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

Dette arbejde blev udført med støtte fra kardiovaskulær kirurgi, First Medical Center, Chinese PLA General Hospital og Laboratory Animal Center, Chinese PLA General Hospital.

Alle de eksperimentelle procedurer og protokoller, der blev anvendt i denne undersøgelse, blev gennemgået og godkendt af Animal Care and Use Committee of PLA General Hospital.
1. Klargøring af Langendorff-apparatet
<ol><li>Forbered Krebs-Henseleit (K-H) bufferen. For at forberede K-H-bufferen tilsættes følgende til destilleret vand: 118,0 mM NaCl, 4,7 mM KCl, 1,2 mMMgSO4, 1,2 mMNaH2PO4, 1,8 mM CaCl2, 25,0 mM NaHCO3,...

<ol>
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(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. 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Denne protokol etablerer en dyremodel for langvarig VF i isolerede rottehjerter, som ikke tidligere er rapporteret. Derudover blev forskellige elektriske stimuleringsbetingelser sammenlignet i denne undersøgelse. Denne undersøgelse giver en model for undersøgelser relateret til ventrikelflimmerstop under hjertekirurgi.
Succesraten for modellen er en meget vigtig indikator, der er relateret til personale, tid og økonomiske omkostninger. I VF-modeller inkluderer succesraten, om VF kan induce...

Hjertekirurgi udføres normalt via thoracotomi, med blokering af aorta og perfusion med en kardioplegisk opløsning for at arrestere hjertet. Gentag hjertekirurgi kan være mere udfordrende end den indledende operation, med højere komplikation og dødelighed 1,2,3. Desuden kan den konventionelle median sternotomi tilgang forårsage skade på broskibene bag brystbenet, den stigende aorta, højre ventrikel og andre vigtige strukturer. Omfattende blødning på grund af adskillelse af bindevæv, brystsårinfektion og sternal osteomyelitis på grund af sternotomi er alle mulige komplikationer. Omfattende dissektion øger risikoen for læsioner og blødning i vitale hjertestrukturer.
Med udviklingen af minimalt invasiv hjertekirurgi er snit blevet mindre, og hjertestop er undertiden vanskeligt at opnå. Gentag hjertekirurgi under ventrikelflimmer (VF)4,5 er sikkert, gennemførligt og kan give bedre myokardiebeskyttelse. Derfor introducerer denne protokol metoden til VF-hjertestop i kirurgi med minimalt invasiv ekstrakorporeal cirkulation. Hjertet mister effektiv sammentrækning under VF, og der er således ikke behov for at sutur og blokere den stigende aorta under operationen, hvilket forenkler proceduren. Men selvom hjertet kontinuerligt perfuseres, kan langvarig VF stadig være skadelig for hjertet.
Da denne metode bliver mere udbredt, bliver spørgsmålet om, hvordan man beskytter hjertet under VF, stadig mere relevant. Dette vil kræve omfattende og dybtgående undersøgelser ved hjælp af dyremodeller for langtids-VF. Tidligere har forskning på dette område for det meste brugt store dyr6,7 og har krævet samarbejde mellem kirurger, anæstesiologer, perfusionister og andre forskere. Disse undersøgelser tog for lang tid, prøvestørrelserne var ofte små, og undersøgelserne fokuserede generelt på hjertefunktion og mindre på mekanistiske og molekylære vurderinger. Til dato har ingen undersøgelse rapporteret en detaljeret protokol til etablering af en langsigtet VF-model.
Denne protokol giver således de detaljer, der er nødvendige for at udvikle en langsigtet VF-rottemodel ved hjælp af Langendorff-apparater. Protokollen er enkel, økonomisk, gentagelig og stabil.

<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

Ventrikelflimmer (VF) er en dødelig arytmi med høj forekomst hos hjertepatienter, men VF-anholdelse under perfusion er en forsømt metode til intraoperativ anholdelse inden for hjertekirurgi. Med de seneste fremskridt inden for hjertekirurgi er efterspørgslen efter langvarige VF-undersøgelser under perfusion steget. Feltet mangler imidlertid enkle, pålidelige og reproducerbare dyremodeller af kronisk ventrikelflimmer. Denne protokol inducerer langvarig VF gennem vekselstrøm (AC) elektrisk stimulering af epikardiet. Forskellige betingelser blev brugt til at inducere VF, herunder kontinuerlig stimulering med lav eller høj spænding for at inducere langvarig VF og stimulering i 5 minutter med lav eller høj spænding for at inducere spontan langvarig VF. Succesraterne for de forskellige tilstande samt antallet af myokardieskade og genopretning af hjertefunktionen blev sammenlignet. Resultaterne viste, at kontinuerlig lavspændingsstimulering inducerede langvarig VF, og at 5 minutters lavspændingsstimulering inducerede spontan langvarig VF med mild myokardieskade og en høj grad af genopretning af hjertefunktionen. Imidlertid havde den lavspændings, kontinuerligt stimulerede langsigtede VF-model en højere succesrate. Højspændingsstimulering gav en højere VF-induktionshastighed, men viste en lav defibrilleringssuccesrate, dårlig genopretning af hjertefunktionen og alvorlig myokardieskade. På baggrund af disse resultater anbefales kontinuerlig lavspændings epikardial AC-stimulering for sin høje succesrate, stabilitet, pålidelighed, reproducerbarhed, lav indvirkning på hjertefunktionen og mild myokardieskade.

