Treatment of Immature Baboons for 28 Days with Early

Nasal Continuous Positive Airway Pressure

Merran A. Thomson, Bradley A. Yoder, Vicki T. Winter, Helen Martin, Deborah Catland, Theresa M. Siler-Khodr,
and Jacqueline J. Coalson
Division 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

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 156day 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.
Keywords: 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 (13).
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
(611). 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

(Received in original form September 12, 2003; accepted in final form February 10, 2004)

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).
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@uthscsa.edu
Am J Respir Crit Care Med Vol 169. pp 10541062, 2004
Originally Published in Press as DOI: 10.1164/rccm.200309-1276OC on February 12, 2004
Internet address: www.atsjournals.org

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 (1315). 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 (2022)
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).

METHODS
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

Timed gestations were determined by observing characteristic sex skin
changes and confirmed by a fetal ultrasound examination at 112115
days of gestation. Pregnant baboon dams (Papio papio) were treated
with 6 mg of intramuscular betamethasone 48 and 24 hours before
elective hysterotomy under general anesthesia. nCPAP study animals
were delivered at 125 2 days (67% of term gestation at 185 days).
At birth infants were weighed, sedated with intramuscular ketamine
hydrochloride (5 mg/kg), and intubated with a 2.0-mm endotracheal
tube. Infants were treated with Curosurf (200 mg/kg; provided by Chiesi

Thomson, Yoder, Winter, et al.: Early nCPAP in Immature Baboons

Farmaceutica, Parma, Italy) (nCPAP group) before the initiation of

ventilator support.
Ventilation was initiated with a humidified, pressure-limited, timecycled 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 5570 mm Hg.
Infants were nursed in a servo-controlled, infrared-warmed, body plethysmograph (VT1000; VitalTrends Technology, New York, NY) set
at 36.9C, capable of continuous tidal volume measurements and computer-regulated intermittent pulmonary function testing.

Respiratory Management
Ventilator adjustments were made on the basis of chest radiograph,
clinical examination, arterial blood gas measurement, and tidal volume
measurement as described below. We used the European 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 reduction of ambient
light and noise (1517, 24, 25) in the care of the infants.
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 5570
mm Hg, PaCO2 at 5060 mm Hg, pH greater than 7.2, and tidal volumes
of 46 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, 1416 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 (1015 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.

1055

Nutritional Management
During the first 24 hours of life the study animals received heparinized
normal saline via the umbilical artery catheter and a 5% dextrosewater
infusion with supplemental calcium via the central venous catheter.
Initial volume intakes for the first day of life were calculated to deliver
250275 ml/kg per day, but subsequently decreased over the first 34
days to 180200 ml/kg per day. Initial fluid requirements were necessary
to maintain electrolyte homeostasis, to provide minimal urine output
at 12 ml/kg per hour, to maintain acceptable blood pressure, and to
minimize metabolic acidosis.
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 4872
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 1030 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 180200 ml/kg/day, 120160 calories/
kg/day, and 3.0 g/kg/day of protein.

Patent Ductus Arteriosus

Animals were monitored by clinical examination and echocardiography
for evidence of patent ductus arteriosus. If the patent ductus arteriosus
was believed clinically to have contributed to the need to continue or
reinstitute ventilation in an animal, the treatment protocol allowed for
the use of volume restriction and dopamine as required to maintain
blood pressure and urine output. Indomethacin and surgical ligation
were treatment options for those with clinical instability (19).

