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Noninvasive Electrocardiography in the Perinatal Mouse

doi: 10.3791/61074 Published: June 12, 2020
Lindsey A. Fitzsimons1,2, Victoria L. Brewer3, John Forrester2, Adrian M. Moran4, Kerry L. Tucker1,2


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).


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., “heart blocks”), 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.

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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 > 70).

1. Device preparations

  1. 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 °C/98.6 °F). The internal heating unit is contained within the measuring unit and heats only the plastic surface. The silver wire electrodes are not heated.
  2. Allow approximately 15 min for the surface to reach the temperature. Use this time to gather and set up animals.
    NOTE: The protocol may be paused at this point and the platform can remain plugged in and heating for an extended period of time. In the absence of a self-heating electrode platform, an animal safe heating pad may also be used to keep mother and pups from becoming hypothermic.

2. Animal preparations

  1. Collect the mother and pups and keep within the housing cage until ready to collect.
  2. Once the measuring unit has heated to the temperature, remove the mouse pup from the cage and wipe the thorax with 70% ethanol sprayed on a wipe. Place the pup on the heated surface of the plastic.
  3. Allow the mouse to acclimate to the surface in the dark for approximately 2-5 minutes.

3. Mouse and electrode platform setup (electrode application)

  1. Use a metal spatula, probe, or wooden dowel to collect a small droplet of adhesive, electrical conducting gel (a quick-drying high-conductivity electrode gel commonly used for placement of rodent electrodes).
    NOTE: Any nonfibrous, solid object can be used to apply the conducting gel, as long as the object will not leave behind synthetic fibers or similar material on the electrodes that could interfere with the quality of the electrical signal.
  2. Using the spatula/dowel, gently touch the top of each of the four, flattened electrode surfaces by pressing gently down and pulling the conducting gel at an oblique angle away from the center of the electrode construct. Make sure that each individual electrode is completely covered with the gel.
    CAUTION: This step is extremely important to ensure that the conductive, electrode gel does not adhere to more than a single electrode. Adhesive strands that form between electrodes can conduct charge and potentially interfere with or short out the desired electrical signal. The protocol should not be paused at this time as the gel will begin to solidify and become adherent. Make sure to set up the mouse to the platform within 5-10 minutes of applying conducting gel (or equivalent conductive electrode gel substitution).
  3. Place the metal spatula or wooden dowel with the remainder of the gel to the side.
  4. Place the neonatal mouse pup sternum down and prone with the head of the pup facing the outgoing USB edge of the platform. Make sure that a portion of the pup’s chest is covering each of the four electrodes. Gently restrain the pup’s forearms by their side while simultaneously holding down for approximately 1 min to allow the conducting gel to set.
  5. Place rubber silicone bumpers on the right and left sides of the pup. Bumpers should secure the pup on each side and provide stability to prevent excessive movements of the mouse but should NOT prevent all movement of the mouse. Once installed, watch the mouse for a moment and adjust bumper placement as needed.
    CAUTION: Do not compress the mouse too tightly as this can interfere with respiratory mechanics and respiratory rate.
  6. Use the dowel that was set aside to apply remaining conducting gel to the grounding tail electrode and place on the rump of the pup. Apply gentle pressure to allow the gel to set before releasing the pup.
  7. Place the final silicon bumper on top of the rump of the mouse to hold the grounding electrode in place.
    CAUTION: Do not apply excessive force while placing the final bumper as this could cause discomfort to the pup and/or displace the grounding electrode.
  8. Grab ahold of the entire platform and gently place inside the Faraday cage.
    CAUTION: Use caution and ensure the top silicone bumper does not become displaced once the Faraday cage is in place.
  9. Prior to recording, make sure the mouse pup is not moving excessively and make sure the body and head of the mouse appears secure.
    CAUTION: Make sure the mouse pup’s head is able to move somewhat freely within the bumpers and is not completely snout down into the platform. The raised platform is designed to elevate the mouse thorax slightly and prevent suffocation, but this should be closely monitored.

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Representative Results

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 pups.

