Induktion og vurdering af iskæmi-reperfusion skade i Langendorff-perfusionerede rottehjerter
Summary
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.
Abstract
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.
Introduction
Belysning af de begivenheder, der ligger til grund hjertekammeret svar på både iskæmi og reperfusion er afgørende at forbedre behandlingen af myokardieinfarkt 1 og hjerte kirurgiske procedurer, der kræver aorta cross-fastspænding 2. Mens in vivo modeller for iskæmisk reperfusionsbeskadigelse tillader meget nyttig endepunktsanalyse, er de ikke så effektive til at studere de funktionelle virkninger af iskæmisk reperfusionsbeskadigelse akut i realtid. Derudover in vivo iskæmisk reperfusion modeller frembringer generelt en betydelig variabilitet i infarktstørrelse, og direkte levering af lægemiddel til hjertet på tidspunktet for reperfusion er udfordrende. Udnyttelsen af et Langendorff isoleret hjerte til undersøgelse iskæmisk reperfusionsbeskadigelse muliggør tidstro funktionel vurdering af farmakologiske behandlinger, ensartet område med infarkt væv og øjeblikkelig levering af lægemiddel direkte til myokardiet.
Først beskrevne by Oscar Langendorff i 1895 3, Langendorff isoleret hjerte er en robust model til at studere iskæmi reperfusionsskade, der er blevet brugt i iskæmireperfusion forskning for de sidste 40 år 4,5. Her er nogle modifikationer foretages for at optimere det isolerede hjerte til funktionel analyse. In situ kanylering af aorta, mens hjertet slår sikrer, at hjertet ikke oplever iskæmisk prækonditionering, der kunne ændre resultaterne af iskæmisk reperfusion forsøg 6. For at lette dette, er en tracheotomi udføres, tillader ventilation og sikring fysiologisk stabilitet af rotte under operationen. Hjertet bliver derefter fastgjort til et glas vandkappe spiral søjlen gennem hvilken Krebs Henseleit puffer leveres via retrograd perfusion direkte ind i aorta. En saltvand-fyldte ballon indsættes i den venstre ventrikel og fæstnet til en tryktransducer, som giver mulighed for tidstro måling af tryk inde fra ventriklen end beregning af flere funktionelle parametre. Ved afslutningen af eksperimentet, hjertet skyllet med koldt saltvand at standse sammentrækning og lynfrosset i flydende nitrogen for at muliggøre nedstrøms analyse af DNA, RNA og protein niveauer. Således ændres, Langendorff perfunderet hjerte fungerer som et effektivt system til direkte overvågning af den fysiologiske effekt af farmakologiske interventioner på noget tidspunkt akut under iskæmi reperfusionsskade.
Protocol
Representative Results
Discussion
Det isolerede perfunderede rotte- hjerte med held kan anvendes til at undersøge virkningen af farmakologisk intervention på kardial forkonditionering i iskæmisk reperfusionsbeskadigelse 9. Der er dog nogle væsentlige skridt til den procedure, der skal standardiseres for at sikre reproducerbare resultater. Fastholdelse af en temperatur på 37,4 ° C i systemet er kritisk, da selv mild hypotermi og hypertermi kan forårsage kardial forkonditionering 10,11. Den samlede tid, der går, fra indsprøj…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Denne publikation blev støttet af South Carolina Clinical & Translationel Research (SCTR) Institute, med en akademisk hjem på Medical University of South Carolina, NIH / NCATS Grant Nummer UL1 TR000062. Yderligere støtte blev leveret af VA hæderspris BX002327-01 til DRM. DJH blev støttet af NIH / NCATS Grant nummer TL1 TR000061 og NIH Grant nummer T32 GM008716. SEA blev støttet af NIH Grant nummer T32 HL07260.
Materials
| Sodium Chloride | Sigma Aldrich | S3014 | |
| Potassium Chloride | Sigma Aldrich | P9541 | |
| Magnesium Sulfate | Sigma Aldrich | 203726 | |
| Potassium Phosphate Dibasic | Sigma Aldrich | RES20765-A7 | |
| Calcium Chloride Dihydrate | Sigma Aldrich | C8106 | |
| Sodium Bicarbonate | Sigma Aldrich | S5761 | |
| D-Glucose | Sigma Aldrich | G8270 | |
| Octanoic Acid | Sigma Aldrich | C2875 | |
| 2,3,5-triphenyltetrazolium chloride | Sigma Aldrich | T8877 | |
| Medical Pressure Transducer | MEMSCAP | SP844 | |
| Masterflex Peristaltic Pump | Cole Parmer | EW-07521-40 | |
| Masterflex Easy Load Pump Head | Cole Parmer | EW-07518-10 | |
| Heated circulating water bath | Lauda | M3 | |
| Tubing Flow Module | Transonic | TS410 | |
| Modular Research Console | Transonic | T402 | |
| Inline flow sensor | Transonic | ME2PXN | |
| PowerLab Data Acquisition Device | AD Instruments | PL3508 | |
| LabChart data acquisition software | AD Instruments | MLU60/8 |
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