AEvD was supported by the Dutch Heart Foundation (DHF-2007B103). The authors would like to thank all mothers and children in the Amsterdam Born Children and their Development (ABCD) study, and the entire development and maintenance team of the VU-AMS system at the Division for Instrumentation - the Department of Psychophysiology (VU University, Amsterdam, the Netherlands).

1. Preparation: Starting Up
<ol>
	<li>You need:
	<ul>
		<li>a VU-AMS5fs ambulatory recording device (including an infrared interface cable that either connects to the RS232 serial port of a PC or to a USB port).</li>
		<li>7 electrodes (we used ConMed 1690-003).</li>
		<li>2 charged AA-batteries.</li>
		<li>an empty CompactFlash memory card (the VU-AMS5fs has been extensively tested with the 1GB 80x CF card from Transcend (TS1GCF80), but other CF cards should work too).</li>
		<li>a laptop or PC with flash card reader and the Data Analysis Management Software (DAMS) suite installed.</li>
		<li>a stopwatch.</li>
		<li>music player with children's stories and headphones and a small self-inflatable air mattress are optional.</li>
	</ul>
	</li>
	<li>Check the time and date settings on the laptop/PC, since these will be recorded as metadata on your files. Put the empty memory card and full batteries in the VU-AMS device (successful placement is signalled by a triple beep). When the device is on standby, the green light will flash twice every ten seconds. This indicates it is ready, but not recording. Now connect the device to the laptop using the provided cable and start up the DAMS program. Initiate communication with the device (select the tab 'device' and choose the appropriate connection mode, infrared cable or bluetooth).</li>
	<li>Have the participant take off his/her upper body wear. In the places where the electrodes will be placed,clean the skin with alcohol-wipes, and place the seven electrodes on the chest and back (Figure 1). Then attach the lead wires following the color scheme, and connect them to the device.</li>
	<li>Check the battery type and battery voltage indication (this should be about 3.4 V for alkaline and about 2.4 V for rechargeable NiMH batteries). Fill out the identification field. The typical sampling frequencies are as shown in the figure (Figure 2).</li>
	<li>Measure the distance between the two chest electrodes in millimeters, and fill this out in the field 'ICG-V distance'. Then click 'send settings' to send the current settings/ID to the device.</li>
	<li>Now, the 'Online' option of the program should be used to display the ECG, &Delta;Z (this is the respiration) and dZ/dt (this is the ICG). The &Delta;Z signal reflects the base impedance across the thorax, which after appropriate filtering can be used to extract the respiration signal with high fidelity 7. The dZ/dt signal is the &Delta;Z differentiated over time and reflects rapid changes in &Delta;Z linked to the ejection of blood from the ventricle into the aorta.
	<ol>
		<li>A clear QRST-complex should be detectable in the ECG. The R-wave should be upward and it should be the peak with the largest (absolute) amplitude in either direction.</li>
		<li>The &Delta;Z should be within -0.5 and +0.5 &Omega; most of the time and dZ/dt between -1 and +1 &Omega;/sec.</li>
		<li>Z0 should always stay within an 8 to 20 &Omega; range. This variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child's height, and the 'wetness' of the thorax column enclosed by the measuring electrodes, differences in body composition (e.g. BMI) can affect the amplitude of the dZ/dt signal (fat mass containing less water than muscle). Individual differences in absolute Z0 amplitude are also reflected in the &Delta;Z signal but this does not affect the determination of systolic time intervals, which are amplitude-independent.</li>
		<li>The &Delta;Z signal should reflect deep breathing of the subject clearly (instruct the child to take a slow deep breath and exhale slowly).</li>
		<li>In the ICG the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable. Light movement of the subject should not distort the dZ/dt signal. If these criteria are not met, re-clean the skin and re-attach the electrodes until satisfactory signals are obtained.</li>
	</ol>
	</li>
	<li>When good signals are attained, start data recording by pressing the 'start' button. You will hear a beep acknowledging the start of the recording and the green light will start flashing once every three seconds. The registration has now started. Close the VU-DAMS program. You can now disconnect the device from the interface cable.</li>
</ol>
2. The Registration Period
<ol>
