Lung Biology Laboratory, Departments of 1 Pediatrics and 2 Medicine, Georgetown University School of Medicine, Washington, District of Columbia 20057-1481; and 3 Institute de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, Strasbourg, France
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ABSTRACT |
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The formation of pulmonary alveoli in mice
and rats by subdivision of alveolar saccules that constitute the
newborn's gas-exchange region ends by approximately postnatal
day 14. However, alveoli continue to form after age 14 days
until age ~40 days by means other than septation of the saccules
present at birth. With the use of morphometric procedures and retinoic
acid receptor (RAR)-+/+ and RAR-
/
mice, we now show the volume of individual alveoli (
a), the number of alveoli (Na), and alveolar surface area (Sa) are the same in
14-day-old RAR-
+/+ and RAR-
/
mice.
However, at age 50 days,
a is larger, and Na and Sa are smaller,
in RAR-
/
than in RAR-
+/+ mice,
although total lung volume is the same in both groups. These findings,
and prior data showing RAR-
is an endogenous inhibitor of alveolus
formation during, but not after, the perinatal period, indicate there
are developmental period-specific regulators of alveolus formation and
that total lung volume and alveolar dimensions may have different regulators.
all-trans retinoic acid; retinoic acid receptor- null
mice; morphometry; lung volume
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ARTICLE |
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THE FORMATION OF PULMONARY ALVEOLI in mice
(1) and rats (4, 5), by subdivision of the
gas-exchange structures (alveolar saccules) present at birth, begins at
approximately postnatal day 3-4, is mainly over by
postnatal day 14 (5, 9, 10, 17), and where
quantitated (rats) is responsible for approximately one-third of
alveoli formed in the first two postnatal weeks (9, 10,
17). Alveolus formation by other than septation of alveolar saccules present at birth continues in mice (7) and rats
(2, 3, 9) after postnatal day 14 until
approximately age 5-6 wk. Alveolus formation by septation of the
alveolar saccules present at birth occurs throughout the lung (5,
9, 10, 17); its cessation, but the continued formation of
alveoli, suggests the molecular regulation of alveolus formation, is,
at least in part, developmental period specific. A developmental period
molecular specificity of the regulation of alveolus formation is
supported by the demonstration that retinoic acid receptor (RAR)- is
an endogenous inhibitor of alveolus formation during, but not after, the perinatal period (15). We now report RAR-
is not
required for alveolus formation in mice during the first two postnatal weeks but is required for the production of alveoli of normal volume
(
a), number (Na), and surface area (Sa) between age 14 and 50 days.
RAR-/
mutant mice were generated in France, as
previously described, on a mixed 129/Sv × C57BL/6 background
(8), shipped to Georgetown University, housed in the
Department of Comparative Medicine, allowed food (Rodent Chow 5001;
Ralston Purina, St. Louis, MO) and water ad libitum, and maintained on
a 12:12-h light-dark cycle at ~22°C. RAR-
/
females were bred with RAR-
+/
males, and
RAR-
+/+ females were bred with RAR-
+/+
males. The day of birth was considered age 1 day. Male mice were killed
at ages 14 or 50 days by cutting the abdominal aorta after inducing a
surgical level of anesthesia by the intraperitoneal injection of
xylazine plus ketamine. Genotyping was performed, as previously
described (8), on a piece of mouse tail. All procedures on
animals were approved by the Georgetown University Animal Care and Use
Committee and conformed to National Institutes of Health and US
Department of Agriculture guidelines for the care and use of animals.
We fixed lungs by the intratracheal infusion of cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at a transpulmonary pressure of 20 cmH2O (10, 11-14). We identified alveoli, estimated their volume, and calculated the number per lung as previously described (10, 12, 13, 15). Thirty alveoli were analyzed per mouse. The volume density of alveolar components and the Sa of alveoli were determined by counting points and intersections (21). The statistical significance between two groups was estimated by using an unpaired two-tailed t-test analysis (19), and P < 0.05 was considered to be statistically significant.
