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Am J Physiol Lung Cell Mol Physiol 288: L450 –L459, 2005. First published December 10, 2004; doi:10.1152/ajplung.00347.2004.

Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease Donald C. McCurnin,1 Richard A. Pierce,2 Ling Yi Chang,3 Linda L. Gibson,4 Sherri Osborne-Lawrence,4 Bradley A. Yoder,5,6 Jay D. Kerecman,7 Kurt H. Albertine,8 Vicki T. Winter,5,6 Jacqueline J. Coalson,5,6 James D. Crapo,3 Peter H. Grubb,7 and Philip W. Shaul4 Departments of 1Pediatrics and 5Pathology, University of Texas Health Science Center; 6The Southwest Foundation for Biomedical Research; 7San Antonio Military Pediatric Center, San Antonio; 4Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas; 2Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; 3Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado; and 8Department of Pediatrics, University of Utah Health Sciences Center, Salt Lake City, Utah Submitted 16 September 2004; accepted in final form 14 November 2004

McCurnin, Donald C., Richard A. Pierce, Ling Yi Chang, Linda L. Gibson, Sherri Osborne-Lawrence, Bradley A. Yoder, Jay D. Kerecman, Kurt H. Albertine, Vicki T. Winter, Jacqueline J. Coalson, James D. Crapo, Peter H. Grubb, and Philip W. Shaul. Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 288: L450 –L459, 2005. First published December 10, 2004; doi:10.1152/ ajplung.00347.2004.—Nitric oxide (NO) serves multiple functions in the developing lung, and pulmonary NO production is decreased in a baboon model of chronic lung disease (CLD) after premature birth at 125 days (d) gestation (term ⫽ 185d). To determine whether postnatal NO administration alters the genesis of CLD, the effects of inhaled NO (iNO, 5 ppm) were assessed in the baboon model over 14d. iNO caused a decrease in pulmonary artery pressure in the first 2d and a greater rate of spontaneous closure of the ductus arteriosus, and lung compliance was greater and expiratory resistance was improved during the first week. With iNO, postmortem pressure-volume curves were shifted upward, lung DNA content and cell proliferation were increased, and lung growth was preserved to equal that which occurs during the same period in utero. In addition, the excessive elastin deposition characteristic of CLD was normalized by iNO, and there was evidence of stimulation of secondary crest development. Thus, in the baboon model of CLD, iNO improves early pulmonary function and alters lung growth and extracellular matrix deposition. As such, NO biosynthetic pathway dysfunction may contribute to the pathogenesis of CLD.

(NO), generated by nitric oxide synthase (NOS), plays a key role in physiological processes in the pulmonary epithelium (5, 19). Studies in the mature airway indicate that the epithelium is the primary source of NO (14) and that NO mediates neurotransmission, smooth muscle relaxation, bacteriostasis, ciliary motility, mucin secretion, and plasma exudation (5, 19, 23, 35). In the perinatal period, epithelium-derived NO is critically involved in the regulation of lung liquid production and of peripheral contractile elements (13, 25). NO also has a well-recognized role in mediating pulmonary vasomotor tone around the time of birth (39). In studies of lungs from fetal baboons, we have previously shown that all three NOS isoforms, neuronal NOS

(nNOS), endothelial NOS (eNOS), and inducible NOS, are principally expressed in proximal respiratory epithelium. In addition, there are maturational increases in the expression of the three NOS isoforms and in NO production during the early third trimester (40). Thus pulmonary NOS expression is upregulated during fetal development in the primate, and this process may be critical to airway, parenchymal, and pulmonary vascular function in the early postnatal period. Chronic lung disease (CLD) is a devastating disorder that arises after premature birth and the use of ventilatory support during the course of hyaline membrane disease or apnea due to prematurity. The clinical course of infants with CLD is often complicated by dramatically increased pulmonary vascular and airway resistance (2). Because NO has a major role in the regulation of pulmonary function in the perinatal period, we recently determined whether there are alterations in NOS isoform expression and activity in proximal lung and changes in NO production in a model of CLD in baboon fetuses delivered at 125 days of gestation (term ⫽ 185 days) and ventilated for 14 days. The baboon model closely mimics the current form of CLD in extremely preterm human infants (12, 48). In contrast to the normal 73% increase in NOS activity that occurs over the same developmental period in utero, there was an 83% decline in activity with CLD due to decreases in nNOS and eNOS expression. In addition, exhaled NO levels at the time of preterm birth at 125 days of gestation were one-third the concentrations observed at birth later in the third trimester, and they remained depressed until day of life 11. As such, there are dramatic declines in nNOS and eNOS expression and activity and a parallel diminution in NO production in the lung in the early postnatal period in CLD (3). However, it is entirely unknown whether NO biosynthetic pathway dysfunction contributes to the functional or structural abnormalities that are characteristic of the disorder. To better understand the potential role of pulmonary NOS and NO in the pathogenesis of CLD associated with preterm birth, we evaluated the effects of continual postnatal NO administration via inhalation on pulmonary vascular and airway function in the baboon model over 14 days. The baboons were born by cesarean section at 0.67 gestation, which is comparable to 27 wk of postconceptual age in humans, and

Address for reprint requests and other correspondence: P. W. Shaul, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

nitric oxide; patent ductus arteriosus THE SIGNALING MOLECULE NITRIC OXIDE

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INHALED NO AND NEONATAL CHRONIC LUNG DISEASE

inhaled NO (iNO) at 5 ppm was begun at 1 h of age. We tested the hypothesis that iNO counteracts the pulmonary hypertension and bronchoconstriction associated with CLD. Because NO also mediates peripheral contractile elements in the newborn lung (25), we also tested the hypothesis that iNO improves dynamic lung compliance and postmortem pressurevolume relationships. In addition, because the current form of CLD is characterized by abnormal elastin deposition and fewer and larger alveoli (21, 41) and NO plays a role in alveolar development and branching morphogenesis (4, 49), we evaluated the effect of iNO on pulmonary growth and structure. Because NO antagonizes smooth muscle cell replication (18), we also assessed possible effects on bronchiolar or pulmonary arterial muscularity. MATERIALS AND METHODS