	<li>Once the registration has started, ask the child to lie down for the first experimental condition. When the child has been in the supine position (without head-up tilt) for two minutes, you shortly (&lt; 2 s) press the small black button on top of the device. Pressing this button marks a special event, and will later on help you identify the start of this condition in your data.</li>
	<li>After four minutes, press the event button again. This signals the end of the lying down condition. Now have the child sit up and repeat the procedure for this second condition. Press the button, wait four minutes and press the button again. The children are instructed to rest quietly during these conditions.</li>
	<li>To stop the measurement, press and hold down the button for at least 3 sec. The light will flash every 10 sec to indicate it has stopped and is in 'stand by' mode. Once the device has stopped, you may disconnect the lead wire plug from the connector and the lead wires from the electrodes.</li>
	<li>Remove the batteries and flash card form the VU-AMS device and place the flash card in the reader unit. Move the acquired files to a designated directory (typically the name of the directory will be identical to the subject identifier used in the identification field).</li>
</ol>
3. Processing the Data
<ol>
	<li>Upon opening the data with VU-DAMS program, the data will be automatically converted from raw data format (extension .5fs) to a new format (extension .amsdata). This is the data file that VU-DAMS will be using in the ensuing steps.</li>
	<li>First extract the Inter Beat Interval time series from the ECG signal. Select the Detect R-peaks tab. An automated algorithm will detect all R-peaks in the ECG signal and select (if present) periods with very low ECG quality for removal. In the upper left hand corner the number of Blue (correct), Yellow (medium suspicious) or RED (highly suspicious) is indicated. By pressing '.' (DOT) the cursor is moved to the next suspicious R-peak and the user can delete or add markers for R-waves by hand. It is recommended that at least all highly suspicious beats are visually inspected.</li>
	<li>The main aim is to obtain a mean value for the heart rate, the preejection period (PEP) and measures of respiratory sinus arrhythmia (RSA, HF, RMSSD) across the experimental conditions used. Therefore proceed by indicating which periods in the raw data correspond to these conditions. This process is called 'data labeling'. Select the Label Data tab. Two panels show the heart rate signal and movement signal respectively, as well as the actual time of the recording.</li>
	<li>Place the mouse cursor in the top bar where it says "click and drag to add labels" at around the start time of your first condition and drag the mouse to the end time of that condition. These times are either obtained from a written record of start and stop times (that you noted down during data collection) or you can use the start and stop markers obtained from pressing the button at the start and end of each condition, which are the vertical lines running across the HR and movement graphs.</li>
	<li>Each label can be given a (unique) identifier to signal a particular condition. In our case we have only one category for our labels: experimental condition. This category has two values: lying down and sitting up.</li>
	<li>VU-DAMS needs to be made aware of the experimental design by a so-called label configuration file (label.cfg). This is an ASCII file that can be opened with most text editors and, for example, looks like this: 
	# exp_condition 
	10 lying down 
	11 sitting up</li>
	<li>By placing the label.cfg file in the directory of the .amsdata files, it will be automatically loaded by the VU-DAMS program. Once a label has been made, a pop up screen will appear with the categories/values listed in the label.cfg file. Select 'lying down' for the first label and 'sitting up' for the second label.</li>
	<li>After labeling, select the 'Impedance scoring' tab to score the PEP in the impedance cardiogram. For each of the conditions an ensemble averaged dZ/dt waveform is displayed, time-locked to the ECG R-peak. An ensemble averaged ECG is presented below the dZ/dt waveform. Place the four vertical cursors in the correct positions: ECG Q-wave onset (start of electrical activity), ICG B-point (start of the ejection phase), ICG dZ/dt-min (maximal ejection speed), and ICG X-point (aortic valve closure - end of ejection phase).</li>
	<li>Next, select the 'Respiration Scoring' tab to score the peak-valley RSA using the respiratory and ECG signals. Automated breath-to-breath scoring of the respiratory interval and the shortest interbeat interval during inspiration and the longest interbeat interval during expiration can now be inspected. Typically the automated detection algorithm should not classify more than 15% of the breaths as deviant - otherwise inspect the respiration signal and tune the parameters of the detection algorithm as needed. 
	 
