Heart perfusion examinations
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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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Advantages of in vitro heart perfusion studies
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


Species for perfusion
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


Heart perfusion preparations
HEART PERFUSION PREPARATIONS

  • LANGENDORFF HEART PERFUSION SYSTEM

  • „WORKING-HEART” PREPARATION DESCRIBED BY NEALY


Langendorff heart preparation
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


Langendorff perfusion of rat heart preparation i

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)


Cannulation of the heart

! Air emboli

! Coronary ostia

! Aortic valves


Langendorff perfusion of rat heart preparation ii
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


Working heart preparation
„Working-heart” Preparation

Advantages:

-filling pressures and afterload can be controlled

Used with hearts from rats, dogs, pigs


Perfusion temperature
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 !


Parameters determined during heart perfusion i
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


Parameters determined during heart perfusion i i
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


Composition of perfusion fluid
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


Oxygen delivery during perfusion
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


  • Blood perfusion
    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?


    Erythrocyte perfusion

    Stability

    Less edema

    Immunological reactions

    ERYTHROCYTE PERFUSION

    • Washed red blood cells

    • Membrane oxygenator

    • Hematocrit of 25-40%

    • Blood cells from different species – sheep


    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 )


    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)



    REPRESENTATIVE 31P NMR SPECTUMS OF HIGH ENERGY PHOSPHATES

    PCr

    Pi

    γ-P

    α-P

    β-P

    ATP

    IR+G

    IR

    I

    N



    Determination of heart function
    DETERMINATION OF HEART FUNCTION LANGENDORFF PERFUSED HEART

    • 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


    89.25 Hgmm LANGENDORFF PERFUSED HEART

    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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