PEDIATRICS Vol. 121 No. 5 May 2008, pp. 945-956 (doi:10.1542/10.1542/peds.2007-2051)
ARTICLE |
Ibuprofen-Induced Patent Ductus Arteriosus Closure: Physiologic, Histologic, and Biochemical Effects on the Premature Lung
a Department of Pediatrics, University of Texas Health Science Center, San Antonio, Texas
b Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado
c Pharmaceutical Discovery Division, SRI International, Menlo Park, California
d Pulmonary Biology, Cincinnati Children's Hospital, University of Cincinnati, Cincinnati, Ohio
e Department of Pediatrics, University of Utah, Salt Lake City, Utah
f Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas
g Cardiovascular Research Institute
h Department of Pediatrics, University of California, San Francisco, California
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ABSTRACT |
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OBJECTIVE. The goal was to study the pulmonary, biochemical, and morphologic effects of a persistent patent ductus arteriosus in a preterm baboon model of bronchopulmonary dysplasia.
METHODS. Preterm baboons (treated prenatally with glucocorticoids) were delivered at 125 days of gestation (term: 185 days), given surfactant, and ventilated for 14 days. Twenty-four hours after birth, newborns were randomly assigned to receive either ibuprofen (to close the patent ductus arteriosus; n = 8) or no drug (control; n = 13).
RESULTS. After treatment was started, the ibuprofen group had significantly lower pulmonary/systemic flow ratio, higher systemic blood pressure, and lower left ventricular end diastolic diameter, compared with the control group. There were no differences in cardiac performance indices between the groups. Ventilation index and dynamic compliance were significantly improved with ibuprofen. The improved pulmonary mechanics in ibuprofen-treated newborns were not attributable to changes in levels of surfactant protein B, C, or D, saturated phoshatidylcholine, or surfactant inhibitory proteins. There were no differences in tracheal concentrations of cytokines commonly associated with the development of bronchopulmonary dysplasia. The groups had similar messenger RNA expression of genes that regulate inflammation and remodeling in the lung. Lungs from ibuprofen-treated newborns were significantly drier (lower wet/dry ratio) and expressed 2.5 times more epithelial sodium channel protein than did control lungs. By 14 days after delivery, control newborns had morphologic features of arrested alveolar development (decreased alveolar surface area and complexity), compared with age-matched fetuses. In contrast, there was no evidence of alveolar arrest in the ibuprofen-treated newborns.
CONCLUSIONS. Ibuprofen-induced patent ductus arteriosus closure improved pulmonary mechanics, decreased total lung water, increased epithelial sodium channel expression, and decreased the detrimental effects of preterm birth on alveolarization.
Key Words: patent ductus arteriosus • cytokines • lung remodeling • bronchopulmonary dysplasia • pulmonary edema • wet/dry ratio • compliance • indomethacin • surfactant • epithelial sodium channel
Abbreviations: BPD—bronchopulmonary dysplasia • PDA—patent ductus arteriosus • ENaC—epithelial sodium channel • BALF—bronchoalveolar lavage fluid • MCP—monocyte chemoattractant protein • MIP—macrophage inflammatory protein • IL—interleukin • TNF—tumor necrosis factor • IFN—interferon
Although a persistent patent ductus arteriosus (PDA) is associated with the development of bronchopulmonary dysplasia (BPD),1,2 its role in causing BPD is currently not known.3–5 The absence of appropriate, randomized, clinical trials has made it difficult to examine a cause-and-effect relationship. Most PDA treatment trials were designed to assess the efficiency of pharmacotherapy on PDA closure, rather than to examine the role of a persistent PDA in neonatal morbidity.3 Only 1 randomized trial, performed 30 years ago, was designed specifically to examine the role of a persistent untreated PDA in neonatal morbidity.6 The investigators found that a persistent PDA prolonged the need for respiratory support. Whether the increased need for respiratory support was indicative of permanent pulmonary pathologic conditions is not known. An appropriate animal model to address this question has not been available until recently.