	Guidelines for visual inspection of the ECG, ICG and respiration signals and interactive PEP and RSA scoring can be found on the VU-AMS website, <a href="http://www.vu-ams.nl" target="_blank">www.vu-ams.nl</a>.</li>
</ol>
<ol start="10">
	<li>Finally, select the Label Information tab. A table with the results appears after calculation. Each row represents the average value of a series of physiological parameters (heart rate, PEP, RSA, RR) for each labeled time period. The first column has the subject identifier (Label_ID). The last column indicates the values of all categories used during labeling (here only a single category 'experimental condition' with two values, 'sitting up' and 'lying down'). Spectral powers of the interbeat interval times series are given only for labels with a minimum length of 4 min (otherwise the missing code is displayed). The spreadsheet in this display can be exported to ASCII or EXCEL for further statistical analyses.</li>
</ol>

<ol>
	<li>de Geus, E. J., Willemsen, G. H., Klaver, C. H., van Doornen, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+measurement+of+respiratory+sinus+arrhythmia+and+respiration+rate.">Ambulatory measurement of respiratory sinus arrhythmia and respiration rate.</a> Biol. Psychol. 41, 205-227 (1995).</li><li>Goedhart, A. D., van der, S. S., Houtveen, J. H., Willemsen, G., de Geus, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+time+and+frequency+domain+measures+of+RSA+in+ambulatory+recordings.">Comparison of time and frequency domain measures of RSA in ambulatory recordings.</a> Psychophysiology. 44, 203-215 (2007).</li><li>Goedhart, A. D., Kupper, N., Willemsen, G., Boomsma, D. I., de Geus, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+stability+of+ambulatory+stroke+volume+and+cardiac+output+measured+by+impedance+cardiography.">Temporal stability of ambulatory stroke volume and cardiac output measured by impedance cardiography.</a> Biol. Psychol. 72, 110-117 (2006).</li><li>Riese, H., Groot, P. F. C., van den Berg, 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=Large-scale+ensemble+averaging+of+ambulatory+impedance+cardiograms.">Large-scale ensemble averaging of ambulatory impedance cardiograms.</a> Behavior Research Methods Instruments &amp; Computers. 35, 467-477 (2003).</li><li>Willemsen, G. H., de Geus, E. J., Klaver, C. H., van Doornen, L. J., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+monitoring+of+the+impedance+cardiogram.">Ambulatory monitoring of the impedance cardiogram.</a> Psychophysiology. 33, 184-193 (1996).</li><li>van Dijk, A. E., van Eijsden, M., Stronks, K., Gemke, R. J., Vrijkotte, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+stress+and+balance+of+the+child's+cardiac+autonomic+nervous+system+at+age+5-6+years.">Prenatal stress and balance of the child's cardiac autonomic nervous system at age 5-6 years.</a> PLoS ONE. 7, e30413 (2012).</li><li>Houtveen, J. H., de Groot, P. F., de Geus, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+variation+in+posture+and+respiration+on+RSA+and+pre-ejection+period.">Effects of variation in posture and respiration on RSA and pre-ejection period.</a> Psychophysiology. 42, 713-719 (2005).</li><li>Goedhart, A. D., Willemsen, G., Houtveen, J. H., Boomsma, D. I., De Geus, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+low+frequency+heart+rate+variability+and+preejection+period:+two+sides+of+a+different+coin.">Comparing low frequency heart rate variability and preejection period: two sides of a different coin.</a> Psychophysiology. 45, 1086-1090 (2008).</li><li>van Dijk, A. E., van Eijsden, M., Stronks, K., Gemke, R. J., Vrijkotte, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardio-metabolic+risk+in+5-year-old+children+prenatally+exposed+to+maternal+psychosocial+stress:+the+ABCD+study.">Cardio-metabolic risk in 5-year-old children prenatally exposed to maternal psychosocial stress: the ABCD study.</a> BMC Public Health. 10, 251 (2010).</li><li>van Lien, R., Goedhart, A., Kupper, N., Boomsma, D., Willemsen, G., de Geus, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underestimation+of+cardiac+vagal+control+in+regular+exercisers+by+24-hour+heart+rate+variability+recordings.">Underestimation of cardiac vagal control in regular exercisers by 24-hour heart rate variability recordings.</a> Int. J. Psychophysiol. 81, 169-176 (2011).</li></ol>

We have nothing to disclose.

We used an ambulatory recording device to measure cardiac autonomic control in 3097 children, aged between 5 and 7 years. Seven electrodes sufficed to measure the ECG and ICG from which the heart rate, heart rate variability and the systolic time intervals were extracted. Heart rate variability in the respiratory frequency band (RSA) is a valid indicator of cardiac parasympathetic activity. The systolic time interval, PEP, by reflecting cardiac contractility, is a valid indicator of cardiac sympathetic activity. The mean values obtained for HR, PEP and RSA, the effects of posture changes and the differences between boys and girls were in line with what would be expected from the literature.
As ambulatory monitoring removed the necessity of assessment in a laboratory our recordings could be done in various locations (e.g. school, sports center, science museum) without differences in signal recording quality. However, it is of crucial importance to standardize within or between subject comparisons for posture and physical load, as afterload and preload effects can co-determine the PEP without any changes in cardiac sympathetic drive 7. We conclude that ambulatory recording of the ECG and ICG in large samples of children is highly feasible and propose the current standardized study design as a useful template for future assessments of cardiac autonomic control in children.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>VU-AMS5fs ambulatory recording device &amp; infrared interface cable</td>
			<td>VU University Amsterdam</td>
			<td>n/a</td>
			<td><a href="http://www.vu-ams.nl" target="_blank">http://www.vu-ams.nl</a></td>
		 </tr>
		 <tr>
			<td>Electrodes</td>
			<td>ConMed</td>
			<td>1690-003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AA-batteries</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>CompactFlash memory card</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laptop/pc with flash card reader</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>VU-DAMS software suite</td>
			<td>VU University Amsterdam</td>
			<td>&nbsp;</td>
			<td>free download, <a href="http://www.vu-ams.nl" target="_blank">http://www.vu-ams.nl</a></td>
		 </tr>
		 <tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Music player &amp; headphones</td>
			<td></td>
			<td></td>
			<td>optional</td>
		 </tr>
		 <tr>
			<td>Self-inflatable air mattress</td>
			<td></td>
			<td></td>
			<td>optional</td>
		 </tr>
		</tbody>
 </table>

