Noninvasive Electrocardiography in the Perinatal Mouse

Podsumowanie

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

Streszczenie

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).

Wprowadzenie

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 disease^1,2. 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 mortality^3,4_. 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 anesthetics^5,6,7. 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.

Protokół

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.

Wyniki

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

Dyskusje

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

Materiały

Name	Company	Catalog Number	Comments
LabScribe4	iWorx	LabScribe4	Software used to record ECG	https://www.iworx.com/users/teaching.php
Neonatal Mouse ECG & Respiration System	iWorx	RS-NMECG : Neonatal Mouse ECG	ECG device	https://www.iworx.com/research/cardiac-function/rs-nmecg/
Tensive Conductive Adhesive Gel	Parker Laboratories, Inc	22-60	Tac-gel used as conductive gel for ECG	https://www.parkerlabs.com/tensive.asp