Animal model. All animal studies were performed at the Southwest Foundation for Biomedical Research (SFBR) Primate Center in San Antonio, TX. All procedures were approved by the Institutional Animal Care and Use Committee at the SFBR. Pregnancies in baboons (Papio papio) were timed with cycle dates, and fetal growth parameters were obtained from prenatal ultrasound examinations performed at 70 and 100 days of estimated fetal gestation. Fetal baboons were delivered at 125 ⫾ 2 days of gestation (term ⫽ 185 days) by cesarean section. At birth the baboons were weighed, sedated, intubated, and given 4 ml/kg of surfactant (Survanta, courtesy of Ross Laboratories, Columbus, OH) before the initiation of ventilator support. Ventilation was provided for 14 days with a humidified, pressure-limited, time-cycled infant ventilator (InfantStar, Infrasonics, San Diego, CA) or a 3100A high-frequency oscillatory ventilator (kindly provided by Sensormedics, Yorba Linda, CA) when necessary. Pulse oximeters were kindly provided by Masimo (Irvine, CA). Details of animal care have been published elsewhere (12). Animals were randomly assigned to either the control group, which received routine care, or to the NO group, which received routine care plus iNO. For selected endpoints, additional studies were done using lung tissue from 125- or 140-day gestation animals killed immediately at delivery. Ventilatory management and NO replacement. The ventilatory approach entailed a strategy to maintain tidal volumes at 4 – 6 ml/kg as determined by a VitalTrends body plethysmograph system (VT1000; Vitaltrends Technology, New York, NY) and to generate adequate chest motion by clinical examination. There was rigorous targeting of arterial blood gas parameters to PaCO2 values ranging from 45 to 55 Torr and PaO2 levels between 55 and 70 Torr. In an attempt to minimize exposure to high FIO2, if the PaO2 level was above target goals, FIO2 was weaned until ⬍0.40, and then modifiers of mean airway pressure or FIO2 were decreased as tolerated. If PaO2 was below target guidelines, a chest radiograph was obtained to evaluate lung inflation. Adjustments in mean airway pressure were made to minimize underinflation or overinflation of the lung. If lung inflation was deemed adequate, FIO2 alone was adjusted. Levels of ventilatory and oxygen support were assessed by determinations of oxygenation index (OI) and ventilation index (VI). The formula utilized for OI was OI ⫽ mean airway pressure 共cm/H2O兲 ⫻ FIO2 ⫻ 100/PaO2 The formula for VI was VI ⫽ peak inspiratory pressure ⫻ ventilator rate ⫻ PaCO2/1,000 One hour after delivery, iNO was administered to the experimental group at a level of 5 ppm with an INOVent according to established procedures (26). The NO gas and INOVent were kindly provided by iNO Therapeutics (Clinton, NJ). NO inhalation was continued at the AJP-Lung Cell Mol Physiol • VOL

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same level until the completion of the study at 14 days of age. This strategy was chosen because lung NO production is depressed in this model until day of life 11 (3). Hemodynamic support. Significant hypotension was defined as a transduced mean blood pressure ⬍28 mmHg accompanied by either increasing base deficit or decreasing urine output. Hypotension was initially treated with additional volume supplementation (20 ml/kg over 1 h) and the use of dopamine (5–20 ␮g 䡠 kg⫺1 䡠 min⫺1). Dobutamine was added (4 –10 ␮g 䡠 kg⫺1 䡠 min⫺1) if mean pressure was not restored to ⬎28 mmHg. In those animals where dobutamine appeared to exacerbate hypotension, epinephrine (0.2–1.0 ␮g 䡠 kg⫺1 䡠 min⫺1) was used in lieu of or as an additional inotrope. If this approach failed to improve mean blood pressure within 2 h, then a stress dose of hydrocortisone (1.0 mg/kg) was administered at 6-h intervals until either mean blood pressure increased to ⬎28 mmHg or a maximum of four doses of hydrocortisone were received. Once mean blood pressure was stable for ⬎12 h, pressor support was weaned in reverse to the order it was initiated. Echocardiography and pulmonary function testing. Echocardiographic studies were performed at 1 and 6 h of age and at 24-h intervals, up to 1 day before necropsy. The echocardiograms were done by one of the authors (D. C. McCurnin) coincident to the pulmonary function tests using previously reported techniques (47). Pulmonary function testing was performed with the VT1000 body plethysmograph (Vitaltrends Technology). This system is a flowthrough whole body plethysmograph for the continuous measurement of gas exchange and ventilation of infants during assisted ventilation (12, 48). Dynamic lung compliance and resistance measurements were of the respiratory system as a whole. For data analysis compliance was corrected for body weight. Patent ductus arteriosus ligation. To control for the presence or absence of a persistent patent ductus arteriosus (PDA) and its potential ramifications on cardiopulmonary function (10), all animals underwent surgical ductal ligation on day of life 6 by standard techniques. Surgical ligation was done regardless of prior spontaneous ductal closure or not. This time point was chosen because previous experience has shown that the animals do not tolerate the surgery before day of life 6. Postmortem pressure-volume measurements. Immediately before termination, the animals breathed 100% oxygen for 5 min, and we degassed the lungs by clamping the trachea at end expiration for 2 min. After the removal of the lungs from the thoracic cavity en bloc, we carried out postmortem quasistatic inflation pressure-volume measurements by inflating the lungs in a stepwise manner (5-cmH2O increments) to a pressure of 20 cmH2O. At each increment, the pressure was held for 30 s, and volume was recorded. The lungs were then inflated to 35 cmH2O for 1 min, and maximal lung volume was recorded. A deflation limb pressure-volume curve was also generated by reducing pressure in steps of 5 cmH2O, with stabilization at each step, and recording of the corresponding volumes (44). Lung growth, cell replication, and apoptosis. Total lung wet weight was determined at termination before any lavage procedure, and the wet weight-to-dry weight ratio was calculated following dessication of an aliquot of tissue from a nonlavaged lobe. After homogenization of a separate tissue sample, the total protein content was determined by Bradford analysis, and DNA was extracted, precipitated, and quantitated by a fluorescent dye technique (8, 46). Cell proliferation was evaluated by immunostaining for the proliferation-associated marker Ki67 (1:50 dilution; Dako Cytomation, Carpenteria, CA), which has been used previously in studies in the baboon CLD model (27). The degree of apoptosis was assessed by staining for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) by end-labeling of 3⬘ DNA fragments with biotinylated uridine (GIBCO-BRL, Grand Island, NY) and signal generation with streptavidin-peroxidase and diaminobenzidine-hydrogen peroxide (29). Starting in the left upper corner of the tissue section on each slide, we photographed the first 10 fields of alveoli subjacent to 288 • MARCH 2005 •

