Physiologic reflexes and their impact on resuscitation of the newborn.

Most depressed newborn infants respond promptly to minimal intervention. When that does not happen, a thorough understanding of the physiologic reflexes that promote and prevent, prompt restoration of heart rate, blood pressure, and respiration is crucial. This article will review data demonstrating that lung inflation, in the purely mechanical sense, is a key element in a successful resuscitation. A complicated resuscitation is most often because of the caretaker's lack of awareness of basic cardiopulmonary physiology than to an unusually depressed infant.

Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus.

Endothelial (e) nitric oxide synthase (NOS) activity modulates pulmonary vascular tone in the normal fetus and decreases pulmonary vascular resistance (PVR) at birth. Mechanisms contributing to sustained elevations of PVR and the failure of postnatal adaptation at birth are uncertain but may include decreased eNOS activity. To test this hypothesis, we studied the effects of chronic intrauterine pulmonary hypertension on lung eNOS content and NOS activity in an ovine model of perinatal pulmonary hypertension and in normal lambs. We measured eNOS mRNA and protein content by Northern and Western blot analyses, respectively. Calcium-dependent and total NOS activities were determined by assaying the conversion of L-[14C]arginine to L-[14C]citrulline from lung homogenates. To determine the effects of intrauterine hypertension on lung eNOS content, fetal lung tissue was harvested 8-12 days after intrauterine closure of the ductus arteriosus (DA) performed at 125-128 days of gestation (term = 147 days). Although positive immunostaining for eNOS persisted in lung vascular endothelium, eNOS protein content was reduced by 48%, as measured by Western analysis (P < 0.001). Chronic hypertension reduced lung eNOS mRNA content by 30% (P < 0.05). Compared with age-matched controls, Ca(2+)-dependent NOS activity was decreased after DA ligation by 75% (P < 0.01). We conclude that chronic intrauterine pulmonary hypertension decreases eNOS in the fetal lung. We speculate that decreased NO production contributes to failure of postnatal adaptation in this experimental model of persistent pulmonary hypertension of the newborn.

Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease.

To determine the effects of inhaled NO (iNO) on pulmonary edema and lung inflammation in experimental hyaline membrane disease (HMD), we measured the effects of iNO on pulmonary hemodynamics, gas exchange, pulmonary edema, and lung myeloperoxidase (MPO) activity in extremely premature lambs (115 d of gestation, 0.78 term). In protocol 1, we measured the effects of iNO (20 ppm) on lung vascular endothelial permeability to 125I-labeled albumin (indexed to blood volume using 57Cr-tagged red blood cells) during 1 h (n = 10) and 3 h (n = 14) of conventional mechanical ventilation with FiO2 = 1.00. In comparison with controls, iNO improved pulmonary hemodynamics and gas exchange, but did not alter lung weight-to-dry weight ratio or vascular permeability to albumin after 1 or 3 h of mechanical ventilation. To determine whether low dose iNO (5 ppm) would decrease lung neutrophil accumulation in severe HMD, we measured lung MPO activity after 4 h of mechanical ventilation with or without iNO (protocol 2). Low dose iNO improved gas exchange during 4 h of mechanical ventilation (PaO2 at 4 h: 119 +/- 35 mm Hg iNO versus 41 +/- 7 mm Hg control, p < 0.05), and reduced MPO activity by 79% (p < 0.05). We conclude that low dose iNO increases pulmonary blood flow, without worsening pulmonary edema, and decreases lung neutrophil accumulation in severe experimental HMD. We speculate that in addition to its hemodynamic effects, low dose iNO decreases early neutrophil recruitment and may attenuate lung injury in severe HMD.

Role of nitric oxide and cGMP system in regulation of ductus arteriosus tone in ovine fetus.

