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>

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C., 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=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.">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.
Our studies included testing of drugs. However, if pharmacological tests are omitted, the step 4.7 can be modified by beginning the recording 5 min after the injection of anesthetics, and the ECG data can be used from 10 to 15 min. ECG values are relatively stable over 15 min post-anesthesia and have been replicated in the same mouse 6 h after the first measurement5.
Autonomic blockade and stimulation by drugs elicit differential responses with regards to heart rate. Several protocols have been used in ECG research. Based on telemetered ECG recordings in mice, atropine, isoprenaline, and propranolol did not significantly change heart rate, whereas carbachol significantly decreased it (wild-type, 739 &#177; 33 bpm; atropine, 726 &#177; 5 bpm; carbachol, 205 &#177; 54 bpm; isoprenaline, 722 &#177; 32 bpm; propranolol, 560 &#177; 21 bpm)9. Based on ECG recordings by the noninvasive system that uses paw-sized conductive electrodes embedded in a platform, atropine and isoprenaline significantly increased heart rate in mice (p &#60; 0.05), whereas propranolol did not change it (p = NS) (wild-type, 706 &#177; 13 bpm; atropine, 727 &#177; 12 bpm; isoprenaline, 12 &#177; 2% increase versus control; propranolol, 584 &#177; 53 bpm)4,10. With this noninvasive ECG system, isoprenaline induced ST segment depression4.
Surface ECG signals (lead II via limb electrodes) are acquired under isoflurane anesthesia during high-resolution transthoracic echocardiography (TTE) with an ultrasound system11. ECG recordings by the TTE suggested that heart rate increased 15 min after the administration of atropine11. Similar to our protocol, 6-lead ECG recordings under anesthesia with tribromoethanol using 5-needle electrodes (1 electrode implanted subcutaneously in each limb and 1 placed in the precordial position) that are connected to a data acquisition system with an amplifier set12. With this method, using 6-lead ECG, carbachol significantly lowered heart rate (p &#60; 0.001) and increased QT interval (p &#60; 0.001), but propranolol did not significantly change either parameter (wild-type, 395 &#177; 65 bpm; carbachol, 177 &#177; 36 bpm; propranolol, 351 &#177; 30 bpm)12. Another report that made 3-lead ECG measurements under anesthesia with tribromoethanol showed that isoprenaline significantly increased heart rate in wild-type mice (p &#60; 0.01) (wild-type, 422 &#177; 17 bpm; isoprenaline, 503 &#177; 27 bpm)13. 14 Overall, the heart rate is lower in ECG measurements under anesthesia than in those in a conscious mouse. Differences between control and drug-treated groups are well reflected in ECG recordings under anesthesia and by the system that uses paw-sized conductive electrodes embedded in a platform, in a conscious mouse, because changes in heart rate and QT interval are detected on treatment with atropine, carbachol, and isoprenaline but not propranolol alone10,11,12,13. In contrast, telemetered ECG recordings detect only changes in heart rate by carbachol9.
This ECG method under anesthesia with tribromoethanol also notes differences in heart rate and QTc interval on administration with atropine, carbachol, and isoprenaline but not propranolol, implying its high sensitivity. Here with autonomic disturbances, we showed changes in heart rate and QTc interval. Further we have published a manuscript with our ECG method that describes a change in PR interval and another one that addresses changes in QRS duration and QTc interval, partially supporting the sensitivity in all of PQRS waves15,16.
The protocol has many advantages comparable to the non-invasive method that allows ECG recording in a conscious mouse with paw-sized electrodes embedded in a platform. However, the major limitation of our protocol is the use of anesthetics such as tribromoethanol. 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 Although ECG recordings in a conscious animal are preferred to those under anesthesia, variations in sympathetic and parasympathetic tone, and relatively high heart rate sometimes make measurements in conscious mice less than ideal for all applications of echocardiography6.
Overall, despite its limitations (e.g., the use of anesthesia), our ECG method has many advantages: (i) it is a technically simple procedure only requiring stable insertion of ECG electrodes under the skin, (ii) has low experimental costs&#8212;the outlay is primarily for the initial hardware setup; (iii) has short measurement times of less than 20 min per mouse, and can be conducted on young mice (&#62;15 g body weight, in our experience)16 and even neonates (postnatal days 2&#8210;4)17. Thus, screening experiments for drugs and various types of mice (e.g., genetically modified, disease models) can be performed quickly and without much cost per mouse, constituting a reliable and sensitive analysis and can be used as an additional supporting data beyond telemetered ECG recordings.

