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 - PowerPoint PPT Presentation

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<ul><li><p>HEART PERFUSION EXAMINATIONS</p></li><li><p>ADVANTAGES OF IN VITRO HEART PERFUSION STUDIESCan be studied quickly and in large numberHighly reproducibleEnables biochemical, physiological, morphological studiesAbsence of confounding effect of organs, systemic circulation, neurohumoral factorsDrugs, hormones can be added exogenously in a controlled mannerDose-response studiesCan be studied for several hours Regional/global ischemia anoxia/hypoxia - studiesInduction of arrhythmiasECG mapping and ablation of conduction pathways</p></li><li><p>SPECIES FOR PERFUSIONMammalian/non-mammalian hearts (frog, bird)Large animal hearts:Pig, monkey, sheep, dogHigh cost, greater variability, large volumes of perfusion fluids, special equipmentMost frequently studied:Rat, rabbit, guinea pig, hamster, ferret, mouseTransgenic technology Mouse hearts: small, high heart rateRat: best characterized, most frequently used, ease of handlingDifficulties:Rat: short action potentialRabbit: anesthesiaGuinea pig: collateralized vasculature</p></li><li><p>HEART PERFUSION PREPARATIONS</p><p>LANGENDORFF HEART PERFUSION SYSTEM</p><p>WORKING-HEART PREPARATION DESCRIBED BY NEALY </p></li><li><p>LANGENDORFF HEART PREPARATIONMODES OF PERFUSATE DELIVERY:-constant flow rate-constant hydrostatic pressure-Shattock electrical feedback systemUsed with hearts from mice, rats, guinea pigs, rabbits</p></li><li><p>LANGENDORFF PERFUSION OF RAT HEARTPREPARATION I.Anesthesia:Inhalation of agents (ether, halothane or metoxyflurane)Injection: (i.p., i.v.) (pentobarbitone)</p><p>Anticoagulation: Heparin </p><p>Excision of the heart from the donor animal</p><p>Immersion of heart in cold perfusion solution (40C)</p></li><li><p>Cannulation of the heart! Air emboli! Coronary ostia! Aortic valves </p></li><li><p>LANGENDORFF PERFUSION OF RAT HEARTPREPARATION II. Washout period for 10 minutesInstrumentation:Contractile function measurements: -Open tip pressure transducer with intraventricular balloon intraventricular pressure, heart rate monitoringPace-bipolar silver wire electrodeECG-stainless steel cannula</p></li><li><p>Working-heart PreparationAdvantages:-filling pressures and afterload can be controlled</p><p>Used with hearts from rats, dogs, pigs</p></li><li><p>PERFUSION TEMPERATURENear or at the normal body temperature37.0-37.50CTemperature control:-Thermostatically regulated cabinet in which warm air is circulated- Thermostatically controlled water-jacketed systemAvoid over-heating !</p></li><li><p>Parameters determined during heart perfusion I.Morphology and vascular anatomyLight/electron microscopyMicrobiopsiesFixation by perfusionBiochemistryArterio-venous differences in substrates, metabolites (lactate, oxygen, proteins, enzymes)Biopsies, NMR spectroscopy: on-line measurement of high energy phosphates, metabolites, ionsMicroelectrodes: ions, pH, action potentialDelivery of vectors in gene transfer studiesCardiac rhythm and electrophysiologyConduction pathway mapping and selective ablation</p></li><li><p>Parameters determined during heart perfusion II. Cardiac contractile functionSystolic, diastolic pressures, cardiac pump functionEcho techniques pressure volume relationships, indices of contractile functionPharmacologyVarious therapeutic agents, dose-response studiesGreat speed and reproducibility, drugs can be easily washoutVascular biologyVascular reactivity, endothelial and smooth muscle functionInterventions on coronary flow and its distribution</p></li><li><p>COMPOSITION OF PERFUSION FLUIDKrebs-HenseleitpH=7.4NaCl: 118.5 mM, NaHCO3: 25.0 mM, KCl: 4.7 mM, MgSO4: 1.2, KH2PO4: 1.2, glucose: 11.0 mM, CaCl2: 2.5 mMCalcium?Calcium and phosphate?Glucose?Fatty acids?Edema?Filtration: 5 m filter</p></li><li><p>OXYGEN DELIVERY DURING PERFUSIONPerfusion with