Name: analyzing long-term electrocardiography recordings to detect arrhythmias in mice
Uploaded: 2021-05-23T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice at JoVE.com

This work was supported by German Research Foundation (DFG; Clinician Scientist Program In Vascular Medicine (PRIME), MA 2186/14-1 to P. Tomsits and D. Sch&#252;ttler), German Centre for Cardiovascular Research (DZHK; 81X2600255 to S. Clauss), the Corona Foundation (S199/10079/2019 to S. Clauss), the ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 to S. Clauss), the Heinrich-and-Lotte-M&#252;hlfenzl Stiftung (to S. Clauss) and the China Scholarship Council (CSC, to R. Xia). The funders had no role in manuscript preparation.

1. Prearrangements
<ol>
	<li>Start Ponemah 6.42 software, and confirm the username and serial number of the software license on the following screen by clicking on Continue.</li>
	<li>Load the experiment containing the ECG of interest
	<ol>
		<li>If Ponemah is started for the first time, note that the Ponemah Get Started dialog opens, offering three options: 1) Create Experiment, 2) Load Experiment, 3) Import Experiment.
		<ol>
			<li>Select Load Experiment to open a f...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+ECG+recording+in+mice.">Ambulatory ECG recording in mice.</a> Journal of Visualized Experiments : JoVE. (39), e1739 (2010).</li><li>Mehendale, A. 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=Unlock+the+information+in+your+data:+Software+to+find,+classify,+and+report+on+data+patterns+and+arrhythmias.">Unlock the information in your data: Software to find, classify, and report on data patterns and arrhythmias.</a> Journal of Pharmacological and Toxicological Methods. 81, 99-106 (2016).</li><li>Hulsmans, M., 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=Macrophages+facilitate+electrical+conduction+in+the+heart.">Macrophages facilitate electrical conduction in the heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Boukens, B. 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The surface ECG is the primary diagnostic tool for patients suffering from heart rhythm disorders, providing insights into many electrophysiological phenomena. Nevertheless, sufficient analysis of cardiac surface ECG pathologies requires knowledge and definition of normal physiologic parameters. Many years of epidemiological research have led to broad consent on what is physiologic in humans and thus enabled physicians worldwide to clearly distinguish the pathologic. However, the analysis of surface ECG data is a major c...

Cardiac arrhythmias are common, affecting millions of patients worldwide1. Ageing populations show a growing incidence and thus a major public health burden resulting from cardiac arrhythmias and their morbidity and mortality2. Current treatment strategies are limited and often associated with significant side effects and remain ineffective in many patients3,4,5,6. Novel and innovative therapeutic strategies that causally target arrhythmia mechanisms are urgently needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary; mice have been proven to be an ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets7,8,9. Continuous ECG recording is a well-established concept in the clinical routine of arrhythmia detection10.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice as they allow continuous recording of the ECG (a common approach is to implant the leads in a lead-II position) over a period of several months in freely moving, awake mice11,12. However, due to the huge number of data points (up to more than 1 million QRS complexes per day) and limited knowledge of murine standard values, the analysis of telemetry data remains challenging. Commonly available telemetry transmitters for mice last up to 3 months, leading to the recording of up to 100 million QRS complexes. This means that pragmatic analysis protocols are much needed to reduce the time spent with each individual dataset and will allow researchers to handle and interpret this huge amount of data. To obtain a clean ECG signal upon recording, transmitter implantation needs to be optimal-the lead positions should be as far apart as possible to allow higher signal amplitudes.
The interested reader may be referred to a protocol by McCauley et al.12 for more information. Further, to minimize noise, cages and transmitters must be placed in a silent environment not prone to any disturbance, such as a ventilated cabinet with controlled environmental factors (temperature, light, and humidity). During the experimental period, lead positioning must be checked regularly to avoid loss of signal due to lead perforation or wound healing issues. Physiologically, there is a circadian alteration in ECG parameters in rodents as in humans, generating the need for a standardized approach to obtaining baseline ECG parameters from a continuous recording. Rather than calculating mean values of ECG parameters over a long period, analysis of a resting ECG similar to that in humans should be performed to obtain basic parameters such as resting heart rate, P wave duration, PR interval, QRS duration, or QT/QTc interval. In humans, a resting ECG is recorded over 10 s, at a normal heart rate of 50-100/min. This ECG includes 8 to 17 QRS complexes. An analysis of 20 consecutive QRS complexes is recommended in the mouse as &#34;resting ECG equivalent&#34;. Because of the above-mentioned circadian alteration, a simple approach is to analyze two resting ECGs per day, one at daytime and one at night time. Depending on the light on/off cycle in the animal facility, suitable times are selected (e.g., 12 AM/PM), and basic parameters are obtained.
Next, a heart rate plot over time is used to detect relevant tachy- and bradycardia, with consecutive manual exploration of these episodes to get a first impression. This heart rate plot then leads to the important parameters of maximum and minimum heart rate over the recorded period as well as heart rate variability over time. After that, the dataset is analyzed for arrhythmias. This article describes a step-by-step approach to obtain these baseline ECG data from long-term telemetry recordings of awake mice over a recording period of up to three months. Further, it describes how to detect arrhythmias using the software, Ponemah version 6.42, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). This version is compatible with both Windows 7 (SP1, 64 bit) and Windows 10 (64 bit).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ponemah Software</td>
			<td>Data Science international</td>
			<td></td>
			<td>ECG Analysis Software</td>
		</tr>
	</tbody>
</table>

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.

