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(Chest. 2004;125:2247-2252.)
© 2004 American College of Chest Physicians

Prolonged Nitric Oxide Inhalation Fails to Regress Hypoxic Vascular Remodeling in Rat Lung*

Bao Hua Jiang, MD; Junko Maruyama, MD; Ayumu Yokochi, MD; Manabu Iwasaki, MD; Homare Amano, MD; Yoshihide Mitani, MD and Kazuo Maruyama, MD

* From the Department of Anesthesiology and the Intensive Care Unit (Drs. Jiang, Yokochi, Iwasaki, Amano, and K. Maruyama), and Departments of Physiology (Dr. J. Maruyama) and Pediatrics (Dr. Mitani), Faculty of Medicine, University of Mie, Japan.

Correspondence to: Kazuo Maruyama, MD, Department of Anesthesiology, Mie University School of Medicine, 2-174, Edobashi, Tsu, Mie, Japan 514-8507; e-mail: masuika{at}clin.medic.mie-u.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: The purpose of present study was to investigate whether long-term nitric oxide (NO) inhalation during the recovery in air might improve the regression of chronic hypoxic pulmonary hypertension (PH) and vascular changes.

Materials and methods: The rats were exposed to 10 ppm of NO in air for 10 days (n = 12) and 30 days (n = 4), or 40 ppm of NO in air for 10 days (n = 6) and 30 days (n = 12) following 10 days of hypobaric hypoxia (380 mm Hg, 10% oxygen). For each NO group, air control rats following hypoxic exposure were studied at the same time (n = 13, 11, 9, and 11, respectively). Normal air rats (n = 6) without hypoxic exposure and rats (n = 7) following 10 days of hypoxic exposure were used as normal and chronic hypoxic control groups, respectively. Muscularization of normally nonmuscular peripheral arteries and medial hypertrophy of normally muscular arteries were assessed by light microscopy. An additional 16 rats were used to investigate the recovery of pulmonary artery pressure with (n = 8) and without NO inhalation (n = 8) after 10 days of hypobaric hypoxia.

Results: Long-term hypoxia-induced PH, right ventricular hypertrophy (RVH), and hypertensive pulmonary vascular changes, each of which regressed partly after recovery in room air. There were no differences among rats with and without NO during each recovery period in RVH, medial wall thickness of muscular artery, and the percentages of muscularized arteries at the alveolar wall and duct levels. Continuous inhaled 40 ppm NO decreased pulmonary artery pressure from 40.1 ± 1.1 to 29.9 ± 3.8 mm Hg (mean ± SE) [n = 8], which was not different in the rats without NO inhalation (n = 8). Urine nitrate level was higher in rats that had inhaled NO.

Conclusion: Continuous NO inhalation showed no effect on regression of pulmonary vascular remodeling in chronic hypoxic PH after returning to room air.

Key Words: hypoxia • nitric oxide • pulmonary artery catheter • pulmonary hypertension • recovery • right ventricular hypertrophy • vascular remodeling • vasodilation


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic hypoxia causes pulmonary hypertension (PH) and hypertensive pulmonary vascular changes in rats.123 Vascular changes include new muscularization of normally nonmuscular peripheral pulmonary arteries, medial hypertrophy of muscular arteries, and an increase in extracellular matrix protein such as collagen and elastin.1234 After recovery in normoxic air, the PH and structural changes of the pulmonary vascular bed regress, which is incomplete and residual PH and vascular remodeling are observed.356

Sodium nitroprusside, a nitric oxide (NO) donor, induces relaxation in isolated pulmonary arteries from patients with COPD7 and chronic hypoxic rats.8 Short-term inhaled NO reduced pulmonary artery pressure in chronic hypoxia-induced pulmonary hypertensive rats9 and in patients with primary pulmonary hypertension.10 Although long-term NO inhalation has been performed in patients with primary PH11 and idiopathic pulmonary fibrosis,12 the effect of inhaled NO on the regression of pulmonary vascular remodeling has not been studied. Since vascular structural changes regress after a reduction in BP1314 and vasodilation might improve the recovery of hypertensive vascular changes,15 if inhaled NO has an effect on pulmonary vascular remodeling it might hasten the recovery of vascular structural changes through the reduction of pulmonary artery pressure. In this study, we investigated whether long-term NO inhalation during the recovery in air after chronic hypoxia might improve the regression of pulmonary vascular remodeling and right ventricular hypertrophy (RVH) in rats.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
10 ppm NO Study: Adult, male, Sprague-Dawley rats weighting 203 to 384 g (Clea; Osaka, Japan) were randomly assigned to one of the following groups: normal rats kept in room air as a normoxic control group (n = 6), chronic hypoxic control rats kept in a hypobaric hypoxia (air at 380 mm Hg) for 10 days (n = 7), rats exposed to air only for 10 days (n = 13) or 30 days (n = 11) after 10 days of hypobaric hypoxia, and rats exposed to air plus 10 ppm of NO for 10 days (n = 12) or 30 days (n = 4) after 10 days of hypobaric hypoxia. Air and NO groups were studied at the same time.

