Published online November 1, 2006
PEDIATRICS Vol. 118 No. 5 November 2006, pp. 2038-2050 (doi:10.1542/peds.2006-0622)
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ARTICLE

Delayed Extubation to Nasal Continuous Positive Airway Pressure in the Immature Baboon Model of Bronchopulmonary Dysplasia: Lung Clinical and Pathological Findings

Merran A. Thomson, MB, ChBa, Bradley A. Yoder, MDb, Vicki T. Winter, BSc, Luis Giavedoni, PhDd,e, Ling Yi Chang, PhDf and Jacqueline J. Coalson, PhDc,d

a Clinical Sciences Division, Imperial College, London, United Kingdom
b Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah
c Department of Pathology, University of Texas Health Science Center, San Antonio, Texas
d Southwest Foundation for Biomedical Research, San Antonio, Texas
e Southwest National Primate Center, San Antonio, Texas
f Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE. Using the 125-day baboon model of bronchopulmonary dysplasia treated with prenatal steroid and exogenous surfactant, we hypothesized that a delay of extubation from low tidal volume positive pressure ventilation to nasal continuous positive airway pressure at 5 days (delayed nasal continuous positive airway pressure group) would not induce more lung injury when compared with baboons aggressively weaned to nasal continuous positive airway pressure at 24 hours (early nasal continuous positive airway pressure group), because both received positive pressure ventilation.

METHODS AND RESULTS. After delivery by cesarean section at 125 days (term: 185 days), infants received 2 doses of Curosurf (Chiesi Farmaceutica S.p.A., Parma, Italy) and daily caffeine citrate. The delay in extubation to 5 days resulted in baboons in the delayed nasal continuous positive airway pressure group having a lower arterial to alveolar oxygen ratio, high PaCO2, and worse respiratory function. The animals in the delayed nasal continuous positive airway pressure group exhibited a poor respiratory drive that contributed to more reintubations and time on mechanical ventilation. A few animals in both groups developed necrotizing enterocolitis and/or sepsis, but infectious pneumonias were not documented. Cellular bronchiolitis and peribronchiolar alveolar wall thickening were more frequently seen in the delayed nasal continuous positive airway pressure group. Bronchoalveolar lavage levels of interleukin-6, interleukin-8, monocyte chemotactic protein-1, macrophage inflammatory protein-1 {alpha}, and growth-regulated oncogene-{alpha} were significantly increased in the delayed nasal continuous positive airway pressure group. Standard and digital morphometric analyses showed no significant differences in internal surface area and nodal measurements between the groups. Platelet endothelial cell adhesion molecule vascular staining was not significantly different between the 2 nasal continuous positive airway pressure groups.

CONCLUSIONS. Volutrauma and/or low-grade colonization of airways secondary to increased reintubations and ventilation times are speculated to play causative roles in the delayed nasal continuous positive airway pressure group findings.

Key Words: nasal continuous positive airway pressure • bronchopulmonary dysplasia • bronchiolitis • alveolization • vasculogenesis • cytokines • chemokines • necrotizing enterocolitis • volutrauma • biotrauma • sepsis

Abbreviations: nCPAP—nasal continuous positive airway pressure • BPD—bronchopulmonary dysplasia • ISA—internal surface area • LV-PPV—low tidal volume positive pressure mechanical ventilation • EnCPAP—early nasal continuous positive airway pressure • DnCPAP—delayed nasal continuous positive airway pressure • PIP—peak inspiratory pressure • PEEP—positive end-expiratory pressure • FIO2—fraction of inspired oxygen • PDA—patent ductus arteriosus • NEC—necrotizing enterocolitis • PV curve—pressure–volume curve • PECAM—platelet endothelial cell adhesion molecule • BAL—bronchoalveolar lavage • BALF—bronchoalveolar lavage fluid • IFN—interferon • IL—interleukin • MCP—monocyte chemotactic protein • MIP—macrophage inflammatory protein • TNF—tumor necrosis factor • a/A ratio—arterial to alveolar oxygen ratio • IQR—interquartile range

