AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L454-L461, 2007. First published October 6, 2006; doi:10.1152/ajplung.00095.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/L454    most recent
00095.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flecknoe, S. J.
Right arrow Articles by Hooper, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flecknoe, S. J.
Right arrow Articles by Hooper, S. B.

Increased lung expansion alters lung growth but not alveolar epithelial cell differentiation in newborn lambs

S. J. Flecknoe,* K. J. Crossley,* G. M. Zuccala, J. E. Searle, B. J. Allison, M. J. Wallace, and S. B. Hooper

Fetal and Neonatal Research Group, Department of Physiology, Monash University, Victoria, Australia

Submitted 15 March 2006 ; accepted in final form 29 September 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although increased lung expansion markedly alters lung growth and epithelial cell differentiation during fetal life, the effect of increasing lung expansion after birth is unknown. We hypothesized that increased basal lung expansion, caused by ventilating newborn lambs with a positive end-expiratory pressure (PEEP), would stimulate lung growth and alter alveolar epithelial cell (AEC) proportions and decrease surfactant protein mRNA levels. Two groups of lambs were sedated and ventilated with either 0 cmH2O PEEP (controls, n = 5) or 10 cmH2O PEEP (n = 5) for 48 h beginning at 15 ± 1 days after normal term birth. A further group of nonventilated 2-wk-old lambs was used for comparison. We determined wet and dry lung weights, DNA and protein content, a labeling index for proliferating cells, surfactant protein mRNA expression, and proportions of AECs using electron microscopy. Although ventilating lambs for 48 h with 10 cmH2O PEEP did not affect total lung DNA or protein, it significantly increased the proportion of proliferating cells in the lung when compared with nonventilated 2-wk-old controls and lambs ventilated with 0 cmH2O PEEP (control: 2.6 ± 0.5%; 0 PEEP: 1.9 ± 0.3%; 10 PEEP: 3.5 ± 0.3%). In contrast, no differences were observed in AEC proportions or surfactant protein mRNA levels between either of the ventilated groups. This study demonstrates that increases in end-expiratory lung volumes, induced by the application of PEEP, lead to increased lung growth in mechanically ventilated 2-wk-old lambs but do not alter the proportions of AECs.

positive end-expiratory pressure; lung maturation; lung growth; alveolar epithelial cells

ALTHOUGH THE EFFECTS OF ALTERED lung expansion on lung growth and maturation are well documented during fetal life, the relationship between lung expansion and lung development after birth is still unclear. During fetal development, the future airways are filled with a liquid that is secreted across the pulmonary epithelium into the lumen and exits the lung via the trachea. As the upper fetal airway restricts the loss of lung liquid, liquid accumulates within the future airways. Liquid accumulation maintains the fetal lungs in an expanded state that is critical for normal fetal lung growth and development (17). Prolonged increases in the basal level of fetal lung expansion stimulate lung cell proliferation, structural development of the alveolar parenchyma (23), and type II-to-type I alveolar epithelial cell (AEC) transdifferentiation (8). Conversely, decreased fetal lung expansion can cause lung growth and structural development to cease while inducing an increase in the proportion of type II AECs (1, 11).

To initiate air breathing at birth, the airways must be cleared of liquid to allow the entry of air, which in turn causes an air-liquid interface to form within the lung. As a result, surface tension forms within the lung, which increases lung recoil despite the presence of surfactant. Thus, in the absence of the distending influence of lung liquid, and, due to the surface tension-mediated increase in lung recoil, the lung partially collapses away from the chest wall after birth, leading to the creation of a subatmospheric intrapleural pressure (3). Because the mechanisms responsible for maintaining basal lung expansion before and after birth are different, it is currently unclear whether alterations in the degree of basal lung expansion affect lung growth and AEC differentiation postnatally as they do prenatally. Previous studies have demonstrated that the compensatory lung growth induced by hemipneumonectomy is expansion dependent, indicating that lung expansion may be an important determinant of postnatal lung growth (4, 27–30). Similarly, the application of continuous positive airway pressure (CPAP) to newborn ferret pups for 2 wk also increases lung weights (32). On the other hand, we have previously found no effect of 12 h of CPAP on postnatal lung growth or surfactant protein mRNA levels in newborn lambs (19). Thus the question of whether lung expansion regulates postnatal lung growth and AEC differentiation after birth is not resolved. Our aim was to determine the effect of increased lung expansion on postnatal lung growth and AEC differentiation by ventilating newborn lambs for 48 h in the presence or absence of a positive end-expiratory pressure (PEEP).

