American Journal of Respiratory and Critical Care Medicine

A borderline viability model of bronchopulmonary dysplasia (BPD)/chronic lung disease of infancy (CLD) with pathophysiologic parameters consistent with those in extremely immature humans with BPD/CLD is described. After prenatal steroid treatment of pregnant dams, 12 premature baboons were delivered by cesarean-section at 125 d (term gestation, 185 d), treated with exogenous surfactant, and maintained on appropriate oxygen and positive pressure ventilation for at least 1 to 2 mo. In spite of appropriate oxygenation (median Fi O2 at 28 d = 0.32; range, 0.21 to 0.50) and ventilatory strategies to prevent volutrauma, the baboons exhibited pulmonary pathologic lesions known to occur in extremely immature humans of less than 1,000 g: alveolar hypoplasia, variable saccular wall fibrosis, and minimal, if any, airway disease. The CLD baboon lungs showed significantly decreased alveolization and internal surface area measurements when compared with term and term + 2-mo air-breathing controls. A decrease in capillary vasculature was evident by PECAM staining, accompanied by dysmorphic changes. Significant elevations of TNF- α , IL-6, IL-8 levels, but not of IL-1 β and IL-10, in tracheal aspirate fluids were present at various times during the period of ventilatory support, supporting a role for mediator-induced autoinflammation. IL-8 levels were elevated in necropsy lavages of animals with significant lung infection. This model demonstrates that impaired alveolization and capillary development occur in immature lungs, even in the absence of marked hyperoxia and high ventilation settings.

Despite the advances in the prevention of respiratory distress syndrome in infants, bronchopulmonary dysplasia (BPD) remains a major complication in premature infants who require prolonged ventilatory support (1, 2). When originally described by Northway and coworkers in 1967 (3), the infants had severe hyaline membrane disease, received high inspired oxygen concentrations, and were mechanically ventilated with high positive airway pressures. Our laboratories successfully used the premature baboon (140 d of gestation; term, 185 d) to model the pathological, clinical, and radiological findings of the classic or original form of BPD described by Northway and colleagues, by using hyperoxia and positive pressure ventilation in a non-surfactant-treated premature baboon (4-9). We also determined that if the premature baboon at 140 d of gestation was treated with appropriate oxygen levels, that even without exogenous surfactant, its respiratory distress syndrome (hyaline membrane disease) would resolve in less than 96 h and the animal could be weaned from the ventilator and survive with no sequelae. This latter finding mirrored the change in the epidemiology of BPD that was being seen in the human infant.

Improvements in respiratory care and management had allowed infants of more than 30 wk of gestation to survive their respiratory distress syndrome, even before the widespread use of exogenous surfactant therapy. This had led to an increased survival of very immature infants, who were at most risk of developing bronchopulmonary dysplasia (10-14). This changed disease spectrum of BPD led Seidner and coworkers to develop a gestationally more immature model of BPD, in which premature baboons were delivered at 125 d of gestation, given exogenous surfactant, treated with appropriate oxygenation and positive pressure ventilation for 14 d, and developed a milder form of bronchopulmonary dysplasia (15).

Now, with the more widespread use of newer treatment modalities, e.g., prenatal steroids and exogenous surfactant, a milder form of BPD, called by some workers neonatal chronic lung disease (CLD) (16-18), is more prevalent. In this article, we describe our efforts to develop a model of CLD incorporating the treatment modalities used in the immature human infant; prenatal steroids, exogenous surfactant, appropriate oxygenation, and volume-sparing ventilatory strategies in the immature 125-d baboon. Pregnant dams were treated with prenatal glucocorticoids, after which borderline viable fetuses were delivered at 125 d. The infants received exogenous surfactant at birth, were maintained on appropriate oxygen and ventilatory support, and survived for at least 1 of 2 mo, and longer. Their treatment protocols, clinical course, pathologic and morphometric findings, and mediator responses in lung fluids are presented, and document that many aspects of the milder disease in the extremely immature human infant with CLD have been approximated.

Delivery and Instrumentation

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 with American Association for Accreditation of Laboratory Animal Care (AAALAC) guidelines. Timed gestations were determined by observing characteristic sex skin changes and confirmed by serial fetal ultrasound examinations. Dams were treated with 6 mg of intramuscular betamethasone 48 and 24 h before elective hysterotomy under general anesthesia. Study infants were delivered at 125 + 6 d (67% of term gestation at 185 d). At birth all animals were weighed, received intramuscular ketamine hydrochloride (10 mg/kg) for anesthesia, and were intubated with a 2.5- to 3.0-mm endotracheal tube. Before the first breath tracheal lung liquid was collected. All animals received exogenous surfactant (Survanta, 100 mg/kg; donated by Ross Laboratories, Columbus, OH) before initiation of ventilator support. Ventilation was initiated with a humidified, pressure-limited, time-cycled infant ventilator (donated by InfantStar; Infrasonics, San Diego, CA) with an initial rate at 40 breaths/min, peak inspiratory pressure (PIP) adequate to move the chest, positive end-expiratory pressure (PEEP) at 4 cm H2O and Fi O2 at 1.00. Animals were instrumented with an umbilical arterial catheter and percutaneous central venous catheter and nursed in a servo-controlled, infrared-warmed, body plethysmograph (VT1000; Vitaltrends Technology, New York, NY) capable of continuous tidal volume measurements and computer-regulated intermittent pulmonary function testing. Subsequent ventilator adjustments were made on the basis of chest radiograph, clinical examination, arterial blood gas measurement, and tidal volume measurement as described below. Intermittent sedation was provided as needed with ketamine (5 mg/kg) and/or diazepam (0.1 mg/kg).

