© 1995 American Heart Association, Inc.
Articles |
Continuous Nitric Oxide Inhalation Reduces Pulmonary Arterial Structural Changes, Right Ventricular Hypertrophy, and Growth Retardation in the Hypoxic Newborn Rat
From the Departments of Anaesthesia (J.D.R., R.C.J., W.M.Z.), Pediatrics (J.D.R.), and Medicine (K.D.B.) and the Cardiovascular Research Center (J.D.R., C.T.R., K.D.B.), Harvard Medical School at Massachusetts General Hospital, Boston.
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Abstract Breathing low oxygen levels for several weeks produces progressive pulmonary artery hypertension and smooth muscle hypertrophy and hyperplasia in many species. Because nitric oxide (NO) is an important regulator of pulmonary vascular tone, we examined whether the continuous inhalation of low levels of NO gas would attenuate pulmonary arterial structural changes in hypoxic rat pups. Nine-day-old rat pups and their mothers continuously breathed at FIO2 0.21 or 0.10 with or without adding 20 ppm (by volume) NO for 2 weeks. Lung tissue was obtained for vascular morphometric analysis, and the hearts were dissected to measure right ventricular weight and levels of mRNA encoding rat atrial natriuretic factor (rANF). In addition, femur and skull length were radiographically determined. Breathing at FIO2 0.10 for 14 days increased pulmonary arterial wall thickness and the proportion of muscular arteries in the lung periphery. Right ventricular weight and right ventricular rANF gene expression increased, whereas body weight and skeletal growth were reduced (all P<.05). Continuous inhalation of 20 ppm NO at FIO2 0.10 for 2 weeks decreased hypoxic pulmonary vascular structural changes and somatic growth retardation and prevented the increase of right ventricular weight and right ventricular rANF mRNA levels. These observations suggest that chronically breathing NO attenuates pulmonary vascular smooth muscle hypertrophy and/or hyperplasia and extension into distal arterial walls, right ventricular hypertrophy, and growth retardation of newborns breathing at a low oxygen level.
Key Words: pulmonary artery hypertension • congenital heart disease • nitric oxide • newborn rats
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Immediately after birth, lung distension and the elaboration of endogenous vasoactive substances precipitously reduce the pulmonary vascular resistance and increase pulmonary blood flow. After birth, the alveoli and associated intra-acinar vessels proliferate (for a review, see Reid1 ). The increased number of intra-acinar vessels causes a further decrease in pulmonary vascular resistance associated with a reduction in right ventricular weight. During infancy, vascular smooth muscle cells proliferate in both preacinar and intra-acinar arteries, thereby increasing arterial wall thickness. In addition, smooth muscle cells develop in the wall of distal pulmonary arteries. Congenital heart lesions, associated with an increased postnatal pulmonary blood flow,2 3 4 cause pulmonary artery hypertension, increased and/or accelerated pulmonary artery smooth muscle hyperplasia, hypertrophy and growth in peripheral vessel walls, right ventricular hypertrophy, and reduced somatic growth. During chronic hypoxia of the rat, pulmonary vessel wall smooth muscle cells similarly undergo hypertrophy and/or hyperplasia and proliferate in the walls of peripheral arteries. Pulmonary vascular remodeling with right ventricular hypertrophy and somatic growth retardation also occurs in human beings residing at high altitudes5 and can be reproduced in newborn and infant rats continuously breathing gases with reduced oxygen tension for several weeks.6 7
Nitric oxide (NO), an endothelium-derived relaxing factor, regulates pulmonary vascular resistance in the perinatal period.8 9 10 11 Children with congenital heart lesions,12 as well as adult rats continuously breathing at a low oxygen level,13 appear to have reduced endothelial NO production. Adding NO to inhaled gas reduces the pulmonary vascular resistance in patients with many forms of congenital heart disease,14 suggesting that NO receptor/effector systems are intact. Several studies also suggest that endothelium-derived factors modulate subjacent smooth muscle growth in vivo15 16 and in vitro.17 18 19 However, it is unknown whether endothelial NO production regulates normal or abnormal pulmonary vascular smooth muscle proliferation in the newborn.
