Name: Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus (Video) | JoVE Journal
Uploaded: 2011-04-05T13:00:00.000+00:00
Duration: 8 min 28 s
Description: Learn ECG evaluation methods to assess cardiac risk and arrhythmia susceptibility in rats following status epilepticus. This technique enables researchers to study seizure-related cardiac dysfunction using electrocardiogram recordings.

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This research was supported by a grant from the Citizens United for Research in Epilepsy (CURE) to SLB.

1. Materials to Construct
 <ol>
	<li>A jugular vein catheter is constructed from a piece (100 mm) of PE-50 polyethylene tubing, beveled at one end, and then filled with heparin saline (50 U heparin/mL saline).</li>
	<li>The ECG recording electrodes are constructed from two 100 mm lengths of insulated silver wire (30AWG). One end of both wires is stripped and soldered to a microconnector, and the insulation is twisted to form a third wire that is used as ground. Five-mm of insulation is stripped from the distal end of the wires and discarded.</li>
</ol>
2. Implanting Jugular Vein Catheter
<ol>
	<li>Anesthetize the animal by administration of Urethane, ip (1.2 g/kg).</li>
	<li>Routinely check (10 min intervals) depth of anesthesia by evaluating the pedal withdrawal response and/or eye reflexes. If needed, supplement anesthesia.</li>
	<li>After the animal is at the appropriate plane of anesthesia, shave the right side of the neck from the clavicle to the chin.</li>
	<li>Make a longitudinal incision (10-15 mm) in the skin above the carotid artery and open the incision.</li>
	<li>Using blunt dissection techniques, spread the underlying muscle to locate and isolate the right jugular vein.</li>
	<li>Place two pieces of surgical silk (#2) under the jugular vein, and position the sutures at the rostral and caudal most portions of the incision. Tie the rostral ligature to discontinue flow through the vein.</li>
	<li>Retract the caudal ligature to lift the vessel off the underlying tissue and make a small cut. Then insert the beveled end of the heparin-filled (50 U/ml saline) PE-50 polyethylene catheter (100 mm in length), and advance the tip approximately 8 mm to the level of the right atrium. </li>
	<li>In order to secure the catheter, tie the caudal ligature around both the vessel and catheter. Finally, close the wound with either wound clips or suture.</li>
	<li>During the implantation of the catheter, the exposed end is either clamped with a hemostat or plugged to prevent back flow of blood prior to attachment to an infusion pump.</li>
</ol>
3. Implanting CG Electrodes
<ol>
	<li>Place the animal on its back and shave the chest. </li>
	<li>For placement of the electrodes, make two longitudinal incisions (≈ 10 mm), one in the upper right and one in the lower left quadrants of the chest, and free the overlying skin to expose the thoracic muscles.</li>
	<li>Suture one of the exposed 5-mm tips of the silver-wire electrodes into the thoracic muscles in each of the exposed areas, and place the ground electrode subcutaneously.</li>
	<li>Close the incisions with wound clips and plug the microconnector into the recording device.</li>
	<li>Wrap the animal loosely in a towel to maintain body temperature during the procedure.</li>
</ol>
4. Recording ECG
<ol>
	<li>Recordings are made using a Powerlab Data Acquisition System and Macintosh computer or similar hardware/software (amplified, 50X; filtered, 1-1000 Hz). </li>
	<li>Following a 20-30 min equilibration period, record ECG for 20 min.</li>
</ol>
5. Inducing Ventricular Arrhythmias
<ol>
	<li>Attach the jugular vein catheter to a remote syringe placed in a programmable infusion pump.</li>
	<li>Initiate the aconitine infusion at a rate to deliver a constant dose of 5 ug/kg/min for 7 minutes. </li>
	<li>Following ventricular fibrillation or the end of the infusion period, euthanize the animal using an approved procedure. </li>
</ol>
6. Analysis of ECG 
<ol>
	<li>The QT interval represents the total duration of ventricular electrical activity. Fig. 1 shows a model ECG recording and the duration of the QT interval. Calculate the mean time between the initiation of the Q-wave and termination of the T-wave for all recorded heartbeats over a five-minute interval obtained during the 20 min recording period. </li>
