Name: a model of long-term ventricular fibrillation in isolated rat hearts
Uploaded: 2023-02-17T14:15:00
Description: Watch this Scientific Journal Video about A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts at JoVE.com

Detta arbete utfördes med stöd av kardiovaskulär kirurgi, First Medical Center, Chinese PLA General Hospital och Laboratory Animal Center, Chinese PLA General Hospital.

Alla experimentella förfaranden och protokoll som användes i denna undersökning granskades och godkändes av djurvårds- och användningskommittén vid PLA General Hospital.
1. Förberedelse av Langendorff-apparaten
<ol><li>Förbered Krebs-Henseleit (K-H) buffert. För att förbereda K-H-bufferten, tillsätt följande till destillerat vatten: 118,0 mM NaCl, 4,7 mM KCl, 1,2 mMMgSO4, 1,2 mMNaH2PO4, 1,8 mM CaCl2, 25,0 mM NaHCO...

<ol>
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J., Kampman, K., Glagov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+fibrillation+during+cardiopulmonary+bypass:+Long-term+effects+on+myocardial+morphology+and+function.">Ventricular fibrillation during cardiopulmonary bypass: Long-term effects on myocardial morphology and function.</a> The Annals of Thoracic Surgery. 27 (3), 230-234 (1979).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pravastatin+attenuates+cardiac+dysfunction+induced+by+lysophosphatidylcholine+in+isolated+rat+hearts.">Pravastatin attenuates cardiac dysfunction induced by lysophosphatidylcholine in isolated rat hearts.</a> European Journal of Pharmacology. 640 (1-3), 139-142 (2010).</li><li>Lang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL10/IP-10+neutralization+can+ameliorate+lipopolysaccharide-induced+acute+respiratory+distress+syndrome+in+rats.">CXCL10/IP-10 neutralization can ameliorate lipopolysaccharide-induced acute respiratory distress syndrome in rats.</a> PLoS One. 12 (1), e0169100 (2017).</li><li>Lubbe, W. F., Bricknell, O. L., Marzagao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+fibrillation+threshold+and+vulnerable+period+in+the+isolated+perfused+rat+heart.">Ventricular fibrillation threshold and vulnerable period in the isolated perfused rat heart.</a> Cardiovascular Research. 9 (5), 613-620 (1975).</li><li>Hottentrott, C. E., Towers, B., Kurkji, H. J., Maloney, J. V., Buckberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hazard+of+ventricular+fibrillation+in+hypertrophied+ventricles+during+cardiopulmonary+bypass.">The hazard of ventricular fibrillation in hypertrophied ventricles during cardiopulmonary bypass.</a> The Journal of Thoracic and Cardiovascular Surgery. 66 (5), 742-753 (1973).</li><li>Hottenrott, C., Maloney, J. V., Buckberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+ventricular+fibrillation+on+the+adequacy+of+regional+myocardial+flow.+I.+Electrical+vs.+spontaneous+fibrillation.">Studies of the effects of ventricular fibrillation on the adequacy of regional myocardial flow. I. Electrical vs. spontaneous fibrillation.</a> The Journal of Thoracic and Cardiovascular Surgery. 68 (4), 615-625 (1974).</li><li>Buckberg, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+hypothermia+on+regional+myocardial+blood+flow+and+metabolism+during+cardiopulmonary+bypass.+I.+The+adequately+perfused+beating,+fibrillating,+and+arrested+heart.">Studies of the effects of hypothermia on regional myocardial blood flow and metabolism during cardiopulmonary bypass. I. The adequately perfused beating, fibrillating, and arrested heart.</a> The Journal of Thoracic and Cardiovascular Surgery. 73 (1), 87-94 (1977).</li><li>Gazmuri, R. J., Berkowitz, M., Cajigas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+effects+of+ventricular+fibrillation+in+the+isolated+rat+heart.">Myocardial effects of ventricular fibrillation in the isolated rat heart.</a> Critical Care Medicine. 