The objective of the present study was to examine whether inhaling low levels of NO reduces the abnormal pulmonary vascular structural changes caused when the rat pup breathes at a low oxygen level. We report that continuous NO inhalation by the hypoxic rat pup reduces thickening of the pulmonary vascular wall and extension of smooth muscle into peripheral lung arteries and reduces growth retardation. In addition, inhalation of NO prevents right ventricular hypertrophy caused by breathing gases with reduced oxygen concentration.
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These investigations were approved by the Subcommittee for Research Animal Studies of the Massachusetts General Hospital.
Animal Model and Exposure Chambers
Starting 9 days after birth, litters of eight
Sprague-Dawley rat pups of both genders and their mothers
(Charles River Laboratories, Wilmington, Mass) continuously breathed at
FIO2 0.10 or 0.21, with or without inhaled
NO, for 14 days. The bedding was changed, and water and food were
replenished every 2 to 3 days. During this time, the rats breathed at
FIO2 0.21 for fewer than 15 minutes.
We chose to use 20 ppm NO because our previous studies of newborn11 and adult lambs20 with hypoxic and U46619-mediated pulmonary vasoconstriction and of infant14 and adult21 human beings with pulmonary artery hypertension have shown that this low level of inhaled NO causes profound reduction of the pulmonary artery hypertension and pulmonary vascular resistance. The gas mixtures were blended by using separately regulated and calibrated flowmeters for oxygen, nitrogen, and NO gases (Airco). The gas mixtures were introduced into identical 40-L acrylic inhalation chambers. This exposure system produced gaseous atmospheres with stable FIO2 and NO levels. The FIO2 was measured by using a polargraphic electrode (Hudson Oxygen Meter 5590), FICO2 was measured with a capnometer, and the NO concentration was measured by chemiluminescence22 (model 14A, Thermo Environmental Instruments Inc). The concentration of nitrogen dioxide and nitrogen with higher oxidation states (NOx) was measured by chemiluminescence after conversion of the NOx to NO by a heated stainless steel oven (750°C) with 98% efficiency (model 100B NOx generator, Thermo Environmental Instruments Inc). High rates of fresh gas flow into the chambers allowed atmospheric exchange every 10 minutes. The high chamber gas turnover rates and adding soda lime (J.T. Baker Chemical Co) to the chambers reduced CO2 and NOx to <1% and 2 ppm, respectively. The gases exiting the exposure chambers, as well as those discharging from the chemiluminescence instrument, were scavenged by use of a Venturi trap maintained at negative atmospheric pressure with the laboratory's central vacuum system.
Lung Morphometry
After exposure, the rat pups were killed with an injection of
100 mg/kg IP sodium pentobarbital. The heart and lungs were removed en
bloc via a midline incision through the sternum. The airway and main
pulmonary artery were cannulated with polyethylene catheters (PE-20 and
PE-100, respectively), and the pulmonary veins were ligated. The
technique we used to distend and identify vessels has been well
characterized.6 7 23 24 25 The pulmonary arteries and airways
were distended to 100 and 23 cm of water pressure, respectively, with
1% glutaraldehyde and 4% formaldehyde in phosphate-buffered saline
and fixed for 7 days. Because fixative is infused via the pulmonary
artery and the pulmonary veins are ligated, as the fixative enters the
pulmonary circulation, blood is washed forward into the veins. This
assists in the identification of arteries and veins. Subsequently, the
lungs were progressively dehydrated in ethanol solutions and embedded
in resin (Historesin, Cambridge Instruments GmbH). Sections (2 µm
thick) were obtained from the left lung and cardiac and diaphragmatic
lobes, treated with 0.5% potassium permanganate and 1% oxalic acid,
and stained with Miller's elastin stain26 and toluidine
blue to demonstrate smooth muscle and elastic laminae of the arteries
and interstitial structures. By staining, smooth muscle cells in
muscular arteries were seen between two elastic laminae.
Lung sections, from each of the three lobes, were examined for the
degree of alveolar collapse, vessel distension, and presence of
intra-alveolar or interstitial inflammatory cells. Morphometric
analysis of the pulmonary arteries was performed by using the
technique of Hislop and Reid27 and Meyrick and
Reid.6 The pulmonary veins were not analyzed since no
obvious changes in them were seen in the lung we examined and because
previous studies of newborn7 and adult27 rats
have shown that they are not structurally changed by chronic hypoxia.