A poor-quality reading has no discernable beats, shows clear interference, and has waves or inconsistency across the reading (Figure 2). To achieve the highest quality ECG, follow instructions carefully. Use caution with application of conducting gel, as it is moderately adhesive, and may require additional time to allow the mouse to accommodate to the device. By doing this, it lowers the risk of the mouse moving, there being a shorting out of electrodes, and for correct use of the device. Mice should be placed on the device so the head is facing the cords that connect the device to the USB-port and in a prone position (Figure 3). The mouse should be secured by rubber bumpers to hold them securely in place, with two on the side and one on the top (Figure 3). These bumpers should secure the mouse, but should not inhibit the mouse from moving its head. The layout of the mouse is important for the reading, as the leads are stationary. The leads are set up so that the front two electrodes are Lead I (Figure 3). The rear two electrodes are Leads II and III, with the ground electrode being on the rump of the pup (Figure 3). Setting the mouse up in this way will allow for better results.

The program used allows for the analysis of the ECG in the program. This provides analysis of key aspects including heart rate, R-R intervals, QRS complex interval, QT interval, and PR interval. Given this ability, it was possible to establish a data set of normative values for a perinatal mouse (Table 1). These normative results were based off mice who were analyzed within one day after birth. It was found that an average heartbeat was 357.2 beats per minute (bpm). The average R-R, QRS, QT, and PR intervals were 169.1, 16.9, 45.4, and 36.3 milliseconds (ms), respectively (Table 1). Importantly, the setup can be used to analyze ECG patterns from neonatal mice suffering from congenital heart defects (Figure S2).

Pup Age Ave/STDEV Heart Rate (bpm) R-R Interval (ms) PR Duration (ms) QRS Duration (ms) QT Duration (ms) ST Duration (ms) T Duration (ms) P Duration (ms)
P1 Averages 357.2 169.1 36.3 16.9 45.4 16.4 18 12.8
Standard Deviation 36.3 20 10.9 5.8 16 7.4 7.2 3.1
P3 Averages 412.4 149.2 46.4 14.5 53 22.3 16.2 14.8
Standard Deviation 55.4 21.4 6.8 11 12.2 6.9 4.6 3.1
P5 Averages 505.5 119.2 46.7 11.7 51.3 20.8 18.8 14.2
Standard Deviation 19.2 4.6 13.3 5.8 8.1 11.4 4.6 2.3
P7 Averages 555.3 108.7 40 9.5 43.6 20.3 13.7 14
Standard Deviation 34.2 7 2.5 0.6 6 7.1 3.2 2.7

Table 1: Representative results of ECG measurements for the average perinatal mouse pup P1, P3, P5, and P7.

Figure 1
Figure 1: Representative electrocardiographic reads from neonatal mice on the first (A, P1.0), third (B, P3.0), and seventh (C, P7.0) postnatal day.
(A-C) Images represent examples of good quality ECG tracings using the 2-lead, noninvasive device, captured in a 1.5 s frame of the reading. Notable characteristics of good ECG reads include clear, discernable beats, as described collectively by the presence of consistent P-waves followed by a QRS complex and subsequent T-waves, visible in both Leads I-II of each postnatal time point. Examples also include a low signal-to-noise ratio (minimal artifact) and a discernable isoelectric line. Top ECG strip (red): Lead I; bottom ECG strip (green): Lead II. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representative ECG read with complications.
This image is representative of a poor-quality ECG reading using the 2-lead, noninvasive device on the first postnatal day (P1.0). The above images were captured in a 1.5 s reading frame. Poor quality ECG tracings are characterized by the absence of discernable beats (and specific cardiac cycle waveforms), along with pronounced artifact (high signal:noise ratio), and notable inconsistencies between Leads I and II from a given mouse pup. To improve this ECG, both the device and the silicone bumpers securing the pup would require repositioning within the Faraday cage. To minimize electromagnetic interference, removal of all moving devices near the apparatus would need to be carried out. The final troubleshooting measure would involve repositioning of the mouse pup on the device electrodes and/or more conductive gel would need to be (re-)applied. Top ECG strip (red): Lead I; bottom ECG strip (green): Lead II. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Placement of the mouse pup and limb lead electrodes for collection of early postnatal ECG.
(A) Left: Anterior perspective of mouse placement on electrode platform within the Faraday cage (black). Right: Lateral view illustrating proper mouse placement on top of raised electrodes/platform; supportive silicone bumpers (not pictured) are placed to either side and across the top of the mouse pup within the Faraday cage. (B) Bipolar limb leads and electrode placement on the neonatal mouse. Illustration depicts the point of contact for each raised electrode on the ventral thoracic surface of the mouse pup. (B,C) Electrode placement, chest lead directionality, and (C) corresponding, representative ECG tracings from a neonatal mouse pup at P1.0 (Lead I (red); Lead II (green)). Please click here to view a larger version of this figure.