Ventilatory Management

The ventilatory approach applied to all animals was based on a strategy to maintain tidal volumes at 4–6 ml/kg as continuously measured by the VitalTrends system and associated with adequate chest motion by clinical examination. The rate was adjusted as required to regulate PaCO2 between 45 and 55 mm Hg. The rate could be increased to a maximum frequency of 60. If a PIP greater than 40 cm H2O was required to achieve desired tidal volumes and PaCO2 goals, the acceptable limit for PaCO2 was increased to 65 mm Hg. High-frequency oscillatory ventilation (HFOV) (3100A; SensorMedics, Anaheim, CA) was instituted as a rescue therapy if air leak developed (pneumothorax, pneumatocele, interstitial emphysema) or if the PaCO2 was greater than 65 mm Hg, with a PIP greater than 40 cm H2O and a rate of 60 breaths/min. The rescue approach to HFOV was similar to that previously described from this laboratory (19).

Target goals for PaO2 were 55–70 mm Hg. Oxygenation was primarily manipulated through changes in PEEP and its effect on mean airway pressure (Paw), and Fi O2 . Lung inflation on chest radiographs was also assessed. In an effort to minimize exposure to high Fi O2 , if PaO2 was above target goals, Fi O2 was initially weaned until < 0.40, and then modifiers of Paw or Fi O2 were decreased as tolerated. If PaO2 was below target guidelines, a chest radiograph was obtained to evaluate lung inflation. Appropriate adjustments in PEEP (Paw) were made in an effort to minimize under- or overinflation of the lung. End-expiratory pressure rarely exceeded 5 cm H2O. If lung inflation was deemed adequate, Fi O2 was adjusted as indicated. HFOV was applied as rescue therapy if PaO2 was below target goals at Paw > 16 cm H2O and Fi O2 = 1.00.

Extubation was attempted after 7 d of age, in the absence of respiratory distress, and if targeted blood gases were maintained with a ventilator rate < 15 per minute, PIP < 20 cm H2O, PEEP < 4 cm H2O, and Fi O2 < 0.30.

Pulmonary Function Testing

Pulmonary function testing was performed using the VT1000 body plethysmograph (Vitaltrends Technology). This system is a flow-through whole body plethysmograph patterned after that reported by Hjalmarson and Olsson (20), and similar to that described by Schulze and colleagues (21). The system uses a differential piezoresistive pressure transducer interfaced with a single-screen pneumotachometer to detect air flow in and out of the sealed plethysmograph. Designed specifically for neonatal use, the tidal volume ranges from 1.0 to 50.0 ml (resolution, 0.1 ml), the frequency response is to 5 Hz, and the flow range is +175 ml/s. The system interfaces with two dedicated microcomputers capable of pattern recognition, data storage, data analysis, and real-time presentation of flow–volume and pressure–volume curves. Tidal volume was monitored continuously for the first 48 h of life. As an esophageal pressure catheter was not used, compliance and resistance measurements, obtained at 6, 12, 18, 24, 36, and 48 h and every 24 h thereafter while intubated, were of the respiratory system as a whole. Ten breaths (meeting five predefined breath selection criteria) were recorded at each time point and averaged for determination of tidal volume, dynamic respiratory system compliance, and expiratory resistance. Variability between measurements was compared by periodic triplicate recording of pulmonary function tests and was consistently < 5%. For data analysis tidal volume and compliance were corrected for body weight. Functional residual capacity was not determined.

Nutritional Management

During the first 24 h of life all animals received heparizined normal saline via the umbilical artery catheter and a 5% dextrose–water infusion with supplemental calcium via the central venous catheter. Initial volume intakes for the first day of life were calculated to deliver 250– 300 (cm3/kg)/d, but subsequently decreased over the first 3–4 d to approximately 175–200 (cm3/kg)/d. These initial fluid requirements were necessary to maintain electrolyte homeostasis, to provide minimal urine output at 1–2 (cm3/kg)/h, to maintain acceptable blood pressure, and to minimize metabolic acidosis. Parenteral nutrition was initiated at 24 h of life with amino acids at 1.25 (g/kg)/d (Trophamine; McGaw, 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 2.5 (g/kg)/d at 48 h of life and l-cysteine [0.60 (mmol/kg)/d] was added at 72 h of life. A 20% lipid emulsion (Intralipid; Pharmacia & Upjohn, Clayton, NC) was initiated on Day 7 of life and increased to 2.5 (g/kg)/d. If clinically stable, enteral nutrition was initiated on Day 7 of life. Donated human breast milk was given by intermittent gastric infusion at an initial volume of 10 (ml/kg)/d, and advanced by 5–10 (ml/kg)/d, as tolerated. Once enteral intakes of 100 (cm3/kg)/d were tolerated, enteral feeding was changed to Primilac (Bio-Serv, Frenchtown, NJ). Nutritional goals included a volume intake of 150– 200 (cm3/kg)/d, 80–120 (cal/kg)/d, and 3.0 (g/kg)/d of protein.

