Apoptosis in Neonatal Murine Lung Exposed to Hyperoxia
Sharon A. McGrath-Morrow and Jennifer Stahl
Department of Pediatrics, Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins Medical Institutions, Baltimore, Maryland
Exposure to high concentrations of oxygen in the neonatal
period may impair lung growth and is a major contributing
factor to the development of bronchopulmonary dysplasia.
Cell death from hyperoxic injury may occur through either an
apoptotic or nonapoptotic pathway, and we were interested
in determining the type of cell death that occurs in the lung of
neonatal mice exposed to hyperoxia. We found increased levels of Bax messenger RNA, a gene associated with apoptosis,
in the lungs of neonatal mice born and raised in 92% hyperoxia. We next determined the extent of apoptosis taking place
in the lungs of neonatal mice exposed to hyperoxia using
terminal deoxyribonucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling in 3.5-, 4.5-, and
5.5-d-old neonatal lung. The number of apoptotic cells in peripheral lung was significantly higher in the 3.5-, 4.5-, and 5.5d-old mice treated with oxygen compared with that in the
room-air control mice. Further, the number of apoptotic cells
in the lung increased with longer exposure duration. In murine lung bronchus cells exposed to hyperoxia, growth arrest
occurred after 48 h of oxygen exposure. Using annexin V
binding, necrotic cell death was found to be the major form of
cell death in these cells after 72 h of hyperoxic exposure. We
conclude that 92% hyperoxia causes significant lung injury in
neonatal mice exposed to hyperoxia, and that the number of
apoptotic cells in the lung increases the longer the duration of
exposure. The increase in apoptosis from hyperoxic exposure
during a critical period of lung development may be an important factor in the impaired lung growth and remodeling that
occur in animals exposed to high oxygen concentrations. Finally, it appears that hyperoxic injured cells in neonatal lung
undergo both apoptotic and nonapoptotic cell death.
Infants with respiratory distress syndrome are frequently
given high concentrations of oxygen to maintain adequate
tissue oxygenation. Exposure to hyperoxia in the neonatal
period, however, is a major contributing factor to the
development of bronchopulmonary dysplasia (BPD) (1).
BPD is characterized by impaired lung growth, airway inflammation, and increased airway resistance. Histologically, exposure to hyperoxia can lead to a decreased number of alveoli and a reduction in lung surface area (2).
Neonatal mice and rats exposed to high concentrations of
oxygen have been shown to develop large terminal air
spaces and decreased numbers of septa, similar to lesions
that are present in the lungs of infants with BPD (3–5). Because the majority of lung growth in children occurs in the
first 2 yr of life, exposure to high levels of oxygen can af(Received in original form September 8, 2000 and in revised form March
15, 2001)
Address correspondence to: Dr. Sharon McGrath-Morrow, Dept. of Pediatric Pulmonary, Johns Hopkins Hospital, Park 316 N. Wolfe St., Baltimore, MD 21287-2533. E-mail: [email protected]
Abbreviations: bronchopulmonary dysplasia, BPD; fluorescein isothiocyanate, FITC; messenger RNA, mRNA; sodium dodecyl sulfate, SDS;
deoxyribonucleotidyl transferase–mediated deoxyuridine triphosphate–
biotin nick-end labeling, TUNEL.