Nasal continuous positive pressure ventilation (nCPAP) was reported to be a successful ventilatory strategy in preterm infants managed at a US institution with a low incidence of bronchopulmonary dysplasia (BPD) in 1987.1 Several European studies during the 1990s stimulated a renewal of interest in the use of nCPAP,2–4 and subsequent studies have further defined the use of nCPAP in the NICU.5–8 However, adequately powered randomized, controlled trials have not yet been performed to confirm a long-term benefit. We have recently shown that the lungs of nCPAP-treated extremely preterm baboons had relatively normal-appearing lung parenchyma and had surface area measurements comparable to those of age-matched intrauterine gestational controls.9 In that study, a 24-hour period of positive pressure ventilation was required before weaning to nCPAP because the baboons did not spontaneously breathe after delivery as a result of the dam's need for sedation before and during the cesarean section. It is reported that premature infants who require ventilation will have impaired alveolization,10 and several animal studies have supported the concept that there is an arrest in lung development in immature lungs subjected to ventilation-induced injury.11–13 However, Cherukupalli et al14 observed a near-normal lung in 1 human infant who had received minimal oxygen and ventilatory support. This observation and our finding that internal surface areas (ISAs) were comparable in animals on nCPAP and the age-matched gestational controls have made us question whether arrest really is inevitable, especially if lung injury can be minimized. Because many practicing neonatologists use low tidal volume positive pressure mechanical ventilation (LV-PPV) for several days to stabilize the extremely premature infant before weaning to CPAP, the purpose of this study was to compare the effects of early nCPAP (EnCPAP) commenced at 24 hours of age to those of conventional LV-PPV for 5 days followed by extubation to nCPAP (delayed nCPAP [DnCPAP]) in the premature baboon treated with prenatal steroids and prophylactic surfactant-replacement therapy. Using a 28-day study period, we hypothesized that these 2 study groups of extremely preterm baboons would show no differences in lung physiology, the incidence of lung infection/inflammation, and subsequent lung development.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal studies were performed at the Southwest Foundation for Biomedical Research in San Antonio, Texas. All animal husbandry, animal handling, and procedures were reviewed and approved by the Southwest Foundation for Biomedical Research's Institutional Animal Care and Use Committee to conform to American Association for Accreditation of Laboratory Animal Care guidelines.

Delivery and Instrumentation
Timed gestations were determined by observing characteristic sex skin changes and confirmed by fetal ultrasound examination. Pregnant baboon dams (Papio cynocephalus) were treated with 6 mg of intramuscular betamethasone 48 and 24 hours before elective hysterotomy under general anesthesia. Study animals were delivered at 125 ± 2 days (67% of term gestation at 185 days). At birth all animals were weighed, intubated, and treated with 200 mg/kg Curosurf (provided by Chiesi Farmaceutica S.p.A., Parma, Italy) before the initiation of ventilator support.

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 per minute, peak inspiratory pressure (PIP) adequate to move the chest, positive end-expiratory pressure (PEEP) at 5 cmH2O, and supplemental oxygen (FIO2) commenced at 0.40. PIP 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. The preterm baboons 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.

Respiratory Management
The respiratory management of both groups of preterm baboons was initially identical and has been previously described in detail.9 We used practices of rapid weaning of ventilation, permissive hypercapnia, careful positioning with meticulous attention to maintenance of patency of the upper airway, early nutrition, minimal handling, and the reduction of ambient light and noise. In both groups, PEEP was maintained constant at 5 cmH2O; PIP, FIO2, and rate were reduced over the first 6 hours of life to achieve target levels of PaO2 at 55 to 70 mmHg, PaCO2 at 50 to 60 mmHg, pH >7.2, and tidal volumes of 4 to 6 mL/kg. A repeat dose of surfactant (Curosurf 100 mg/kg) was administered routinely at 6 hours of age to both groups. All received caffeine citrate (20 mg/kg) intravenously at 1 and 12 hours of age and daily thereafter (10 mg/kg). Sedation was kept to a minimum, especially in the 12 hours before extubation; however, if the animal experienced distress, chloral hydrate (10–15 mg) or ketamine (2.5 mg/kg) was administered as required.

