The Laboratory Animal Care Committee at the State University of New York at Buffalo

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Merilyn Boyd
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1 METHODS The Laboratory Animal Care Committee at the State University of New York at Buffalo approved all procedures and protocols in this study. PRENATAL LIGATION OF THE DUCTUS ARTERIOSUS: The technique for creating pulmonary hypertension by prenatal ligation of the ductus arteriosus has been described previously (1, 2, 11). Briefly, time-dated pregnant ewes (mixed breed) were operated on at 127 days of gestation (term = 146 days). Anesthesia was induced with 20 cc of a 5% solution of sodium thiopental and maintained with 1.5 to 2.0% halothane. The fetal head and left foreleg were delivered through a hysterotomy. A left lateral thoracotomy was performed in the fourth intercostal space of the fetus and the ductus arteriosus was ligated. The chest was closed and the fetus returned to the uterus. Postoperatively the ewe was treated with intramuscular ampicillin (300 mg/day) and gentamicin (40 mg/day) for 48 hours. The ewe was allowed to recover for 9 days. ISOLATED VESSEL PREPARATION: Following delivery by Cesarean section under the same anesthesia as described above, the umbilical cord was clamped. Prior to the first breath, lambs were sacrificed by rapid exsanguination through a direct cardiac puncture. We have described the subsequent techniques previously in detail (11, 12) Briefly, the heart and lungs were removed en bloc from the thorax immediately after death and placed in Krebs-Ringer solution (in mm = NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, KH 2 PO NaHCO , glucose 5.6, and calcium disodium ethylenediamine-tetraacetate 0.026). Fifth generation intralobar pulmonary arteries (13) with inside diameters of < 500 µ were isolated, dissected with care to preserve the integrity of the endothelium, and cut into rings approximately 2 mm wide and 0.7 to 1.5 mg in weight. Wet tissue weights were obtained at the end of each experiment after blotting the rings dry on gauze pads. The force of contraction was normalized by the weight of each ring and expressed as grams/gram of tissue (g/g). Vessel rings were mounted on stainless steel hooks and placed in

2 SOD in Combination with Inhaled NO Page 2 water-jacketed chambers. Tissues were bathed with 6 ml of the Krebs-Ringer solution, which was maintained at 37 o C, and aerated with a gas mixture of 94% O 2 and 6% CO 2 to maintain a ph of 7.40, a PCO 2 of 38 torr and a PO 2 of greater than 500 torr. A continuous recording of isometric force generation was obtained by tying each vessel ring to a force-displacement transducer (Statham UC 2; Statham Instruments, Hato Rey, PR) that was connected to an oscillographic recorder. Once mounted, the vessel rings were allowed to equilibrate for 20 minutes in the bathing solution. A micrometer was then used to stretch the tissues repeatedly in small increments over the next 45 minutes until resting tone remained stable at a passive tension of 1.0 grams for control arteries and 1.2 grams for arteries from hypertensive lambs. Preliminary experiments determined that this is the optimal length for generation of active tone in response to exogenous norepinephrine (11). The following pharmacological agents were used: indomethacin, L-norepinephrine (NE), DL-propranolol hydrochloride, polyethylene glycol-sod (SOD), polyethylene glycol-catalase (CAT), and S-nitrosyl-acetyl-penicillamine (SNAP). All drugs were purchased from Sigma Chemical Company (St. Louis, MO). All drugs were dissolved in distilled H 2 O except for indomethacin, which was dissolved in ethanol. Preliminary experiments showed ethanol had no effect in the concentrations used in the tissue bath studies. Drugs were made fresh daily. ISOLATED VESSEL PROTOCOL: Pulmonary arteries from hypertensive lambs were pretreated for 20 minutes with 10-6 M propranolol to block beta-adrenergic receptors, and with 10-5 M indomethacin to prevent the formation of vasoactive prostaglandins. They were preconstricted with an EC 50 concentration of norepinephrine (NE, 10-6 M), as determined from preliminary studies in which cumulative concentration-response curves for NE (10-8 to 10-5 M) were developed. Once the response to NE had reached a steady level, cumulative concentrationresponse curves to SNAP ( U/mL) were obtained by increasing its bath concentration in successive steps. The next concentration was added only when the response to the prior

3 SOD in Combination with Inhaled NO Page 3 concentration had reached a plateau. Some vessels were incubated for 20 minutes with polythethylene glycol-bound SOD (37.5 U/mL) and catalase (1200 U/mL) prior to relaxation with SNAP. NEWBORN LAMB PROTOCOL Postnatal Care: At days (nine days after ductal ligation), the pregnant ewe was fasted and anesthetized as described above, and the fetal head exposed through a hysterotomy. The subsequent techniques have previously been described in detail (14, 15). The fetal trachea was intubated with a 4.0 mm cuffed endotracheal tube. The carotid artery and jugular vein were exposed in the neck, polyvinyl catheters inserted, and advanced into the aorta and the right atrium, respectively. The fetal chest was exposed and a left thoracotomy was performed. Polyvinyl catheters were placed in the main pulmonary artery and left atrium, and a 10.0 mm ultrasonic transit time flow transducer was placed around the main pulmonary artery. The umbilical cord was ligated and the lamb delivered. The lamb was wrapped in a homeothermic servo controlled warming blanket (Harvard Apparatus, Edenbridge, KY) and placed under an infant warmer (Air Shields, Vickers) to maintain temperature at 39 o C. Ventilation was initiated with a time cycled, pressure control ventilator (Servo 900C; Siemens, Sweden) at an FiO , ventilator rate (IMV) = 60 breaths/minute, peak inspiratory pressure (PIP) = 30 cm H 2 O, peak end expiratory pressure (PEEP) = 4 cm H 2 O, and an inspiratory time (Ti) = 33%. The IMV and PIP were adjusted to maintain PaCO 2 between mm Hg. Tris-hydroxymethyl aminomethane (THAM) was used to correct any metabolic acidosis (defined as a base deficit > 10). Systemic hypotension, defined as a decrease in mean systemic blood pressure of greater than 15 mm Hg, or an initial hemoglobin of less than 11 gm/dl, was corrected by administration of 10 ml/kg of maternal blood. The newborn lamb was sedated with 10 mg/kg of ketamine hydrochloride as needed. A total of 60 minutes of stabilization occurred after clamping of the umbilical cord, and before initial hemodynamic recordings were performed. During each protocol no blood or