40 ppm NO Study: In another experiment using 40 ppm of NO, rats were also randomly assigned to one of the following groups: rats exposed to air only for 10 days (n = 9) or 30 days (n = 11) after 10 days of hypobaric hypoxia, and rats exposed to air plus 40 ppm of NO for 10 days (n = 6) or 30 days (n = 12) after 10 days of hypobaric hypoxia.

NO Exposure Chamber
NO was obtained from Sumitomo Seika (Chiba, Japan) as a mixture of 10,000 ppm in pure N2. The mixture was introduced into the inlet of a glass exposure chamber (air at 760 mm Hg, 21% oxygen) with a constant flow of air (Fig 1 ). The air flow was obtained with an electrically driven vacuum pump so that immediately before it was introduced into the glass exposure chamber, the gas was diluted with fresh air and the oxidation of NO to NO2 in the glass exposure chamber was avoided as far as possible. The NO and NO2 concentration in the chamber was measured using a chemiluminescence analyzer (CLA-510SS; Horiba; Kyoto, Japan). The NO2 concentration in the glass chamber was < 0.5 ppm. The NO concentration was < 0.01 ppm in the control group glass chamber. Three rats were housed in each chamber, and control chambers were studied at the same time. The chambers were opened for < 1 h once a day so that the daily food and water intake, and stool and urine volume could be measured. Rats were also weighed every 2 days.



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  		Figure 1.. NO exposure chamber. NOx = NO, NO2.

 
Measurement of Urine Nitrate
Inhaled NO reacts with hemoglobin, and the greater part of the metabolites are excreted in the urine in the form of nitrate.16 To confirm the effective supplementation of inhaled NO in our system, we determined the nitrate concentration in urine. Urine samples from hypoxic rats exposed to air only (n = 5) and 40 ppm of NO (n = 5) were analyzed by high-pressure liquid chromatography with a high-pressure cadmium column.17

Pulmonary Artery Pressure
To investigate the recovery of pulmonary artery pressure after long-term hypoxic exposure, we used additional 16 rats to measure mean pulmonary artery pressure (mPAP) every day. After 9 days of hypoxia, the rats were anesthetized with pentobarbital sodium (45 mg/kg) administered intraperitoneally. A pulmonary artery catheter (Silastic tubing, 0.31-mm inner diameter and 0.64-mm outer diameter; Kaneka Medix Corporation; Osaka, Japan) was inserted via the right external jugular vein into the pulmonary artery by a closed-chest technique, as previously described.1318 Three to 4 h hours later, while fully awake, the rats were returned to hypobaric hypoxia again for another 24 h, so that a total of 10 days of hypoxia was performed. Immediately after returning to normoxia, mPAP was measured. Then the rats were kept in room air with or without 40 ppm NO. The rats were removed from the NO exposure chamber every day for 15 min, and the mPAP was recorded with a physiologic transducer and an amplifier system (AP 620G; Nihon Kohden; Tokyo, Japan) once the rats were calm.

Structural Studies
Preparation of Lung Tissue for Morphometric Analysis: Lung section for morphometric analysis of vasculature was prepared using a barium injection method that has been reported in detail.131819 A blood sample for the hematocrit was obtained from the heart. Lung sections were stained for elastin by the Van Gieson method. The right ventricle of the heart was dissected from the left ventricle plus septum and weighed separately. The heart weight ratio (right ventricle/left ventricle plus septum) [RV/LV + S] was calculated.