Pulmonary arteries, cut in true or oblique section, were classified
according to wall structure and by their location, ie, either as
preacinar or intra-acinar. Smooth muscle cells were identified in the
vessel wall by their staining and by their position between two elastic
laminae. If 
75% of the circumference of a blood vessel was encircled
by two elastic laminae, it was classed as muscular; if less, it was
categorized as partially muscular. Vessels with no elastic lamina and
15 µm external diameter (ED) were classed as nonmuscular; vessels
15 µm were considered capillaries and not analyzed. The preacinar
arteries were readily identified from the preacinar veins by their wall
structure and location; the arteries travel with airways and have a
thicker wall. Within the acinus, the arteries were differentiated from
venules and from veins, both by their wall structure and by the absence
of blood in the lumen. For all muscularized arteries, the minimum ED
through the major axis was measured; on the same axis, the
thickness of the arterial wall was measured from the external elastic
lamina to the basement membrane of the endothelium. The percent wall
thickness (%WT) was calculated: %WT=100x2xWT/ED, where WT was the
average thickness of the smooth muscle between the internal and
external elastic lamina of two sides of muscular arteries or the
thickness on one side of partially muscular arteries. In each lung
section, all arteries associated with bronchioli and terminal and
respiratory bronchioli were analyzed. Similarly, the first 25 alveolar
arteries encountered, in distended lung areas, that were associated
with ducts or the walls were analyzed. The proportion of muscular,
partially muscular, or nonmuscular alveolar arteries was calculated. A
total of 1290 pulmonary arteries were analyzed from a total of 16 rat
pups, with 4 pups in each of the four groups breathing at either
FIO2 0.21 or 0.10 with and without NO.
Lung Water Content
The effect of 2 weeks of continuous breathing at
FIO2 0.10 with 20 ppm NO and <2 ppm
NOx or without NO on lung water content was determined
gravimetrically. After dissection of the lung from the hilar
structures, the wet weight was determined by using an analytical scale.
The lung was subsequently dried to a constant weight at 65°C for 72
hours and then reweighed. The percent lung water was determined: %
water=100x(wet weight-dry weight)/wet weight.
Measurement of Ventricular Weight and rANF mRNA Levels
We measured the effect of breathing at
FIO2 0.10 and 0.21, with and without inhaled
NO, on right ventricular hypertrophy for 59 rat pups by using the
methodology of Fulton et al.28 After the heart had been
fixed for 7 days in 1% glutaraldehyde and 4% formaldehyde in
phosphate-buffered saline, the atria and large vessels were removed,
and the right ventricular free wall was carefully dissected from the
left ventricle and interventricular septum. The right ventricle and
left ventricle with septum were separately weighed.
To measure right ventricular rANF mRNA levels, right ventricular muscle was dissected to exclude epicardial and atrial tissue, and total RNA was isolated by using the guanidine isothiocyanate–cesium chloride method.29 Ten micrograms of RNA were fractionated in a 1.3% agarose–formaldehyde gel, transferred to charged nylon membranes (MSI Inc), and cross-linked to the membranes with UV light. Membranes were hybridized with a 32P-labeled Pst 1 restriction fragment derived from rat rANF cDNA at 42°C.30 Membranes were washed with 30 mmol/L NaCl, 3 mmol/L sodium citrate, and 0.1% sodium dodecyl sulfate at 65°C and exposed to radiographic films. To ensure equal RNA transfer onto the membranes, they were hybridized with a 32P-labeled oligonucleotide probe (5'-ACG GTA TCT GAT CGT CTT CGA ACC-3') complementary to rat 18S ribosomal RNA.31 The membranes were washed with 0.9 mol/L NaCl and 90 mmol/L sodium citrate with 0.05% sodium pyrophosphate at 37°C and then exposed to radiographic films.
Studies of Somatic Growth
All rat pups were weighed before and after the 2-week exposure
to FIO2 0.21 or 0.10 with and without NO.
