Name: Electrocardiogram Recordings in Anesthetized Mice using Lead II (Video) | JoVE Journal
Uploaded: 2020-06-20T15:00:00.000+00:00
Duration: 4 min 16 s
Description: Learn a simple, cost-effective ECG protocol for sensitive cardiac recordings in anesthetized mice using Lead II. This method enables pharmacological and genetic screening in cardiovascular research.

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5 minutes

This work was supported by the Basic Science Research Programs that are managed by the National Research Foundation of Korea (NRF) (2015R1C1A2A01052419 and 2018R1D1A1B07042484).

All animal procedures were approved by the local committee for the Care and Use of Laboratory Animals, Kyung Hee University (license number: KHUASP(SE)-18-108) and conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Experimental animals
<ol>
	<li>Keep all mice (39 mice, Balb/c, male, 7&#8210;9 weeks old) in a pathogen-free facility as per the guide for the care and use of laboratory animals.</li>
	<li>Maintain the mice on a 12 h light/dark cycle at constant temperature with free access to food and water.</li>
</ol>
2. Preparation of anesthetics
NOTE: Tribromoethanol is used over ketamine combinations and isoflurane, based on the stability of heart rate and the reproducibility of echocardiography in tribromoethanol-anesthetized mice1,5,6
<ol>
	<li>Make a stock solution of 2,2,2-tribromoethanol at a concentration of 1 g per 1 mL tertiary amyl alcohol. Warm at 40&#8210;45 &#176;C for 24 h. Store at 4 &#176;C for 12 months.</li>
	<li>For working solution, dilute 0.5 mL of stock solution in 19.5 mL of saline (0.9% NaCl) to 25 mg/mL. Warm at 40&#8210;45 &#176;C for 1 h. Store at 4 &#176;C for 1 month.</li>
</ol>
3. ECG system setup
<ol>
	<li>Make sure to set up the system such that there is no noise or vibration within 2 m since ECG signals in a mouse are sensitive to the environmental noise and movement.</li>
	<li>Prepare the hardware setup: a data acquisition system, a bio amplifier, and a computer that is installed with an ECG data analysis software.
	<ol>
		<li>Connect the data acquisition system to the mains (AC) using the power cable.</li>
		<li>Connect the data acquisition system to the computer using a USB cable.</li>
		<li>Connect the signal output on the rear panel of the bio amplifier to an analog input on the front panel of the data acquisition system using a cable.</li>
		<li>Connect the I2C output of the data acquisition system to the I2C input of the bio amplifier using the I2C cable.</li>
		<li>Connect the 3-lead bio amplifier cable to the 6-pin input socket on the front panel of the bio amplifier.</li>
		<li>Turn on the data acquisition system using the switch on the back panel. 
		NOTE: In brief, the signals are amplified through a bio amplifier and recorded using a computerized data acquisition and analysis system with the following channel settings: sampling rate of 2 k/s, range of 20 mV, and low-pass filter setting of 200 Hz.</li>
	</ol>
	</li>
	<li>Open the analysis software program and set it up for ECG data acquisition.
	<ol>
		<li>Go to Setup | Channel Settings. Set the Sample Rate to 2 k/s. Set the Range to 20 mV. Set the Input Amplifier to 200 Hz of Low Pass.</li>
		<li>Go to ECG Analysis | ECG Settings. Choose &#8220;Mouse&#8221; in the Preset of Detection and Analysis settings.</li>
		<li>In the Averaging panel, choose to concatenate N (e.g., 4 beats or 60 s) consecutive cardiac cycles into a single average signal for Averaging View and Table View.</li>
		<li>In the QTc panel, select &#8220;Bazett&#8221; method, which is defined as the heart rate-corrected value of QT interval: QTc = QT / (RR/100)0.5, RR interval = 60 / heart rate7.</li>
	</ol>
	</li>
</ol>
4. ECG measurement
<ol>