Other Care Plans

Arterial blood gases were measured hourly for the first 24 hours, every
2 hours between 24 and 48 hours, every 4 hours from 48 to 96 hours,
and then every 612 hours as determined by clinical needs. Electrolytes
and hematocrit were monitored every 12 to 24 hours. Complete chemistries and blood counts were performed weekly. To maintain hematocrit
between 35 and 45%, packed red blood cells were administered periodically, using fresh heparinized blood obtained from adult baboons.
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
Four 125-day gestational control lungs were used to determine the
baseline developmental parameters of the delivered animals. To assess
for intrauterine developmental changes that would occur with approximately 1 month of further growth and development, four 156-day gestational control animals were used. Air-breathing term control animals
(n 6) were naturally delivered animals that survived for 1 to 2 days,
and their histologic characteristics and morphometric values are given
for reference parameters only.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004

Pathology Methods
Before the planned necropsy, each animal was ventilated with 100%
oxygen for 5 minutes, and deep anesthesia was induced by the slow
infusion of pentobarbital to decrease 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 pressurevolume curve was measured by increasing the pressure on the lung to 35 cm H2O in 5-cm H2O pressure
increments, using a syringe and manometer, and then decreasing the
pressure with measurement of volume after 30 seconds at each pressure
(26). The volumes were corrected for the compression volumes of the
measurement system.
After acquisition of the pressurevolume curve, the right lower
lobe was removed, weighed, and intrabronchially fixed with phosphatebuffered 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 m, 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
10 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 pointcounting 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 40. 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.

Station, TX) and repeated measures analysis of variance. For the pathology data, SPSS version 9.0 (SPSS, Chicago, IL) was used.

RESULTS
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

In Table 1, some clinical variables of the nCPAP animals are

depicted. None required pressor support or steroid treatment
for hypotension. Although the presence of patent ductus arteriosus was uniformly seen in the nCPAP infants, none required
medical or surgical treatment. None developed pulmonary air
leaks. Two infants in the nCPAP group had suspected sepsis
(culture negative); they recovered fully with antibiotic treatment.
Enteral feeding in the nCPAP group was swiftly introduced and
maintained; five achieved full enteral feeds, requiring no further
hyperalimentation. Five nCPAP infants were clinically stable on
Day 28. In spite of our attempt to achieve better enteral nutrition
in the nCPAP animals, body weights at death were significantly
less than those of the 156-day control animals (p 0.001).

Bronchoalveolar Lavage

Pulmonary Course

Bronchoalveolar lavage (BAL) was performed at necropsy in the 125day and 150- to 160-day gestational control and nCPAP groups. After
necropsy, a preweighed lobe of lung was lavaged with 0.9% NaCl
(pH 7.4) with a recovery of 7080% of the instilled volume. Lavage
specimens for cell counts and differentials were centrifuged for 10
minutes at 1,500 rpm, and cell counts and differentials were done. A
portion of the supernatant was aliquoted in 1.0-ml aliquots and then
frozen at 70C for cytokine/chemokine studies.

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, 312.6 days) being supported with nCPAP,
and a median of 17.8 days (range, 1224 days) of breathing
without the need for either ventilation or nCPAP.

Cytokine/Chemokine Assays
Interleukin (IL)-6 concentrations were determined in BAL fluid aliquots by specific and sensitive radioimmunoassays. IL-6 was measured
with a specific antiserum to human IL-6 (Sigma, St. Louis, MO) at a
final dilution of 1:100,000, radiolabeled human IL-6 (PerkinElmer Life
Sciences, Boston, MA), 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. An enzyme immunoassay (PerSeptive Diagnostics/
Applied Biosystems, Cambridge, MA) was used to measure IL-8. Assay
sensitivity was 10 pg/ml and the intra- and interassay coefficients of
variation were 100 pg/ml and 10 and 24% for IL-8. This method involved
a two-site solid-phase procedure.

Statistical Analysis
Clinical data are presented as median and either range or interquartile
range unless otherwise indicated. Pathologic data are presented as median and standard deviation unless otherwise stated. A p value of 0.05
or less was required for significance. Statistical results for clinical and
physiologic data were generated with Stata version 7.0 (Stata, College

TABLE 1. GROUP CHARACTERISTICS

Age at birth, d (median [range])

Birth weight, g
Median (range)
Interquartile range
Male:female
Days to establish full enteral
feeds, 150 ml/kg per d
(median [range])
Weight at necropsy, g
Median (range)
Interquartile range

nCPAP
(n 5)

156 Day Gestation

(n 4)

126 (125127)

156

368 (322453)
329393
1:4

698 (644747)
681713
3:1

14.5 (1417.1)

417 (304485)*
326423

698 (644747)*
681713

Definition of abbreviation: nCPAP nasal continuous positive airway pressure.

