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.

Questo studio ha rivelato che, all&#39;interno di un campione di 18 mesi di età i bambini, di riposo standardizzate e attività sfida progettati per ottenere risposte da una serie di domini (sussultare, sensoriale, sociale, di riposo) ha portato ad una serie di risposte ANS. Gli ANS medi risposte trovati in queste condizioni di riposo e di sfida erano simili ai valori ANS riportati da altri che hanno misurato ANS reattività con bambini che usano compiti simili 8,11,27,29. I livelli medi di misure ANS trovat...

Le differenze individuali nel sistema nervoso autonomo per bambini (ANS) reattività svolgono un ruolo importante nello sviluppo e nella manutenzione di problemi di salute fisica e mentale 12-16. Un crescente corpo di evidenze rivela che le differenze individuali nelle misure ANS a riposo e reattività sono predittivi di variazioni nei bambini di internalizzazione e di esternalizzazione psicopatologia 4,9,17-19 e 7,20 salute fisica. Oltre a questi effetti diretti o principali di ANS funzionamento, un organismo di accumulo di studi hanno trovato che la reattività dei bambini può interagire con i fattori di rischio contestuali, come ad esempio eventi avversi o conflitto coniugale, per moderare gli effetti sul benessere dei bambini e la salute (vedi riferimento 21 per una rassegna). Nonostante questo crescente evidenza che la reattività autonomica dei bambini gioca un ruolo nella salute in tutto il corso della vita, e l&#39;interesse che accompagna in questi processi, ciè la necessità di ulteriori ricerche esamina l&#39;andamento delle ANS funzionamento nei bambini sotto i tre anni di età. L&#39;ANS è costituito da due rami, il sistema nervoso simpatico e parasimpatico. Si regola la funzione degli organi interni (ad esempio, la frequenza cardiaca, la respirazione, la digestione e l&#39;eccitazione sessuale), in gran parte attraverso meccanismi inconsci, in risposta a tutti i giorni e negative esperienze 1. Il sistema nervoso parasimpatico (SNP) è la &#39;di riposo e ristoratore&#39; il sistema che mantiene un basso tasso cardiaca a riposo e lo stato rigenerante quando dorme o rilassare il sistema nervoso simpatico (SNS) è il sistema di &#39;lotta o fuga&#39; che risponde alle emergenze o minacciare situazioni di accelerare la frequenza cardiaca 23. Respiratorio aritmia sinusale (RSA) è un indice affidabile dell&#39;influenza PNS sul funzionamento cardiaco, e il periodo di pre-espulsione (PEP) è un indice affidabile dell&#39;influenza SNS sulla vetturaDiac funzionante. Sia RSA e PEP sono stati trovati per essere misure valide attraverso sperimentale blocco farmacologico in campioni di adulti 24,25. Più di recente, questo lavoro è stato esteso in campioni di bambini e adolescenti, stabilire ulteriori RSA e PEP come misure valide e affidabili di attività ANS durante lo sviluppo 8,26,27. Misure di impedenza cardiography affidabili (PEP) utilizzando elettrodi band e Spot sono stati utilizzati per un tempo considerevole in campioni adulti 28. Più di recente, le tecniche di misurazione e di analisi parallele hanno anche dimostrato di essere affidabile e valida in campioni di bambini 11,29,30, anche se la raccolta delle misure PEP nei bambini è rara. Vi è un grande corpo esistente di letteratura sulla PNS funzionanti nei neonati e nei bambini piccoli, tra cui il tono vagale e le misure del seno aritmia respiratoria. Ci sono meno studi del SNS utilizzando PEP, e pochissimi contemporaneamente esaminando sia lasistema nervoso simpatico e parasimpatico, che è fondamentale per la costruzione di comprensione del loro funzionamento altamente integrato. Tra i pochi studi di Pep dei bambini piccoli, la maggior parte trova che i loro protocolli non producono significativi cambiamenti a livello di gruppo in PEP in risposta alle sfide 8,31. Gli studi hanno dimostrato che non vi è la stabilità in misure PNS a partire nell&#39;infanzia riposo e hanno rapporti con il temperamento, il comportamento e la salute, 13,16,26,32,33 ma c&#39;è una comprensione limitata di SNS rispondere e la sua stabilità nel tempo, relazioni per lo sviluppo, ei fattori che determinano che la sua traiettoria di sviluppo. Risposte PNS sono stati attribuiti principalmente per l&#39;impegno sociale 16 e le risposte SNS proposto di riflettere &quot;lotta o fuga risposte&quot; 1 così come la sensibilità ricompensa (vedi riferimento 34 per una rassegna). Nei bambini piccoli, è difficile simulare le sfide specifiche in un laborary e per coinvolgerli a causa della loro capacità di attenzione brevi. Ci sono anche le sfide di misura per i più piccoli che possono sperimentare il disagio con i sette elettrodi in loco necessari per misurare PNS e SNS contemporaneamente. Inoltre, all&#39;interno dei campi della scienza dello sviluppo, ci pochi standard su come suscitare &quot;stress reattività&quot; sufficientemente rispettando anche le esigenze di sviluppo dei bambini e l&#39;etica nel lavoro con i bambini. Questo articolo presenta le informazioni sulla gestione e il punteggio di un protocollo standardizzato composto da condizioni di riposo e impegnativi progettati per suscitare le risposte del sistema nervoso simpatico e parasimpatico da 18 mesi di età i bambini. ANS reattività è tipicamente concepito come la risposta fisiologica ad una discreta esterna stimolo rispetto ad un confronto o stato di riposo, che varia tra i singoli organismi 22. L&#39;esame dei primi eziologia vita mentale e fisica cheproblemi Alth ha portato gli scienziati a essere particolarmente interessato a comprendere la reattività dei bambini a situazioni che evocano adattivo rispondere o possono essere pensati come sfide stressanti. La preponderanza della letteratura si riferisce a questo fenomeno come &quot;reattività allo stress&quot;, anche se incontrando stimoli difficili ha il potenziale per ottenere risposte in una vasta gamma di settori. Così, il protocollo descritto di seguito è stato progettato per ottenere risposte ANS in più domini, tra cui due forme di riposo (durante l&#39;ascolto di una ninna nanna rilassante e durante la visione di una calma, il video neutro) e tre sfide evolutivamente-appropriati per 18 mesi di età: anticipazione / spaventare (jack-in-the-box), sensoriali (succo di limone), e socioemotional (l&#39;ascolto di un grido infantile malato). Il protocollo è stato adattato dai nostri protocolli esistenti per 12 mesi di età 8 e 3-5 anni 11, al fine di renderlo evolutivamente impegnativo, coinvolgente e tollerabile per i più piccoli (18-21 Mesi). Ecco a voi la Developmental sfide Protocol (DCP) e ANS dati del bambino di 18 mesi visita di studio dello stress, Mangiare e presto lo sviluppo (SEED) Studio di donne in gravidanza e la loro prole. partecipanti materno con gravidanze sane sono stati reclutati nel corso 2013-14 per uno studio di aumento di peso gestazionale e lo stress prenatale e erano in sovrappeso, basso reddito, e razzialmente ed etnicamente diversificata. il consenso informato dei genitori per lo studio della sua prole è stata ottenuta solo dopo la nascita e di nuovo prima dell&#39;inizio della raccolta dei dati per lo studio riportato qui, quando la prole erano 18-21 mesi di età durante il 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