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terminal bronchioles branching into respiratory bronchioles at ⫻10 on Kodak Gold film. In a manner similar to that previously used to assess capillary density (12), a point-counting method in which the lung parenchymal tissue served as the volume of reference was employed to determine the volume fraction of Ki67 and TUNEL immunoreactive sites. Elastin deposition. To evaluate elastin deposition, sections of paraformaldehyde-fixed, paraffin-embedded lung were deparaffinized, hydrated, and stained by Hart’s method as previously described (33). Quantitative analysis of the area of elastin deposition in parenchyma, terminal bronchioles, and accompanying small pulmonary arteries was performed (7). mRNA abundance and distribution for tropoelastin, the soluble precursor of elastin, were assessed by in situ hybridization (33). To evaluate alveolar myofibroblast distribution, immunofluorescence was done for ␣-smooth muscle actin (28). Morphometric-histopathologic analyses. To quantifiably assess alveolarization, digital image analysis of the lung parenchyma was performed by the methods of Tschanz and Burri (43). We took 27–33 gray-scale photographs of hematoxylin and eosin-stained paraffin sections of the right lower lobe with a ⫻10 objective following a stratified random sampling procedure (9). We analyzed alveolarization by measuring the number of secondary septal crests. The algorithm described by Tschanz and Burri was adapted into a macro for ImagePro 4.5 (Media Cybernetics, Silver Spring, MD). Each photographic image was processed with the macro to thin, or skeletonize, the alveolar septa on the two-dimensional section into a network of lines that were a single pixel in thickness (43). Figure 1 shows a composite of alveolar walls and their skeletal network. A counting frame that incorporated the concept of forbidden lines for unbiased counting was imposed on the alveolar skeleton. The number and length of primary septal segments and secondary crests were tallied and analyzed. The following specific parameters were evaluated: mean length of primary septal segments and secondary crests, numerical frequency of primary septal segments and secondary crests per mm2 of alveolar area on the section, number of secondary crests per mm of primary septal length, and length ratio of secondary crests to primary septa. Secondary crests were also categorized according to their lengths into four groups: ⬍5 ␮m, 5–10 ␮m, 10 –25 ␮m, and ⬎25 ␮m. Mean length, numerical frequency, and length frequency for each length category were calculated. Gross histopathologic analysis was also done following previously published procedures (12). Hematoxylin and eosin-stained lung sections were analyzed for the presence or absence of secondary crests/ alveoli, the extent of saccular/alveolar wall fibrosis, the degree of

atelectasis, and the presence or lack of airway epithelial metaplasia and smooth muscle hypertrophy or infection. Three blinded raters used a panel of standards representing the spectrum of changes from the most normal (score of 1) to the most abnormal (score of 4) to evaluate a minimum of three photomicrographs from each control or iNO animal. Generalized ␬, measuring the overall level of agreement among the three raters, was moderate for the complete set (␬ ⫽ 0.680), moderate within the control group (␬ ⫽ 0.631), and high within the NO replacement group (␬ ⫽ 0.795). The three ratings were averaged for each photomicrograph, and ratings for control and iNO groups were compared. To evaluate capillary density, lung sections were immunostained for platelet endothelial cell adhesion molecule (PECAM, CD31; Dako, Via Real, CA), an endothelial cell marker, and a point counting method employing the lung parenchymal tissue as the volume of reference was used to determine the fraction of immunoreactive sites (12). One of the authors (J. J. Coalson) blinded to the experimental groups reviewed all samples. Using previously reported approaches (7), we performed quantitative morphometric analysis to evaluate the muscularity of distal airways and small pulmonary arteries. Hart’s elastic fiber stain was used to reveal lung structural features, and analyses were performed exclusively on circular (cross-sectional) profiles of terminal bronchioles and accompanying small pulmonary arteries. To assess airway smooth muscle abundance, the external perimeter (bronchiole area) and the internal perimeter of the muscular layer (epithelium-pluslumen area) were traced, and the epithelium-plus-lumen area was subtracted from the bronchiole area to obtain the muscle area. Results are expressed as the ratio of muscle area to bronchiole area. In a similar manner, to determine vascular smooth muscle abundance, the external perimeter (vessel area) and the internal perimeter of the medial layer (endothelium-plus-lumen area) were traced, and the endothelium-plus-lumen area was subtracted from the vessel area to obtain the medial area. Results for vascular smooth muscle abundance are expressed as the ratio of medial area to vessel area. We inspected 6 –10 terminal bronchioles and accompanying small pulmonary arteries per animal. Statistical analysis. Longitudinal between-group differences over the full course of study were compared by two-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc testing at individual time points. Repeated measures was not performed because values for individual animals were occasionally unobtainable due to technical difficulties or unavailability of the echocardiographer. The frequency of unavailable data points was 4% for pulmonary function tests and 8% for echocardiographic parameters. Single comparisons between two groups were performed with nonpaired Student’s t-tests or Mann-Whitney (nonparametric) for contiguous data and by Fisher’s exact test for categorical data. Comparisons between multiple groups were done by one-way ANOVA with Newman-Keuls post hoc testing. Wilcoxon signed-ranks test was used for the panel of standards histopathological analysis, and the postmortem pressure-volume curves were assessed by two-way repeated-measures ANOVA followed by Newman-Keuls. Significance was accepted at the 0.05 level of probability. All results are expressed as means ⫾ SE. RESULTS