Extubation to nCPAP was attempted at either 24 hours (EnCPAP) or 5 days (120 hours) of age (DnCPAP). Target ventilatory support at the time of extubation was FIO2 < 0.4, PIP < 18 cmH2O, and a rate of <25 breaths per minute. nCPAP was maintained with the Infant Flow Generator nCPAP delivery device (provided by ElectroMedical Equipment Ltd, Brighton, United Kingdom) via nasal prongs and occasionally nasal mask with an initial pressure of 7 cmH2O. Humidification of the circuit was accomplished with the Fisher & Paykel 850 humidifier (provided by Fisher & Paykel Healthcare, Inc, Huntington Beach, CA). nCPAP was continued as long as there was an adequate respiratory drive. Criteria for reventilation included FIO2 >0.5 with a pH <7.20 and recurrent apnea requiring mask ventilation on 2 or more occasions in 6 hours; no limit was set for PCO2 provided the pH was maintained. Because data-storage facilities on the cardiorespiratory monitoring equipment and a pneumotachograph were not available, upper airway obstruction was monitored by the presence of audible air entry throughout inspiration and expiration. During periods of apnea, desaturation, or respiratory failure, if air entry was poor, the animal was carefully repositioned to maximize upper airway patency before reintubation was considered. If the nCPAP treatment failed, the animal was reintubated and ventilated with the least support to achieve adequate gas exchange and chest inflation as described above. Once ventilatory requirements had decreased to the above-described levels, extubation was attempted again. When the baboon had minimal oxygen requirements (FIO2 < 0.25), good respiratory effort, and no chest retractions, nCPAP was discontinued and the animal was placed in humidified supplementary oxygen or air. Nasal CPAP was reinstated if inspired FIO2 exceeded 0.25 or poor respiratory effort or chest retractions were observed. The management of nutrition, patent ductus arteriosus (PDA), and hypotension have been previously described.9,11 Briefly, during the first 24 hours animals received heparinized normal saline and 5% dextrose–water infusion with supplemental calcium, 250 to 275 mL/kg per day, as necessary to maintain electrolyte homeostasis, to provide minimal urine output at 1 to 2 mL/kg per hour, to maintain acceptable blood pressure, and to minimize metabolic acidosis. Fluid intake decreased over the first 3 to 4 days to 180 to 200 mL/kg per day. Parenteral nutrition commenced at 24 hours of life and enteral nutrition usually at 24 to 48 hours; the initial feed volume of 10 mL/kg per day advanced by 10 to 30 mL/kg per day, as tolerated. Supplemental vitamins were added at 20 mL/kg per day once enteral feeding was tolerated. Nutritional goals included 502 to 670 J/kg per day and 3.0 g/kg per day of protein. PDA was monitored by clinical examination and echocardiography. If believed clinically to be contributing to the need to continue or reinstitute ventilation, it was considered for treatment. Volume restriction and dopamine to maintain blood pressure and urine output and ibuprofen, but not surgical ligation, were treatment options for those animals with clinical instability. Significant hypotension, defined as a transduced mean blood pressure of <25 mmHg accompanied by either increasing base deficit or decreasing urine output, was treated by the stepwise use of additional volume, dopamine and/or dobutamine, and finally hydrocortisone.

Control Animals
To determine the baseline developmental parameters of delivered animals at 125 days, lungs of 4 fetuses delivered by cesarean section from dams that had received the standard prenatal-steroid treatment at 123 and 124 days of gestation were used. Their morphometric values are given for reference parameters only. To assess for intrauterine developmental changes that would occur with ~1 month of additional growth and development, 4 dams were treated with prenatal steroids at 123 and 124 days of gestation, and the fetuses were delivered at 153 days. Reference to an earlier group of 156-day gestational controls refers to animals that did not receive antenatal steroid treatment.

Pathology and Morphometry
All necropsies were performed at 28 days postdelivery unless it was determined that an early necropsy was needed to prevent organ damage and suffering when an animal developed a life-threatening complication such as necrotizing enterocolitis (NEC) or major sepsis. Before the planned necropsy, the animal was ventilated with 100% oxygen for 5 minutes, and deep anesthesia was induced by the slow infusion of pentobarbital to decrease the blood pressure by 50%. The endotracheal tube was clamped to allow for adsorption atelectasis, and after 2 minutes the heart was stopped with additional pentobarbital. The chest was opened and a pressure–volume (PV) curve was measured by increasing the pressure on the lung to 35 cmH2O in 5-cmH2O pressure increments, using a syringe and manometer, and then decreasing the pressure with measurement of volume after 30 seconds at each pressure.15 The volumes were corrected for the compression volumes of the measurement system.

After the acquisition of the PV curve, the right lower lobe was removed, weighed, and intrabronchially fixed with phosphate-buffered 4% paraformaldehyde at 20 cm H2O constant pressure for 24 hours. After fixation, the volume of the right lower lobe was determined by volume displacement. The lobe was cut into 3 serial, equally spaced horizontal tissue sections. The entire cut surfaces of all 3 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. Ten random fields of lung parenchyma per animal were photographed at x10 magnification. Parenchymal areal density was determined by dividing the number of points falling on lung parenchyma by the total number of points falling on the entire section. Total parenchymal volume was calculated by multiplying the total lung volume by the parenchymal areal density.16 Airway inflammatory lesions were graded using a modification of the method of Cimolai et al.17 Using the 3 stained sections per lung, grades were assigned and included a mild designation (near-normal or minimal collections of mural inflammatory cells in >75% of terminal and respiratory bronchioles), moderate (mural accumulations of inflammatory cells 2–4 layers thick in >50% of the terminal and respiratory bronchioles), and severe (>4 layers of inflammatory cells in >50% of the terminal and respiratory bronchioles).

Total ISA, wall thickness, and surface-to-volume ratios were determined by standard methods on 10 micrographs of paraffin-embedded sections, photographed at x10 magnification.18 A digital imaging analysis according to methods described by Tschanz and Burri19 and Crapo et al20 was used to determine the extent of alveolization, the number and length of primary septal segments and secondary crests, and the surface density of primary alveolar septa and secondary crests. In addition, secondary crests were categorized according to their lengths into 4 groups: <5, 5 to 10, 10 to 25, and >25 µm mean length, and the numerical frequency for each length category was calculated.