Figure 4
Figure 4: Representative ECG tracings of neonatal mice at multiple postnatal time points.
Representative ECG reads (top 2 traces) and illustrated cardiac cycles (bottom row) from neonatal mouse pups on the first (A, P1.0), third (B, P3.0), and seventh (C, P7.0) postnatal day. Each image represents an exemplary ECG tracing using the 2-lead, noninvasive device, captured in a 1.5 second frame of the reading (A-C, Lead I (top/red); Lead II (bottom/green)). While individual waveforms do appear to undergo morphological changes with increasing age, notable and consistent characteristics include clear, discernable beats, as described collectively by the presence of consistent P-waves followed by a QRS complex and subsequent T-waves, visible in both Leads I-II of each postnatal time point. Please click here to view a larger version of this figure.

Figure S1: Illustration of traditional limb lead electrodes for noninvasive collection of early postnatal ECG. (A, left) Lateral view of mouse and electrode placement within Faraday cage (box). (B) Traditional self-stick skin electrodes are positioned on the dorsal surface of the pup. (A, right) ECG-like signal may be interpreted with the use of traditional electromyography transducer to produce a minimalistic ECG tracing discernable only in Lead II (C, bottom). (B-C) Electrode placement, chest lead directionality, and corresponding, representative ECG tracing from a neonatal mouse pup at P1.0 (Lead II; purple). Please click here to download this figure.

Figure S2: Comparative electrocardiographic reads from littermate control pups and mutant pups with congenital heart disease on the first postnatal day (P1.0). (A,B) Images represent examples of good quality ECG tracings from healthy neonatal pups (A, CONTROL) compared to pups born with CHD (B, CHD MUT) at P1.0. The 2-lead, noninvasive device was used to capture ECG tracings at 10.0 (A,B, top) and 1.5-second intervals (A, B, bottom). Noticeable differences in heart rate are apparent in the CHD MUT (B), as visualized by the decreased number of cardiac cycles (complexes) visible in the given time frame. Comparison also reveals irregularities in the general morphology of QRS waveforms, frequency, and overall regularity of cardiac cycles in the CHD MUT (B) when compared to the control (A). Lead I (red); Lead II (green)). Please click here to download this figure.

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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 electrocardiogram protocols in that there is no physical trauma to the mouse. The protocol is totally noninvasive, does not require the use of anesthesia, and is optimal for mice immediately after birth. No other electrocardiogram device allows for pups this young to be analyzed in this manner9,10,11. This protocol aims to establish a reliable reference method to generate normative data specific to the neonatal mouse population, but applicable to human pediatric populations.

When performing an electrocardiogram on such a small animal, it is important to be cautious with all steps. However, there are a few key steps that can change the quality of the results. The first is applying the conductive gel. If there is too much gel, there will be a higher chance for the electrodes to connect and short. If there is not enough gel, there will not be a secure connection. The best method to apply the gel is to approach the electrode from the outside corner and roll the gel over the top of the electrode. It is very important that extreme caution is taken to assure there are no threads between electrodes, which would interfere with the presence and/or quality of the electrical activity. It may be useful to take a thin tool (e.g., forceps), and run it between the electrodes to collect any stray threads which may not be apparently visible. While not formally required as part of the protocol, this extra step could serve as an additional precaution to ensure optimal conduction and minimal noise.

