This&#160;research was supported by NIH 1 U01 HL097973, NHLBI 5 R01 HL116511-02, UCSF-CTSI Grant Number UL1 TR000004, the Robert Wood Johnson Health and Society Scholars Program, and the Lisa and John Pritzker Family Foundation. The authors also wish to acknowledge Michelle Stephens for her assistance with scoring the ANS data as well as Vanessa Tearnan, Marialma Gonzales-Cruz, Yurivia Cervantes, Amy Engler, Stephanie Grover and Karen Jones-Mason for their assistance in collecting the ANS data, and Michael Coccia for his help with the data. We are also thankful to the families for their generous participation in this research and to the volunteers who helped us to test and refine this protocol, including the first author&#8217;s two children. We want to acknowledge our mentors who taught us about measuring ANS and why measuring ANS responsivity is meaningful in young children: W. Thomas Boyce, Gary Bernston, and Dave Lozano.

<ol>
	<li>Berntson, G., Quigley, K., Lozano, D., Cacioppo, J. T., Tassinary, L. G., Bernston, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8,+Cardiovascular+Psychophysiology.">Chapter 8, Cardiovascular Psychophysiology.</a> Handbook of Psychophysiology. , (2007).</li><li>Boyce, W. T., 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=Early+father+involvement+moderates+biobehavioral+susceptibility+to+mental+health+problems+in+middle+childhood.">Early father involvement moderates biobehavioral susceptibility to mental health problems in middle childhood.</a> J. Am. Acad. Child Adolesc. 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The authors declare that they have no competing financial interests.

Cette étude a révélé que, dans un échantillon de 18 mois les enfants âgés, repos standardisé et des tâches de provocation visant à obtenir des réponses à partir d&#39;une gamme de domaines (effaroucher, sensoriel, social, repos) a conduit à une série de réponses du SNA. Les ANS moyennes réponses trouvées dans ces conditions de repos et de provocation étaient similaires aux valeurs ANS rapportés par d&#39; autres qui ont mesuré la réactivité ANS avec de jeunes enfants qui utilisent des tâches simil...

