Name: noninvasive electrocardiography in the perinatal mouse
Uploaded: 2020-06-12T13:30:00
Description: Watch this Scientific Journal Video about Noninvasive Electrocardiography in the Perinatal Mouse at JoVE.com

The authors acknowledge generous support from the Saving tiny Hearts Society (KLT), the UNE COBRE Program (NIGMS grant number P20GM103643; LAF), and the SURE Fellowship Program at the University of New England (VLB), as well as patient technical support from Ashish More (iWorx, Dover, NH). Figure 3, Figure 4, and Figure S1 were created with Biorender software.

The following protocol follows the standards of the Institutional Animal Care and Use Committee of the University of New England. Close observation of the protocol should deliver satisfactory ECG reads in all examined neonates (n &#62; 70).
1. Device preparations
<ol>
	<li>Plug the device into the USB port of a computer with the ECG software downloaded on it. The measuring device will automatically begin heating up to (37 &#176;C/98.6 &#176;F). The internal heating unit is contained within t...

<ol>
	<li>Pappano, A. J., Wier, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular Physiology. 11, 40-41 (2019).</li><li>Kaese, S., Verheule, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+electrophysiology+in+mice:+A+matter+of+size.">Cardiac electrophysiology in mice: A matter of size.</a> Frontiers in Physiology. 3, 1-19 (2012).</li><li>Sisakian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyopathies:+Evolution+of+pathogenesis+concepts+and+potential+for+new+therapies.">Cardiomyopathies: Evolution of pathogenesis concepts and potential for new therapies.</a> World Journal of Cardiology. 6 (6), 478-494 (2014).</li><li>London, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Arrhythmias:+From+(Transgenic)+Mice+to+Men.">Cardiac Arrhythmias: From (Transgenic) Mice to Men.</a> Journal of Cardiovascular Electrophysiology. 12 (9), 1089-1091 (2001).</li><li>Zehendner, C. M., Luhmann, H. J., Yang, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+and+Novel+Method+to+Monitor+Breathing+and+Heart+Rate+in+Awake+and+Urethane+Anesthetized+Newborn+Rodents.">A Simple and Novel Method to Monitor Breathing and Heart Rate in Awake and Urethane Anesthetized Newborn Rodents.</a> PLoS ONE. 5, 62628 (2013).</li><li>Zhao, Y., 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=Dry-contact+microelectrode+membranes+for+wireless+detection+of+electrical+phenotypes+in+neonatal+mouse+hearts.">Dry-contact microelectrode membranes for wireless detection of electrical phenotypes in neonatal mouse hearts.</a> Biomedical Microdevices. 17 (2), 40 (2015).</li><li>Cao, H., 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=Wearable+multi-channel+microelectrode+membranes+for+elucidating+electrophysiological+phenotypes+of+injured+myocardium.">Wearable multi-channel microelectrode membranes for elucidating electrophysiological phenotypes of injured myocardium.</a> Integrative Biology. 6 (8), 789 (2014).</li><li>Ho, D., 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=Heart+rate+and+electrocardiography+monitoring+in+mice.">Heart rate and electrocardiography monitoring in mice.</a> Current Protocols in Mouse Biology. 1 (1), 123-139 (2011).</li><li>Heier, C. R., Hampton, T. G., Wang, D., DiDonato, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+electrocardiogram+intervals+during+growth+of+FVB/N+neonate+mice.">Development of electrocardiogram intervals during growth of FVB/N neonate mice.</a> BMC Physiology. 10, 16 (2010).</li><li>Heier, C. R., DiDonato, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECG+in+neonate+mice+with+spinal+muscular+atrophy+allows+assessment+of+drug+efficacy.">ECG in neonate mice with spinal muscular atrophy allows assessment of drug efficacy.</a> Frontiers Biosciences (Elite Ed). 7, 107-116 (2015).</li><li>Chu, V., 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=Method+for+noninvasively+recording+electrocardiograms+in+conscious+mice.">Method for noninvasively recording electrocardiograms in conscious mice.</a> BMC Physiology. 1, 6 (2001).</li><li>Patel, S. I., Souter, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equipment-related+electrocardiographic+artifacts:+causes,+characteristics,+consequences,+and+correction.">Equipment-related electrocardiographic artifacts: causes, characteristics, consequences, and correction.</a> Anesthesiology. 108 (1), 138-148 (2008).</li><li>Castellan, R. F. P., Thomson, A., Moran, C. M., Gray, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocardiogram-gated+kilohertz+visualisation+(EKV)+ultrasound+allows+assessment+of+neonatal+cardiac+structural+and+functional+maturation+and+longitudinal+evaluation+of+regeneration+after+injury.">Electrocardiogram-gated kilohertz visualisation (EKV) ultrasound allows assessment of neonatal cardiac structural and functional maturation and longitudinal evaluation of regeneration after injury.</a> Ultrasound in Medicine and Biology. 46 (1), 167-179 (2020).</li></ol>