	<li>To correct the QT interval for differences in heart rate (QTc), use Bazett’s formula, QTc=QT/(RR)½, where RR is the mean interval between heartbeats. The RR interval is calculated as the duration between successive Q-waves obtained during the analyzed period.</li>
	<li>The QT dispersion (QTd) is calculated by subtracting the minimum QT from the maximum QT recorded for each animal.</li>
</ol>
7. Analysis of Heart Rate Variability
<ol><li>Software programs (i.e. LabChart 7, ADInstruments, Inc.) that analyze variability in the RR intervals obtained during the period of analysis are used for automated calculation of both temporal and/or spectral variance in heart rate. </li></ol>
8. Analysis of Susceptibility to Ventricular Arrhythmias
<ol><li>Calculate the latency from the initiation of aconitine infusion to the first premature ventricular contraction (PVC, Fig. 2, Panel 1), ventricular tachycardia (VT, Fig, 2, Panel 2) and ventricular fibrillation (VF, Fig. 2 Panel 3). </li></ol>
9. Representative Results
Fig. 3 illustrates one measure of HRV in the spectral domain (root mean squared of the standard deviation of the RR 
interval, RMSSD) in control rats and in animals that underwent SE one week prior to testing. This measure of HRV, which represents changes in the 
parasympathetic control of cardiac function 26, is significantly decreased in animals following status epilepticus. Fig. 4 shows that both 
corrected QT interval (QTc, Panel A) and QT dispersion (QTd, Panel B) are significantly increased two weeks following SE27. Taken together, these 
data predict that SE increases risk of ventricular arrhythmias. This prediction is confirmed by the findings that the periods of aconitine infusion 
necessary to induce the experimental arrhythmias (PVC, VT, VF) observed in these studies were significantly smaller in animals following SE (Fig. 5)27.
<img src="/files/ftp_upload/2726/2726fig1.jpg" alt="Electrocardiogram diagram, QT interval measurement, cardiac cycle phases PQRST." data-altupdatedat="1788235205000" data-alt="figure 1"> Figure 1.Model ECG recording of a normal heartbeat showing P, QRS, and T waves. The QT interval is calculated as the time between the dashed lines.
<img src="/files/ftp_upload/2726/2726fig2.jpg" alt="ECG waveforms, three-channel, cardiac activity analysis, labeled peaks A, B, C, D, measurement chart." data-altupdatedat="1788235205000" data-alt="figure 2"> Figure 2. Panel 1) Premature ventricular contractions (PVCs) are isolated contractions of the ventricles (B) that occur between normal heartbeats (A). PVC recordings are characterized by the absence of P and T waves. 
Panel 2 Ventricular tachycardia (VT) is a series of multiple ventricular contractions (C) that occur with no intervening normal heartbeats (A).
Panel 3 Ventricular fibrillation (VF) is the lack of discernable rhythm and no association between QRS and P waves (D).
<img src="/files/ftp_upload/2726/2726fig3.jpg" alt="RMSSD analysis bar graph, comparison of control vs SE group." data-altupdatedat="1788235205000" data-alt="figure 3"> Figure 3. The root mean squared of the standard deviation (RMSSD; msec) of the intervals between heartbeats observed in control rats and in animals undergoing status epilepticus (SE) 1 wk prior to testing. **p&lt;0.01 compared to Control.
<img src="/files/ftp_upload/2726/2726fig4.jpg" alt="QT interval comparison; bar graph; QTc, QTd measurements; Control vs. SE group results." data-altupdatedat="1788235205000" data-alt="figure 4"> Figure 4. Mean corrected QT interval (Panel A) and QT dispersion (Panel B) calculated from ECG recordings obtained from control animals (Cont) and from rats undergoing status epilepticus (SE) two weeks prior to testing. *p&lt;0.05 compared to Cont. (Metcalf, et al., Am. J. Physiol. Heart and Circ, 2009, Am Physiol Soc, used with permission from 27).
<img src="/files/ftp_upload/2726/2726fig5.jpg" alt="Latency comparison bar graph; Control vs. SE in PVC, VT, VF; experiment results." data-altupdatedat="1788235205000" data-alt="figure 5"> Figure 5. Latencies (sec) between the initiation of aconitine infusion and the 1st premature ventricular contraction (PVC), ventricular tachycardia (VT), and ventricular fibrillation (VF) in control animals (Cont) and rats undergoing status epilepticus (SE) two weeks prior to testing. *p&lt;0.05, **p&lt;0.01 compared to Cont. (Metcalf, et al., Am. J. Physiol. Heart and Circ, 2009, Am Physiol Soc, used with permission from 27).