I alt 57 rotter blev brugt i forsøgene, hvoraf 30 opfyldte inklusionskriterierne. De inkluderede dyr blev opdelt i fem grupper med seks dyr i hver gruppe: kontrolgruppen (gruppe C), den lavspændingskontinuerligt stimulerede langsigtede VF-gruppe (gruppe LC), den højspændingsstimulerede kontinuerligt langsigtede VF-gruppe (gruppe HC), den lavspændingsinducerede spontane langsigtede VF-gruppe (gruppe LI) og den højspændingsinducerede spontane langsigtede VF-gruppe (gruppe HI). Den eksperimentelle proces for hver gru...

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

Denne protokol præsenterer en model af langvarig ventrikelflimmer i rottehjerter induceret af kontinuerlig stimulering med lavspændingsvekselstrøm. Denne model har en høj succesrate, er stabil, pålidelig og reproducerbar, har en lav indvirkning på hjertefunktionen og forårsager kun mild myokardieskade.

En model af langvarig ventrikelflimmer i isolerede rottehjerter

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

Denne protokol giver et detaljeret behov for at udvikle en langsigtet ventrikelflimmer rottemodel ved hjælp af Langendorff-apparatet. Det udfylder hullet af langvarig ventrikelflimmer i smådyrsmodellen. Det tekniske er enkelt, økonomisk, gentageligt og stabilt.Denne teknik kan anvendes i undersøgelsen af ventrikelflimmerstop under perfusion, som udføres i visse kliniske situationer inden for hjertekirurgiske sessioner og operationer. Til at begynde med skal du forberede Krebs-Henseleit- eller KH-bufferen ved at tilsætte 118 millimolært natriumchlorid, 4,7 millimolært kaliumchlorid, 1,2 millimolært magnesiumchlorid, 1,2 millimolært natriumdihydrogenphosphat, 1,8 millimolært calciumchlorid, 25 millimolært natriumbicarbonat, 11,1 millimolær glucose og 0,5 millimolær EDTA i destilleret vand. Forbered derefter det modificerede Langendorff-perfusionssystem.Kolben indeholdende KH-buffer gasses kontinuerligt med 95 % ilt og 5 % kuldioxid ved et tryk på ca. 80 millimeter kviksølv. Anbring den ene ende af perfusionsrøret i KH-bufferen, før midten af perfusionsrøret gennem vandbadet, og fastgør en stump 20 gauge nål til den anden ende af perfusionsrøret. Efter ophængning af nålen på et trådstativ skal du justere temperaturen på vandbadet, så temperaturen på KH-bufferen fra enden af perfusionssystemet er 37 grader Celsius.Brug en fysiologisk signaloptager til at digitalisere og optage alle de analoge signaler. Brug to nåleelektroder i rustfrit stål til at optage et bipolært elektrokardiogram eller EKG og to nåleelektroder i rustfrit stål til elektrisk stimulering. Tilslut den ene ende af de fire elektroder til den fysiologiske signaloptager og den anden ende tæt på det område, hvor hjertet vil blive placeret efter fastgørelse til apparatet.Brug derefter den bærbare software til automatisk at genkende, justere og registrere bipolære EKG og hæmodynamiske parametre. Indstil de elektriske stimulatorparametre til 30 hertz vekselstrøm, hvor lavspændingsgruppen modtager 2 volt og højspændingsgruppen modtager 6 volt. Med den bedøvede rotte forbundet til en ventilator efter cervikal dissektion og trakeal intubation, løft huden af xiphoidprocessen med tandede tang og lav et tre centimeter tværgående snit i huden med vævsaks.Udvid hud- og ribbensnittene til axillen på begge sider i V-form. Brug stump dissektion til at adskille brachiocephalic bagagerummet fra det omgivende væv. Klem derefter den brachiocephalic bagagerum med buede tang for at lette fjernelsen af hjertet.Nedsænk straks hjertet i en petriskål med KH-buffer ved 0 til 4 grader Celsius for at vaske og pumpe det resterende blod ud. Overfør hjertet til en anden petriskål. Afskær det overflødige væv og identificer aorta.Brug to oftalmiske tang til at løfte aorta og indsæt aorta i Langendorff-apparatets stumpe nål. Derefter bindes en knude med en 0 suturtråd ved hjælp af en assistent. Tænd for perfusionsflowregulatoren.Skub ballonen gennem mitralventilen ind i venstre ventrikel. Fyld ballonen med destilleret vand for at opnå et diastolisk sluttryk på 