Patent Ductus Arteriosus

All animals were monitored by clinical examination and echocardiography for evidence of patent ductus arteriosus (PDA). Management of PDA included attempted volume restriction and use of dopamine as required to maintain blood pressure (BP) and urine output. Indomethacin (0.2 mg/kg every 12 h for three doses, followed by 0.1 mg/ kg every 24 h for three doses) was initially attempted in several animals but a PDA frequently recurred. Operative ligation within 48 h of the onset of clinical signs of PDA, with echocardiographic confirmation of significant left-to-right shunt, has been the therapy of choice during the last year of study.

Other Care Plans

Arterial blood gases were measured hourly for the first 24 h, every 2 h between 24 and 48 h, every 4 h from 48 to 96 h, and then every 6–12 h as determined by clinical needs. Electrolytes and hematocrit were monitored every 12 to 24 h. Complete chemistries and blood counts were performed weekly. To maintain the hematocrit between 30 and 45%, packed red blood cells were administered periodically in the form of fresh heparinized blood from adult donors.

All animals were initially treated with ampicillin [100 (mg/kg)/d] and gentamicin [2.5 (mg/kg)/d] for the first 3 d of life. Owing to early colonization with Pseudomonas, antibiotics were changed to amikacin/ceftazidime or amikacin/pipericillin for the remainder of the first week of life and then discontinued. Subsequent antibiotic use, as needed for clinically suspected sepsis, included the use of vancomycin and the preceding anti-Pseudomonas regimen. Owing to several early deaths associated with Candida sepsis, prophylactic fluconazole was initiated in all animals as a 6.0-mg/kg dose at 12, 96, and 168 h of age. Doses were then given every other day until Day 28 of life. Prophylactic intravenous immunoglobulin (Sandoglobulin; Swiss Red Cross, Berne, Switzerland) was also given at a dose of 400 mg/kg on Days 5 and 21 of life.

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. Hypotension was initially treated with additional volume supplementation (10–20 ml/kg at least twice over a 1-h period) and the use of dopamine (5–20 μg/kg · min). Dobutamine was occasionally used but had a tendency to further decrease mean BP. Cardiac output measurements were not available. If this approach failed to improve mean blood pressure, then a stress dose of hydrocortisone (1.0 mg/kg) was administered at 6-h intervals until either mean blood pressure increased to more than 25 mm Hg or a maximum of four doses of hydrocortisone were received. Twenty-five percent of the animals required the use of corticosteroids.

Eleven of the 12 animals survived from 27 to 71 d (CLD 1- to 2-mo group), and the twelfth animal survived for 8 mo. Histopathologic, immunocytochemical, morphometric, and selected ultrastructural findings are presented for the 11 CLD 1- to 2-mo survivors, and cell count and cytokine data are presented for all 12 animals (CLD study group).

To assess for intrauterine development changes, 156-d gestational controls (n = 5) were used (31 d of additional gestational development). To assure scheduled deliveries and avoid spontaneous deliveries, an additional five near term gestation fetuses at 175 ± 2 d (50 d of additional fetal lung development) (175-d control group) were used. Air-breathing term controls (n = 5) were naturally delivered animals that survived for 1 d (term control group). Four colony-reared animals underwent right lower lobectomies at 2 mo of age and comprised the air-breathing control group for the long-term CLD animals (term + 2-mo controls).

Pathology: Light Microscopy and Immunocytochemistry

At the time of the lobectomy or necropsy, the right lower lobe was removed, weighed, and intrabronchially fixed with phosphate-buffered 4% paraformaldehyde and 0.1% glutaraldehyde at 20 cm H2O constant pressure for 24 h. 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 microscopy 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. Mean linear intercept (22) and total internal surface area (TISA) were determined by standard methods (22-24), on 10 micrographs of resin-embedded sections, photographed at ×10 magnification. Platelet endothelial cell adhesion molecule (CD31, PECAM; Dako, Carpinteria, CA), a marker for endothelial cells, was used to immunostain lungs from 125-d gestation, 175-d near term, term + 2-mo controls, and 1- to 2-mo CLD specimens. A monoclonal elastin antibody (Sigma, St. Louis, MO) and Miller's van Gieson elastica stain were used to stain for elastic fibers. A point-counting method, in which the lung parenchymal tissue served as the volume of reference, was used to determine the volume fraction (Vv) of immunoreactive sites (25, 26). A grid with 216 points was superimposed on color photographs taken from 10 random, noncontiguous fields per lung specimen at a magnification of ×40. The number of points falling on immunoreactive sites and on lung parenchyma was recorded. The Vv (positive stained profile/parenchyma) was calculated as the ratio of the number of points falling on immunoreactive PECAM sites to points on lung parenchyma.

