Published ahead of print on February 12, 2004, doi:10.1164/rccm.200309-1276OC
© 2004 American Thoracic Society Treatment of Immature Baboons for 28 Days with Early Nasal Continuous Positive Airway PressureDivision of Paedatrics, Hammersmith Hospital, London, United Kingdom; Department of Medicine and Physiology, Southwest Foundation for Biomedical Research; Departments of Pathology, Pediatrics, and Obstetrics and Gynecology, University of Texas Health Science Center-San Antonio; and Pediatrix Medical Group, San Antonio, Texas Correspondence and requests for reprints should be addressed to Jacqueline J. Coalson, Ph.D., Department of Pathology, 7703 Floyd Curl Drive, UTHSCSA, San Antonio, TX 78229. E-mail: coalson{at}uthscsa.edu
Using the 125-day baboon model of long-term bronchopulmonary dysplasia, we hypothesized that early use of nasal continuous positive airway pressure (nCPAP), a noninvasive ventilatory method, combined with prophylactic surfactant therapy would permit continuation of alveolar and vascular development in the lung. Retrospective human studies have shown that infants treated with nCPAP spend less time on mechanical ventilation and thereby sustain less volutrauma. After delivery by cesarean section at 125 days (term, 185 days), the infants received two doses of surfactant (Curosurf) and daily caffeine citrate. Weaning from low-volume positive pressure ventilation to nCPAP was attempted at 24 hours of age. Serial physiological parameters were recorded. Lung histopathology and morphometric measurements of nCPAP animals were done after necropsy at 28 days and data were compared with 125- and 156-day gestational controls. Documented episodes of clinical sepsis and pneumonia at postmortem examination were absent. nCPAP lungs showed enlarged thin-walled air spaces with minimal fibroproliferation and scattered secondary crests. Internal surface area and surface-to-volume ratio dimensions were similar to those of 156-day gestational control lungs, the intrauterine developmental control. nCPAP is an effective noninvasive ventilatory technique that minimizes lung injury in baboons at risk of developing bronchopulmonary dysplasia.
Key Words: alveolarization • cytokines • pneumonia • sepsis • vasculogenesis In spite of numerous pharmacologic and technical advances in neonatal lung care, bronchopulmonary dysplasia (BPD) remains a cause of serious morbidity in surviving preterm infants (1–3). This "new BPD" differs from that originally described by Northway and coworkers (4) in that it affects predominantly those infants born between 24 and 28 weeks of gestation with birth weights less than 1,000 g, many of whom will have received antenatal glucocorticoids, minimal "gentle ventilation," and exogenous surfactant therapy (5). A variety of factors including surfactant deficiency, volutrauma, oxygen exposure, antenatal exposure to proinflammatory cytokines, postnatal infection, patent ductus arteriosus, and inadequate postnatal nutrition are thought to play a role in the pathogenesis of neonatal BPD (6–11). Metaanalysis has shown a significant decrease in the risk of mortality of BPD for neonates born at less than 30 weeks of gestation when surfactant is given prophylactically (12). The single greatest predictor for BPD appears to be the initiation of mechanical ventilation in the very low birth-weight infant (3, 13, 14). Retrospective studies have suggested that the early application of nasal continuous positive airway pressure (nCPAP) reduces the need for subsequent endotracheal intubation, mechanical ventilation, and surfactant therapy (13–15). Verder and coworkers (16, 17) have demonstrated that surfactant replacement therapy coupled with nCPAP in the early stage of respiratory distress syndrome is more effective than nCPAP alone; it improves oxygenation and reduces the need for mechanical ventilation in preterm infants. We have developed an immature primate model for neonatal BPD that approximates the human situation in terms of lung development and long-term ventilator support (18), and has clinical, biochemical, and histopathologic features comparable to those described in extremely immature human infants with BPD (19). Sustained mechanical ventilation with or without prophylactic surfactant therapy is accompanied by interrupted alveolar (20–22) and capillary development, the consistent histopathologic findings of neonatal BPD in mechanically ventilated premature infants. Although the application of early nCPAP and surfactant therapy has been associated with decreased rates of neonatal BPD, there are no studies describing its effect on subsequent lung development. Similarly, the effect of combining early surfactant replacement therapy and early nCPAP has not previously been reported in a long-term immature animal model of neonatal BPD. The purpose of this initial study was to establish whether it was possible to minimize the need for mechanical ventilation in the immature baboon by combining prophylactic surfactant with nCPAP therapy. We hypothesized that early extubation to nCPAP would result in acceptable gas exchange during the phase of acute lung injury and result in less inflammation or infection, and thereby enhance alveolarization over a 28-day study period. Some of the results of this study have been previously reported in an abstract (23).