Premature baboons, delivered at 125 days of gestation (term: 185 days), have a neonatal course similar to that of premature human infants delivered between 26 and 27 weeks of gestation.7 They both develop respiratory distress and failure of PDA closure after birth. Despite prenatal glucocorticoid and surfactant treatments, low-tidal volume ventilation, and low supplemental oxygen administration during the first 2 weeks after delivery, premature baboons develop pulmonary histopathologic changes that are similar to those described for premature human infants with BPD.8,9 We previously examined the effects of surgical ligation of the PDA on the development of BPD in this model.10 We found no evidence that surgical ligation altered the evolution of histologic BPD. Because the trauma of surgical ligation itself might have obscured the effects of PDA closure on lung development, we designed the following study to examine the effects of pharmacologic PDA closure on the development of BPD during the first 2 weeks after delivery.
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METHODS |
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Animals and Procedures
Studies were performed at the Southwest National Primate Research Center, Southwest Foundation for Biomedical Research (San Antonio, TX), and were approved by the institutional animal care and use committee. Details of animal care were published elsewhere.8–10 Briefly, timed pregnant baboon (Papio papio) dams were treated with 6 mg of intramuscularly administered betamethasone 48 and 24 hours before elective delivery at 125 ± 2 days of gestation (term: 185 days). At birth, the infants were weighed, sedated, intubated, given surfactant (Survanta, courtesy of Ross Laboratories, Columbus, OH) before initiation of ventilator support (InfantStar ventilator; Infrasonics, San Diego, CA), and ventilated for 14 days. Ventilator adjustments were made on the basis of chest radiographic findings, clinical examination results, arterial blood gas measurements, and tidal volume measurements.9 Target goals for PaO2 were 55 to 70 mmHg, for PaCO2 were 45 to 55 mmHg, and for tidal volume were 4 to 6 mL/kg. Nutritional, fluid, transfusion, antibiotic, and blood pressure management were described previously.9 None of the animals received postnatal steroid treatment.
Preterm newborns received 1 of 2 treatment protocols, that is, ibuprofen (n = 8) or no treatment (control; n = 13). Ibuprofen was administered intravenously (over 20 minutes) according to the following schedule: 10 mg/kg (24 hours), 5 mg/kg (48 hours), 5 mg/kg (72 hours), 5 mg/kg (96 hours), and 5 mg/kg (120 hours). We studied the newborns for the first 14 days after birth because there was a high likelihood that the animals would develop septicemia and/or pneumonia beyond 14 days.8 Because sepsis plays a significant role in the development of chronic lung disease in preterm infants, the presence of septicemia or pneumonia in the animals would significantly compromise our ability to detect the effects of a PDA on the evolution of BPD. Chest radiographs were obtained daily, and surveillance cultures were obtained while the animals were alive; histologic examinations were performed at necropsy. None of the animals in our control or treatment groups developed septicemia or pneumonia during the study period.
Pulmonary function testing was performed with a VitalTrends plethysmograph system (VT1000; Vitaltrends Technology, New York, NY).9 Compliance measurements were of the respiratory system as a whole and were corrected for body weight. Oxygenation index (oxygenation index = mean airway pressure x fraction of inspired oxygen x 100/PaO2) and ventilation index (ventilation index = peak inspiratory pressure x ventilator rate x PaCO2/1000) were calculated at the same times.
A complete echocardiographic examination, including assessment of ductal patency, was performed daily by using an 8-MHz transducer interfaced with a Biosound AU3 echocardiographic system (Biosound, Genoa, Italy).11,12 Tracheal aspirates were collected after instillation of 1 mL of sterile normal saline solution through the endotracheal tube at 24 hours, 72 hours, 5 to 7 days, 9 to 11 days, and 14 days, as described previously.9 Tracheal aspirate samples were available from 10 control and 8 ibuprofen-treated newborns. For selected end points (see below), additional studies were performed with lung tissue from fetuses (at 125, 140, and 175 days of gestation) delivered through cesarean section and killed before breathing and from 1-day-old, spontaneously delivered, term, newborn baboons.
