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Inotropes in neonatal asphyxiaJoynt et al
Milrinone, Dobutamine or Epinephrine Use in Asphyxiated Newborn Pigs ResuscitatedWith 100% Oxygen
METHODS
Mixed breed piglets (1-3d; 1.5-2.3 kg) were obtained on the day of experimentation. Piglets were excluded for weight < 1.5 kg, evidence of preexisting illness, or hemoglobin < 60 g/L. The protocol followed the guidelines of the Canadian Council on Animal Care (2000) and approved by the institutional Animal Care and Use Committee.
Animal Preparation
Animal preparation and H-R protocol have been previously described.12 Briefly, the piglets were anesthetized with isofluorane, which was then switched to intravenous midazolam (0.1-0.2 mg/kg/h), fentanyl (5-50 µg/kg/h) and pancuronium (0.05-0.1 mg/kg/h) to provide anesthesia, analgesia and paralysis, respectively. Additional boluses of fentanyl (10 µg/kg) and acepromazine (0.25 mg/kg) were given as necessary. Pancuronium was discontinued after thoracotomy. Oxygen saturation, measured by pulse oximetry (Nellcor, Hayward, CA), was kept between 90-100% by adjusting the fractional inspired oxygen concentration (0.21-0.24). Infusions of 10% dextrose (10 mL/kg/h, venous) and of 0.9% normal saline (2 mL/kg/h, arterial) were maintained during the experiment.
Through femoral vessels, 5F catheters (Sherwood Medical Co., St Louis, MO) were placed at right atrium and the infrarenal aorta to measure central venous pressure and mean arterial pressure (MAP), respectively. Via a tracheostomy, animals were mechanically ventilated (ventilator model IV-100, Sechrist Industries Inc., Anaheim, CA) at a rate of 18-20 breaths/min and pressure of 19/4 cm H2O.
Through a left anterior thoracotomy, the main pulmonary artery was exposed and cannulated with a 20G catheter (Arrow International, Reading, PA) to measure pulmonary arterial pressure (PAP). A Transonic® flow probe (6SB, Transonic Systems Inc, Ithica, NY) was placed around the pulmonary artery to monitor the blood flow as a surrogate of CO. The Ductus Arteriosus was ligated.
Via a neck incision, a 2SS Transonic® flow probe was placed to measure blood flow across the left common carotid artery. The abdominal aorta was exposed retroperitoneallyvia a flank incision. Superior mesenteric and left renal arteries were isolated and encircled byTransonic® flow probes (3SB and 2SB, respectively) to measure blood flows.
Incisions were covered and kept moist to minimize evaporative losses.Body temperature was kept at 38.5-39.5°C.
Hypoxia-Reoxygenation and Treatment Procedure
Piglets were block-randomized to 4 groups (n=6/group), which underwent H-R. A sham-operated group (n=4) had no H-R or medication delivery.
Alveolar hypoxia was induced atfractional inspired oxygen concentrationof 0.10-0.15 (PaO2 of 20-40 mmHg) for 2h. Piglets were then reoxygenated with 100% oxygen for 1h and a further 3h of 21% oxygen. Arterial PCO2 was maintained between 35-45 mmHg during the experimental period. Previous studies have determined that in this piglet model, this degree of normocapnic hypoxemia will produce clinical hypoxia followed by significant cardiac dysfunction and systemic hypotension at 2h of reoxygenation.24,25 All piglets received a 10 mL/kg Ringer’s lactate bolus 30 min prior to medication delivery. At 2h of reoxygenation, piglets received an intravenous infusion of placebo (0.9% NaCl), milrinone (0.75 μg/kg/min)(Pharmaceutical Partners of Canada Inc., Richmond Hill, ON), dobutamine (20 μg/kg/min)(Sandoz Canada, Boucherville, QC) or epinephrine (0.5 μg/kg/min)(Erfa Canada Inc., Westmount, QC) at 1 mL/kg/h for 2h in a blinded-randomized fashion. We previously demonstrated that these dosages optimally improved CO after 2h infusion in H-R piglets.12,13,24
Medication Preparation and Delivery
To maintain blinding, all infusions (clear, odorless) were identified by number only and were reconstituted in a standard volume of 0.9% NaCl solution by a technician uninvolved in the study. A loading milrinone bolus (75 μg/kg), or an equivalent 0.9% NaCl bolus (for the other medications), which was prepared in separate loading syringes and preflushed tubing by the technician, was given at 1.6 mL/kg over 15 min to the animals prior to the medication infusions.
Hemodynamic and Oxygen Transport Measurements
Heart rate, MAP, PAP, central venous pressure, pulmonary artery and regional blood flows were monitored continuously and recorded at normoxic baseline (0 min), at 2h of hypoxia (120 min), at 0.5h of reoxygenation (150 min) and 2h of reoxygenation (240 min, pretreatment), and every 30 min following medication or placebo delivery. Simultaneous arterial and venous blood samples were taken for blood gas analyses by ABL500 and OSM3 Hemoximeter (Radiometer, Copenhagen, Denmark). Hemodynamic variables were calculated as a mean over 2 min at these time-points. CO and carotid, SMA and renal flows were indexed for piglet weight (CAF, SMAF and RAF, respectively). Stroke volume, VR and oxygen transport were calculated using standard formula. The pulmonary VR was estimated by PAP÷CO because the left atrial pressure was not measured.
Arterial blood samples (1 mL) were taken at specified time points, centrifuged at 15,000 rpm for 10 min, and the supernatant collected and frozen at –80ºC for subsequent biochemical analysis. Less than 5% of the piglet blood volume was collected as bloodwork and replacements with Ringer’s lactate were given accordingly.
