Name: optimization of transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice
Uploaded: 2022-06-29T15:00:00
Description: Watch this Scientific Journal Video about Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice at JoVE.com

Figure 2 was created with BioRender.com. This work was supported by grants from the National Heart, Lung, and Blood Institute at the National Institutes of Health (HL096844 and HL133127); the American Heart Association (2160035, 18SFRN34230125 and 903918 [MBM]); and the National Center for Advancing Translational Sciences of the National Institute of Health (UL1 TR000445).

This procedure was approved by the Vanderbilt Institutional Animal Care and Use Committee and is consistent with the Guide for the Care and Use of Laboratory Animals. The protocol was developed using both genetic9 and acquired10 (e.g., hypertension) mouse models of AF susceptibility. The operator was blinded to the phenotype of the mouse under study.
1. Animal selection
<ol>
	<li>For genetic models, subject mice to biweekly (i....

<ol>
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Transesophageal atrial pacing not only allows serial studies in the same animal, but its duration is typically shorter than intracardiac studies (~20 min), thus minimizing anesthetic use and its effects on electrophysiologic parameters. 
It is critical to optimize the methods initially for each individual mouse model. Aging increases AF inducibility in normal mice18,19, and individual genetic models may demonstrate AF inducibility over...

Atrial fibrillation (AF) represents a final common pathway for multiple acquired and genetic risk factors. For studies investigating the pathophysiologic mechanisms of the AF substrate, mouse models are advantageous given the ease of genetic manipulation and the fact that, in general, they reproduce the AF susceptibility observed in humans for different clinical phenotypes1,2,3. However, mice rarely develop spontaneous AF4, necessitating the use of provocative atrial pacing studies.
Programmed electrical stimulation (PES) can be performed to assess murine atrial electrophysiology and AF susceptibility using either intracardiac5 or transesophageal6 pacing. While the transesophageal approach is particularly advantageous as a survival procedure, its use is complicated by the numerous published experimental protocols7,8 and sources of variability that can hinder reproducibility9. Moreover, limited reported protocol comparisons make selecting an appropriate pacing protocol challenging.
The current protocol aims to utilize a systematic strategy to develop model-specific transesophageal PES methods for assessing murine AF susceptibility in order to increase reproducibility. Importantly, initial pilot studies are performed to optimize the pacing protocol by accounting for age, sex, and pacing mode variability, with pacing designed to minimize inadvertent parasympathetic stimulation that can confound results9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>27 G ECG electrodes</td>
			<td>ADInstruments</td>
			<td>MLA1204</td>
			<td></td>
		</tr>
		<tr>
			<td>2-F octapolar electrode catheter</td>
			<td>NuMED</td>
			<td>CIBercath</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated carbon canister</td>
			<td>VetEquip</td>
			<td>931401</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>ADInstruments</td>
			<td>LabChart v8.1.13</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological amplifier</td>
			<td>ADInstruments</td>
			<td>FE231</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition hardware</td>
			<td>ADInstruments</td>
			<td>PowerLab 26T</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>MWI Veterinary</td>
			<td>NC1886507</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>DPIP</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Bloom Associates</td>
			<td>DTU-210</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>Model A365</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice

Mouse models of genetic and acquired risk factors for atrial fibrillation (AF) have proven valuable in investigating the molecular determinants of AF. Programmed electrical stimulation can be performed using transesophageal atrial pacing as a survival procedure, thus enabling serial testing in the same animal. However, numerous pacing protocols exist, which complicates the reproducibility. The present protocol aims to provide a standardized strategy to develop model-specific experimental parameters to improve reproducibility between studies. Preliminary studies are performed to optimize the experimental methods for the specific model under investigation, including age at the time of the study, sex, and parameters of the pacing protocol (e.g., mode of pacing and definition of AF susceptibility). Importantly, care is taken to avoid high stimulus energies, as this can cause stimulation of the ganglionic plexi with inadvertent parasympathetic activation, manifested by exaggerated atrioventricular (AV) block during pacing and often associated with artifactual AF induction. Animals demonstrating this complication must be excluded from the analysis.

Transesophageal atrial pacing studies assess the electrophysiologic properties of the SA and AV nodes by determining the SNRT and AVERP, as well as AF susceptibility6 (Figure 1). ECG recording enables measurements of P wave duration, PR interval, QRS duration, and QT/QTc intervals. Continuous recording of the ECG during rapid atrial pacing can provide the following measurements of AF vulnerability: the number of episodes induced during the study, cumulative and averag...