In one series of experiments (see below), we compared the effects of different cyclooxygenase inhibitors (ibuprofen and indomethacin) on lung epithelial sodium channel (ENaC) expression. For these experiments, preterm newborns (125 days of gestation) were treated with either ibuprofen (see schedule above) or indomethacin (0.1 mg/kg per dose; see schedule in ref 11). The newborns were necropsied after only 6 days of mechanical ventilation.
Preparation of Total RNA, Reverse Transcription, and Quantitative Polymerase Chain Reaction
At necropsy, total lung wet weight was determined. Tissue from the right middle lobe was frozen immediately in liquid nitrogen for RNA isolation, as described previously.13 TaqMan universal polymerase chain reaction master mixture (PE Applied Biosystems, Foster City, CA) was used to quantify the expression of the genes of interest. Taqman probes were designed by using the Primer Express program (Applied Biosystems) and were labeled with the fluorophores 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, as reporter and quencher dyes, respectively. An ABI Prism 7500 sequence detection system (Applied Biosystems) was used to determine the cycle threshold. Reactions were conducted in triplicate. Data were analyzed by using by the Sequence Detector 1.6.3 program. Malate dehydrogenase was used as an internal control, for normalization of the data.14
Wet/Dry Ratio
An aliquot of tissue from the right middle lobe was desiccated and used to calculate the wet weight/dry weight ratio.15,16
Western Analysis for 
-ENaC
A piece of the right middle lobe was homogenized in lysis buffer, and Western blotting for -ENaC protein was performed with an affinity-purified chicken polyclonal antibody raised against peptides corresponding to the NH2-terminal 22 amino acid residues of the rabbit -ENaC protein (Affinity BioReagents, Golden, CO), as described previously.16
Bronchoalveolar Lavage for Measurement of Saturated Phoshatidylcholine, Total Protein, Surfactant Proteins, and Cytokines/Chemokines
At necropsy, bronchoalveolar lavage of the left lower lobe was performed with 0.9% NaCl solution. Cells were removed from the bronchoalveolar lavage fluid (BALF) through centrifugation, as described previously.17 Aliquots were saved for measurement of saturated phoshatidylcholine, total protein, surfactant proteins (A, B, C, and D), and cytokines.18,19 By using left lower lobe/total lung weight ratios, results of the BALF measurements were calculated for the total lung and normalized to kilograms of body weight. Saturated phoshatidylcholine was also measured in tissue homogenates of the left lower lobe after lavage.
Tracheal aspirates and aliquots of BALF supernatants were diluted with 1 volume of 0.1% bovine serum albumin in phosphate-buffered saline and used for the simultaneous determination of 29 cytokines and chemokines with Luminex technology, as described previously.20 The coated bead/biotinylated antibody combinations used were for granulocyte colony-stimulating factor (Linco-plex human G-CSF; Linco Research, St Charles, MO), granulocyte/macrophage colony-stimulating factor (Beadlyte human GM-CSF; Upstate USA, Charlottesville, VA), interferon (IFN)- (anti-human IFN- clones MMHA-11 and MMHA-2; PBL Biomedical Laboratories, Piscataway, NJ), IFN-
(Beadlyte primate IFN-; Upstate), interleukin (IL)-1β (Beadlyte human IL-1β; Upstate), IL-1 receptor antagonist (Fluorokine MAP human IL-1R antagonist/IL-1F3; R&D System, Minneapolis, MN), IL-2 (Beadlyte primate IL-2; Upstate), IL-4 (Linco-plex human IL-4; Linco), IL-5 (Linco-plex human IL-5; Linco), IL-6 (Linco-plex human IL-6; Linco), IL-8 (Beadlyte human IL-8; Upstate), IL-9 (Beadlyte human IL-9; Upstate), IL-10 (anti-human IL-10 clones BN-10 and QS-10; Cell Sciences, Canton, MA), IL-12 (p40) (anti-human IL-12 clones IL-12I and IL-12II; Mabtech, Mariemont, OH), IL-13 (Beadlyte human IL-13; Upstate), IL-17 (human IL-17; Biosource International, Camarillo, CA), IL-18 (anti-human IL-18 clones 125-2H and 159-12B; MBL International, Woburn, MA), monocyte chemoattractant protein (MCP)-1 (human MCP-1; Biosource), macrophage-derived chemokine (Beadlyte human MDC; Upstate), macrophage inflammatory protein (MIP)-1 (human MIP-1; Biosource), MIP-1β (human MIP-1β; Biosource), regulated on activation, normal T expressed and secreted cytokine (Beadlyte human RANTES [regulated upon activation, normal T cells expressed and secreted]; Upstate), tumor necrosis factor (TNF)- (Beadlyte human TNF-; Upstate), and TNF-β (Beadlyte human TNF-β; Upstate).