Tracheal Aspirates

Tracheal aspirates (TAs) were collected at 24, 48, 72, and 96 h, and at 6–8, 9–10, 13–15, and 16–44 d. Animals were disconnected from the ventilator, and 1 ml of sterile normal saline was instilled through the endotracheal tube (ET). The animal was reconnected to the ventilator and allowed to breathe 4 or 5 breaths. The animal was disconnected again, and a sterile 5- or 6F suction catheter was inserted through the endotracheal tube to 1–2 cm past the tip of the ET until it reached the carina. The suction catheter was connected to a sterile suction trap and suction applied until there was no longer a liquid return. The animal was then reconnected to the ventilator. The suction catheter was rinsed with about 0.5 ml of sterile normal saline, so the total volume of aspirate was 1 ml.

If the aspirate was used for a cell count and differential, it was transferred to a Nunc (Roskilde, Denmark) tube, placed in wet ice, and transferred to the laboratory. If the aspirate was banked, it was centrifuged for 10 min at 2,500 rpm; the supernatant was removed and aliquoted in 0.25–ml aliquots, and then frozen at −70° C.

Cytokine/Chemokine Assays

Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) concentrations were determined in TA aliquots by specific and sensitive radioimmunoassays. TNF-α was measured with a specific antiserum to human TNF-α (Caltag Laboratories, South San Francisco, CA) at a final dilution of 1:100,000, radiolabeled human TNF-α (New England Nuclear, Boston, MA), and purified human TNF-α for the standard (Collaborative Research, Bedford, MA). Assay sensitivity was 16 pg/tube and the intra- and interassay coefficients of variation were 5.5 and 6.9%, respectively. IL-6 was measured using a specific antiserum to human IL-6 (Sigma) at a final dilution of 1:100,000, radiolabeled human IL-6 from New England Nuclear, and purified human IL-6 for the standard (Austral Biologicals, San Ramon, CA). Assay sensitivity was 0.6 pg/tube and the intra- and interassay coefficients of variation were 6.5 and 11.9%, respectively. Enzyme immunoassays (PerSeptive Diagnostics, Framingham, MA) were used to measure IL-1β and IL-8. Assay sensitivities were 10 pg/ml and the intra- and interassay coefficients of variation were 4.8 and 12%, respectively, for IL-1β, and 100 pg/ml and 10 and 24%, respectively, for IL-8. IL-10 was measured using the TiterZyme E1A procedure (PerSeptive Biosystems). This method used a two-site solid-phase enzyme assay methodology. The standard was recombinant IL-10 and assay sensitivity was 4 pg/ml with 100 μl of sample. Intraassay and interassay coefficients of variation were 4.6 and 11%, respectively. Although there is no consensus as to the appropriate reference protein, protein determinations were done using the bicinchonic acid (BCA) protein assay (Pierce, Rockford, IL).

Data Analyses

Data for the clinical parameters are presented as means plus the standard deviation (SD), unless otherwise noted. Intergroup differences for timed data were compared by analysis of variance for repeated measures. For total and differential cell counts, cytokine, and mean linear intercept (Lm) and internal surface area (ISA) analyses, comparisons across the various time points were made using one-way ANOVAs and multiple Student t tests, using the Bonferroni adjustment to the level of significance. Statistical results were generated using SAS (Cary, NC) software.

Group Characteristics

During the first year of development of this long-term model, only one of nine animals survived past 27 d; however, during years 2 and 3, survival to 27 d reached 71 and 60%, respectively. Twelve of 31 animals survived to at least 27 d. Group characteristics are shown in Table 1. Hypotension was a common problem for these immature animals. A majority of animals received volume and pressor support in the first 48 h of life. Eight of the 31 (26%) animals died before 10 d of age, 3 from severe vascular compromise secondary to major vessel thrombosis, 2 with severe air leak problems, 2 with positive blood cultures for Pseudomonas, and 1 with sepsis syndrome but negative cultures. Another 11 animals died between 10 and 27 d of age. Deaths after 10 d of age were due to acute cardiopulmonary decompensation consistent with a clinical picture of sepsis syndrome and/or positive blood cultures in 10 of the 13 animals.


Body weight, g383 + 48*
Gestation, d125 + 6
Ratio, male/female12/19
Dopamine27 (87%)
Steroid15 (48%)
Air leak 3 (10%)
HFO rescue12 (39%)
Cholestasis10 (33%)
PDA18 (60%)
Death < 240 h8 (26%)

Definition of abbreviations: HFO = High-frequency oxygen; PDA = patent ductus arteriosus.

*Mean + SD.

Pulmonary Course

A comparison of changes in ventilator support, arterial blood gases, and pulmonary function studies of the 12 survivors over the first 28 d is shown in Table 2. After prophylactic surfactant therapy the respiratory distress syndrome was typically mild to moderate. Maximum resolution occurred by Day 5, and was followed by a gradual increase over the next 1–3 wk in supplemental oxygen needs. Pressure requirements gradually increased or plateaued to maintain PaCO2 . Tidal volume and rate were not significantly changed over any time of the study (data not shown). Although ventilator support requirements increased slightly over time, dynamic respiratory system compliance significantly improved by 14 d of age compared with 24–48 h of age.


