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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Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering 
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac 
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4. In animal models, it is valuable to compare 
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
 been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5 and 
 hypertension 6, following exercise 7 and exposure to air pollutants 8, as well as determination of the antiarrhythmic efficacy of pharmacological 
 agents 9,10. 
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a 
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that 
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min 
11,12 11,12, and results in mortality in 20% of cases 13. Many individuals survive the SE, but die within 30 days 14,15.
 The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these 
 deaths 14-17. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature 
 sudden and unexpected death associated with epilepsy (SUDEP) 18. As with SE, the precise mechanisms mediating SUDEP are not known. 
 It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22.
 To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is 
 necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders 
 23-25. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
 collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
 These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical 
 activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden 
 cardiac death.

Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described.

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Blind Endotracheal Intubation in Neonatal Rabbits

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical characteristics, which is further complicated in the case of the newborn. Of the different methods of advanced airway management, endotracheal intubation is less aggressive than tracheostomy, and is more feasible than supraglottic management given the lack of supraglottic devices of such a small size. As direct glottis visualization is very difficult in animals this size, this blind intubation model is presented as an effective alternative, especially for experiments requiring prolonged anesthesia. Using this method, we performed blind intubations with a 90% success rate.

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...