Platelet endothelial cell adhesion molecule(PECAM, CD31; Dako Corporation, Carpinteria, CA), a marker for endothelial cells, was used to immunostain lung sections from prenatal-steroid–treated 125-day (baseline control) and 153-day gestation (intrauterine developmental control) and both groups of nCPAP-treated animals. Color photographs from 10 random, noncontiguous fields per lung specimen were obtained at a magnification of x40 and analyzed. Areal density of the air-exchange parenchyma was determined by dividing the number of points falling on air-exchange parenchyma by the number of points falling on the entire section. The total volume of air-exchange parenchyma was determined by multiplying the total parenchymal volume by the areal density of the air-exchange parenchyma. Areal density of PECAM immunoreactive sites was calculated as the ratio of the number of points falling on immunoreactive PECAM sites to points on air-exchange parenchyma. Total microvascular endothelial cell volume was calculated by multiplying the areal density of PECAM immunoreactive sites by the total volume of air-exchange parenchyma.16,21

Bronchoalveolar Lavage
Bronchoalveolar lavages (BALs) were performed at necropsy in the prenatal-steroid–treated 125-day and 153-day gestational controls and both nCPAP study groups. After necropsy, a preweighed lobe of lung was lavaged with 0.9% NaCl (pH 7.4) with a recovery of 70% to 80% of the instilled volume.22 Lavage specimens for cell counts and differentials were centrifuged for 10 minutes at 1500 rpm, and cell counts and differentials were performed. Portions of the supernatant were aliquoted in 1.0-mL aliquots then frozen at –70°C for cytokine/chemokine studies.

Cytokine/Chemokine Assays
BAL fluid (BALF) supernatants were used for the simultaneous determination of 22 cytokines and chemokines with the Luminex technology. The following coated bead/biotinylated antibody combinations were used: granulocyte colony-stimulating factor (Linco-plex human granulocyte colony-stimulating factor, Linco Research, St Charles, MO), granulocyte-macrophage colony-stimulating factor (Beadlyte human granulocyte-macrophage colony-stimulating factor, Upstate USA, Charlottesville, VA), growth-regulated oncogene-{alpha} (Upstate USA), interferon [IFN]-{alpha} (anti-human IFN-{alpha} clones MMHA-11 and MMHA-2, PBL Biomedical Laboratories, Piscataway, NJ), IFN-{gamma} (Beadlyte primate interleukin [IL]-2, Upstate USA), IL-1ß (Beadlyte human IL-1ß, Upstate USA), IL-1Ra (Fluorokine MAP human IL-1Ra/IL-1F3, R&D System, Minneapolis, MN), IL-2 (Beadlyte primate IL-2, Upstate USA), IL-4 (Linco-plex human IL-4, Linco Research), IL-5 (Linco-plex human IL-5, Linco Research), IL-6 (Linco-plex human IL-6, Linco Research), IL-8 (Beadlyte human IL-8, Upstate USA), IL-9 (Beadlyte human IL-9, Upstate USA), IL-10 (anti-human IL-10 clones BN-10 and QS-10, Cell Sciences Inc, Canton, MA), IL-13 (Beadlyte human IL-13, Upstate USA), 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), MCP-1 (Beadlyte anti-human MCP-1, Upstate USA), MIP-1{alpha} (Beadlyte human MIP-1{alpha}, Upstate USA), MIP-1ß (human MIP-1ß, Biosource International), RANTES (Beadlyte human RANTES [regulated on activation, normal T expressed and secreted], Upstate USA), tumor necrosis factor [TNF]-{alpha} (Beadlyte human TNF-{alpha}, Upstate USA), and TNF-ß (Beadlyte human TNF-ß, Upstate USA). BALF supernatants were diluted with 1 volume of 0.1% bovine serum albumin in phosphate-buffered saline, and the cytokine concentrations were determined in duplicate as described before,23 using human cytokines as standards.

Data and Statistical Analyses
A sample size of 6 EnCPAP-treated and 5 DnCPAP-treated baboons was proposed for study. The set of hypotheses to be tested was whether one treatment resulted in changes relative to the other treatment. Assuming the observed or log-transformed outcomes to be compared are parametric, if any baboon population treatment differences in which the mean difference between treatments for a parameter is at least twice as large as the SD of the parameter, then the sample size of 11 baboons would be sufficient to detect the population difference using unpaired Student's t test at the .05 level with a power of 83%. Statistical results for clinical and physiologic data were generated using Stata 7.0 (Stata Corporation, College Station, TX), and for the repeated measures analysis of variance and the pathology data, SPSS 14.0 (SPSS, Inc, Chicago, IL) was used. A P value of ≤.05 was required for significance.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 shows the EnCPAP and DnCPAP group characteristics and several clinical variables. Weaning to nCPAP was successful in all preterm baboons studied; however, it was easier to achieve in the EnCPAP group when extubation was attempted at 24 hours. In the DnCPAP group when extubation was delayed to day 5, the animals had more difficulty maintaining a patent upper airway and exhibited a clinically observed weaker respiratory drive manifested by increased desaturations associated with apnea, frequently of a mixed type.


