Development of the pulmonary vasculature in newborn lambs: structure-function relationships

Our objectives were 1) to describe the quantitative light microscopy and ultrastructure of newborn lamb lungs and 2) to correlate hemodynamic changes during normoxia and hypoxia with the morphology. By light microscopy, we measured the percent muscle thickness (%MT) and peripheral muscularization of pulmonary arteries and veins from 25 lambs aged less than 24 h, 2-4 days, 2 wk, and 1 mo. At the same ages, lungs were isolated and perfused in situ and, after cyclooxygenase blockade with indomethacin, total, arterial (delta Pa), middle (delta Pm), and venous pressure gradients at inspired O2 fractions of 0.28 (mild hyperoxia) and 0.04 (hypoxia) were determined with inflow-outflow occlusion. During mild hyperoxia, delta Pa and delta Pm fell significantly between 2-4 days and 2 wk, whereas during hypoxia, only delta Pm fell. The %MT of all arteries (less than 50 to greater than 1,000 microns diam) decreased, and peripheral muscularization of less than 100-microns-diam arteries fell between less than 4 days and greater than 2 wk. Our data suggest that 1) the %MT of arteries determines normoxic pulmonary vascular resistance, because only arterial and middle segment resistance fell, 2) peripheral muscularization is a major determinant of hypoxic pulmonary vasoconstriction, because we observed a fall with age in peripheral muscularization of less than 100-micron-diam arteries and in delta Pm with hypoxia, and 3) the arterial limit of the middle segment defined by inflow-outflow occlusion lies in 100- to 1,000-microns-diam arteries.     

Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Developmental change in isoproterenol-mediated relaxation of pulmonary veins of fetal and newborn lambs

-Adrenergic agonists are important regulatorsof perinatal pulmonary circulation. They cause vasodilation primarilyvia the adenyl cyclase-adenosine 3',5'-cyclic monophosphate(cAMP) pathway. We examined the responses of isolated fourth-generationpulmonary veins of term fetal (145 ± 2 days gestation)and newborn (10 ± 1 days) lambs to isoproterenol, a -adrenergicagonist. In vessels preconstricted with U-46619 (a thromboxaneA₂ analog), isoproterenol inducedgreater relaxation in pulmonary veins of newborn lambs than in those offetal lambs. The relaxation was eliminated by propranolol, a-adrenergic antagonist. Forskolin, an activator of adenyl cyclase,also caused greater relaxation of veins of newborn than those of fetallambs. 8-Bromoadenosine 3',5'-cyclic monophosphate, a cellmembrane-permeable analog of cAMP, induced a similar relaxation of allvessels. Biochemical studies show that isoproterenol and forskolininduced a greater increase in cAMP content and in adenyl cyclaseactivity of pulmonary veins in the newborn than in the fetal lamb.These results demonstrate that -adrenergic-agonist-mediatedrelaxation of pulmonary veins increases with maturation. An increase inthe activity of adenyl cyclase may contribute to the change.

     

Umbilical cord compression produces pulmonary hypertension in newborn lambs: a model to study the pathophysiology of persistent pulmonary hypertension in the newborn

We investigated the effects of chronic intrauterine hypoxaemia produced by prolonged partial umbilical cord compression on the circulation shortly after birth in lambs. Vascular catheters were inserted in 10 fetal sheep at 120 to 130 days gestation to measure descending aortic blood gases, arterial pH, and arterial O2 saturation. An inflatable silicone rubber balloon cuff was also placed around the umbilical cord. After recovery and the return of descending aortic blood gases to the normal range, the balloon was gradually inflated, decreasing the PaO2 from 21.2 +/- 3.6 to 17.5 +/- 1.3 mm Hg and the arterial O2 saturation from 57.1 +/- 9.2% to 37.2% +/- 5.2. After 14.3 +/- 3.7 days of partial umbilical cord compression, the lambs were delivered by Caesarean section, instrumented to measure systemic and pulmonary arterial, right atrial and pulmonary arterial wedge pressures, pulmonary and systemic blood flows, and mechanically ventilated. Five normal lambs were also studied. From 60 to 120 min after delivery, when compared to normal lambs, the umbilical compression lambs had an increased pulmonary arterial pressure (P less than 0.05) pulmonary vascular resistance (P less than 0.05), and right atrial pressure (P less than 0.05) with similar arterial blood gases. In both groups, hypoxic ventilation produced an increase in pulmonary arterial pressure (P less than 0.05) which on return to room air ventilation decreased to baseline in the normal lambs but not in the umbilical cord compression lambs (P less than 0.05). Prolonged partial umbilical cord compression produces chronic fetal hypoxaemia and pulmonary arterial hypertension after birth. This may represent a model to study the pathophysiology of persistent pulmonary hypertension syndrome.     

