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Circulation Research. 1995;76:215-222

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(Circulation Research. 1995;76:215-222.)
© 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

Jesse D. Roberts, Jr, Carole T. Roberts, Rosemary C. Jones, Warren M. Zapol, Kenneth D. Bloch

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.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
 down arrowReferences
 
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 {chi}2 analysis. When significant differences were detected by ANOVA or {chi}2 analysis, a post hoc Scheffé's F test34 was used. Significance was determined at P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down. 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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  		Figure 1. Photomicrograph of pulmonary arteries in a hypoxic rat pup. Miller's elastin and toluidine blue stains allow clear definition of the external (white arrow) and internal (black arrow) elastic laminae and endothelial cells (*). Bar=10 µm; original magnification x680.

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 2Down). 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 3Down; {chi}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 3Down, top). The fraction of partially muscular alveolar wall arteries was also increased (P<.05; Fig 3Down, bottom).



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  		Figure 2. Bar graph showing the effect of 2 weeks of continuous inhalation of nitric oxide (NO) on pulmonary artery percent wall thickness in rat pups breathing at FIO2 0.21 or 0.10. Lung tissue was fixed at constant pressure, sectioned, and stained as detailed in the text. For a total of 1290 pulmonary arteries from 16 rat pups, the minimum external diameter through the major axis of the vessel and associated wall thickness(es) allowed calculation of percent wall thickness. Breathing at FIO2 0.10 significantly increased the percent wall thickness in preacinar and respiratory bronchiolar arteries between 500 to 1000 µm and alveolar duct arteries 15 to 50 µm in external diameter. Inhaling 20 ppm NO at FIO2 0.10 reduced percent vessel wall thickness to air-breathing control levels. Furthermore, inhaling NO at FIO2 0.21 did not alter wall thickness compared with breathing at FIO2 0.21 without NO. Values are mean±SD. *P<.05 vs FIO2 0.21 without NO; {dagger}P<.05 vs FIO2 0.21 with inhaled NO; and §P<.05 vs FIO2 0.10 with inhaled NO.



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  		Figure 3. Bar graphs showing the effects of 2 weeks of continuous inhalation of nitric oxide (NO) on the proportion of muscular (M), partially muscular (PM), and nonmuscular (NM) pulmonary arteries in hypoxic rat pups breathing at FIO2 0.21 or 0.10. Top, Breathing a low oxygen concentration increased the proportion of M and PM alveolar duct arteries. Continuous inhalation of NO at FIO2 0.10 reduced the proportion of M arteries compared with breathing at FIO2 0.10 without NO. Bottom, Continuous breathing at FIO2 0.10 increased the proportion of PM alveolar wall arteries. Inhalation of NO at FIO2 0.10 reduced the proportion of M alveolar wall arteries to levels observed in rat pups breathing air. The proportion of M, PM, and NM arteries associated with alveolar ducts or in the alveolar wall was unaltered by NO inhalation at FIO2 0.21. Values are mean±SD. *P<.05 vs FIO2 0.21 without NO; {dagger}P<.05 vs FIO2 0.21 with inhaled NO; {ddagger}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 2Up and 3Up). 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 1Down). 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 4Down).


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  		Table 1. Effect of 2 Weeks of Continuous Exposure to FIO2 0.21 or 0.10 With or Without Nitric Oxide on Right Ventricular Hypertrophy and Somatic Growth



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  		Figure 4. Effects of 2 weeks of continuously breathing at FIO2 0.21 or 0.10 with (+) or without (-) 20 ppm nitric oxide (NO) on rat atrial natriuretic factor (ANF) mRNA levels in the right ventricular wall of newborn rat pups as determined by RNA blot hybridization. Ten micrograms of RNA extracted from the right ventricular free wall were fractionated on a formaldehyde-agarose gel, transferred to a charged membrane, and cross-linked with UV light. The membrane was incubated with a 32P-labeled restriction fragment prepared from rat ANF cDNA, washed at high stringency, and subjected to autoradiography (top). This allowed identification of a 0.90-kb mRNA encoding rat ANF. Subsequently, the membrane was stripped of the previous probe and then hybridized with a 32P-labeled 24-mer oligonucleotide corresponding to 18S RNA and again subjected to autoradiography (bottom).

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 1Up, Fig 5Down). 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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  		Figure 5. Line graph showing the effect of continuous breathing of nitric oxide (NO) for 2 weeks on somatic growth of the hypoxic rat pup. Breathing at FIO2 0.10 caused growth retardation. Inhaling 20 ppm NO at FIO2 0.10 produced growth intermediately between that caused by breathing at FIO2 0.21 and 0.10 without NO. Each group consisted of eight rat pups. Values are mean±SD. *P<.05 vs FIO2 0.21 without NO; {dagger}P<.05 vs FIO2 0.21 with inhaled NO; and §P<.05 vs FIO2 0.10 with inhaled NO.

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 2Down). 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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  		Table 2. Effect of 2 Weeks of Continuous Exposure to FIO2 0.21 or 0.10 With or Without Nitric Oxide on Blood Values


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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.


*    Acknowledgments
 
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.


*    Footnotes
 
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.


*    References
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 *References
 

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