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</ol>

We have nothing to disclose.

Two aspects of the described procedures are of critical importance. Firstly, the rate of aconitine administration to the 
heart must be equivalent across animals. This requires consistent placement of the jugular vein catheter tips relative to the heart, and careful 
adjustment of the infusion rate. The rate of aconitine delivery to the hearts must be equal in order to appropriately evaluate the onset of 
ventricular arrhythmias relative to altered susceptibility. If aconitine delivery varies, then the latency to arrhyt...

methods for ecg evaluation of indicators of cardiac risk, and susceptibility to aconitine-induced arrhythmias in rats following status epilepticus

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 ...

The overall goal of this procedure is to evaluate cardiac, electrical function and susceptibility to ventricular arrhythmias in experimental animals following seizures. This is accomplished by first inserting an infusion catheter into a jugular vein of an animal that has undergone seizure activity. Next, electrodes are implanted in thoracic muscles to record cardiac electrocardiograms or ECGs.ECGs are then recorded under control conditions and during jugular vein infusion of an agent that induces cardiac arrhythmias. Finally, ECG records are evaluated. Ultimately, results can be obtained that show seizures alter electrical cardiac function, and increased risk of lethal ventricular arrhythmias.Through analysis of ECG recordings, the seizure evoked changes in ECG can serve as a prognostic indicator of the risk of sudden cardiac death. The main advantage of this technique is that these procedures, which are non-invasive in humans, produce recordings of electrical activity to the heart that can be used to predict the risk of lethal cardiac arrhythmias. Furthermore, in animal models, changes in cardiac activity can be directly related to susceptibility of arrhythmias.This method can be used to address the key question in the field of seizure disorders. That is the relationship between changes in electrical activity of the heart and sudden cardiac death following status epilepticus and an epilepsy. These techniques have implications for the treatment of seizure disorders as individuals identified as being at risk for cardiac dysfunction resulting from seizures.Using this non-invasive procedure could be placed on cardioprotective therapy to reduce the risk of sudden cardiac death. Though this method can provide insights into cardiac dysfunctions associated with seizure disorders, it can also be applied to any pathological condition, which can be characterized by lethal ventricular arrhythmias and which can be modeled in animals. Generally, individuals familiar with the fundamental and surgical techniques of rodents or other small animals should not have any problems with this method.We first had the idea for using this method and we were looking for a fast, inexpensive, and easily performed procedure to evaluate cardiac function that was also applicable to diagnosis in humans Before implanting ECG electrodes into the rat for data collection and induction of arrhythmias during IV administration of an arrhythmogenic agent such as aconitine, construct a jugular vein catheter from a 100 millimeter long piece of PE 50 polyethylene tubing by beveling one end, and then blunting the bevel. Then fill the catheter with heparin saline. Next construct ECG recording electrodes from two 100 millimeter lengths of insulated silver wire.Strip one end of both wires and solder them to a micro connector. Then twist the insulation to form a third wire that is used as a ground After soldering wires are sealed with epoxy. Strip five millimeters of insulation from the distal end of the wires and discard to implant the jugular vein catheter.First anesthetize the animal by an intraperitoneal injection of urethane. This dose is sufficient to maintain the animal in the appropriate plane of anesthesia for the duration of the procedure. This is a non survival surgery.Ensure that the animal is at the appropriate plane of anesthesia by performing a toe pinch. Then shave the right side of the neck from the clavicle to the chin and shave the chest. Next, disinfect the shaved portion of the neck with iodine alcohol and make a longitudinal incision in the skin above the carotid artery and open the incision using fine tipped curved dissecting forceps.Gently separate the skin from the underlying muscle and then the muscles that overlay the jugular vein. Finally, using similar techniques, free the jugular vein from the underlying tissue. Place two pieces of number two, surgical silk under the jugular vein, and position the sutures at the rostral and coddle.Most portions of the incision tie the rostral ligature to discontinue flow through the vein. Retract the al ligature to lift the vessel off the underlying tissue and using micro scissors or a 23 gauge needle. Make a small cut.Then insert the blunted beveled end of the previously constructed heparin filled PE 50 polyethylene catheter and advance the tip approximately eight millimeters to the level of the right atrium. Secure the catheter by tying the al ligature around both the vessel and catheter. Finally, close the wound with either wound clips or sutures.Once the catheter is implanted, place the animal on its back and disinfect the shaved portion of the chest. For placement of the electrodes, make two 10 millimeter longitudinal incisions, one in the upper right and one in the lower left quadrants