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A major barrier to therapeutic development in neonates is a lack of standardized drug response measures that can be used as clinical trial endpoints. The ability to quantify treatment response in a way that aligns with relevant downstream outcomes may be useful as a surrogate marker for new therapies, such as those for bronchopulmonary dysplasia (BPD).To construct a measure of clinical response to dexamethasone that was well aligned with the incidence of severe BPD or death at 36 weeks' postmenstrual age.Retrospective cohort study.Level IV Neonatal Intensive Care Unit.Infants treated with dexamethasone for developing BPD between 2010 and 2020.Two models were built based on demographics, changes in ventilatory support, and partial pressure of carbon dioxide (pCONinety-five infants were treated with dexamethasone before 36 weeks. Compared to the baseline support and demographic data at the time of treatment, changes in ventilatory support improved ordinal model sensitivity and specificity. For the binary classification, BPD incidence was well aligned with risk levels, increasing from 16% to 59%.Incorporation of response variables as measured by changes in ventilatory parameters and pCO

Objective: To describe effectiveness of repeat dexamethasone for bronchopulmonary dysplasia (BPD) and to evaluate adverse effects on growth. Study design: Retrospective study of infants treated with 1 or 2 courses of dexamethasone for BPD. Effectiveness was defined as successful step-down in respiratory support by end of treatment. Adverse effects on growth were analyzed and compared to untreated controls. Results: A total of 132 dexamethasone-treated infants were identified. In total, 52% (69/132) of infants treated with initial dexamethasone achieved step-down in respiratory support compared to 38% (20/52) of infants with repeat dexamethasone. Growth trajectory did not significantly differ among infants treated with 1 or 2 courses of dexamethasone compared with controls (weight: P = 0.23, length: P = 0.68, and head circumference: P = 0.77). Conclusions: Repeat dexamethasone for BPD was less effective in weaning respiratory support compared to initial course. Changes in growth parameters to discharge were comparable between controls and infants treated with 1 or 2 dexamethasone courses.

Objective To determine if multidisciplinary team-based care of severe BPD/CLD infants improve survival to discharge. Design/methods Retrospective review of severe BPD/CLD infants cared for by dedicated multidisciplinary CLD team using consensus-driven protocols and guidelines. Results Total of 267 patients. Median gestational age was 26 weeks (IQR 24, 32); median birth-weight was 0.85 (IQR 0.64, 1.5). Twenty-four percent were preterm with severe BPD, 46% had other primary respiratory diseases (none BPD diseases). Total number of patients, proportion of patients with tracheostomy, prematurity, and genetic diagnoses increased over time. 88.8% survived to discharge. Unadjusted logistic regression showed that tracheostomy was not associated with odds of death; secondary pulmonary hypertension was associated with odds of tracheostomy (OR = 1.795 p value = 0.0264), or death (OR = 8.587 p value =

Objective The aim of this study is to determine patterns of neurally adjusted ventilatory assist (NAVA) use in ventilator-dependent preterm infants with evolving or established severe bronchopulmonary dysplasia (sBPD) among centers of the BPD Collaborative, including indications for its initiation, discontinuation, and outcomes. Study Design Retrospective review of infants with developing or established sBPD who were placed on NAVA after ≥4 weeks of mechanical ventilation and were ≥ 30 weeks of postmenstrual age (PMA). Results Among the 13 sites of the BPD collaborative, only four centers (31%) used NAVA in the management of infants with evolving or established BPD. A total of 112 patients met inclusion criteria from these four centers. PMA, weight at the start of NAVA and median number of days on NAVA, were different among the four centers. The impact of NAVA therapy was assessed as being successful in 67% of infants, as defined by the ability to achieve respiratory stability at a lower level of ventilator support, including extubation to noninvasive positive pressure ventilation or support with a home ventilator. In total 87% (range: 78–100%) of patients survived until discharge. Conclusion We conclude that NAVA can be used safely and effectively in selective infants with sBPD. Indications and current strategies for the application of NAVA in infants with evolving or established BPD, however, are highly variable between centers. Although this pilot study suggests that NAVA may be successfully used for the management of infants with BPD, sufficient experience and well-designed clinical studies are needed to establish standards of care for defining the role of NAVA in the care of infants with sBPD.