Morphometric Analysis of Pulmonary Arteries: Light microscope slides were analyzed, without previous knowledge of treatment groups. All barium-filled arteries in each tissue section were examined at 400 x, for an average of 353 arteries per section (range, 206 to 570 arteries per section). Each artery was identified as being one of two structural types for the presence of muscularity: muscularized (with a complete medial coat, incomplete medial coat, or only a crescent of muscle being present), and nonmuscular (no muscle apparent). The percentage of muscularized arteries at the alveolar wall level (%AW) and percentage of muscularized arteries at the alveolar duct level (%AD) were calculated. For normally muscular arteries between 100 µm and 200 µm in diameter (6 to 19 arteries were found per section), the wall thickness of the media (distance between external and internal elastic laminae) was measured along the shortest curvature, and the percentage of medial wall thickness (%MWT) was calculated.131819

Statistical Analysis
All values are expressed as means ± SE. Changes in mPAP during the recovery period were analyzed using repeated-measures analysis of variance. Differences between the two groups were determined by the unpaired Student t test. When more than two means were compared, one-way analysis of variance was used. If a significant difference was found, the Dunnett test was used to identify which groups were different. Differences were reported as significant at p < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Weight Gain, Food and Water Intake, Stool and Urine Volume
No significant differences were found in weight gain at each time point among rats exposed to air only, air plus 10 ppm of NO, and air plus 40 ppm of NO. Average food and water intake, and total urine and stools volume per day were similar in all groups throughout the course.

Recovery of Vascular Structural Changes and RVH in PH Rats
Chronic hypoxia increased %AW, %AD, %MWT, and RV/LV + S compared with normal control rats (Fig 2, 3 ). After 10 days and 30 days of recovery, all of these values decreased but remained higher than in control rats, except the value of the RV/LV + S, which returned to normal after 30 days of recovery. No differences were found between air for 10 days and air for 30 days of recovery in the %AW, %AD, %MWT, except the RV/LV + S. Continuous 10 ppm (Fig 2) and 40 ppm (Fig 3) NO inhalation for 10 days and 30 days showed no effect on the recovery.



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  		Figure 2.. 10 ppm NO study, showing recovery of vascular structural changes and RVH in PH rats. Top left, I: %AW; top right, II: %AD; bottom left, III: %MWT of muscular arteries (diameter, approximately 100 to 200 µm); bottom right, IV: RV/LV + S. CH = chronic hypoxic control rats kept in a hypobaric hypoxia (air at 380 mm Hg) for 10 days; Air-1 = rats exposed to air only after 10 days of hypoxic exposure in the 10 ppm NO study. N.S. = not significant. *p < 0.05 vs normal, #p < 0.05 vs chronic hypoxia. Values are means ± SE.

 


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  		Figure 3.. 40 ppm NO study, showing recovery of vascular structural changes and RVH in PH rats. Top left, I: %AW; top right, II: %AD; bottom left, III: %MWT of muscular arteries (diameter, approximately 100 to 200 µm); bottom right, IV: RV/LV + S. Air-2 = rats exposed to air only after 10 days of hypoxic exposure in the 40 ppm study. Values are means ± SE. See Figure 2 legend for expansion of abbreviations.

 
Hematocrit
Chronic hypoxia increased the hematocrit value from 38.1 ± 0.3% (normal, n = 6) to 56.9 ± 1.0% (chronic hypoxia, n = 7) [p < 0.05]. The values had fallen significantly after 10 days and 30 days of recovery in room air, but were still higher than normoxic control values. Continuous 10 ppm and 40 ppm NO inhalation for 10 days and 30 days did not hasten these recoveries.

Recovery of Pulmonary Artery Pressure: mPAP in PH Rats Exposed to 40 ppm of NO
During the 7-day recovery period, mPAP decreased from 40.1 ± 1.1 to 29.9 ± 3.8 mm Hg in rats with 40 ppm of NO inhalation (n = 8), which was similar to the rats without NO inhalation (n = 8). We could not maintain the pulmonary artery catheter in the pulmonary artery for > 7 days because of technical difficulties.

Urine Nitrate Concentration
Average value of the nitrate concentration in urine samples was significantly higher in the NO group (28.0 ± 4.3 mmol/L, n = 5) than in the air group (0.5 ± 0.05mmol/L, n = 5) [p < 0.01].