27 (8), 1542-1550 (1999).</li><li>Clasen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+approach+for+programmed+electrical+stimulation+in+mice:+Inducibility+of+ventricular+arrhythmias.">A modified approach for programmed electrical stimulation in mice: Inducibility of ventricular arrhythmias.</a> PLoS One. 13 (8), e0201910 (2018).</li><li>Diaz-Maue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cardiac+rhythm+management+by+applying+optogenetic+multi-site+photostimulation+in+murine+hearts.">Advanced cardiac rhythm management by applying optogenetic multi-site photostimulation in murine hearts.</a> Journal of Visualized Experiments. (174), e62335 (2021).</li><li>Jungen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+intracardiac+neurons+on+cardiac+electrophysiology+and+arrhythmogenesis+in+an+ex+vivo+Langendorff+system.">Impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo Langendorff system.</a> Journal of Visualized Experiments. 135, e57617 (2018).</li><li>Koretsune, Y., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+calcium+in+the+pathophysiology+of+ventricular+fibrillation+and+in+the+pathogenesis+of+postarrhythmic+contractile+dysfunction.">Cell calcium in the pathophysiology of ventricular fibrillation and in the pathogenesis of postarrhythmic contractile dysfunction.</a> Circulation. 80 (2), 369-379 (1989).</li><li>Brazier, J. R., Cooper, N., McConnell, D. H., Buckberg, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+hypothermia+on+regional+myocardial+blood+flow+and+metabolism+during+cardiopulmonary+bypass.+III.+Effects+of+temperature,+time,+and+perfusion+pressure+in+fibrillating+hearts.">Studies of the effects of hypothermia on regional myocardial blood flow and metabolism during cardiopulmonary bypass. III. Effects of temperature, time, and perfusion pressure in fibrillating hearts.</a> The Journal of Thoracic and Cardiovascular Surgery. 73 (1), 102-109 (1977).</li><li>von Planta, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+resuscitation+in+the+rat.">Cardiopulmonary resuscitation in the rat.</a> Journal of Applied Physiology. 65 (6), 2641-2647 (1988).</li><li>Luo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+increases+cardiac+electrical+remodelling+in+rats+and+mice+via+NOX4/ROS/CaMKII-mediated+calcium+signalling.">Ageing increases cardiac electrical remodelling in rats and mice via NOX4/ROS/CaMKII-mediated calcium signalling.</a> Oxidative Medicine and Cellular Longevity. 2022, 8538296 (2022).</li><li>Hohnloser, S., Weirich, J., Antoni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+direct+current+on+the+electrical+activity+of+the+heart+and+on+its+susceptibility+to+ventricular+fibrillation.">Influence of direct current on the electrical activity of the heart and on its susceptibility to ventricular fibrillation.</a> Basic Research in Cardiology. 77 (3), 237-249 (1982).</li><li>Xie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-energy+defibrillation+increases+the+severity+of+postresuscitation+myocardial+dysfunction.">High-energy defibrillation increases the severity of postresuscitation myocardial dysfunction.</a> Circulation. 96 (2), 683-688 (1997).</li><li>Manoach, M., Netz, H., Erez, M., Weinstock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+self-defibrillation+in+mammals:+Age+and+drug+dependence.">Ventricular self-defibrillation in mammals: Age and drug dependence.</a> Age and Ageing. 9 (2), 112-116 (1980).</li><li>Filippi, S., Gizzi, A., Cherubini, C., Luther, S., Fenton, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+hypothermic+ventricular+fibrillation:+The+role+of+temperature+and+tissue+size.">Mechanistic insights into hypothermic ventricular fibrillation: The role of temperature and tissue size.</a> Europace. 16 (3), 424-434 (2014).</li></ol>