Les différences individuelles dans le système nerveux autonome des enfants (ANS) réactivité jouent un rôle important dans le développement et l&#39; entretien des problèmes de santé physique et mentale 12-16. Un nombre croissant de preuves montre que les différences individuelles dans les mesures du SNA au repos et la réactivité sont prédictifs de variations chez les enfants de intériorisation et d&#39; extériorisation psychopathologie 4,9,17-19 et 7,20 de la santé physique. En plus de ces effets directs ou principales de fonctionnement du SNA, un organisme accumulant des études ont montré que la réactivité des enfants peuvent interagir avec des facteurs de risque contextuels, tels que les événements indésirables ou les conflits conjugaux, pour modérer les effets sur le bien - être et la santé des enfants (voir référence 21 pour une revue). En dépit de cette évidence croissante que la réactivité autonome des enfants joue un rôle dans la santé tout au long de la vie, et l&#39;intérêt d&#39;accompagnement dans ces processus, ilest le besoin de recherches supplémentaires examinant le développement du fonctionnement du SNA chez les enfants de moins de trois ans. Le SNA se compose de deux branches, les systèmes nerveux parasympathique et sympathique. Il régule la fonction des organes internes (par exemple, la fréquence cardiaque, la respiration, la digestion, et l&#39; excitation sexuelle), en grande partie grâce à des mécanismes inconscients, en réponse à tous les jours et les effets indésirables des expériences 1. Le système nerveux parasympathique (PNS) est le système «réparatrice repos et« qui maintient une fréquence cardiaque de repos faible et de l&#39;état de restauration lors du sommeil ou de détente Le système nerveux sympathique (SNS) est le système de «combat ou fuite» qui répond aux urgences ou menaçant situations en accélérant son rythme cardiaque 23. sinus respiratoire arythmie (RSA) est un indice fiable de l&#39;influence PNS sur le fonctionnement cardiaque, et la période de pré-éjection (PEP) est un indice fiable de l&#39;influence SNS sur la voiturediac fonctionnement. Les deux RSA et la PPE ont été jugées valides par le biais de mesures blocage pharmacologique expérimental dans des échantillons d&#39;adultes 24,25. Plus récemment, ce travail a été étendue dans des échantillons de l&#39; enfant et de l&#39; adolescent, en établissant en outre RSA et la PPE en tant que mesures valides et fiables de l&#39; activité du SNA au cours du développement 8,26,27. Mesures d&#39; impédance cardiographie fiables (PEP) à l&#39; aide de la bande et ponctuelles électrodes ont été utilisées pour un temps considérable dans des échantillons d&#39;adultes 28. Plus récemment, des techniques de mesure et d&#39; analyse parallèles ont également été montré pour être fiable et valable dans des échantillons d&#39;enfants 11,29,30, bien que la collecte de mesures de PEP chez les jeunes enfants est rare. Il y a un grand corps existant de la littérature sur les SNP fonctionnant chez les nourrissons et les jeunes enfants, y compris le tonus vagal et des mesures d&#39;arythmie sinusale respiratoire. Il y a moins d&#39;études du SNS utilisant PEP, et très peu d&#39;examiner simultanément lasystèmes nerveux parasympathique et sympathique, ce qui est essentiel pour la construction de la compréhension de leur fonctionnement hautement intégré. Parmi les quelques études de PEP des jeunes enfants, la plupart trouvent que leurs protocoles ne produisent pas d&#39; importants changements au niveau du groupe à PEP en réponse aux défis 8,31. Des études ont démontré qu&#39;il existe une stabilité au repos mesures PNS à partir de la petite enfance et ils ont des relations avec le tempérament, le comportement et la santé, 13,16,26,32,33 mais il y a une compréhension limitée de SNS répondant et sa stabilité dans le temps, relations au développement, et les facteurs qui déterminent que sa trajectoire de développement. Les réponses du SNP ont été attribuées principalement à l&#39; engagement social et 16 réponses SNS proposé pour refléter «combat ou fuite» des réponses 1, ainsi que la sensibilité de récompense (voir référence 34 pour une revue). Chez les jeunes enfants, il est difficile de simuler des défis spécifiques dans un laborary et les engager en raison de leur courte durée d&#39;attention. Il y a aussi des défis de mesure pour les tout-petits qui peuvent éprouver un malaise avec les sept électrodes ponctuelles nécessaires pour mesurer PNS et SNS simultanément. En outre, dans les domaines de la science du développement, il y a peu de normes sur la façon d&#39;obtenir &quot;la réactivité du stress&quot; suffisamment tout en respectant les besoins de développement des enfants et l&#39;éthique dans le travail avec les enfants. Cet article présente des informations sur l&#39;administration et la notation d&#39;un protocole standardisé composé de conditions de repos et stimulantes conçues pour obtenir des réponses du système nerveux parasympathique et sympathique de 18 mois les enfants âgés. ANS réactivité est généralement conçue comme la réponse physiologique à un stimulus externe discret par rapport à une comparaison ou un état ​​de repos, qui varie selon les 22 organismes individuels. L&#39;examen de la vie étiologie précoce du mental et physique, ilproblèmes de ALTH a conduit les scientifiques à être particulièrement intéressés à comprendre la réactivité des enfants à des situations qui évoquent d&#39;adaptation de répondre ou peuvent être considérés comme des défis de stress. La prépondérance de la littérature se réfère à ce phénomène comme &quot;la réactivité du stress&quot;, bien que la rencontre des stimuli difficiles a le potentiel pour obtenir des réponses à travers un large éventail de domaines. Ainsi, le protocole décrit ci-dessous a été conçu pour obtenir des réponses du SNA dans plusieurs domaines, y compris deux formes de repos (tout en écoutant une berceuse apaisante et tout en regardant un calme, vidéo neutre) et trois défis de développement appropriés pour les enfants de 18 mois: l&#39;anticipation / effaroucher (jack-in-the-box), sensoriel (jus de citron), et socioaffectif (écouter un cri d&#39;enfant malade). Le protocole a été adapté de nos protocoles existants pour 12 enfants âgés de mois 8 et 3-5 ans 11, afin de le rendre au stade de développement stimulant, engageant et tolérable pour les tout - petits (18-21 Mois). Nous présentons ici le Protocole de développement Défis (DCP) et ANS données à partir du 18 mois visite d&#39;étude du stress, alimentation et développement précoce (SEED) Étude des femmes enceintes et leur progéniture. participants maternels avec des grossesses en santé ont été recrutés au cours de 2013-14 pour une étude de gain de poids gestationnel et le stress prénatal et étaient en surpoids, faible revenu, et de diversité raciale et ethnique. le consentement éclairé des parents pour l&#39;étude de sa progéniture a été obtenue juste après la naissance et de nouveau avant le début de la collecte de données pour l&#39;étude rapportée ici, quand la progéniture étaient 18-21 mois pendant 2014-2015.