4 SOD in Combination with Inhaled NO Page 4 THAM infusions were given and ventilator settings remained constant. Following each study, the lambs were sacrificed by a lethal dose of sodium pentobarbital. Materials: Recombinant human superoxide dismutase (rhsod) was obtained from Bio- Technology General Corporation (Iselin, NJ). NO was obtained as 1,000 ppm in nitrogen (Matheson Gas Products Inc., Twinsburg, OH), and blended with oxygen to obtain 0.5 or 5 ppm. NO was continuously analyzed from the inspired gas immediately prior to the endotracheal tube by a chemiluminescence analyzer (Model 42H/42S; Thermoenvironmental Instruments, Franklin, MA). Experimental Protocol: (n = 18 newborn lambs with prenatal ligation of ductus arteriosus, gestational age days). Animals were selected to receive rhsod alone (5 mg/kg diluted in 1 ml/kg with saline), ino alone (5 and 80 PPM), or their combination in a randomized block fashion. In animals receiving rhsod, it was delivered as a single bolus through the endotracheal tube immediately after intubation of the trachea. Hemodynamic and blood gas values were measured at one hour of life (pre-ino) and following 30 minutes of ino at 5 ppm (1.5 hours). ino was discontinued, and hemodynamics were allowed to return to baseline for a 30 minute period (2 hours). ino was then resumed at 80 ppm, and hemodynamics and blood gas values were measured following 30 minutes of ino at 80 ppm (2.5 hours). MEASUREMENTS: Phasic pulmonary arterial, left atrial, aortic, and airway pressures were measured by Gould Statham physiologic pressure transducers (P-23 XL; Gould Electronics, Cleveland, OH) which were calibrated at the start of each experiment with a mercury column manometer. Pulmonary blood flow was measured by an ultrasonic transit time flow transducer (10.0 mm around the main pulmonary artery; Model T 101; Transonic Systems, Ithaca, NY) and processed by a digital flowmeter. These data were recorded continuously on a physiologic amplifier-recorder system (Gould Electronics). Pulmonary vascular resistance (PVR) was

5 SOD in Combination with Inhaled NO Page 5 calculated as: PVR = (MPAP - LAP) / Qp and reported in units of (mmhg kg min/ml). Aortic blood was collected for measurement of ph, po 2, pco 2, hemoglobin, and base status (Acid-Base Laboratory 3; Radiometer Medical A/S, Copenhagen, Denmark). ANALYSIS FOR rhsod CONCENTRATION: SOD concentration analyses were performed using a monoclonal antibody specific for rhsod and radioimmunoassay (RIA) as previously-described in detail (16). The concentration of rhsod has been shown to directly correlate with activity of the enzyme. Briefly, the RIA was performed by first labeling 100 µg rhsod with 125 I using 1 mci Bolton Hunter Reagent (Amersham, Arlington Heights, IL). The solution was then diluted to 15 ml with phosphate buffered saline with 0.1% bovine serum albumin. A total of 100 µl of sample or standard ( µg rhsod/l) was added to 100 µl of the 125 I-labeled rhsod solution and 100 µl of mouse anti-rhsod antibody (Bio-Technology General Corp., Iselin, New Jersey). After the solution was incubated at 4 o C for 12 h, 50 µl of rabbit anti-mouse whole serum (Sigma Chemical Company; 1:40 dilution) was added. This solution was incubated at room temperature for 30 min, and then 300 µl of 20% polyethylene glycol 8000 (Sigma Chemical Company) was added. After the solution was incubated at room temperature for 20 min, centrifugation was performed at 1500 X g for 20 min. The supernatant was counted in an LKB 1282 Compugamma RIA program. DATA ANALYSIS: All data are expressed as mean ± SE, with n representing the number of animals studied. Statistical analysis was performed with the Statview 4.5 software package (Abacus Concepts, Berkley, CA). Statistical comparisons for normally-distributed data within groups were performed using ANOVA for repeated measures, followed, if necessary, by Student- Newman-Keuls post hoc testing for multiple comparisons. A Wilcoxon signed rank test was used to compare groups of data that were not normally distributed. A one-way ANOVA was

6 SOD in Combination with Inhaled NO Page 6 performed to determine differences in hemodynamic responses between groups of intact lambs. A p value < 0.05 was considered significant.

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