Immunohistochemical and Lung Morphometric Analyses
The right lower lobe was removed, weighed, and intrabronchially fixed with phosphate-buffered 4% paraformaldehyde at constant pressure of 20 cmH2O for 24 hours. After fixation, the volume of the right lower lobe was determined through volume displacement and the lobe was processed for light microscopy.9 The right lower lobe was cut into 4 pieces of equal thickness. Tissue sections from each of the right lower lobe pieces were obtained by following a stratified random-sampling procedure.21
Several different morphometric approaches (described in detail elsewhere22–25) were used to quantify alveolar secondary septation and surface area. First, the volume density of secondary septa, that is, the ratio of the number of coherent-square grid points overlying alveolar secondary septa to the number of points overlying lung parenchyma, was measured. The same numbers of points overlaying lung parenchyma were counted per tissue section. Second, the tissue area, expressed as the tissue area/(tissue area + airspace area) ratio, was measured. Color thresholding was used to determine the calibrated pixel area over the tissue or airspace in 15 calibrated fields per tissue section (1 tissue section per baboon). Third, the width of the distal airspace walls was measured by drawing a line perpendicularly across the distal airspace walls, at the midpoint between the junctions of adjacent airspace walls. Five width measurements were made in each of 15 calibrated fields per tissue section (1 tissue section per baboon). Fourth, digital image analysis of alveolarization and surface area was performed. Twenty-seven to 33 grayscale photographs of tissue sections were obtained with a x10 objective, by following a stratified random-sampling procedure.21 Digital image analysis was performed by adapting the algorithm described by Tschanz and Burri26 into a macro for ImagePro 5.0 (Media Cybernetics, Silver Spring, MD). Each photographic image was processed with the macro to thin, or skeletonize, the alveolar septa on the 2-dimensional section into a network of lines that were a single pixel in thickness.26 The number and length of primary septal segments and secondary septa were tallied and summed to obtain the total alveolar surface area for the right lower lobe. The number of branch points (junctions of primary segments and/or secondary septa) per square millimeter of alveolar area on the section was used to determine the degree of alveolar complexity.22
Macrophage accumulation was assessed in lung parenchyma or distal airspace by determining the number of immunolabeled macrophages per unit area of the respective reference space. Macrophages were identified through immunolocalization of CD16 (mouse monoclonal antibody, catalog No. NCL-CD16; Novocastra Laboratories, Newcastle Upon Tyne, United Kingdom).24 The number of immunolabeled macrophages was counted in lung parenchyma or airspace, and the calibrated pixel area over lung parenchyma or airspace was automatically determined through color thresholding on 15 calibrated fields per tissue section (1 tissue section per baboon).
