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.

המחקר גילה כי, בתוך מדגם של 18 ילדים בן חודש, מנוחה סטנדרטית ומשימות אתגר שנועדו לעורר תגובה ממגוון תחומים (להבהיל, חושית, חברתית, מנוחה) הביא מגוון של תגובות ANS. תגובות ANS הממוצע נמצאות בתנאים נחים ואתגר אלה היו דומות לערכי ANS שדווחו על ידי אנשים אחרים שיש להם נמדדים היענ...

הבדלים אינדיבידואליים מערכת העצבים האוטונומית של הילדים (ANS) תגובתיות לשחק תפקיד חשוב בפיתוח והתחזוקה של בעיות בריאות גופניות ונפשיות 12-16. גוף גדל והולך של ראיות מגלה כי הבדלים אינדיבידואליים אמצעי ANS במנוחת תגובתיות הם חזויים של וריאציות בילדים של ההפנמה והחצנת פסיכופתולוגיה 4,9,17-19 ו 7,20 בריאות גופנית. בנוסף להשפעות הישירות או עיקריים אלה של תפקוד ANS, גוף של מחקרים צבירת מצאו כי תגובתיות של ילדים יכולים לקיים אינטראקציה עם גורמי סיכון הקשר, כגון תופעות לוואי או סכסוך משפחתי, למתן השפעות אלו על רווחתם של ילדים ובריאות (ראה התייחסות 21 לסקירה). למרות ראיות הולכות ומצטברות זה כי תגובתיות האוטונומית של ילדים משחקת תפקיד חשוב בבריאות על פני מהלך החיים, לבין האינטרס המלווה בתהליכים אלה, ישצורך במחקר נוסף לבחינת התפתחות תפקוד ANS בילדים מתחת לגיל שלוש. ANS מורכב משני סניפים, מערכות העצבים הפאראסימפתטית ואוהד. זה מסדיר את תפקוד האיברים הפנימיים (למשל, קצב לב, נשימה, עיכול, ועוררות מיניות), בעיקר באמצעות מנגנונים מודעים, בתגובה לחוויות היומיום ושליליות 1. מערכת העצבים הפאראסימפתטית (PNS) היא &#39;המנוחה מבריאה&#39; מערכת מקיימת קצב לב במנוחה נמוכה ומדינה משקמת כאשר ישן או מרגיע מערכת העצבים הסימפתטית (SNS) היא המערכת &#39;הילחם או ברחו&#39; שמגיבה חירום או מאיים במצבים ידי האצת קצב הלב של אחד 23. הפרעות קצב סינוס נשימה (RSA) היא מדד אמין של שפעת PNS על תפקוד לב, ותקופה-פליטה מראש (PEP) הוא מדד אמין של שפעת SNS על מכוניתDIAC מתפקד. שניהם RSA ו PEP נמצאו להיות אמצעי בתוקף עד המצור תרופתי ניסיוני בדגימות של מבוגרים 24,25. לאחרונה, עבודה זו הוארכה לתוך דגימות ילד ונער, עוד הקמת RSA ו PEP כאמצעים תקפים ומהימנים של פעילות ANS ברחבי פיתוח 8,26,27. העכבה אמינה מדידות רִשׁוּם הַלֵב (PEP) באמצעות אלקטרודות להקה במקום שמשו במשך זמן ניכר בדגימות מבוגרות 28. לאחרונה, טכניקות מדידה אנליטית במקביל גם הוכחו להיות מהימן ותקף בדגימות של ילדים 11,29,30, למרות אוסף של צעדים PEP אצל ילדים צעירים הוא נדיר. ישנו גוף גדול קיים של ספרות על PNS מתפקד אצל תינוקות וילדים צעירים, כולל טון מחנק ואמצעי הפרעות קצב סינוס נשימה. ישנם מחקרים פחות של SNS באמצעות PEP, ומעט מאוד לבחון בו זמנית הן אתמערכות עצבים הפאראסימפתטית ואוהד, שהוא קריטי לבניית הבנה של התפקוד המשולב ביותר שלהם. בודדי המחקרים של PEP ילדים צעירים, רובם מוצאים כי הפרוטוקולים שלהם אינם מייצרים שינויים ברמת קבוצה משמעותיים PEP בתגובה לאתגרי 8,31. מחקרים הוכיחו כי יש יציבות נח אמצעי PNS החל בגיל רך ויש להם קשרים עם טמפרמנט, התנהגות, ובריאות, 13,16,26,32,33 אבל יש הבנה מוגבלת של SNS להגיב ויציבות לאורך זמן, יחסים להתפתחות, ואת הגורמים מעצבים כי מסלול ההתפתחותיים שלו. תגובות PNS יוחסו בעיקר מעורבות חברתית 16 ותגובות SNS מוצעות כדי לשקף &quot;הילחמו או תגובות טיסה&quot; 1, כמו גם רגיש גמול (ראה התייחסות 34 לסקירה). בילדים צעירים, זה מאתגר כדי לדמות אתגרים ספציפיים בתוך laboratoר&quot;י ו להעסיק אותם בשל טווח הקשב הקצר שלהם. ישנם גם אתגרי מדידה לפעוטות אשר עלול להיתקל אי נוחות עם שבע האלקטרודות במקום צורך למדוד PNS ו SNS זמנית. בנוסף, בתוך תחומי המדע התפתחותית, יש כמה סטנדרטים על איך לעורר &quot;תגובתיות מתח&quot; מספיק זמן גם כיבוד הצרכים ההתפתחותיים של הילדים ואת האתיקה בעבודה עם ילדים. מאמר זה מציג מידע על הממשל ועל הניקוד של פרוטוקול סטנדרטי מורכב תנאים המנוחים ומאתגר שנועדו לעורר תגובות מערכת עצבים הפאראסימפתטית ואוהד מ -18 ילדים בן חודש. תגובתיות ANS נתפסים בדרך כלל כמו התגובה הפיזיולוגית לקרובו גירוי חיצוני דיסקרטית כדי השוואה או במצב מנוחה, אשר משתנה על פני אורגניזמים בודדים 22. בחינת האטיולוגיה חייו המוקדמת של נפשי וגופני הואבעיות alth הובילו מדענים להתעניייין במיוחד בהבנת תגובתיות ילדים למצבים מעוררים אדפטיבית להגיב או ניתן לחשוב על אתגרים מלחיצים כמו. העליונות של הספרות מתייחסת לתופעה זו כאל &quot;תגובתיות לחץ&quot;, למרות מפגש עם גירויים מאתגרים יש הפוטנציאל לעורר תגובות על פני מגוון רחב של תחומים. לפיכך, הפרוטוקול מתואר להלן נועד לעורר תגובות ANS בדומיינים המרובים כולל שתי צורות של מנוחה (תוך כדי האזנה לשיר ערש מרגיע בעת שצפו רגוע, וידאו ניטראלי) ושלושה אתגרים מבחינה התפתחותית-מתאימים בני 18 חודשים: ציפייה / להבהיל (שקע-in-the-box), חושי (מיץ לימון), ו socioemotional (האזנת זעקת תינוק חולה). פרוטוקול הותאם מתוך הפרוטוקולים הקיימים שלנו עבור 12 ילדים בני 8 חודשים ו 3-5 ילדים בגילאי 11, על מנת להפוך אותו מבחינה התפתחותית-מאתגר, מרתק נסבל לפעוטות (18-21 חודשים). כאן אנו מציגים את פרוטוקול אתגרים התפתחותיים (DCP) ו ANS נתונים מהביקור מחקר בן 18 חודשים של מתח, אכילה ופיתוח הקדומה (SEED) לחקר נשים בהריון וצאצאיהם. משתתפים אימהיים עם הריונות בריאים גויסו במהלך 2013-14 לצורכי מחקר על עלייה במשקל הריון ומתח לפני ואחרי סבלו מעודף משקל, הכנסה נמוכה, גזעי ומגוון מבחינה אתנית. הורים &#39;הסכמה מדעת לחקר צאצאיה הושגה רק לאחר לידה ושוב לפני תחילת איסוף הנתונים למחקר דיווח כאן, כאשר הצאצאים היו 18-21 חודשים של גיל במהלך 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