PEEP is commonly used in neonatal ventilation to maintain end-expiratory lung volumes. As PEEP prevents lung collapse at end expiration, it reduces the risk of atelectatic lung trauma and markedly improves arterial oxygen saturation levels, although this can be at the expense of reducing pulmonary blood flow (12, 25). We aimed to determine the effects of increased levels of lung expansion (experienced during ventilation with PEEP) on lung growth and epithelial cell proportions and hypothesized that PEEP-induced increases in postnatal lung expansion would lead to increased lung growth and altered AEC phenotypes, specifically, an increase in the proportion of type I AECs and a decrease in the proportion of type II AECs with a concomitant decrease in surfactant protein mRNA levels.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experiments conducted on animals were approved by the Monash University Committee for Ethics in Animal Experimentation. Two groups of twin, term-born, healthy Merino x Border Leicester lambs were randomly assigned to mechanical ventilation for 48 h with an applied PEEP of either 0 (0 PEEP; n = 5) or 10 cmH2O (increased lung expansion or 10 PEEP, n = 5). An additional group of unsedated, nonventilated 2-wk-old control lambs (2-wk control; n = 4) was used to identify any effects of sedation and mechanical ventilation. At 11 ± 1 day postnatal age (PNA), aseptic surgery was performed on ventilated lambs to implant polyvinyl vascular catheters. Anesthesia was induced by inhalation of 5% halothane (Fluothane; Zeneca, ICI Operations, Australia) in O2 (medical grade oxygen, BOC Gases) and was maintained after tracheal intubation with 0.5–2.0% halothane in O2. Polyvinyl catheters were implanted into the femoral artery (single catheter) and vein (2 catheters) to regularly collect arterial blood samples and to infuse glucose (5% dextrose) and anesthetic agents during the 48-h experimental period. All catheters were tunneled subcutaneously and exteriorized through an incision in the lamb's right flank. The lambs were allowed ~4 days to recover before the experiment began.

On the day of the experiment (15 ± 1 days PNA), lambs were anaesthetized with an intravenous injection of sodium thiopentone (0.4 mg/kg Pentothal; Boehringer Ingelheim, New South Wales, Australia), intubated with a cuffed endotracheal tube, and connected to a mechanical ventilator (Babylog 8000+; Dräger Medical International, Lübeck, Germany). The ventilator was set in volume guarantee mode and delivered warm, humidified room air at a tidal volume of 7.5 ml/kg to both groups of lambs for the duration of the experiment. Inspiratory and expiratory times were set at ~0.6 and 1.2 s, respectively. Throughout the experiment, blood gas and respiratory parameters were recorded at least every hour (every 10 min for the 1st hour, every 30 min for the next 2 h, and hourly thereafter). The lamb's oxygenation status was measured by calculating the alveolar-arterial difference in oxygen (AaDO2) using the formula AaDO2 = [Pressure(barometric) – Pressure(H2O)] x FIO2 – (PaCO2/0.93) – PaO2 (26). Urine output was also monitored, and heart rate, blood pressure, peak inspiratory pressures, PEEP, and mean airway pressure were recorded digitally throughout the experiment (Powerlab/SSP; ADIndustries, Castle Hill, Australia). The lambs were maintained in a sedated state with either pentobarbitone sodium (Nembutal; Rhone Merieux) or alfaxalone (10 mg/ml; Alfaxan-CDRTU, Jurox, New South Wales, Australia). Alfaxalone was used to minimize the suppressive effects of long-term sedation with Nembutal on heart rate and blood pressure; the combination of the two forms of anesthesia proved to be very successful. The level of sedation was carefully monitored, and the rate of delivery of anesthesia was altered accordingly. Long-acting antibiotics (50 mg·kg–1·day–1, Ampicillin) were administered intravenously at 12 hourly intervals.

Upon completion of the 48-h experimental period, lambs were humanely killed with an overdose of pentobarbital sodium (135 mg/kg iv). The body weight of the lamb was recorded before the lungs, heart, brain, liver, and kidneys were dissected out and weighed. Portions of the left lung were rapidly frozen in liquid nitrogen for biochemical analysis, and the right lung was fixed at 20 cmH2O using 4% paraformaldehyde in phosphate buffer before postfixation in Zamboni fixative or 2% glutaraldehyde and subsequent processing for light and transmission electron microscopy (TEM), respectively.

Lung Growth Parameters

Wet and dry lung weights as well as total protein (soluble) and DNA contents of the fetal lung were determined using established techniques (16, 22). Wet lung weights were determined at postmortem examination, whereas dry lung weights were calculated by drying pieces of lung tissue (~400 mg) in a 60°C oven until no change in weight could be detected (~3 days). DNA contents and concentrations were measured using a standard fluorometric assay (15). Lung tissue samples (0.5–1 g) were homogenized in ice-cold sodium phosphate buffer (3 M NaCl, 0.05 M Na2HPO4, 0.05 M NaH2PO4 x 2H2O, 0.002 M EDTA, pH 7.4) and centrifuged at 3,500 rpm for 5 min at 4°C. A 1:5 dilution of the supernatant was used to measure DNA concentrations. DNA standards (calf thymus DNA, Sigma) were dissolved in the same phosphate buffer and diluted to give concentrations of 72.5, 36.3, 18.1, 9.1, 4.6, and 0 µg/ml. An EDTA solution (0.002 M, 850 µl) and the fluorochrome (Hoechst 33258; 600 µl and 2.5 µg/ml in 0.125 M in NaH2PO4 x 2H2O, 3 M NaCl, pH 7.4) were added to 50 µl of either standard (in triplicate) or diluted sample (in duplicate). The standards and samples were then incubated together at room temperature in the dark before measuring the fluorescence using an excitation wavelength of 356 nm and an emission wavelength of 480 nm (15).