Tissue sections stained with Hart's elastic fiber stain were used to assess smooth muscle abundance in terminal bronchioles and their adjacent pulmonary artery.22,25 With the use of terminal bronchioles as the reference structure, the measurements were restricted to the same generation of resistance airway and artery. The area of the airway and pulmonary artery smooth muscle was determined by measuring the total area of the lumen plus smooth muscle and subtracting the measured area of the lumen alone. Results are expressed as the smooth muscle area/total area ratio. We made 2 to 5 area measurements per tissue section for terminal bronchioles and for the adjacent pulmonary artery (1 tissue section per baboon). To be included in the measurement process, the profile of terminal bronchioles and their adjacent pulmonary artery had to be nearly circular, defined as a maximal width/maximal length ratio of >0.70.23,25
Statistical Analyses
Data are presented as mean ± SD. Between-group differences were compared with analysis of variance, unpaired t test, or Mann-Whitney rank-sum test, where appropriate. Statistical results were generated by using Statview software (SAS Institute, Cary, NC). Significant differences were defined by P < .05.
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RESULTS |
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Study Groups and Clinical Course
Twenty-one animals (control: 13 animals; ibuprofen: 8 animals) were ventilated for 14 days. There were no differences between the 2 groups in birth weight (control: 355 ± 12 g; ibuprofen: 367 ± 7 g), gender (control: 31% male; ibuprofen: 50% male), gestation (control: 126 ± 1 days; ibuprofen: 125 ± 2 days), central blood cell counts (performed on days 7 and 14), or metabolic panel results (glucose, creatinine, albumin, globulin, alkaline phosphatase, lactate dehydrogenase, bilirubin, calcium, and phosphorus levels and anion gap; measured at birth and days 3, 6, and 14) (data not shown).
Cardiovascular Function
During the first 24 hours (before ibuprofen treatment was started), the 2 groups had similar systemic blood pressures, left-right shunts through the PDA (reflected by the pulmonary/systemic blood flow ratio), and indices of left ventricular performance, namely, shortening fraction (not shown) and rate-corrected velocity of circumferential fiber shortening (Fig 1). The PDA in the ibuprofen group either closed and remained closed (n = 5) or closed and then intermittently reopened (n = 3) after ibuprofen treatment (mean pulmonary/systemic blood flow ratio from 24 hours through 14 days: 1.1 ± 0.1) (Fig 1). In contrast, all of the animals in the control group had a PDA that remained open throughout the entire 14-day experiment; the average pulmonary/systemic blood flow ratio for the control group fluctuated between 1.5 and 2.2 from 24 hours through 14 days (mean: 1.8 ± 0.2; P < .001, compared with the ibuprofen group) (Fig 1).
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After the start of treatment, the ibuprofen group had significantly lower left ventricular end diastolic diameters and higher mean (as well as systolic and diastolic) systemic blood pressures (Fig 1). The group also had significantly lower pulmonary/systemic systolic pressure ratios than did the control group (data not shown).
The 2 groups had similar left ventricular performance indices (Fig 1); there were no differences between the 2 groups in base deficit, serum bicarbonate levels, or need for dopamine/dobutamine administration during the 14-day treatment course (data not shown). There were also no differences in fluid intake and urine output between the 2 groups (Fig 2).
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Pulmonary Function
During the first 24 hours (before ibuprofen treatment was started), the 2 groups had similar measurements of compliance, as well as ventilation and oxygenation indices (Fig 2). After the start of ibuprofen treatment, the ibuprofen group had significantly lower ventilation index and higher pulmonary compliance, compared with the control group (between days 1 and 14) (Fig 2). The 2 groups differed significantly in compliance and ventilation index during the early respiratory distress period (days 1 through 4: compliance, P = .004; ventilation index, P = .027) and during the later part of the experimental course (days 4.5 through 14: compliance, P = .013; ventilation index, P = .049).
Surfactant Amounts
At the time of necropsy, there was no difference between the 2 groups in the amounts of saturated phosphatidylcholine in either the lung tissue or the BALF (Fig 3). Similarly, there were no differences between the 2 groups in the amounts of surfactant proteins B, C, or D (Fig 3). In contrast, surfactant protein A levels were significantly greater in the ibuprofen group, compared with the control group (Fig 3).