All animal studies were performed at the Southwest Foundation for Biomedical Research (San Antonio, TX). All animal husbandry, animal handling, and procedures were reviewed and approved to conform to American Association for Accreditation of Laboratory Animal Care guidelines.
Delivery and Instrumentation Ventilation was initiated with a humidified, pressure-limited, time-cycled Infant Star ventilator (provided by Infrasonics, San Diego, CA). The initial rate was set at 40 breaths/minute, peak inspiratory pressure (PImax) adequate to move the chest, positive end-expiratory pressure (PEEP) at 5 cm H2O, and FIO2 commenced at 0.40. Peak inspiratory pressure was aggressively weaned to maintain minimal but not excessive chest wall motion during subsequent instrumentation with an umbilical arterial catheter and percutaneous central venous catheter. First PaO2 values ranged from 44 to 96 mm Hg. After this initial measurement, FIO2 was adjusted to achieve target levels of PaO2 of 55–70 mm Hg. Infants were nursed in a servo-controlled, infrared-warmed, body plethysmograph (VT1000; VitalTrends Technology, New York, NY) set at 36.9°C, capable of continuous tidal volume measurements and computer-regulated intermittent pulmonary function testing.
Respiratory Management To minimize potential lung damage and optimize extubation to nCPAP, the following criteria were used: PEEP was maintained constant at 5 cm H2O; PImax, FIO2, and breathing rate were reduced quickly over the first 6 hours of life to achieve target levels of PaO2 at 55–70 mm Hg, PaCO2 at 50–60 mm Hg, pH greater than 7.2, and tidal volumes of 4–6 ml/kg (monitored by the VitalTrends system), while ensuring there was still minimal yet visible chest wall movement. A chest radiograph was used to help assess lung inflation. Ventilation parameters of FIO2, less than 0.3; PImax, 14–16 cm H2O; PEEP, 5 cm H2O; and breathing rate, 20 breaths/minute were targets for the first 24-hour study period. A repeat dose of surfactant (Curosurf, 100 mg/kg) was administered routinely at 6 hours of age. Caffeine citrate (20 mg/kg) was given intravenously at 1 and 12 hours of age, and daily thereafter (10 mg/kg). Further sedation was kept to a minimum, but if the infant experienced distress, chloral hydrate suppositories (10–15 mg) were administered as required. The infants were nursed prone or full on the left or right side, but never supine, in an environment with low levels of light and noise. Extubation to nCPAP was attempted at 24 hours of age if the animal had an FIO2, less than 0.4, PImax less than 18 cm H2O, and a breathing rate less than 25 breaths/minute. The required sedation to insert the umbilical artery and percutaneous central venous catheters resulted in the infants having a poor respiratory drive initially; extubation before 24 hours failed. All infants were maintained on a single type of nCPAP delivery device, the Infant Flow Generator (provided by ElectroMedical Equipment, Brighton, UK), via nasal prongs and occasionally nasal mask with an initial pressure of 7 cm H2O. Care was taken to ensure an adequate seal between the prongs/mask and the nares, and a patent upper airway was maintained by the use of positioning and suction. To cope with the high gas flow rate of the Infant Flow Generator, the humidification of the circuit was accomplished with the Fisher and Paykel 850 humidifier (provided by Fisher & Paykel Healthcare, Laguna Hills, CA). An oro- or nasogastric tube was used frequently to aspirate swallowed air from the stomach. Each infant continued on nCPAP as long as there was adequate respiratory drive, the criteria for which included an FIO2 less than 0.5, pH greater than 7.20, with no limit set for PaCO2 provided the pH was maintained. If the nCPAP treatment failed, the infant was reintubated and ventilated with the least support to achieve adequate gas exchange and chest inflation as described above. If the infant had minimal oxygen requirements (FIO2 less than 0.25), good respiratory effort, and no chest retractions, nCPAP was discontinued and the animal was placed in humidified supplementary oxygen or air. nCPAP was reinstated if inspired FIO2 exceeded 0.25 or poor respiratory effort or chest retractions were observed.