At the end of experiment, piglets were euthanized with intravenous pentobarbital (100 mg/kg). Samples of left ventricle and distal ileum were freeze-clamped in liquid nitrogen and stored at –80ºC orpreserved in 10% formalin for subsequent biochemical and histologic analysis, respectively.
Plasma Cardiac Troponin I,Plasma and Tissue Lactate Levels Determination
Plasma levels of porcine cardiac troponin I were assayed using a commercially available ELISA kit (Life Diagnostics, West Chester, PA)according to manufacturer’s recommendations.
To determine the level of lactate in plasma, enzyme-coupled NADH colorimetry method was used with spectrophotometry at 340 nm (Spectramax 190, Molecular Devices, Sunnyvale, CA).26For left ventricle and small intestine tissue lactate determination, frozen tissue (50 mg) was homogenized in 6% perchloric acid/ 0.5mM EGTA (50 μL). Following the addition of 5M potassium carbonate (1:10, v:v), the supernatant was prepared and substituted for plasma in the lactate assay described above.
Histopathology
Left ventricle and intestinal specimens underwent hematoxylin and eosin staining, and were evaluated by 2 independent pathologists (GC, LJ) blinded to treatment group. Pathologic injury was graded based on previously established criteria.27,28
Statistics
Results are expressed as mean±SD. Hemodynamic variables were analyzed by 2-way repeated measuresANOVA. Repeated measures ANOVA on ranks and Kruskal-Wallis test were used to analyze differences within and between groups, respectively, if normality failed. Post-hoc pairwise comparisons with Tukey or Dunnett’s method were studied when indicated. Analysis was performed using Sigma Stat (Version 2.0, Jandel Scientific, San Rafael, CA). Significance was defined as p<0.05.
ESM-Table 1 Arterial blood gases and plasma lactate levels in hypoxia- reoxygenated piglets treated with placebo (normal saline), epinephrine, dobutamine, or milrinone infusion. (n=6 per group).
Baseline / End of hypoxia / 1 hour of reoxygenation / Pretreatment / 2 hours of treatmentpH
Placebo-control / 7.40±0.02 / 6.99±0.08† / 7.26±0.09† / 7.37±0.06 / 7.37±0.06
Epinephrine / 7.44±0.04 / 7.04±0.04† / 7.31±0.07† / 7.40±0.06 / 7.38±0.05
Dobutamine / 7.42±0.04 / 7.02±0.06† / 7.30±0.1† / 7.41±0.06 / 7.41±0.05
Milrinone / 7.43±0.03 / 7.06±0.1† / 7.32±0.08† / 7.41±0.02 / 7.41±0.04
PaO2 (mmHg)
Placebo-control / 60±7 / 36±8† / 370±20† / 64±9 / 64±13
Epinephrine / 69±5 / 32±5† / 400±73† / 63±5 / 69±8
Dobutamine / 64±5 / 38±8† / 404±62† / 60±9 / 71±13
Milrinone / 62±7 / 31±6† / 373±60† / 62±6 / 58±4
PaCO2 (mmHg)
Placebo-control / 42±3 / 42±6 / 36±6 / 37±4 / 40±3
Epinephrine / 39±2 / 44±6 / 38±3 / 36±3 / 41±3
Dobutamine / 41±3 / 42±7 / 39±6 / 37±3 / 41±3
Milrinone / 39±3 / 42±5 / 39±5 / 37±4 / 40±3
HCO3- (mmol/L)
Placebo-control / 26±3 / 10±3† / 16±2† / 21±2† / 24±5
Epinephrine / 26±2 / 12±2† / 19±2† / 22±3† / 24±4
Dobutamine / 26±3 / 11±3† / 19±3† / 23±3† / 25±3
Milrinone / 25±3 / 12±4† / 20±3† / 23±3† / 25±2
Base Excess (mmol/L)
Placebo-control / 1.3±3.1 / -19.2±3.5† / -10.1±3.3† / -3.4±2.7 / -0.8±5.3
Epinephrine / 1.5±2.0 / -16.7±2.4† / -6.5±2.7† / -2.1±3.1† / -1.0±4.0
Dobutamine / 1.5±3.4 / -17.8±3.6† / -6.8±4.1† / -1.5±3.7† / 0.8±3.2
Milrinone / 1.3±2.8 / -16.2±4.9† / -5.5±3.5† / -1.2±3.2 / 0.6±2.6
Lactate (mmol/L)
Placebo-control / 4.0±1.1 / 13.7±6.2† / Not done / 6.4±2.1 / 4.5±3.0
Epinephrine / 4.8±1.1 / 13.1±5.8† / Not done / 3.7±3.1 / 5.4±1.8
Dobutamine / 4.6±0.7 / 15.9±1.4† / Not done / 5.9±1.3 / 3.3±1.3§
Milrinone / 3.8±1.4 / 12.3±5.7† / Not done / 4.2±0.6 / 2.4±1.2*§
*p<0.05 vs. placebo-controls
†p<0.05 vs. baseline
§p<0.05 vs. epinephrine-treated group
ESM-Figure Legends
Figure 4The effect of 2h of epinephrine, dobutamine, or milrinone infusion on regional vascular resistance (B) and oxygen delivery (C) (left carotid, superior mesenteric and left renal arteries) in newborn piglets during hypoxia and reoxygenation compared to placebo-controls (n=6 per group).
*p<0.05 all medication-treated groups vs. placebo-controls
#p<0.05 individual group vs. milrinone treated group
ESM-Figure 4B:
ESM-Figure 4C:
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