Fig. 1. Composite image of fetal baboon lung alveolar walls and their skeletal tracing. Image was created using ImagePro with a custom macro following the algorithm described by Tschanz and Burri (43). Primary septal segments (arrows) are represented by lines between nodal points (F). Secondary crests (arrowheads) are represented by lines between end points (E) and nodal points. AJP-Lung Cell Mol Physiol • VOL

Study groups and clinical course. The clinical characteristics of the control and NO groups are presented in Table 1. Sixteen animals were entered in the control group, and 10 were entered in the NO group. More animals were in the control group than in the NO group because additional controls were available during the course of the study from shared, noninterventional protocols in the Bronchopulmonary Dysplasia Consortium. There were four deaths in control animals, which occurred at 24, 26, 35, and 168 h of age, and there were two deaths in NO 288 • MARCH 2005 •

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Table 1. Clinical characteristics of study groups

Initial study groups, n Deaths, n Longitudinal study groups, n Male/female, n Gestational age, d Birth weight, g

Control

NO

16 4 12 8/4 125⫾2 405⫾26

10 2 8 6/2 125⫾1 418⫾36

NO, nitric oxide; d, day.

animals at 26 and 120 h of age. All deaths were related to respiratory failure and unresponsive hypotension, and the death rate was similar in the two groups. Because most of the deaths occurred within 48 h of birth and all took place within the first 7 days of study, these animals were excluded from longitudinal analyses. The number of males vs. females, the gestational ages at delivery, and the birth weights were similar in the control and NO animals, both in the initial and the longitudinal study groups. There were no differences between groups for daily fluid intake, daily urine output, or daily weights over the course of the study. The use of pressor support was common. Whereas the number of animals treated with dopamine was similar between groups (Table 2), the duration of dopamine use was greater in NO animals. Although dobutamine use was comparable, more NO animals than controls received epinephrine. Hydrocortisone was administered to the majority of animals in both groups (10 of 12 controls and 7 of 8 NO animals). Pressor support requirements after 96 h were similar in control and NO animals. Pulmonary and systemic hemodynamics. Estimates of pulmonary artery pressure were feasible by analysis of the peak velocity of the PDA jet until the time of surgical ductal ligation on day 6 of life. The ratio of pulmonary to systemic arterial pressure was lower in the NO group vs. control on days 1 and 2 of life (Fig. 2A). Pulmonary arterial pressure alone did not differ between groups throughout the study period (data not Table 2. Pressor support requirements

Dopamine Number treated Age initiated, h Duration of use, h Maximum dose, ␮g䡠kg⫺1䡠min⫺1 Number treated at 96 h Dose at 96 h, ␮g䡠kg⫺1䡠min⫺1 Dobutamine Number treated Age initiated, h Duration of use, h Maximum dose, ␮g䡠kg⫺1䡠min⫺1 Number treated at 96 h Dose at 96 h, ␮g䡠kg⫺1䡠min⫺1 Epinephrine Number treated Age initiated, h Duration of use, h Maximum dose, ␮g䡠kg⫺1䡠min⫺1 Number treated at 96 h

Control (n ⫽ 12)

NO (n ⫽ 8)

11 27 (21–30) 67 (46–92) 12 (9–14) 7 2 (2–3)

7 24 (10–31) 115 (68–153)* 14 (10–16) 6 4 (2–9)

5 32 (22–35) 37 (22–72) 5 (3–6) 1 2

5 30 (14–38) 42 (13–49) 2 (1–4) 0

1 36 45 1.0 0

5* 33 (31–40) 31 (12–44) 0.8 (0.4–0.8) 0

Values are expressed as median (25–75 percentile), *P ⬍ 0.05 vs. control. AJP-Lung Cell Mol Physiol • VOL

Fig. 2. Inhaled nitric oxide (iNO) alters pulmonary and systemic hemodynamics during the 1st postnatal week in chronic lung disease (CLD). A: pulmonary artery pressure (PAP) was estimated noninvasively by measuring the peak velocity of the patent ductus arteriosus (PDA) jet, and the ratio of PAP to systemic arterial pressure (SAP) was calculated. B: left and right ventricular output was calculated from the mean velocity and used to determine pulmonary-to-systemic blood flow ratio (Qp/Qs). C: mean SAP was measured via an arterial catheter up to the day before necropsy. Values are means ⫾ SE, n ⫽ 12 and 8 for control and NO groups, respectively. Statistical comparisons between groups were made by 2-way ANOVA (P ⬍ 0.05 for all results shown) followed by Newman-Keuls post hoc testing at individual time points. *P ⬍ 0.05 vs. control.

shown). The ratio of pulmonary to systemic blood flow (Qp/ Qs) was lower in the NO group on day 5 of age (Fig. 2B). There was a greater rate of spontaneous ductus arteriosus closure in the NO group (6 of 8 animals) vs. controls (2 of 12 animals, P ⬍ 0.05 by ␹2 analysis) before elective surgical ligation at 6 days of age. In the NO group, the ductal closures occurred as follows: one between days 0 and 1, two between days 1 and 2, one between days 2 and 3, one between days 4 and 5, and one between days 5 and 6. After ligation, Qp/Qs values were near 1.0 and similar in control and NO groups. Mean systemic arterial pressure was similar in the two groups except on days 5 and 6 of life, when it was greater in the NO animals (Fig. 2C). This may have been related to the lower rate of ductal patency and therefore less left-to-right shunting in the 288 • MARCH 2005 •