Nutritional Management To provide enough energy for spontaneous breathing, nutrition was commenced earlier and increased more aggressively than in previous baboon models of BPD (19). Parenteral nutrition was initiated at 24 hours of life with amino acids at 1.5 g/kg per day (Trophamine; B. Braun Medical, Irvine, CA), electrolytes, vitamins (Pediatric MVI [Astra, Westborough, MA] or Cernevit [Clintec, Deerfield, IL]), and trace elements (MTE-5; Fujisawa USA, Deerfield, IL). Amino acid intake was increased to 3.0 g/kg per day at 48 hours of life and l-cysteine (0.60 mmol/kg per day) was added at 72 hours of life. A 20% lipid emulsion (Intralipid; Pharmacia and Upjohn, Clayton, NC), was initiated on Day 2 at 1.5 g/kg per day, and was increased to 3.0 g/kg per day by Day 5 if tolerated. Enteral nutrition was initiated once bowel gas was noted on abdominal radiographs and stool had been passed, usually at 48–72 hours. Primilac (Bio-Serv, Frenchtown, NJ) was given by intermittent gastric infusion at an initial volume of 10 ml/kg per day and advanced by 10–30 ml/kg per day, as tolerated. Supplemental vitamins were given enterally (Poly-Vi-Sol, 0.25 ml/day; Mead Johnson Nutritionals, Evansville, IN) once enteral feeds were tolerated at 20 ml/kg/day. Nutritional goals included a volume intake of 180–200 ml/kg/day, 120–160 calories/kg/day, and 3.0 g/kg/day of protein.
Patent Ductus Arteriosus
Other Care Plans All animals were treated with antibiotics for the first 10 days of life, with subsequent antibiotic use as needed for clinically suspected infection. Prophylactic fluconazole was initiated in all animals (dose, 6.0 mg/kg) at 12, 96, and 168 hours of age. Doses were then given twice a week until Day 28. Significant hypotension was defined as a transduced mean blood pressure less than 25 mm Hg accompanied by either increasing base deficit or decreasing urine output. The protocol for management of hypotension was as previously described (19), and included the stepwise use of additional volume, dopamine and/or dobutamine, and finally hydrocortisone.
Control Animals
Pathology Methods After acquisition of the pressure–volume curve, the right lower lobe was removed, weighed, and intrabronchially fixed with phosphate-buffered 4% paraformaldehyde at a constant pressure of 20 cm H2O for 24 hours. After fixation, the volume of the right lower lobe was determined by volume displacement. The lobe was cut into three serial, equally spaced horizontal tissue sections. The entire cut surfaces of all three horizontal sections were processed for light microscopic study. These specimens were dehydrated in alcohol, embedded in paraffin, cut at 4 µm, and stained with hematoxylin and eosin. The presence or absence of secondary crests/alveoli, the extent of saccular/alveolar wall fibrosis, if present, and the presence or lack of airway involvement were assessed subjectively in all animals. Total internal surface area and surface-to-volume ratios were determined by standard methods on the basis of 10 micrographs of resin-embedded sections, photographed at x10 magnification (27). Platelet endothelial cell adhesion molecule (PECAM, CD31; DakoCytomation, Carpinteria, CA), a marker for endothelial cells, was used to immunostain lungs from 125-day gestation (baseline control), 156-day gestation (intrauterine developmental control), and 28-day nCPAP-treated animals. A semiquantitative point-counting method in which the lung parenchymal tissue served as the volume of reference was used to determine the volume fraction of immunoreactive sites (28). A grid with 216 points was superimposed on color photographs taken from 10 random, noncontiguous fields per lung specimen at a magnification of x40. The number of points falling on immunoreactive sites and on lung parenchyma was recorded. The volume fraction was calculated as the ratio of the number of points falling on immunoreactive PECAM sites to points on lung parenchyma.