To identify factors leading to readmission for tracheostomized, ventilator-dependent infants2 years of age.Retrospective cohort study of 117 tracheostomized, ventilator-dependent infants followed through 2 years of age.Home ventilator use (at hospital discharge, 6 and 12 months of age), inhaled steroid use (at 12 and 24 months of age), oxygen dependence (at 6 and 12 months of age) and tracheostomy (at discharge, 6 and 12 months of age) were increased risks for rehospitalization. Equipment malfunction throughout the first 2 years also contributed to readmissions. Viral infection, with rhinovirus/enterovirus the most commonly identified pathogen, was the most common etiology for rehospitalization. Diuretic use and initial comorbid diagnoses were not associated with increased risk of rehospitalization.The risk for rehospitalization for infants requiring tracheostomy and ventilator support was affected by prolonged oxygen use, prolonged ventilator dependence, inhaled steroid use and equipment malfunction, and was equally distributed throughout the first 2 years of life.

The objective of the study was to compare the effect of two different dexamethasone regimens on respiratory outcomes of ventilator-dependent preterm infants. Retrospective study of ventilated preterm infants

Background Bronchopulmonary Dysplasia (BPD) is an orphan disease and advances in prevention and treatment are lacking. Clinical efficacy of systemic corticosteroid therapy to reduce the severity of lung disease and BPD is highly variable. Our objective was to assess whether candidate SNPs in corticosteroid metabolism and response genes are associated with short-term phenotypic response to systemic corticosteroids in infants at high risk for BPD. Methods Pharmacogenetic analysis of data from a large randomized controlled trial (TOLSURF) in infants treated with dexamethasone or hydrocortisone using multivariate linear regression. The primary outcome was change in Respiratory Severity Score (RSS, mean airway pressure × FiO2) at day 7 of corticosteroid treatment. Results rs7225082 in the intron of CRHR1 is significantly associated with the magnitude of decrease in RSS 7 days after starting treatment with systemic corticosteroid (meta-analysis p=2.8×10−4). Each T allele at rs7225082 is associated with a smaller absolute change in RSS at day 7, i.e. less response to systemic corticosteroids. Conclusion Genetic variability is associated with corticosteroid responsiveness with regard to respiratory status in preterm infants. Identification of genetic markers of corticosteroid responsiveness may allow for therapeutic individualization, with the goal of optimizing the risk to benefit ratio for an individual child.

Objective To report on the population of infants receiving a tracheostomy, identify acute post-tracheostomy clinical decompensations, and seek predictive markers associated with acute complications following the placement of a tracheostomy. Study Design Retrospective deidentified clinical data was provided by the Infant Pulmonary Data Repository at Children's Mercy Hospital, Kansas City. Data from infants undergoing tracheostomy from January 1, 2008 through September 30, 2016 were divided into one of two study groups based on clinical correlations: (1) no acute decompensations within 72 hours post-tracheostomy or (2) acute clinical decompensation defined as sustained escalation of respiratory care within the 72 hours following tracheostomy. Results Thirty-four percent of infants undergoing tracheostomy during this period developed acute post-tracheostomy clinical decompensations. Elevated pre-tracheostomy positive end expiratory pressure, mean airway pressure, and echocardiogram findings suggestive of pulmonary hypertension (PH) or ventricular dysfunction were associated with acute post-tracheostomy clinical decompensations. Additionally acute post-tracheostomy clinical decompensation was associated with higher rate of death prior to discharge. Conclusion Infants requiring higher respiratory support and infants with PH or ventricular dysfunction are at risk of acute post-tracheostomy clinical decompensation, thus identifying these patients may lead to better pre-tracheostomy counseling and potentially targeted treatments to decrease this risk.

Objective The objective of this study was to assess the outcomes of preterm infants who failed to extubate following initial treatment with steroids. Materials and Methods This is a retrospective cohort study of ventilator-dependent preterm infants treated with first course of systemic steroids to facilitate extubation. Outcomes of infants who successfully extubated were compared with infants who failed to extubate. Results In this study, 74 infants (mean gestation 25.4 ± 1.4 weeks and mean birth weight 764 ± 163 g) met inclusion criteria. Of these, 41 (55%) were successfully extubated and 33 (45%) were not. Baseline demographics were similar between the two groups. The primary outcome of severe bronchopulmonary dysplasia (BPD) or death at 36 weeks was higher among infants who failed to extubate (94 vs. 63%, p = 0.002). Severe BPD remained significantly higher even after adjustment for potential confounders (odds ratio: 12.2, 95% confidence interval: 2.1–70.5, p = 0.005). Extubation failure was also associated with substantially higher rate of tracheostomy (32 vs. 5%, p = 0.003) and gastrostomy tube placement (61 vs. 22%, p = 0.001), as well as longer days of hospitalization (179 ± 72 vs. 129 ± 44 days, p = 0.001) and mechanical ventilation (112 ± 89 vs. 52 ± 42 days, p Conclusion Failure to extubate after first course of systemic steroids for BPD is associated with poor prognostic implications.

