Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs.

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.

Chronic lung injury in preterm lambs: abnormalities of the pulmonary circulation and lung fluid balance.

Chronic lung disease of early infancy, or bronchopulmonary dysplasia, is a frequent complication of prolonged mechanical ventilation after premature birth. Pulmonary hypertension and edema are common features of this condition, which is often attributed to long-term, repetitive overinflation of incompletely developed lungs. The overall objective of this work was to examine the effects on the pulmonary circulation and lung fluid balance of different ventilation strategies using large versus small inflation volumes in an animal model of bronchopulmonary dysplasia. We studied 16 newborn lambs that were delivered prematurely (124+/-3 d gestation, term = 147 d) by cesarean section and mechanically ventilated for 3 to 4 wk. Ten lambs were ventilated at 20 breaths/min, yielding a tidal volume of 15+/-5 mL/kg, and six lambs were ventilated at 60 breaths/min, yielding a tidal volume of 6+/-2 mL/kg. All lambs received surfactant at birth and had subsequent surgery for closure of the ductus arteriosus and catheter placement to allow serial measurements of pulmonary vascular resistance and lung lymph flow. Chronic lung injury, documented by serial chest radiographs and postmortem pathologic examination, developed in all lambs irrespective of the pattern of assisted ventilation. Pulmonary vascular resistance, which normally decreases during the month after birth at term, did not change significantly from the first to the last week of study. Lung lymph flow, an index of net transvascular fluid filtration, increased with time in lambs that were ventilated at 20 breaths/min, but not in lambs ventilated at 60 breaths/min. Lymph protein concentration decreased with time, indicative of increased fluid filtration pressure, without evidence of a change in lung vascular protein permeability. Postmortem studies showed interstitial lung edema, increased pulmonary arteriolar smooth muscle and elastin, decreased numbers of small pulmonary arteries and veins, and decreased capillary surface density in distal lung of chronically ventilated lambs compared with control lambs that were killed either 1 d (same postconceptional age) or 3 wk (same postnatal age) after birth at term. Thus, chronic lung injury from prolonged mechanical ventilation after premature birth inhibits the normal postnatal decrease in pulmonary vascular resistance and leads to lung edema from increased fluid filtration pressure. These abnormalities of the pulmonary circulation may contribute to the abnormal respiratory gas exchange that often exists in infants with bronchopulmonary dysplasia.

Chronic lung injury in preterm lambs. Disordered respiratory tract development.

The cause of chronic lung disease of early infancy, often called bronchopulmonary dysplasia (BPD), remains unclear, partly because large-animal models that reliably reproduce BPD have not been available. We developed a model of BPD in lambs that are delivered prematurely and ventilated for 3 to 4 wk after birth to determine whether the histopathology of chronic lung injury in premature lambs mimics that which occurs in preterm infants who die with BPD, and to compare two ventilation strategies to test the hypothesis that differences in tidal volume (VT) influence histopathologic outcome. The two ventilation strategies were slow, deep ventilation (20 breaths/min, 15 +/- 2 ml/kg body weight VT; n = 5) or rapid, shallow ventilation (60 breaths/min, 6 +/- 1 ml/kg body weight VT; n = 5). Lambs were delivered at 125 +/- 4 d gestation (term = 147 d), treated with surfactant, and mechanically ventilated with sufficient supplemental oxygen to maintain normal arterial oxygenation (60 to 90 mm Hg). Quantitative histologic analysis revealed lung structural abnormalities for both groups of experimental lambs compared with lungs of control term lambs that were < 1 d old (matched for developmental age; n = 5) or 3 to 4 wk old (matched for postnatal age; n = 5). Compared with control lambs, chronically ventilated preterm lambs had pulmonary histopathology characterized by nonuniform inflation patterns, impaired alveolar formation, abnormal abundance of elastin, increased muscularization of terminal bronchioles, and inflammation and edema. Slow, deep ventilation was associated with less atelectasis, less alveolar formation, and more elastin when compared with rapid, shallow ventilation. We conclude that prolonged mechanical ventilation of preterm lambs disrupts lung development and produces pulmonary histopathologic changes that are very similar to those that are seen in the lungs of preterm infants who die with BPD. This chronic lung disease is not prevented by surfactant replacement at birth, does not appear to require arterial hyperoxia, and is influenced by VT.

