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 arbeidet ble utført med støtte fra kardiovaskulær kirurgi, First Medical Center, Chinese PLA General Hospital og Laboratory Animal Center, Chinese PLA General Hospital.

Alle eksperimentelle prosedyrer og protokoller som ble brukt i denne undersøkelsen ble gjennomgått og godkjent av Animal Care and Use Committee of PLA General Hospital.
1. Klargjøring av Langendorff-apparatet
<ol><li>Forbered bufferen Krebs-Henseleit (K-H). For å forberede K-H-bufferen, tilsett følgende til destillert vann: 118,0 mM NaCl, 4,7 mM KCl, 1,2 mM MgSO 4, 1,2 mM NaH 2 PO4, 1,8 mM CaCl2, 25,0 mM NaHCO3, 11,1 mM glukose og 0,5 mM...

<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. 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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 protokollen etablerer en dyremodell av langsiktig VF i isolerte rottehjerter som ikke tidligere er rapportert. I tillegg ble forskjellige elektriske stimuleringsforhold sammenlignet i denne studien. Denne studien gir en modell for studier relatert til ventrikkelflimmerstans under hjertekirurgi.
Suksessraten til modellen er en svært viktig indikator som er relatert til personell, tid og økonomiske kostnader. I VF-modeller inkluderer suksessraten om VF kan induseres i hjertet og om hjert...

Hjertekirurgi utføres vanligvis via torakotomi, med blokkering av aorta og perfusjon med en kardioplegisk løsning for å arrestere hjertet. Gjentatt hjertekirurgi kan være mer utfordrende enn den første operasjonen, med høyere komplikasjoner og dødelighet 1,2,3. Videre kan den konvensjonelle median sternotomi-tilnærmingen forårsake skade på brokarene bak brystbenet, den stigende aorta, høyre ventrikkel og andre viktige strukturer. Omfattende blødninger på grunn av separasjon av bindevev, sternal sårinfeksjon og sternal osteomyelitt på grunn av sternotomi er alle mulige komplikasjoner. Omfattende disseksjon øker risikoen for lesjoner og blødninger i vitale hjertestrukturer.
Med utviklingen av minimalt invasiv hjertekirurgi har snittene blitt mindre, og hjertestans er noen ganger vanskelig å oppnå. Gjentatt hjertekirurgi under ventrikkelflimmer (VF)4,5 er trygt, gjennomførbart og kan gi bedre myokardbeskyttelse. Derfor introduserer denne protokollen metoden for VF-hjertestans i kirurgi med minimal invasiv ekstrakorporeal sirkulasjon. Hjertet mister effektiv sammentrekning under VF, og det er derfor ikke nødvendig å suturere og blokkere den stigende aorta under operasjonen, noe som forenkler prosedyren. Men selv om hjertet er kontinuerlig perfundert, kan langsiktig VF fortsatt være skadelig for hjertet.
Etter hvert som denne metoden blir mer utbredt, blir spørsmålet om hvordan man beskytter hjertet under VF stadig mer relevant. Dette vil kreve omfattende og grundige studier ved bruk av dyremodeller av langsiktig VF. Tidligere har forskning på dette feltet stort sett brukt store dyr6,7 og har krevd samarbeid mellom kirurger, anestesileger, perfusjonister og andre forskere. Disse studiene tok for lang tid, utvalgsstørrelsene var ofte små, og studiene fokuserte generelt på hjertefunksjon og mindre på mekanistiske og molekylære vurderinger. Til dags dato har ingen studier rapportert en detaljert protokoll for å etablere en langsiktig VF-modell.
Denne protokollen gir dermed detaljene som trengs for å utvikle en langsiktig VF-rottemodell ved hjelp av Langendorff-apparat. Protokollen er enkel, økonomisk, repeterbar 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

Ventrikkelflimmer (VF) er en dødelig arytmi med høy forekomst hos hjertepasienter, men VF-stans under perfusjon er en neglisjert metode for intraoperativ stans innen hjertekirurgi. Med de siste fremskrittene innen hjertekirurgi har etterspørselen etter langvarige VF-studier under perfusjon økt. Feltet mangler imidlertid enkle, pålitelige og reproduserbare dyremodeller av kronisk ventrikkelflimmer. Denne protokollen induserer langsiktig VF gjennom vekselstrøm (AC) elektrisk stimulering av epikardiet. Ulike forhold ble brukt for å indusere VF, inkludert kontinuerlig stimulering med lav eller høy spenning for å indusere langvarig VF og stimulering i 5 minutter med lav eller høy spenning for å indusere spontan langsiktig VF. Suksessratene for de forskjellige tilstandene, samt frekvensen av myokardskade og gjenoppretting av hjertefunksjon, ble sammenlignet. Resultatene viste at kontinuerlig lavspenningsstimulering induserte langvarig VF og at 5 min lavspenningsstimulering induserte spontan langvarig VF med lett myokardskade og høy restitusjonsgrad av hjertefunksjon. Den lavspente, kontinuerlig stimulerte langsiktige VF-modellen hadde imidlertid en høyere suksessrate. Høyspenningsstimulering ga høyere VF-induksjonsrate, men viste lav suksessrate for defibrillering, dårlig restitusjon av hjertefunksjonen og alvorlig myokardskade. På grunnlag av disse resultatene anbefales kontinuerlig lavspent epikardial AC-stimulering for sin høye suksessrate, stabilitet, pålitelighet, reproduserbarhet, lav innvirkning på hjertefunksjonen og mild myokardskade.