Bronchoalveolar Lavage
Cytokine/Chemokine Assays
Statistical Analysis
During this initial study to assess the feasibility of nCPAP in the 125-day primate model, six animals were studied; five survived to 28 days (672 hours). A sixth nCPAP animal had a birth weight of 279 g, the smallest preterm animal ever delivered at the BPD Resource Center. This female animal was successfully extubated to nCPAP at 27 hours of life and required no treatment for hypotension or patent ductus arteriosus. She remained on nCPAP for a total of 13.6 days and her respiratory condition was stable enough for her to spend 2.7 days without any respiratory support. However, she was the only animal in the nCPAP group who could not be established on full enteral feeds. She developed cholestasis a few days before developing necrotizing enterocolitis for which reventilation was required, and she was necropsied on Day 19. In this study, only the data of the five 28-day nCPAP survivors were compared with the control gestational groups.
nCPAP Group Characteristics
Pulmonary Course The nCPAP animals were successfully extubated at a median of 26 hours of life (range, 24 to 29 hours). Two short periods of reventilation for suspected sepsis (blood cultures were negative) were required. The duration of ventilation was 51 and 53 hours in these two animals. The five 28-day survivors spent a median of 9.04 days (range, 3–12.6 days) being supported with nCPAP, and a median of 17.8 days (range, 12–24 days) of breathing without the need for either ventilation or nCPAP. The requirement for supplementary oxygen was low throughout the study period (Figure 1A) . Measurements of arterial blood gases were not made after 14 days of life, as the umbilical artery catheter was removed from all animals. The arterial-to-alveolar O2 ratio was used to measure effective oxygen exchange, and it was consistently greater than 0.45 throughout the study in animals extubated early and placed on nCPAP (Figure 1A).
Figure 1B represents serial pH and PaCO2 measurements over the first 14 days of life. The pH is lowest between 1 and 3 days (median, 7.29; range, 7.15–7.36), during which time the PaCO2 ranged from 36 to 58 mm Hg (median, 43.0 mm Hg). This coincided with the initial period of stabilization on nCPAP. The pH rose to 7.3 and higher by 4 days and remained at that level throughout the rest of the study period, with the PaCO2 remaining fairly constant. The repeated measures analysis of variance test showed no significant differences over time in any variable except pH, which decreased over the first 24 hours of life (p = 0.04), but was still within the defined normal range for the study. Respiratory system mechanics are shown for the first 24 hours of life in Figures 2A and 2B , after which the animals were extubated to nCPAP. The peak inspiratory pressures required to maintain target tidal volume and PaCO2 (Figure 1B) fell over this time period and were consistent with the improvement in dynamic respiratory compliance (Figure 2B). Expiratory airway resistance (Figure 2B) was low in the nCPAP group. These animals therefore had respiratory function compatible with minimal lung injury and had minimal ventilatory requirements in the first 24 hours of life before extubation. The pressure–volume curves obtained at necropsy (Figure 3) confirmed that was still the case at the end of the study. Overall, nCPAP animals were generally well, needed only minimal respiratory support, had good respiratory physiology, and did not acquire serious postnatal lung infections or sepsis.
