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HEART PERFUSION EXAMINATIONS

HEART PERFUSION EXAMINATIONS. 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

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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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