Il sistema nervoso autonomo (ANS) è costituito da due rami, il sistema nervoso simpatico e parasimpatico, e controlla la funzione degli organi interni (ad esempio, la frequenza cardiaca, la respirazione, la digestione) e risponde a tutti i giorni e le esperienze avverse 1. Misure ANS nei bambini sono stati trovati per essere correlato a problemi di comportamento, regolazione delle emozioni, e la salute 2-7. Pertanto, la comprensione dei fattori che influenzano lo sviluppo ANS durante la prima infanzia è importante. Entrambi i rami degli ANS influenzano risposte cardiovascolari dei bambini piccoli agli stimoli e sono stati misurati in maniera non invasiva, tramite apparecchiature di monitoraggio esterno, utilizzando misure valide e affidabili del cambiamento fisiologico 8-11. Tuttavia, vi sono pochi studi su bambini molto piccoli con misure simultanee del sistema nervoso simpatico e parasimpatico, che limita la comprensione del funzionamento integrato dei due sistemi. Inoltre, il majority degli attuali studi di bambini riferiscono sulle misure di riposo ANS infanti o la loro reattività al usata comunemente paradigmi di interazione madre-bambino, e meno si sa circa ANS reattività ad altre condizioni difficili. Presentiamo un progetto di studio e protocollo standardizzato per la valutazione non invasiva e rapida del controllo autonomico cardiaco a 18 mesi di età i bambini. Descriviamo i metodi per il monitoraggio continuo dei rami simpatico e parasimpatico degli ANS in condizioni di riposo e di sfida nel corso di una visita a domicilio o di laboratorio e di fornire risultati descrittivi dal nostro campione di 140 bambini etnicamente diversi che utilizzano attrezzature convalidato e software di punteggio. I risultati hanno rivelato che questo protocollo in grado di produrre una serie di risposte fisiologiche sia per il riposo e le condizioni evolutivamente impegnative, come indicato da cambiamenti nella frequenza cardiaca e gli indici di attività simpatico e parasimpatico. Gli individui hanno dimostrato variabilità riposo livelli, responses alle sfide e reattività sfida, che fornisce ulteriori prove che questo protocollo è utile per l&#39;esame delle differenze individuali ANS per i più piccoli.