5 til 10 millimeter kviksølv. Tilslut EKG og elektriske stimuleringselektroder til hjertet.Placer hjertet i midten af det kappede glaskammer, der opretholdes ved 37 grader Celsius. Lad hjertet ekvilibrere i 20 minutter inde i kammeret. Juster derefter vandbadstemperaturen for at holde temperaturen i det kappede glaskammer på 30 grader Celsius.Når temperaturen har nået det ønskede niveau, skal du aktivere den elektriske stimuleringskontakt på den bærbare software. Hvis dyret er en del af den kontinuerligt stimulerede langsigtede ventrikelflimmer eller VF-gruppe, skal du tillade 90 minutters elektrisk stimulering. Efter 90 minutters VF skal du bruge elektroder til at give 0,1 joule jævnstrømsdefibrillering.Samtidig reguleres vandbadstemperaturen for at lade temperaturen stige langsomt i det kappede glaskammer til 37 grader Celsius i 10 minutter. Efter defibrillering skal du lade hjertet slå i 60 minutter og derefter stoppe slagene ved langsom perfusion med 10% kaliumchlorid ved ca. 37 grader Celsius. Fjern hjertet for kreatinkinase eller CK-MB-analyse og histologisk analyse.VF-hastighederne, succesraten for defibrillering og succesraten for VF-modellen vises her. Gruppe LC og gruppe HC modtog kontinuerlig elektrisk stimulering, og VF opstod således med en succesrate på 100%. Men gruppe HC viste lavere succesrater for defibrillering.Gruppe LI og gruppe III, hvor den elektriske stimulering blev slukket efter fem minutter, havde forskellige VF-hastigheder, men VF-hastigheden var langsommere i begge grupper sammenlignet med gruppe LC og gruppe HC. Både gruppe LC og gruppe LI havde bedre succesrater for defibrillering. Men samlet set havde Group LC den højeste model succesrate, mens Group LI havde en lavere model succesrate. Puls eller HR, koronar flow eller CF og venstre ventrikulær trykforskel eller LVPD-restitutionshastigheder er vist her.Hemodynamikken i gruppe C forblev stabil under eksperimentet og viste et lille fald i HR, CF og LVPD. De to grupper med lavspændingsinduceret VF havde lignende ydeevne og en god genvindingshastighed. HR og LVPD var ikke signifikant forskellige i disse grupper sammenlignet med gruppe C.Men genvindingsgraden for CF var signifikant bedre end i gruppe C.In derimod var den hæmodynamiske genvindingshastighed for de to grupper med højspænding induceret til langvarig VF dårlig, og den højspændingsstimulerede langsigtede VF-gruppe viste den værste genvindingsrate.Analysen af koronar effusionsvæsker viste, at CK-MB-niveauerne var højere i begge højspændingsgrupper. Der blev ikke fundet forskelle mellem de to lavspændingsgrupper, og gruppe C.Hematoxylin og eosinfarvning viste et elektrodeforbrændingsområde i gruppe HC. Denne protokol er velegnet til undersøgelse relateret til ventrikelflimmerstop og perfusion i kardiovaskulær 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.

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 fluorescensimagografi af Isoleret Biventricular Arbejde kaninhjerter

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.

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.

Metode til isoleret<em&gt; Ex vivo</em&gt; Lung Perfusion i en rotte Model: Erfaringer fra at udvikle en rotte 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 rotte model af ventrikelflimren og genoplivning ved konventionel Closed-bryst Teknik

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 og vurdering af iskæmi-reperfusion skade i Langendorff-perfusionerede rottehjerter

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 af Katetre til Langsigtet motionsture Afprøvning i Swine

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.

Langsigtet blodtryksmåling i Frit Flytning Mus Brug Telemetri

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 omvendte hjertemodel for interstitiell transudatsamling fra det isolerede rottehjerte

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.

Standardiseret model af ventrikulær fibrillering og avanceret hjerteliv støtte i svin

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