OBJECTIVE. We compared serial measurements of inflammatory mediators and markers in infants treated with inhaled nitric oxide or placebo to assess the effects of inhaled nitric oxide therapy on lung inflammation during bronchopulmonary dysplasia. We investigated relationships between respiratory severity scores and airway concentrations of inflammatory markers/mediators.METHODS. As part of the Nitric Oxide (to Prevent) Chronic Lung Disease trial, a subset of 99 infants (52 placebo-treated infants and 47 inhaled nitric oxide-treated infants; well matched at baseline) had tracheal aspirate fluid collected at baseline, at 2 to 4 days, and then weekly while still intubated during study gas treatment (minimum of 24 days). Fluid was assessed for interleukin-1β, interleukin-8, transforming growth factor-β, N-acetylglucosaminidase, 8-epi-prostaglandin F2α, and hyaluronan. Results were normalized to total protein and secretory component of immunoglobulin A.RESULTS. At baseline, there was substantial variability of each measured substance and no correlation between tracheal aspirate fluid levels of any substance and respiratory severity scores. Inhaled nitric oxide administration did not result in any time-matched significant change for any of the analytes, compared with the placebo-treated group. There was no correlation between any of the measured markers/mediators and respiratory severity scores throughout the 24 days of study gas administration. In the posthoc analysis of data for inhaled nitric oxide-treated infants, there was a difference at baseline in 8-epi-prostaglandin F2α levels for infants who did (n = 21) and did not (n = 26) develop bronchopulmonary dysplasia at postmenstrual age of 36 weeks.CONCLUSIONS. Inhaled nitric oxide, as administered in this study, seemed to be safe. Its use was not associated with any increase in airway inflammatory substances.

OBJECTIVE This study aims to determine risk factors for rehospitalization in extremely premature infants. STUDY DESIGN A retrospective cohort study of 157 infants born

Objective. The pathogenesis of chronic lung disease (CLD) involves inflammation with proteolytic damage to lung extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that, acting in concert with their tissue inhibitors, tightly orchestrate extracellular matrix morphogenesis and repair after injury. Imbalances in their levels relative to that of their inhibitors have been implicated in diseases characterized by matrix disruption and remodeling. We investigated the possibility that imbalances in MMP-9 and MMP-2 relative to their tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, respectively, in tracheal aspirates of preterm infants may be involved in the development of CLD.Methods. Serial tracheal aspirates collected from birth until extubation in 49 ventilated preterm infants (24-32 weeks’ gestations) were analyzed for MMP-2, MMP-9, TIMP-1, and TIMP-2. Data normalized by TA values of free secretory component of immunoglobulin A were compared for CLD (n = 22) versus no CLD (n = 27). Also, known clinical predictors of CLD (gestational age, birth weight, and sex) were assessed for both groups. Association of predictors with the outcome CLD was assessed by logistic regression.Results. Mean gestational age was lower in CLD infants, but birth weight and gender were comparable for both groups. CLD infants had significantly lower TIMP-1 level with higher MMP-9/TIMP-1 ratio during the first 2 weeks of life and low TIMP-2 and MMP-2 levels during the first 3 days of life compared with no-CLD infants. Logistic regression analysis indicated that the findings are predictive of CLD.Conclusions. We conclude that low tracheal aspirate levels of TIMPs, with a high MMP-9/TIMP-1 ratio early in life, are associated with subsequent development of CLD.