Am. J. Respir. Cell Mol. Biol. Vol. 25, pp. 150–155, 2001
Internet address: www.atsjournals.org
fect ultimate lung growth and function (6). We were interested in determining whether apoptosis was the predominant form of cell death in neonatal lung subjected to
hyperoxic stress. During both normal prenatal and postnatal lung development, apoptosis has been shown to be involved in lung remodeling in both rat lung and human fetal lung explants (7, 8). Prenatally, mesenchymal cells have
been shown to undergo apoptosis, presumably as a mechanism to thin the septa and establish an adequate pulmonary alveolar–capillary interface (7). Postnatally, apoptosis appears to be involved in ridding the lung of excess
numbers of fibroblasts and type 2 epithelial cells to increase lung surface area (8). Apoptosis requires the activation of the caspases, a family of cytosolic proteases. During
apoptosis Bax, a proapoptotic gene, releases cytochrome c
from the mitochondria, which in turn induces caspase activation (9). Morphologically, the cell undergoes a series of
changes before death, including cell shrinkage, chromatin
condensation, active membrane blebbing, and the formation of apoptotic bodies. In contrast to necrosis or nonapoptotic cell death, apoptosis is believed to occur in the absence of inflammation (10). It is not known whether apoptosis
is in part responsible for the impaired lung growth found
in developing lung exposed to hyperoxia. Previous studies
in adult mice have shown that apoptosis is significantly increased in the lungs of mice exposed to hyperoxia and is
associated with increased mortality (11, 12). Ward and colleagues found that transgenic mice that overexpress interleukin (IL)-6 had increased survival (12). In their model,
IL-6 protected lung from the effect of hyperoxia and this
was associated with a decrease in apoptotic cell number in
the lung. They also found that the transgene-positive mice
had an increase in Bcl-2 protein. Others have found that
Bcl-2 and Bcl-xL are able to block the cytochrome c mitochondrial release induced by Bax (9). Neonatal mice have
previously been shown to be more resistant to hyperoxic
stress (13). This was partially secondary to an increase in
lung antioxidant enzyme activity and a difference in chemokine response to hyperoxic stress when compared with
adult animals (13). Also, the form of cell death that neonatal murine lungs undergo in response to hyperoxic stress
may influence survival in the neonatal mouse; for example, apoptosis versus a nonapoptotic cell death. In the
present study we were interested in determining whether
apoptosis was a primary mechanism of cell death in the
lungs of newborn mice exposed to hyperoxic stress. Therefore, we studied the effect of hyperoxia on the lungs of
newborn mice in an effort to understand the role of apoptosis during hyperoxic stress in the neonate.
We previously found that 3.5-d-old CD-1 mice raised in
92% oxygen had impaired lung growth, as demonstrated
by a 3-fold decrease in bromodeoxyuridine uptake (14).
Using this model, we isolated lung sections from mice
McGrath-Morrow and Stahl: Apoptosis in Hyperoxic Exposed Murine Lung
raised in 92% oxygen for 3.5, 4.5, and 5.5 d, and compared
these with the lungs of mice raised in room air. Apoptotic
cells in the lung were identified in situ using terminal deoxyribonucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling (TUNEL).
Materials and Methods
RNA Extraction and Northern Blot Analysis
Timed pregnant CD-1 mice (Charles River, Wilmington, MA) were
placed in a hyperoxic chamber at 18.5 d of gestation. Mice were
born into hyperoxic conditions (92% FiO 2) the following morning. Mothers were rotated every 12 to 24 h to prevent death from
acute oxygen toxicity. These experiments were done according to
the animal protocol approved by the Animal Care Use Committee of the Johns Hopkins University School of Medicine. Lung
tissue was harvested at specific time points from both hyperoxicexposed and age-matched control neonatal mice raised in room
air. RNA was isolated by the RNAzol method. Total RNA was
used for Northern blot analysis.
The quantity of 15 to 20 g of total RNA was run on a 1%
agarose/formaldehyde gel (equal amounts were loaded per blot),
transferred to a filter (genescreen plus; Dupont, Boston, MA), and
baked for 2 h at 80C in a vacuum oven. Murine Bax (ATTC clone)
and -actin (gift of Dr. S.-J. Lee, Johns Hopkins University) complementary DNA clones were labeled with 32P and random primed
as previously described (15). Blots were hybridized with the solution of 5 saline sodium phosphate ethylenediamenetetraacetic
acid; 10% dextran sulfate; 50% formamide; 1% sodium dodecyl
sulfate (SDS); 200 mg/ml salmon DNA; and 0.1% each of bovine
serum albumin, ficoll, and polyvinylpyrrolidone, at 42 C overnight; then washed in 0.1% SDS and 0.2% saline sodium citrate
five times for 20 min each time at 55 C. RNA signals were quantified using a phosphorimager program (ImageQuant v3.2).