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Biochemical Indicators of Inflammation and Remodeling
Compared with the nonventilated fetuses, both groups of premature newborns had elevated concentrations of proinflammatory molecules (IFN-
, IL-8, MCP-1, and MIP-1/β) in their necropsy BALF (Table 1). Although there were modest differences between the ibuprofen and control groups in IFN-, IL-1 receptor antagonist, and MIP-1β concentrations in both the necropsy BALF and the daily tracheal aspirates, there were no consistent differences between the ibuprofen and control groups in the cytokines most commonly associated with the development of BPD (IL-1β, IL-6, IL-8, and TNF-)27–31 (Tables 1 and 2). There was no difference in pulmonary protein permeability between the 2 groups (total protein content of the BALF: control: 241 ± 106 mg/kg body weight, n = 13; ibuprofen: 246 ± 93 mg/kg body weight, n = 8).
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Compared with the nonventilated fetuses, both groups of premature newborns had altered messenger RNA expression of several genes involved in inflammation, vascular development, and lung remodeling. Several genes had increased expression (CD3
, cyclooxygenase 2, and TNF-), whereas others had decreased expression (endothelial nitric oxide synthase, endothelin receptor A, vascular endothelial growth factor, transforming growth factor-β2, transforming growth factor-β3, and very late activation antigen-4). However, none of the genes seemed to be altered by ibuprofen-induced PDA closure (Table 3).
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Lung Fluid Balance
After birth, fluid was rapidly removed from the term baboon lung. Approximately one half of the late-gestation fetal lung wet weight was lost within 24 hours after birth (Fig 4). In contrast, the rate of net fluid removal from the premature lung was much slower (Fig 4). The lung wet weight decreased by <30% during the first 14 days after premature delivery. Baboons in the ibuprofen group had a small but significant decrease in total lung wet weight, compared with baboons in the control group (Fig 4).
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We hypothesized that a difference in the rate of fluid movement out of the alveolar compartment and into the lung interstitium might contribute to the difference in pulmonary mechanics between the ibuprofen and control groups. Because the
-subunit of ENaC seems to be critical for alveolar fluid clearance at birth,32 we examined its expression in the preterm baboon lungs (Fig 5). ENaC expression increased 2.5-fold in the lungs of newborns that were treated with ibuprofen (or indomethacin, another nonselective cyclooxygenase inhibitor). In contrast, ENaC expression in the control newborn lungs (125-day fetus) did not change after birth (Fig 5).
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Morphometric and Histopathologic Findings
The lung tissue had similar histologic appearances in the ibuprofen and control groups (Fig 6). The distal airspace walls were thin, alveolar secondary septa were present, and alveolar macrophages were sparse in both groups of baboons. Smooth muscle abundances around terminal bronchioles and their neighboring pulmonary arteries were also similar between the 2 groups (Table 4).
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We performed digital image analysis to determine the total alveolar surface area for the right lower lobe (by using the algorithm described by Tschanz and Burri26). As the gestation advanced from 125 and 140 days, there was a significant increase in the total alveolar surface area of the fetal lung (Fig 7). The ibuprofen-treated newborns, which were ventilated during the same 14-day period, also had a significant increase in total alveolar surface area after birth. The total alveolar surface area in the ibuprofen-treated newborns was significantly larger than that of the fetuses at 125 days of gestation (and their surface area was not significantly different from the area of the 140-day gestation fetal lung) (Fig 7).