Lung cell proliferation rates were determined by immunohistochemistry using a marker for proliferating cells (Ki67). Ki67 is an antigen expressed by proliferating cells during all phases of the active cell cycle (7). Paraffin sections (4 µm) were mounted onto Superfrost Plus glass slides, with each slide containing lung tissue collected from controls or lambs exposed to either 0 cmH2O PEEP or 10 cmH2O PEEP. The sections were deparaffinized and then rehydrated in a series of ethanol washes. Endogenous peroxidases were blocked using 3% hydrogen peroxide before the sections were washed in PBS (pH 7.4) and microwaved in 0.01 M sodium citrate (pH 6) for 20 min to enhance antigen retrieval. Nonspecific binding was blocked by incubating slides in a blocking buffer [10% normal goat serum (NGS); NGS/01% Triton X-100/0.05 M Tris·HCl, pH 7.2]. Slides were then incubated at room temperature for 90 min with primary antibody (Ki67, anti-human antigen, monoclonal mouse, Dako Cytomation) diluted 1:150 in Dako antibody solution (Dako antibody diluent with background reducing component, Dako). Following washes in PBS/0.1% Tween 20, sections were incubated with secondary antibody (Biotinylated goat anti-mouse IgG, Vector Laboratories) diluted 1:700 in PBS/0.1% Tween 20. The sections were washed in PBS/0.1% Tween 20, incubated in ABC (Avidin/Biotin/horseradish peroxidase complex, Vector Laboratories), diluted 1:150 in PBS, and then washed in PBS. Diaminobenzidene solution was applied to the slides and incubated for 7 min. After a final wash in PBS, the sections were lightly counterstained using hematoxylin and were coverslipped.

Three sections, each from different regions of the lung, were used from each lamb; tissue blocks and sections were chosen at random. To calculate the proportion of cells positively labeled for Ki67, 10 images were randomly captured from each of the three different tissue sections using ImagePro Plus version 4.5 software package. At least 1,800 cells were counted per animal at a magnification of x100, and a labeling index was determined by expressing the number of Ki67-labeled cells as a percentage of all cells counted. Larger airways and blood vessels were excluded from the analysis so that only cells in the perialveolar region were counted.

AEC Differentiation

Analysis of AEC phenotypes. We chose to identify AECs using morphological criteria identified by TEM, as previously described (8, 10, 11), rather than by light microscopy in conjunction with staining for specific cell markers. We chose this method of analysis because it is currently unclear which markers should be used to categorically identify AECs in sheep (see Ref. 9). Given that previous studies have demonstrated that the nuclear diameters of type I and type II AECs are similar (2, 5), the chances of counting a nuclear profile of each cell type are equal using TEM; our observations (unpublished) confirm the findings of these earlier studies. Of particular importance are the findings that the nuclear diameters of type I and type II AECs are not affected by changes in the basal level of lung expansion (Flecknoe and Hooper, unpublished observations).

Following fixation, the right lung was processed for TEM as previously described (8–11). Briefly, the right lung was cut into 5-mm slices. With the use of a grid, six sections were chosen at random and cut into cubes (2 x 2 x 2 mm), taking care to avoid major airways and blood vessels. The tissue cubes were then washed in 0.1 M cacodylate buffer, incubated in 2% OsO4 (in 0.1 M cacodylate buffer), dehydrated in a series of ethanol and propylene oxide washes, and embedded in epoxy resin. At least three epoxy resin/tissue blocks were randomly chosen from each animal. Ultrathin sections (70–90 nm) were cut using a diamond knife, mounted on 200-mesh copper grids, and stained with aqueous uranyl acetate and Reynolds lead citrate. All sections were coded, and the observer was blinded to the experimental group.

Using a transmission electron microscope (Joel 100s), a minimum of 100 AECs were classified per animal, and the proportions of each phenotype were determined by counting the number of nuclear profiles of each type (8, 11). At least three different sections were viewed per animal, ensuring that only one section per tissue block was analyzed. Identification of AECs depended on clear visualization of the basement membrane with all AECs localized on the luminal surface of this membrane. AECs were categorized as one of three phenotypes: type I AECs, type II AECs, and intermediate AECs. Undifferentiated AECs, which are easily identified by their abundant cytoplasmic glycogen, are not present at this stage of development (10). Type I AECs had flattened cytoplasmic extensions, flattened nuclei, little perinuclear cytoplasm, and few cytoplasmic organelles. Type II AECs were rounded in shape with a rounded nucleus and had microvilli on their apical surface and abundant cytoplasmic organelles, including lamellar bodies. The intermediate cells were a heterogenous group that displayed characteristics of both type I and type II AECs. Their classification depended on the presence of marked cytoplasmic extensions, but these cells also contained lamellar bodies and usually had apical surface microvilli (8).