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study was that continuous NO inhalation during recovery did not alter the regression process in the percentages of muscularized artery at the alveolar wall and duct levels, medial wall thickness of normally muscular artery, hematocrit, and RVH. Regression of vascular structural changes without NO during the recovery period was observed, similar to that of an earlier report.3

Inhaled NO did not show significant effect on body weight, food and water intake, and urine and stool volume, consistent with the previous report.19 The significant higher concentration of nitrate in urine in the NO group than in the control group suggested the effective supplementation of inhaled NO.

Vasoconstriction might exist during the development and recovery of chronic hypoxia-induced pulmonary vascular changes, so that vasodilators have been investigated to see the effect on regression. Nifedipine lessened the PH and vascular remodeling produced by chronic hypoxia, and it also reversed medial wall thickness of established PH.15 Although acute pulmonary vasodilation was induced by isoproterenol,5 long-term administration of isoproterenol did not enhance the regression of vascular changes. These vasodilators have effects on both systemic and pulmonary vasculature. Inhaled NO is used as a selective pulmonary vasodilator in experimental and clinical PH. Since pulmonary endothelial NO synthase and protein expression are upregulated during recovery from chronic hypoxia,20 supplementing endogenous NO with the inhaled NO gas during the recovery period might enhance vasodilation in the pulmonary artery. Channick et al10 reported that there were significant improvement effects on mPAP and pulmonary vascular resistance in patients with primary PH during short-term inhaled NO, and speculated that long-term inhaled NO might have a role in these patients. Yung et al12 reported the improvement effects of continued inhaled NO on the exercise capacity of a patient with idiopathic pulmonary fibrosis. But the effect of prolonged NO inhalation on regression of pulmonary vascular remodeling during the recovery period is unclear. Therefore, we designed this study to determine whether long-term inhaled NO could improve the regression during the recovery period in long-term hypoxia-induced PH. If inhaled NO had a favorable effect on the recovery process, the following was expected: inhaled NO improves the degree of the maximal recovery, maximal recovery was achieved earlier with NO inhalation than without NO inhalation, or both of the above. We could not detect any of these at least after 10 days and 30 days of the recovery period with inhaled NO.

The effect of continuous NO inhalation on the development and recovery of pulmonary hypertensive vascular remodeling might be different. During long-term hypoxic exposure, the continuous inhalation of NO reduces the development of pulmonary hypertensive changes.2122 During the development phase, there is hypoxia, but during the recovery phase there is no hypoxia, suggesting the magnitude of pulmonary vasoconstriction is less during the recovery period. The absence of hypoxia during the recovery phase might explain the difference in the effect of inhaled NO on vascular remodeling between development and recovery. Even if inhaled NO had an effect on the recovery process, it might be earlier, before 10 days of recovery. However, this might be unlikely or very subtle, if at all, because inhaled NO did not hasten the reduction of mPAP during the early recovery period between 1 day and 7 days after cessation of hypoxic exposure. Although short-term NO inhalation did reduce the mPAP by approximately 13% from the baseline pulmonary artery pressure in pulmonary hypertensive rats induced by 10 days chronic hypoxia,9 this level of reduction might have no effect on the regression of vascular changes.

Inhaled NO is used in postoperative patients with congenital heart disease complicated with PH.23 After surgical repair, hypertensive vascular changes do regress.24 If inhaled NO hastened this recovery process, postoperative NO inhalation would benefit patients by not only reducing functional pulmonary vasoconstriction but also by improving structural changes. We could not detect these additional effects of NO on the recovery process at least in a chronic hypoxia model of PH.


   Footnotes
 
Abbreviations: %AD = percentage of muscularized arteries at the alveolar duct level; %AW = percentage of muscularized arteries at the alveolar wall level; %MWT = percentage of medial wall thickness; mPAP = mean pulmonary artery pressure; NO = nitric oxide; PH = pulmonary hypertension; RVH = right ventricular hypertrophy; RV/LV + S = right ventricle/left ventricle plus septum

This work was supported in part by Grants-In-Aid for Scientific Research 09470326, 12470319, and 14370484 from the Japanese Ministry of Education, Science and Culture, and Grant for Pediatric Research 8C-02 and 11C-02 from the Ministry of Health and Welfare.

Dr. Jiang is supported by FY2002 JSPS (Japan Society for the Promotion of Science) postdoctoral fellowship for foreign researcher.

Received for publication December 26, 2002. Accepted for publication October 30, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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