Westphal M, et al.
Effects of AVP in acute lung injury
1
Cardiopulmonary effects of low-dose arginine vasopressin
in ovine acute lung injury
Martin Westphal, MD, PhD; Sebastian Rehberg, MD; Marc O. Maybauer MD, PhD; Dirk
M. Maybauer, MD, PhD; Perenlei Enkhbaatar, MD, PhD; Beena B. Westphal-Varghese,
MD; Frank C. Schmalstieg, MD, PhD; Naoki Morita, MD, PhD; Robert A. Cox, PhD;
Lillian D. Traber, RN; Hal Hawkins, MD, PhD; Elbert Whorton, PhD, Daniel L. Traber,
PhD
Supplemental digital content
Westphal M, et al.
Effects of AVP in acute lung injury
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Materials and Methods
Instrumentation and surgical procedures. Briefly, a right femoral artery catheter
(18-GA, 36 inches; Parke-Davis, Sandy, UT, USA), a left atrial catheter (0.062 inch inner
diameter, 0.125 inch outer diameter; Dow Corning, Midland, MI, USA) and a Swan-Ganz
thermal dilution catheter (REF 131F7; Edwards Lifesciences LLC, Irvine, CA, USA) were
placed. All intravascular catheters were connected to pressure transducers with
continuous flushing devices (model PX3X3, Baxter-Edwards Critical Care, Irvine, CA).
The animals were then allowed to recover for one week with free access to water and
food.
Mechanical ventilation. All animals were mechanically ventilated (Servo
Ventilator 900C, Siemens-Elema, Sweden) with a tidal volume of 15 mL·kg-1 and a
positive end-expiratory pressure of 5 cmH2O throughout the entire experiment. These
ventilator settings are in accordance with previous studies using the same animal model
(1-3). In this context, it is important to consider that the pulmonary compliance in sheep
is higher than in humans. Therefore, higher tidal volumes than in humans are required
to guarantee adequate ventilation. The inspiratory oxygen fraction (FiO2) was set at
100% for the first 3 h post injury, and was then adjusted to maintain sufficient
oxygenation (arterial oxygen saturation > 95%, partial pressure of oxygen > 90 mmHg),
whenever possible. The respiratory rate was initially set at 30 breaths·min-1 and was
then adjusted according to individual blood gas analyses.
Hemodynamics and fluid balance. Heart rate, mean arterial pressure, mean
pulmonary arterial pressure, central venous pressure, pulmonary artery occlusion
pressure, and left atrial pressures were recorded on a hemodynamic monitor (model
Westphal M, et al.
Effects of AVP in acute lung injury
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7830A, Hewlett Packard, Santa Clara, Calif). Cardiac output was measured in triplicate
with the thermodilution technique (Monitor 9530; Baxter-Edwards Critical Care, Irvine,
CA), using 10 mL of ice-cold 5% dextrose solution as indicator. Cardiac index,
pulmonary and systemic vascular-resistance indices as well as stroke volume index, leftand right ventricular stroke work indices were calculated using standard equations (4).
Hemodynamic measurements were performed at baseline and repeated at 1, 3, 6, 12,
18, and 24 h.
Since sheep do not perspire and the inhaled gases were humidified, fluid balance
was calculated as the difference of fluid input and urinary output (2). During the
experiment, the animals had free access to food, but not to water to accurately
determine fluid intake.
Pulmonary gas exchange, acid-base balance and white blood cell count
Corresponding to the hemodynamic measurements, samples of arterial and matched
mixed venous blood were analyzed for gas tensions, oxygen saturation, pH and base
excess using a blood gas analyzer (System 1302; Instrumentation Laboratory,
Lexington, MA); all measurements were corrected for core body temperature.
Pulmonary shunt fraction and PaO2/FiO2 ratio were calculated using standard formulas
(4). Plasma protein concentrations were measured using a refractometer (National
Instrument, Baltimore, MD, USA). Plasma oncotic pressures were determined through a
semi-permeable membrane in a colloid osmometer (Model 4420; Wescor, Logan, UT,
USA). Hematocrit was measured by blood centrifugation in heparinized microhematocrit
capillary tubes (Fisher Brand, Pa, USA).
Westphal M, et al.
Effects of AVP in acute lung injury
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Blood was sampled under sterile conditions in tubes containing EDTA as
anticoagulant. White blood cell count was immediately determined with a Hemavet
850™ (Drew Scientific, Inc., Oxford, CT) adjusted for sheep blood. Plasma
concentrations of nitric oxide and its metabolites were evaluated by measuring nitric
oxide, nitrate and nitrite (NOx) at baseline, 1, 3, 6, 12, 18, and 24 h using an established
protocol (5, 6).
Obstruction scores of bronchi and bronchioles. Samples of the right lung (middle
and upper lobe) of each animal were excised, injected, and immersed with 10%
phosphate-buffered formalin. Fixed samples were then embedded in paraffin, sectioned
into 6-μm slices, and stained with hematoxylin-eosin. A pathologist unaware of the study
protocol evaluated the degree of airway obstruction according to a standardized
protocol. In brief, 100–200 bronchi and 20–50 bronchioles were investigated per sheep,
and the percentage of cross-sectional area obstructed by casts was determined (0–
100%). Finally, a mean value for airway obstruction of both bronchi and bronchioles was
calculated as described in detail by Cox and colleagues (7).
Westphal M, et al.
Effects of AVP in acute lung injury
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References
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Westphal M, Enkhbaatar P, Schmalstieg FC, et al. Neuronal nitric oxide synthase
inhibition attenuates cardiopulmonary dysfunctions after combined burn and smoke
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Enkhbaatar P, Connelly R, Wang J, et al. Inhibition of neuronal nitric oxide synthase
in ovine model of acute lung injury. Crit Care Med 2009;37(1):208-214.
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contents.
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Cox RA, Burke AS, Soejima K, et al. Airway obstruction in sheep with burn and
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