Pathology: Light Microscopy, Immunocytochemistry, and Transmission Electron Microscopy At necropsy, the nCPAP 28-day survivors did not have any gross evidence of lung or extrapulmonary infection or sepsis. The lungs were well inflated and normal in appearance, similar to the gross appearance of the term controls. The 125- and 156-day gestational control lungs showed even inflation after fixative instillation. Determinations of right lower lobe lung displacement volumes showed no significant differences between the nCPAP and 156-day gestational control groups (data not shown). Light microscopically, 125-day gestation lungs showed rounded air spaces and widened alveolar walls (Figure 4A) . The interstitium contained scattered cells and clear or pale-staining connective tissue matrix. Ultrastructural features of the lung at 125 days of gestation showed thick saccular walls that contained abundant undifferentiated mesenchymal cells with clear or glycogen-containing cytoplasm, whereas others were densely filled with fibrillar elements and actin filaments (Figure 4B). Capillaries were difficult to identify unless a portion of a lumen could be visualized; however, occasional centrally located vessels were identified in the interstitium. The saccular walls were lined by glycogen-containing progenitor epithelial Type 2 cells with absent cytoplasmic lamellar bodies (Figure 4B).
The 156-day gestation lung had thinner saccular/alveolar walls than did the 125-day gestational control lung, along with numerous secondary crests (Figure 5A) . Ultrastructurally, the alveolar Type 2 cells still had abundant cytoplasmic glycogen stores, but only rare lamellar bodies were seen. Transitional Type 2 cells (flattened Type 1 epithelial cells in appearance but microvilli still present) were evident (Figure 5B). The interstitium contained predominantly subepithelially placed capillaries. Mesenchymal cells, some with clear cytoplasm and others with numerous mitochondria and rough endoplasmic reticulum, were present in the interstitium. Myofibroblasts and/or capillaries could be identified in some of the secondary crest formations (Figure 5B), and elastin deposits were evident in the tips of the secondary crests.
nCPAP lung specimens showed evenly inflated thinned saccular walls with minimal interstitial cellularity and fibroproliferation (Figure 6A) . Scattered secondary crests were evident in the expanded air spaces and a few alveolar structures were present (Figure 6A). The bronchi and bronchioles did not show epithelial changes, and the pulmonary arteries and arterioles were normal in appearance. Ultrastructurally, the saccular/alveolar walls showed variable numbers of interstitial cells that had dense cytoplasm and no glycogen stores (Figure 6B). Some had features of monocytes or macrophages, but most were undifferentiated. Myofibroblasts were sparse. Focally, the connective tissue matrix had a vacuolated appearance. In Figure 6B, the saccular/alveolar wall shows several outgrowths along the surface that likely represent secondary crest formation.
Term plus 1- to 2-day lungs are shown to depict the features of the lung after uninterrupted gestational maturation. Light microscopically, they show more abundant and complex elongated secondary crests and alveoli (Figure 7A) . Electron microscopically, capillaries were seen located in a subepithelial configuration on the thin, fused side of the air–blood barrier. Alveolar Type 2 cells contained variable numbers of cytoplasmic lamellar bodies, but the abundant cytoplasmic stores of glycogen were absent. Within the air spaces, free surface material was present (Figure 7B). The interstitium was attenuated focally, but focal sites of several mononuclear cells and connective tissue matrix were evident. Elongated secondary crests/alveoli were present, usually with some portion(s) of the capillary endothelium and/or circulating red or white blood cells evident (Figure 7B).
Morphometric determinations of alveolar wall thickness substantiated the light microscopic findings in that 125-day gestational control lungs had significantly thickened saccular/alveolar walls when compared with nCPAP and 156-day gestational control specimens (p < 0.01). Although nCPAP lungs tended to have thicker walls, there were no significant differences when compared with 156-day gestation control lungs. Internal surface area measurements were significantly greater in 156-day gestational control and nCPAP lungs than in 125-day gestational control lungs (p < 0.01), but internal surface area measurements were not significantly different between the 156-day gestational control and nCPAP study groups (Figure 8) . Surface-to-volume ratios values were significantly less in the 125-day gestational controls when compared with the other two groups (p < 0.001) (Figure 9) . Point-count determinations of PECAM immunostaining are shown in Figure 10 . As expected during development, PECAM vascular staining increased and parenchymal values decreased as birth draws near. The 125-day gestation group had significantly less PECAM staining when compared with the other study groups (p  0.0005). PECAM and total parenchyma values of the 156-day gestation and nCPAP groups were not significantly different.