Age(d )
Fi O2 0.38 ± 0.090.46 ± 0.160.43 ± 0.170.33 ± 0.120.27 ± 0.090.38 ± 0.110.30 ± 0.060.37 ± 0.090.36 ± 0.17
PIP, cm H2O24 ± 326 ± 626 ± 5 22 ± 320 ± 324 ± 222 ± 320 ± 724 ± 7
Paw, cm H2O9 ± 211 ± 310 ± 29 ± 28 ± 39 ± 27 ± 17 ± 3 7 ± 1
PaO2 , mm Hg65 ± 873 ± 2462 ± 868 ± 12* 69 ± 1074 ± 1575 ± 1368 ± 1477 ± 8
PaCO2 , mm Hg48 ± 846 ± 1050 ± 944 ± 444 ± 748 ± 1147 ± 949 ± 942 ± 9
Compliance,  (ml/cm H2O)/kg0.28 ± 0.120.32 ± 0.190.29 ± 0.140.36 ± 0.180.42 ± 0.150.39 ± 0.130.49 ± 0.23* 0.41 ± 0.140.52 ± 0.24*
Resistance,  (cm H2O/ml)/s254 ± 90279 ± 97266 ± 99289 ± 85190 ± 52252 ± 95184 ± 74223 ± 56253 ± 98

Definition of abbreviations: Paw = mean airway pressure; PIP = Peak inspiratory pressure.

*p < 0.05 versus 1–3 d.

Characteristic progression of chest radiographs is shown in Figures 1a–1d. Early radiographs show consistent low lung volumes, diffuse ground glass appearance, and air bronchograms. By Day 6 the chest radiograph appeared relatively clear with good lung expansion. Random patchy densities became apparent between Days 5 and 10, occasionally associated with decreased lung volumes until pressure support was increased. The chest radiograph typically remained abnormal through the remainder of the clinical course, with wandering patchy densities, intermittent labor atelectasis, and occasional areas of hyperinflation.

Patent Ductus Arteriosus

Patent ductus arteriosus was identified by echocardiographic techniques in the majority of infants during the first week of life. Among 12 survivors to 1 mo, significant clinical findings, including widened pulse pressure, a murmur, and enlarge cardiothymic shadow, were noted in 6. Eight of the 12 infants were treated with indomethacin between 3 and 10 d of age. Owing to recurrence of the PDA, four were subsequently ligated.


Beyond the chronic pulmonary insufficiency of prematurity that this model is designed to study, several other problems frequently managed in immature humans manifested in this long-term baboon model. These included difficulty in establishing tolerance of enteral nutrition, intermittent line sepsis, cholestatic jaundice, and, as previously mentioned, patent ductus arteriosus. It is not the purpose of this article to described these problems, but it is important to note their occurrence as further evidence of the similarity of this model to the immature human infant.

Pathology: Light and Electron Microscopy and Immunocytochemistry

Twelve CLD animals survived for 27 to 71 d before succumbing to an infection-induced sacrifice or death. They were killed when therapy failed to alter the clinical course, so that the lungs would not have extensive superimposed disease. In Figure 2, the developmental lung appearance at 125 d of gestation, term + 1 d, and term + 2 mo are shown. At 125 d of gestation, the baboon lung is in the canalicular and very early saccular stage of lung development (Figure 2a). This stage of lung development is comparable to the 24- to 26-wk human infant. Depending on the fixation method, the distal parenchymal compartment of the lung at this time appeared thick walled, with minimal air spaces in immersion-fixed specimens versus a thinner, but cellular wall and rounded air spaces in intrabronchially instilled specimens. These rounded distal saccules were simplified in appearance, with scattered protuberances (seen as slight elevations or outpouchings) along their walls that were the progenitor secondary crest ridges that will evolve into the alveoli (Figure 2a). Ultrastructurally, at 125 d of gestation, the interstitial compartment of primitive saccular/alveolar wall contained abundant cells, extracellular matrix, and vessels that were situated centrally and subjacent to the epithelial basement membranes in some sites (Figure 3a). Light microscopically, the lining epithelial cells had clear cytoplasm and apical nuclei (Figure 2a), and ultrastructurally were the progenitor alveolar epithelial cells that contained abundant glycogen, rare microvilli, and an absence of lamellar body inclusions (Figure 3a).

Similar to the human, the baboon lung showed early alveolization at term gestation (Figure 2b). The width of the saccular/alveolar walls at term was markedly thinned when compared with that of the 125-d gestation lung (Figures 2a and 2b, and 3a and 3b), but subjectively appeared to have more alveolar wall cells than the term + 2-mo specimens (Figure 2c). The term + 2-mo controls had numerous thin-walled alveoli (Figures 2c and 3c).

The classic inflation pattern of alternating zones of overinflation and atelectasis was not a major feature of the pathology in the CLD animals. The CLD lung specimens showed large “simplified” distal saccules with variable degrees of fibrosis, and an obvious decrease in the number of alveoli whether they had survived for 1 or 2 mo (Figures 4a–4c). The severity of cellular fibroproliferation in the interstitium did not correlate with the length of time receiving ventilator support, e.g., Figure 4a is from a 71-d survivor, Figure 4b represents a 30-d survivor, and Figure 4c is from a 39-d survivor. Two of four survivors with diffuse saccular interstitial fiboproliferation, as shown in Figure 4c, had experienced several episodes of suspected pneumonia and sepsis during their clinical courses, and died with evidence of focal bronchopneumonia. Conversely, the two animals with the pattern of predominantly thinned saccular walls, as seen in Figure 4a, had experienced fewer clinical episodes of lung infection or sepsis, and did not exhibit lung infection at necropsy. However, in the remaining eight animals, suspected clinical episodes of clinical infections and lung changes at necropsy findings could not be correlated. The three animals that received stress doses of hydrocortisone did not exhibit fewer alveoli than the remaining eight CLD baboons.