Role of neutrophils in lung vascular injury and edema after premature birth in lambs.

To investigate the role of neutrophils in the pathogenesis of respiratory distress after premature birth, we assessed the relationship between circulating neutrophil concentration and neutrophil accumulation in the lung, lung lymph and pleural liquid flow, and extravascular lung water in 10 chronically catheterized preterm lambs (127 +/- 1 days gestation) that were mechanically ventilated for 8 h after birth. Circulating neutrophil concentration transiently decreased within 2 h after birth and then returned to prenatal values by 6-8 h. The decrease in circulating neutrophil concentration was related directly to the accumulation of neutrophils in the air spaces, drainage of liquid and protein from the lung 6-8 h after delivery, and postmortem extravascular lung water. In additional studies, we intravenously administered mechlorethamine to 5 fetal lambs to reduce circulating neutrophils before delivery (neutrophil concentration before birth: 9 +/- 11 cells/microl). Compared with control lambs, neutrophil-depleted lambs had significantly less drainage of liquid (7.8 +/- 5.9 vs. 2.6 +/- 1.9 ml/h, respectively) and protein (116 +/- 74 vs. 42 +/- 27 mg/h, respectively) from the lung 6-8 h after birth and significantly less extravascular lung water at postmortem (6.5 +/- 0. 8 vs. 4.8 0.6 g/g dry lung, respectively). Thus neutrophils contribute to the pathogenesis of respiratory distress after premature birth by increasing lung vascular protein permeability and promoting lung edema.

Chronic lung injury in preterm lambs: disordered pulmonary elastin deposition.

Prolonged mechanical ventilation of premature neonates is often associated with abnormal morphological development of the lung and chronic lung disease, sometimes called bronchopulmonary dysplasia (BPD). Impaired alveolar development is a hallmark of this disease. To better understand the effects of mechanical ventilation on lung elastin expression, we studied lung tissue from 10 preterm lambs (gestation = 125 days; term = 148 days) mechanically ventilated for 3-4 wk at a respirator rate of 20 breaths/min and tidal volume of 15 +/- 5 ml/kg (n = 5) or 60 breaths/min and tidal volume of 5 +/- 2 ml/kg (n = 5). Histopathology showed increased elastin accumulation and abnormal morphological development in the ventilated groups. Postmortem lung desmosine content was increased significantly in the 20 breaths/min group. Tropoelastin mRNA expression was increased in both ventilated groups. In situ hybridization localized increased tropoelastin mRNA expression to sites of accumulated elastin in extended alveolar walls with scant, attenuated secondary crests. Lung collagen content, as assessed by the amount of hydroxyproline in lung tissue, was similar to controls. These data suggest that excessive production and accumulation of elastin is associated with chronic lung injury from prolonged mechanical ventilation after premature birth.

Methods for retrovirus-mediated gene transfer to fetal lung.

With the advent of improved vectors for DNA delivery, somatic gene therapeutic approaches have expanded rapidly in the last few years. The vast majority of applications include ex vivo and in vivo protocols in patients postnatally. Nonetheless there is increasing interest and compelling reasons to consider prenatal application of somatic gene therapy (1,2. In the current chapter, we will review theoretical, ethical, and experimental support for in utero gene therapy and then outline the methodology and large animal model we are currently using to consider retrovirus-mediated gene transfer to fetal lung. In this latter regard, the candidate inherited disorder is cystic fibrosis and the reader is referred to Chapters 1 and 12 in this volume.

Retrovirus-mediated gene transfer in lungs of living fetal sheep.

In utero somatic gene transfer may be a useful therapeutic strategy for a variety of inherited disorders. In the present study, we demonstrate transgene expression in the airways of fetal lamb lungs, 2-3 weeks after injection of Moloney murine leukemia retrovirus based vectors containing cDNA for beta-galactosidase (lacZ) or human interleukin receptor antagonist protein (IRAP), into the fluid filled future airspace of fully catheterized twin fetal lambs (104-117 days gestational age; term 147 days). Expression of lacZ or IRAP was limited to the twin that received the respective vector and was apparent, at light microscopic level, in the epithelium and submucosal space of proximal airways, and to a lesser extent, in the respiratory epithelium of the distal airways. These data demonstrate for the first time that transfer of foreign DNA to fetal lung can be accomplished. These findings support the use of retroviral vectors for somatic lung DNA transfer and suggest that inherited disorders such as cystic fibrosis may be approached therapeutically via gene transfer, in utero.