Surfactant protein analysis. Fetal lung surfactant protein A (SP-A), SP-B, and SP-C mRNA levels were quantified by Northern blot analysis (18). Briefly, total RNA was extracted from fetal lung tissue, and 20 µg was denatured and electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde. The RNA was then transferred to a nylon membrane (Duralon; Stratagene, La Jolla, CA) by capillary action and cross-linked to it using ultraviolet light (Hoeffer UVC 500, Amrad). The membrane was incubated in hybridization buffer [50% (vol/vol) deionized formamide, 7% (wt/vol) SDS, 5x saline-sodium phosphate-EDTA, and 0.1 mg/ml denatured and fragmented salmon sperm DNA] for 3–4 h at 42°C. This was followed by hybridization with the 32P-labeled SP-A, SP-B, or SP-C cDNA probe (2 x 106 counts·min–1·ml–1) for 24–48 h at 42°C in the same hybridization buffer (18). The membranes were then washed, sealed in airtight bags, and exposed to a storage phosphor screen for 24–48 h at room temperature. To standardize the amount of total RNA loaded onto each lane, the blot was stripped and then reprobed with a 32P-labeled ovine cDNA probe for 18S rRNA. The relative levels of SP-A, SP-B, and SP-C mRNA were quantified by measuring the total integrated density of each band using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis. All data are expressed as the means ± SE, and the level of significance used was P < 0.05, unless otherwise stated. A two-way ANOVA for repeated measures was used to determine differences in arterial blood gas measurements and ventilator parameters over time. Differences between individual data points were then identified using a post hoc least significant difference test. One-way ANOVAs were used to compare the body weights, organs weights, cellular proliferation, DNA and protein concentrations and content, whereas Student's unpaired t-tests compared SP mRNA levels, differences in the proportions of type I, type II, and intermediate AEC types between 0 cmH2O PEEP and 10 cmH2O PEEP groups (differences between each cell type were not compared). The total integrated density of each SP-A, SP-B (the density of the two SP-B transcripts were summed), and SP-C transcript on the Northern blot was divided by the total integrated density of the 18S rRNA band for that sample (lane) to account for minor differences in total RNA loading between lanes. As a result, the band densities are presented as a ratio of the 18S rRNA band density, with the increased lung expansion group (10 PEEP) expressed as a percentage of the mean value obtained from the control (0 PEEP) group of lambs.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lambs were born from twin pregnancies at term (145 ± 1 days) with a mean birth weight of 3.45 ± 0.1 kg. No differences were detected in arterial pH, PaO2, or SaO2 between animals in either of the ventilated groups for the duration of the experiment. However, animals in the 10 cmH2O PEEP group experienced marginally higher PaCO2 levels for the last 10 h (0 PEEP: 31.8 ± 0.9 mmHg vs. 10 PEEP: 40.9 ± 0.8 mmHg) and demonstrated a lower AaDO2 between 8 and 12 h (0 PEEP: 25.6 ± 1.4 vs. 10 PEEP: 10.3 ± 0.9 mmHg) and 25 and 48 h (0 PEEP: 34.5 ± 0.7 vs. 10 PEEP: 14.9 ± 0.5 mmHg) of the ventilation period; a lower AaDO2 is indicative of improved oxygenation. As expected, peak inspiratory pressures (PIP) and mean airway pressures (Paw) were higher in the 10 cmH2O PEEP group than control (PIP: 20.45 ± 0.45 vs. 14.3 ± 1.8 cmH2O; Paw: 13.2 ± 0.03 vs. 4.2 ± 0.02 cmH2O, respectively) (Fig. 1), but tidal volume and minute ventilation were not different between groups. Although the mean heart rate decreased over the 48-h period [from 194 ± 14 and 185 ± 8 beats per minute (bpm) to 164 ± 5 and 165 ± 5 bpm after 48 h in 0 PEEP and 10 PEEP lambs, respectively], there was no difference between the groups. Similarly, urine output was not different between animals ventilated with 0 cmH2O or 10 cmH2O PEEP (15.8 ± 2.8 ml·kg–1·h–1 vs. 14.1 ± 1.2 ml·kg–1·h–1, respectively).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Mean (±SE) peak inspiratory pressures and mean airway pressures in lambs ventilated with 0 (open symbols) or 10 cmH2O (solid symbols) positive end-expiratory pressure (PEEP). *Values in 10 PEEP group are significantly different to those from 0 PEEP lambs.

 
Body and lung weights. At postmortem, the body weights of nonventilated control lambs and those exposed to either 0 cmH2O or 10 cmH2O PEEP were similar (Table 1). There was no difference in the wet (2-wk control: 165.9 ± 19.0 g; 0 PEEP: 183.4 ± 30.8 g; 10 PEEP: 171.8 ± 15.8 g) and dry (2-wk control: 32.7 ± 3.8 g; 0 PEEP: 28.1 ± 2.7 g; 10 PEEP: 28.7 ± 2.9 g) lung weights between controls and lambs exposed to 0 and 10 cmH2O PEEP. Similarly, when expressed as a percentage of body weight, wet and dry lung weights were not different between groups (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Body weights (kg) and wet and dry lung weights expressed as a proportion of body weight (g/kg) of lambs ventilated for 48 h with a PEEP of either 0 or 10 cmH2O

 
Lung protein content and concentration. There was no difference in the protein concentration or content of lung tissue collected from nonventilated control lambs (content: 3,674.4 ± 808.57 mg/kg; concentration: 137.9 ± 12.03 mg/g) or lambs exposed to either 0 (content: 2,371.0 ± 259.8 mg/kg; concentration: 91.4 ± 9.6 mg/g) or 10 cmH2O PEEP (content: 3,100.8 ± 306.7 mg/kg; concentration: 113.7 ± 9.7 mg/g) (Fig. 2).