	<li>Place a mouse on a precision scale and record its weight.</li>
	<li>Induce anesthesia in the mouse by intraperitoneal (i.p.) injection of a working solution of tribromoethanol (18 mL of working solution per kg body weight (b.w.)).</li>
	<li>Place an anesthetized mouse in supine position. Ensure that the mouse is completely anesthetized (less than 2 min).</li>
	<li>Insert the electrodes with acupuncture needles subcutaneously into the right and left forelimbs and the left hindlimb according to the lead II ECG scheme and fix them with tape (Figure 1). Ensure that the depth and position of inserted electrodes are consistent throughout the experiments.</li>
	<li>Connect the other ends of the electrodes by clicking them into the three snap connectors at the other end of the lead wires of the 3-lead bio amplifier cable.</li>
	<li>Inject drugs (i.p.) 3 min after the anesthetics have been delivered (Figure 2).</li>
	<li>Begin recording the ECG 10 min after injecting anesthetics. Once the recording is completed, use the ECG data from 12 to 17 min after injection of anesthetics for analysis.</li>
	<li>At the end of the ECG recording session, carefully remove the electrodes.</li>
</ol>
5. ECG data analysis
<ol>
	<li>Go to ECG Analysis | Averaging View and ensure that the software correctly identifies the start and end of the P wave, QRS complex, and T wave in individual beats. If necessary, manual correction of these waves and intervals is possible by moving misplaced cursors to the appropriate positions. 
	NOTE: As depicted in Figure 3A, the PR interval spans the onset of the P wave to that of the QRS complex (mostly missing Q wave in a mouse ECG). The QRS duration extends from the onset of the Q wave (primarily an R wave in a mouse ECG) to the end of the S wave. The QT interval comprises the onset of the Q wave (mostly the R wave in a mouse ECG) to the end of the T wave. Note the shorter duration and absence of a Q wave and ST segment in the mouse ECG relative to the human ECG8.</li>
	<li>Go to ECG Analysis | Table View and select the correctly identified ECG data by checking individual beats in the Averaging View window. 
	NOTE: Figure 3 shows several examples of actual mouse ECG signals. Figure 3A represents a normal wild-type signal that has been correctly identified with regard to P wave, QRS complex, and T wave. Computerized selection of PQRS waves can incur erroneous misplacements, such as in Figure 3B a normal wild-type signal that misplaces the onset of the P wave. In Figure 3C an ECG signal that misplaces the end of the QRS complex, resulting in an overestimation of QRS duration. In Figure 3D an ECG signal that misplaces the end of the QRS complex, resulting in underestimation of the QRS complex due to the ambiguous T wave and Figure 3E an ECG signal with an unidentifiable T wave. Without exclusion or manual corrections, PQRS intervals can be over or underestimated. Be sure to select the ECG signals that have been correctly identified and the signals that do not miss the target peaks. Consequently, such cases, including B, C, D, and E (Figure 3), are excluded in accurately estimating ECG parameters in general.</li>
	<li>Select the ECG data of interest in Table View, and copy/paste them to a spreadsheet file.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Perform the statistical analysis using a statistics program. Analyze the data with the experimental conditions blinded. Perform Student&#8217;s t-test and Mann-Whitney U-test for 2-group comparisons. The numbers in each figure indicate the number of mice that is used for each group. Report the results as mean &#177; SEM.</li>
	<li>Consider differences with p &#60; 0.05 by U-test to be statistically significant: *, p &#60; 0.05; **, p &#60; 0.01; and ***, p &#60; 0.005 versus respective controls.</li>
</ol>