asanguinous perfusion fluidsPerfusion with bloodPerfusion with oxygen-carrying hemoglobin substitutes </p><p>Gassed perfusion solution:95% oxygen + 5% CO2Required to the correct pHIn case of addition of fatty acids or proteins membrane oxigenator is recommended</p><p>Parabiotic preparation with support rat</p></li><li><p>BLOOD PERFUSION-Decrease of hematocrit to 28-30% with Gelofusine solution-Ventilation of support rat with 95% oxygen-Control of body temperature, blood pressure, breathingLess edemaStable heart functionAlmost physiological coronary flow rateBlood elements (neu)Support animal?</p></li><li><p>ERYTHROCYTE PERFUSIONWashed red blood cellsMembrane oxygenatorHematocrit of 25-40% Blood cells from different species sheep</p></li><li><p>Ischemia blood supply is less than the required amountReperfusion restoration of blood flow after coronary occlusionIschemic preconditioning short repeated ischemic episodes evokes the preconditioning of the heart (that means cardioprotection during the next longer ischemic period )31P NMR SPECTROSCOPY/LANGENDORFF HEART PERFUSION</p></li><li><p>ENERGY METABOLISM IN THE STRIATED AND HEART MUSCLEStriated muscleHeart muscleMitochondria++ +++++Basic act. FFA (adipose tissue)keton bodies (liver)Medium act.FFAFFA +++keton bodies blood glucose +blood glucoseketon bodies +Max. act.+fermentation of glycogen+ creatine-phosphate +creatine-phosphateEnergy metabolism.mostly aerobicfully aerobicmax. act. - anaerobicEnergy poolsglycogen creatine-phosphate creatine-phosphate (glycogen )</p></li><li><p>CREATINE/PHOSPHOCREATINE TRANSFORMATION</p></li><li><p>PiPCr-P-P-PATPIR+GIRINREPRESENTATIVE 31P NMR SPECTUMS OF HIGH ENERGY PHOSPHATES</p></li><li><p>RECOVERY OF CREATINE PHOSPHATE AFTER ISCHEMIA-REPERFUSION IN LANGENDORFF PERFUSED HEART</p><p>Chart1</p><p>100100100.25000000</p><p>99.810499.52.42.43.73.70.90.9</p><p>99.299.6598.252.72.74.54.52.82.8</p><p>24.233.31204.84.84.24.25.45.4</p><p>01.155.1000000</p><p>000000000</p><p>18.123.8740.54.24.23.73.73.13.1</p><p>24.245.4546.95.75.75.45.45.15.1</p><p>22.142.1546.14.54.55.85.85.45.4</p><p>21.338.43415.25.26.26.24.84.8</p><p>19.834.636.13.83.84.54.54.54.5</p><p>Kontroll IR</p><p>IR+L-2286 10 M</p><p>IR+L-2286 20 M</p><p>perfzis id (perc)</p><p>CrP (normoxis rtk %-a)</p><p>Sheet1</p><p>IRL-2286 10L-2286 20L- 10%KORRL-20KORR</p><p>3100100100.250001444107.04225352111001388100100</p><p>699.810499.50.92.43.7133598.9621942179103110979.899135446799</p><p>999.299.6598.252.82.74.5134910010097970.533141210497</p><p>324.233.31205.44.84.229321.719792438821.71178.42939481278</p><p>1501.155.1000000755.40345821335</p><p>30000000000443.17002881840</p><p>318.123.8740.53.14.23.744933.283914010433.259542.867435158542.8</p><p>624.245.4546.95.15.75.462346.182357301746.265447.118155619647.1</p><p>922.142.1546.15.44.55.852638.99184581173965647.262247838647.3</p><p>1221.338.43414.85.26.236326.908821349135.562845.244956772345.2</p><p>1519.834.636.14.53.84.544032.616753150532.656140.417867435240.4</p><p>2/12/04</p><p>L-10L-20</p><p>21591001002268100.5319148936100.5</p><p>2268105.0486336267105224499.468085106499.5</p><p>214499.305233904699.393241.312056737611</p><p>97044.928207503544.9226011.5248226955.2</p><p>502.31588698472.31506.64893617020</p><p>0001175.186170212838.2</p><p>31414.54377026414.540046.7</p><p>96644.742936544744.71466.471631205744.9</p><p>97945.345067160745.30036.8</p><p>89341.36174154741.3686138.16489361731.8</p><p>79136.637332098236.6105346.6755319149</p><p>101344.9024822695</p><p>83136.835106383</p><p>71831.8262411348</p><p>100100100</p><p>103105104</p><p>10099.399.65100100.5100.25</p><p>21.744.9233.319910099.5</p><p>02.31.159799.598.25</p><p>0008119.5</p><p>33.214.5423.8755.25.1</p><p>46.244.745.45000</p><p>3945.342.1542.838.240.5</p><p>35.541.3638.4347.146.746.9</p><p>32.636.634.647.344.946.1</p><p>45.236.841</p><p>40.431.836.1</p><p>Sheet1</p><p>000000000</p><p>0002.42.43.73.70.90.9</p><p>0002.72.74.54.52.82.8</p><p>0004.84.84.24.25.45.4</p><p>000000000</p><p>000000000</p><p>0004.24.23.73.73.13.1</p><p>0005.75.75.45.45.15.1</p><p>0004.54.55.85.85.45.4</p><p>0005.25.26.26.24.84.8</p><p>0003.83.84.54.54.54.5</p><p>IR</p><p>IR+4OHQ</p><p>IR+HO</p><p>perfusion