of the chest and free the overlying skin to expose the thoracic muscles using relatively fine surgical silk suture. One of the exposed five millimeter tips of the silver wire electrodes into the thoracic muscles in each of the exposed areas.Close the incisions with wound clips and plug the micro connector into the recording device. ECG recordings are made using a power lab data acquisition system and Macintosh computer or similar hardware and software following a 20 to 30 minute equilibration period during which the animal is placed on a grounded metal sheet and loosely wrapped in a towel. To maintain its body temperature record ECG for 20 minutes, attach the jugular vein catheter to a remote syringe placed in a programmable infusion pump.Initiate the aconitine infusion to deliver a constant dose of five micrograms per kilogram per minute For seven minutes, denote the starting point of the infusion on the computer record following ventricular fibrillation or the end of the infusion period. Euthanize the animal using an approved procedure. The QT interval represents the total duration of ventricular electrical activity.This figure shows a model ECG recording and the duration of the QT interval. Calculate the mean time between the initiation of the Q wave and termination of the T-wave for all recorded heartbeats over a five minute interval obtained during the 20 minute control recording period to correct the QT interval for differences in heart rate. Use bass's formula shown here where RR is the mean interval between heartbeats.The RR interval is calculated as the duration between successive Q waves obtained during the analyzed period. The QT dispersion is calculated by subtracting the minimum QT from the maximum QT recorded for each animal. To analyze heart rate variability, use a software program such as lab chart seven that analyzes variability in the RR intervals obtained during the period of analysis and calculates both temporal and or spectral variants in heart rate.For the analysis of susceptibility to ventricular arrhythmias, calculate the latency from the initiation of aconitine infusion to the first premature ventricular contraction, ventricular tachycardia, and ventricular fibrillation. This figure illustrates one measure of HRV in the spectral domain. The root means squared of the standard deviation of the RR interval or R-M-S-S-D in control rats and in animals that underwent SE one week prior to testing.This measure of HRV, which represents changes in the parasympathetic control of cardiac function, is significantly decreased in animals following se. This figure shows that both corrected QT interval and QT dispersion are significantly increased. Two weeks following se taken together these data predict that se increases risk of ventricular arrhythmias.This prediction is confirmed by the findings that the periods of aconitine infusion necessary to induce the experimental arrhythmias observed in these studies were significantly smaller in animals following SE.Generally, this procedure can be done in about an hour and a half when it is performed properly. While attempting this procedure, it's important to remember that the jugular vein catheter must be kept clear and that the ECG electrodes are constructed and implanted in such a way to produce the most noise free signal possible. After watching this video, you should have a good understanding of how to evaluate electrical activity of the heart and relate it to susceptibility to lethal ventricular arrhythmias.In rodent models of pathologies associated with sudden cardiac death.

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In this video, we describe simultaneous recording of video, EEG, ECG and respiratory cues in a rabbit model in response to orally administered drug to understand the sequence of events leading to seizure. The model can be used for testing potential drugs for their neuro-cardiac safety.

Multisystem Monitoring in Rabbit Model: A Method to Assess Neuro-Cardiac and Respiratory Parameters to Study the Oral Drug-induced Triggers for  Arrhythmia and Seizures

In this video, we demonstrate a multisystem monitoring method to evaluate neurological and cardiorespiratory abnormalities during intravenous injection of a seizure-inducing drug. The simultaneous monitoring of video EEG, ECG, oximetry, and capnography parameters helps to understand the real-time physiological changes associated with epileptic seizures.

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Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

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Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

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Inducing an Acute Electroconvulsive Seizure in a Rat Model

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

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Electrocardiogram Recordings in Anesthetized Mice using Lead II

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.

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

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.

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Learn ECG evaluation methods to assess cardiac risk and arrhythmia susceptibility in rats following status epilepticus. This technique enables researchers to study seizure-related cardiac dysfunction using electrocardiogram recordings.

Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus (Video) | JoVE Journal

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

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