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SUPPLEMENTAL DIGITAL CONTENT 1Rabbit anesthesiaAfter subcutaneous infiltration of 1% lidocaine each rabbit had a 22-gauge catheter inserted in the left ear vein and anesthesia was intravenously induced with 5 to 10?mg/kg propofol (Fresofol 1%, Pharmatel Fresenius Kabi, Austria). The rabbit was intubated and mechanically ventilated with 100% oxygen, initial tidal volumes of 6?ml/kg, and a rate of 60 breaths/min on a small animal ventilator 7025, Ugo Basile, Comerio, Italy. After induction, rabbits received anesthesia according to the assigned treatment protocol as described. Rabbits anesthetized with volatile anesthesia alone received 0.9% saline with infusion rates of 3.5 to 7 ml/kg/hour to match the volumes of infusion received in the intravenous anesthesia protocols. Anesthesia concentration was adjusted in anticipation of painful events such as skin incision or sternotomy to ensure that depth of anesthesia was sufficient, whereas anesthesia was run at the prescribed level, when surgical stimulation was absent. Adequate depth of anesthesia was assessed at regular intervals by deep paw pinch, and by looking for presence of apnea in situations when ventilation was intermittently ceased ventilation during surgical access to the heart. Rabbit surgerySaline-filled catheters were inserted into the right internal carotid artery and into the right atrium through the right internal jugular vein for monitoring of systemic arterial and right atrial pressures, respectively. Arterial blood gasses were sampled and repeated every hour in order to adjust ventilation parameters to maintain pH = 7.4, PaCO2 = 40 to 45 mmHg, and PaO2 > 100 mmHg. The heart was exposed via a median sternotomy and a Doppler flow probe (T206, Transonic Systems Inc., Ithaca, New York, USA) was placed around the ascending aorta for recording of cardiac output and heart rate. A 3-French combined micro-manometer pressure catheter and 10-electrode dual-field conductance catheter (SPR-877, Millar Instruments, Texas, USA) was inserted into the left ventricle via an apical stab. A silicone sling was placed around the inferior vena cava in order to perform acute preload reduction as described below. Following the initial surgery, the rabbit received a bolus of 30 ml intravenous 0.9% NaCl solution and afterwards a period of 30 minutes was allowed for the preparation to stabilize prior to baseline recordings. During the procedure, a body temperature of 39°C was maintained using a regulated surface heating blanket (Harvard Apparatus, Holliston, Massachusetts, USA).If a rabbit was randomized to the ischemia-reperfusion, a silk suture was ligated around the proximal left descending artery (LAD) at a point one third of the distance from the origin of the LAD to the apex of the heart. LAD occlusion was confirmed by distal blanching of the myocardium. Data acquisition in the in vivo rabbit experimentsBefore data collection, the pressure-volume loop catheters were calibrated using α correctional value, and parallel wall conductance was determined using the hypertonic saline method, as previously described in detail and set out below.1 Left ventricular (LV) volumes were calculated based on electrical conductance measured using 5 segments of the LV conductance catheter, and high-fidelity LV pressures were simultaneously acquired with the same LV catheter. Recordings were performed at the following time points: 0, 2, 5, 10, 20, and 30 minutes (ischemia or time control), and then at 32, 35, 40, 50, 60, 70, 80, 90, 110, 130, and 150 minutes (reperfusion or time control). Right atrial pressure (RAP), mean arterial blood pressures (MAP), and heart rate (HR) were recorded on PowerLab, (8SP, AD Instruments, Sydney, Australia), and digitally stored using Chart v5.5.1, (AD Instruments, Sydney, Australia). The pressure-volume loops were recorded using Conduct NT v2.8 (CD Leycom, Zoetermeer, Netherlands), and analyzed offline using a CFL-512 Cardiac Function Laboratory (CD Leycom, Zoetermeer, Netherlands), to produce the following hemodynamic parameters: cardiac index (CI), systemic vascular resistance index (SVRI), preload-recruitable stroke-work (PRSW), and end-diastolic pressure-volume relationship (EDPVR). The SVRI was calculated as 80*(MAP-RAP)/CI, whereas PRSW and EDPVR were calculated by least mean of squares regression using the first 10 to 15 PV loops immediately following acute preload reduction (r2 > 0.9 was considered acceptable regression). After acquisition of hemodynamic data, rabbits were sacrificed, and myocardial tissue damage was assessed as previously described in detail.2 Pressure-Volume Loop Catheter (PV) CalibrationThe left ventricular (LV) conductance catheter, used to construct the PV loops, contains a 10-electrode dual-field system that has a flat frequency-response up to 10 kHz. When placed within the long axis of the left ventricle it allows high-fidelity recording of LV volume, along with simultaneous pressure measured through a pressure sensor at the distal tip of the catheter. Briefly, estimation of ventricular volume utilises the continuous measurement of electrical conductance in the blood by passing an alternating current (30μA RMS, 20kHz) in parallel and in series between electrodes