Western Blot Analysis
Whole-cell lysates were solubilized in 2% SDS. Protein concentrations were determined using the Biorad DC protein assay kit (BioRad Laboratories, Hercules, CA). Lysates were loaded and run on
a 12% SDS–polyacrylamide gel. Each protein gel contained equal
amounts of protein per lane (20 g/lane). Gels were then transferred to nitrocellulose and Western blot analysis was performed
using an anti-Bax monoclonal antibody (sc-7480; Santa Cruz Biotechnologies, Santa Cruz, CA) at 1:100 dilution in 5% blotto, phosphate-buffered saline (PBS) and .05% Tween overnight at 4 C.
The blots were then washed three times in PBS-Tween, incubated
with a mouse immunoglobin, horseradish peroxidase–linked whole
antibody (RPN 2108; Amersham, Arlington Heights, IL) for 1 h at
a 1:500 dilution, then washed and developed using enhanced chemiluminescence (RPN 2106; Amersham).
Cell Lines
Murine lung bronchus cells were obtained from American Type
Culture Collection (#CRL-6382; Rockville, MD) and grown to
50% confluence. The cell line is from normal murine lung and
bronchus. The cells were grown in Dulbecco’s modified Eagle’s
medium (GIBCO BRL Life Technologies, Rockville, MD) with
10% fetal bovine serum, penicillin /streptomycin, and 4 mM
L-glutamine. The cells were seeded at 110,000 cells per 25-mm
flask. Cells were either placed in a modular incubator chamber
(Billups-Rothenberg, Del Mar, CA) and exposed to 95% O 2 and
5% CO2, or placed in room air and 5% CO 2. Cells were gassed
every day and media were changed daily. Cells were then har-
151
vested at specific time periods for counting, protein, and RNA
extraction for Western and Northern blot analysis.
Assays for Apoptosis
The lungs of neonatal mice were perfused with 4% paraformaldehyde through the trachea until the lungs were fully distended,
then placed in 4% paraformaldehyde overnight, dehydrated in
ethanol, paraffin-embedded, and cut into 10- m sections. Apoptosis was determined by in situ labeling using terminal deoxynucleotidyl transferase (In situ cell death detection kit, fluorescein;
Boehringer Mannheim, Indianapolis, IN; catalog #1-684-795). Cells
were then counterstained with 4,6-diamidino-2-phenylindole (DAPI)
to identify the nuclei. Fluorescein isothiocyanate (FITC)–labeled
apoptotic cells were detected under fluorescent microscopy. Under 40 magnification, three areas of lung parenchyma were systematically and randomly photographed from each animal. Murine lung bronchus cells were grown on glass coverslips and kept
in room air and 5% CO2 or exposed to 95% O2 and 5% CO2 for
72 h. The cells were then stained for apoptosis using an Oncor apoptag peroxidase kit (S7100-kit; Oncor, Gaithersburg, MD). Murine lung bronchus cells were grown in T25 flasks and left in
room air and 5% CO2 or 95% O2 and 5% CO2 for 72 h. Cells
were then counted and annexin V binding was determined using
TACS Annexin V–FITC (catalog #TA4638; R&D Systems, Minneapolis, MN), following the manufacturer’s protocol. Cells were
stained with propidium iodide and annexin V–FITC, which allowed determination of viable, early apoptotic, or late apoptotic/
necrotic cells.
Statistical Analysis
Statistical calculations were performed using the SPSS 8.0 statistical package for Windows (SPSS, Chicago, IL) and Microsoft
Excel 97 statistical package for Windows. Differences in measured variables between experimental and control groups were
determined using comparison of the means using one-way analysis of variance (ANOVA) statistical calculations and Student’s
t test (two-tailed). Statistical difference was accepted at P 0.05.
