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.

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The authors declare that they have no competing financial interests.

この研究は、18ヶ月齢の子どものサンプル内、ドメインの範囲からの応答を引き出すために設計された標準化された休息と挑戦のタスクが（、感覚、社会的、休憩を驚愕）ANS応答の範囲につながった、ことを明らかにしました。これらの安静時および課題条件で見つかった平均ANSの応答は、同様のタスク8,11,27,29を使用して、幼い子供たちとANSの応答性を測定している他のユーザーに?...

子どもの自律神経系の個体差（ANS）は、反応性物理的および精神的健康の問題12-16の開発と維持に重要な役割を果たしています。証拠の成長体は休息と反応性でANS対策には個人差が子供が内在化し、精神病理学4,9,17-19と身体の健康の7,20を外部の変動を予測することが明らかになりました。 ANSの機能のこれらの直接的または主効果に加えて、研究の蓄積体は子供たちの反応性が子どもの福祉と健康に関するそれらの影響を緩和するために、そのような有害事象や夫婦紛争などの文脈的危険因子と相互作用することができることを見出している（参照21を参照レビューのため）。子どもの自律神経反応性があり、ライフコース全体の健康における役割、およびそれらのプロセスにおける伴う関心を果たしていることが、この成長している証拠にもかかわらず、3歳未満の子供にANSの機能の開発を検討する追加の研究のために必要です。 ANSは、二つのブランチ、副交感神経と交感神経のシステムで構成されています。それは、日常と有害事象1に応答して、大部分は無意識のメカニズムによって、内部器官（ 例えば、心拍数、呼吸、消化、および性的覚醒）の機能を調節します。副交感神経系（PNS）」の休息と修復」交感神経系を寝たりリラックスしたときに低安静時心拍数と修復の状態を維持するシステム（SNS）は「戦いまたは飛行」緊急事態に応答するシステムまたは脅迫的です自分の心拍数23を加速することによって状況。呼吸性洞性不整脈（RSA）は、心臓の機能上のPNSの影響の信頼できる指標であり、前出期間（PEP）は、車上のSNSの影響の信頼できる指標でありますダイアック機能。 RSAおよびPEPの両方が大人24,25のサンプルにおける実験薬理学的遮断を介して有効な対策であることが判明しています。最近では、この作業は、さらなる発展8,26,27を通じてANS活動の有効性と信頼性対策としてRSAおよびPEPを確立し、子供や思春期のサンプルに拡張されました。バンドやスポットの電極を使用した信頼性の高いインピーダンスカルジオグラフィの測定値（PEP）は、成人のサンプル28にかなりの時間のために使用されています。幼児におけるPEP対策のコレクションはまれですが、最近では、並列測定および分析技術はまた、子どもたち11,29,30のサンプルで信頼性が高く、有効であることが示されています。 迷走神経緊張と呼吸性洞性不整脈の措置を含む幼児や子供で機能PNS、に関する文献の現存大きな体が存在します。同時に両方を調べる少ないPEPを使用してSNSの研究、および非常に少数があります。その高度に統合された機能の理解を構築するために重要である副交感神経と交感神経系、。幼児のPEPの少数の研究のうち、ほとんどは彼らのプロトコルが課題8,31に応じてPEPの有意なグループレベルの変化を生じないことがわかります。研究は、そこに安定性が幼児期に始まるPNS対策を休んでいると、彼らは気質、行動、および健康との関係を持って、13,16,26,32,33が、時間をかけて応答し、その安定性SNSの限られた理解があることが実証されています開発との関係、およびその発達軌道ことを形作る要因。 PNS応答は社会エンゲージメント16とSNS応答に主に起因している1と同様に報酬感度（レビューのために34を参照する参照）」を戦うまたは飛行応答」を反映することを提案しました。幼児では、laboratoで具体的な課題をシミュレートするために挑戦されていますRY及びそれらの短い注意スパンにそれらを従事します。同時にPNSとSNSを測定するために必要な7スポット電極と不快感を経験することが幼児のための測定の課題もあります。また、発達科学の分野内で、そこにも子どもたちとの作業に子どもの発達ニーズと倫理を尊重しつつ、十分に「ストレス反応」を引き出す方法についていくつかの規格。この記事では、18ヶ月の子供から副交感神経と交感神経系の応答を誘発するように設計された休息と厳しい状況で構成される標準化されたプロトコルの管理およびスコアリングに関する情報を提示します。 ANSの反応性は、典型的には、個々の生物22にわたって変化する比較または休止状態への個別の外部刺激の相対的に生理的反応として概念化されています。精神的、肉体的に彼の初期の生活の病因の検討ALTHの問題は、適応応答を呼び起こすかのようなストレスの課題と考えることができる状況に子どもたちの反応を理解する上で特に興味があることが科学者をリードしてきました。挑戦的な刺激に遭遇すると、ドメインの広い範囲にわたって応答を誘発する可能性を秘めているが、文学の圧倒的多数は、「ストレス反応」として、この現象をいいます。期待/：このように、プロトコルは、以下に説明するが、2（なだめるような子守唄を聴きながら落ち着いを見ながら、ニュートラルビデオ）残りのフォームと18ヶ月児のための3つの発達に応じた課題を含む複数のドメイン間でのANS応答を誘発するように設計されました驚愕（イン・ザ・ボックスジャック）、感覚（レモン汁）、および社会情緒的（病気の幼児の叫びを聞いて）。プロトコルは、それは、発生的に挑戦幼児のために従事し、許容させるために、12ヶ月児8と3-5歳児11のための当社の既存のプロトコルから適合させた（18-21ヶ月）。 ここでは、ストレス、食事や早期開発（SEED）妊婦の研究とその子孫の18ヶ月の研究訪問から発達課題プロトコル（DCP）とANSデータを提示します。健康な妊娠を持つ母親の参加者は、人種や民族的に多様な妊娠の体重増加と出生前ストレスの研究のために2013から14の間に募集し、太りすぎ、低所得であった、としました。子孫が2014年から2015年の間に、年齢の18から21ヶ月だったとき、彼女の子孫の研究のための保護者のインフォームドコンセントは、ここで報告された研究のためだけに出産した後に得られると、再び以前のデータ収集の開始にしました。