time (min)</p><p>CrP (% of normoxic value)</p><p>Sheet2</p><p>kontrollL-2286 10L-2286 20L10L20L10</p><p>30.72.50.860000018.70.541558065505.03978779842.5198938992</p><p>61.36.20.090.81.83.769.652.1055018138002.105501813810.26904130356.1872715587</p><p>90.541.12.042.52.34.5631.9044740024001.90447400240.37893141341.1417027079</p><p>350.3545.78555.24.64.2126338.1801692866149543.295684911738.180169286653.391436150145.7858027183</p><p>1571.670.9672.460.0100220366.5961305925206359.745149145766.596130592575.331564986770.9638477896</p><p>3010010010000033081003453100100100100</p><p>361.562.4662.652.82.63.7221767.0193470375186554.011004923367.019347037557.900719969762.4600335036</p><p>645.145.9931.65.14.62.6152546.100362757108031.277150304146.10036275745.888594164545.9944784607</p><p>935.4637.331.994.54.53.2133340.2962515115112032.435563278340.296251511534.255399772637.2758256421</p><p>123834.1325.264.85.32.9118335.761789601129537.503620040535.76178960132.512315270934.137052436</p><p>1543.132.527.794.53.82.5113334.2503022975111432.261801332234.250302297530.920803334632.585552816</p><p>1335.0397877984291.18706508390.54155806551.18706508390.8643115747</p><p>27110.26904130354.80.19647973800.1964797380.098239869</p><p>100.37893141341004.093327875604.09332787562.0466639378</p><p>140953.3914361501188076.954564060643.295684911776.954564060660.1251244861</p><p>198875.3315649867208185.182153090559.745149145785.182153090572.4636511181</p><p>26391002443100100100100</p><p>152857.9007199697174271.305771592354.011004923371.305771592362.6583882578</p><p>121145.888594164578031.927957429431.277150304131.927957429431.6025538667</p><p>90434.255399772677131.559557920632.435563278331.559557920631.9975605994</p><p>85832.512315270931813.016782644337.503620040513.016782644325.2602013424</p><p>81630.920803334657023.331968890732.261801332223.331968890727.7968851114</p><p>L5 azaz kontroll</p><p>280.7637752319</p><p>481.3093289689</p><p>200.545553737</p><p>186350.8183306056</p><p>262771.6584833606</p><p>3666100</p><p>198854.2280414621</p><p>165445.1172940535</p><p>130035.4609929078</p><p>129838</p><p>158243.1533006001</p><p>Sheet2</p><p>100100100.25000000</p><p>99.810499.52.42.43.73.70.90.9</p><p>99.299.6598.252.72.74.54.52.82.8</p><p>24.233.31204.84.84.24.25.45.4</p><p>01.155.1000000</p><p>000000000</p><p>18.123.8740.54.24.23.73.73.13.1</p><p>24.245.4546.95.75.75.45.45.15.1</p><p>22.142.1546.14.54.55.85.85.45.4</p><p>21.338.43415.25.26.26.24.84.8</p><p>19.834.636.13.83.84.54.54.54.5</p><p>Kontroll IR</p><p>IR+L-2286 10 mcM</p><p>IR+L-2286 20 mcM</p><p>perfzis id (perc)</p><p>CrP (normoxis rtk %-a)</p><p>Sheet3</p><p>000000000</p><p>0000.80.81.81.83.73.7</p><p>0002.52.52.32.34.54.5</p><p>0005.25.24.64.64.24.2</p><p>0000.010.010000</p><p>000000000</p><p>0002.82.82.62.63.73.7</p><p>0005.15.14.64.62.62.6</p><p>0004.54.54.54.53.23.2</p><p>0004.84.85.35.32.92.9</p><p>0004.54.53.83.82.52.5</p><p>Kontroll IR</p><p>IR+L-2286 10 mcM</p><p>IR+L-2286 20 mcM</p><p>perfzis id (perc)</p><p>Pi (Ischaemia vgi rtk %-a)</p></li><li><p>DETERMINATION OF HEART FUNCTIONInsertion of a latex balloon into the left ventricleEnd-diastolic pressure 8-12 mmHgSelection of hearts: on the basis of the stability of high-energy phosphates (assessed by NMR)Normoxia 15min, ischemia 25 min, reperfusion 45 minFunctional data:LVEDP=left ventricular end-diastolic pressureLVDP=levt ventricular developed pressureRPP=rate pressure productHR=heart ratedP/dt</p></li><li><p>LVDP=levt ventricular developed pressureLVEDP=left ventricular end-diastolic pressureHR=heart rateRPP=rate pressure productdP/dtDOXORUBICIN-INDUCED DETERIORATION OF HEART FUNCTION</p></li></ul>