Four litters of 8 pups were weighed initially and then reweighed after
3.5, 7, 10.5, and 14 days of exposure. For 39 pups, the crown-to-rump
lengths were measured. In addition, after the pups were killed, supine
x-ray films were obtained by use of a cathode tube with 20 kV for 3
minutes (Faxitron 43805N, Hewlett Packard) and radiographic film
(X-OMAT AR, Kodak) to assess skeletal growth. Femur lengths were
determined by measuring the rectilinear distance between the most
proximal border of the greater trochanter to the distal border of the
lateral femoral condyle; skull anteroposterior lengths were determined
by measuring the distance from the occiput to the maxillary ridge. No
correction was made for parallax because the magnification of the
skeletons was 0.2%.
In addition, to examine the effect of NO inhalation on growth retardation due to breathing at a low oxygen concentration in the absence of the mother, twelve 23-day-old weaned infant rats of both genders breathed at FIO2 0.10 with either 0 or 20 ppm NO for up to 14 days. Their weights were determined as described above.
Analysis of Blood Components
After 14 days of breathing at FIO2
0.21 or 0.10, with or without 20 ppm NO added, blood from 30 rat pups
was sampled by cardiac puncture after death for determination of
hematocrit and plasma total protein, albumin, and globulin levels. In
addition, after 2 weeks, blood was sampled for optical determination of
methemoglobin32 from 15 pups breathing at
FIO2 0.21, with and without 20 ppm NO.
Statistical Methods
The observer was unaware of the specimen's treatment group
during the morphometric analysis of the lung, heart dissection and
measurement of ventricular weight, body x-ray, and measurement of
skeletal length. All data are presented as the mean±SD. The mean
%WT values of pulmonary arteries and proportions of muscular,
partially muscular, and nonmuscular arteries of each of the four
animals in each of the four groups were compared by a factorial model
of ANOVA.6 27 33 Therefore, the values from each animal
within a treatment group were weighted equally. In comparing the
distribution of the nominal incidence of muscular, partially muscular,
and nonmuscular arteries, the total number of arteries that were either
muscular, partially muscular, or nonmuscular of all the rat pups within
each group were compared by 
2 analysis. When
significant differences were detected by ANOVA or
2 analysis, a post hoc Scheffé's F
test34 was used. Significance was determined at
P<.05.
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General Appearance of the Animals
Rat pups breathing at FIO2 0.10, with or without NO, appeared hyperpneic compared with those breathing air. Those breathing at FIO2 0.10 without NO were less active than those breathing 20 ppm NO at the same FIO2 level. In addition, pups breathing at FIO2 0.10 without NO exhibited little grooming activity; the grooming and fur of those breathing at FIO2 0.10 with 20 ppm NO were indistinguishable from air-breathing rat pups.
Pulmonary Vascular Structural Changes
In a qualitative survey of pulmonary vessel structure, the
pulmonary arteries of rat pups breathing at
FIO2 0.10 without inhaled NO were obviously
thick compared with arteries in pups that breathed at
FIO2 0.10 with inhaled NO or at
FIO2 0.21 with or without NO. Furthermore, in
agreement with the reported data of others,7 27 no
difference between the thickness of the walls of pulmonary veins in
hypoxic and normoxic pups was observed. Pulmonary arteries with a
muscular, partially muscular, or nonmuscular wall were observed in each
lung section. Representative partially muscular arteries are shown
in Fig 1
. In addition, we observed no intimal damage,
angiomatoid lesions, or interstitial hemorrhage in the lungs of animals
who breathed at FIO2 0.10 or 0.21, with or
without NO.
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Quantitative analysis confirmed that breathing at
FIO2 0.10 caused marked structural changes in
the pulmonary arteries of pups, consisting of an increase in percent
wall thickness, reduction in the internal diameter of both preacinar
and intra-acinar arteries, and an increase in the muscularization of
intra-acinar arteries that normally were nonmuscular.6 7
The percent wall thickness of muscular and partially muscular
bronchiolar and terminal and respiratory bronchiolar arteries (500 to
1000 µm ED), as well as alveolar duct arteries (15 to 50 µm ED),
increased twofold after 14 days of breathing at
FIO2 0.10 (P<.05, Fig 2). The external diameter of the pulmonary arteries was
not altered by breathing at FIO2 0.10; the
internal diameter of muscular and partially muscular alveolar wall
arteries was reduced from 42.3±9.4 to 27.6±3.7 µm
(P<.05) by increasing the wall thickness. Of interest,
breathing at FIO2 0.10 did not change the
%WT of preacinar arteries between 51 and 499 µm ED (%WT, 7.1±2%
[FIO2 0.21] and 8.6±3.1%
[FIO2 0.10]; P=.47). This lack
of structural change, however, may be due to the wide variation of %WT
versus arterial vessel ED observed in the rat.35 Breathing
at FIO2 0.10 also caused extension of smooth
muscle cells into arteries associated with alveolar ducts and the
alveolar wall (Fig 3; 2=23.7, P<.05).