<ol><li>Ho, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+rate+and+electrocardiography+monitoring+in+mice%5BTitle%5D)+AND+%22Current+Protocols+in+Mouse+Biology%22%5BJournal%5D)">Heart rate and electrocardiography monitoring in mice.</a> Current Protocols in Mouse Biology. 1, 123-139 (2011).</li>
<li>Vatner, S. F., Takagi, G., Asai, K., Shannon, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+physiology+in+mice%3A+Conscious+measurements+and+effects+of+anesthesia%5BTitle%5D)+AND+%22Cardiovascular+Physiology+in+the+Genetically+Engineered+Mouse%22%5BJournal%5D)">Cardiovascular physiology in mice: Conscious measurements and effects of anesthesia.</a> Cardiovascular Physiology in the Genetically Engineered Mouse. , 257-275 (2002).</li>
<li>Cesarovic, N., Jirkof, P., Rettich, A., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantation+of+radiotelemetry+transmitters+yielding+data+on+ecg%2C+heart+rate%2C+core+body+temperature+and+activity+in+free-moving+laboratory+mice%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">Implantation of radiotelemetry transmitters yielding data on ecg, heart rate, core body temperature and activity in free-moving laboratory mice.</a> Journal of visualized experiments : JoVE. (57), (2011).</li>
<li>Chu, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+non-invasively+recording+electrocardiograms+in+conscious+mice%5BTitle%5D)+AND+%22BMC+Physiology%22%5BJournal%5D)">Method for non-invasively recording electrocardiograms in conscious mice.</a> BMC Physiology. 1, 6(2001).</li>
<li>Kim, M. J., Lim, J. E., Oh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+non-invasive+method+for+electrocardiogram+recording+in+mouse+using+lead+ii%5BTitle%5D)+AND+%22Biomedical+Science+Letters%22%5BJournal%5D)">Validation of non-invasive method for electrocardiogram recording in mouse using lead ii.</a> Biomedical Science Letters. 21, 135-143 (2015).</li>
<li>Roth, D. M., Swaney, J. S., Dalton, N. D., Gilpin, E. A., Ross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+anesthesia+on+cardiac+function+during+echocardiography+in+mice%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Impact of anesthesia on cardiac function during echocardiography in mice.</a> American Journal of Physiology. Heart and Circulatory Physiology. 282 (6), 2134-2140 (2002).</li>
<li>Mitchell, G. F., Jeron, A., Koren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+heart+rate+and+q-t+interval+in+the+conscious+mouse%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Measurement of heart rate and q-t interval in the conscious mouse.</a> The American Journal of Physiology. 274 (3), 747-751 (1998).</li>
<li>Farraj, A. K., Hazari, M. S., Cascio, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+utility+of+the+small+rodent+electrocardiogram+in+toxicology%5BTitle%5D)+AND+%22Toxicological+sciences+%3A+an+official+journal+of+the+Society+of+Toxicology%22%5BJournal%5D)">The utility of the small rodent electrocardiogram in toxicology.</a> Toxicological sciences : an official journal of the Society of Toxicology. 121 (1), 11-30 (2011).</li>
<li>Gehrmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+parasympathetic+heart+rate+control+in+mice+with+a+reduction+of+functional+g+protein+betagamma-subunits%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Impaired parasympathetic heart rate control in mice with a reduction of functional g protein betagamma-subunits.</a> American Journal of Physiology. Heart and Circulatory Physiology. 282 (2), 445-456 (2002).</li>
<li>Chu, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrocardiographic+findings+in+mdx+mice%3A+A+cardiac+phenotype+of+duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Muscle+%26+Nerve%22%5BJournal%5D)">Electrocardiographic findings in mdx mice: A cardiac phenotype of duchenne muscular dystrophy.</a> Muscle & Nerve. 26 (4), 513-519 (2002).</li>
<li>Merentie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+ecg+findings+in+aging%2C+with+conduction+system+affecting+drugs+and+in+cardiac+pathologies%3A+Development+and+validation+of+ecg+analysis+algorithm+in+mice%5BTitle%5D)+AND+%22Physiological+Reports%22%5BJournal%5D)">Mouse ecg findings in aging, with conduction system affecting drugs and in cardiac pathologies: Development and validation of ecg analysis algorithm in mice.</a> Physiological Reports. 3 (12), (2015).</li>
<li>Calvillo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Propranolol+prevents+life-threatening+arrhythmias+in+lqt3+transgenic+mice%3A+Implications+for+the+clinical+management+of+lqt3+patients%5BTitle%5D)+AND+%22Heart+Rhythm+%3A+the+Official+Journal+of+the+Heart+Rhythm+Society%22%5BJournal%5D)">Propranolol prevents life-threatening arrhythmias in lqt3 transgenic mice: Implications for the clinical management of lqt3 patients.</a> Heart Rhythm : the Official Journal of the Heart Rhythm Society. 11 (1), 126-132 (2014).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+atrial+arrhythmogenicity+and+altered+ca%282%2B%29+homeostasis+in+murine+ryr2-p2328s+hearts%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Acute atrial arrhythmogenicity and altered ca(2+) homeostasis in murine ryr2-p2328s hearts.</a> Cardiovascular Research. 89 (4), 794-804 (2011).</li>
<li>Kmecova, J., Klimas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+rate+correction+of+the+qt+duration+in+rats%5BTitle%5D)+AND+%22European+Journal+of+Pharmacology%22%5BJournal%5D)">Heart rate correction of the qt duration in rats.</a> European Journal of Pharmacology. 641 (2-3), 187-192 (2010).</li>
<li>Kim, H. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Garem1+regulates+the+pr+interval+on+electrocardiograms%5BTitle%5D)+AND+%22Journal+of+Human+Genetics%22%5BJournal%5D)">Garem1 regulates the pr interval on electrocardiograms.</a> Journal of Human Genetics. 63 (3), 297-307 (2018).</li>
<li>Nam, J. M., Lim, J. E., Ha, T. W., Oh, B., Kang, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac-specific+inactivation+of+prdm16+effects+cardiac+conduction+abnormalities+and+cardiomyopathy-associated+phenotypes%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Cardiac-specific inactivation of prdm16 effects cardiac conduction abnormalities and cardiomyopathy-associated phenotypes.</a> American Journal of Physiology. Heart and Circulatory Physiology. 318 (4), 764-777 (2020).</li>
<li>Knollmann, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isoproterenol+exacerbates+a+long+qt+phenotype+in+kcnq1-deficient+neonatal+mice%3A+Possible+roles+for+human-like+kcnq1+isoform+1+and+slow+delayed+rectifier+k%2B+current%5BTitle%5D)+AND+%22The+Journal+of+Pharmacology+and+Experimental+Therapeutics%22%5BJournal%5D)">Isoproterenol exacerbates a long qt phenotype in kcnq1-deficient neonatal mice: Possible roles for human-like kcnq1 isoform 1 and slow delayed rectifier k+ current.</a> The Journal of Pharmacology and Experimental Therapeutics. 310 (1), 311-318 (2004).</li>
</ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

