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.

이 연구는 도메인 (, 감각, 사회, 휴식을 놀라게)의 범위에서 반응을 유도하도록 설계 18개월 된 어린이, 표준화 된 휴식과 도전 과제의 샘플에서 ANS 응답의 범위로 이끄는 것으로 나타났습니다. 이러한 휴식과 도전 조건에서 발견 된 평균 ANS 응답은 유사한 작업 8,11,27,29를 사용하여 어린 자녀와 함께 ANS 응답 속도를 측정 한 다른 사람에 의해보고 된 ANS 값 유사했다. 우리의 샘플에서 발견 ?...

어린이 자율 신경계 개인차 (ANS) 반응성 신체적 정신적 건강 문제 12-16의 개발과 유지에 중요한 역할을한다. 증거의 성장 몸은 휴식과 반응성에서 ANS 조치 개인차가 어린이가 내면화 및 정신 병리학 4,9,17-19 및 신체 건강에 7,20을 외부화의 변화의 예측 있음을 알 수있다. ANS의 기능의이 직접 또는 주 효과뿐만 아니라, 연구의 축적 몸이 아이들의 반응은 아이들의 복지와 건강에 그 영향을 완화하는 부작용 또는 배우자의 충돌 등의 상황에 맞는 위험 인자와 상호 작용할 수 있음을 발견 하였다 (참조 참조 (21) 검토). 아이들의 자율 반응이, 삶의 과정에서 건강에 중요한 역할, 그리고 그 과정에서 동반 관심을한다는 것을이 성장하는 증거에도 불구하고3 세 미만의 어린이 ANS의 기능의 개발을 검사하는 추가 연구가 필요합니다. ANS는 두 가지, 부교감 및 교감 신경계로 구성되어 있습니다. 또한 일상 및 이상 반응 1에 응답하여 무의식적 메커니즘을 통해 내부 기관 (예, 심장 박동, 호흡, 소화, 성적 흥분)의 기능을 조절한다. 부교감 신경계 (PNS)는 수면 또는 교감 신경계 (SNS)를 휴식 할 때 낮은 휴식 심박수와 회복 상태를 유지 &#39;휴식과 회복&#39;시스템 비상 사태 또는 위협에 응답하는 &#39;싸움이나 비행&#39;시스템입니다 하나의 심장 박동 (23)을 가속화하여 상황. 호흡기 동 부정맥 (RSA)는 자동차의 SNS 영향력의 안정적인 인덱스 신뢰할 수있는 심장 기능에 PNS의 영향 인덱스 및 예비 토출 기간 (PEP)입니다DIAC가 작동. RSA와 PEP 모두 성인 (24, 25)의 샘플 실험 약리학 봉쇄를 통해 유효한 조치 것으로 밝혀졌다. 최근이 작품은 발전 8,26,27에 걸쳐 ANS 활동의 유효하고 신뢰할 수있는 방법으로 RSA와 PEP를 구축, 아동 및 청소년 샘플로 확장되었습니다. 밴드 스폿 전극을 사용하여 안정적인 임피던스 심전도 측정 (PEP)은 성인의 샘플 (28) 상당 기간 동안 사용되어왔다. 최근 병렬 측정 및 분석 기술은 또한 어린 아이들의 PEP 조치의 컬렉션 드문 있지만, 신뢰성과 어린이 11,29,30의 샘플에서 유효한 것으로 밝혀졌다. 유아와 미주 톤과 호흡 공동 부정맥 조치를 포함하여 어린이에서 작동 PNS에 문학의 현존 큰 몸이있다. 거기 PE​​P를 사용 SNS 적은 연구하고, 거의 동시에 검사 두자신의 고도로 통합 된 기능의 이해를 구축하기위한 중요한 부교감 및 교감 신경계. 