Totalt 57 rotter ble brukt i forsøkene, hvorav 30 oppfylte inklusjonskriteriene. De inkluderte dyrene ble delt inn i fem grupper, med seks dyr i hver gruppe: kontrollgruppen (gruppe C), den lavspente kontinuerlig stimulerte langtids VF-gruppen (gruppe LC), den høyspente kontinuerlig stimulerte langsiktige VF-gruppen (gruppe HC), den lavspenningsinduserte spontane langsiktige VF-gruppen (gruppe LI) og den høyspenningsinduserte spontane langsiktige VF-gruppen (gruppe HI). Den eksperimentelle prosessen for hver gruppe er...

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

Denne protokollen presenterer en modell for langvarig ventrikkelflimmer i rottehjerter indusert av kontinuerlig stimulering med lavspent vekselstrøm. Denne modellen har en høy suksessrate, er stabil, pålitelig og reproduserbar, har liten innvirkning på hjertefunksjonen og forårsaker bare mild myokardskade.

En modell for langvarig ventrikkelflimmer i isolerte 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 protokollen gir et detaljert behov for å utvikle en langsiktig ventrikkelflimmerrottemodell ved hjelp av Langendorff-apparatet. Det fyller gapet av langvarig ventrikkelflimmer i smådyrmodellen. Det tekniske er enkelt, økonomisk, repeterbart og stabilt.Denne teknikken kan brukes i studiet av ventrikulær fibrilleringsstans under perfusjon, som utføres i visse kliniske situasjoner innen hjertekirurgi økter og operasjoner. For å begynne å forberede Krebs-Henseleit eller KH-bufferen ved å tilsette 118 millimolært natriumklorid, 4, 7 millimolært kaliumklorid, 1, 2 millimolært magnesiumklorid, 1, 2 millimolært natriumdihydrogenfosfat, 1, 8 millimolært kalsiumklorid, 25 millimolært natriumbikarbonat, 11, 1 millimolar glukose og 0, 5 millimolar EDTA i destillert vann. Forbered deretter det modifiserte Langendorff perfusjonssystemet.Gass kontinuerlig kolben som inneholder KH-buffer med 95 % oksygen og 5 % karbondioksid ved et trykk på ca. 80 millimeter kvikksølv. Plasser den ene enden av perfusjonsrøret i KH-bufferen, pass midten av perfusjonsrøret gjennom vannbadet, og fest en stump 20 gauge nål til den andre enden av perfusjonsrøret. Etter å ha suspendert nålen på et trådstativ, juster temperaturen på vannbadet slik at temperaturen på KH-bufferen fra enden av perfusjonssystemet er 37 grader Celsius.Bruk en fysiologisk signalopptaker til å digitalisere og registrere alle analoge signaler. Bruk to rustfritt stål nål elektroder for å registrere en bipolar elektrokardiogram eller EKG og to rustfritt stål nål elektroder for elektrisk stimulering. Koble den ene enden av de fire elektrodene til den fysiologiske signalopptakeren og den andre enden nær området der hjertet skal plasseres etter festing til apparatet.Deretter bruker du den bærbare programvaren til automatisk å gjenkjenne, justere og registrere bipolar EKG og hemodynamiske parametere. Sett de elektriske stimulatorparametrene til 30 hertz vekselstrøm med lavspenningsgruppen som mottar 2 volt og høyspenningsgruppen mottar 6 volt. Med den bedøvede rotta koblet til en ventilator etter cervikal disseksjon og trakeal intubasjon, løft huden av xiphoidprosessen med tanntang og lag et tre centimeter tverrsnitt i huden med vevsaks.Forleng hud- og ribbesnittene til aksillene på begge sider i en V-form. Bruk stump disseksjon for å skille brakiocephalic stammen fra det omkringliggende vevet. Klem deretter den brakiocephaliske stammen med buede tang for å lette fjerning av hjertet.Senk hjertet umiddelbart i en petriskål med KH-buffer ved 0 til 4 grader Celsius for å vaske og pumpe ut restblodet. Overfør hjertet til en annen petriskål. Klipp av overflødig vev og identifisere aorta.Bruk to oftalmiske tang for å løfte aorta og sett aorta inn i den stumpe nålen til Langendorff-apparatet. Deretter knytter du ved hjelp av en assistent en knute med en 0 suturtråd. Slå på perfusjonsstrømningsregulatoren.Skyv ballongen gjennom mitralventilen inn i venstre ventrikel. Fyll ballongen med destillert