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

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 stimulation, respectively9.
Heart rate increased significantly in atropine- (p &#60; 0.05) and isoprenaline-treated mice (p &#60; 0.05) and fell with carbachol (p &#60; 0.005) as compared with vehicle (vehicle, 391 &#177; 13 bpm versus atropine, 487 &#177; 15 bpm versus carbachol, 158 &#177; 7 bpm; vehicle, 382 &#177; 14 bpm versus isoprenaline, 548 &#177; 8 bpm; vehicle, 404 &#177; 25 bpm versus propranolol, 303 &#177; 16 bpm) (Figure 4). In addition, QTc interval rose in atropine- (p &#60; 0.05) and isoprenaline-treated mice (p &#60; 0.05) and decreased in carbachol-treated mice (p &#60; 0.005) versus vehicle (vehicle, 46.5 &#177; 0.6 ms versus atropine, 51.1 &#177; 1.3 ms versus carbachol, 29.4 &#177; 1.0 ms; vehicle, 41.8 &#177; 1.2 ms versus isoprenaline, 57.5 &#177; 3.5 ms) (Figure 4). Figure 5 shows representative Chart Views and Averaging Views for the ECG signals in atropine-, carbachol-, and vehicle-treated mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61583/61583fig01.jpg" /> 
Figure 1: ECG lead placement. 
Acupuncture needle electrodes are inserted subcutaneously according to the lead II ECG scheme (right and left forelimbs and the left hindlimb) and are fixed with tape. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61583/61583fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61583/61583fig02.jpg" /> 
Figure 2: Scheme of anesthetic and drug treatments. 
Three minutes after injection of anesthetics (e.g., tribromoethanol), administer drugs (e.g., atrotpine, carbachol, isoprenaline, and propranolol; i.p.). Ten minutes after the anesthetics have been delivered, start recording the ECG. Collect ECG data from 12&#8210;17 min after the injection of anesthetics. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61583/61583fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61583/61583fig03.jpg" /> 
Figure 3: Examples of mouse ECG signals. 
(A) A normal wild-type signal that is correctly identified with regards to the P wave, QRS complex, and T wave. (B) A normal wild-type signal that misplaces the onset of the P wave. (C) An ECG signal that misplaces the end of the QRS complex. (D) An ECG signal that misplaces the end of the QRS complex due to an ambiguous T wave. (E) An ECG signal with an unidentifiable T wave. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61583/61583fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61583/61583fig04.jpg" /> 
Figure 4: ECG measurements in mice treated with agonists and antagonists of the autonomic nervous system. 
(A) Administration of atropine (1 mg/kg) increases heart rate and QTc interval. (B) Carbachol (0.5 mg/kg) decreases heart rate and QTc interval. (C) Isoprenaline (1 mg/kg) increases heart rate and QTc interval. (D) Propranolol (1 mg/kg) does not change any ECG parameters. *, p &#60; 0.05; ***, p &#60; 0.005. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61583/61583fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61583/61583fig05.jpg" /> 
Figure 5: Representative ECG signals of mice treated with agonists and antagonists of the parasympathetic nervous system. 
(A) ECG signals of vehicle-treated mouse acquired from Chart Views and Averaging Views (a data analysis program). (B) Signals of atropine-treated mouse. (C) Signals of carbachol-treated mouse. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61583/61583fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Electrocardiogram Recordings in Anesthetized Mice using Lead II at JoVE.com

Electrocardiogram Recordings in Anesthetized Mice using Lead II

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

12.5K Views.  Kyung Hee University. 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.

Video: Electrocardiogram Recordings in Anesthetized Mice using Lead II

Programmed Electrical Stimulation in Mice

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ventricular arrhythmias, and sudden cardiac death.1 Intracardiac pacing studies can be performed in mice using a 1.1F octapolar catheter inserted into the jugular vein, and advanced into the right atrium and ventricle. Here, we illustrate the steps involved in performing programmed electrical stimulation in mice. Surface ECG and intracardiac electrograms are recorded simultaneously in the atria, atrioventricular junction, and ventricular myocardium, whereas intracardiac pacing of the atrium is performed using an external stimulator. Thus, programmed electrical stimulation in mice provides unique opportunities to explore molecular mechanisms underlying conduction defects and cardiac arrhythmias.

Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates.

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ...

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

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

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

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.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Biology

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

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).

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 ...

Topographical EEG Recordings of Visual Evoked Potentials in Mice using Multichannel Thin-film Electrodes

Visual evoked potentials (VEP) allow the characterization of visual function in preclinical mouse models. Various methods exist to measure VEPs in mice, from non-invasive EEG, subcutaneous single-electrodes, and ECoG to fully invasive intracortical multichannel visual cortex recordings. It can be useful to acquire a global, topographical EEG-level characterization of visual responses previous to local intracortical microelectrode measurements in acute experimental settings. For example, one use case is&#160;to assess global cross-modal changes in VEP topography in deafness models before studying its effects on a local intracortical level. Multichannel epicranial EEG is a robust method to acquire such an overview measure of cortical visual activity.&#160;Multichannel epicranial EEG provides comparable results through a standardized, consistent approach to, for example, identify cross-modal, pathological, or age-related changes in cortical visual function. The current study presents a method to obtain the topographical distribution of flash-evoked VEPs with a 32-channel thin-film EEG electrode array in anesthetized mice. Combined with analysis in the time and frequency domain, this approach allows fast characterization and screening of the topography and basic visual properties of mouse cortical visual function, which can be combined with various acute experimental settings.

The present protocol describes a simple procedure to acquire and analyze the topography of epicranial visual evoked potentials with 32-multichannel thin-film electrodes in the mouse.

Visual evoked potentials (VEP) allow the characterization of visual function in preclinical mouse models. Various methods exist to measure VEPs in mice, ...

Neuroscience

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 ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

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.

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. ...