어린 아이들의 PEP의 몇몇 연구 중 대부분은 자신의 프로토콜이 문제 8,31에 대한 응답으로 PEP에 상당한 그룹 수준의 변화를 생성하지 않는 것을 발견. , 연구는 안정성이 초기 단계에 시작 PNS 조치를 휴식에 있다는 것을 증명하고있다 그리고 그들은 기질, 행동, 건강, 13,16,26,32,33과의 관계를 가지고 있지만 그 안정성은 시간이 지남에 따라이 응답 SNS의 ​​제한된 이해하고 개발 관계, 그 발달 궤적 것을 형성 요인. PNS 응답은 사회 참여 (16)에 주로 기인하고 SNS 응답은 (검토를 위해 34 참조 참조) 반영 일뿐만 아니라 보상 감도 &quot;응답 싸움이나 비행&quot;을 제안했다. 어린 아이에서는 laborato 특정 문제를 시뮬레이션하는 도전공예와 그들의 짧은주의 집중 기간에 참여하기. 동시에 PNS와 SNS를 측정하는 데 필요한 일곱 자리 전극과 불편 함이 발생할 수 유아에 대한 측정 문제도 있습니다. 또한, 발달 과학의 분야에서,이 또한 아이들과 함께 작업 아동의 발달 적 요구와 윤리를 존중하면서 충분히 &quot;스트레스 반응&quot;을 유도하는 방법에 대한 몇 가지 기준. 이 문서 18 개월 된 어린이의 부교감 및 교감 신경계 반응을 유도하도록 설계 휴식과 도전적인 조건들로 구성된 표준화 된 프로토콜의 관리 및 채점에 대한 정보를 제공합니다. ANS 반응성는 일반적으로 개별 생물체 (22)에 걸쳐 변화 비교하거나 휴식 상태로 분리 된 외부 자극에 대하여 생리 반응으로 개념화된다. 심신의 초기 삶의 원인의 시험 그는alth 문제가 응답 적응을 연상하거나 스트레스 도전으로 간주 될 수있는 상황에 아이들의 반응성을 이해하는데 특히 관심이 과학자를 주도하고있다. 도전적인 자극을 발생하는 도메인의 넓은 범위에 걸쳐 반응을 유도 할 수있는 잠재력을 가지고 있지만 문학의 우세는 &quot;스트레스 반응성&quot;로이 현상을 말한다. 따라서, 프로토콜은 아래의 나머지 두 가지 형태를 포함한 여러 도메인에서 ANS 반응을 유도하도록 설계되었습니다 기술 된 18 개월짜리 세 발달에 적합한 과제 (부드러운 자장가 듣고 진정, 중립 비디오를 보면서 동안) : 기대 / 놀라게 (잭 - 더 - 박스에서) 감각 (레몬 주스), 및 (아픈 유아의 외침을 듣고) socioemotional. 프로토콜은, 발달-도전 유아에 대한 참여 및 허용하게하기 위해, 12 개월 어린이 (8) 3-5 세 (11)에 대한 기존의 프로토콜에서 적응했다 (18-21 개월). 여기에 우리가 제시 발달 과제 프로토콜 (DCP) 및 ANS 스트레스의 18 개월 된 연구 방문의 데이터, 식사 및 임신 여성과 그들의 자손의 조기 개발 (SEED) 연구. 건강한 임신과 산모 참가자들은 인종과 다​​양한 인종 임신 체중 증가와 태아 스트레스의 연구 2013-14 동안 모집 및 과체중, 저소득이었다 하였다. 그녀의 자손의 연구를위한 부모의 동의는 출생 후 자손이 시대의 18-21개월이 2,014에서 2,015 사이되었음을 여기에보고 된 연구에 대한 데이터 수집의 시작 다시 이전 얻었다.