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Mean (±SE) lung protein and DNA concentration (mg/g lung tissue; A and C, respectively) and content (mg/kg body wt; B and D, respectively) in nonventilated lambs (open bars) and those ventilated with 0 (hatched bars) or 10 cmH2O (solid bars) PEEP. Non-vent cont, nonventilated control.

 
Lung DNA content and concentration. There was no difference in the DNA concentration or content of lung tissue collected from nonventilated control lambs (content: 83.3 ± 8.0 mg/kg; concentration: 3.6 ± 0.8 mg/g; n = 2) or lambs exposed to either 0 (content: 104.6 ± 7.2 mg/kg; concentration: 4.0 ± 0.2 mg/g) or 10 cmH2O PEEP (content: 105.1 ± 8.0 mg/kg; concentration: 3.8 ± 0.2 mg/g) (Fig. 2).

Lung cell proliferation. Expressed as a proportion of the total number of cells counted, the percentage of proliferating cells stained positive with Ki67 was significantly higher in lambs exposed to 10 cmH2O PEEP (3.5 ± 0.3%) compared with that in nonventilated control lambs (2.6 ± 0.5%) and lambs exposed to 0 cmH2O PEEP (1.9 ± 0.3%). The proportion of proliferating cells was not different between nonventilated control lambs and those ventilated with 0 cmH2O PEEP (Fig. 3).


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 3. A: mean (±SE) percentage of proliferating cells in nonventilated control lambs (open bar) or those ventilated with 0 (hatched bar) or 10 cmH2O (solid bar) PEEP. Values that do not share a common letter are significantly different (P < 0.05) from each other. B–D: representative light micrographs taken from lung tissue of nonventilated lambs and lambs ventilated with 0 or 10 cmH2O PEEP, respectively. Proliferating cells appear brown in color as they are positive for Ki67 immunolabeling.

 
AEC Differentiation

Proportion of each AEC phenotype. The proportions of type I (0 PEEP 54.8 ± 3.6%; 10 PEEP 54.8 ± 4.6%), type II (0 PEEP 43.2 ± 3.1%; 10 PEEP 45.5 ± 1.3%), and intermediate AECs (0 PEEP 2.0 ± 0.8%; 10 PEEP 2.6 ± 0.8%) were similar in lambs ventilated with 10 cmH2O PEEP and lambs ventilated with 0 cmH2O PEEP (Fig. 4). Too few undifferentiated AECs were observed at this stage for statistical comparison (<1% in both experimental groups).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. The relative proportions of type I (A), type II (B), and intermediate (C) alveolar epithelial cells (AECs) in lung tissue collected from lambs ventilated with 0 (open bars) or 10 cmH2O (solid bars) PEEP, respectively. AEC proportions were not different between groups.

 
Surfactant protein gene expression. In lambs exposed to 10 cmH2O PEEP for 48 h, lung SP-A (10 PEEP 158.1 ± 29.2% vs. 0 PEEP 100.0 ± 9.1%), SP-B (10 PEEP 124.0 ± 13.1% vs. 0 PEEP 100.0 ± 6.2%), and SP-C (10 PEEP 148.5 ± 22.5% vs. 0 PEEP 100.0 ± 8.6%) mRNA levels tended to be increased compared with lambs exposed to 0 cmH2O PEEP (Fig. 5). However, the differences observed between the two groups were not statistically significant (P = 0.119, 0.151, and 0.099, respectively).


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 5. Surfactant protein A (SP-A) (A), SP-B (B), and SP-C (C) mRNA levels in lung tissue collected from lambs ventilated with 0 (open bars) or 10 cmH2O (solid bars) PEEP. Values are presented as a percentage of control (0 PEEP) values. No statistically significant differences were found between groups.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The basal level of lung expansion is a potent stimulus for growth and development of the fetal lung (1, 20, 22, 23) and has profound effects on the phenotype of AECs (1, 8, 9, 11). Increased lung expansion in the fetus stimulates an increase in pulmonary DNA content, DNA synthesis rates, lung weights, and protein contents and stimulates the transdifferentiation of type II AECs into type I AECs. As a result, the proportion of type II cells decreases from ~30% to <2% of the total number of AECs after 10 days (8). This decrease in the proportion of type II AECs is likely to account for the marked reduction in the expression of the surfactant proteins following an increase in fetal lung expansion (11, 18). In view of these previous findings, we hypothesized that increased levels of lung expansion after birth would 1) increase postnatal lung growth and 2) alter AEC proportions by increasing type I AECs and decreasing type II AECs. Our results are consistent with the first hypothesis and show that the application of PEEP for 48 h stimulated an increase in cellular proliferation rates in 2-wk-old lambs. In contrast, following exposure to increased postnatal lung expansion, the proportion of each AEC phenotype was not altered, and surfactant protein gene expression was not reduced. This study demonstrates that increasing end-expiratory lung volumes by applying a PEEP of 10 cmH2O promotes lung growth but does not alter AEC differentiation or surfactant protein gene expression in 2-wk-old lambs.