Detta protokoll etablerar en djurmodell av långvarig VF i isolerade råtthjärtan som inte tidigare har rapporterats. Dessutom jämfördes olika elektriska stimuleringsförhållanden i denna studie. Denna studie ger en modell för studier relaterade till ventrikelflimmerstopp under hjärtkirurgi.
Modellens framgångsgrad är en mycket viktig indikator som är relaterad till personal, tid och ekonomiska kostnader. I VF-modeller inkluderar framgångsgraden om VF kan induceras i hjärtat och om ...

Hjärtkirurgi utförs vanligtvis via thorakotomi, med blockering av aorta och perfusion med en kardioplegisk lösning för att arrestera hjärtat. Upprepad hjärtkirurgi kan vara mer utmanande än den initiala operationen, med högre komplikationer och dödlighet 1,2,3. Dessutom kan den konventionella mediansternotomimetoden orsaka skador på brokärlen bakom bröstbenet, den stigande aortan, höger kammare och andra viktiga strukturer. Omfattande blödning på grund av separation av bindväv, sternal sårinfektion och sternal osteomyelit på grund av sternotomi är alla möjliga komplikationer. Omfattande dissektion ökar risken för lesioner och blödningar i vitala hjärtstrukturer.
Med utvecklingen av minimalt invasiv hjärtkirurgi har snitt blivit mindre och hjärtstillestånd är ibland svårt att uppnå. Upprepad hjärtkirurgi under ventrikelflimmer (VF)4,5 är säker, genomförbar och kan ge bättre hjärtskydd. Därför introducerar detta protokoll metoden för VF-hjärtstillestånd vid kirurgi med minimalt invasiv extrakorporeal cirkulation. Hjärtat förlorar effektiv sammandragning under VF, och därmed finns det inget behov av sutur och blockera den stigande aortan under operationen, vilket förenklar proceduren. Men även om hjärtat kontinuerligt perfuseras kan långvarig VF fortfarande vara skadligt för hjärtat.
När denna metod blir mer utbredd blir frågan om hur man skyddar hjärtat under VF alltmer relevant. Detta kommer att kräva omfattande och fördjupade studier med djurmodeller av långtids-VF. Tidigare har forskningen inom detta område mestadels använt stora djur 6,7 och har krävt samarbete mellan kirurger, anestesiologer, perfusionister och andra forskare. Dessa studier tog för lång tid, provstorlekarna var ofta små och studierna fokuserade i allmänhet på hjärtfunktion och mindre på mekanistiska och molekylära bedömningar. Hittills har ingen studie rapporterat ett detaljerat protokoll för att upprätta en långsiktig VF-modell.
Detta protokoll ger således de detaljer som behövs för att utveckla en långsiktig VF-råttmodell med hjälp av Langendorff-apparaten. Protokollet är enkelt, ekonomiskt, repeterbart och stabilt.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0 Non-absorbable suture</td>
			<td>Ethicon, Inc.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>95% O2 + 5% CO2</td>
			<td>Beijing BeiYang United Gas Co., Ltd.&#160;</td>
			<td></td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>AcqKnowledge software</td>
			<td>BIOPAC Systems Inc.</td>
			<td>Version 4.2.1</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Automatic biochemistry analyzer</td>
			<td>Rayto Life and Analytical Sciences Co., Ltd.</td>
			<td>Chemray 800</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>BIOPAC research systems</td>
			<td>BIOPAC Systems Inc.</td>
			<td>MP150</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Blunt needle (20 G, TWLB)</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10005861</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>CK-MB assay kits&#160;</td>
			<td>Changchun Huili Biotech Co., Ltd.</td>
			<td>C060</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>Curved forcep</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009717</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>BIOPAC Systems Inc.</td>
			<td>STEMISOC</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>63005518</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Tianjin Biochem Pharmaceutical Co., Ltd.</td>
			<td>H120200505</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>21082201</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20025118</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Needle electrodes</td>
			<td>BIOPAC Systems Inc.</td>
			<td>EL452</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Ophthalmic clamp</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Perfusion tube</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10016318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10018960</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10019318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20040718</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sprague-Dawley (SD) rats</td>
			<td>SPF (Beijing) biotechnology Co., Ltd.</td>
			<td>Male, 300-350g</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Jiangsu Jingchuang Electronics Co., Ltd.</td>
			<td>GSP-6</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Tissueforceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Toothed forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Chengdu Instrument Factory</td>
			<td>DKX-150</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Water bath1</td>
			<td>Ningbo Scientz Biotechnology Co.,Ltd.</td>
			<td>SC-15</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Water bath2</td>
			<td>Shanghai Yiheng Technology Instrument Co., Ltd.</td>
			<td>DK-8D</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
	</tbody>
</table>

a model of long-term ventricular fibrillation in isolated rat hearts

Ventrikulärt flimmer (VF) är en dödlig arytmi med hög förekomst hos hjärtpatienter, men VF-stopp under perfusion är en försummad metod för intraoperativt stopp inom hjärtkirurgi. Med de senaste framstegen inom hjärtkirurgi har efterfrågan på långvariga VF-studier under perfusion ökat. Fältet saknar dock enkla, tillförlitliga och reproducerbara djurmodeller av kroniskt ventrikelflimmer. Detta protokoll inducerar långvarig VF genom växelström (AC) elektrisk stimulering av epikardiet. Olika betingelser användes för att inducera VF, inklusive kontinuerlig stimulering med låg eller hög spänning för att inducera långvarig VF och stimulering i 5 minuter med låg eller hög spänning för att inducera spontan långsiktig VF. Framgångsgraden för de olika tillstånden, liksom graden av hjärtskada och återhämtning av hjärtfunktionen, jämfördes. Resultaten visade att kontinuerlig lågspänningsstimulering inducerade långvarig VF och att 5 min lågspänningsstimulering inducerade spontan långvarig VF med mild myokardskada och en hög grad av återhämtning av hjärtfunktionen. Den lågspända, kontinuerligt stimulerade långsiktiga VF-modellen hade dock en högre framgångsgrad. Högspänningsstimulering gav en högre frekvens av VF-induktion men visade en låg defibrillering framgång, dålig återhämtning av hjärtfunktionen och svår hjärtskada. På grundval av dessa resultat rekommenderas kontinuerlig lågspännings epikardiell AC-stimulering för dess höga framgångsgrad, stabilitet, tillförlitlighet, reproducerbarhet, låg påverkan på hjärtfunktionen och mild myokardskada.