<table>
	<tbody>
		<tr>
			<td>ANS equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop and power cord&#160;</td>
			<td>Dell</td>
			<td>60-0107-3.0</td>
			<td>Mindware BioLab software requires a PC with 2 USB ports</td>
		</tr>
		<tr>
			<td>ANS data collection software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>BioLab acquisition program</td>
		</tr>
		<tr>
			<td>ANS data acquisition unit and cords</td>
			<td>Mindware</td>
			<td>50-3711-08</td>
			<td>BioNex 8-slot Chassis</td>
		</tr>
		<tr>
			<td>Snap leads</td>
			<td>Mindware</td>
			<td>93-0498-00</td>
			<td>2 white, 2 red, 2 brown, 1 black</td>
		</tr>
		<tr>
			<td>Spot electrodes</td>
			<td>Kendall</td>
			<td>31112496</td>
			<td>130 model; 7 for each assessment + extras-to be sealed in a ziplock bag at all times)</td>
		</tr>
		<tr>
			<td>ANS scoring/editing software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>HRV Analysis, version 3.1.0F and IMP Analysis, version 3.1.0I</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protocol equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tablet computer</td>
			<td>Apple</td>
			<td></td>
			<td>iPad; for playing audio and video clips</td>
		</tr>
		<tr>
			<td>portable speaker</td>
			<td>Polariod</td>
			<td>PBT510</td>
			<td></td>
		</tr>
		<tr>
			<td>Jack in the Box</td>
			<td>Schylling</td>
			<td>SMJB</td>
			<td>Sock Monkey theme</td>
		</tr>
		<tr>
			<td>Lemon juice</td>
			<td>ReaLemon</td>
			<td>98077</td>
			<td>0.14oz shelf-stable packets</td>
		</tr>
		<tr>
			<td>Pasture pipettes</td>
			<td>Grainger</td>
			<td>6FAV9</td>
			<td>1 ml size, disposable</td>
		</tr>
		<tr>
			<td>Portion cups</td>
			<td>Dart Solo Con</td>
			<td>250PC</td>
			<td>2.5 oz, disposable, plastic</td>
		</tr>
		<tr>
			<td>audio files of baby cry</td>
			<td>Alkon lab</td>
			<td></td>
			<td>30 second clip recorded in a neonatal intensive care unit</td>
		</tr>
		<tr>
			<td>audio file of baseline lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Across the Universe&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>audio file of final lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Here Comes the Sun&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>video file of neutral video</td>
			<td></td>
			<td></td>
			<td>2 minutes of Baby Einstein video</td>
		</tr>
		<tr>
			<td>Retractable measuring tape</td>
			<td>Rollfix</td>
			<td>TM04</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surge protector</td>
			<td></td>
			<td></td>
			<td>to plug in all electronics</td>
		</tr>
		<tr>
			<td>2-prong adapter for surge protector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony</td>
			<td></td>
			<td>Handycam HDR-CX440</td>
		</tr>
		<tr>
			<td>Video Encoder, power and input cords</td>
			<td>Mindware</td>
			<td>50-8614-01</td>
			<td>optional; to sync video and ANS data files</td>
		</tr>
		<tr>
			<td>Baby wipes and gauze</td>
			<td></td>
			<td></td>
			<td>for cleaning and drying oily skin</td>
		</tr>
		<tr>
			<td>Toys</td>
			<td></td>
			<td></td>
			<td>to distract children during assessment</td>
		</tr>
		<tr>
			<td>Visual aids</td>
			<td></td>
			<td></td>
			<td>pictures of child with electrodes on, description of protocol to show participants</td>
		</tr>
		<tr>
			<td>Script of protocol for research assistants</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charging cords for tablet and speaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Doll and reinforcer</td>
			<td></td>
			<td></td>
			<td>for demonstration if necessary</td>
		</tr>
	</tbody>
</table>

measuring cardiac autonomic nervous system (ans) activity in toddlers - resting and developmental challenges