In this study, we used neonatal ventilators to deliver a guaranteed tidal volume (Vt) of 7.5 ml/kg, as we have previously shown that the Vt in healthy newborn lambs is ~7.5 ml/kg (6); this Vt is also the most effective for ventilating preterm lambs (26). We chose to use a set Vt to ensure that the degree of phasic lung expansion associated with tidal breathing was identical between groups. Therefore, any differences observed could only be attributed to differences in the basal level of lung expansion experienced. Using this protocol, we successfully ventilated lambs for 48 h, maintaining their blood gas values within targeted physiological ranges considered normal for 2-wk-old lambs (6). Although the PaCO2 and AaDO2 were slightly higher over the last 10 h in lambs ventilated at 10 cmH2O PEEP, we consider it unlikely that these small changes could have affected lung growth rates, type II AEC proportions, and surfactant protein mRNA levels.

Although lung weight and DNA and protein contents were not altered, cell proliferation rates were significantly elevated by ventilation for 48 h with 10 cmH2O PEEP. These data suggest that ventilation with an increase in basal lung expansion by 10 cmH2O PEEP can accelerate lung growth after birth and are consistent with previous findings demonstrating that prolonged increases in basal lung expansion stimulate lung growth both before and after birth (1, 17, 22, 32). It is interesting to note that the increase in DNA synthesis rates induced by 48 h of 10 cmH2O PEEP (~35% increase above nonventilated controls and 77% increase above lambs ventilated with 0 cmH2O PEEP) in lambs is less than that which occurs after 48 h of tracheal obstruction (TO) in late gestation fetal sheep (~800% increase; Ref. 22). Although it is difficult to compare between experiments due to differences in lung maturity, the increase in basal lung expansion, and the method of measuring cell proliferation rates, it is likely that the growth response is more rapid in response to PEEP. This is because the application of PEEP causes an immediate stepwise increase in basal lung expansion, whereas it takes many hours for the secreted liquid to accumulate and increase basal lung expansion following TO (22). Thus it is possible that there was a much greater increase in lung cell proliferation rates at an earlier time point than was measured in this experiment.

Although newborn infants are thought to generate an intrinsic PEEP through expiratory breaking to maintain or increase functional residual capacity, the available evidence in lambs demonstrates that expiratory laryngeal adductor activity only occurs during non-rapid eye movement sleep and leads to an increase in expiratory time and a reduction in respiration rate (14); we have observed similar patterns in newborn rabbit pups (unpublished observations). Indeed, increases in tracheal pressure only occur during adductor activation, which does not persist throughout expiration (14), suggesting that 2-wk-old lambs tend not to generate an intrinsic PEEP. For this reason, we chose to use a PEEP of zero in our ventilated control animals, as we believe that this most accurately represents the situation in control lambs. However, to address the issue of intubation and ventilation per se on postnatal lung growth, we included a nonventilated, nonintubated control group in our analyses and found no difference between these control groups. Thus the effect of 10 cmH2O of PEEP on postnatal lung growth is most likely due to increased basal lung expansion.

Although lung weight was not increased following 48 h of ventilation with PEEP, the application of CPAP to newborn ferret pups for 2 wk increased total lung weight (32). Because increasing airway pressure can markedly reduce pulmonary blood flow (PBF) and increase pulmonary vascular resistance (12, 25), it is also possible that the increase in lung weight observed resulted from pulmonary edema. However, in our study we observed similar wet and dry lung weights in lambs exposed to 0 and 10 cmH20 PEEP, indicating that 48 h of PEEP did not induce pulmonary edema.

A second hypothesis of this study was that an increase in postnatal lung expansion, caused by ventilating with 10 cmH2O PEEP, would stimulate type II-to-type I AEC transdifferentiation, leading to a decrease in both type II cell proportions and SP-A, SP-B, and SP-C mRNA levels. Contrary to our hypothesis, we found that 10 cmH2O PEEP did not alter the proportions of type I or type II AECs. Furthermore, we found that SP-A, SP-B, and SP-C mRNA levels tended to increase in lambs ventilated with 10 cmH2O PEEP for 48 h, although this increase was not quite significant. These results demonstrate that the relationship between lung expansion and surfactant protein gene expression in the liquid-filled fetal lung is very different compared with the air-filled lung after birth.

There are several explanations that may account for the inability to induce AEC transdifferentiation and a reduction in surfactant protein gene expression in response to 48 h of ventilation with a PEEP of 10 cmH2O. It is possible that 48 h of PEEP was insufficient time to stimulate changes in AEC phenotypes and surfactant protein gene expression. However, we believe this explanation to be unlikely as previous studies have shown that 5–10 h of ventilation using 3 cmH2O PEEP alters mRNA levels for SP-A and SP-B in preterm lambs (31). Furthermore, in the late gestation fetus, 48 h of TO is sufficient to markedly reduce surfactant protein gene expression (18) and type II cell proportions, and, as indicated above, this stimulus is only applied gradually over the first 24 h (22). Alternatively, it is likely that the mechanical forces imposed on AECs are much more complex in an air-filled lung with an air/liquid interface than they are in a liquid-filled fetal lung. Indeed, the transmission of force onto the alveolar epithelium caused by an increase in internal distending pressure may be markedly less in an air-filled lung due to the presence of surface tension, which would oppose the distending influence of the applied PEEP. As a result, it is possible that significantly less strain is applied to individual epithelial cells for a given increase in basal luminal pressure. Thus a much greater increase in PEEP may be required to influence AEC phenotypes and surfactant protein gene expression in the air-filled lung after term birth. However, as PEEP levels in excess of 10 cmH2O are rarely used in neonatal ventilation, further increases in PEEP would have little clinical relevance.