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NO group during this time period. There was no difference between groups in the rate-corrected velocity of circumferential fiber shortening, an index of left ventricular function (data not shown). Pulmonary function. Findings for dynamic lung compliance are shown in Fig. 3A. Compliance was greater in NO animals compared with controls on days 1, 4, and 6. After elective surgery to close the ductus on day 6 of life, differences in dynamic lung compliance between groups were less apparent. Results for expiratory resistance are given in Fig. 3B. Resistance was lower in the NO group compared with controls on days 4, 5, and 6. After surgical ductal ligation on day of life 6, a difference in resistance was only evident on day 10. Although the care providers were not blinded to the control vs. iNO status of the animals, protocols for the use of ventilator and oxygen support were strictly guided by predetermined goals for blood gas values (12). The OI for the experimental groups is shown in Fig. 4A. There was no difference in OI between control and iNO animals. However, the VI was lower in the NO group vs. controls on day 6 of life before the thoracotomy for ductal ligation (Fig. 4B). VI was similar between study groups following the surgical procedure. Pressure-volume measurements and lung weights. Findings for the postmortem pressure-volume measurements for the control and NO groups are provided in Fig. 5. Both the inspiratory and expiratory curves were shifted upward in the NO group vs. the control group. In addition, the lung volume at maximal distending pressure of 35 cmH2O was increased by 45% with iNO. We evaluated overall lung growth by determining the lung weights at termination. There was a 19% increase in lung

Fig. 3. iNO improves dynamic lung compliance and expiratory resistance during the 1st postnatal week in CLD. Compliance (ml 䡠 cmH2O⫺1 䡠 kg⫺1, A) and expiratory resistance (cmH2O 䡠 ml⫺1 䡠 s⫺1, B) were measured by whole body plethysmography. Reported values are for the respiratory system as a whole. Values are means ⫾ SE, n ⫽ 12 and 8 for control and NO groups, respectively. Statistical comparisons were made between groups by 2-way ANOVA (P ⬍ 0.05 for all results shown) followed by Newman-Keuls post hoc testing at individual time points. *P ⬍ 0.05 vs. control. AJP-Lung Cell Mol Physiol • VOL

Fig. 4. iNO does not alter oxygenation index (A), but ventilation index (B) is improved late in the 1st week. Values are means ⫾ SE, n ⫽ 12 and 8 for control and NO groups, respectively. Statistical comparisons were made between groups by 2-way ANOVA (not significant for A, P ⬍ 0.05 for B) followed by Newman-Keuls post hoc testing at individual time points. *P ⬍ 0.05 vs. control.

weight relative to body weight with NO (Fig. 6A). Body weight at necropsy was similar in the control and NO-treated animals. To provide additional perspective to the effects of iNO on overall lung growth, we made comparisons between the lung weights of gestational controls and those of the two CLD study groups. As expected with normal fetal growth, the weights of lungs from 140-day gestational controls were greater than

Fig. 5. iNO causes marked changes in postmortem pressure-volume curves in CLD. Quasistatic inspiratory pressure-volume measurements were performed up to 20 cmH2O, lung volume at maximal distending pressure of 35 cmH2O was recorded, and expiratory curves were also generated. Values are means ⫾ SE, n ⫽ 12 and 8 for control and NO groups, respectively. Both inspiratory and expiratory curves were different in control vs. NO groups (2-way repeatedmeasures ANOVA P ⬍ 0.05 and Newman-Keuls post hoc testing P ⬍ 0.05 at all pressures except for inspiratory curve at 5 and 10 cmH2O). 288 • MARCH 2005 •

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Fig. 6. iNO stimulates lung growth in CLD. A: lung wet weights at the end of study were calculated relative to body weight (BW), and the DNA and protein content was measured and expressed relative to wet weight (WW). Using immunohistochemistry for the cell cycle marker Ki67, we evaluated cell proliferation in lungs from control (B) and NO replacement animals (C). Cell proliferation in the airway (D) was greater in the NO group vs. controls (Con). Magnification in B and C is ⫻120, bar ⫽ 50 um. Values are means ⫾ SE, n ⫽ 12 and 8 for Con and NO groups, respectively, *P ⬍ 0.05 vs. Con.

those for 125-day gestation controls (Table 3). In contrast, following extrauterine existence over the same time period, the lungs from the control, nontreated CLD group had weights that were not different from those of 125-day gestation fetal animals. However, postnatal NO administration during that interval yielded lung weights that were greater than those of 125-day gestation controls and similar to those of 140-day gestation controls. When not corrected for body weight as was done in Fig. 6A, the difference in lung weights between control and NO-treated animals approached but did not reach statistical significance (P ⫽ 0.057). The wet-to-dry weight ratios were similar for lungs from control and NO-treated CLD animals, being 5.99 ⫾ 0.29 and 6.17 ⫾ 0.19, respectively. To further evaluate the basis for increased total lung capacity and lung weight in the NO animals, we measured the DNA and protein contents of the lungs (Fig. 6A). The DNA content was 2.2-fold greater in the NO group. In contrast, the protein content was unaltered. Because an increase in DNA content represents greater cellularity, relative levels of apoptosis and cell proliferation were evaluated. TUNEL staining yielded rare positive cells, and no difference was noted in control and NO lungs (data not shown). However, Ki67 staining revealed that compared with control lungs (Fig. 6B), cell proliferation was markedly increased in iNO animals (Fig. 6C). This was particularly evident in the terminal bronchioles, in which the percentage of positive cells was twofold greater in NO lungs AJP-Lung Cell Mol Physiol • VOL