measuring cardiac autonomic nervous system (ans) activity in children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

In the Amsterdam Born Children and their Development study, a Dutch prospective, longitudinal birth cohort, the measurement protocol was started in 3,097 children 6. Approval was obtained from the Academic Medical Center Medical Ethical Committee, the VU University Medical Center Medical Ethical Committee and the Registration Committee of Amsterdam. All participating mothers gave written informed consent for themselves and their children.
As the monitors are lightweight and unobtrusive, the children tolerated these measurements very well. We do not have data on the refusal rate, but experience taught us that only a few children resisted the placement of the electrodes and thereby obstructed further assessment. Of the 3,097 registrations, 0.7% were lost due to either equipment failure or misplacement of files. Out of the 3,074 registrations left, 98.7% were of children who completed the entire protocol (n = 3,056). Within each of the labelled time periods (we originally labelled four time periods, but later summarized these to two), we encountered unclear ICG signals, meaning PEP could not be determined. This led to a loss of 1.5% in the first out of four labelled periods, 2.4% in the second, 2.8% in the third and 4.1% in the fourth period. Complete data on PEP in all time periods was available in 2,797 cases (91.5%, thus 8.5% loss due to unclear ICG signals). Complete data on heart rate (HR), pre-ejection period (PEP) and respiratory sinus arrhythmia (RSA), as well as sex and age, was available from 2,761 children; in this final step, 1.3% data loss occurred, due to unknown reasons. Overall, 89.2% of the started registrations led to full subject data. The mean age of the children was 5.7 years (SD 0.5; interquartile range 5.0:6.5), and their BMI was 15.5 kg/m2 (SD 1.5; interquartile range 13.9:17.2).
The mean values of the major outcome variables HR, PEP, and RSA are given in Table 1 and graphically depicted in Figure 3, separately for boys and girls. HR (both lying down and sitting up) and PEP (only sitting up) were higher in girls than in boys (both postures). RSA was lower in girls than in boys (both postures). The higher values for HR in girls are likely to be caused by the lower vagal (parasympathetic) cardiac control. Their sympathetic cardiac control was not different or even lower than that in boys (sitting up).
In both sexes, HR was higher when sitting up compared to lying down, whereas RSA was lower when sitting up. This reflects the lower vagal control when sitting up. PEP was shorter lying down then sitting up. This effect was also as expected, and it reflects the outcome of opposite processes: lower sympathetic activity (lengthens PEP) while lying down with increased preload (shortens PEP)7.
<table border="1" class="jove_content_small" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td rowspan="3"> </td>
 <td colspan="6">Boys</td>
 <td colspan="5">Girls</td>
 </tr>
 <tr>
 <td colspan="3">Lying down</td>
 <td colspan="3">Sitting up</td>
 <td colspan="2">Lying down</td>
 <td colspan="3">Sitting up</td>
 </tr>
 <tr>
 <td>Mean</td>
 <td colspan="2">SD</td>
 <td>Mean</td>
 <td>SD</td>
 <td>&nbsp;</td>
 <td>Mean</td>
 <td>SD</td>
 <td>Mean</td>
 <td>SD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heart rate (bpm)</td>
 <td>83.9</td>
 <td>9.5</td>
 <td>*</td>
 <td>89.1</td>
 <td>10</td>
 <td>*&dagger;</td>
 <td>86.9</td>
 <td>10.1</td>
 <td>92.4</td>
 <td>10.4</td>
 <td>&dagger;</td>
 </tr>
 <tr>
 <td>Pre-ejection period (msec)</td>
 <td>76.9</td>
 <td>11.8</td>
 <td>&nbsp;</td>
 <td>78.5</td>
 <td>12.2</td>
 <td>*&dagger;</td>
 <td>77.7</td>
 <td>10.3</td>
 <td>81</td>
 <td>11.7</td>
 <td>&dagger;</td>
 </tr>
 <tr>
 <td>Respiratory sinus arrythmia (msec)</td>
 <td>127.0</td>
 <td>60.4</td>
 <td>*</td>
 <td>115.7</td>
 <td>55.8</td>
 <td>*&dagger;</td>
 <td>121.7</td>
 <td>56.8</td>
 <td>108.7</td>
 <td>51.9</td>
 <td>&dagger;</td>
 </tr>
 </tbody>
</table>
Table 1. Cardiac autonomic nervous system measures in boys and girls, by posture on posture difference. p &lt; 0.05 for one sample T-test on sex difference. &dagger; p &lt; 0.05 for paired samples T-test.
<img alt="Figure 1" src="/files/ftp_upload/50073/50073fig1.jpg" /> 
 Figure 1. The seven electrodes should be placed on the participant's chest and back. The first ECG electrode (V-) is placed slightly below the right collar bone 4 cm to the right of the sternum. The second ECG electrode (V+) is placed at the apex of the heart over the ninth rib on the left lateral margin of the chest approximately at the level of the processus xiphodius. The third ECG electrode (GND) is a ground electrode and is placed on the right side, between the lower two ribs at the right abdomen. The first ICG measuring electrode (V1) is placed at the top end of the sternum, between the tips of the collar bones. The second ICG measuring electrode is placed at the xiphoid complex of the sternum, where the ribs meet. The two current electrodes are placed on the back: I- on the spine over the cervical vertebra C4, at least 3 cm (1 in) above the ICG measuring elec-trode V-, and I+ between thoracic vertebrae T8 and T9 on the spine, at least 3 cm (1") below the ICG measuring elec-trode V2. The ICG electrode placement takes into account that the largest part of the left ventricle driven change in thorax impedance is captured by the column between the suprasternal notch and the processus xiphoideus.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50073/50073fig2highres.jpg" src="/files/ftp_upload/50073/50073fig2.jpg" /> 
 Figure 2. The typical settings used for a recording as displayed by the DAMS software after connecting to the VU-AMS5fs device. <a href="https://www.jove.com/files/ftp_upload/50073/50073fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/50073/50073fig3.jpg" fo:content-width="3.5in" fo:src="/files/ftp_upload/50073/50073fig3highres.jpg"/> 
 Figure 3. Cardiac autonomic nervous system measures in boys and girls, by posture. * indicates p &lt; 0.05 for one sample T-test on sex difference. # indicates p &lt; 0.05 for paired samples T-test on posture difference.

