Name: impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system
Uploaded: 2018-05-22T14:00:00
Description: Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

Forfatterne vil gjerne takke Hartwig Wieboldt for hans utmerket kundestøtte og UKE mikroskopi Imaging anlegget (Umif) den University Medical Center Hamburg-Eppendorf for å gi mikroskop og støtte. Denne forskningen ble finansiert bythe Förderverein des Universitären Herzzentrums Hamburg e.V. og DZHK (tysk Centre for hjerte forskning) [FKZ 81Z4710141].

Alle prosedyrer som involverer dyr ble godkjent av lokale myndigheter i staten of Hamburg, Universitetet i Hamburg dyr omsorg og bruk komiteer.
1. forberedelse av Langendorff apparater
Merk: Et kommersielt tilgjengelig Langendorff perfusjon systemet brukes.
<ol><li>Forberede en modifisert Krebs-Henseleit løsning (119 mM av natriumklorid, 25 mM av natrium bikarbonat, 4.6 mM av kalium klorid, 1,2 mM kalium fosfat monobasic, 1.1 mM magnesium sulfat, 2,5 av kalsium k...

<ol>
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I dette manuskriptet er den velkjente Langendorff ex vivo hjertet perfusjon systemet presentert som et verktøy for å studere virkningen av intracardiac neurons cardiac electrophysiology og arrhythmogenesis ved hjelp av ulike kartlegging og stimulering teknikker inkludert endocardial og epicardial tilnærminger.
Flere deler av protokollen er avgjørende for oppsettet. Først er det viktig å bruke en forberedelse teknikk som atrial fett pads forblir intakt eller fjernes raskt uten sk...

Langendorff ex vivo hjertet perfusjon systemet fortsetter å være et relevant verktøy for å utføre et bredt spekter av fysiologiske, morfologiske og biokjemiske og farmakologiske studier i sentralt denervated hjerter1,2 ,3,4,5 siden sin oppfinnelse i slutten 19th århundre6. Hittil dette systemet er fortsatt mye brukt for ulike emner (f.eks., iskemi reperfusion) eller å studere cardiac farmakologiske effekter7,8, og er et grunnleggende verktøy i hjerte forskning. Levetiden til denne metoden resultater fra flere fordeler (f.eks., mål utføres uten påvirkning av det sentrale nervesystemet eller andre organer, systemisk sirkulasjon eller sirkulerende hormoner). Eventuelt legemidler kan legges på en kontrollert måte til perfusjon bufferen eller brukes til spesifikke strukturer direkte. Eksperimenter er reproduserbare, og et relativt høyt antall eksperimenter kan utføres i en kort periode. (Delvis) åpne natur oppsettet kan gjøre temperaturregulering vanskelig og må tas i betraktning. Selv om Langendorff systemet brukes også i større arter9, mindre dyr blir hovedsakelig brukt som eksperimentelle oppsettet er mindre kompleks, og en større biologisk variasjon (f.eks., transgene mouse modeller) kan brukes.
I eksperimentelle oppsett av denne protokollen er påvirker det intracardiac autonome nervesystemet grunnleggende elektrofysiologiske parametere, ventrikulær arrhythmogenesis, epicardial ledning og syklisk adenosin monofosfat (cAMP) dynamics evaluert. Et stort antall intracardiac Ganglion, som er hovedsakelig lokalisert i atrial fett pads og er nå kjent å kontrollere cardiac elektrofysiologi uavhengig av sentral nevrale kontroll, er enten venstre intakt eller manuelt fjernet med forsiktig mekaniske disseksjon. En farmakologisk modulering av det autonome nervesystemet utføres enten globalt ved å legge til legemidler perfusjon bufferen eller lokalt målrettede modulering av atrial Ganglion. Etter eksperimentene er hjerter godt egnet for å bedømme immunohistological som alle blod celler har blitt fjernet på grunn av kontinuerlig perfusjon, som kan øke kvaliteten på flekker.
Det overordnede målet med beskrevet teknikker er å tilby nye perspektiver for detaljerte studier om effekten av det autonome nervesystemet cardiac electrophysiology og arrhythmogenesis i musen hjertet. En grunn til å bruke denne teknikken er at det er mulig å studere og endre det autonome nervesystemet uten virkningen av sentralnervesystemet. En stor fordel er lett sysselsetting farmakologiske eksperimenter, i hvilke potensielle pro- eller antiarrhythmic egenskaper av gamle og nye agenter kan testes. I tillegg er transgene og knockout musen modeller av ulike hjerte sykdommer tilgjengelig å undersøke mekanismene bak arytmi, hjertesvikt eller metabolske sykdommer. Denne tilnærmingen har forbedret vår forståelse av hvordan det autonome nervesystemet på atrial nivå kan påvirke ventrikkel cardiac electrophysiology og induksjon av arytmier.