Totalt användes 57 råttor i försöken, varav 30 uppfyllde inklusionskriterierna. De inkluderade djuren delades in i fem grupper, med sex djur i varje grupp: kontrollgruppen (grupp C), den lågspänningsstimulerade kontinuerligt stimulerade långsiktiga VF-gruppen (grupp LC), den högspänningsstimulerade kontinuerligt långsiktiga VF-gruppen (grupp HC), den lågspänningsinducerade spontana långsiktiga VF-gruppen (grupp LI) och den högspänningsinducerade spontana långsiktiga VF-gruppen (grupp HI). Försöksprocess...

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A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Detta protokoll presenterar en modell av långvarigt ventrikelflimmer i råtthjärtan inducerade av kontinuerlig stimulering med lågspänningsväxelström. Denna modell har en hög framgångsgrad, är stabil, pålitlig och reproducerbar, har låg inverkan på hjärtfunktionen och orsakar endast mild hjärtskada.

En modell av långvarigt ventrikelflimmer i isolerade råtthjärtan

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

Detta protokoll ger ett detaljerat behov av att utveckla en långsiktig ventrikelflimmermodell med hjälp av Langendorff-apparaten. Det fyller gapet av långsiktigt ventrikelflimmer i smådjursmodellen. Det tekniska är enkelt, ekonomiskt, repeterbart och stabilt.Denna teknik kan användas i studien av ventrikelflimmerstopp under perfusion, vilket utförs i vissa kliniska situationer inom hjärtkirurgiska sessioner och operationer. För att börja, förbered Krebs-Henseleit eller KH-bufferten genom att tillsätta 118 millimolar natriumklorid, 4,7 millimolar kaliumklorid, 1,2 millimolar magnesiumklorid, 1,2 millimolar natriumdivätefosfat, 1,8 millimolar kalciumklorid, 25 millimolar natriumbikarbonat, 11,1 millimolar glukos och 0,5 millimolar EDTA i destillerat vatten. Förbered sedan det modifierade Langendorff-perfusionssystemet.Gasa kolven innehållande KH-buffert kontinuerligt med 95 % syre och 5 % koldioxid vid ett tryck av cirka 80 millimeter kvicksilver. Placera ena änden av perfusionsröret i KH-bufferten, passera mitten av perfusionsröret genom vattenbadet och fäst en trubbig 20 gauge nål i den andra änden av perfusionsröret. Efter upphängning av nålen på ett trådstativ, justera temperaturen på vattenbadet så att temperaturen på KH-bufferten från perfusionssystemets ände är 37 grader Celsius.Använd en fysiologisk signalinspelare för att digitalisera och spela in alla analoga signaler. Använd två nålelektroder i rostfritt stål för att registrera ett bipolärt elektrokardiogram eller EKG och två nålelektroder i rostfritt stål för elektrisk stimulering. Anslut ena änden av de fyra elektroderna till den fysiologiska signalskrivaren och den andra änden nära det område där hjärtat kommer att placeras efter fastsättning på apparaten.Använd sedan den bärbara programvaran för att automatiskt känna igen, justera och registrera bipolära EKG och hemodynamiska parametrar. Ställ in de elektriska stimulatorparametrarna till 30 hertz växelström med lågspänningsgruppen som tar emot 2 volt och högspänningsgruppen som tar emot 6 volt. Med den bedövade råttan ansluten till en ventilator efter cervikal dissektion och trakeal intubation, lyft huden från xiphoidprocessen med tandade pincett och gör ett tre centimeter tvärgående snitt i huden med vävnadssax.Förläng huden och revbenssnitten till axillorna på båda sidor i en V-form. Använd trubbig dissektion för att separera brakiocefalisk stam från den omgivande vävnaden. Kläm sedan fast brachiocefaliska stammen med böjda pincett för att underlätta avlägsnandet av hjärtat.Sänk omedelbart hjärtat i en petriskål med KH-buffert vid 0 till 4 grader Celsius för att tvätta och pumpa ut restblodet. Överför hjärtat till en andra petriskål. Skär av den överflödiga vävnaden och identifiera aortan.Använd två oftalmiska pincett för att lyfta aortan och sätt in aortan i Langendorff-apparatens trubbiga nål. Bind sedan en knut med en 0 suturtråd med hjälp av en assistent. Slå på perfusionsflödesregulatorn.Tryck ballongen genom mitralventilen in i vänster kammare. Fyll ballongen med destillerat vatten för att uppnå ett diastoliskt sluttryck på 5 till 10 millimeter kvicksilver. Anslut EKG och elektriska stimuleringselektroder till hjärtat.Placera hjärtat i