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  		TABLE 1 Group Characteristics

 
Figure 1 summarizes the respiratory support required by each group. Three animals in each group required reventilation for poor respiratory drive with respiratory failure on 1 or more occasions. In the EnCPAP group, the duration of additional positive pressure ventilation beyond the 24 hours was short (median: 51.3 hours). In 2 of the EnCPAP animals, the need for reventilation was associated with suspected sepsis (blood culture–negative), but recovery was rapid with extubation again to nCPAP. In a third EnCPAP animal, reventilation was prompted by the development of NEC requiring necropsy on day 19. Blood cultures in this animal were negative. Five EnCPAP animals survived to 28 days.


Figure 1
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  		FIGURE 1 Reasons and timing of additional ventilation or early death are indicated on the bar chart. A indicates reventilated for poor respiratory drive with respiratory failure; CNS, coagulase negative staphylococcal sepsis; SS, suspected culture negative sepsis presenting as poor respiratory drive with respiratory failure requiring reventilation; NEC, necrotizing enterocolitis; PDA, patent ductus arteriosus which failed to respond to medical treatment; PDA + PH, pulmonary hemorrhage secondary to PDA responding to medical treatment.

 
The pulmonary course of the DnCPAP group was different (Fig 1). Four of 5 were successfully extubated on day 5; the fifth was extubated on day 10 after treatment for a PDA with pulmonary hemorrhage on day 2. The duration of additional positive pressure ventilation beyond 5 days in the DnCPAP group was longer (median: 122 hours; P = .01). Reventilation was required in 3 animals on 6 occasions for poor respiratory drive with respiratory failure. One animal required reventilation 3 times (days 7, 12, and 19); blood cultures were negative on all 3 occasions, but this animal developed coagulase- negative staphylococcal sepsis requiring necropsy on day 21. A second animal was reventilated twice (days 7 and 24). Sepsis was suspected on day 24, but blood cultures were negative, and the animal had recovered and was ready for extubation on the day of necropsy (day 28). The third animal was reventilated on day 12, a PDA apparent on day 14 was treated, and repeated attempts to extubate to nCPAP failed because of continued poor respiratory drive, apnea, and respiratory failure. Two animals in the DnCPAP group developed NEC, 1 requiring necropsy on day 22 (blood culture–positive for Klebsiella) and 1 on day 28 (blood culture–negative). Three DnCPAP animals survived to 28 days.

The requirement for FIO2 (Fig 2A) fell in both groups during the first 12 hours and remained consistently low throughout the study in the EnCPAP group. In the DnCPAP group, FIO2 remained significantly higher until day 6, after which the oxygen requirement was reduced to a value similar to that of the EnCPAP infants by day 28.


Figure 2
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  		FIGURE 2 A, FIO2 requirement of DnCPAP group significantly higher between 24 hours and 6 days of age (P = .0026). B, a/A ratio values of DnCPAP group significantly lower than EnCPAP group between 24 hours and 6 days (P = .0031). C, PCO2 in DnCPAP group significantly higher than EnCPAP group between 3 and 14 days (P = .0169). Median and 25th to 75th interquartile ranges are depicted.

 
Measurement of arterial blood gases was not consistently performed after 10 days of life, because the umbilical arterial catheter was removed in some animals. The arterial to alveolar oxygen ratio (a/A ratio) was used to compare effective oxygen exchange between the 2 groups (Fig 2B). When placed on nCPAP at 24 hours, the a/A ratio of the EnCPAP group was consistently >0.45 throughout the study. However, in the DnCPAP preterm baboons the a/A ratio worsened as ventilation continued to day 5, and then improved to be similar to the EnCPAP group by day 10.

Figure 2C represents serial PCO2 measurements of the 2 groups over the study period. Values up to day 10 were measured using umbilical arterial blood samples and thereafter measured using free-flowing capillary samples. The PCO2 values again remained consistently low in the EnCPAP animals (median: 44 mm Hg [IQR: 42-48]). In the DnCPAP group, PCO2 rose from day 2 to reach a maximum at day 6 (median: 71 mm Hg [IQR: 68-74]), 24 hours after extubation to nCPAP.

The PIPs used during the initial ventilatory period of 24 hours in the EnCPAP group (median: 16 [interquartile range, IQR: 15-18]) and 5 days in the DnCPAP group (median: 16 [IQR: 15-17]) were not different.

The PV curves obtained at necropsy reflect static compliance (Fig 3). In the EnCPAP group, the opening pressure is lower and a significantly higher volume is obtained at maximum pressure; this higher lung volume is maintained through most of the deflation curve. At maximum pressure, the compliance seems to be approximately twice as high in the EnCPAP group when compared with the DnCPAP group (P = .0253).