* p 0.001.

Thomson, Yoder, Winter, et al.: Early nCPAP in Immature Baboons

Figure 1. Sequential changes in FIO2 and arterial-to-alveolar O2 ratio (a/

A ratio) in the nCPAP group (median and 25th75th interquartile range)
are shown in (A ). In (B ), sequential changes in PaCO2 and pH in the
nCPAP group (median and 25th75th interquartile range) are depicted.

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, 7.157.36), 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 pressurevolume
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.

1057

Figure 2. Measurement of pulmonary mechanics. Sequential changes

in first 24 hours of life in (A ) peak inspiratory pressure (cm H2O) during
pulmonary function testing and tidal volume (ml/kg). (B ) Expiratory
airway resistance (cm H2O/ml per second) and respiratory system dynamic compliance (ml/cm H2O per kg per VT). Data are shown as median
and 25th75th interquartile range.

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

Figure 3. Pressurevolume curves at necropsy. For comparison only,

the pressurevolume curves for term plus 1- to 2-day-old spontaneously
breathing control animals, and animals delivered at 125 days that were
ventilated with low tidal volume positive pressure ventilation (LV-PPV)
for 27 days or longer (19), have been included. Data are shown as
medians and 25th75th interquartile ranges.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004

Figure 4. A 125-day gestation specimen. In (A ),

the bronchus (labeled B )
and pulmonary artery (labeled A ) are surrounded
by rounded air spaces,
some of which are terminal
bronchioles (Tb), respiratory bronchioles, and alveolar ducts. Early secondary
crest formation is noted
(arrows). (B ) Ultrastructural
features of the lung at 125
days of gestation. The interstitium (I) of the saccule
walls contains undifferentiated mesenchymal cells
with clear or glycogen-containing cytoplasm. Capillaries are inconspicuous, but a
likely pericyte is evident (*).
Large progenitor Type 2
epithelial cells (AT2) that
contain varying amounts
of cytoplasmic glycogen
line the saccular walls. Cytoplasmic lamellar inclusions are not present. AS
air space, either saccular or
alveolar space. (A ) Hematoxylin and eosin; original
magnification, 310. (B )
Uranyl acetate and lead citrate; original magnification, 2000.

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 5. A 156-day gestation specimen. In (A ),

the lung shows considerable maturation light microscopically when compared with the 125-day
gestation specimen. The
saccular walls are considerably thinner. Progenitor respiratory bronchioles
and alveolar ducts show
varying lengths of secondary crests (arrows) and an
occasional alveolus (*). A
pulmonary arteriole. (B )
Ultrastructurally, alveolar
epithelial Type 2 cells
(AT2) are more flattened
and still show abundant
cytoplasmic glycogen and
extremely rare lamellar inclusion bodies. Some have
differentiated into Type 1
epithelium but are transitional in appearance, that
is, Type 1 epithelial thinned
cytoplasm but with microvilli characteristic of the
Type 2 epithelial cell. There
is a small capillary (c ) with
an enclosed red blood cell
to one side of the saccular
wall. The interstitium contains a few undifferentiated
cells (M ). An erupting secondary crest, containing a
myofibroblast (mf) with
abundant cytoplasmic filaments and dense bodies, is
evident. AS air space. (A)
Hematoxylin and eosin;
original
magnification,
310. (B ) Uranyl acetate
and lead citrate; original
magnification, 2000.