producing an electric field in the LV cavity. This raw conductance signal is then converted to a calculated segmental volume by taking into account the specific conductivity of blood and the inter-electrode distances. Hence, time-varying conductance Gi(t) is measured between electrodes and conductance-derived segmental volume between each electrode [Vi(t)] is calculated:Such that, Vi(t) = [1/?i][Li2/?b][Gi(t)-GiP]Where,?i = segmental dimensionless slope constantLi = inter-electrode distance?b = electrical conductivity of bloodGiP = segmental parallel conductance of myocardium and surrounding tissuesFor each segment between electrodes, conductance-derived volume is determined by the total conductance of cavity blood, myocardial wall, and fluids surrounding the heart. This introduces an offset in the relation between true LV intra-cavity segmental volume and the conductance-derived volume. True LV intra-cavity segmental volume is therefore determined by subtraction of the offset volume from the raw conductance-derived volume. Calculation of offset volume is determined by measurement of GiP by the introduction of 0.07?ml?kg-1 10% saline solution into the right atrial catheter with simultaneous recording of PV calibration loops, which were downloaded into individual volume calibration (Vc) files and used to correct for volume offset in subsequent offline analysis.3 In this way, hypertonic saline transiently changes the conductivity of blood without changing GiP thus allowing the latter to be calculated simultaneously during the passage of the bolus through the left ventricle. Total conductance-derived volume [V(t)] was then calculated on the basis of a stacked-cylinder model of individual segmental volumes.Such that, V(t) = [1/?][L2/?b][G(t)-GP]Where,? = average dimensionless slope constantL = total electrode distance of all segments?b = electrical conductivity of bloodGP = total parallel conductance of myocardium and surrounding tissues of all segmentsDuring acquisition of each PV loop, the ratio of the cardiac output measured by the LV conductance catheter to the cardiac output as determined by the aortic flow probe was used to determine ?. Five ml of rabbit blood was withdrawn to determine specific blood resistance (ρ).Where, ?b = 1/ ρ.Determination of Rabbit Myocardial Tissue DamageFollowing data collection, animals that had received the ischemia-reperfusion perfusion protocol were administered heparin (1000 I.U. I.V.) and the LAD artery was permanently tied-off at the same position where the temporary ligature had been placed. Rabbits were then killed with a lethal dose of sodium pentobarbitone (140 mg kg-1) and the heart was excised en bloc with surrounding great vessels. The aortic root was cannulated and directly injected with 1 ml of 2% Evans Blue dye. This resulted in blue staining of all the myocardium except for the region supplied by the vascular bed distal to the ligated LAD (area-at-risk). The left ventricle was then separated from the rest of the heart and frozen. The left ventricle was weighed, and then sectioned with a sharp razor blade, parallel to the atrio-ventricular groove, into 2 mm thick slices. The unstained portion of the left ventricle (area-at-risk) was separated from the blue stained portion (area- not- at- risk) and weighed. Area-at-risk heart slices were then incubated in 2,3,5 triphenyl 2H-tetrazolium chloride (TTC) in phosphate buffer (pH 7.4) at 37°C for 15 min.4 NADH, the co-enzyme present in viable tissue, is stained brick-red by TTC5, and can be easily differentiated from infarcted tissue which takes on a pale bleached appearance. All portions of the left ventricle were stored in 4% formaldehyde for 1-2 days before area-at-risk and infarct size were sizes were determined. Digital photographs were recorded and the area of infarction, in area-at-risk, on both sides of the tissue slices were then traced and averaged using ImageJ software v1.44c for Mac (National Institutes of Health, USA). Area-at-risk was then calculated by the sum of the ratios of the weights of the non-Evans Blue stained sections to total weight of each LV slice. Similarly, the infarct to area-at-risk ratio was then calculated by dividing the area of the pale tissue by the area-at-risk.REFERENCES1.Erb TO, Craig DM, Gaskin PM, Cheifetz IM, Resai Bengur A, Sanders SP: Preload recruitable stroke work relationship in the right ventricle: simultaneous assessment using conductance catheter and sonomicrometry. Crit Care Med 2002; 30: 2535-412.Andrews DT, Royse AG, Royse CF: Functional comparison of anaesthetic agents during myocardial ischaemia-reperfusion using pressure-volume loops. Br J Anaesth 2009; 103: 654-643.Steendijk P, Lardenoye JW, van der Velde ET, Schalij MJ, Baan J: Evaluation of a new transcardiac conductance method for continuous on-line measurement of left ventricular volume. Crit Care Med 2000; 28: 1599-6064. Khalil PN, Siebeck M, Huss R, Pollhammer M, Khalil MN, Neuhof C, Fritz H: Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts. J Pharmacol Toxicol Methods 2006; 54: 307-125.H. H. Klein M.D. SP, J. Schaper, W. Schaper: The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Virchows Archiv A 1981; 393: 287-297 ................
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