vs. controls (Fig. 6D). Cell counts in tracheal aspirates during the course of the study and in terminal lavage fluid were similar in control and NO-treated animals (data not shown). Elastin deposition. In human infants and in animal models of CLD, there is disturbed elastin deposition (33, 41). Using Hart’s staining, we found control lungs displayed coarse elastin fibers distributed in brush-like patterns along thickened, shortened secondary crests (Fig. 7A). In contrast, elastin deposition was limited to a punctate pattern at the tips of secondary crests in lungs from iNO animals (Fig. 7B). Quantification revealed 70% greater elastin deposition in the parenchyma of control vs. NO animals (Fig. 7C). Elastin deposition in airways and arteries was unchanged with iNO (Fig. 7D). The mRNA abundance and distribution of tropoelastin, the soluble precursor of elastin, was assessed by in situ hybridization. There was consistently less tropoelastin mRNA detected in the alveolar walls of NO-treated vs. control animals (Fig. 7, E and F). ␣-Smooth muscle actin distribution was also evaluated by immunofluorescence. Signal was observed diffusely along the alveolar wall of control lungs (Fig. 7G). In contrast, intense ␣-smooth muscle actin staining was limited primarily to single cells at the tips of alveolar septa in lungs from iNO animals (Fig. 7H). Morphometric and histopathologic analyses. Digital image analysis was performed to quantify changes in alveolarization. The length of secondary crests was increased by NO (Fig. 8). Other quantified parameters were unaltered by iNO (Table 4). Gross histopathologic analysis by a panel-of-standards approach yielded similar scores in control and NO groups, and volume density for total parenchyma was unchanged (Table 5). The density of vascularity assessed by PECAM immunostaining was also similar (Table 5). Morphometric analyses were performed to assess the degree of muscularization of terminal bronchioles and accompanying small pulmonary arteries (Table 5). Expressed as the ratio of muscle area to bronchiole area, the muscularization of the airways was unchanged by iNO. Pulmonary arterial muscularization expressed in a comparable manner was also unaltered by NO. DISCUSSION

To determine whether NO biosynthetic pathway dysfunction contributes to the pathogenesis of CLD, the present study assessed the effects of NO administration via iNO in the baboon CLD model. The continuous provision of NO gas at 5 ppm beginning at 1 h of life caused a decrease in pulmonary artery pressure during days 1 and 2. Other functional features of CLD were also altered by iNO during the early course, lung growth was increased, and there was a major impact on elastin deposition. These observations in the primate model are critical to our understanding of NO biology in the developing human lung. Table 3. Effect of iNO on lung weight Study Group, n

125d 140d 125d 125d

gestation (15) gestation (22) ⫹ 14d (CLD) (12) ⫹ 14d (CLD) ⫹ iNO (8)

Lung Wet Weight, g

9.70⫾0.50 12.59⫾0.44* 11.10⫾0.77 13.16⫾0.43*

iNO, inhaled NO; CLD, chronic lung disease. *P ⬍ 0.05 vs. 125d gestation. 288 • MARCH 2005 •

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Fig. 8. iNO increases the length of secondary septal crests. Photographic images of the lung parenchyma from Con (n ⫽ 7) and NO-treated animals (n ⫽ 6) were processed to skeletonize the alveolar septa (Fig. 1), and the length of secondary septal crests was analyzed. Values are means ⫾ SE, *P ⬍ 0.05 vs. Con.

Fig. 7. iNO modifies elastin deposition in CLD. A: Hart’s staining revealed coarse elastin fibers (dark stain) distributed in brush-like patterns along thickened, shortened alveolar extensions in Con lung. B: elastin in NO-treated lung was concentrated in the tips of alveolar septa. Findings in A and B are representative of those in 5– 8 animals/group. Quantification of elastin deposition revealed decreased elastin in the parenchyma of NO-treated vs. Con lungs (C) and similar elastin content in control vs. NO group airway and arteries (D). In C and D, quantification was performed in 6 –10 sections per animal, on 3 animals per group, and values are means ⫾ SE, *P ⬍ 0.05 vs. control. In situ hybridization for tropoelastin revealed more intense distribution in the alveolar walls of control lungs (E) vs. NO-treated lungs (F). Alveolar myofibroblast distribution was assessed by immunofluorescence for ␣-smooth muscle actin. Signal was diffusely distributed along the alveolar walls of Con lungs (G). In contrast, lungs from NO-treated animals displayed ␣-smooth muscle actin staining in discrete single cells at the tips of alveolar septa (H). Findings in G and H are representative of those in 5– 8 animals per group. Magnification: A, B ⫽ ⫻400; E, F ⫽ ⫻100; G, H ⫽ ⫻1,000, bar ⫽ 50 ␮m.

Along with the perhaps predictable early decline in pulmonary artery pressure, there was a surprisingly greater rate of spontaneous ductus arteriosus closure in the NO group vs. controls, with 75 vs. 17% closure, respectively. Prior studies in the preterm baboon model have suggested that endogenous NO contributes to ductal patency (37). In the term ductus, initial functional closure is due to the constriction of ductal smooth muscle, and ensuing anatomic closure entails the loss of cells from the muscular medial layer and the development of neointimal mounds composed of proliferating endothelial cells. In contrast, there is less medial cell loss and less endothelial cell proliferation in the preterm ductus (11). Whereas the initial oxygen-induced constriction would be predicted to be attenuated by a possible increase in bioactive circulating NO metabolites with NO gas administration (17), smooth muscle cell loss AJP-Lung Cell Mol Physiol • VOL