Fourteen-day-old RAR-+/+ mice weighed 6.6 ± 0.2 g (N = 4) and were 54 ± 0.7 mm long
(nose to rump); same-aged RAR-
/
mice weighed
6.5 ± 0.2 g (n = 4) and were 52 ± 0.7 mm long (P > 0.05 between groups for the same
parameter). At age 50 days, RAR-
+/+ mice weighed
24.1 ± 1.1 g and were 88 ± 0.9 mm long
(n = 6); same-aged RAR-
/
mice
weighed 24.5 ± 1.6 g (n = 4) and were
87 ± 1.7 mm long (P > 0.05 between groups for
the same parameter).
Lung volume (18) of RAR-+/+ mice was
0.34 ± 0.01 cm3 (n = 4) and 0.65 ± 0.01 cm3 (n = 6) at ages 14 days and 50 days, respectively. In RAR-
/
mice, lung volume was
0.33 ± 0.01 cm3 (n = 4) and 0.65 ± 0.02 cm3 (n = 4) at ages 14 and 50 days,
respectively (P > 0.05 for the same parameter between
groups at each age). We did not find differences in the volume density
of alveolar air, alveolar duct air, or alveolar wall between
RAR-
+/+ and RAR-
/
mice or in the
absolute volume of each of these parameters at either age (not
shown). At age 14 days,
a, Na, and Sa, respectively, did not
exhibit intergroup differences (Fig. 1).
However, at age 50 days,
a was larger, and Na and Sa were
smaller, in RAR-
/
mice than in
RAR-
+/+ mice (Fig. 1).
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Among species that differ in body mass by several logs, lung volume is
proportional to body mass. However, alveolar size (distance between
alveolar walls) is inversely related to the organism's body
mass-specific O2 consumption, and Sa is directly
proportional to the organism's total O2 consumption
(20). Thus lung volume and alveolar dimensions seem to
have different regulators. This is consistent with the present study in
which a, Na, and Sa, but not lung volume, were affected by the
absence of RAR-
. Similarly, the presence or absence of RAR-
(15) or RAR-
(16) does not affect lung
volume, although their absence affects alveolar dimensions (15,
16).
The genetic dissection of regulatory events may be influenced by the
organism's genetic background. Therefore, the universality of the
present findings, and similar prior findings (15, 16), depend on, in part, obtaining the same results in mice with a different
genetic background. Furthermore, in the RAR-/
mice
we used, there is a high postnatal lethality (8). However, to the extent that the mice we studied at ages 14 and 50 days reflect
the lungs of the dead mice, abnormal alveolar structure is not a cause
of the lethality.
The genetic analysis of RAR regulation of alveolus formation in mice
indicates: 1) RAR- influences alveolus formation between ages 14 and 50 days but not in the perinatal period; 2)
RAR-
is an endogenous inhibitor of alveolus formation in the early postnatal period but not thereafter (15); and
3) RAR-
is required for normal alveolus formation at
least up to age 28 days (16). Thus endogenous
all-trans retinoic acid (ATRA), by virtue of its ability to
bind RARs (6), could regulate alveolus formation in
neonatal and in adult rodents. This possibility is supported by the
evidence that exogenous ATRA induces alveolus formation in newborn as
well as adult rodents (12-14). The differential
regulation of alveolus formation by RARs, and in the case of RAR-
and RAR-
, possible complementary induction of alveolus formation,
should be considered in attempts to use ATRA, other oxidation and
isomerization products of retinol, or synthetic retinoid agonists and
antagonists for therapeutic purposes in lung disease.
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ACKNOWLEDGEMENTS |
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We thank Dr. L. B. Clerch for reviewing the manuscript and Zofia Opalka for technical help.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, and HL-60115.
G. D. Massaro and D. Massaro were Senior Fellows of Lovelace Respiratory Institute when this study was performed. D. Massaro is Cohen Professor at Georgetown University School of Medicine. G. D. Massaro and D. Massaro hold patents for the use of retinoids in lung diseases.
Address for reprint requests and other correspondence: G. D. Massaro, Lung Biology Laboratory, Box 571481, 3900 Reservoir Rd., NW, Washington, DC 20057-1481 (E-mail: massarog{at}georgetown.edu).
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.
10.1152/ajplung.00245.2002
Received 24 July 2002; accepted in final form 1 November 2002.
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