There are several critical steps in the protocol. The surrounding environment should be free from noise and vibration. The ECG electrodes must be inserted under the skin stably and consistently of which the insertion step requires preliminary experiments until the researcher is technically experienced. Further, the anesthetic should be prepared and stored appropriately and used at the proper dose. Finally, the PQRS waves should be located appropriately in individual ECG beats in the Averaging View window.
<p class="j...

The electrocardiogram (ECG), a test that measures the electrical activity of one&#8217;s heartbeat, is a valuable tool for evaluating the cardiac conduction system. The parameters that are measured by an ECG include heart rate, PR interval, QRS duration, and QT interval. In brief, PR interval corresponds to the time that is required for an electrical impulse to travel from the atrial sinus node through the atrioventricular node to the Purkinje fibers; QRS duration is the time for ventricular depolarization to occur through the Purkinje system and ventricular myocardium; and QT interval is the duration of ventricular repolarization.
ECG recordings in mice have helped researchers examine cardiac function and determine the physiological and pathophysiological mechanisms of cardiac phenotypes, such as arrhythmia, atrial fibrillation, and heart failure. Most cardiovascular research has involved studies in genetically engineered mouse models. It is often challenging to obtain meaningful data on ECG recordings from small mice that have been genetically manipulated.
There are several methods for performing ECGs in mice1. Studies suggest that ECG recordings in conscious animals are preferred over anesthetized animals when possible since the effects of anesthesia on cardiac function have been well established2. Two protocols that record ECG in conscious mice are of note1. The ECG radiotelemetry system is the gold standard for continuous long term monitoring of ECG in conscious mice1,3. Despite their strength in being recorded in a conscious state, radiotelemetry-coupled ECG measurements have several limitations, including the high expense for setup and for the implant, its requirement of a highly experienced operator, a stabilization period of over 1 week, its need for large mice (&#62; 20 g), and acquisition of only a single lead of ECG recording1. Another system that uses paw-sized conductive electrodes embedded in a platform allows ECG recordings in conscious mice without anesthesia or implants1,4. This non-invasive system is an alternative method in situations in which radiotelemetry systems are unavailable since it has many advantages: no requirement of surgical treatment, no need of anesthesia, low cost per mouse (only the initial setup is expensive), short time for measurement, and affordability of neonates1,4. The main disadvantage of this system is that it is not suited for continuous long term monitoring1.
Here we introduce another inexpensive, simple, and fast ECG recording method in anesthetized mice and demonstrate its validity and sensitivity by performing an ECG after autonomic blockade/stimulation of the cardiac conduction system. We suggest this ECG method for screening the effects of pharmacological agents, genetic modifications, and disease models in mice.