mitten av den mantlade glaskammaren som hålls vid 37 grader Celsius. Låt hjärtat balansera i 20 minuter inne i kammaren. Justera sedan vattenbadets temperatur för att hålla temperaturen i den mantlade glaskammaren vid 30 grader Celsius.När temperaturen har nått önskad nivå, aktivera den elektriska stimuleringsomkopplaren på den bärbara programvaran. Om djuret är en del av den kontinuerligt stimulerade långsiktiga ventrikelflimmer eller VF-gruppen, tillåt 90 minuters elektrisk stimulering. Efter 90 minuters VF, använd elektroder för att ge 0,1 joule likströmsdefibrillering.Reglera samtidigt vattenbadets temperatur så att temperaturen stiger långsamt i den mantlade glaskammaren till 37 grader Celsius i 10 minuter. Efter defibrillering, låt hjärtat slå i 60 minuter och stoppa sedan slagen genom långsam perfusion med 10% kaliumklorid vid cirka 37 grader Celsius. Ta bort hjärtat för kreatinkinas eller CK-MB-analys och histologisk analys.Graden av VF, framgångsgraden för defibrillering och framgångsgraden för VF-modellen visas här. Grupp LC och grupp HC fick kontinuerlig elektrisk stimulering och därmed inträffade VF med en 100% framgångsgrad. Men grupp HC visade lägre framgångsgrader för defibrillering.Grupp LI och grupp HI där den elektriska stimuleringen stängdes av efter fem minuter hade olika hastigheter av VF, men VF-frekvensen var långsammare i båda grupperna jämfört med grupp LC och grupp HC. Både Group LC och Group LI hade bättre defibrillering framgångsgrad. Men totalt sett hade Group LC den högsta framgångsgraden för modeller, medan Group LI hade en lägre framgångsgrad för modeller. Hjärtfrekvensen eller HR, koronarflöde eller CF och tryckskillnad i vänster kammare eller LVPD-återhämtningshastigheter visas här.Hemodynamiken i grupp C förblev stabil under experimentet och visade en liten minskning av HR, CF och LVPD. De två grupperna med lågspänningsinducerad VF hade liknande prestanda och en god återhämtningsgrad. HR och LVPD var inte signifikant annorlunda i dessa grupper jämfört med grupp C.Men återvinningsgraden för CF var signifikant bättre än i grupp C.In kontrast, den hemodynamiska återhämtningsgraden för de två grupperna med högspänning inducerad till långvarig VF var dålig och högspännings kontinuerligt stimulerad långsiktig VF-grupp visade den sämsta återhämtningsgraden.Analysen av koronar effusionsvätskor visade att CK-MB-nivåerna var högre i båda högspänningsgrupperna. Inga skillnader hittades mellan de två lågspänningsgrupperna och grupp C.Hematoxylin och eosinfärgning visade en elektrodbrännregion i grupp HC. Detta protokoll är lämpligt för studier relaterade till ventrikelflimmerstopp och perfusion vid kardiovaskulär kirurgi.

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Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bedömning av hjärtfunktionen och Energetics i isolerade Mus Hearts Använda 31 P NMR-spektroskopi

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH Fluorescens avbildning av isolerade biventrikulär Arbeta kaninhjärtan

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Minskning av vänster kammares Wall Stress och renovering i funktion i avsaknad Hearts använder Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Metod för Isolerad<em&gt; Ex vivo</em&gt; Lung Perfusion i en råttmodell: Lärdomar från Utveckla en Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

En Rat modell för ventrikelflimmer och Resuscitation av konventionell Stängd-bröst Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Induktion och Bedömning av ischemi-reperfusion skada i Langendorff-perfusion Rat Hearts

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

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

Kirurgisk placering av katetrar för långsiktigt konditionsträning Testning i svin

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

Långsiktig blodtrycksmätning i fritt rörliga Möss Använda Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

Den inverterade hjärtmodellen för interstitiell transudatsamling från det isolerade råtthjärtat

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

Standardiserad modell av ventrikulärt förmaksflimmer och avancerad hjärtlivsstöd i svin

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...