Recording long-term ECGs results in huge data sets. The options for further analyses are manifold and depend on the individual research project. This protocol provides a description of some very basic readouts that can be used by most researchers, especially for screening experiments, e.g., when characterizing a transgenic mouse line or when investigating the effects of a specific treatment in a disease model. A previous project involved the study of a novel drug candidate to determine whether it possessed cardi...

Watch this Scientific Journal Video about Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice at JoVE.com

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

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 ...

Analysis of large ECG data sets, especially when you screen for any arrhythmia, is very challenging and labor intensive. Our semi-automated approach allows for much faster and more convenient screening for arrhythmias in mice. This semi-automated approach leads to more accurate results.The procedure will be demonstrated by Aparna Chivukula, a PhD student in my lab. Begin by analyzing basic ECG parameters. Click on Subject Setup or Channel Details to select any of the derived parameters.As mice follow a circadian rhythm, analyze two resting ECGs per day. One at daytime and one at nighttime to control for circadian effects, selecting suitable times depending on the light on or off cycle in the animal facility. Select a section of the ECG with good signal quality and a stable heart rate in the heart rate trend graph within a defined reasonable timeframe around this time point.Confirm the accuracy of the validation masks, or adjust manually to 20 consecutive QRS complexes, and add missing validation marks. For further calculations and visualizations, mark the lines containing the values of these 20 consecutive QRS complexes in the derived parameter list, and copy to a spreadsheet or statistics software. Open data insights by clicking on Experiment Data Insights and observe the search panel at the top.Then display the Results panel. Observe the number of search hits displayed as a histogram in the bottom of the panel. Given that the normal heart rate of a mouse is 500 to 724 beats per minute, define a search rule, Bradycardia, to detect bradycardia.Right-click within the search list and select Create New Search to open the search entry dialogue. Right-click within the white box and select Add New Clause. Using the dropdown menus and text fields, define the search rule, Bradycardia Single, as value less than 500, and click on Okay to add this search rule to the list.Apply this search rule by clicking and dragging it to the channel of interest on the left. As bradycardia requires more than one long RR interval, define an additional search rule Bradycardia, to account for this. Click on Okay to add this search rule to the list.Apply this search rule by clicking and dragging it to the channel of interest on the left. To confirm bradycardia, and to reject false results, review each result manually by left-clicking on the waveform. Press STRG plus R to reject the selected result, which will then disappear from the list.To define a search rule to detect tachycardia, right-click within the search list and select Create New Search to open the search entry dialogue. Then right-click within the white box and select Add New Clause. Using the dropdown menus and text fields, define the search rule, Tachycardia Single, as value greater than 724, and click on Okay to add this search rule to the list.Apply this search rule by clicking and dragging it to the channel of interest to the left. As tachycardia requires more than one short RR interval, define an additional search rule, Tachycardia, to account for this, and click on Okay to add this search rule to the list. Apply this search rule by clicking and dragging it to the channel of interest on the left.To confirm tachycardia, and to reject false results, review each result manually by left-clicking on the waveform and use the shortcut STRG plus R to reject the selected result. To detect sinoatrial and atrioventricular blocks, right-click within the search list and select Create New Search to open the search entry dialogue. Then right-click within the white panel and select Add New Clause.Using the dropdown menus and text fields, define the search rule, Pause, as value greater than 300, and click on Okay to add this search rule to the list apply this search rule by clicking and dragging it to the channel of interest to the left. To confirm a pause, decide if the pause is a sinoatrial or atrio ventricular block, and reject false results. Review each result manually by left-clicking on the waveform and press STRG plus R to reject the selected result, which will disappear from the list of results.This approach allowed the detection of periods of reduced heart rate, increased atrioventricular and ventricular conduction, as well as altered repolarization in mice treated with a new drug. Furthermore, the protocol was used to determine the time course of arrhythmia occurrence in the form of traces represented at normal sinus rhythm, sinus pause, three degrees of atrioventricular block, and atrial fibrillation. Final readouts are manifold and depend on study design and research topic.In atrial fibrillation, total disease burden has been established as a prognostic marker and could be calculated from data obtained with this protocol.

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Research

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Medicine

Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering 
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac 
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4. In animal models, it is valuable to compare 
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
 been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5 and 
 hypertension 6, following exercise 7 and exposure to air pollutants 8, as well as determination of the antiarrhythmic efficacy of pharmacological 
 agents 9,10. 
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a 
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that 
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min 
11,12 11,12, and results in mortality in 20% of cases 13. Many individuals survive the SE, but die within 30 days 14,15.
 The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these 
 deaths 14-17. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature 
 sudden and unexpected death associated with epilepsy (SUDEP) 18. As with SE, the precise mechanisms mediating SUDEP are not known. 
 It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22.
 To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is 
 necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders 
 23-25. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
 collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
 These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical 
 activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden 
 cardiac death.

Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described.

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

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 ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

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 ...

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.

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 ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).