Results
Survival of Neonatal Mice in Hyperoxia
At 3.5 d of life, neonatal mice born and raised in 92% oxygen had a minimal mortality rate (range, 0 to 11%). Mortality within the litters, however, increased with each subsequent day of exposure to hyperoxia. At 5.5 d of life mice
exposed to hyperoxia had mortality rates between 14 and
34%, and at 6.5 d of life mice exposed to hyperoxia had
mortality rates ranging between 18 and 47%. Survival was
significantly higher in mice at 3.5 d of hyperoxia compared
with mice at 6.5 d of hyperoxia (P 0.03) (Figure 1).
We next weighed the mice and found that the weights
of mice born and raised in 92% hyperoxia were significantly lower at 4.5 d (O2, 2.35 g 0.5; room-air mice,
2.92 g 0.51; P 0.00002) and 5.5 d (O2, 2.67 g 0.64; room
air, 3.14 g 0.47; P 0.00003) when compared with mice
raised in room air (Figure 2). These findings are similar to
those of Northway and colleagues, who found a significant
difference in body weights between O2-treated and roomair mice after 120 h of 100% hyperoxia in C57BL/Ka neonatal mice (16).
We then allowed mice exposed to 92% hyperoxia for
6.5 d to recover in room air for 3 d. Their weights were
compared with the weights of mice raised in room air for
9.5 d. We found no significant difference in body weights
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 25 2001
Figure 1. Percentage of neonatal mice surviving in 92% hyperoxia. Mice were born into 92% hyperoxia. Each group consisted
of two litters that were fed by the same group of mothers. Average size of each group was 29 animals 2.6. The mortality rate at
6.5 d of hyperoxia was significantly greater than the mortality
rates at 2.5 and 3.5 d (†P .02, P .03, respectively). Comparisons of the means were done by one-way ANOVA; n 3.
between the two groups (6.5 d O2/3 d room air, 3.93 g .61; room air, 4.0 g 0.75; P .87).
Apoptosis in Neonatal Murine Lung Exposed to Hyperoxia
A TUNEL assay was performed to determine the number
of cells undergoing apoptosis in the lungs of neonatal mice
exposed to hyperoxia for 3.5, 4.5, and 5.5 d or room air.
From the lungs of each animal, FITC-labeled cells, representing apoptotic cells, were counted from three randomly
selected lung fields (Figure 3). An increase in the number
of cells undergoing apoptosis (apoptotic cells/lung field)
was found in the hyperoxic-treated group at 3.5 d (O2treated, 18.3 cells 11.7; room air, 4.9 cells 1.8; P .0002), 4.5 d (O2-treated, 18.2 cells 8.0; room air, 4.1 cells 2.8; P .00001), and 5.5 d of age (O2-treated, 30.3 cells 13.8; room air, 1.9 cells 1.3; P .00001) which was significantly greater than the number of apoptotic cells found
in the lungs of mice of the same age raised in room air
(Figure 4). The number of cells undergoing apoptosis increased the longer the exposure time to oxygen, with the
number of apoptotic cells in 3.5-d oxygen-treated lung being significantly less than the number of apoptotic cells in
5.5-d oxygen-treated lung (P .03).
In mice exposed to 92% oxygen for 6.5 d and allowed to
recover for 3 d in room air, no difference in the number of
apoptotic cells in the lungs was found compared with mice
raised in room air for 9.5 d (6.5 d O2/3 d room air, 2.3 cells 0.9; room air, 1.8 cells 0.8; P .2). We did, however, notice that the size of the alveolar air spaces in the mice
treated with oxygen and recovered in room air appeared
larger and less complex compared with mice raised in room
air, suggesting an inhibition of lung growth in the oxygentreated animals (Figure 5). This is similar to findings reported by Randell and associates, who showed, using morphometric measurements, that rats receiving 7 d of 95%
hyperoxia at birth had abnormally enlarged alveolar ducts
and decreased alveolar surface area at 40 d of age (17).