Microvascular development is critical for normal lung maturation. The aims of this study were (1) to quantitatively and qualitatively assess lung microvascular growth in the human fetus, from 22 to 40 weeks’ gestation, and (2) to compare development in these infants to those with mild, moderate and severe chronic lung disease (CLD). Using 1- and 4-µm thick sections and electron microscopy, lungs were morphometrically assessed for surface density of distal air spaces; volume density of parenchymal vessels having an air-blood barrier (ABB); percent of distal air space wall having an ABB, and capillary loading, defined as ABB/mm2 of epithelial surface area. The percent of vessels with ABB increased in controls during development in parallel with increasing lung parenchyma. Infants with severe CLD had fewer ABBs and less capillary loading than controls up to 34 weeks’ post-conceptional age (PCA), but by 36–40 weeks, showed catch-up growth. Microvasculature vessel diameter, septal thickness, and air sac diameter at 36–40 weeks’ PCA were increased with severe CLD, and vessels were more distant from the air surface. We conclude that infants with severe CLD have both stunted secondary septation and microvascular development, but over time, the primary septal wall adapts by thinning and increasing the number of ABBs, thereby taking on the function of secondary septa.

Pulmonary hyperoxic injury manifests as widespread alveolar-epithelial and microvascular endothelial cell necrosis, resolution of which requires angiogenesis. We investigated the hypothesis that inhaled nitric oxide (iNO) and hyperoxia each decreases lung vascular endothelial growth factor (VEGF) expression but increases endostatin and that concurrent administration of both gases will show a greater effect. Piglets were randomized to breathe for 5 d room air (RA); RA + NO (RA + 50 ppm NO), O(2) (hyperoxia, F(I)O(2) >0.96), O(2) + NO, or O(2) + NO + REC (O(2) + NO plus recovery in 50% O(2) for 72 h. After the piglets were killed, we measured lung capillary leak, VEGF mRNA, VEGF, and endostatin protein in homogenates, plasma, and lavage. VEGF mRNA decreased significantly with O(2) and O(2) + NO compared with breathing RA (p < or = 0.05). VEGF protein declined in the experimental groups with a significant reduction in the recovery group compared with the RA group (p < or = 0.05). Similar but more dramatic, endostatin declined in all groups relative to the RA group (p < 0.001). Lavage fluid VEGF protein and lung capillary leak rose significantly with O(2) and O(2) + NO compared with RA, but endostatin was unchanged. At 72 h of recovery from hyperoxia, VEGF mRNA and lavage fluid VEGF but not lung VEGF protein had normalized. Hyperoxia and iNO suppresses lung endostatin expression, but iNO unlike hyperoxia alone does not alter lung VEGF production. Hyperoxia paradoxically raises lavageable VEGF levels. This latter effect and that on VEGF mRNA level but not protein is abrogated by recovery in reduced F(I)O(2) for 72 h.

OBJECTIVE To correlate intrauterine and postnatal growth with bronchopulmonary dysplasia (BPD) classification at 36 weeks postmenstrual age (PMA). STUDY DESIGN A retrospective cohort design reviewing medical records for infants

Clinical use of nitric oxide (NO) is usually in conjunction with high oxygen concentrations, the effects of which may include lung neutrophil accumulation, apoptosis and upregulation of antioxidant enzyme activity. To define the effects of NO on neutrophils from young piglets and its relationship to lung neutrophil dynamics during hyperoxia we exposed thirty piglets to room air (RA), RA+NO (50 ppm NO), O2 (FiO2≥0.96) or O2+NO for 5 days. Ten additional animals breathed RA+NO or O2+NO, then recovered in RA for 3 days before sacrifice. Neutrophil CD18 and intracellular oxidant production were measured by flow cytometry. Lung apoptosis were assessed by TUNEL assay. Lung myeloperoxidase, SOD and catalase were measured biochemically. When compared to RA group, there was significant reduction in neutrophil CD18 and intracellular oxidant production in the RA+NO group, but lung MPO was unchanged. The O2 and O2+NO groups did not differ in CD18 expression or in intracellular oxidant production, but had significant increase in lung myeloperoxidase compared to the RA group. Apoptosis increased significantly only in the O2+NO group. The O2 group showed significantly increased lung SOD and catalase activity compared to the RA group, whereas the RA+NO and O2+NO groups did not. We conclude that inhaled NO at 50 ppm decreases neutrophil CD18 expression as well as intracellular oxidant production. However, this effect does not impact lung neutrophil accumulation during concurrent hyperoxia. The combination of NO and O2 exposure produces an increase in lung apoptosis. Finally, NO may prevent upregulation of SOD and catalase activity during hyperoxia, potentially increasing injury.