Watch this Scientific Journal Video about Measuring Cardiac Autonomic Nervous System ANS Activity in Children at JoVE.com

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

measuring-cardiac-autonomic-nervous-system-ans-activity-in-children

Research

JoVE Journal

Medicine

20.6K Views.  Academic Medical Center - University of Amsterdam. Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

Video: Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal organs (e.g., heart rate, respiration, digestion) and responds to everyday and adverse experiences 1. ANS measures in children have been found to be related to behavior problems, emotion regulation, and health 2-7. Therefore, understanding the factors that affect ANS development during early childhood is important. Both branches of the ANS affect young children&#8217;s cardiovascular responses to stimuli and have been measured noninvasively, via external monitoring equipment, using valid and reliable measures of physiological change 8-11. However, there are few studies of very young children with simultaneous measures of the parasympathetic and sympathetic nervous systems, which limits understanding of the integrated functioning of the two systems. In addition, the majority of existing studies of young children report on infants&#8217; resting ANS measures or their reactivity to commonly used mother-child interaction paradigms, and less is known about ANS reactivity to other challenging conditions. We present a study design and standardized protocol for a non-invasive and rapid assessment of cardiac autonomic control in 18 month old children. We describe methods for continuous monitoring of the parasympathetic and sympathetic branches of the ANS under resting and challenge conditions during a home or laboratory visit and provide descriptive findings from our sample of 140 ethnically diverse toddlers using validated equipment and scoring software. Results revealed that this protocol can produce a range of&#160;physiological responses to both resting and developmentally challenging conditions, as indicated by changes in heart rate and indices of parasympathetic and sympathetic activity. Individuals demonstrated&#160;variability in resting levels, responses to challenges, and challenge reactivity, which provides additional evidence that this protocol is useful for the examination of ANS individual differences for toddlers.

Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

We describe the methods for continuous monitoring of the autonomic nervous system under resting and challenge conditions with 18 month old children. Results revealed that this protocol can produce meaningful physiological responses in both branches of the autonomic nervous system and elicit significant individual variability in patterns of responses.

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...