Watch this Scientific Journal Video about Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice at JoVE.com

Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice

The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.

Mouse models of genetic and acquired risk factors for atrial fibrillation (AF) have proven valuable in investigating the molecular determinants of AF. ...

Programmed electrical stimulation using transesophageal atrial pacing can determine electrophysiologic properties and susceptibility to atrial fibrillation in mice. Here we described a standardized procedure for protocol development that can optimize reproducibility. The transesophageal approach for atrial pacing is typically a survival procedure, thus enabling serial testing in the same animal.Important components for protocol development include initial pallet studies to optimize experimental parameters for the model under study and the identification of arrhythmias caused by pacing-induced parasympathetic stimulation. For genetic models, subject mice to biweekly atrial pacing, starting at eight weeks of age to determine the optimal period of AF susceptibility, study both sexes as one may not develop an AF phenotype. For acquired models, conduct pacing after mice achieved physical maturity.While maturity is typically achieved at 12 weeks, adjust age as necessary for experimental models. Perform both burst and decremental pacing to determine the optimal pacing mode. Separate each procedure by a minimum of 24 hours.Analyze the data using multiple definitions of AF susceptibility. Use the optimized model specific parameters and definition of AF susceptibility for subsequent studies on additional mice. After the loss of the pedal reflex, place the anesthetized mouse in a supine position on a heating pad designed to maintain body temperature at approximately 37 degrees Celsius with the hindlimbs taped to the pad surface.Obtain a surface ECG lead one by the subcutaneous placement of 27 gauge ECG needle electrodes connected to a biological amplifier and data acquisition hardware into the forelimbs with a ground electrode in the hindlimb. Insert a two-French octapolar electrode catheter connected to a stimulator and stimulus isolator into the esophagus. Insert to a depth that approximates the distance from the mouth to just above the xiphoid cartilage with the neck extended.Begin data acquisition with continuous recording of ECG lead one using the analysis software. Properly position the catheter within the esophagus to enable capture. To do so, apply a 1.5 milliampere stimulus with a pulse width of two milliseconds at a cycle length slightly shorter than the sinus cycle length.Carefully position the catheter until consistent atrial capture is obtained. To determine the atrial diastolic capture threshold, initiate pacing at 1.5 milliamperes with a pulse width of two milliseconds at the cycle length used for atrial capture. Decrease the stimulus amplitude by 0.05 milliampere increments until the loss of atrial capture with subsequent increase until capture.Obtain the lowest possible pacing threshold to minimize inadvertent pacing-induced parasympathetic stimulation. Adjust the stimulus amplitude to twice the threshold. Measure electrophysiologic parameters, including the sinus node recovery time, Wenckebach cycle length, and atrioventricular effective refractory period prior to rapid atrial pacing for AF induction.Perform pacing at twice the threshold with a pulse width of two milliseconds using either burst pacing at different cycle lengths or decremental pacing as determined from initial studies. For burst pacing, pace at an initial cycle length of 50 milliseconds for 15 seconds, followed by trains at cycle lengths of 40, 30, 25, 20, and 15 milliseconds. For decremental pacing, perform three to five trains and pace at a cycle length of 40 milliseconds that is reduced by two milliseconds every two seconds until termination at 20 milliseconds.Terminate the procedure after 30 seconds of sinus rhythm following the last pacing train or after a 10 minute episode of AF, whichever comes first. Gently remove catheter and ECG electrodes. In the case of serial testing, wait for a minimum of 24 hours before repeating the pacing procedure.ECG Recording enabled measurements of P wave duration, PR interval, QRS duration, and QT and QTC intervals. Continuous recording of the ECG during rapid atrial pacing provided a recording of each episode of AF induced during the study. From these data, the cumulative and average duration of the episodes and the number of sustained AF episodes were recorded.Episodes of excessive atrioventricular block during pacing indicate periods of pacing-induced parasympathetic stimulation, signifying that any associated AF was an artifact of this phenomenon rather than the pathophysiology of the model itself. This was caused by inadvertent stimulation of the ganglionic plexi located on the posterior left atrium, resulting in parasympathetic activation. This type of arrhythmia induction increases the incidence of AF in control mice and causes greater arrhythmia variability within an experimental group.Given these contaminating features, animals that experience AF under these conditions should be excluded from the analysis. When using this experimental strategy, initial pilot studies and the recognition of parasympathetic stimulation causing inadvertent AF induction are particularly important for protocol optimization and study reproducibility.

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