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense-cored vesicles; membrane specializations were not well-developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to "parasynaptic" [Schmitt (1984) Neuroscience, 13:991-1001] or "volume" [Agnati et al. (1995) Neuroscience, 69:711-726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles. Previous studies from our laboratory [Chronwall et al. (1987) Endocrinology, 120:1201-1211; Chronwall et al. (1988) Endocrinology, 123:1992:1202] demonstrated heterogeneity in proopiomelanocortin (POMC) biosynthesis among individual melanotropes, prompting the hypothesis that the degree of innervation could govern the expression of certain molecules. We combined immunohistochemistry and in situ hybridization histochemistry to evaluate whether melanotrope molecular heterogenity is spatially correlated with axons and terminals. Tentatively, melanotropes expressing low levels of POMC and alpha1A subunit P/Q type Ca2+ channel mRNAs often were apposed to axons, whereas those with low levels of D2L receptor mRNA rarely were contacted by axons, suggesting that innervation could be one of the factors inducing and maintaining heterogeneity.

Non-cyclooxygenase-derived prostaglandin F2- (PGF2)-like compounds can be formed by membrane lipid peroxidation. We sought to characterize one member of this class of compounds, 8-epi-PGF2α, for its biological properties on the pulmonary vasculature in young piglets. We first compared 8-epi-PGF2α to a thromboxane (Tx) mimetic, U46,619, to determine relative pulmonary vasoconstrictive effects. We next determined if the vasoconstriction induced by both agonists would be reversed by 50 ppm of inhaled nitric oxide (NO). We also determined the degree of inhibition of U46,619 and of 8-epi-PGF2α by a Tx receptor antagonist, SQ 30,741. Anesthetized, ventilated piglets (18 ± 2 days, 3.7 ± 0.5 kg) were infused with randomly selected doses of U46,619 and of 8-epi-PGF2α to describe dose-response curves plotting pulmonary vascular resistance (PVRi) against dose. When a maximal dose was achieved, inhaled NO (50 ppm) was administered. Results for the calculated PVRi (mm Hg/l/min/kg) are as follows (mean ± SD): for U46,619, baseline value: 43 ± 10, peak dose: 165 ± 46, and peak dose with NO: 73 ± 33; for 8-epi-PGF2α, baseline value: 38 ± 9, peak dose: 138 ± 30, and peak dose with NO: 72 ± 25, and for both drugs, PVRi at peak dose was greater than at baseline (p < 0.001). PVRi at peak dose plus NO also remained elevated above baseline (p < 0.05; repeated measures ANOVA). The Tx receptor antagonist SQ 30,741 (1–10 mg/kg) inhibited U46,619-induced pulmonary vasoconstriction completely; however, the 1 mg/kg dose provided only 90% inhibition against 8-epi-PGF2α. We conclude that 8-epi-PGF2α can contribute to pulmonary vasoconstriction in young piglets in a dose-dependent reversible manner and that it acts primarily via pulmonary vascular Tx receptors.

Burn care and research have significantly improved over the past years. However, insurance coverage of such treatments does not reflect the improvements in this multi-disciplinary field. Government insurance policies in first world countries renown for burn care treatment, such as Switzerland and the United States, have not adapted to the complexity and longitudinal nature of burn care. Using case studies from both countries, we have analyzed both the institutional and policy approach to pediatric burn treatment coverage. Subsequently, by presenting the Shriners burn care model, we offer a policy recommendation to both the Swiss and the American governments to better their present legislation and infrastructure on pediatric burn coverage.

A prostaglandin F2-like compound, 8-epi-PGF2alpha, formed from oxidation of arachidonate, has been proposed as an indicator of lipid peroxidation. We determined whether tracheal aspirate or urinary 8-epi-PGF2alpha levels would differ over time or between infants in a control group and infants with severe respiratory failure. We correlated tracheal aspirate 8-epi-PGF2alpha levels with the fraction of inspired oxygen and with mean airway pressures at 24 and 48 hours of life. Levels in tracheal aspirates were in the range of 0 to 36 pg/microg of fSC of IgA and were higher in infants with severe pulmonary disorders compared with those in infants in the control group (p < 0.02). Urinary concentrations did not discriminate between sick infants and infants in the control group.