If the presence of noise of static causes the ECG to be unreadable (Figure 2), it may be useful to remove all electronic devices from the immediately (table-top) vicinity. This is especially helpful if any of the electronic devices present nearby are moving, as this movement can be picked up by the ECG recording device12. It is also important not to introduce any outside movements during data collection. Outside movements that could interfere with the quality of the ECG could include setting objects down on the same nearby surface, and must be avoided until after the reading is complete. In addition to outside devices, very active mouse pups may also cause electrical interference associated with excessive body movements. The likelihood of this type of musculoskeletal interference increases as the pups mature, which should be considered when selecting ages for data collection. In the event that the pup shifts from the electrodes in a way that significantly compromises the quality of the ECG reading, repositioning of the pup should be considered. Repositioning the mouse before opting to reapply the electrode gel can provide improved results in most cases and save additional time and reagents. Before repositioning the pup, select the pause button in the software. Pausing the run will stop active recording of the ECG but will continue to track time. Of note, when the recording is resumed, the ECG will appear at a later time than paused at. Slide the device platform out from the Faraday age with the mouse still positioned between the bumpers. Remove the bumpers surrounding the mouse, and gently lift the pup off of the electrodes. Reposition pup on the electrodes following the same protocol of gently holding the mouse in place for 1 min for gel to adhere (step 3.4-3.5). Try to reposition the mouse so that the electrodes are on the thorax between the upper limbs (Figure 3). While designed as an ideal, noninvasive method for collecting ECG in neonatal mice, one limitation associated with this protocol would be the increased mobility associated with data collection on an un-anesthetized mouse, as the mouse may also move and shift on the device which will affect the quality of the reading. While movement may be limited with positioning of silicone bumpers, this cannot be prevented without the use of sedation or anesthesia.

In the situation in which an ECG recording comes with heavy interference (Figure 2) despite having minimized all electrical interference, the next step that should be taken is to reposition the external wiring connecting the recording platform to the Faraday cage. It is very important that the external wiring remains properly connected to the recording platform during data acquisition. If external wiring is repositioned, be sure to reattach this wiring carefully at both ends, until a clearer recording can be obtained. If the use of the Faraday cage provided with the device is not suitable, the device can be used in other Faraday cages.

If the recording is not clear or the mouse has moved from the electrodes, remove the mouse from the device and clean the electrodes by taking forceps and removing all the conductive gel. Because the conducting gel is water-soluble, one can also use warm water to gently remove excess gel from the skin of the pup. Reapply the gel and reposition the pup.

To obtain the best results make sure the device is properly cleaned before and after each use. The gel does dry and can be removed using forceps to pull it from the device, but the gel is water soluble, so a damp cloth can be used to clean the electrodes of the recording platform.

Older mice have been more active in the recording process, so it is important to closely monitor them as they often move from the electrodes and can even move off the device platform. While a clear read may not happen right away, with troubleshooting and repositioning, there has been success in getting usable recordings with this device (Figure 1). Active mice may need to be returned to their mother and reanalyzed after a break. They can also be held in the palm of a hand and gently covered to provide heat and darkness until the pup settles down.

This device is designed to collect ECG data on mouse pups from the age of birth to P10 (Figure 4). Pups older than P10 will likely not be able to fit into the device with the Faraday cage, an essential component to maximizing signal to noise ratios. Even at P10, positioning adjustments will likely need to be made to accommodate a larger body size into the device. Use extreme caution when moving the device into and out of the Faraday cage. Removal of the top bumper will allow the mouse to lay on the electrode platform with the surrounding Faraday cage. Given that the mice at this age are more active, they are more apt to move off the electrodes without the stabilization of the top bumper. The top bumper can also be placed in front of the pup to help discourage the pup moving off the device.

The novelty of this device and corresponding protocol include optimization for use immediately after birth, the ability of the system to accommodate a broader age range (P1-P10) and the need addressed by this method to expand the translational applications of in vivo research methods in the field of cardiovascular physiology and beyond. Although sophisticated devices utilizing echocardiography to quantify cardiac cycles in neonate mice are available13, one great advantage of this protocol is that it allows for a relatively simple and affordable means to address basic electrophysiological parameters, which is very attractive in the current parlous scientific funding environment.

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The authors report no conflicts of interest.


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.


Name Company Catalog Number Comments
LabScribe4 iWorx LabScribe4 Software used to record ECG
Neonatal Mouse ECG & Respiration System iWorx RS-NMECG : Neonatal Mouse ECG ECG device
Tensive Conductive Adhesive Gel Parker Laboratories, Inc 22-60 Tac-gel used as conductive gel for ECG



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Cite this Article

Fitzsimons, L. A., Brewer, V. L., Forrester, J., Moran, A. M., Tucker, K. L. Noninvasive Electrocardiography in the Perinatal Mouse. J. Vis. Exp. (160), e61074, doi:10.3791/61074 (2020).More

Fitzsimons, L. A., Brewer, V. L., Forrester, J., Moran, A. M., Tucker, K. L. Noninvasive Electrocardiography in the Perinatal Mouse. J. Vis. Exp. (160), e61074, doi:10.3791/61074 (2020).

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