Bax Expression in Neonatal Murine Lung and Murine Lung
Bronchus Cells
Lung from mice treated with 92% oxygen was analyzed
for Bax messenger RNA (mRNA) expression and compared with room-air controls. The proapoptotic gene Bax
Figure 2. Comparison of body weights between neonatal mice in
hyperoxia and room air. Mice in both room air and hyperoxia
were weighed daily. At 4.5 d the average weight of the oxygentreated mice (2.35 g 0.5) was significantly less than that of the
room-air mice (2.92 g 0.51, *P .00002). At 5.5 d the average
weight of the oxygen-treated mice (2.67 g 0.64) was significantly less than that of the room-air mice (3.14 g 0.47; **P .00003). The 9.5-d time point represents mice exposed to 92% oxygen for 6.5 d and allowed to recover in room air for 3 d. There
was no significant difference in body weight between the oxygentreated/recovery group and the room-air group. Error bars represent standard error of the mean. At each time point from .5 to 5.5 d
the weights of each litter were averaged. Each time point had between two and eight litters. For the 9.5-d mice: O 2/recovery group
n 6, room air n 5.
has previously been shown to be induced during hyperoxic
injury in adult murine lung (11). We found that Bax
mRNA levels were induced in neonatal murine lung at as
early as 1.5 d of hyperoxic exposure, with levels increasing
2.5-fold above baseline by 4.5 d of hyperoxic exposure
(Figure 6a). After 2 d of room-air recovery, however, Bax
mRNA levels returned to baseline (Figure 6b). This suggests a correlation between increasing numbers of apoptotic cells and increasing mRNA levels of Bax during exposure to hyperoxia in neonatal lung.
We then exposed murine lung bronchus cells to 95% O2
and 5% CO2 and found that cells underwent growth arrest
by 48 h of hyperoxia (Figure 7a). We then evaluated Bax
protein levels in room air and hyperoxia. At 24 and 48 h,
Bax protein levels were increased in cells exposed to hyperoxia; however, at later time points Bax protein levels
appeared to decrease. In room-air cells Bax protein was
increased at later time points, possibly representing an increase in apoptosis from cell overgrowth (Figure 7b). Using a TUNEL assay, we found at 72 h a population of cells
from both room-air and hyperoxic-treated cells that were
apoptotic (Figure 8a). The majority of cells, however, did
not stain for apoptosis (data not shown). We next used annexin V binding to determine whether necrotic cell death
was the primary mode of cell death in oxygen-treated murine lung bronchus cells. Annexin V binding was done to
determine the percentage of viable, apoptotic, or necrotic/
late apoptotic cells in room-air and hyperoxic-treated
cells. The viability of room-air cells by annexin V binding
at 72 h was 60.4% 7.0%, and for O2-treated cells 29.1% 1.4% (P .002). In the oxygen-treated cells, 43.3% 18.5% of the cells were undergoing necrotic cell death by
72 h, in contrast to only 11.1% 6.1% of the room-air
cells (P .05) (Figure 8b).
McGrath-Morrow and Stahl: Apoptosis in Hyperoxic Exposed Murine Lung
Figure 3. Apoptotic cells in the peripheral airways of hyperoxicexposed neonatal mice. On the left, 10-m sections of paraffinembedded peripheral lung from newborn mice born and raised in
92% hyperoxia for either 3.5, 4.5, or 5.5 d; on the right, peripheral
lung sections from mice born and raised in room air for 3.5, 4.5,
or 5.5 d. Apoptotic cells are labeled with FITC (green) and nuclei
are labeled with DAPI (blue). The white arrows point to apoptotic cells in the peripheral lung. An increase in number of apoptotic cells is found in the hyperoxic-exposed lung.