<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

自律神経系（ANS）は二つの分岐、副交感神経と交感神経のシステムで構成され、内部器官（ 例えば、心拍数、呼吸、消化）の機能を制御し、日常と有害事象1に応答します。子供のANS対策が問題行動、感情の調節、および健康2-7に関連することが見出されています。したがって、幼児期の間にANSの発達に影響を与える要因を理解することが重要です。 ANSの両方のブランチは、刺激に対する幼児の心血管応答に影響を与え、生理学的変化8-11の有効かつ信頼性の尺度を使用して、外部の監視装置を経由して、非侵襲的に測定されています。しかし、2つのシステムの統合機能の理解を制限する副交感神経と交感神経系の同時措置、と非常に若い子供たちのいくつかの研究があります。また、マユォリ幼児の既存研究のtyが、一般的に母子相互作用のパラダイムを使用すると、より少ないが、他の厳しい状況にANS反応性について知られている乳児の静止ANS措置またはそれらの反応性について報告します。私たちは、18ヶ月の子供の心臓の自律制御の非侵襲的かつ迅速な評価のための研究デザインと標準化されたプロトコルを提示します。私たちは、自宅や研究室訪問中に休息とチャレンジ条件下でANSの副交感神経と交感神経の枝を連続的に監視するための方法を説明し、検証済みの機器やスコアリングソフトウェアを使用して140民族的に多様な幼児の私達のサンプルから記述的な知見を提供しています。結果は、心拍数の変化や副交感神経と交感神経活動の指標によって示されるように、このプロトコルは、休息と発達厳しい状況の両方に対する生理学的反応の範囲を生成することができることを明らかにしました。個人はRESPONS、安静時のレベルの変動を示しました課題に対するエス、と挑戦反応、このプロトコルは、幼児のためのANS個人差の検討に有用であることが、追加の証拠を提供します。

SEEDの研究では、162母子ダイアド（; 30％ラティーナ; 16％白; 17％、他のまたは多民族の37％アフリカ系アメリカ人）を登録しました。 18月の訪問のために、私たちは140人の子供（在籍したサンプルの87％）で、人の評価を完了しました。 6参加者が離れて移動し、残りの参加者が到達不能またはこの訪​​問のため利用できませんでした。訪問のANSデータ収集コンポーネントの拒否率は、この?...

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

私たちは、18ヶ月の子供たちと一緒に休息と挑戦の条件で自律神経系を連続的に監視するための方法を説明します。結果は、このプロトコルは、自律神経系の両方の分岐に意味のある生理学的応答を生成し、応答のパターンに有意な個体差を誘発することができることを明らかにしました。

幼児に心臓自律神経系（ANS）活性の測定 - 休憩と発達課題

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

measuring-cardiac-autonomic-nervous-system-ans-activity-toddlers

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Unit 1

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

Biopsychology

The Autonomic Nervous System

SCIENTISTS IN ACTION

15.2K ビュー.  University of California, San Francisco. 私たちは、18ヶ月の子供たちと一緒に休息と挑戦の条件で自律神経系を連続的に監視するための方法を説明します。結果は、このプロトコルは、自律神経系の両方の分岐に意味のある生理学的応答を生成し、応答のパターンに有意な個体差を誘発することができることを明らかにしました。 

ビデオ: 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.

定量的な自律神経系検査

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

医学

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.

小児の測定心臓自律神経系（ANS）活動

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.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

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.

新生児脳波に結合されたフラット先端プローブを用いた有害誘発脳活動の電気生理学的測定

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.

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

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.

心臓電気生理学、不整脈原性Ex Vivo蛍光システムで心臓内のニューロンの影響

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.

低酸素/再酸素化後心筋細胞におけるミトコンドリア膜電位を測定するためのフロー フローサイトメトリーを用いたアッセイ

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;

ラットの心筋梗塞を誘導する半最小侵襲法と、単離した働く心臓システムによる心機能の評価

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.

GCaMP3レポーターによる心臓特異的カルシウムフラックス測定による心臓リプログラミングのインビトロ評価

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

EV リッチ血漿の血液凝固亢進の評価

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.

著者スポットライト:外傷性脳損傷患者における凝固障害の解読 

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.

EV活性化凝固時間(EV-ACT)を用いた細胞外小胞(EV)の凝固促進活性の決定

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