The proportion of muscular and partially muscular alveolar duct
arteries increased from 4.6±3.4% to 43.2±5.4% and 22.6±12.1% to
46.9±9.1%, respectively (Fig 3, top). The fraction of
partially muscular alveolar wall arteries was also increased
(P<.05; Fig 3, bottom).
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P<.05 vs FIO2 0.21 with inhaled
NO; and §P<.05 vs FIO2 0.10 with
inhaled NO.
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P<.05 vs FIO2 0.10 without
inhaled NO; and §P<.05 vs FIO2
0.10 with inhaled NO.
Inhaling 20 ppm NO prevented the pulmonary vascular changes found in
rat pups breathing at a low oxygen concentration (Figs 2
and 3).
Breathing NO at FIO2 0.10 diminished, by
nearly 35%, the increase in percent wall thickness of both 15- to
50-µm and 500- to 1000-µm ED pulmonary arteries observed in rat
pups breathing at FIO2 0.10 without NO. The
%WT of partially muscular and nonmuscular pulmonary arteries of rats
breathing at FIO2 0.10 without and with
inhaled NO, respectively, was 16.4±2.5 versus 10.8±2.1% for arteries
15 to 50 µm ED and 7.5±0.3% versus 4.9±0.8% for arteries of 500
to 1000 µm ED. Inhaling NO prevented the reduction in internal
diameter of muscular and partially muscular alveolar wall arteries.
Furthermore, inhalation of NO while breathing at a low oxygen
concentration attenuated the muscularization of distal arteries: in
animals breathing NO at FIO2 0.10, the
proportion of muscular alveolar duct arteries was intermediate between
those observed in animals breathing at FIO2
0.21 or 0.10, whereas the percentage of partially muscular arteries was
similar to that in air-breathing control animals. In addition, the
proportion of partially muscular arteries in alveolar wall arteries was
similar to that of rat pups breathing air.
Breathing NO at FIO2 0.21 did not alter the typical development of structures in the rat pup lungs. In addition, NO breathing did not appear to induce pulmonary injury: neither interstitial blood nor inflammatory cells infiltrating lung vessels, airways, or the interstitium were observed. Others have shown that hypoxia does not alter the extravascular lung water of spontaneously breathing but anesthetized rats.36 Breathing NO at FIO2 0.10 for 2 weeks did not alter the fraction of lung water (0 ppm NO, 83±6% [n=11]; 20 ppm NO, 86±5% [n=8]; P=.40). These findings suggest that continuous breathing of NO with low levels of NOx did not cause lung injury.
Measurement of Ventricular Weight and rANF mRNA
Two weeks of breathing at FIO2 0.10
caused right ventricular hypertrophy (Table 1). Right
ventricular weight and the ratio of the weight of the right ventricle
to the weight of the left ventricle with intraventricular septum
[RV/(LV+S)] were increased by nearly 80%. The increase of RV/(LV+S)
was proportional to the increase in wall thickness of pulmonary
arteries of 15 to 50 µm ED. Breathing at
FIO2 0.10 increased the abundance of rANF
mRNA in the cells of the right ventricular free wall (Fig 4).
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Inhaling 20 ppm NO prevented the increase in right ventricular weight and RV/(LV+S) that was due to breathing at FIO2 0.10. In addition, continuous breathing of NO reduced right ventricular rANF mRNA levels. Breathing 20 ppm NO at FIO2 0.21 reduced right ventricular rANF mRNA levels even though it did not alter ventricular weight.