Vasopressin effects on lung liquid volume in fetal sheep.

The normal switch from placental to pulmonary gas exchange at birth requires rapid removal of liquid from the lungs. Previous reports contend that vasopressin may be important in regulating this process, but this notion derives from studies in which fetal sheep received very large doses of vasopressin that yielded plasma concentrations at least 10 times greater than those that have been measured during normal labor. To study the physiologic effects of vasopressin on lung liquid volume in fetal sheep, we made three sets of experiments. First, we measured plasma vasopressin concentrations [VP] in 15 late-gestation fetal sheep, five of which were at various stages of spontaneous labor. [VP] in these fetuses ranged from < 1 (prelabor) to 31 (during labor) microU/mL; postmortem extravascular lung water (EVLW) ranged from 4.5 to 14.5 g/g dry lung tissue. In a second series of studies, we measured EVLW in five sets of near-term (138 +/- 1 d, term = 147 d) twin fetal sheep that received an 8-h i.v. infusion of either isotonic saline (control twin) or AVP (AVP-treated twin) at a rate of approximately 1 (mU/kg)/min. This dose was chosen to mimic [VP] measured in fetuses that had been studied during labor. [VP] did not change in the control twins, whereas [VP] increased from 1.8 +/- 1.0 to 27.7 +/- 3.5 microU/mL in treated twins. There was a small, statistically significant difference in EVLW between twins that received AVP and untreated twins (11.9 +/- 1.8 versus 14.6 +/- 2.8 g/g dry lung).(ABSTRACT TRUNCATED AT 250 WORDS)

Surfactant treatment at birth reduces lung vascular injury and edema in preterm lambs.

To study the effect of surfactant administration on fluid balance in the premature lung, we measured pulmonary vascular pressures, lung lymph and pleural liquid flow, and concentrations of protein in lymph, pleural liquid, and plasma before and after birth in 12 chronically catheterized preterm lambs (127-128 d gestation) treated with either placebo or surfactant just before surgical delivery. Eight lambs received intrapulmonary saline (placebo), and four lambs received surfactant; all lambs were mechanically ventilated with O2 for 8 h after birth. In control lambs, lung lymph and pleural liquid flow increased from 2.7 +/- 0.4 mL/h during the 2-4 h before birth to 9.2 +/- 2.1 mL/h by 6-8 h after birth; lymph and pleural space protein drainage increased from 58 +/- 7 mg/h during the 2-4 h before birth to 134 +/- 25 mg/h by 6-8 h after birth. In lambs treated with surfactant, there was no significant increase in lymph and pleural liquid flow after birth (before birth, 2.3 +/- 0.3 mL/h; 6-8 h after birth, 3.4 +/- 0.9 mL/h); likewise, lymph and pleural space protein drainage did not change after birth (before birth, 54 +/- 6 mg/h; 6-8 h after birth, 50 +/- 8 mg/h). Postmortem extravascular lung water was significantly less in lambs treated with surfactant compared with control lambs (control, 6.5 +/- 0.3 g/g dry lung; surfactant-treated, 5.0 +/- 0.2 g/g dry lung).(ABSTRACT TRUNCATED AT 250 WORDS)

Lung vascular protein permeability in preterm fetal and mature newborn sheep.

The purpose of this study was to see whether there are developmental differences in the protein permeability of the pulmonary circulation that might contribute to the abnormal lung fluid balance seen in premature lambs with respiratory failure. In one series of experiments, we measured albumin turnover time, which reflects the escape rate of radiolabeled albumin from the pulmonary circulation, of five preterm fetal lambs (125 +/- 1 days gestation) and five newborn lambs (19 +/- 9 days old). Turnover time was not significantly different in fetuses (160 +/- 38 min) and newborns (141 +/- 54 min), implying a similar protein permeability of the pulmonary circulation. In additional experiments, we measured pulmonary hemodynamic and lung lymph flow responses to intravenous saline infusion in seven preterm fetal lambs (130 +/- 3 days gestation) and seven newborn lambs (14 +/- 3 days old). During saline infusion, calculated fluid filtration pressure increased by a similar amount in fetuses and newborns (3.4 +/- 0.8 and 2.8 +/- 0.9 Torr, respectively), resulting in a similar change in lung lymph flow in fetuses and newborns (0.59 +/- 0.27 and 0.55 +/- 0.25 ml.h-1.kg body wt-1, respectively). The results of these studies indicate that protein permeability of the pulmonary circulation does not change significantly during late fetal and early postnatal development.