<table><tbody><tr><td>2,2,2-tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-25G</td><td>anesthetics, Avertin</td></tr><tr><td>Animal</td><td>Japan SLC, Inc., Shizuoka, Japan</td><td></td><td>Balb/c mice, male, aged 7-9 weeks</td></tr><tr><td>Atropine</td><td>Sigma-Aldrich</td><td>A0123</td><td>parasympathetic antagonist</td></tr><tr><td>BioAmp</td><td>AD Instruments, Bella Vista, Australia</td><td>ML132</td><td>bio amplifier</td></tr><tr><td>Carbachol</td><td>Sigma-Aldrich</td><td>C4382</td><td>parasympathetic agonist</td></tr><tr><td>Electrodes with acupuncture needles</td><td>DongBang Acupuncture Inc., Sungnam, Korea</td><td>DB106</td><td>0.20 x 15 mm</td></tr><tr><td>Isoprenaline</td><td>Sigma-Aldrich</td><td>I2760</td><td>sympathetic agonist</td></tr><tr><td>LabChart 8</td><td>AD Instruments, Bella Vista, Australia</td><td></td><td>data analysis software</td></tr><tr><td>Mouse food</td><td>LabDiet, St. Louis, MO, USA</td><td>5L79</td><td>Mouse diet</td></tr><tr><td>PowerLab 2/28</td><td>AD Instruments, Bella Vista, Australia</td><td></td><td>data acquisition system</td></tr><tr><td>Propranolol</td><td>Sigma-Aldrich</td><td>P0884</td><td>sympathetic antagonist</td></tr><tr><td>SPSS Statistics program</td><td>SPSS</td><td>SPSS 25.0</td><td>statistics program</td></tr></tbody></table>

electrocardiogram recordings in anesthetized mice using lead ii

Electrocardiogram Recordings in Anesthetized Mice using Lead II

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and pharmacological information regarding the electrocardiogram. However, making electrocardiogram measurements in small animals in vivo, such as mice, has been challenging. To this end, we used an electrocardiogram recording method in anesthetized mice with many advantages: it is a technically simple procedure, is inexpensive, has short measuring time, and is affordable, even in young mice. Despite the limitations with using anesthesia, comparisons between control and experimental groups can be performed with enhanced sensitivity. We treated mice with agonists and antagonists of the autonomic nervous system to determine the validity of this protocol and compared our results with previous reports. Our ECG protocol detected increased heart rates and QTc intervals on treatment with atropine, decreased heart rates and QTc intervals after carbachol treatment, and higher heart rates and QTc intervals with isoprenaline but did not note any change in ECG parameters on administration of propranolol. These results are supported by previous reports, confirming the reliability of this ECG protocol. Thus, this method can be used as a screening approach to making ECG measurements that otherwise would not be attempted due to high cost and technical difficulties.

Pharmacological experiments
To determine whether our noninvasive ECG measurement reflects the influence of autonomic modulation on the cardiac conduction system, normal Balb/c mice were challenged with agonists and antagonists of the autonomic nervous system (ANS). Atropine and carbachol were used to effect parasympathetic autonomic blockade and stimulation, respectively, whereas propranolol and isoprenaline were administered to elicit sympathetic autonomic blockade and stimul...

We present an ECG protocol that is technically easy, inexpensive, fast, and affordable in small mice, and can be performed with enhanced sensitivity. We suggest this method as a screening approach for studying pharmacological agents, genetic modifications, and disease models in mice.

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and ...

electrocardiogram-recordings-in-anesthetized-mice-using-lead-ii

Research

JoVE Journal

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Learn a simple, cost-effective ECG protocol for sensitive cardiac recordings in anesthetized mice using Lead II. This method enables pharmacological and genetic screening in cardiovascular research.

Electrocardiogram Recordings in Anesthetized Mice using Lead II

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