153
Figure 4. Resolution of apoptosis in the lungs of neonatal mice
previously exposed to hyperoxia. Top panel: peripheral lung
from 9.5-d-old mouse raised in room air. Bottom panel: peripheral lung from 9.5-d-old mouse exposed to 92% hyperoxia for
4.5 d and allowed to recover for 3 d in room air. No increase in
apoptotic-labeled cells was found in the lung previously exposed
to hyperoxia compared with the room-air control lung. The alveolar air spaces of the previously O 2-treated lung appear larger
and less complexed than the room-air lung.
Discussion
In this study we found that high concentrations of oxygen
led to an increase in apoptosis in the peripheral lung of
neonatal mice. The number of cells undergoing apoptosis
increased the longer the exposure to oxygen, and the number of apoptotic cells correlated with the degree of lung injury. The increase in apoptosis during hyperoxia was associated with an induction of Bax mRNA expression. Although
it was not possible to identify specific cell types undergoing apoptosis, the apoptotic cells were primarily found along
the alveolar surface of the peripheral airways. We also
studied the effect of hyperoxia on murine lung bronchus
cells. These cells underwent both growth arrest and cell
death when exposed to high concentrations of oxygen. The
murine lung bronchus cells, however, exhibited primarily
necrotic cell death when exposed to 72 h of hyperoxia.
Previous studies have reported varying results when
they have examined the role of apoptosis during hyperoxic
exposure. In mature lung and in alveolar type 2 epithelial
cells grown in culture, exposure to hyperoxia has been
shown to induce apoptosis (11, 12, 18). Mantell and coworkers, using adult murine lung, found as we did that the
number of apoptotic cells in lung exposed to hyperoxia
correlated well with the extent of lung injury (19). Hyperoxic injury, however, may cause both apoptotic and necrotic cell death (19). We found that Bax mRNA levels in
neonatal lung increased and correlated with the number of
apoptotic cells in neonatal lung exposed to hyperoxia. These
observations, however, do not necessarily mean that apop-
Figure 5. Apoptosis in neonatal murine lung exposed to 92% hyperoxia and room air. Apoptotic cells, in neonatal murine lung
exposed to 92% hyperoxia and room air, were identified by in
situ labeling using TUNEL. TUNEL cells were counted from
three randomly selected areas of lung under fluorescent microscopy at 40 magnification. Significant increases in the number of
apoptotic-labeled cells were found at 3.5, 4.5, and 5.5 d in the
oxygen-treated lung compared with room-air murine lung (*P .0002, **P .00001, and ***P .00001). *9.5: 9.5-d oxygen-treated
mice were exposed to 92% O2 for 6.5 d and allowed to recover in
room air for 3 d. No significant difference in the number of apoptotic-labeled cells was found in the lungs of these mice compared
with room-air controls. Error bars represent standard deviations.
n 6 for 3.5-d O2-treated mice and 4.5- and 5.5-d room-air mice;
n 5 for 4.5- and 5.5-d O2-treated mice; n 4 for 3.5-d room-air
mice; and n 3 for 9.5-d-old mice.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 25 2001
Figure 7. Growth of murine lung bronchus cells in 95% hyperoxia and room air. (a) Growth of murine bronchus cells in room
air (filled diamonds) and 95% O2 and 5% CO2 (gray squares).
Cells in hyperoxia underwent growth arrest by 48 h of 95% oxygen. (b) Protein expression of Bax and p21 in murine lung bronchus cells exposed to hyperoxia and room air.