Figure 3
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  		FIGURE 3 PV curves at necropsy. Lungs of EnCPAP animals achieved significantly higher volumes at maximum pressure (P = .0253). Median and 25th to 75th interquartile ranges are depicted. PV curve for the term animals are included for reference only. a P = .0253.

 
Pathology and Morphometry
At necropsy, the lungs of the EnCPAP and DnCPAP preterm baboons did not show any gross evidence of lung or extrapulmonary infection. Right lower lobe weights were similar, and determinations of the right lower lobe lung displacement volumes showed no significant differences between the EnCPAP and DnCPAP groups (data not shown).

In Fig 4, 125- and 153-day gestational controls and the 2 nCPAP lungs are shown. The 125-day lung depicts the baseline appearance at delivery and shows thick saccular walls and early secondary crest formation (Fig 4A). In 153-day gestational controls, the intrauterine developmental control (125 ± 28 days), thin-walled branching respiratory bronchioles, and abundant alveolar ducts showed secondary crests and some alveoli (Fig 4B). Light microscopically, EnCPAP lung specimens showed evenly inflated distal airspaces, and the respiratory bronchioles and alveolar ducts showed minimal interstitial cellularity and fibroproliferation (Fig 4C). Secondary crests and alveolar structures were evident in the expanded air spaces. In the DnCPAP animals, the distal lung showed similar thin-walled respiratory bronchioles and alveolar ducts (Fig 4D) except for sites of subjacent alveolar wall thickening around scattered terminal and respiratory bronchioles (see Fig 5B). The thickened alveolar walls contained an increased number of cells, including mononuclear cells and occasional neutrophils, and probably resulted in the trend for an increase in alveolar wall thickness in the DnCPAP lung specimens when compared with those of the EnCPAP animals (P = .088). Bronchi showed only rare mural inflammatory cells, and the pulmonary arteries and arterioles were normal in appearance.


Figure 4
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  		FIGURE 4 A, 125-day gestational control. Bronchioles (a) and rounded saccular spaces with thickened walls can be seen. Bulges or protuberances from walls into air spaces are progenitor secondary crests. B, 153 day gestational control. Air spaces larger than 125-day control and thinned saccular walls have abundant secondary crests and alveolar structures (b). C, EnCPAP 28-day specimen. Air spaces well expanded and thinned saccular walls show increase in alveolar complexity (ie, elongated branching walls with secondary crests and alveolar formation). D, DnCPAP 28-day specimen. Walls of thinned saccular structures may be slightly more cellular than EnCPAP, but ongoing alveolar formation is present. (Hematoxylin and eosin: original magnification, x10.

 

Figure 5
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  		FIGURE 5 A, EnCPAP 28-day specimen. Terminal bronchiole (a) branches into several respiratory bronchioles. Walls of bronchiolar structures as thin as those of subjacent saccular/alveolar walls. Mural inflammation minimal, grade mild. B, DnCPAP 28-day specimen. Compared with A, terminal bronchiole and branches show thickened, cellular walls with interstitial widening of saccular/alveolar walls. C, DnCPAP 28-day specimen. The wall of the bronchiole infiltrated with inflammatory cells, a cellular bronchiolitis, grade moderate to severe. D, DnCPAP 28-day specimen. Mural cellular infiltrate contains abundant mononuclear inflammatory cells with scattered lymphoplasmacytic cells (b). Hematoxylin and eosin: A and B, x10; C, x20; D, x40, original magnifications.

 
Accumulations of mononuclear inflammatory cells including lymphocytes were seen in walls of terminal and respiratory bronchioles of both nCPAP groups. The infiltrate varied in cell density, from a mild grade of minimal or scattered cells (Fig 5A) to moderate and severe grades up to 4 to 6 cell layers (Fig 5 B–D). This lesion, designated as cellular bronchiolitis, was substantively increased in the DnCPAP lung specimens. Sixty-seven percent of the EnCPAP-graded airways had mild grades of severity compared with only 22% of the airways in the DnCPAP lungs, with the remainder having moderate-to-severe grades. Surface-to-volume ratio (Fig 6A) and ISA measurements (Fig 6B) were not significantly different between the nCPAP groups using standard morphometric techniques (P = .0575 and .956, respectively), but the surface-to-volume ratio of the DnCPAP group was significantly less than that of the 153-day gestational control group value (P = .0189). The digital imaging analysis for the 153-day gestational control and 2 nCPAP study groups also showed no significant difference in the ISA measurement, nor were there significant differences in the number and length of primary septal segments and secondary crests, surface density of primary alveolar septa and secondary crests and the numerical frequency and lengths of the secondary crests (see Table 3, which is published as supporting information at www.pediatrics.org/content/full/118/5/2038).