Necropsy BAL Fluid Cytokine/Chemokine Levels In the nCPAP group the IL-6 median concentration was 49.7 pg/ml (range, 30.4–83.7 pg/ml) versus a median of 166.3 pg/ml (range, 27.6–728.1 pg/ml) in the 150- to 160-day gestational controls (p < 0.001). For IL-8 there was no difference between the two groups; in the nCPAP group the median was 50 pg/ml (range, 28.2–93.3 pg/ml) versus a median of 25 pg/ml (range, 20–117.8 pg/ml) in the 150- to 160-day gestational controls.
The standards of care commonly applied in neonatal intensive care units include prenatal steroid treatment of the mother and postnatal treatment of the infant with exogenous surfactant and the use of a low tidal volume ventilatory strategy. Since 1994, when Verder and coworkers published the first randomized trial combining the use of nCPAP and surfactant therapy (17), the technique has been used in some U.S. neonatal units (29, 30) and more widely in Europe (16, 31). This mode of treatment seems to be more successful if combined with prenatal steroid administration (16). When we originally described the arrest in alveolar and capillary development in animals ventilated with low-volume positive pressure ventilation (PPV), we hoped that "gentler" ventilatory modalities would allow noninjured lungs to alveolarize further. We hypothesized that the early use of nCPAP, combined with very early surfactant therapy, would improve alveolar and vascular development. When we completed analysis of the nCPAP group and compared the pathology findings with those of the previously published PPV-ventilated group, we were impressed with the "evenness" of the inflation of nCPAP lungs when compared with PPV-ventilated lungs. The latter group frequently had shown striking dilatation of alveolar ductal sites with adjacent smaller saccular spaces. As surface-to-volume ratio determinations had been used as a shape estimator of gas-exchanging parenchyma in an interesting report by Silva and coworkers (32), it was used in this study as it combines the point count method for volume estimation and the mean linear intercept method for surface density determination (27). Our data in this study indicate that nCPAP does not cause an arrest in alveolar development, as internal surface area and surface-to-volume ratios are similar in the nCPAP and 156-day gestational control lungs (in utero developmental control). These results differ from our earlier study in which baboons ventilated with low volume-positive pressure for a similar period of time or longer showed significantly reduced internal surface area measurements when compared with 156-day gestational controls (19). However, these animals received a lower dose of a different surfactant (Survanta), were not as vigorously fed, were not successfully weaned to CPAP for appreciable periods of time, and acquired postnatal infections. To maximize the chance of extubation to nCPAP we chose to use a combination of surfactant and caffeine as a respiratory stimulant to enable preterm neonates to be maintained on nCPAP. This approach had been shown to be successful in preterm infants in a randomized trial (33) and in studies from Europe (16, 17). We were disappointed when the animals could not be successfully extubated before 24 hours as their respiratory drive was poor, probably related to the slow excretion of sedative drugs that are required initially to ensure adequate pain relief for the dam and infant. We were concerned that the delay in extubation to nCPAP from a low-volume ventilatory strategy might expose the lungs to sufficient injury to prevent further lung development. Another concern involved how well we would be able to feed the infants to provide adequate nutrition for growth. Despite our attempts to feed early and aggressively, it still took 2 weeks to achieve full enteral feeds. The weight at necropsy in the nCPAP group was above birth weight in all but one animal. The weight gain, however, was slow and suboptimal when compared with the 156-day gestation control infants. Clearly, further attempts in future to improve postnatal growth must be made. In spite of these concerns, morphometric assessments of internal surface area and surface-to-volume ratios indicate that lung development did continue in the nCPAP animals. This finding indicates that volutrauma-induced injury was decreased, but also indicates that the lack of postnatally acquired infection may