Figure 6. Induction of Bax mRNA levels in murine lung exposed
to 92% hyperoxia. (a) Total RNA from neonatal murine lung
was isolated from mice born and raised in either 92% hyperoxia
or room air, at different time points. Northern blot analysis was
performed using Bax and -actin probes. Levels of Bax mRNA
were normalized to -actin levels. By 4.5 d, mRNA levels of Bax
were increased 2.5-fold above room-air controls. (b) mRNA levels of Bax returned to baseline in the lungs of mice exposed to
4.5 d of hyperoxia and allowed to recover in room air for 2 d.
tosis is the primary cause of cell death in neonatal lung exposed to hyperoxia. The majority of cells in the peripheral
lung exposed to hyperoxia were not apoptotic by TUNEL.
Indeed, death by necrosis may be the major mode of cell
death caused by hyperoxia. This is supported by our findings in murine lung bronchus cells. We found that although some cells exposed to hyperoxia underwent apoptosis by TUNEL staining, the majority of cells were not
apoptotic. Bax protein levels were initially increased in
murine lung bronchus cells exposed to hyperoxia, however
protein levels appeared to decrease the longer the expo-
sure to oxygen. Further, as cells in room air grew to confluency, they had increases in Bax protein levels. The increase in Bax protein in the room-air cells may correlate
with apoptosis from cell overgrowth.
Using annexin V binding in the murine lung bronchus
cells, we found that the majority of cells were undergoing
necrotic cell death when exposed to hyperoxia for 72 h.
This suggests that, at least with longer exposure to high
concentrations of oxygen, necrosis is a major cause of cell
death in these cells. Kazzaz and colleagues also found that
A549 cells, a distal lung epithelial cell line, underwent necrotic cell death when exposed to hyperoxia (20). Therefore, it is likely that a combination of apoptosis and necrosis is occurring in neonatal lung exposed to high concentrations
of oxygen. Factors that may influence whether a cell undergoes apoptosis include adenosine triphosphate (ATP)
levels in the cell and the type of insult to the cell. It has
been shown that the level of cellular ATP can determine
the balance between apoptotic and necrotic cell death
(21). Allen and White found that cellular ATP levels were
decreased in A549 cells exposed to 95% hyperoxia and depleted of glucose (22).
Although we have shown that apoptosis is increased in
neonatal lung exposed to hyperoxia, it is not clear how important this is to lung development. An increase in apoptotic cells in the lung during development may affect ultimate growth of the lung, whereas an increase in apoptosis
in adult lung may not. Under normal circumstances, apoptosis is involved in remodeling of the lung prenatally and
postnatally (7). The timing of apoptosis in the development
of many organs has been shown to be important. In murine
lung, postnatal lung growth is delayed until after Day 2 of
life (16, 23). Maximal cell proliferation takes place at Day
4, then cell proliferation declines until Day 10 (23). Scanning electron microscopy studies of neonatal murine lung
have shown that an increase in alveolar number occurs until Day 14, at which time the lung begins to histologically
resemble the adult lung (24). It is possible that an increase
McGrath-Morrow and Stahl: Apoptosis in Hyperoxic Exposed Murine Lung
155
neonatal lung during a crucial period of lung development
may adversely affect future lung growth. Additional studies will be needed to evaluate this further.
Acknowledgments: This work was supported by NIH-KO8 award #HL03624
and an American Lung Association Research Grant to one author (S.A.M.).
References
Figure 8. Annexin V binding and apoptosis in murine lung bronchus cells. (a) Apoptotic cells in both hyperoxic and room-air
murine lung bronchus cells. Arrows point to apoptotic cells. Left
panel: cells grown in 95% O2 and 5% CO2 for 72 h. Right panel:
cells grown in room air and 5% CO 2 for 72 h. (b) Annexin V
binding in murine lung bronchus cells grown in 95% hyperoxia
and room air for 72 h. *P .002, **P .05, n 3.
in apoptosis from hyperoxia, however minimal, during this
critical phase of development may interfere with normal
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shown to have markedly decreased numbers of alveoli compared with age-matched controls (4). We observed in neonatal mice initially exposed to hyperoxia and then allowed
to recover in room air that their lungs had larger and less
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Nutritional status may also affect lung growth during
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