and endothelial cell proliferation would be enhanced due to the antimitogenic effects of NO bioactivity in the former and the promitogenic and promigratory effects in the latter cell type (18, 30). To address these possibilities, comparisons of ductal anatomy in control and NO-treated animals during the first week of life are now warranted. Along with the observed differences in pulmonary artery pressure in the two study groups, there were modest effects on systemic hemodynamics. Mean systemic arterial pressure was similar in the two groups except on days 5 and 6 of life, when it was greater in the iNO animals. This may be related to the lower rate of ductal patency and therefore less left-to-right shunting in the NO group during that time period. It is notable that pressor support requirements were greater in the NOtreated animals, but this difference was apparent only during the first 96 h. Echocardiographic evaluation of the rate-corrected velocity of circumferential fiber shortening was not altered by NO, indicating that left ventricular function was not impaired. It has been demonstrated that iNO can cause cGMP accumulation and vasodilation in extrapulmonary vascular beds (24). This may be due to NO binding to vacant heme sites on hemoglobin (Hb) or to reduced plasma thiols to form Fe-nitrosyl- or S-nitroso-Hb or a plasma S-nitrosothiol, thereby retaining NO bioactivity which is capable of causing systemic vasodilation (17). We speculate that in the context of prematurity and the accompanying frailty in systemic hemodynamic status (12, 38), the nonpulmonary effects of iNO may be more apparent. Detailed studies of NO metabolism should be undertaken during this period of development. In addition to the changes in hemodynamics, iNO modified pulmonary function during the first week of life. Dynamic lung compliance was frequently increased in the NO-treated group, and following initial stabilization expiratory resistance was decreased from day 4 to 6. In addition, although the OI was similar in control and NO-treated animals, the VI was imTable 4. Digital analysis of alveolarization Control

NO

n 7 6 Lung volume, cm3 6.74⫾0.56 7.77⫾0.93 Number of primary septal segments, #/mm2 687⫾46 716⫾68 Mean length of primary septal segments, ␮m 16.65⫾0.50 16.64⫾0.77 Number of secondary crests, #/mm2 169⫾13 146⫾16 Number of secondary crests/mm primary septa 15⫾1 13⫾1 Length ratio of secondary crest to primary septa 8.0⫾0.6 9.6⫾1.5 288 • MARCH 2005 •

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Table 5. Histopathological and morphometric analyses

Panel of standards scores Volume density, total parenchyma Volume density, PECAM staining Bronchiole smooth muscle area Pulmonary artery smooth muscle area

Control

NO

2.1⫾0.2 0.30⫾0.02 0.38⫾0.02 0.15⫾0.01 0.67⫾0.03

1.9⫾0.3 0.32⫾0.03 0.36⫾0.03 0.17⫾0.01 0.73⫾0.03

PECAM, platelet endothelial cell adhesion molecule.

proved with iNO on day 6. These findings are consistent with the known role of NO in the regulation of both bronchomotor tone and peripheral contractile elements in the developing lung (22, 25). The effects of iNO on pulmonary function were less apparent during wk 2, after the elective ductal ligation on day 6. This is potentially related to adverse effects of the thoracotomy. Now knowing that iNO actually promotes ductal closure, we can contemplate new experiments to assess pulmonary function after wk 1 without the impact of invasive thoracic surgery. We evaluated the persistent impact of iNO on lung function by performing postmortem quasistatic inflation and deflation pressure-volume measurements. Both the inspiratory and expiratory curves were shifted dramatically upward in the NO group vs. the control group. These findings provide strong evidence of an improvement in lung function related to iNO that remains apparent at 14 days. In addition, the lung volume at maximal distending pressure was increased by 45% with iNO. Furthermore, the wet and dry weights of the lungs relative to body weight were both increased 19% in the NO group, indicating an increase in lung parenchymal volume not related to differences in lung water. In fact, the wet weights of the lungs from NO-treated animals were increased compared with 125-day gestation controls, and they were similar to those of 140-day gestation fetal baboons, which are their postconceptual age in utero controls. In contrast, the wet weights of nontreated CLD lungs remained comparable to those of 125day gestation fetuses. The DNA content of the lungs per unit of wet weight was remarkably 2.2-fold greater in the NO-treated group. This related neither to changes in apoptosis with iNO nor to a change in inflammatory cell infiltration as assessed by tracheal aspirate cell counts; instead it was related to an increase in pulmonary cell replication as indicated by Ki67 staining, particularly in the distal airways. There is accumulating evidence that NO enhances growth and differentiation in the developing lung (4, 49), and the present findings indicate that iNO has marked mitogenic effects on the lung in the CLD model. Disturbed elastin deposition is a pathologic hallmark of the current form of CLD in premature infants (41) and in models of the disease in preterm lambs and baboons (12, 33). Elastin is normally abundant in the tips of alveolar septa, which form alveolar entrance rings when viewed in three dimensions (32). Alveolar myofibroblasts, which have the morphology of fibroblasts but contain contractile elements and express ␣-smooth muscle actin, are believed to be the source of septal elastin in the form of its soluble precursor tropoelastin (31), and elastin is thought to play a key role in alveogenesis (15). In CLD in humans and animal models, there are increased amounts and abnormal distribution of elastin in short, blunted alveolar AJP-Lung Cell Mol Physiol • VOL