Changes in lung lipid during spontaneous labor in fetal sheep.

The goals of this study were 1) to examine changes in lung liquid formation and composition during spontaneous labor in fetal lambs and 2) to determine the importance of beta-adrenergic stimulation and transepithelial Na+ flux in removing liquid from the lung lumen near birth. We measured net production of lung liquid (Jv), lung liquid composition, and transpulmonary electrical potential difference (PD) before and during labor in fetal sheep with chronically implanted tracheal and vascular catheters. We determined Jv by measuring rate of change in lung liquid concentration of 125I-albumin, an impermeant tracer that was mixed in lung liquid at the start of each study. In 17 paired experiments, Jv decreased from 11 +/- 2 ml/h (Jv > 0 = secretion) before labor to -1 +/- 2 ml/h (Jv < 0 = absorption) during labor; in 5 paired experiments, PD changed from -7 +/- 1 mV (lumen negative) before labor to -12 +/- 1 mV during labor. To determine whether absorption of lung liquid during labor is the result of beta-adrenergic stimulation, we studied the effect of propranolol on Jv during labor. When propranolol (40 microM) was added to lung liquid during active labor, Jv decreased from -2 +/- 2 to -8 +/- 3 ml/h (n = 9). Thus, propranolol did not inhibit lung liquid absorption during labor. To determine whether transepithelial Na+ movement provides the driving force for lung liquid clearance during labor, we tested the effects of amiloride, an Na+ transport inhibitor, on Jv and PD.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoproteinemia slows lung liquid clearance in young lambs.

To determine whether hypoproteinemia slows the rate at which liquid is cleared from the lung lumen, we studied 36 lambs, 18 of which underwent repeated plasmapheresis, reducing plasma protein concentration by 37% and plasma protein osmotic pressure by 39%. We killed 29 lambs (14 hypoproteinemic and 15 normoproteinemic) and removed their lungs 1, 2, or 6 h after intratracheal instillation of isotonic saline (6 ml/kg body wt). We measured extravascular lung water and determined the percentage of tracheally instilled liquid that was cleared from the lungs by comparison with control lambs that did not receive saline into their airways. The percent liquid cleared from the lungs after 1 and 2 h was significantly less in hypoproteinemic than in normoproteinemic lambs (37 vs. 65% at 1 h, 58 vs. 75% at 2 h, respectively). By 6 h nearly all the liquid (> 92%) was cleared from the lungs of all lambs. Thus hypoproteinemia slows the initial rate of clearance of liquid from the lungs of lambs. To determine whether reduced plasma protein osmotic pressure might redirect this liquid into lung lymphatics, we measured lung lymph flow (Q1) in five lambs (7.7 +/- 1.4 kg, 19 +/- 4 days old) for > or = 2 h before and 6 h after tracheal instillation of saline. In each lamb, paired studies were done 3-6 days apart; between studies the lambs underwent plasmapheresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion transport regulation of lung liquid secretion in foetal lambs.

To test the hypothesis that liquid formation in the foetal lung reflects the balance between Cl- secretion and Na+ absorption by the respiratory tract epithelium, we studied the independent and combined effects of selective ion transport inhibitors on basal production of lung liquid in foetal lambs. We prepared 19 foetal lambs (gestation 125 +/- 4, term = 147 days) with chronic indwelling catheters for subsequent measurement of luminal liquid production over time (JV). Using an impermeant tracer technique, we measured JV before and after tracheal instillation of 2 different inhibitors of ion transport: bumetanide, a Na(+)-K(+)-2Cl- co-transport inhibitor, and amiloride, a Na+ transport inhibitor. In 7 foetuses we sequentially added bumetanide (10(-4) M) and 2 different concentrations of amiloride (10(-6) M, 10(-4) M) to the liquid within the lung lumen. After we gave bumetanide, JV decreased from 12 +/- 4 ml/h to 0 +/- 5 ml/h and subsequently increased during the 2 periods of amiloride exposure (10(-6) M: 6 +/- 5 ml/h; 10(-4) M: 7 +/- 7 ml/h). In 5 control studies we gave bumetanide, followed by only amiloride vehicle. JV for all time periods in the control studies was similar to the experimental group, demonstrating no effect of amiloride. In 5 foetuses we administered the 2 concentrations of amiloride before bumetanide. There was no change in JV with either concentration of amiloride (baseline: 13 +/- 2 ml/h; 10(-6) M amiloride: 15 +/- 5 ml/h; 10(-4) M amiloride: 13 +/- 6 ml/h).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased pulmonary vascular filtration pressure does not alter lung liquid secretion in fetal sheep.