Figure 6
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  		FIGURE 6 A, Surface-to-volume ratio is a shape estimator of gas-exchanging parenchyma. DnCPAP value is significantly less than that in the153-day gestation group. Means ± SD, Bonferroni-adjusted Student's t test. a P = .0189 vs DnCPAP group. B, ISA determinations are not significantly different among both nCPAP and 153-day groups. 125-day gestation group is for reference only. Means ± SD, Bonferroni adjusted Student's t test.

 
Stereologic determinations of PECAM endothelial cell immunostaining are shown in (Fig 7). Endothelial cell staining was not significantly different between the 2 nCPAP groups or between the 2 nCPAP groups and the 153-day developmental controls.


Figure 7
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  		FIGURE 7 Stereologic volumetric determinations of PECAM-stained microvascular compartments were not significantly different among the 3 study groups. Means ± SD, Student's t test with Boniferoni's adjustment.

 
Necropsy Total Cell and Differential Cell Counts and BALF Cytokine/Chemokines
BAL specimens from both study groups revealed a significant increase of neutrophils in the DnCPAP group when compared with the EnCPAP samples (P = .04; Fig 8). There were no significant differences in total cell counts or in the other cell differential counts. Of the 22 cytokines and chemokines tested, 5 were significantly different between the 2 study groups (Table 2). The DnCPAP group had elevated concentrations of IL-6 (P = .0035), IL-8 (P = .0093), MCP-1 (P = .001), MIP-1{alpha} (P < .0331), and growth-regulated oncogene-{alpha} (P < .0019) when compared with the values of the EnCPAP group. The levels of the other cytokines and chemokines in the 2 study groups showed no significant differences.


Figure 8
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  		FIGURE 8 Total cell and differential cell counts of necropsy lavage fluids show that neutrophils were significantly increased in DnCPAP group. Means ± SD, Student's t test. a P = .0394 vs EnCPAP group.

 

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  		TABLE 2 Luminex 22-Plex Cytokine/Chemokine Data

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study indicate that LV-PPV sustained for a 5-day period followed by weaning to nCPAP elicited substantive detrimental pathophysiological effects in extremely preterm baboons when compared with those ventilated only 24 hours before extubation to nCPAP. The 5-day ventilation period in the DnCPAP animals resulted in worse physiologic measurements and increased episodes of apnea that required more reintubations and additional time on LV-PPV. The incidence of bronchopneumonia was no different between the 2 nCPAP groups, but the lungs of the DnCPAP animals had more cellular bronchiolitis. DnCPAP animals had more episodes of NEC with or without sepsis, and BALF specimens from the DnCPAP group contained a number of elevated inflammatory cytokines/chemokines. Developmentally, lung measurements of ISA and total endothelial cell volume were comparable in the 2 nCPAP study groups and the 153-day intrauterine developmental control group.

All preterm baboons were successfully weaned to nCPAP, but it was much easier to achieve in the EnCPAP group. Continued ventilation for an additional 4 days was associated with an increased oxygen requirement, lower a/A ratio and higher PCO2, all commonly used clinical indicators of lung injury and disease. Although the oxygen requirement and a/A ratio improved in the DnCPAP animals after extubation to nCPAP, the PCO2 remained elevated likely reflecting increased lung injury and the observed poor respiratory drive. Apnea with associated respiratory failure was the prime cause for reintubation and ventilation in both groups, but in the EnCPAP animals, reventilation was short and benign except for 1 animal that developed NEC. Idiopathic apnea of prematurity is a common problem in preterm infants, and it was not surprising that extremely preterm baboons would exhibit a similar problem.

Clinically symptomatic PDA, sepsis, and NEC were more common in the DnCPAP group. In all animals but 1, these conditions did not prevent initial extubation to nCPAP at 5 days or contribute to the need for reintubation early in the study period. In the animal that was unsuccessfully treated with ibuprofen, the continued left-to-right shunt through the PDA probably contributed to the continued need for ventilation. However, the degree of lung injury and inflammation was no different from that seen in DnCPAP animals who required less ventilation, and sepsis, linked with both the development of BPD and reopening of PDA,24 was not present in this particular animal. NEC is not an infrequent finding in this extremely preterm animal model when survived past 2 weeks of age, so it is difficult to make any interpretation of this finding in 3 of the study animals. The presence of PDA in all animals, NEC in 3, and sepsis in 2, did not seem to affect lung structure. This may be attributable in part to the policy of doing immediate necropsies in animals diagnosed with NEC and/or sepsis, obviating the adverse effects of the complicating injury and its supportive treatment.