Somatic Growth
There was no difference in the initial body weight of the
9-day-old rat pups in the four groups. Breathing at
FIO2 0.10 reduced the body weight and length
and femur and skull lengths of the hypoxic rat pups compared with
control rat pups that breathed air (Table 1, Fig 5).
During the first week of breathing at FIO2
0.10, there was little growth of the newborn rat pups. Subsequently,
the growth rate increased slightly. Breathing at
FIO2 0.10 with 20 ppm NO increased rat pup
body weight and body and skeletal lengths to levels intermediately
between those breathing at FIO2 0.21 or 0.10
without inhaled NO.
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Breathing at a low oxygen level reduced the growth rates of infant rats that had been weaned from their mothers before exposure to NO. After 12 days of hypoxia, the body weight of the weaned infant rats breathing NO at FIO2 0.10 was 23% greater than those breathing this low oxygen level without added NO (data not shown).
Blood Components
Compared with breathing air, 2 weeks of breathing at
FIO2 0.10 produced a 27% increase in the
hematocrit in the rat pups. In addition, breathing at a low oxygen
level without NO caused a 14% reduction in plasma albumin
concentration compared with breathing air, without altering globulin
concentration (Table 2). Inhaling 20 ppm NO at
FIO2 0.10 did not alter the hematocrit
compared with breathing at FIO2 0.10 without
inhaled NO. NO inhalation at FIO2 0.10
prevented the reduction in plasma albumin concentration. NO inhalation
did not increase methemoglobin levels in rat pups breathing air (0 ppm
NO, 5.2±1.8% [n=7]; 20 ppm NO, 6.2±1.6% [n=8];
P=.31).
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In the present study, breathing low levels of NO reduced pulmonary vascular changes in hypoxic rat pups. In agreement with the findings of others,6 7 continuous hypoxia increased the pulmonary arterial wall thickness. In addition, breathing at FIO2 0.10 increased the proportion of muscular alveolar arteries, suggesting extension of smooth muscle into their walls. We found that breathing 20 ppm NO at FIO2 0.10 prevented the increase in wall thickness of pulmonary arteries and partially reduced the distal extension of smooth muscle cells into alveolar duct and wall arteries. Inhaling NO also prevented right ventricular hypertrophy and decreased growth retardation caused by chronic hypoxia. Furthermore, 2 weeks of NO inhalation did not produce any evidence of pulmonary or systemic toxicity: no significant methemoglobinemia, pulmonary interstitial inflammation, hemorrhage, or edema was observed, nor was any obvious alteration of typical postnatal pulmonary development detected.
The mechanisms producing neomuscularization of arteries during continuous breathing at low oxygen levels are unknown. In the adult lung, differentiation of pericytes and intermediate cells in the region of alveolar wall vessels leads to the development of smooth muscle cells during hypoxia.37 In the postnatal lung, the pulmonary arteries thicken and neomuscularization occurs in distal vessels.25 Stimuli caused by hypoxia in the postnatal lung may accelerate such cellular changes and also cause smooth muscle hypertrophy and hyperplasia. The stimuli for proliferation may include transduction of increased pulmonary vascular pressure and/or shear stress, low oxygen tension, and/or modulation of mitogenic substances such as platelet-derived growth factor (PDGF),38 insulin-like growth factor I,39 epidermal growth factor, basic fibroblast growth factor,40 and vascular endothelial growth factor.41 It is unknown precisely how low levels of NO reduce pulmonary vascular alterations caused by breathing low levels of oxygen. Breathing NO acutely reduces pulmonary artery hypertension during hypoxic or U46619-mediated pulmonary vasoconstriction in newborn11 and adult20 animals and in human beings42 with normal pulmonary vascular structure. In addition, NO inhalation reduces pulmonary artery hypertension in newborn animals43 44 and in human beings14 21 45 with a structurally altered pulmonary vascular bed. In newborn rat pups breathing at a low oxygen level, it is possible that NO inhalation reduced pulmonary artery hypertension and thereby reduced vascular smooth muscle changes. It is also possible that inhaled NO altered cellular factors modulating growth within the vascular wall. This latter possibility is supported by preliminary studies using cells in culture that suggest that NO is antimitogenic18 19 46 and modulates the expression of genes encoding growth factors including endothelin 1 and PDGF-B.47
