and lower than the rest of its body.With a second investigator spreading the fur to expose the underlying skin, subdermally insert 35-degree angle bent electrodes into each axilla. Place leads on the chest, posterior to the right and left forelimbs. And on the abdomen, anterior to the left hind limb.Then place a ground pin electrode anterior to the right hind limb on the abdomen. When all of the ECG leads have been properly placed, secure the rabbit in an appropriately sized restrainer with the hind limbs under the animal. Using a 45-degree angle approach, insert subdermal straight-pin electrodes into the scalp.Place one EEG lead on the right anterior, left anterior, right occipital, and left occipital regions of the head. And place a central reference lead at the central point between the other four leads. Run the wires between the ears, and loosely tether them to the restrainer behind the head.To monitor respiration during the experiment, attach a pulse oximeter to one ear over the marginal ear vein, and gently secure a face mask with capnography tubing over the mouth and nose of the animal. Then attach the other end of the tubing to the vital signs monitor. To record video EEG-ECG, open an appropriate commercially-available EEG software program and adjust the video such that all of the rabbits can be observed within the field of view.Perform a baseline recording for the animal for a minimum of 10 to 20 minutes, or until the heart rate stabilizes to 200 to 250 beats per minute for at least five minutes. Set the low frequency filter to one hertz and the high frequency filter to 59 hertz. Add time-locked notes in real-time to indicate the timing of interventions, neuro-cardiac events, and motor or investigator artifacts.For photic stimulation, place a light source with a circular reflector 30 centimeters in front of the rabbit at eye level with the flash intensity set to the maximum. And place two mirrors on each side of the head, and one mirror behind the rabbit, so that the light enters the rabbit's eyes. Attach the light to a controller with an adjustable rate, intensity, and duration.With the photic stimulator set to one hertz and the flash on, record the response for 30 seconds. Cover the rabbit's eyes with a mask to simulate or cause eye closure and stimulate for another 30 seconds. After recording the eyes open and eyes closed response at each frequency, turn off the photic stimulator for 30 seconds and set the controller to the next frequency setting.After recording for 60 seconds at each frequency from one to 25 hertz in two hertz intervals, decrease the frequency from 60 to 25 hertz in five hertz increments, and record for 30 seconds with the eyes open and 30 seconds with the eyes closed. At the end of the experiment, remove the EEG and ECG leads from the rabbit and return the animals to their home cages for routine care by the husbandry staff. Before administering the medication, collect 10 to 20 minutes of baseline EEG-ECG video from the untreated rabbit as demonstrated.For the oral administration of a medication of interest, mix 0.3 milligrams per kilogram of the drug of interest in three milliliters of food-grade applesauce, and load the mixture into a three milliliter oral syringe. Gently lifting the upper lip of the rabbit, slide the tip of the syringe into the side of the mouth that is unobstructed by the rabbit's teeth, and inject the entire volume of drug-supplemented applesauce into the rabbit's mouth. Then collect two hours of EEG-ECG video data before returning the rabbit to its home cage for routine care.Before recording the EEG-ECG response to intravenous medication administration, shave the posterior surface of the rabbit's ear and use 70%ethanol to disinfect the site, and to dilate, the marginal ear vein. Carefully, cannulate the marginal ear vein with a 25-gauge angiocatheter, and place an injection plug at the end of the catheter. Tape a splint created from three rolled pieces of gauze to the ear to secure the catheter in place and to hold the ear upright.Inject one milliliter of 10 USP units per milliliter of heparinized saline to keep the catheter patent. When baseline recording has finished, inject saline supplemented with one to 10 milligrams per kilogram of the drug of interest into the catheter once every 10 minutes. After each dose, carefully monitor the video EEG-ECG, oximetry capnography, for any neuro, cardiac, electrical and respiratory abnormalities or visual evidence of epileptiform activity.Note these changes in real-time, as well as during post analysis. To analyze the ECG video, use an appropriate commercially-available software to identify periods of tachycardia, brachycardia, ectopic beats, or other arrhythmias within the ECG data. To reduce the amount of data to review, create a tachogram to increase the ease with which periods of tachycardia, bradycardia, or irregularities of the RR interval can be identified.For video EEG analysis after a drug administration experiment, visually scroll through the EEG tracing to distinguish epileptic versus non-epileptic movements for at least one minute after each dose of drug. For video EEG analysis after a photic stimulation experiment, create a spectral analysis plot in an appropriate EEG analysis software program. Then analyze the occipital leads of the EEG for the presence and absence of the occipital driving rhythm.The occipital driving rhythm will create a peak in the spectral analysis that corresponds to the frequency of the photic stimulator. Assessment of the ECG morphology allows the detection of abnormal heart rates, conduction, and ECG wave forms. The traces can also be used to quantify the heart rate, RR interval, PR interval, P duration, QRS interval, QT interval, QTc, JT interval, and T-peak to T-end interval.Recordings from the occipital EEG leads typically exhibit a higher amplitude than that observed for frontal lead data. And the dominant frequency in all of the leads are commonly measured in the Delta range. Here, sleep spindle waves from a representative rabbit experiment are shown.Multiple EEG montages of one period of sleep demonstrate that these waves arise from the center of the head, which is consistent with human findings. In addition to normal EEG changes, various conscious, non-epileptic rabbit movements can also be observed during baseline recordings that can be used to distinguish these data from epileptiform discharges. It is important to place the ECG lead securely into the skin to keep the wires away from the legs and to take care that the electrodes remain attached when transferring rabbits into the restrainer.This multi-system recording apparatus will enable future drug safety and efficacy studies, and facilitate a comprehensive understanding of various acquired and inherited diseases. This technique captures the course of potentially fatal, multiorgan dysfunction after a seizure, which will lead to a better understanding of the mechanism of SUDEP.

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