vann for å oppnå et diastolisk trykk på 5 til 10 millimeter kvikksølv. Koble EKG og elektriske stimuleringselektroder til hjertet.Plasser hjertet i midten av det jakkede glasskammeret som opprettholdes ved 37 grader Celsius. La hjertet balansere i 20 minutter inne i kammeret. Juster deretter temperaturen i vannbadet for å opprettholde temperaturen i det kappede glasskammeret ved 30 grader Celsius.Etter at temperaturen har nådd ønsket nivå, aktiverer du den elektriske stimuleringsbryteren på den bærbare programvaren. Hvis dyret er en del av den kontinuerlig stimulerte langsiktige ventrikkelflimmer eller VF-gruppen, la 90 minutter med elektrisk stimulering. Etter 90 minutter med VF, bruk elektroder for å gi 0,1 joule likestrøm defibrillering.Reguler samtidig vannbadtemperaturen slik at temperaturen stiger sakte i det jakkede glasskammeret til 37 grader Celsius i 10 minutter. Etter defibrillering, la hjertet slå i 60 minutter og deretter stoppe slagene ved langsom perfusjon med 10% kaliumklorid ved ca. 37 grader Celsius. Fjern hjertet for kreatinkinase eller CK-MB-analyse og histologisk analyse.Frekvensen av VF, suksessraten for defibrillering og suksessraten for VF-modellen er vist her. Gruppe LC og gruppe HC fikk kontinuerlig elektrisk stimulering, og dermed oppstod VF med en suksessrate på 100 %. Men gruppe HC viste lavere suksessrate for defibrillering.Gruppe LI og gruppe HI der den elektriske stimuleringen ble slått av etter fem minutter, hadde forskjellige frekvenser av VF, men VF-frekvensen var langsommere i begge gruppene sammenlignet med gruppe LC og gruppe HC. Både gruppe LC og gruppe LI hadde bedre suksessrater for defibrillering. Men samlet sett hadde gruppe LC den høyeste suksessraten for modellene, mens gruppe LI hadde en lavere suksessrate for modellen. Hjertefrekvens eller HR, koronar flow eller CF, og venstre ventrikkel trykkforskjell eller LVPD utvinningsgrad er vist her.Hemodynamikken i gruppe C forble stabil under forsøket og viste en liten reduksjon i HR, CF og LVPD. De to gruppene med lavspenningsindusert VF hadde lik ytelse og god utvinningsgrad. HR og LVPD var ikke signifikant forskjellige i disse gruppene sammenlignet med gruppe C.Men utvinningsgraden av CF var signifikant bedre enn i gruppe C.In kontrast, den hemodynamiske utvinningsgraden for de to gruppene med høyspenning indusert til langsiktig VF var dårlig og høyspenningen kontinuerlig stimulert langsiktig VF-gruppe viste den verste utvinningsgraden.Analysen av koronar effusjonsvæske viste at CK-MB-nivåene var høyere i begge høyspenningsgruppene. Det ble ikke funnet forskjeller mellom de to lavspenningsgruppene og gruppe C.Hematoksylin- og eosinfarging viste et elektrodebrenningsområde i gruppe HC. Denne protokollen er egnet for studier relatert til ventrikkelflimmerstans og perfusjon ved 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 Fluorescens Imaging av isolerte Biventricular Arbeide 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 ...

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.

Måte Isolert<em&gt; Ex Vivo</em&gt; Lung Perfusjons i en Rat Modell: Erfaringer fra å utvikle 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 av ventrikkelflimmer og Resuscitation av Konvensjonell Lukket brystet 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.

Induksjon og vurdering av iskemi-reperfusjon Injury in Langendorff-perfuserte 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 Plassering av katetre for langsiktig kardiovaskulær trening Testing 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.

Langsiktig blodtrykksmåling i fritt bevegelige Mus Bruke 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 inverterte hjertemodellen for interstitial transudatsamling fra det isolerte rottehjertet

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.

Standardisert modell av ventrikulær fibrillasjon og avansert hjertelivsstøtte i svin

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