מערכת העצבים האוטונומית (ANS) מורכבת משני סניפים, מערכות העצבים הפאראסימפתטית ואוהד, ושולטת תפקוד האיברים הפנימיים (למשל, קצב לב, נשימה, עיכול) ומגיבה היומיום וחוויות שליליות 1. אמצעי ANS בילדים נמצאו להיות קשור לבעיות התנהגות, ויסות רגשות, ובריאות 2-7. לכן, הבנת הגורמים המשפיעים פיתוח ANS במהלך הילדות המוקדמת חשוב. שני הענפים של ANS להשפיע תגובות קרדיווסקולריות של ילדים צעירים לגירויים ו נמדדו פולשנית, באמצעות ציוד בקרה חיצונית, תוך שימוש באמצעים תקף ומהימן של שינוי פיזיולוגי 8-11. עם זאת, ישנם כמה מחקרים של ילדים צעירים מאוד עם אמצעים סימולטני של מערכת העצבים הפאראסימפתטית ואוהד, אשר מגביל את ההבנה של תפקוד משולב של שתי המערכות. בנוסף, Majority של קיימים מחקרים של ילדים צעירים מדווחים על צעדי ANS המנוחה &#39;תינוקות או תגובתיות שלהם נפוץ פרדיגמות אינטראקציה אם-ילד, ופחות ידוע על תגובתיות ANS לתנאים מאתגרים אחרים. אנו מציגים עיצוב לימוד פרוטוקול סטנדרטי עבור הערכה לא פולשנית ומהירה שליטה אוטונומית לב בילדים בן 18 חודש. אנו מתארים שיטות לניטור רציף של הסניפים הפאראסימפתטית ואוהד של ANS בתנאים המנוחים ואתגר במהלך ביקור בבית או במעבדה ולספק ממצאים תיאוריים מהמדגם שלנו של 140 פעוטות מגוונות מבחינה אתנית באמצעות ציוד תוקף ותוכנת ניקוד. התוצאות הראו כי פרוטוקול זה יכול לייצר מגוון של תגובות פיזיולוגיות הן מנוחה מבחינה התפתחותית בתנאים מאתגרים, כפי שצוין על ידי שינויים בקצב הלב ומדדי הפעילות הפאראסימפתטית ואוהד. אנשים הפגינו השתנות רמות נחות, responses לאתגרים, תגובתיות אתגר, אשר מספק ראיות נוספות כי פרוטוקול זה שימושי לבחינת הבדלים אישיים ANS לפעוטות.