Whether the saccular walls of the CLD survivors appeared thin with few cells or thickened and hypercellular (Figure 5a and 5b), ultrastructurally they were not normal, and contained increased mesenchymal cells, mononuclear cells, and focal deposition of elastin and collagen fibers (Figures 6 and 7). Some saccular walls showed blunted “abortive” outpouchings/ secondary crests (Figure 8a). The elastic fiber stains (anti-elastin and Miller's van Gieson elastica) showed elastin staining focally within these protuberances along some saccular walls (Figure 8b) and in the tips of rudimentary secondary crests/ alveoli. Only occasional aggregates of increased elastic deposition were identified in the saccular walls. Mean linear intercepts (Figure 9) and total internal surface area (Figure 10) determinations showed that the CLD specimens had significantly decreased alveolization and decreased internal surface area measurements when compared with the 156-d gestation and term + 2-mo controls.

At 125 d of gestation, the baboon lung had minimal, if any, secondary crests, and vascular development, although present, was limited to the primitive saccular walls (Figure 11a). From 125 to 185 d of gestation, the capillaries, comparably stained with PECAM (Figures 11b, 12a and 12b), arborized from corner precapillary arterioles, gradually thinned in diameter, and at term were abundant within the saccular/alveolar walls, a pattern that persisted postnatally in the term + 2-mo controls. When compared with the 175-d gestation (near term) and term + 2-mo air-breathing controls, the CLD specimens showed an adaptive, but dysmorphic, pattern of vascular organization. Hematoxylin and eosin and PECAM staining of the CLD lungs showed corner precapillary arterioles with either adjacent dilated vessels and/or thinned capillaries, haphazardly configured in the saccular walls (Figures 13a and 13b). The increased wall thicknesses in the 125-d gestation control and the CLD specimens were reflected in significantly higher volume density determinations of total parenchyma when compared with the 175-d gestation (near term) and term + 2-mo controls (p ⩽ 0.05) (Figure 14). Conversely, the 175-d gestation and term + 2-mo controls had increased PECAM staining when compared with the 125-d gestation control and CLD study groups (p ⩽ 0.05) (Figure 14). Although the CLD animals had severe capillary vascular hypoplasia, they did have a significantly higher PECAM-stained volume than that of the 125-d gestation group, suggesting that vasculogenesis, although interrupted and largely arrested, had progressed after premature delivery and maintenance with appropriate oxygenation and ventilatory support.

As determined by light microscopy, the 8-mo survivor had a pathologic appearance similar to that of the 1- to 2-mo survivors, i.e., large air spaces with minimal alveoli. However, mean linear intercept and internal surface area measurements were similar to those of 156-d gestation and term controls, respectively, suggesting that some alveolization had occurred over the additional 6 mo of survival.

Tracheal Aspirate Cell Counts and Differentials

Total cell counts of the tracheal aspirates collected on Days 2, 6, 10, and 22 are shown in Figure 15. Although the number of cells tended to increase steadily until Day 10, no significant differences were noted owing to the variability. A similar trend was seen in the differential cell counts, e.g., the alveolar macrophages and neutrophils (Figure 15). Total cell counts in the CLD tracheal aspirates of more than 1 million cells by Day 2 was surprising, in view of our experience with tracheal aspirates in the 140-d pro re nada (PRN) baboon model, which does not receive exogenous surfactant and develops hyaline membrane disease. In Figure 16, the total cell counts, and macrophage and neutrophil differentials, are compared at 48 h postdelivery and PRN ventilatory support in the 125- and 140-d gestation models. Total cells were significantly increased, more than twofold, in the CLD animals when compared with the 140-d PRN animals (p ⩽ 0.05). The macrophage-monocyte population in the CLD animals was increased more than 10-fold when compared with 140-d PRN animals (p ⩽ 0.05), while no difference was evident in the neutrophil counts.

Tracheal Aspirate Chemokine/Cytokines

Normalization of tracheal aspirate data for protein did not affect the overall differences in antigen concentrations. The cytokine/chemokine values determined in the tracheal aspirates are plotted as scattergrams in Figures 17A–17E. Term infant and normal adult values are included for comparison, but were not included in the statistical analyses. The median level of IL-1β was higher in term infants when compared with that of CLD animals, and TNF-α median values in the normal adults were higher than any of the CLD and the term infant values. Within the CLD study times, TNF-α values were significantly elevated on Days 6–8, 9–10, and 16–44 when compared with the 48- to 72-h study time (p ⩽ 0.05) (Figure 17a). There were no significant differences in IL-1β and IL-10 values at any of the study times (Figures 17b and 17e, respectively). IL-8 values on Days 6–8 and 9–10 were significantly increased when compared with those at 24 h, 48–72 h, and 16–44 d (p ⩽ 0.05 (Figure 17c). IL-6 values were significantly higher at 9–10 and 16–44 d when compared with 48- to 72-h levels (p ⩽ 0.05) (Figure 17d).