Hypoxia-induced periodic breathing in newborn lambs

This study was designed to elucidate the effect of hypoxia on the breathing rhythmicity and the effect of hypoxia on periodic breathing (PB) in two groups of newborn lambs (less than 2 days and 10 days of age). Lambs undergoing a hypoxic ventilatory test [0.08 inspired O2 fraction (FIo2) for 13 min] experienced no apnea or PB in hypoxia, but all developed PB during the 1-min period immediately after their abrupt return to 0.21 FIo2. This PB occurred when alternation of arterial PO2 and PCO2 in mild hypoxic and hypocapnic conditions induced an overshoot-undershoot response of the chemical drive to breathe. The magnitude of PB was found to be greater in the animals with a higher peripheral chemoreflex sensitivity to hypoxia but ceased altogether when the hypoxic-hypocapnic conditions were resolved. When these conditions were removed more quickly, that is, when the animals were returned either to 0.50 FIo2 or to 0.03 FIco2, no PB was observed. To clarify the role of hypoxia as a central depressant on the genesis of PB, we tested to determine whether additional central tissue hypoxia, using carboxyhemoglobin (30%), would worsen the episodes of PB. No effect on breathing rhythmicity was observed. These findings suggest not only that, in newborn animals and adults, the mechanisms of post-hypoxia-induced PB are identical but that the PB elicited in mild hypoxic conditions is a peripheral chemoreflex-mediated event rather than a centrally mediated one.     

Enhanced systolic function of the right ventricle during respiratory distress syndrome in newborn lambs

Respiratory distress syndrome (RDS) causes pulmonary hypertension. It is often suggested that this increased afterload for the right ventricle (RV) might lead to cardiac dysfunction. To examine this, we studied biventricular function in an experimental model. RDS was induced by lung lavages in seven newborn lambs. Five additional lambs served as controls. Cardiac function was quantified by indexes derived from end-systolic pressure-volume relations obtained by pressure-conductance catheters. After lung lavages, a twofold increase of mean pulmonary arterial pressure (from 15 to 34 mmHg) was obtained and lasted for the full 4-h study period. Stroke volume was maintained (5.2 +/- 0.6 ml at baseline and 6.1 +/- 1.4 ml at 4 h of RDS), while RV end-diastolic volume showed only a slight increase (from 6.5 +/- 2.3 ml at baseline to 7.7 +/- 1.3 ml at 4 h RDS). RV systolic function improved significantly, as indicated by a leftward shift and increased slope of the end-systolic pressure-volume relation. Left ventricular systolic function showed no changes. In control animals, pulmonary arterial pressure did not increase and right and left ventricular systolic function remained unaffected. In the face of increased RV afterload, the newborn heart is able to maintain cardiac output, primarily by improving systolic RV function through homeometric autoregulation.     

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.     

Laryngeal response to nasal ventilation in nonsedated newborn lambs.

Although endoscopic studies in adult humans have suggested that laryngeal closure can limit alveolar ventilation during nasal intermittent positive pressure ventilation (nIPPV), there are no available data regarding glottal muscle activity during nIPPV. In addition, laryngeal behavior during nIPPV has not been investigated in neonates. The aim of the present study was to assess laryngeal muscle response to nIPPV in nonsedated newborn lambs. Nine newborn lambs were instrumented for recording states of alertness, electrical activity [electromyograph (EMG)] of glottal constrictor (thyroarytenoid, TA) and dilator (cricothyroid, CT) muscles, EMG of the diaphragm (Dia), and mask and tracheal pressures. nIPPV in pressure support (PS) and volume control (VC) modes was delivered to the lambs via a nasal mask. Results show that increasing nIPPV during wakefulness and quiet sleep led to a progressive disappearance of Dia and CT EMG and to the appearance and subsequent increase in TA EMG during inspiration, together with an increase in trans-upper airway pressure (TUAP). On rare occasions, transmission of nIPPV through the glottis was prevented by complete, active glottal closure, a phenomenon more frequent during active sleep epochs, when irregular bursts of TA EMG were observed. In conclusion, results of the present study suggest that active glottal closure develops with nIPPV in nonsedated lambs, especially in the VC mode. Our observations further suggest that such closure can limit lung ventilation when raising nIPPV in neonates.     

Effect of artificial surfactant on pulmonary function in preterm and full-term lambs

We hypothesized that agents very different from surfactant may still support lung function. To test this hypothesis, we instilled FC-100, a fluorocarbon, and Tween 20, a detergent, which have higher minimum surface tensions and less hysteresis than surfactant, into 15 full-term and 14 preterm lambs. FC-100 and Tween 20 were as efficient as natural surfactant in improving gas exchange and compliance in preterm lambs with respiratory failure. Dynamic compliance correlated with the equilibrium surface tension of the alveolar wash in both full-term (P less than 0.02) and preterm (P less than 0.008) lambs. Functional residual capacity in full-term and preterm lambs was lower after treatment with the two test agents than with surfactant, findings consistent with qualitative histology. Oxygenation in full-term lambs correlated with mean lung volumes (P less than 0.003), suggesting that the hysteresis and/or low minimum surface tension of surfactant may improve mean lung volume, and hence oxygenation, by maintaining functional residual capacity. The effects of the test agents suggest that agents with biophysical properties different from surfactant may still aid lung expansion.     