Because of their small size, it is difficult to directly measure pulmonary artery pressure in the awake 9-day-old rat or the 23-day-old rat after 14 days of treatment. However, other investigators have shown that the level of right ventricular rANF mRNA is increased by elevated ventricular wall stretch.48 49 50 In the adult rat, Stockman et al51 observed that hypoxia increased right ventricular weight and levels of rANF mRNA and immunoreactivity. Therefore, we reasoned that right ventricular rANF mRNA levels reflect the degree of right heart afterload in the newborn rat and thus provide us with an indirect means to confirm the effect of pulmonary artery pressure on breathing at a low oxygen level with and without NO. We observed that breathing at FIO2 0.10 increased rANF mRNA level of the right ventricle, whereas breathing NO at the same oxygen tension prevented the increase in rANF gene expression. Although we carefully excluded atrial and epicardial tissue from the right ventricular free wall during our dissection of the heart, the tissue preparation did not allow determination of whether the rANF mRNA in the right ventricular free wall was from endocardial and/or myocardial cells. Of interest, breathing NO at FIO2 0.21 reduced the level of right ventricular rANF mRNA below that measured in air-breathing control rats. This suggests that NO inhalation reduces ventricular rANF mRNA levels52 by accelerating the normal postnatal fall in pulmonary artery pressure.
Chronic hypoxia reduces the somatic growth rate of both newborn6 and young rats.7 53 54 The present studies showed that breathing at FIO2 0.10 inhibited somatic growth in the newborn rat for the first 7 days of exposure and that growth subsequently was slower than in animals breathing air. In the newborn rats breathing at FIO2 0.10 with 20 ppm NO, growth was only reduced for the first 3.5 days; thereafter, growth increased to rates observed in air-breathing pups. Nevertheless, because of the initial reduction of growth, body weight after 2 weeks of breathing at a low oxygen level with NO remained less than at air-breathing levels. Although breathing at FIO2 0.10 with NO significantly reduces pulmonary vascular structural changes and right ventricular hypertrophy, it is possible that breathing higher concentrations of NO could further reduce hypoxia-associated growth retardation.
Breathing at FIO2 0.10 reduced pup plasma albumin levels, suggesting that it caused poor feeding and protein-calorie malnutrition. When pups breathed NO at FIO2 0.10, their plasma albumin levels remained at levels found in rat pups breathing air. NO breathing may have reduced hypoxia-associated growth retardation by improving nursing efficiency or maternal lactation. However, since NO inhalation reduced hypoxia-mediated growth retardation in young rats that were weaned before continuous breathing at FIO2 0.10, the primary salutary effects of NO breathing may be independent of nursing effects.
In summary, 2 weeks of continuous inhalation of NO by the hypoxic rat pup inhibited pulmonary arterial smooth muscle hypertrophy and/or hyperplasia and smooth muscle extension in the walls of distal arterial arteries, right ventricular hypertrophy, and somatic growth retardation. We speculate that continuous inhalation of NO might prevent pulmonary vascular structural changes and pulmonary artery hypertension in infants with congenital heart disease causing increased pulmonary blood flow. It is also possible that inhaling NO may be beneficial to some infants residing at high altitudes who suffer from pulmonary vascular structural alterations due to hypobaric hypoxia. Continuous NO inhalation by infants with congenital heart disease may diminish growth retardation and thereby decrease the need for early, and often only palliative, surgical or catheterization procedures. In addition, inhalation of NO by infants with hypobaric hypoxia and severe growth retardation may increase their growth and survival rates.
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Acknowledgments |
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This study was supported by US Public Health Service grant HL-42397; a Grant-in-Aid from the American Heart Association, with funds contributed in part by the Massachusetts Affiliate, Inc; and a grant to the Cardiovascular Research Center from Bristol Myers Squibb Pharmaceuticals. We are also indebted to Melahat Kavosi and Karen Rock for technical assistance and to Dr Donald Bloch for helpful comments on the manuscript.
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Footnotes |
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Reprint requests to Jesse Roberts Jr, MD, Department of Anesthesia, Massachusetts General Hospital, Boston, MA 02114. E-mail roberts@helix.mgh.harvard.edu.
Received June 6, 1994; accepted October 21, 1994.
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