In five of the survivors, the presence of multiple foci of bronchopneumonia at death was associated with an elevated level of IL-8 recovered from the bronchoalveolar fluid (BAL) at necropsy (⩾ 10,000 pg/ml; range, 11,929–> 20,000 pg/ml). In animals with minimal lung infection or sepsis without lung involvement, IL-8 values ranged from 120 to 1,810 pg/ml, well below the values in the more heavily infected animals. Only one animal had an IL-8 value between 1,810 and > 20,000 pg/ ml (7,094 pg/ml) with no histopathological evidence of lung infection. On gross examination the left lower lobe, used for lavage in this animal, appeared grossly hemorrhagic and more diseased than the right lower lobe, used for histopathologic study. Increased IL-8 levels in tracheal aspirates obtained during the clinical course appeared to correlate when lung infection was suspected clinically in several of the animals. However, comparable increases in IL-8 levels were documented when atelectasis or a PDA was identified, with no other signs of infection present. Elevations of IL-6, TNF-α, IL-1β, and IL-10, in either tracheal aspirates or necropsy lavage fluids, did not correlate with any specific clinical parameter. In two of the four more heavily infected animals, there was, subjectively on light microscopy evaluation, more interstitial fibroproliferation as noted above in the pathologic findings.

BPD is now a disease that is seen primarily in preterm newborns of less than 1,000 g and less than 28 wk in gestational age, considerably more immature than the older infants of 31– 34 wk of gestation with classic BPD described by Northway and coworkers (3). Lung development in infants of 24–27 wk of gestation is in the canalicular stage (presecondary crest formation), and at 31–34 wk of gestation in the saccular stage, during which the cylindrical saccules are subdivided by secondary crests that become alveoli (27). The use of prenatal steroids and postnatal exogenous surfactant, coupled with advances in critical care management in diminishing volutrauma and oxygen injury, have resulted in the present clinical disease in the extremely immature infant, which some have chosen to call chronic lung disease of infancy (16-18). Its pathogenesis involves extreme lung immaturity, treatment-induced oxygen and volutrauma injuries, an autoinflammatory response, and a disorganized reparative response.

The CLD baboon model described in this study was designed to incorporate the features now seen in the human disease: use of prenatal steroids, infants born with lung development still in the canalicular stage of development, treatment with postnatal exogenous surfactant, use of oxygen and volume-sparing treatment strategies, and good nutrition. In spite of maintaining these immature infants in an ideal newborn intensive care unit (NICU) environment, they exhibited a number of physiologic characteristics similar to the extremely immature human also at risk for neonatal CLD. These included an early cardiovascular instability (typically accompanied by mild to moderate metabolic acidosis, oliguria, and low blood pressure) that was often poorly responsive to volume and pressor support, difficulty in establishing optimal enteral nutrition, a propensity for nosocomial infection, development of cholestasis after several weeks of parenteral nutrition (usually precipitated by an infection), and persistence of a patent ductus arteriosus (poorly responsive to indomethacin therapy). Each of these, alone or in combination, may contribute to the risk for development of chronic lung injury in the immature infant. Their occurrence in this immature baboon model emphasizes the similarity to the human condition, and underscores the multiple factors that must be accounted for in understanding the mechanisms for lung injury in the immature infant.

At 1 to 2 mo of age, the CLD baboons exhibited alveolar hypoplasia, similar to the alveolar simplification, alveolar hypoplasia, or arrested acinar development described in more recent BPD pathology reports (28-33). This lack of alveolization was also present in the CLD 8-mo survivor. In addition, a hypoplasia of capillary development within the saccular walls was identified in the CLD baboons, a finding that has not been published in connection with human infants with BPD.

Although present in some human infants at 32 wk of gestation, alveoli are not uniformly present until 36 wk. At birth, about 50 million alveoli are present in the human newborn lung (27). Thurlbeck (34) has reported that children exhibit new complete alveolization of the lung at 2 yr of age. However, Burri (35) has stated that on the basis of morphological criteria alone, he suspects bulk alveolar formation to be completed by about 6 mo of age. Cumulative pathologic data show that a decrease in alveolization is a predictable outcome in any infant who undergoes premature delivery and initiates pulmonary gas exchange with oxygen and positive pressure ventilation. It was a consequence common to the gestationally older infants who were treated with high levels of supplemental oxygen and positive pressure ventilation described in early reports in the late 1960s and 1970s (3, 36-39), the extremely immature infants who received less oxygen and better ventilatory support during the 1980s (29-32), and the extremely immature infants who received exogenous surfactant treatment during the late 1980s and early 1990s (33). On the basis of the persistent lack of alveolization in the 8-mo CLD survivor (comparable to human age 22 to 28 mo), alveolar hypoplasia may be the outcome in human infants with CLD, i.e., infants of extreme immaturity, treated with prenatal steroids, surfactant therapy, and appropriate oxygen and small tidal volume management.