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In contrast, there was no increase in total alveolar surface area after birth in the control newborns (with a PDA). The surface area of the control newborn lung was significantly smaller than the area of the fetal lung at 140 days gestation. Similar findings were noted when the degree of alveolar complexity (expressed as the number of branch points, ie, junctions of primary segments and/or secondary septa) was examined (Fig 7).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Lung injury superimposed on an immature lung leads to BPD. Numerous factors have been shown to alter the development of BPD, including degree of mechanical ventilation, fluid overload and edema, infection, inflammation, oxidant/antioxidant balance, protease/antiprotease balance, surfactant depletion or dysfunction, nitric oxide, nutrition angiogenesis, and genetic factors.33 The increased use of prenatal steroid treatment, exogenous surfactant therapy, adequate nutrition, and low-volume ventilation has altered the histopathologic appearance of BPD in recent years. BPD is now characterized primarily by impaired alveolar and vascular growth, rather than by extensive fibrosis, smooth muscle proliferation, and regional heterogeneity.8
PDA closure was shown previously to improve pulmonary mechanics34–36 and to decrease the need for mechanical ventilation.6 We observed similar results in the preterm baboons treated with ibuprofen. Ibuprofen decreased left-right PDA shunting and improved pulmonary compliance and ventilation indices (Figs 1 and 2). Cardiac performance was not affected by ibuprofen treatment (Fig 1). The novel findings of the current study relate to the impact ibuprofen treatment has on lung morphologic features and fluid clearance.
Digital image analysis showed an arrest in alveolar development in the lungs of control newborns that were exposed to a moderate-size left-right PDA shunt for 14 days (Fig 7). In the control newborn lungs, the alveolar surface area and branching were both significantly decreased, compared with fetal lungs that remained in utero until 140 days. In contrast, no significant arrest in alveolar development was observed when the PDA was closed with ibuprofen (Fig 7). These effects on lung histologic features were observed with digital image analysis; other assessments of gross histopathologic features, alveolarization, vascularity, and smooth muscle changes did not seem to differ between the 2 groups (Table 4). This discrepancy between different morphometric techniques was observed previously.22 It suggests that a single morphometric index of alveolar formation may not be sufficient to detect differences in preterm ventilated lungs.
The changes in pulmonary mechanics and alveolar surface area did not seem to be attributable to changes in surfactant levels. There were no differences between the groups in saturated phosphatidylcholine or surfactant protein B, C, or D levels (Fig 3). There was also no difference in the total amount of protein in the airspace (which might inhibit surfactant activity37).
We did observe a significant decrease in surfactant protein A content in the BALF of the control animals (Fig 3). This has been observed in both animal and human newborns who progress to BPD.17,38–40 It is doubtful that the decrease in surfactant protein A content had any effect on lung mechanics during our 14-day experiment.41 Surfactant protein A does opsonize bacteria and viruses and is thought to participate in the lung's defense. Decreased amounts of surfactant protein A might have increased the risk for pulmonary infection in the control animals. However, both experimental groups received comparable prophylactic antibiotic therapy and neither group had any clinical, radiographic, bacteriologic, hematologic, or histologic evidence of infection or pneumonia (data not shown). We hypothesize that the decreased amount of surfactant protein A in the control group is likely a reflection of the decreased alveolar surface area (resulting from epithelial cell damage/loss), rather than a factor contributing to the damage/loss.
Premature delivery and mechanical ventilation have been shown to increase levels of proinflammatory cytokines/chemokines (IL-1β, IL-6, IL-8, MCP-1, and TNF-) without affecting levels of antiinflammatory cytokines (IL-1 receptor antagonist, IL-4, IL-10, IL-12, IL-13, and IL-18) in the lung.18,27–31,42,43 Similarly, we found that the expression of several proinflammatory cytokines/chemokines and mediators of lung remodeling was altered by premature birth (Tables 1–3). Ibuprofen-induced PDA closure, however, did not affect the molecules most commonly associated with the development of BPD (IL-1β, IL-6, IL-8, and TNF-)27–31 (Tables 1–3). It is possible that our inability to detect differences between the 2 experimental groups may result from the small number of animals in each group; however, detectable differences in cytokine expression have been observed after other therapeutic maneuvers in this same experimental model.9
The improved pulmonary mechanics in the ibuprofen group were associated with an increase in ENaCs in the lung and a decrease in total lung water. In contrast to term newborns, preterm newborns have a slow rate of fluid clearance from the lungs (Fig 4). The presence of a PDA increases the rate of hydrostatic fluid filtration into the lung's interstitium44 and has been shown to increase the incidence of pulmonary edema and hemorrhage in preterm baboons and humans.45,46 However, detectable differences in total lung water do not become apparent until the lungs have been exposed to a PDA for several days15,44 (Fig 4).