The data points collected in perinatal day 1 mouse pups are slightly below the average expected values for adult mice (500-700 beats per minute).8 There is an increase in heart rate as the mouse ages, which falls more in line for the expected values (Table 1). However, it is important to emphasize that neonatal values were on the lower end of this range, supporting the idea that normative values should be documented in an age-specific manner. This method is different than other el...

Cardiac function can be measured in different ways, the most common of which includes the use of electrocardiography (ECG) to analyze the conduction of electric current through the heart as well as its overall cardiac cycle and function1. Electrocardiography continues to be a useful diagnostic tool for identifying and characterizing cardiac anomalies in both human and animal models of disease1,2. Irregularities in an electrocardiogram reading can be found in abnormal cardiac development (i.e., congenital heart disease (CHD)), and can include arrhythmias manifesting as changes in heart rate (e.g., bradycardia), and rhythm (e.g., &#8220;heart blocks&#8221;), suggestive of defects in the integrity and/or function of the underlying myocardium. Changes such as these may predispose patients to life-threatening cardiac dysfunction (e.g., congestive heart failure and/or cardiac arrest) and increased mortality3,4. Given the high rates of mortality with severe and untreated CHD, developing a standardized and repeatable method for collecting ECG during this early postnatal period is critical.
Although we are not the first to address this problem, previous methods of collecting ECG on a mouse pups have traditionally included invasive procedures (subcutaneous needle or wire electrodes) and/or the use of anesthetics5,6,7. Advantages of performing noninvasive ECG analysis include minimizing pain and undo stress on the animal. While the experimenter must still be cautious about causing the pup stress, the device is designed to avoid common stressors in order to produce accurate data. In the context of evaluating cardiac function, introducing anesthesia to animals that may have cardiopulmonary abnormalities could potentially mask or even exacerbate underlying conditions. Anesthetics may affect the electrical conduction by altering depolarization and/or repolarization of the cells. Finally, the use of anesthesia can put the newborn pup at an increased risk for hypothermia, which could further confound any inherent pathology. The following protocol does not introduce any anesthetics, invasive procedures, or pronounced discomfort to the pup. Once equipment setup is finalized, device setup and data collection involving the animal can be completed efficiently, after which the pups can be returned to their mother. Additionally, this system allows for repeat and/or serial analyses to be performed, which is ideal for experiments requiring analysis over time, introduction of pharmacological therapies, etc.