As compared with infants with old BPD pathology findings, human infants with new BPD/CLD and the long-term baboon with CLD do not have airway changes of squamous metaplasia and peribronchial fibrosis (30, 33), severe alveolar septal fibrosis (31), or hypertensive vascular changes (30). However, airway muscle thickening and derangements in elastic fiber architecture and arrangement persist as abnormalities in new BPD (29-31). Loosli and Potter in 1959 (40), implicated the role of elastogenesis in alveolar formation. Elastic fibers are identified with the appearance of septal primordial cells in developing sheep lung (41), and tropoelastin gene expression is observed at sites of septation in the rat (42). The presence of the small elastin fibers in the saccular walls of the CLD-injured baboon lungs may indicate that a “catch-up” in alveolar formation might be possible, if an appropriate therapy were developed.

Quantification of the capillary vasculature has not been reported in prior BPD pathology reports (29-32). The capillary hypoplasia present in the CLD baboon may reflect an arrest in vasculogenesis that is ongoing during the canalicular stage of lung development. The hallmark of the canalicular phase is vascularization, during which capillaries form in the mesenchyme and fuse with previously formed and developing pulmonary arteries and veins (43). Many of the capillaries in the primitive air space walls destined to be saccules, and then alveoli, are still dispersed and situated centrally, at 24–26 wk of gestation in the human and at 125 d of gestation in the baboon. By term, an extensive capillary network has developed in the baboon, in striking contrast to that in the lungs of the CLD animals. The overall decrease in PECAM staining was not unexpected in the CLD infants because of the lack of alveolization, but its extent and the dysmorphic changes, demonstrated by the PECAM staining, were quite marked and not readily identified on routine H&E specimens. Comparable vascular findings have now been documented in the lungs of human infants with BPD (our personal observations, 1998).

A number of investigators have proposed that an inflammatory autoinjury, possibly initiated after oxygenation and ventilatory volutrauma, occurs and then persists during the evolution of CLD (44-46). Workers have identified that the influx of inflammatory cells obtained by tracheal aspirate or bronchoalveolar lavage served as a marker of lung damage (47, 48). In utero, premature baboons at 125 d of gestation have minimal, if any, alveolar macrophages, comparable to other premature species. Mononuclear cells enter the saccules/ alveoli just before and after parturition in mammals, including the human (49-52), and their appearance correlates with the appearance of surfactant material in newborn monkeys (53). In this study, an increased influx of alveolar macrophages was identified in the air spaces of the surfactant-treated CLD baboons within 48 h, a finding not evident in the 48-h tracheal aspirates of the 140-d baboon model of hyaline membrane disease (HMD)/BPD, which does not receive exogenous surfactant treatment. In human infants, exogenous surfactant treatment induced an increased cell count in BAL fluid within the first 3 d of life (54) and was associated with a higher percentage of neutrophils in Day 7 lavage samples of surfactant-treated infants in another study (55).

Most investigators believe the “alveolitis”of recruited polymorphonuclear cells (PMNs) and alveolar macrophages and/ or monocytes, coupled with the combined oxidative/volutrauma-induced injury to saccular wall cells, likely result in the array of mediators identified in lung secretions of human infants with CLD/BPD (reviewed in References 56–58). Comparable cytokine elevations were identified in lung fluids of the CLD animals, e.g., baboon infants with CLD had significant elevations of TNF-α concentrations during Days 6 through 10 and elevated IL-6 levels during Days 9 and 10.

High concentrations of IL-8 in lung fluid samples of CLD baboons were also evident, comparable to the human infant with CLD (reviewed in References 56–58). When severalfold increases above preceding measurements of IL-8 occurred during the clinical course of individual animals, some could be correlated with radiographic, microbiologic, and clinical data of suspected pneumonia, but other increases could not be related to underlying infection. The most consistent association was the finding of multiple foci of bronchopneumonia in the lung at necropsy and IL-8 levels exceeding 10,000 pg/ml in BAL fluid (BALF). Animals with sepsis and minimal to no infection in the lung did not show elevated IL-8 levels in BALF at necropsy. Our data concerning the potential association of pulmonary infection and more severe interstitial fibroproliferation are too limited to allow any conclusions.

In summary, a model of CLD has been developed in the very immature baboon. The affected baboons have clinical findings, histopathologic features, and inflammatory cell profiles and mediators in airway secretions comparable to those described in extremely immature human infants with CLD. Alveolar and capillary hypoplasia are the striking histopathologic lesions documented in this model, as is an inflammatory cellular and mediator profile believed to play a role in the disease development and/or reflect underlying infection. As the immature state of the lung has now become the major factor in the pathogenesis of BPD, further research will focus on how premature birth and survival alter the fetal lung adaptation to extrauterine development. Now that less oxygen and small tidal volumes are being used to manage immature infants with lung disease, future work needs to determine if alveolar and vascular hypoplastic lesions are permanently irreversible, or could be reversed with new and yet to be defined treatment modalities.

The authors thank the BPD Resource Center personnel: the animal husbandry group led by Drs. D. Carey and M. Leland, the NICU technicians and the Wilford Hall neonatal fellows who care for the infants, and the pathology staff who perform the necropsies and do the morphometry. Dr. J. Schoolfield is thanked for biostatistical assistance.

Supported by NIH Grants HL52636 and HL52646.

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Correspondence and requests for reprints should be addressed to J. J. Coalson, Ph.D., Department of Pathology, 7703 Floyd Curl Drive, UTHSC-SA, San Antonio, TX 78284. E-mail:


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