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Fetal Ductus Arteriosus LigationPulmonary Vascular Smooth MuscleBiochemical and Mechanical Changes

Jaques Belik, A.J. Halayko, K. Rao, and Newman L. Stephens

To evaluate the smooth muscle mechanical and biochemical changes associated with persistent pulmo-nary hypertension syndrome of the newborn, we studied 31 fetal sheep in which the ductus arteriosus wasligated at 125 days of gestation. Sixty-one noninstrumented and six sham-operated fetuses served ascontrols. All animals were delivered by cesarean section at 137-140 days of gestation, and theexperimental group had the ductus arteriosus ligated for 123 days. The ligated group demonstrated ahigher mean (SEM) pulmonary artery pressure (72.33.8 versus 54.12 mm Hg, p

Belik et al Pulmonary Vascular Muscle and Fetal Ductus Arteriosus Ligation 589

pertension in the rat- in which excessive pulmonaryvascular muscularization is also seen, the pulmonaryvascular muscle was shown to generate less force than inthe control condition.'2 Although morphological sub-strate differences between the fetal sheep and adult ratlung do exist, these data suggest that the medial hyper-trophy of the pulmonary vasculature in the rat model ofpulmonary hypertension consists of hypertrophiedsmooth muscle cells with reduced contractility.

Therefore, to ascertain the role of muscle contractionin the maintenance of a high resistance to blood flowafter birth in the PPHN syndrome, we evaluated bio-chemical and mechanical property changes of pulmo-nary vascular smooth muscle after ductus arteriosusligation in fetal sheep.

Materials and MethodsAnimal Preparation

Pregnant ewes between 123 and 127 days of gestation(term, 145 days) were fasted for at least 24 hours beforesurgery. The ewes were anesthetized with intravenouspentobarbital sodium (loading dose, 20 mg/kg; mainte-nance, 1 mg/kg every 30 minutes). Under sterile condi-tions, the fetal head and upper chest were deliveredthrough a uterine incision. A left fetal thoracotomy wasperformed, exposing the heart and great vessels. Theductus arteriosus was identified and ligated with 2-0 silksuture in 31 fetuses (ligated group). The chest wasclosed in layers. The fetus was carefully returned to theuterus, the wall of which was closed in layers. Particularcare was taken to avoid any significant amniotic fluidloss. Catheters, when placed, were tunneled to the flankof each ewe, and ampicillin (1 g) was injected into theamniotic sac and abdominal cavity at the time ofsurgical closure. Thereafter, both ewe and fetus re-ceived antibiotics for 4 days. The ewe received 1x 106units penicillin and 50 mg gentamicin intramuscularlytwice daily, and the fetus received one half of this doseintravenously once a day when a catheter was in place.All wounds were cleaned daily, and the catheters werefilled with fresh heparin solution (1,000 units/ml) toprevent clotting.

Six ewes were subjected to the same surgical proce-dure, but the ductus arteriosus was only visualized andnot ligated (sham-operated group). In twelve of theligated and in all sham-operated animals, polyvinylcatheters (5F) were placed into the carotid artery andjugular vein for hemodynamic measurements. In thesefetuses, blood samples (0.3 ml) were obtained dailyfrom the carotid artery for blood gas and pH measure-ments carried out with a blood gas analyzer (modelABL-3, Radiometer, Copenhagen). In the ductus arte-riosus-ligated fetuses in which no intravascular cathe-ters were placed, daily two-dimensional real time ultra-sound images were obtained to confirm that the fetuseswere alive by documenting the presence of heart beats.Sixty-one noninstrumented sheep fetuses served ascontrols (noninstrumented group).

Experimental DesignBetween 137 and 140 days of gestation the noninstru-

mented, sham-operated and ligated ewes were sub-jected to a cesarian section under pentobarbital sodium

and a 5F Swan-Ganz catheter was advanced from thejugular vein into the pulmonary artery. Sequentialmeasurements of mean right atrial, ventricular, andpulmonary arterial pressures were obtained. Systemicarterial pressure was also recorded by the carotid arterycatheter. Vascular and intracardiac pressures weremeasured with pressure transducers (Gould Inc., Ox-nard, Calif.) and continuously recorded on paper. Mea-surements of heart rate were derived from the pressure-wave frequency. Subsequently, the fetus was subjectedto a left thoracotomy, and a precalibrated electromag-netic flow probe (flowmeter and probe, Carolina Med-ical Electronics, Inc., King, N.C.) of adequate size (lessthan 20% constriction of the vessel) was placed aroundthe main pulmonary artery immediately proximal to theductus arteriosus; then a measurement of right ventric-ular output was obtained. Finally, the left atrial pressurewas obtained by direct puncture of the left atrium. Allfetuses were rendered apneic at the time of cesareansection by the pentobarbital sodium anesthesia admin-istered to the ewe; thus, the lungs were never inflated oroxygenated.