Determinants of heat production in newborn lambs

Measurement of summit metabolism (the maximum rate of heat production) in lambs aged 1 or 4h revealed considerable between animal variation. Summit metabolism per unit body weight decreased as body weight increased whereas summit metabolism per unit body surface area was independent of body weight. Severe pre-partum hypoxia was apparently associated with a low summit metabolism at 1 or 4h of age which made such lambs very susceptible to hypothermia. This deficiency in heat production capacity did not appear to be a permanent featuresince most lambs so affected recovered full thermoregulatory ability by 12h of age. Feeding of colostrum conferred an immediate 18% increase in summit metabolism. The significance of these findings to the prevention of hypothermia in the newborn lamb is discussed.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Distribution of pulmonary vascular pressure as a function of perinatal age in lambs

The distribution of pulmonary vascular resistance (PVR) with respect to compliance was determined using vascular occlusion in isolated lungs from lambs at five ages, from 2 wk before birth to 1 mo of age. The major change in PVR occurred in the pressure gradient across the middle compliant region (delta Pm), which dropped sharply at birth, remained low for 2 wk, and increased at 1 mo. Pulmonary vasoreactivity also varied with ages. Lungs at 0-4 days did not respond to hypoxia and responded poorly to prostaglandin F2 alpha (PGF2 alpha). In contrast, lungs at 13-33 days had significant increases in delta Pm and the gradient across relatively indistensible arterial vessels during hypoxia and increases in all gradients with PGF2 alpha. Ventilation of fetal lungs reduced PVR, mainly because of a 50% reduction in delta Pm. Our results demonstrate that the magnitude and distribution of PVR relative to compliance varied as a function of perinatal age and that pulmonary vasoreactivity depended on postnatal age. The major effect of ventilating fetal lungs was on the middle region.     

Effects of hypoxia and hypercapnia on nonnutritive swallowing in newborn lambs.

The aim of the present study was to investigate the effect of hypercapnia and hypoxia on apnea and nonnutritive swallowing (NNS) frequency, as well as on the coordination between NNS and phases of the respiratory cycle in newborn lambs, while taking into account the potential effects of states of alertness. Six lambs were chronically instrumented for recording electroencephalogram, eye movements, diaphragm and thyroarytenoid muscle (a glottal adductor) activity, nasal airflow, and electrocardiogram. Polysomnographic recordings were performed in nonsedated lambs exposed to air (control), 10% O(2), and 5% CO(2) in a random order at 3, 4, and 5 days of age. Although hypercapnia decreased apnea frequency in wakefulness and active sleep (P = 0.002 vs. air and hypoxia), hypoxia had no significant effect on apnea. In addition, although hypercapnia increased NNS frequency during wakefulness and quiet sleep (P < 0.005 vs. air and hypoxia), hypoxia tended to decrease NNS frequency. Finally, only hypercapnia altered NNS-breathing coordination by increasing NNS at the transition from inspiration to expiration (ie-type NNS; P < 0.001 vs. air and hypoxia). In conclusion, whereas hypercapnia increases overall NNS frequency by specifically increasing ie-type NNS, hypoxia has the inverse tendency. Results were identical in all three states of alertness.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Response of newborn lambs to CO-induced hypoxia

This study was undertaken to measure the neonate's response to CO-induced hypoxia in the first 10 days of life. CO breathing was used to induce hypoxia because CO causes tissue hypoxia with no or minimal chemoreceptor stimulation. An inspired gas mixture of 0.25 to 0.5% CO in air was used to raise the blood carboxyhemoglobin (HbCO) progressively from 0 to 60% over approximately 20 min. The study, conducted in awake conscious lambs aged 2 and 10 days, consisted in measuring the response of ventilation and the change in arterial blood gases during the rise of HbCO. The results showed that the 2- and 10-day-old lambs tolerated very high HbCO levels without an increase in minute ventilation (VE) and without metabolic acidosis. At both ages, HbCO caused no VE change until HbCO levels rose to between 45 and 50% after which the VE change was exponential in some animals but minimal in others. The VE change was brought about by a rise in tidal volume and respiratory frequency. During the period of maturation from 2 to 10 days, there was a small shift to the right in the VE-HbCO response. In the 10-day-old lambs the VE response to high HbCO was greater than that of the 2-day-olds because of the lambs' higher respiratory frequency response. Six of the 10-day-old lambs but only two of the 2-day-old lambs showed a hypoxic tachypnea to HbCO of 55-65%. None of the lambs developed periodic breathing, dysrhythmic breathing, or recurrent apneas with an HbCO level as high as 60%.(ABSTRACT TRUNCATED AT 250 WORDS)