Lo studio ha arruolato 162 SEED diadi madre-bambino (37% afro americani, il 30% Latina, 16% bianchi, 17% altri o multirazziale). Per la visita 18 mesi, abbiamo completato la valutazione di persona con 140 bambini (87% del campione iscritto); 6 partecipanti si allontanarono, e le restanti partecipanti erano irraggiungibili o non disponibili per questa visita. Il tasso di rifiuto per ANS componente la raccolta dei dati della visita è stato 3/140 (2%) per le madri disponibili per questa visita. Tre visite sono state effet...

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

Descriviamo i metodi per il monitoraggio continuo del sistema nervoso autonomo sotto di riposo e di provocazione condizioni con 18 mesi di età i bambini. I risultati hanno rivelato che questo protocollo può produrre risposte fisiologiche significative in entrambi i rami del sistema nervoso autonomo e suscitare notevole variabilità individuale nei modelli di risposte.

Misurazione cardiaca Sistema Nervoso Autonomo (ANS) Attività in Toddlers - Riposo e di sviluppo sfide

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 Views.  University of California, San Francisco.  Descriviamo i metodi per il monitoraggio continuo del sistema nervoso autonomo sotto di riposo e di provocazione condizioni con 18 mesi di età i bambini. I risultati hanno rivelato che questo protocollo può produrre risposte fisiologiche significative in entrambi i rami del sistema nervoso autonomo e suscitare notevole variabilità individuale nei modelli di risposte. 

Video: 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.

Quantitativa test autonomici

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

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Medicina

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.

Misurazione cardiaca Sistema Nervoso Autonomo (SNA) Attività nei bambini

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.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

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.

Elettrofisiologici misurazione dell'attività cerebrale nocive-evocato in neonati usando una sonda di piatto-punta accoppiata per elettroencefalografia

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 modello di ferita Drosophila In Vivo per lo studio della neurorigenerazione la periferica e del sistema nervoso centrale

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.

Impatto dei neuroni intracardiaci elettrofisiologia cardiaca e aritmogenesi in un sistema di 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'analisi basata su citometria a flusso per misurare il potenziale di membrana mitocondriale in miociti cardiaci dopo ipossia/riossigenazione

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;

Metodo semi-minimo invasivo per indurre l'infarto miocardico nei ratti e la valutazione della funzione cardiaca da parte di un sistema cardiaco funzionante isolato

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.

Valutazione in vitro della riprogrammazione cardiaca mediante misurazione del flusso di calcio specifico cardiaco con un GCaMP3 Reporter

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

Valutazione dell'ipercoagulazione del sangue del plasma ricco di 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.

Autore in primo piano: Decifrare i disturbi della coagulazione nei pazienti con lesioni cerebrali traumatiche 

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.

Determinazione dell'attività procoagulante della vescicola extracellulare (EV) utilizzando il tempo di coagulazione attivato da EV (EV-ACT)

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