מחקר SEED נרשם 162 צמדי אמא-ילד (37% אפרו-אמריקאי, 30% לטינה; 16% לבנים; 17% נוספים או גזעיים). עבור הביקור 18 חודש, השלמנו את הערכה פנים אל פנים עם 140 ילדים (87% מהמדגם רשום); 6 משתתפים התרחקו, והמשתתפים הנותרים לא היו נגישות או לא זמינים עבור הביקור הזה. שיעור הסירוב עבור רכיב איסוף נת...

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

אנו מתארים את שיטות לניטור רציף של מערכת העצבים האוטונומית בתנאים מנוחה אתגר עם 18 ילדים בני חודש. התוצאות הראו כי פרוטוקול זה יכול לייצר תגובות פיזיולוגיות משמעותיות בשני הענפים של מערכת העצבים האוטונומית ולעורר השתנות הפרט משמעותי בדפוסי התגובות.

מדידת מערכת העצבים האוטונומית לב (ANS) פעילות פעוטות - Resting ואתגרים התפתחותית

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.  אנו מתארים את שיטות לניטור רציף של מערכת העצבים האוטונומית בתנאים מנוחה אתגר עם 18 ילדים בני חודש. התוצאות הראו כי פרוטוקול זה יכול לייצר תגובות פיזיולוגיות משמעותיות בשני הענפים של מערכת העצבים האוטונומית ולעורר השתנות הפרט משמעותי בדפוס...

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.

בדיקה כמותית האוטונומית

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

Measuring Cardiac Autonomic Nervous System (ANS) Activity in 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.

מערכת מדידת פעילות לב עצבים אוטונומית (ANS) בילדים

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.

חליטות עורק הראשי לpharmacokinetic וpharmacodynamic ניתוח של taxanes בעכברים

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.

מדידות אלקטרופיזיולוגיות של פעילות המוח יתמודד עורר Neonates באמצעות בדיקה שטוח-טיפ מצמידים אלקטרואנצפלוגרם

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.

מודל פציעה דרוזופילה In Vivo ללמוד Neuroregeneration ב ההיקפית ואת מערכת העצבים המרכזית

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.

ההשפעה של נוירונים Intracardiac על הלב אלקטרופיזיולוגיה, Arrhythmogenesis במערכת Langendorff Vivo לשעבר

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.

וזמינותו מבוססי Cytometry זרימה מודדים פוטנציאל ממברנה מיטוכונדריאלי של תאי שריר הלב לאחר היפוקסיה/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 ...

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;

חצי מינימלי שיטה פולשנית כדי לגרום אוטם שריר הלב בחולדות הערכה של תפקוד הלב על ידי מערכת לב עבודה מבודדת

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.

הערכה במבחנה של תכנות לב מחדש על ידי מדידת שטף סידן ספציפי לב עם כתב GCaMP3

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

הערכת קרישת יתר בדם של פלזמה עשירה ברכב חשמלי

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.

מחבר זרקור: פענוח הפרעות קרישה בנפגעי מוח טראומטיים 

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.

קביעת פעילות הקרישה של שלפוחית חוץ-תאית (EV) באמצעות זמן קרישה המופעל על ידי EV (EV-ACT)

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