<table>
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sodium hydrogencarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>401676</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sodium pyruvate bioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Carbogen (95% O2 / 5% CO2)</td>
			<td colspan="2">SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sterile filter steritop-GP 0.22</td>
			<td>EMD Millipore</td>
			<td>SCGPT05RE</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Atropine sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A0257</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Hexamethonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>H2138</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Nicotine free base 98-100%</td>
			<td>Sigma-Aldrich</td>
			<td>N3876</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Formalin solution neutral buffered 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution 30% (w/w) in H2O</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>D8418</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline tablets</td>
			<td>Gibco / Invitrogen</td>
			<td>18912-014</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Triton-x-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Albumin bovine fraction V</td>
			<td>Biomol, Hamburg, Germany</td>
			<td>11924.03</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Chicken anti neurofilament</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Rabbit anti tyrosine hydroxylase</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Goat anti choline acetyltransferase</td>
			<td>EMD Millipore</td>
			<td>AP144P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; rabbit IgG Alexa 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; goat IgG Alexa 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11057</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; chicken IgY Alexa 647</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>AP194SA6</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated donkey &#945; rabbit igG</td>
			<td>R&#38;D Systems</td>
			<td>AP182B</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated donkey &#945; goat igG</td>
			<td>R&#38;D Systems</td>
			<td>AP192P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated goat &#945; chicken igY</td>
			<td>R&#38;D Systems</td>
			<td>BAD010</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>H-1000</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Vectastain ABC kit</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>PK-4000</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Steady DAB/Plus</td>
			<td>Abcam plc, Cambridge, UK</td>
			<td>ab103723</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>HistoClear</td>
			<td>DiaTec, Bamberg, Germany</td>
			<td>HS2002</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>BisBenzimide H33342 trihydrochloride (Hoechst)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B2261</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Vectashield HardSet mounting medium</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>VEC-H-1400</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Perfusion system</td>
			<td>HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH, March-Hugstetten, Germany</td>
			<td>&#160;73-4343</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr>
			<td>Data acquisition system and corresponding software for catheter and physiological parameter</td>
			<td>Powerlab 8/30 &#38; Labchart, ADInstruments, Dunedin, New Zealand</td>
			<td>PL3508 PowerLab 8/35</td>
			<td>Langendorff setup</td>
		</tr>
		<tr>
			<td>Octapolar catheter</td>
			<td>CIB&#8217;ER Mouse, NuMed Inc., Hopkinton, NY, USA</td>
			<td>custom</td>
			<td>Langendorff setup</td>
		</tr>
		<tr>
			<td>Stimulus generator</td>
			<td>STG4002, Multi Channel Systems, Reutlingen, Germany</td>
			<td>STG4002-160&#181;A</td>
			<td>Stimulation setup</td>
		</tr>
		<tr>
			<td>Stimulation software</td>
			<td>Multi Channel Systems, Reutlingen, Germany</td>
			<td>MC_Stimulus II</td>
			<td>Stimulation setup</td>
		</tr>
		<tr>
			<td>Data acquisition system and corresponding software for epicardial electrograms</td>
			<td>ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany</td>
			<td>USB-ME128-System</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Multi-electrode array</td>
			<td>MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany</td>
			<td>EcoFlexMEA36</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Multi-electrode array recording software</td>
			<td>Multi Channel Systems, Reutlingen, Germany</td>
			<td>MC_Rack</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>15003-08</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14575-09</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14110-15</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Dumont SS Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11203-25</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>London Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11080-02</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11003-13</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>Parafilm M, Bemis NA, based in Neenah, WI, United States</td>
			<td></td>
			<td>Consumable Materials</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany</td>
			<td></td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED, Andover, UK</td>
			<td>pE-100</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>DualView</td>
			<td>Photometrics, Tucson, AZ, USA</td>
			<td>DV2-SYS</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>DualView filter set</td>
			<td>Photometrics, Tucson, AZ, USA</td>
			<td>05-EM</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>optiMOS scientific CMOS camera</td>
			<td>Qimaging, Surrey, BC, Canada</td>
			<td>01-OPTIMOS-R-M-16-C</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>Imaging software &#160;</td>
			<td>Micro-Manager; Vale Lab, University of California San Francisco, CA, USA</td>
			<td></td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>Analysis Software</td>
			<td>Image J software; Public Domain, NIH, USA</td>
			<td></td>
			<td>FRET</td>
		</tr>
	</tbody>
</table>

impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system

Siden sin oppfinnelse late 19th -tallet fortsetter Langendorff ex vivo hjertet perfusjon systemet å være et relevant verktøy for å studere et bredt spekter av fysiologiske, biokjemiske, morfologiske og farmakologiske parametere i sentralt denervated hjerter. Her beskriver vi et oppsett for modulering av intracardiac autonome nervesystemet og vurdering av dens innflytelse på grunnleggende elektrofysiologi, arrhythmogenesis og syklisk adenosin monofosfat (cAMP) dynamics. Intracardiac autonome nervesystemet modulert av mekanisk dissection atrial fett pads-i hvilke murine Ganglion ligger hovedsakelig- eller ved bruk av globale samt målrettet farmakologiske intervensjoner. En octapolar elektrofysiologiske kateter brukes på høyre atrium og høyre ventrikkel og epicardial plassert flere elektrode matriser (MEA) for høy oppløsning tilordning brukes til å fastsette cardiac electrophysiology og arrhythmogenesis. Han selvmord resonans energioverføring (bånd) imaging utføres for sanntids overvåking av leiren i enkelte regioner. Neuromorphology er studert ved hjelp av antistoff-basert flekker av hele hjerter med neuronal markører for å veilede identifikasjon og modulering av konkrete mål av intracardiac autonome nervesystemet i utført studiene. Ex vivo Langendorff oppsettet gir et høyt antall reproduserbar eksperimenter på kort tid. Likevel, den delvis åpne naturen oppsettet (f.eks., under MEA målinger) gjør konstant temperaturkontroll vanskelig og bør holdes til et minimum. Denne beskrevet metoden gjør det mulig å analysere og modulere intracardiac autonome nervesystemet i desentraliserte hjerter.

Figur 1 viser et bilde av Langendorff inkludert 2 flere elektrode matriser (tiltak). Før eksperimentet plasseres intracardiac kateter nær kanyle å lette en rask og enkel innsetting i høyre atrium/høyre ventrikkel og sikre en kort tidsperiode før balanse kan starte. Den nedre delen av kammeret kan bli forsterket (se pilene i figur 1) slik at kammeret er helt lukket og garanterer en stabil temperatur.
<p class="jove_conte...

Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Her presenterer vi en protokoll for modulering av intracardiac autonome nervesystemet og vurdering av dens innflytelse på grunnleggende elektrofysiologi, arrhythmogenesis og leiren dynamics ved hjelp av en ex vivo Langendorff oppsett.

Virkningen av Intracardiac Neurons Cardiac Electrophysiology og Arrhythmogenesis i en 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 ...

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Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

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The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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Morsmelk forbedrer veksten av Enteroids: en Ex Vivo modell av celle spredning

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The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

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Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...