Although endogenous nitric oxide (NO) modulates basal tone in the fetal pulmonary and systemic circulations, little is known about its role in regulating ductus arteriosus (DA) tone. Immunostaining of DA tissue from late-gestation fetal lambs demonstrated strong staining for endothelial NO synthase (eNOS) in DA endothelium. To study the physiological role of the NO and guanosine 3',5'-cyclic monophosphate (cGMP) system in the DA in vivo, we measured the hemodynamic effects of NG-nitro-L-arginine (L-NNA; 30 mg), a NOS inhibitor, methylene blue (40 mg), a guanylate cyclase inhibitor, and indomethacin (0.8 mg), a cyclooxygenase inhibitor, in 10 chronically prepared late-gestation fetal lambs. L-NNA increased main pulmonary artery (MPA) and aortic pressures (P < 0.05 vs. baseline) but did not change the pressure gradient between the MPA and the aorta. L-NNA caused a small decrease in DA flow and a slight rise in resistance across the DA. Methylene blue increased both MPA pressure and the pressure gradient between the MPA and the aorta from 0.3 +/- 0.2 (baseline) to 7.0 +/- 2.7 mmHg (P < 0.05). Indomethacin increased both MPA pressure and the pressure gradient between the MPA and the aorta from 1.1 +/- 0.4 (baseline) to 6.3 +/- 1.5 mmHg (P < 0.05) after 40 min. Indomethacin decreased DA flow and increased DA resistance. We conclude that eNOS is in fetal DA endothelial cells and that NOS inhibition causes constriction of the DA in vivo. DA constriction after NOS inhibition is minimal, especially in comparison with cyclooxygenase inhibition. Methylene blue also constricts the DA, suggesting that guanylate cyclase activity contributes to DA relaxation. We speculate that, although the NO and cGMP system modulates DA tone, prostaglandins may play a greater role.

Maturation-related changes in endothelial nitric oxide synthase immunolocalization in developing ovine lung.

It is unknown whether high fetal pulmonary vascular tone is due in part to absent or decreased endothelial nitric oxide synthase (eNOS), the enzyme that produces nitric oxide in the vascular endothelium. To determine the timing of appearance and maturational changes of eNOS in the developing pulmonary circulation, we performed immunohistochemistry in lungs from fetal, neonatal, and adult sheep. Using a mouse monoclonal antibody against bovine aortic eNOS, we found immunoreactive eNOS selectively in the endothelium and it was present at all fetal ages. Immunoreactivity was seen as early as 29% gestation in the developing capillaries coursing through fetal mesenchyme. By 6 days after birth, immunoreactivity was decreased in most vessels and nearly absent in the distal pulmonary arteries of adult animals. We conclude that immunoreactive eNOS is present very early in fetal life and appears to decrease postnatally. We speculate that the early presence of eNOS in the fetal lung supports a possible role for endogenous nitric oxide activity in the regulation of vascular tone or angiogenesis in the developing pulmonary circulation.

Agarose infiltration improves morphology of cryostat sections of lung.

BACKGROUND: Optimal morphology and immunohistochemistry of the lung requires the organ to remain fully distended. When the lung is prepared for frozen sections, especially during the stage of cryoprotection, liquid fixatives leak out and the lung tends to collapse. We sought to develop a new technique to improve the morphology of frozen sections of the lung while facilitating tissue handling and immunoreactivity. EXPERIMENTAL DESIGN: Human, fetal sheep and adult rat lungs were distended with 1% low temperature melting agarose and vessels were perfused with a 1% paraformaldehyde solution. RESULTS: The agarose infiltration permitted easy handling of the lung tissue, and maintained lung architecture during cryoprotection. Agarose infiltration and paraformaldehyde fixation provided excellent morphology, that was comparable to paraffin-embedded tissue. The structural preservation was especially noticeable with human lung that remained well distended with the agarose technique. Because of the mild fixation and good morphology, precise localization of antigenic sites was possible for the surfactant proteins SP-A, SP-C and SP-D, cytokeratin, and alveolar macrophage markers. CONCLUSIONS: This procedure can improve special fixation and embedding protocols for lung immunocytochemistry and immunofluorescence.