The purpose of this study was to determine whether an increase in pulmonary vascular filtration pressure affects net production of liquid within the lumen of the fetal lung. We studied 14 chronically catheterized fetal lambs [130 +/- 3 (SD) days gestation] before, during, and after a 4-h rapid (500 ml/h) intravenous infusion of isotonic saline. In seven fetuses we measured pulmonary arterial and left atrial pressures, lung lymph flow, and protein osmotic pressures in plasma and lymph. In eight lambs with a chronically implanted tracheal loop cannula, we measured the change in luminal lung liquid volume over time by progressive dilution of tracheally instilled 125I-albumin, which stays within the lung lumen. Saline infusion increased pulmonary vascular pressures by 2-3 mmHg and decreased the plasma-lymph difference in protein osmotic pressure by 1 mmHg. Lung lymph flow increased from 1.9 +/- 0.6 to 3.9 +/- 1.2 (SD) ml/h; net production of luminal lung liquid did not change (12 +/- 5 to 12 +/- 6 ml/h). Thus an increase in net fluid filtration pressure in the pulmonary circulation, which was sufficient to double lung lymph flow, had no significant effect on luminal lung liquid secretion in fetal sheep.

Developmental changes in lung epithelial ion transport and liquid movement.

During fetal life, the lungs are filled with liquid that flows from the pulmonary circulation across the epithelium in response to the osmotic force generated by Cl- secretion of airway and distal lung epithelial cells. As birth approaches, net Cl- secretion across the respiratory tract epithelium decreases, and this is associated with a reduction in the flow of liquid into the lung lumen. The cause for this change is unknown, but several recent studies indicate that it may be related to alterations in the hormonal milieu to which the lung epithelium is exposed late in gestation. The switch from placental to pulmonary gas exchange at birth requires rapid removal of liquid from the lung lumen. During labor and the immediate postnatal period, the pulmonary epithelium changes from a predominantly Cl-(-)secreting membrane to a predominantly Na(+)-absorbing membrane, with resultant reversal of the direction of flow of lung liquid. There is considerable evidence that this change reflects an active metabolic process involving increased Na(+)-K(+)-ATPase activity in lung epithelial cells, which drives liquid from the lung lumen into the interstitium, with subsequent absorption into the pulmonary circulation. This Na(+)-K(+)-ATPase-dependent process persists in the bronchopulmonary epithelium of the mature lung and probably has an important role in clearance of alveolar edema associated with heart failure or lung injury.

Intrapulmonary terbutaline and aminophylline decrease lung liquid in fetal lambs.

To see if phosphodiesterase inhibition might enhance the effect of beta-adrenergic stimulation on fetal lung liquid secretion, we studied the independent and combined effects of intrapulmonary terbutaline and aminophylline on net production of lung luminal liquid over time (Jv) in fetal lambs with chronically placed tracheal loop catheters. We calculated Jv during baseline and experimental periods (90-120 min each) by measuring serial concentrations of 125I-albumin, an impermeant tracer that was well mixed in the luminal liquid. In 21 experiments, tracheal instillation of terbutaline (10(-5) M) decreased Jv from 11 +/- 1 (mean +/- SEM) to -3 +/- 2 mL/h. In six other studies, aminophylline (10(-3) M) alone had no significant effect on Jv. In 12 experiments, we gave the two drugs sequentially: terbutaline decreased Jv from 11 +/- 2 to -3 +/- 2 mL/h and aminophylline further decreased Jv to -8 +/- 2 mL/h. Amiloride (10(-4) M), an inhibitor of epithelial Na+ transport, reversed the combined effect of terbutaline and aminophylline, increasing Jv to 8 +/- 1 mL/h. Thus, phosphodiesterase inhibition enhances the beta-adrenergic effect of terbutaline on Na(+)-dependent absorption of liquid from the lung lumen of fetal lambs.