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HEART PERFUSION EXAMINATIONS

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  1. HEART PERFUSION EXAMINATIONS

  2. ADVANTAGES OF IN VITRO HEART PERFUSION STUDIES • Can be studied quickly and in large number • Highly reproducible • Enables biochemical, physiological, morphological studies • Absence of confounding effect of organs, systemic circulation, neurohumoral factors • Drugs, hormones can be added exogenously in a controlled manner • Dose-response studies • Can be studied for several hours • Regional/global ischemia – anoxia/hypoxia - studies • Induction of arrhythmias • ECG – mapping and ablation of conduction pathways

  3. SPECIES FOR PERFUSION • Mammalian/non-mammalian hearts (frog, bird) • Large animal hearts: • Pig, monkey, sheep, dog • High cost, greater variability, large volumes of perfusion fluids, special equipment • Most frequently studied: • Rat, rabbit, guinea pig, hamster, ferret, mouse • Transgenic technology • Mouse hearts: small, high heart rate • Rat: best characterized, most frequently used, ease of handling • Difficulties: • Rat: short action potential • Rabbit: anesthesia • Guinea pig: collateralized vasculature

  4. HEART PERFUSION PREPARATIONS • LANGENDORFF HEART PERFUSION SYSTEM • „WORKING-HEART” PREPARATION DESCRIBED BY NEALY

  5. LANGENDORFF HEART PREPARATION MODES OF PERFUSATE DELIVERY: -constant flow rate -constant hydrostatic pressure -Shattock electrical feedback system Used with hearts from mice, rats, guinea pigs, rabbits

  6. 18 LANGENDORFF PERFUSION OF RAT HEARTPREPARATION I. • Anesthesia: • Inhalation of agents (ether, halothane or metoxyflurane) • Injection: (i.p., i.v.) (pentobarbitone) • Anticoagulation: Heparin • Excision of the heart from the donor animal • Immersion of heart in cold perfusion solution (40C)

  7. Cannulation of the heart ! Air emboli ! Coronary ostia ! Aortic valves

  8. LANGENDORFF PERFUSION OF RAT HEARTPREPARATION II. • Washout period for 10 minutes • Instrumentation: • Contractile function measurements: -Open tip pressure transducer with intraventricular balloon –intraventricular pressure, heart rate monitoring • Pace -bipolar silver wire electrode • ECG -stainless steel cannula

  9. „Working-heart” Preparation Advantages: -filling pressures and afterload can be controlled Used with hearts from rats, dogs, pigs

  10. PERFUSION TEMPERATURE • Near or at the normal body temperature • 37.0-37.50C • Temperature control: -Thermostatically regulated cabinet in which warm air is circulated - Thermostatically controlled water-jacketed system • Avoid over-heating !

  11. Parameters determined during heart perfusion I. • Morphology and vascular anatomy • Light/electron microscopy • Microbiopsies • Fixation by perfusion • Biochemistry • Arterio-venous differences in substrates, metabolites (lactate, oxygen, proteins, enzymes) • Biopsies, NMR spectroscopy: on-line measurement of high energy phosphates, metabolites, ions • Microelectrodes: ions, pH, action potential • Delivery of vectors in gene transfer studies • Cardiac rhythm and electrophysiology • Conduction pathway mapping and selective ablation

  12. Parameters determined during heart perfusion II. • Cardiac contractile function • Systolic, diastolic pressures, cardiac pump function • Echo techniques – pressure volume relationships, indices of contractile function • Pharmacology • Various therapeutic agents, dose-response studies • Great speed and reproducibility, drugs can be easily washout • Vascular biology • Vascular reactivity, endothelial and smooth muscle function • Interventions on coronary flow and its distribution

  13. COMPOSITION OF PERFUSION FLUID • Krebs-Henseleit • pH=7.4 • NaCl: 118.5 mM, NaHCO3: 25.0 mM, KCl: 4.7 mM, MgSO4: 1.2, KH2PO4: 1.2, glucose: 11.0 mM, CaCl2: 2.5 mM • Calcium? • Calcium and phosphate? • Glucose? • Fatty acids? • Edema? • Filtration: 5 μm filter

  14. OXYGEN DELIVERY DURING PERFUSION • Perfusion with asanguinous perfusion fluids • Perfusion with blood • Perfusion with oxygen-carrying hemoglobin substitutes • Gassed perfusion solution: 95% oxygen + 5% CO2 • Required to the correct pH • In case of addition of fatty acids or proteins membrane oxigenator is recommended • Parabiotic preparation with support rat

  15. BLOOD PERFUSION -Decrease of hematocrit to 28-30% with Gelofusine solution -Ventilation of support rat with 95% oxygen -Control of body temperature, blood pressure, breathing Less edema Stable heart function Almost physiological coronary flow rate Blood elements (neu) Support animal?

  16. Stability Less edema Immunological reactions ERYTHROCYTE PERFUSION • Washed red blood cells • Membrane oxygenator • Hematocrit of 25-40% • Blood cells from different species – sheep

  17. 31P NMR SPECTROSCOPY/ LANGENDORFF HEART PERFUSION Ischemia – blood supply is less than the required amount Reperfusion– restoration of blood flow after coronary occlusion Ischemic preconditioning – short repeated ischemic episodes evokes the preconditioning of the heart (that means cardioprotection during the next longer ischemic period )

  18. ENERGY METABOLISM IN THE STRIATED AND HEART MUSCLE Striated muscle Heart muscle Mitochondria ++ +++++ Basic act. FFA (adipose tissue) keton bodies (liver) Medium act. FFA FFA +++ keton bodies blood glucose + blood glucose keton bodies + Max. act.+fermentation of glycogen + creatine-phosphate +creatine-phosphate Energy metabolism.mostly aerobic fully aerobic max. act. - anaerobic Energy pools glycogen creatine-phosphate creatine-phosphate (glycogen)

  19. CREATINE/PHOSPHOCREATINE TRANSFORMATION

  20. REPRESENTATIVE 31P NMR SPECTUMS OF HIGH ENERGY PHOSPHATES PCr Pi γ-P α-P β-P ATP IR+G IR I N

  21. RECOVERY OF CREATINE PHOSPHATE AFTER ISCHEMIA-REPERFUSION IN LANGENDORFF PERFUSED HEART

  22. DETERMINATION OF HEART FUNCTION • Insertion of a latex balloon into the left ventricle • End-diastolic pressure 8-12 mmHg • Selection of hearts: on the basis of the stability of high-energy phosphates (assessed by NMR) • Normoxia 15min, ischemia 25 min, reperfusion 45 min • Functional data: • LVEDP=left ventricular end-diastolic pressure • LVDP=levt ventricular developed pressure • RPP=rate pressure product • HR=heart rate • dP/dt

  23. 89.25 Hgmm 9.25 Hgmm 1000 msec 0 msec 25.5 Hgmm 8.5 Hgmm 0 msec 1000 msec • LVDP=levt ventricular developed pressure • RPP=rate pressure product HR=heart rate dP/dt • LVEDP=left ventricular end-diastolic pressure DOXORUBICIN-INDUCED DETERIORATION OF HEART FUNCTION

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