Le système nerveux autonome (SNA) se compose de deux branches, les systèmes nerveux parasympathique et sympathique, et contrôle la fonction des organes internes (par exemple, la fréquence cardiaque, la respiration, la digestion) et répond à tous les jours et les effets indésirables 1. Mesures du SNA chez les enfants ont été trouvés être liés à des problèmes de comportement, la régulation des émotions et de la santé 2-7. Par conséquent, la compréhension des facteurs qui influent sur le développement ANS pendant la petite enfance est importante. Les deux branches du SNA affectent les réponses cardiovasculaires chez les jeunes enfants à des stimuli et ont été mesurés de manière non invasive, par l&#39; intermédiaire de l&#39; équipement de surveillance externe, en utilisant des mesures valides et fiables des changements physiologiques 8-11. Cependant, il existe peu d&#39;études sur les très jeunes enfants avec des mesures simultanées du système nerveux parasympathique et sympathique, ce qui limite la compréhension du fonctionnement intégré des deux systèmes. En outre, la majority d&#39;études des jeunes enfants existants rapport sur les mesures de repos SNA de nourrissons ou de leur réactivité à l&#39;couramment utilisé paradigmes d&#39;interaction mère-enfant, et moins est connu au sujet ANS réactivité à d&#39;autres conditions difficiles. Nous présentons un modèle d&#39;étude et protocole normalisé pour une évaluation non invasive et rapide de contrôle autonome cardiaque chez les 18 mois les enfants âgés. Nous décrivons des méthodes de surveillance continue des branches parasympathique et sympathique du SNA dans des conditions de repos et de provocation lors d&#39;une maison ou d&#39;un laboratoire visite et de fournir des résultats descriptifs de notre échantillon de 140 origines ethniques diverses tout-petits en utilisant un équipement validé et logiciel de notation. Les résultats ont révélé que ce protocole peut produire une gamme de réponses physiologiques à la fois au repos et les conditions de développement difficiles, comme indiqué par les changements de la fréquence cardiaque et les indices de l&#39;activité parasympathique et sympathique. Les individus ont démontré la variabilité des niveaux de repos, responsabes aux défis, et le défi réactivité, ce qui fournit une preuve supplémentaire que ce protocole est utile pour l&#39;examen des différences individuelles SNA pour les tout-petits.

L&#39;étude SEED inscrit 162 dyades mère-enfant (37% d&#39;Afro-américains, 30% Latina, 16% blancs, 17% autres ou multiraciales). Pour la visite de 18 mois, nous avons terminé l&#39;évaluation en personne avec 140 enfants (87% de l&#39;échantillon inscrits); 6 participants se sont éloignés, et les autres participants étaient inaccessible ou indisponible pour cette visite. Le taux de refus des ANS composante de collecte de données de la visite était 3/140 (2%) pour les mères disponibles pour cette visite. Tro...

Watch this Scientific Journal Video about Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges at JoVE.com

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

Nous décrivons les méthodes de surveillance continue du système nerveux autonome sous repos et de provocation conditions avec 18 mois les enfants âgés. Les résultats ont révélé que ce protocole peut produire des réponses physiologiques significatives dans les deux branches du système nerveux autonome et de susciter la variabilité individuelle importante dans les modèles de réponses.

Mesure du système nerveux cardiaque Autonomic (ANS) Activité in Toddlers - Repos et développement Défis

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

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

Biopsychology

The Autonomic Nervous System

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15.2K vues.  University of California, San Francisco.  Nous décrivons les méthodes de surveillance continue du système nerveux autonome sous repos et de provocation conditions avec 18 mois les enfants âgés. Les résultats ont révélé que ce protocole peut produire des réponses physiologiques significatives dans les deux branches du système nerveux autonome et de susciter la variabilité individuelle importante dans les modèles de réponses. 

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

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.

Des tests quantitatifs Autonomic

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

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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>).

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.

Système de mesure (ANS) Activité Nervous Autonomic Cardiac in Children

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

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusions de l&#39;artère carotide pour pharmacocinétiques et pharmacodynamiques Analyse des taxanes chez la souris

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

Mesures électrophysiologiques de l’activité cérébrale évoquée par nocives chez les nouveau-nés en utilisant une sonde à embout plat couplé à l’électroencéphalographie

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

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.

Un modèle de blessure de Drosophila In Vivo pour étudier la neurogenèse dans le périphérique et le système nerveux Central

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.

Impact des neurones intracardiaques sur l’électrophysiologie cardiaque et arythmogenèse dans un système de Langendorff Ex Vivo

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.

Un test Flow Cytometry pour mesurer le potentiel de Membrane Mitochondrial dans les Myocytes cardiaques après l’hypoxie/réoxygénation

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;

Méthode invasive semi-minimale pour induire l’infarctus du myocarde chez les rats et l’évaluation de la fonction cardiaque par un système cardiaque de travail isolé

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

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.

Évaluation in vitro de la reprogrammation cardiaque en mesurant le flux de calcium spécifique cardiaque avec un rapporteur GCaMP3

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

Évaluation de l’hypercoagulation sanguine du plasma riche en EV

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Pleins feux sur l’auteur : Déchiffrer les troubles de la coagulation chez les patients ayant subi un traumatisme crânien 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

Détermination de l’activité procoagulante de la vésicule extracellulaire (EV) à l’aide du temps de coagulation activé par EV (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...