<table>
	<tbody>
		<tr>
			<td>LabScribe4</td>
			<td>iWorx</td>
			<td>LabScribe4</td>
			<td>Software used to record ECG</td>
			<td>https://www.iworx.com/users/teaching.php</td>
		</tr>
		<tr>
			<td>Neonatal Mouse ECG &#38; Respiration System</td>
			<td>iWorx</td>
			<td>RS-NMECG : Neonatal Mouse ECG</td>
			<td>ECG device</td>
			<td>https://www.iworx.com/research/cardiac-function/rs-nmecg/</td>
		</tr>
		<tr>
			<td>Tensive Conductive Adhesive Gel</td>
			<td>Parker Laboratories, Inc</td>
			<td>22-60</td>
			<td>Tac-gel used as conductive gel for ECG</td>
			<td>https://www.parkerlabs.com/tensive.asp</td>
		</tr>
	</tbody>
</table>

noninvasive electrocardiography in the perinatal mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

An ideal ECG would have a clear, prominent signal that allows all waves to be analyzed in several different time frames (Figure 1). The laboratory initially employed a custom application of an electromyography apparatus to produce ECGs of an unsatisfactory quality, which only allowed us to analyze basic parameters such as heart rate (Figure S1). This inspired work with a company to develop a novel prototype ECG device specifically for the analysis of early postnatal mouse pu...

Watch this Scientific Journal Video about Noninvasive Electrocardiography in the Perinatal Mouse at JoVE.com

Noninvasive Electrocardiography in the Perinatal Mouse

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

This unique protocol allows the assessment of neonatal mouse cardiovascular function using electrocardiography technology immediately after birth in a noninvasive manner without the use of anesthetics. Assessing murine cardiovascular function in the early postnatal period will expand our understanding of the neonatal heart in a manner that could directly correlate with human clinical data. To prepare the measuring device for the analysis, plug the device into the USB port of a computer.The measuring device will automatically begin heating up to 37 degrees Celsius. After about 15 minutes, remove a mouse pup from its home cage and wipe the thorax with 70%ethanol. Place the pup on the heated surface of the plastic and allow the animal to acclimate to the surface for approximately two to five minutes in the dark.While the mouse is acclimating, collect a small droplet of adhesive electrical conducting gel and gently press the droplet down onto the top of each of the four flattened electrode surfaces, carefully pulling the conducting gel away at an oblique angle from the center of the electrode construct after contact. When each electrode is completely covered with gel, place the neonatal mouse pup onto the platform in the prone position with the head of the pup facing the outgoing USB edge of the platform with each electrode covering a portion of the pup's upper thorax. Gently restrain the forearms while simultaneously applying gentle pressure to the electrodes for approximately one minute.When the conducting gel is set, secure the pup on the right and left sides with rubber silicone bumpers. Monitor the mouse for a moment adjusting the bumper placement as needed. When the pup is secure, apply any remaining conducting gel to the grounding tail electrode and use gentle pressure to place the electrode on the rump of the pup to allow the gel to set before releasing the pup.Carefully place the final silicone bumper on top of the rump to hold the grounding electrode in place and gently place the entire platform into the Faraday cage, then check that the pup is not moving excessively and that the body and head of the mouse appear secure. An ideal ECG will have a clear prominent signal that allows all of the waves to be analyzed in several different timeframes. A poor quality reading has no discernible beats, shows clear interference, and exhibits waves or inconsistencies across the reading.The program provides analysis of the key aspects of the ECG reading, including the heart rate, RR intervals, QRS complex interval, QT interval, and PR interval. Using these data to establish a set of normative values, in this representative analysis, it was determined that pups analyzed in the first postnatal day had an average heart rate of 357.2 beats per minute. For a pup in the first postnatal day, the average RR, PR, QRS, and QT intervals were 169.1, 36.3, 16.9, and 45.4 milliseconds respectively.Importantly, the setup can be used to analyze ECG patterns from neonatal mice suffering from congenital heart defects. The device is effective and reliable, but also highly sensitive to surrounding electrical interference. Make sure to always remove any unnecessary equipment including laptop chargers and smart devices prior to recording.Additional possible measurements include respiration and lead III, which is calculated from leads I through II.Additional variables could allow for further overlap with other physiological symptoms and/or specific cardiac arrhythmias.

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