Inhaled nitric oxide effects on lung structure and function in chronically ventilated preterm lambs

Abstract

Rationale: Inhaled nitric oxide (iNO) can reverse neonatal pulmonary hypertension and bronchoconstriction and reduce proliferation of cultured arterial and airway smooth muscle cells.

Objectives: To see if continuous iNO from birth might reduce pulmonary vascular and respiratory tract resistance (PVR, RE) and attenuate growth of arterial and airway smooth muscle in preterm lambs with chronic lung disease.

Methods: Eight premature lambs received mechanical ventilation for 3 weeks, four with and four without iNO (5-15 ppm). Four term lambs, mechanically ventilated without iNO for 3 weeks, served as additional control animals.

Measurements: PVR and RE were measured weekly. After 3 weeks, lung tissue was processed for quantitative image analysis of smooth muscle abundance around small arteries (SMart) and terminal bronchioles (SMtb). Radial alveolar counts were done to assess alveolar number. Endothelial NO synthase (eNOS) protein in arteries and airways was measured by immunoblot analysis.

Main results: At study's end, PVR was similar in iNO-treated and untreated preterm lambs; PVR was less in iNO-treated preterm lambs compared with term control animals. RE in iNO-treated lambs was less than 40% of RE measured in preterm control animals. SMart was similar in iNO-treated and both groups of control lambs; SMtb in lambs given iNO was significantly less (approximately 50%) than in preterm control animals. Radial alveolar counts of iNO-treated lambs were more than twice that of preterm control animals. eNOS was similar in arteries and airways of iNO-treated preterm lambs compared with control term lambs.

Conclusions: iNO preserves structure and function of airway smooth muscle and enhances alveolar development in preterm lambs with chronic lung disease.

Figure 1.

Respiratory tract and pulmonary vascular resistances (mean and SD) in four preterm lambs that received iNO at 5–15 ppm (dark bars) compared with four preterm lambs that did not receive inhaled nitric oxide (iNO; white bars) and four term lambs that did not receive iNO (hatched bars) during 3 weeks of mechanical ventilation. *Significant difference compared with the group of four term lambs, p < 0.05; ^†significant difference compared with the group of four preterm lambs given iNO, p < 0.05. BW = bodyweight.

Figure 2.

Airway and pulmonary artery smooth muscle area (mean and SD) in four preterm lambs that received iNO at 5–15 ppm (dark bars) compared with four preterm lambs that did not receive iNO (white bars) and four term lambs that did not receive iNO (hatched bars) during 3 weeks of mechanical ventilation. *Significant difference compared with the group of four preterm lambs not given iNO, p < 0.05; ^†significant difference compared with the group of four term lambs, p < 0.05.

Figure 3.

Representative photomicrographs showing smooth muscle wall thickness between the internal and external elastic laminae (arrows; Hart's elastin stain) of terminal bronchioles (TB) in lungs of preterm lambs that were mechanically ventilated for 3 weeks either without iNO (control, left) or with iNO (5–15 ppm, right).

Figure 4.

Lung tissue images illustrating differences in radial alveolar counts for preterm lambs that were mechanically ventilated for 3 weeks without iNO (left) or with iNO (right). Radial alveolar counts averaged 1.5 ± 0.3 alveoli/terminal bronchiole in lambs that did not receive iNO and 3.6 ± 1.5 alveoli/terminal bronchiole in lambs that received iNO. The arrow points to a secondary crest (alveolar septa). TRU = terminal respiratory unit, which is the landmark for measuring radial alveolar counts.

Figure 5.

Radial alveolar count and capillary surface density (mean and SD) in lungs of four preterm lambs that received iNO at 5–15 ppm (dark bars) compared with four preterm lambs that did not receive iNO (white bars) and four term lambs that did not receive iNO (hatched bars) during 3 weeks of mechanical ventilation. *Significant difference compared with the group of four term lambs, p < 0.05; ^†significant difference compared with the group of four preterm lambs not given iNO, p < 0.05.

Figure 6.

Immunoblot results for endothelial NO synthase (eNOS) in dissected third- and fourth-generation intrapulmonary airways (left) and arteries (right) excised from lungs of 1-day-old term lambs and preterm lambs given iNO, 5–15 ppm, during 3 weeks of mechanical ventilation. Term lambs were at the same postconceptional age as the preterm lambs, but did not receive mechanical ventilation or iNO. Densitometry showed no significant differences for either airways or arteries.

Figure 7.

Summary data (mean and SD) for eNOS protein measured by immunoblot analysis of excised third- and fourth-generation intrapulmonary arteries (dark bars) and airways (light bars) from four groups of lambs: unventilated 1-day-old and 3-week-old lambs born at term (controls), and 3-week-old preterm lambs that received mechanical ventilation for 3 weeks either without iNO (third group from left) or with iNO (right). Results are plotted relative to 1-day-old term lambs (left) that were at the same postconceptional age as the ventilated preterm lambs. Note that eNOS protein abundance was similar in arteries and airways obtained from term control lambs and from preterm lambs that received iNO, whereas eNOS protein was significantly less in airways and arteries of preterm lambs that were ventilated for 3 weeks without iNO. Reference 36 displays all immunoblot results that are depicted in this figure except for those of iNO-treated preterm lambs, which are shown in Figure 6. *Significant difference compared with 1-day-old and 3-week-old term lambs and preterm lambs that were mechanically ventilated with iNO for 3 weeks, p < 0.05.