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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)
slide7

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
slide19

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 )

slide20

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)

determination of heart function
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
slide25

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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