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secondary crests and also in greater numbers of fibers parallel to the axis of extended alveolar walls (12, 33, 41), tropoelastin expression is increased (33), and there is a greater number of ␣-smooth muscle actin-positive cells (42). In the present study, iNO caused dramatic effects on elastin deposition, resulting in a punctate distribution at the tips of secondary crests instead of coarse thickened fibers, and there was a decrease in parenchymal elastin volume of ⬎40%. In the control group, the robust ongoing expression of the soluble precursor for elastin, tropoelastin, indicates that excess elastin deposition continues even after 14 days of mechanical ventilation in this CLD model. In contrast, tropoelastin expression was attenuated in the iNO group, providing an explanation for the decrease in elastin deposition. In addition, iNO yielded ␣-smooth muscle actin-positive staining alveolar cells, presumed to be myofibroblasts, which were discretely localized to alveolar septa instead of diffusely distributed along the alveolar walls. Thus multiple lines of evidence indicate that iNO causes dramatic attenuation of the elastosis that is characteristic of CLD, and this is consistent with the marked changes in postmortem pressure-volume curves. Direct actions of NO on alveolar myofibroblasts must be considered since it has been observed that NO attenuates both myofibroblast accumulation in vivo during inflammatory processes and the differentiation of cultured fibroblasts to myofibroblasts in vitro (45). Along with the impact of iNO on elastin deposition in the developing alveoli, the intervention caused quantifiable lengthening of secondary crests. Other alveolarization parameters, as well as assessments of gross histopathology, vascularity, and smooth muscle changes, showed no difference with iNO. However, the observed effect on secondary crests assessed at only 2 wk of postnatal age suggests that later alveolarization may be modified by iNO. We postulate that the combination of the clear impact on lung growth and elastin deposition and the potential effect on later alveolarization may have dramatic net consequences on lung structure and function during ensuing postnatal life. In the present study, the role of NO in CLD was tested by the provision of the molecule in the form of inhaled gas. The cumulative observations suggest that NO biosynthetic pathway dysfunction may contribute to pathogenesis of the disorder. The early improvements in pulmonary function and VI with iNO or the potent, persistent impact on lung growth and elastin deposition may explain the decrease in the incidence of death or CLD in a recent single-center study of iNO in preterm infants (36). These findings also support the current efforts to complete multicenter trials in high-risk preterm infants (1, 34). However, it should be noted that the high levels of NO with the inhaled gas react with O2 and reactive oxygen species (ROS) including superoxide to yield higher oxides of nitrogen and peroxynitrite, which is capable of damaging alveolar epithelium (6). In contrast, under normal conditions, endogenous NO preferably complexes with thiols to form S-nitrosothiols that serve as a key reservoir of NO-related bioactivity that is resistant to toxic reactions with superoxide and other ROS (20). The present work provides new insights about the impact of exogenous NO on the genesis of CLD. However, further studies are now warranted to determine whether interventions that upregulate endogenous NO production or replace NO in a manner favoring physiological metabolism, or that combine 288 • MARCH 2005 •

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iNO with strategies to reduce ROS (9), result in even greater amelioration of this devastating disease. ACKNOWLEDGMENTS The authors thank all the personnel that support the Bronchopulmonary Disease (BPD) Resource Center: the animal husbandry group led by Drs. D. Carey and M. Leland; the neonatal intensive care unit staff (H. Martin, S. Ali, D. Correll, L. Kalisky, L. Nicley, R. Degan, S. Gamez); the Wilford Hall Medical Center neonatal fellows and D. Catland, who assist in the care of the animals; and the University of Texas Health Science Center San Antonio pathology staff (L. Buchanan, H. Dixon, A. Schreiner), who perform necropsies and obtain biological specimens. The authors also thank M. Dixon for assistance in the preparation of the manuscript. GRANTS This work was supported by National Institutes of Health (NIH) Grants HL-63399 and HD-30276 (P. W. Shaul), HL-63387 (R. A. Pierce), HL-63397 (J. D. Crapo), and HL-56401 and HL-62875 (K. H. Albertine). Additional backing was provided by NIH Grants HL-52636 (BPD Resource Center) and P51RR-13986 for facility support. REFERENCES 1. Abman SH. Monitoring cardiovascular function in infants with chronic lung disease of prematurity. Arch Dis Child Fetal Neonatal Ed 87: F15–F18, 2002. 2. Abman SH and Groothius JR. Pathophysiology and treatment of bronchopulmonary dysplasia. Current issues. Pediatr Clin North Am 41: 277–315, 1994. 3. Afshar S, Gibson LL, Yuhanna IS, Sherman TS, Kerecman JD, Grubb PH, Yoder BA, McCurnin DC, and Shaul PW. Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease. Am J Physiol Lung Cell Mol Physiol 284: L749 –L758, 2003. 4. Balasubramaniam V, Tang JR, Maxey A, Plopper CG, and Abman SH. Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse. Am J Physiol Lung Cell Mol Physiol 284: L964 –L971, 2003. 5. Barnes PJ. Nitric oxide and airway disease. Ann Med 27: 389 –393, 1995. 6. Beckman JS and Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol Cell Physiol 271: C1424 –C1437, 1996. 7. Bland RD, Albertine KH, Carlton DP, Kullama L, Davis P, Cho SC, Kim BI, Dahl M, and Tabatabaei N. Chronic lung injury in preterm lambs: abnormalities of the pulmonary circulation and lung fluid balance. Pediatr Res 48: 64 –74, 2000. 8. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248 –254, 1976. 9. Chang LY, Subramaniam M, Yoder BA, Day BJ, Ellison MC, Sunday ME, and Crapo JD. A catalytic antioxidant attenuates alveolar structural remodeling in bronchopulmonary dysplasia. Am J Respir Crit Care Med 167: 57– 64, 2003. 10. Clyman RI. Recommendations for the postnatal use of indomethacin: an analysis of four separate treatment strategies. J Pediatr 128: 601– 607, 1996. 11. Clyman RI, Chan CY, Mauray F, Chen YQ, Cox W, Seidner SR, Lord EM, Weiss H, Waleh N, Evans SM, and Koch CJ. Permanent anatomic closure of the ductus arteriosus in newborn baboons: the roles of postnatal constriction, hypoxia, and gestation. Pediatr Res 45: 19 –29, 1999. 12. Coalson JJ, Winter VT, Siler-Khodr T, and Yoder BA. Neonatal chronic lung disease in extremely immature baboons. Am J Respir Crit Care Med 160: 1333–1346, 1999. 13. Cummings JJ. Nitric oxide decreases lung liquid production in fetal lambs. J Appl Physiol 83:1538 –1544, 1997. 14. Dweik RA, Laskowski D, Abu-Soud HM, Kaneko F, Hutte R, Stuehr DJ, and Erzurum SC. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 101: 660 – 666, 1998. 15. Emery JL. The postnatal development of the human lung and its implications for lung pathology. Respiration 27, Suppl: 41–50, 1970. 16. Forster RE II, DuBois AB, Briscoe WA, and Fisher AB. Mechanics of breathing. In: The Lung: Physiologic Basis of Pulmonary Function Tests, edited by Scott SS and Kelly KM. Chicago, IL: Year Book Medical, 1986, p. 65–114. AJP-Lung Cell Mol Physiol • VOL

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