<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

자율 신경계 (ANS)는 두 가지, 교감 및 부교감 신경계 구성 및 내부 기관 (예, 심장 박동, 호흡, 소화)의 기능을 제어하고, 매일 및 이상 반응 (1)에 응답한다. 어린이 ANS 대책 행동 장애, 감정 조절, 및 건강 2-7에 관련된 것으로 밝혀졌다. 따라서, 유아 동안 ANS 개발에 영향을 미치는 요인을 이해하는 것이 중요합니다. ANS의 두 가지 자극에 어린 아이의 심장 혈관 반응에 영향을 미치는 생리적 변화 8-11의 유효하고 신뢰할 수있는 조치를 사용하여, 외부 모니터링 장비를 통해 비 침습적으로 측정되었다. 그러나 두 시스템의 통합 기능의 이해를 제한하는 부교감 및 교감 신경계의 동시 측정, 아주 어린 아이들의 몇몇 연구가있다. 또한, majori어린이의 연구는 유아 &#39;휴식 ANS 조치 또는 반응성보고 기존의 타이는 일반적으로 어머니 - 자녀 상호 작용 패러다임을 사용하고, 이하 다른 도전 조건에 ANS 반응성에 대한 알려져있다. 우리 18 개월 된 소아 심장 자율 제어의 비 침습적과 신속한 평가를위한 연구 설계와 표준화 된 프로토콜을 제시한다. 우리는 가정이나 실험실 방문하는 동안 휴식과 도전 조건에서 ANS의 부교감 신경과 교감 신경 가지를 지속적으로 모니터링하는 방법을 설명하고 검증 장비 및 채점 소프트웨어를 사용하여 140 다양한 인종 유아의 샘플에서 설명하는 결과를 제공합니다. 결과는 심장 박동의 변화 및 ​​부교감 신경과 교감 신경 활동의 지표로 나타낸 바와 같이이 프로토콜은 휴식과 발달 어려운 조건 모두에 생리적 반응의 범위를 생산할 수있는 것으로 나타났습니다. 개인은 휴식 수준, 응 답에 변화를 보여도전에 말이지,이 프로토콜은 유아를위한 ANS 개인차의 시험에 유용있는 추가 증거를 제공 도전 반응성.

SEED 연구는 162 어머니 - 자식 댓구 (; 30 % 라티 나, 16 % 흰색, 17 % 기타 또는 다민족 37 % 아프리카 계 미국인)를 등록. 18 월 방문을 위해, 우리는 140 명 (등록 된 샘플의 87 %)와 대면 평가를 완료; 6 참가자들은 멀리 이동하고, 나머지 참가자는 도달 할 수없는 또는이 방문 사용할 수 있었다. 방문 ANS 데이터 수집 구성 요소에 대한 거부 비율이 방문 할 수 어머니에 대한 1백40분의 3 (2 %)이었다. 세 방문 대?...

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

우리 18 개월 된 아이들과 함께 휴식과 도전 조건에서 자율 신경계의 지속적인 모니터링을위한 방법을 설명합니다. 결과는이 프로토콜은 자율 신경계의 두 가지 의미있는 생리적 반응을 생산 및 응답의 패턴에 상당한 개별 다양성을 유도 할 수 있습니다 것으로 나타났습니다.

유아에서 심장 자율 신경계 (ANS) 활동을 측정 - 휴식 및 발달 과제

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 조회수.  University of California, San Francisco.  우리 18 개월 된 아이들과 함께 휴식과 도전 조건에서 자율 신경계의 지속적인 모니터링을위한 방법을 설명합니다. 결과는이 프로토콜은 자율 신경계의 두 가지 의미있는 생리적 반응을 생산 및 응답의 패턴에 상당한 개별 다양성을 유도 할 수 있습니다 것으로 나타났습니다. 

비디오: 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.

양적 자율 테스트

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

연구

의학

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.

아이들의 측정 심장 자율 신경계 (ANS) 활동

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.

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.

Electroencephalography 결합 플랫 팁 프로브를 사용 하 여 신생아에 갖는 유해 두뇌 활동의 electrophysiological 측정

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.

주변에 Neuroregeneration 중앙 신 경계를 연구 하 고 대 한 Vivo에서 초파리 부상 모델

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.

심장 전기 생리학 및 전 비보 Langendorff 시스템에서 Arrhythmogenesis Intracardiac 뉴런의 영향

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.

Hypoxia/Reoxygenation 후 심장 Myocytes에서 미토 콘 드리 아 막 잠재력을 측정 하기 위한 교류 Cytometry 기반 분석

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;

쥐의 심근 경색과 격리 된 작업 심장 시스템에 의한 심장 기능 평가를 유도하는 반 최소 침습 적 방법

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 리포터와 심장 특정 칼슘 플럭스를 측정하여 심장 재프로그래밍의 체외 평가

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

EV-Rich Plasma의 혈액 과응고 평가

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.

저자 스포트라이트: 외상성 뇌 손상 환자의 응고 장애 해독 

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-ACT(Activated Clotting Time)를 사용한 세포외 소포(EV)의 응고 활성 측정

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