It is also possible that the lack of an effect of PEEP on AEC phenotypes and surfactant protein gene expression may relate to differences in the type of stretch applied to the postnatal lung compared with the fetal lung in utero. During fetal life, the lung is maintained in a distended state by the accumulation of lung liquid within the future airways (13, 17). These volumes are relatively static and undergo little change in response to individual fetal breathing movements that are essentially isovolumetric (<0.1 ml/kg). This is because the resistance to the movement of large volumes (e.g., equivalent to postnatal Vt) of lung liquid with each movement is too great for the very compliant fetal chest wall (14). As a result, sections of the fetal chest wall partially collapse with each contraction of the diaphragm, leading to little change in lung volume (15). However, after birth, phasic expansion of the lung markedly increases with the onset of air breathing, with Vt increasing to ~7 ml/kg. Previous in vitro studies have shown that phasic stretch stimulates DNA synthesis rates in type II cells (24). Thus increased type II AEC proliferation may account for our inability to detect a decrease in type II AEC proportions. Indeed, many type II AECs were observed in close association with neighboring type II AECs when viewed under TEM, which is not common in the fetal lung. In addition, it is also well established that phasic stretch stimulates SP-C gene expression in cultured type II AECs (21). Thus it is possible that increased phasic stretch associated with ventilation modifies the effect of constant stretch on AEC proliferation, differentiation, and surfactant protein gene expression in vivo. For this reason, we administered a set Vt in both groups of lambs; however, the application of 10 cmH2O PEEP caused these lambs to be ventilated within a different region of the pressure volume curve compared with control lambs. The specific effect of this on surfactant protein mRNA levels and AEC differentiation is unknown.

Although PEEP is widely used in neonatal ventilation to improve oxygenation and prevent atelectatic lung injury in preterm infants, the findings of this study indicate that ventilation with PEEP has additional beneficial effects on the neonatal lung. Indeed, we have demonstrated that 10 cmH2O of PEEP can stimulate significant lung growth in the absence of type II AEC depletion or a decrease in surfactant protein gene expression. However, this level of PEEP markedly reduces PBF, increases pulmonary vascular resistance, and increases shunting through the ductus arteriosus in prematurely delivered lambs (25). The effect of 10 cmH2O PEEP on PBF in lambs used in this study is unknown, although it is likely to have been reduced. However, no effect on pulmonary edema, cardiac output (as indicated by heart rate and arterial pressure), and oxygenation could be detected. Furthermore, it is unlikely that the effect of PEEP on lung growth and cardiopulmonary physiology after birth in normal lambs with compliant lungs can be directly related to very preterm lambs with incompliant lungs. Thus it would be of interest to ascertain whether similar postnatal effects as those observed in this study occur in the less mature lung of very preterm lambs.