(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

Thomson, Yoder, Winter, et al.: Early nCPAP in Immature Baboons

1059

Figure 6. nCPAP 28-day

survivor. In (A ), the air
spaces are more dilated,
because of air breathing,
than those seen in the 156day gestation developmental control. Note the
variation in the number of
secondary crests and alveoli,
with more seen in the middle of the field. Similar to the
156-day gestation control
lung, a few secondary crest
elongations have additional
crests and alveoli forming
from the sides (curved lines),
whereas others have more
blunted secondary crest formation. pa pulmonary artery; br bronchiole. In (B),
electron micrograph shows
alveolar Type 2 cells (AT2),
some capillaries (c), and an
interstitium that contains
several nucleated cells with
dense cytoplasm, but also
some extracellular matrixlike material (*). Note the
two outgrowths that are
likely secondary crest formations (arrows). AS saccular or alveolar space. (A )
Hematoxylin and eosin;
original
magnification,
310. (B ) Uranyl acetate
and lead citrate; original
magnification, 1300.

secondary crests and alveoli (Figure 7A). Electron microscopically, capillaries were seen located in a subepithelial configuration
on the thin, fused side of the airblood 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). Pointcount determinations of PECAM immunostaining are shown in
Figure 10. As expected during development, PECAM vascular
staining increased and parenchymal values decreased as birth

Figure 7. Term plus 1- to

2-day specimen. In (A ), the
lung at term shows larger
air spaces with a substantive increase in complexity
of the secondary crest outgrowths (curved lines).
Many have side branches
and elongated distinct alveoli (*). A portion of a terminal bronchiole (Tb) and
the pulmonary artery (PA)
are evident. In (B ), the
thinned alveolar wall is
lined by several Type 2 epithelial cells and Type 1 cytoplasmic extensions in
the electron micrograph of
the term plus 1- to 2-day
lung. Cytoplasmic lamellar
bodies are present in the
Type 2 cells (AT2), and free
surfactant material (arrow)
is in the alveolar spaces
(AS). The alveolar macrophage (AM), which is infrequently seen, contains
phagocytosed surfactant
material. Capillaries are
identified in the alveolar
wall and the emerging alveolar branch (c ). (A ) Hematoxylin and eosin; original
magnification, 310. (B )
Uranyl acetate and lead citrate; original magnification, 1300.

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 156day 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, 30.483.7 pg/ml) versus a median of 166.3 pg/ml
(range, 27.6728.1 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, 28.293.3 pg/ml) versus a median of 25 pg/ml (range,
20117.8 pg/ml) in the 150- to 160-day gestational controls.

DISCUSSION
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).

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004

Figure 8. Internal surface area determinations show the expected increase in surface area of the gestational controls as term is approached.
The 125-day gestational control lung values are significantly different
from the nCPAP and 156-day values (*p 0.01). The nCPAP group and
156-day gestational control (GC) group (intrauterine developmental
control) show no significant difference in internal surface area. Data are
shown as means and standard deviation (SD).

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

Figure 9. Surface-to-volume determinations show no significant differences in nCPAP and 156-day gestational control lung values. The 125day control lungs were significantly different from the two other study
groups (*p 0.001). Data are shown as means and standard deviation
(SD).

Figure 10. Volume density determinations of PECAM-stained vasculature (solid columns) expressed as a percentage of total parenchyma
(dotted columns) reveal that PECAM-stained vessel volume densities were
comparable in the nCPAP and 156-day gestational control groups and
were significantly increased when compared with 125-day gestational
control values (*p 0.005). Parenchymal counts in the nCPAP and 156day control groups were significantly lower when compared with the
more immature 125-day gestational control lungs (p 0.01). Data
are shown as means and standard deviation (SD). Vv volume fraction.

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

Thomson, Yoder, Winter, et al.: Early nCPAP in Immature Baboons

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-1 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).
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.
Acknowledgment : 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.

1061

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6.

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8.

9.

10.

11.

12.

13.

14.

15.
16.

17.

18.

19.

20.
21.

22.

23.

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