be a substantive contributor to the improved outcome as well. The lack of documented episodes of sepsis and pneumonia during the clinical courses of the infants, plus the low IL-8 BAL levels at necropsy, support this thesis. Lung development in the baboon at 125 days of gestation is in the late canalicular stage, and is similar to development of the human infant lung at 24 to 26 weeks of gestation. Both show beginning secondary crest formation, early vasculogenesis in the primordial alveolar walls, and a lack of an alveolar macrophage population and other immune cells. Jobe has reviewed two factors that can impact the fetal lung before preterm birth and thereby initiate processes that may progress to BPD: antenatal glucocorticoid treatments and fetal exposure to inflammation/infection (34). Jobe reviewed clinical and experimental model data supporting the idea that subjecting the lung antenatally to either or both of these exposures serves as the first "hit" or insult to the fetal lung, and primes it for more ventilator-induced injury and thus inflammation after delivery (34). This nCPAP baboon model uses treatment with antenatal steroids, but does not undergo an experimental induction of an intrauterine inflammatory response. Jobe and coworkers documented in a 2-hour study that conventionally ventilated preterm lambs have 6.6 times more neutrophils and hydrogen peroxide in alveolar washes than do lambs treated with CPAP (35). Our study design did not include collecting tracheal aspirates for inflammatory cell counts and cytokine analyses, so we do not know whether nCPAP blunted a rise in inflammatory cells and proinflammatory cytokines over the first 10 days of life. We have documented increases in IL-8, IL-6, and IL-1ß over this time period in earlier studies (19, 36). Perhaps the lack of an intrauterine infectious/inflammatory process plus only a short exposure to conventional ventilation partially accounts for continued maturation of the lung seen in our nCPAP animals. Our results support that a total arrest in lung development may not be inevitable in all infants born very early. The seminal study by Hislop and coworkers established this tenet in human infants with respiratory distress syndrome who were not ventilated and progressed to normal alveolarization (21). In spite of the need to ventilate the baboons for 24 hours before they could be extubated and put on nCPAP, it appears that the use of a "gentler ventilation" minimized the risk of development of BPD. This finding supports the notion that the nCPAP-treated lung may be able to continue to form alveoli over the 2-year time period that alveolar development is known to persist in humans (37).
The authors thank BPD Resource Center personnel: the animal husbandry group led by Drs. D. Carey and M. Leland, the NICU technicians, and the Department of Pathology staff. Dr. J. Schoolfield is thanked for biostatistical support.
Supported by National Institutes of Health (NIH) grant HL52636 and NIH grant P51 RR13986 for facility support; Chiesi Farmaceutica (surfactant), Infrasonics (ventilator), ElectroMedical Equipment (nCPAP generator and accessories), Fisher & Paykel Healthcare (humidifier). Conflict of Interest Statement: M.A.T. was reimbursed by Chiesi Pharmaceuticals UK for travel and accommodation expenses to attend several conferences in Europe and participated as a speaker in scientific meetings and study days in the UK organized and partly financed by Chiesi Pharmaceuticals UK receiving $225 in 2002 and $225 in 2003 and participated as a speaker in scientific meetings organized and financed by Dey Pharmaceuticals in the USA receiving $1,500 in 2000 and $1,000 in 2001 and received $4,500 for serving on a scientific advisory committee for Chiesi Pharmaceuticals UK in 2003 and a consultancy fee of £375 for preparation of teaching material used by Chiesi Pharmaceuticals UK in 2002; B.A.Y. has no declared conflict of interest; V.T.W. has no declared conflict of interest; H.M. has no declared conflict of interest; D.C. has no declared conflict of interest; T.M.S-K. has no declared conflict of interest; J.J.C. has no declared conflict of interest. Received in original form September 12, 2003; accepted in final form February 10, 2004
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ABSTRACT
METHODS
p < 0.01). Data are shown as means and standard deviation (SD). Vv = volume fraction.