Clearance of fluid from the alveolar airspaces requires the presence of transepithelial sodium transport, through amiloride-sensitive ENaCs.32 ENaC expression increases with advancing gestation (Fig 5) and can be regulated by oxygen and glucocorticoids.47 We found that ibuprofen-treated preterm baboons had a significant increase in pulmonary ENaC expression, compared with control animals. Indomethacin, another nonselective cyclooxygenase inhibitor, had a similar effect on ENaC expression (Fig 4). The effects of ibuprofen and indomethacin on ENaC seem to be attributable to their ability to inhibit cyclooxygenase activity, rather than their effect on PDA closure; ibuprofen exposure (10–7 M) can increase the expression of ENaC twofold in isolated distal lung epithelial cells (MLE-12 cells) in culture (J.P, and S.S., unpublished observations, 2007).
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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We found that pharmacologically induced PDA closure seemed to prevent some of the detrimental effects of preterm birth on alveolar complexity, surface area, and lung water clearance. These improvements occurred as early as 14 days after birth. Our studies cannot determine, however, whether the beneficial effects of ibuprofen were attributable to its effect on the PDA left-right shunt or were attributable to some other pharmacologic effect that might promote alveolarization and lung liquid clearance. We speculate that increased ENaC expression in the ibuprofen-treated animals may lead to increased fluid movement (between the alveolar and interstitial compartments) and this may explain the improved pulmonary mechanics. We further speculate that the improved pulmonary mechanics, and the resultant decreased need for mechanical ventilation, may result directly in improved alveolarization.27,48 Whether these changes lead to long-term consequences for lung structure and function during later life remains to be determined.
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ACKNOWLEDGMENTS |
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This research was supported in part by National Institutes of Health grants HL63399, HL56061, and HL52636 (BPD Resource Center), P51RR13986 (primate center facility support), R24 RR023345 (Dr Giavedoni), HL62875 and HL56401 (Dr Albertine), HL63397 (Dr Chang), HL63329 (Dr Ikegami), and HL46691 and HL56061 (Dr Clyman), Children's Health Research Center, University of Utah (Dr Albertine), and a gift from the Jamie and Bobby Gates Foundation (Dr Clyman). L. Darko (Farmacon) donated the ibuprofen for the experiments.
We thank all of the personnel who support the BPD Resource Center, including the animal husbandry group led by Drs D. Carey and M. Leland, the NICU staff members (H. Martin, D. Correll, L. Kalisky, L. Nicley, R. Degan, S. Salazar, and S. Ali), Deborah Catland, NNP, the Wilford Hall Medical Center neonatal fellows who assist in the care of the animals, and the University of Texas Health Science Center at San Antonio pathology staff members (L. Buchanan, K. Symank, Y. Valdes, and K. Mendoza) who perform necropsies. Albert Wint (University of Utah) helped with the image analysis studies. L. M. Parodi helped with the Luminex assays. Francoise Mauray performed the artwork. We especially thank Vickie Winter, who has skillfully managed to categorize and to keep track of all of the tissue samples and animal data over the years, and Dr Jackie Coalson, who provided invaluable leadership, guidance, and scientific oversight for the BPD Resource Center and with whom we have had many thought-provoking discussions.
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FOOTNOTES |
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Accepted Sep 17, 2007.
Address correspondence to Ronald I. Clyman, MD, Box 0544, HSW 1408, University of California, 513 Parnassus Ave, San Francisco, CA 94143-0544. E-mail: clymanr{at}peds.ucsf.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
| What's Known on This Subject Although a persistent PDA is associated with the development of BPD, its role in causing BPD is not known.
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| What This Study Adds We offer the first evidence that pharmacologic treatment of a persistent PDA may minimize the alveolar growth arrest that characterizes BPD in preterm infants.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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