With the exception of more prevalent and severe cellular bronchiolitis and patchy peribronchiolar wall thickening in the DnCPAP group, well-inflated lung parenchyma with abundant secondary crests and alveolar formation were similar findings in both nCPAP groups. The cellular bronchiolitis, characterized by a mononuclear inflammatory infiltrate in the walls of the small airways, was an unexpected finding and its etiology is undetermined, but microbial colonization and/or low-grade infection are reasonable considerations. Pneumonia was not clinically suspected in any of the study animals, and lung cultures, when obtained at necropsy in both study groups, were negative for bacteria and fungi. However, perhaps a low-grade colonization of the airways secondary to the increased ventilation time and the increased number of reintubations could be responsible for the cellular bronchiolitis. Friedland et al25 demonstrated a time-dependent colonization of the trachea in ventilated neonates; intubation for longer than 4 days yielded an 80% positive culture rate, whereas, intubation for 4 days or less had a 39% positive rate. In the 140-day ventilated baboon model, a sequence of colonization with coagulase-negative staphylococci was noted initially within 2 to 4 days in the nasopharynx, followed by tracheal colonization.26 We suspect that the prolonged initial ventilatory time of 5 days initiated and resulted in an airway injury that permitted microbial colonization and induced a release of increased proinflammatory cytokine/chemokines. The repeated endotracheal reintubations increased the risks of additive airway injury and pathogen introduction.27

A variety of cytokines and chemokines, including those we have analyzed, have been associated with lung-injury in animal and human studies. None of these biomarkers are likely to be specific to a mode of injury but rather are part of the inflammatory milieu associated with mechanical ventilation, oxygen exposure, infection, nutritional deficiencies, and other contributors to lung injury.11,28–38 Volutrauma has been shown to induce a number of deleterious lung injury responses, including the loss of epithelial and endothelial cell integrity, inflammatory cell influx, and increased lung concentrations of inflammatory mediators (reviewed in refs 39–41). Most of the studies applied extreme tidal volumes, were of very short duration, used ex vivo lung preparations, and were performed in adult animals. In this 125-day baboon model, a low tidal volume ventilatory strategy is used to minimize volutrauma, but an elevated proinflammatory cytokine response is seen in tracheal aspirates beginning after a few days of ventilation.11 Because the DnCPAP group spent significantly more time on mechanical ventilation, volutrauma may be a primary contributor to the observed BALF inflammatory changes. Even when the analyte values of the NEC and sepsis-affected animals were excluded from the Luminex analyses (data not shown), the remaining 3 DnCPAP animals still exhibited significantly elevated cytokine and chemokine levels. Overall, the data suggest that it is unlikely that sepsis and/or NEC (ie, source of systemic endotoxin) contributed via a permeability injury to the BALF cytokine differences, and that volutrauma-induced lung injury and the suspected low-grade colonization of airways seem to be the most likely explanations for the significant increases in neutrophils and cytokine/chemokine concentrations in BALF specimens of the DnCPAP group.

The lack of a significant difference in ISA measurements between the nCPAP study groups was documented by the 2 separate morphometric methodologies. In our earlier report, the EnCPAP group was compared with non–prenatal-steroid-treated, 156-day gestational controls (intrauterine developmental control), and the surface areas were not significantly different, supporting that nCPAP treatment allowed alveolar development to progress.9 The ISA value of the prenatal-steroid–treated 153-day gestation control likewise is not significantly different from those of the 2 nCPAP groups. Stereologic volumetric methodology verified that microvascular development was also sustained in both nCPAP groups and was comparable to that seen in the 153-day developmental controls. Also, the microvasculature of nCPAP-treated lungs did not exhibit the immature and/or dysmorphic changes reported in the LV-PPV baboons treated with continuous mechanical ventilation for 1 to 2 months9 and in human infants with BPD.12,16,42,43

To our knowledge, this study is unique in that the use of nCPAP has been administered as soon as possible at 24 hours in a very immature animal model or purposely delayed using an accepted low tidal volume ventilatory strategy for 5 days. Although the number of animals in this study is small, on the basis of our experience with these 2 models, the findings in the DnCPAP group, including the decreased respiratory drive, need for additional reintubations, and resulting increased mechanical ventilation, are undesirable outcomes that could be largely avoided. The different responses between the 2 study groups were consistent and must be considered and further investigated in the human NICU setting. Our baboon study supports the findings of 2 recent clinical reports involving preterm human infants. In each study, infants ventilated from birth had the highest rate of chronic lung disease, neonates treated with CPAP only had the lowest rate, and neonates who failed an initial trial of CPAP and then were ventilated had intermediate rates of chronic lung disease.44,45 The collective data support efforts to avoid ventilating immature infants if at all possible after delivery, or, when ventilation is necessary, to wean them as soon as possible to nCPAP and thereby minimize ventilator-related lung injury and the subsequent development of BPD.


   ACKNOWLEDGMENTS
 
We thank the BPD Resource Center personnel: the animal husbandry group led by Drs D. Carey and M. Leland, and the NICU and pathology staffs. We thank Dr J. Schoolfield for biostatistical support.


   FOOTNOTES
 
Accepted Jul 10, 2006.

Address correspondence to Jacqueline J. Coalson, PhD, Department of Pathology, UTHSCSA, 7703 Floyd Curl Dr, San Antonio, TX 78229. E-mail: coalson{at}uthscsa.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics

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