After the hemodynamic measurements, the fetus wasrapidly killed with an intravenous dose of potassiumchloride, and after the body weight was obtained, thelungs and heart were removed en bloc.

Confirmation of the effective ligature of the ductusarteriosus was always sought by probing the ductusarteriosus from the pulmonary artery side during thepostmortem examination.

Organ WeightsThe hearts from all animals were dissected to obtain

the free wall weights of the right and left ventricles,septum, and atria. In addition, the liver weights werealso obtained from all animals. These measurementswere obtained to evaluate the impact of chronic in-creased afterload on the fetal heart. Total fresh wetlung weight was obtained as a crude measurement oflung edema.

Vascular Smooth Muscle MechanicsA segment of the second generation intralobar pul-

monary artery of randomly selected ligated and nonin-strumented animals was carefully dissected and placedin ice-cold Krebs-Henseleit solution. Using our leversystem to obtain pulmonary vascular muscle mechanicalmeasurements in pilot experiments in sheep, we ob-served that only second-generation intralobar pulmo-nary arterial strips yielded consistent results. Smallerarteries (third and fourth generations) showed poorforce development and shortening (authors' unpub-lished data), likely related to the deleterious effect oftissue dissection and handling on the contractility of thesmooth muscle cells. Thus, in the present study themechanical properties of only the second-generationintralobar pulmonary arterial strips were delineated.The pulmonary vessel rings were transversely opened,

yielding a 2-mm-wide rectangular strip. The strips wereattached to a clamp fixed to the bottom of a 10-ml bathcontaining Krebs-Henseleit solution at 37C, pH 7.4,P02 of 600 mm Hg, and Pco2 of 40 mm Hg. The upperend was tied by 7- 0 graded silk to the magnesium arm of

anesthesia. The fetal head was carefully exteriorized, an electromagnetic muscle lever system.

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590 Circulation Research Vol 72, No 3 March 1993

The following mechanical measurements were ob-tained: 1) Length-force curves were initially obtained toestablish the optimal length and the maximum isometrictension. 2) Maximal shortening capacity was obtainedby the quick-release method, which consisted of releas-ing the muscle to a quasi-zero load (0.5 mN) underpassive and active contraction. For the passive contrac-tions, the degree of muscle shortening after passiverecoil (releasing the muscle to a quasi-zero load from itsresting tension without stimulation) was measured. Forthe measurement of active contraction, 2 seconds afterthe onset of the electrical stimulation (18 V, 10 sec-onds), the muscle was released to a quasi-zero load andallowed to shorten maximally. The maximal active short-ening capacity was represented by the difference be-tween total and passive shortening and expressed as apercentage of the optimal length. 3) Isometric half-relaxation time was measured as the time for the forcegenerated during an electrically stimulated isometriccontraction to decrease by 50%. 4) Isotonic half-timerelaxation was measured by a previously describedtechnique to obtain a load and initial contractile ele-ment-length independent index of isotonic relaxationtime, based on the fact that muscle relaxation dependson the load that it is subjected to, the state of muscleactivation, and the length of the muscle contractileelement at the onset of relaxation.1314 Briefly, at theirideal lengths, the muscle strips were supramaximallyelectrically stimulated to elicit maximal isotonic short-ening. The stimulus was turned off at peak shortening,and the muscle strips were allowed to elongate. Imme-diately after the termination of the electrical stimula-tion, a series of load clamps were applied. The applica-tion of instantaneous load clamps at the same point intime for each contraction ensures that the muscle stripswill begin to relax at the same length of the musclecontractile element even under different loads. For eachof the loads, the time to elongate 50% of the initiallength (half-time) was determined. The half-times foreach curve with their respective loads were fit by linearregression, and the zero-load half-time was extrapo-lated. For all vessel strips, the correlation coefficient forthe load versus half-time was 0.95 or higher (p

Belik et al Pulmonary Vascular Muscle and Fetal Ductus Arteriosus Ligation 591

TABLE 1. Fetal Arterial Blood Gases and pH After SurgerySham-operated (n =6) Ligated (n= 12)

Time after surgery (hours) 24 72 24 72pH 7.300.01 7.38+0.00 7.25+0.03 7.35+0.00*Paco2 (mm Hg) 49.11.2 47.31.4 57.7+3.3t 53.01