In conclusion, this study has provided strong evidence that increased levels of basal lung expansion, induced by the application of 10 cmH2O of PEEP, stimulates lung growth as indicated by increased cell proliferation. Our findings also demonstrate that the acceleration in lung growth was not achieved at the expense of detrimental changes in AEC proportions or surfactant protein gene expression that are commonly observed following increased lung expansion in the fetus. Thus this study provides a new understanding that highlights the potential benefits of ventilating term newborn infants with a positive distending pressure that maintains lung expansion at end-expiration.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Health and Medical Research Council of Australia.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the surgical assistance of Megan Cock and Alex Satragno and the expert technical assistance of Alison Thiel and Valerie Zahra.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Flecknoe, Dept. of Physiology, PO Box 13F, Monash Univ., VIC 3800, Australia (e-mail: sharon.flecknoe{at}med.monash.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. J. Flecknoe and K. J. Crossley share equally as first authors of this work. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123: 649–660, 1977.[ISI][Medline]
  2. Alcorn DG, Adamson TM, Maloney JE, Robinson PM. A morphologic and morphometric analysis of fetal lung development in the sheep. Anat Rec 201: 655–667, 1981.[CrossRef][Medline]
  3. Avery ME, Cook CD. Volume-pressure relationships of lungs and thorax in fetal, newborn, and adult goats. J Appl Physiol 16: 1034–1038, 1961.[Abstract/Free Full Text]
  4. Buhain WJ, Brody JS. Compensatory growth of the lung following pneumonectomy. J Appl Physiol 35: 898–902, 1973.[Free Full Text]
  5. Crapo JD, Barry BE, Gehr P, Bachofen M, Weibel ER. Cell number and cell characteristics of the normal human lung. Am Rev Respir Dis 126: 332–337, 1982.[ISI][Medline]
  6. Davey MG, Johns DP, Harding R. Postnatal development of respiratory function in lambs studied serially between birth and 8 weeks. Respir Physiol 113: 83–93, 1998.[CrossRef][ISI][Medline]
  7. Endl E, Gerdes J. The Ki-67 protein: fascinating forms and an unknown function. Exp Cell Res 257: 231–237, 2000.[CrossRef][ISI][Medline]
  8. Flecknoe S, Harding R, Maritz G, Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol 278: L1180–L1185, 2000.[Abstract/Free Full Text]
  9. Flecknoe SJ, Boland RE, Wallace MJ, Harding R, Hooper SB. Regulation of alveolar epithelial cell phenotypes in fetal sheep: roles of cortisol and lung expansion. Am J Physiol Lung Cell Mol Physiol 287: L1207–L1214, 2004.[Abstract/Free Full Text]
  10. Flecknoe SJ, Wallace MJ, Cock ML, Harding R, Hooper SB. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol 285: L664–L670, 2003.[Abstract/Free Full Text]
  11. Flecknoe SJ, Wallace MJ, Harding R, Hooper SB. Determination of alveolar epithelial cell phenotypes in fetal sheep: evidence for the involvement of basal lung expansion. J Physiol 542: 245–253, 2002.[Abstract/Free Full Text]
  12. Fuhrman BP, Smith-Wright DL, Kulik TJ, Lock JE. Effects of static and fluctuating airway pressure on intact pulmonary circulation. J Appl Physiol 60: 114–122, 1986.[Abstract/Free Full Text]
  13. Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol 81: 209–224, 1996.[Abstract/Free Full Text]
  14. Harding R, Johnson P, McClelland ME. Respiratory function of the larynx in developing sheep and the influence of sleep state. Respir Physiol 40: 165–179, 1980.[CrossRef][ISI][Medline]
  15. Harding R, Liggins GC. Changes in thoracic dimensions induced by breathing movements in fetal sheep. Reprod Fertil Dev 8: 117–124, 1996.[CrossRef][Medline]
  16. Hooper SB, Han VKM, Harding R. Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep. Am J Physiol Lung Cell Mol Physiol 265: L403–L409, 1993.[Abstract/Free Full Text]
  17. Hooper SB, Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22: 235–247, 1995.[ISI][Medline]
  18. Lines A, Nardo L, Phillips ID, Possmayer F, Hooper SB. Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol 276: L239–L245, 1999.[Abstract/Free Full Text]
  19. Lines AL, Davey MG, Harding R, Hooper SB. Effect of increased lung expansion on surfactant protein mRNA levels in lambs. Pediatr Res 50: 720–725, 2001.[ISI][Medline]
  20. Moessinger AC, Harding R, Adamson TM, Singh M, Kiu GT. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86: 1270–1277, 1990.[ISI][Medline]
  21. Nakamura T, Liu M, Mourgeon E, Slutsky A, Post M. Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression. Am J Physiol Lung Cell Mol Physiol 278: L974–L980, 2000.[Abstract/Free Full Text]
  22. Nardo L, Hooper SB, Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res 43: 184–190, 1998.[ISI][Medline]
  23. Nardo L, Maritz G, Harding R, Hooper SB. Changes in lung structure and cellular division induced by tracheal obstruction in fetal sheep. Exp Lung Res 26: 105–119, 2000.[CrossRef][ISI][Medline]
  24. Pasternack M Jr, Liu X, Goodman RA, Rannels DE. Regulated stimulation of epithelial cell DNA synthesis by fibroblast-derived mediators. Am J Physiol Lung Cell Mol Physiol 272: L619–L630, 1997.[Abstract/Free Full Text]
  25. Polglase GR, Morley CJ, Crossley KJ, Dargaville P, Harding R, Morgan DL, Hooper SB. Positive end-expiratory pressure differentially alters pulmonary hemodynamics and oxygenation in ventilated, very premature lambs. J Appl Physiol 99: 1453–1461, 2005.[Abstract/Free Full Text]
  26. Probyn ME, Hooper SB, Dargaville PA, McCallion N, Crossley K, Harding R, Morley CJ. Positive end expiratory pressure during resuscitation of premature lambs rapidly improves blood gases without adversely affecting arterial pressure. Pediatr Res 56: 198–204, 2004.[CrossRef][ISI][Medline]
  27. Rannels DE. Role of physical forces in compensatory growth of the lung. Am J Physiol Lung Cell Mol Physiol 257: L179–L189, 1989.[Abstract/Free Full Text]
  28. Rannels DE, Rannels SR. Compensatory growth of the lung following partial pneumonectomy. Exp Lung Res 14: 157–182, 1988.[ISI][Medline]
  29. Rannels DE, White DM, Watkins CA. Rapidity of compensatory lung growth following pneumnoectomy in adult rats. J Appl Physiol 46: 326–333, 1979.[Abstract/Free Full Text]
  30. Watkins CA, Burkhart LR, Rannels DE. Lung growth in response to unilateral pneumonectomy in rapidly growing rats. Am J Physiol Endocrinol Metab 248: E162–E169, 1985.[Abstract/Free Full Text]
  31. Woods E, Ohashi T, Polk D, Ikegami M, Ueda T, Jobe AH. Surfactant treatment and ventilation effects on surfactant SP-A, SP-B, and SP-C mRNA levels in preterm lamb lungs. Am J Physiol Lung Cell Mol Physiol 269: L209–L214, 1995.[Abstract/Free Full Text]
  32. Zhang S, Garbutt V, McBride JT. Strain-induced growth of the immature lung. J Appl Physiol 81: 1471–1476, 1996.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/L454    most recent
00095.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flecknoe, S. J.
Right arrow Articles by Hooper, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flecknoe, S. J.
Right arrow Articles by Hooper, S. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.