It was our objective to quantify platelet endothelial cell adhesion molecule-1 (PECAM-1) by immunohistochemistry in control infants of 22-50 weeks postconceptual age, and to correlate it with varying degrees of neonatal chronic lung disease (CLD). We tested the hypothesis that the density of PECAM-1 staining will positively correlate with increasing gestational age (GA) and the inflammatory process in CLD. A library of postmortem lung tissue of infants receiving ventilator care was accessed. The population consisted of 35 control infants exposed briefly to oxygen and positive pressure ventilation, and 31 infants who were 23-30 weeks GA with mild to severe CLD. A monoclonal anti-human PECAM-1 antibody was used to stain 5-microm paraffin sections. The slides were viewed at a magnification of x 40 by a blinded examiner. Twenty consecutive fields from standardly expanded tissue samples were viewed, and the volume density of PECAM-1 (V(V PECAM)) per parenchyma was measured, using point counting. In addition, 1-microm sections from 15 controls and 5 infants with CLD were stained with Toluidine blue and viewed under oil at a magnification of x 100, and the volume density of capillaries (V(V CAP)) and capillary load (CL) were calculated. The V(V PECAM) increased significantly with GA in controls (r = 0.72, P < 0.001). There was no relationship between V(V PECAM) and severity of CLD. Both V(V CAP) and CL increased significantly with GA (r = 0.93, P < 0.001; r = 0.94, P < 0.001, respectively). The infants with CLD had a normal or increased V(V CAP) and CL compared to controls. In summary, V(V PECAM), V(V CAP), and CL increased significantly with gestational age in control infants, but the postconceptional age range in CLD infants was too short to determine whether the V(V PECAM) changed. Infants with CLD had normal or increased V(V CAP) and CL compared to controls. The PECAM-1 immunostain does not appear to be a sensitive method for assessing capillary density in infants with CLD. These findings of normal or increased capillary load may represent a vascular adaptation for the lack of secondary septation and decreased surface area in CLD.

The interaction of two vasoactive substances, the vasodilator nitric oxide (NO) and the complex-acting peptide endothelin-1 (ET-1), may help explain the pathophysiology of pulmonary hypertension, an important part of many pulmonary disorders in neonates. To understand better the interactions of inhaled NO and ET-1, we investigated the effects of ET-1 infusions with and without inhaled NO in two groups of piglets, one group pretreated with L-nitro-arginine methylester (L-NAME) and the other not pretreated. Inhaled NO (60 ppm) was administered during infusion of 1.0 µg/kg or 2.5 µg/kg of ET-1. In animals not pretreated with L-NAME, the increase in PVR and in SVR induced by either dose of ET-1 was not reduced with administration of NO. The increase in systemic vascular resistance with ET-1 was greater (mean increase of 50% above baseline with 1.0 µg/kg ET-1 and 100% with 2.5 µg/kg ET-1 by 5 min) than the increase in PVR, but the PVR/SVR ratio did not change during ET-1 administration. In contrast, animals pretreated with L-NAME did demonstrate inhibition of ET-1-induced increase in PVR with NO. No differences in effects on SVR were noted. We conclude that ET-1-induced increases in PVR are not diminished by 60 ppm of inhaled NO unless there has been inhibition of endogenous NO production.

Substantial evidence indicates that the major precursors for retinal pigment epithelial (RPE) lipofuscin are molecular components of the photoreceptor outer segments. Previous experiments have demonstrated that the retinoids required for transduction by the photoreceptors promote RPE lipofuscin fluorophore formation. Animals deficient in these retinoids accumulate lipofuscin in the RPE at greatly reduced rates. The protein components of the photoreceptor outer segments also appear to be involved in the formation of RPE lipofuscin fluorophores. When degradation of phagocytosed photoreceptor outer segment proteins by the RPE is blocked, this tissue rapidly becomes engorged with phagosome-derived inclusions that have fluorescence properties similar to lipofuscin. Experiments were conducted to determine whether the development of lipofuscin-like fluorescence in these inclusions was dependent on the availability of retinoids. Rats were fed diets containing vitamin A either in the form of retinyl palmitate (+ A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (− A), whichdoes not support visual function. After the retinas of the − A animals had been depleted of retinoids involved in vision, animals from both groups were given intraocular injections of the protease inhibitor leupeptin. Two days later, the amounts of lipofuscin-like autofluorescence from the RPEs of rats in each group were determined. Leupeptin treatment produced an increase in this fluorescence in RPEs of the + A animals, but not in the RPEs of the rats fed the − A diet, despite the fact that phagosome-like inclusions accumulated in the RPE in both dietary groups. This finding suggests that retinoids involved in the visual process are probably directly involved in RPE lipofuscin fluorophore formation.

Inhaled nitric oxide (NO) is now established as a selective pulmonary vasodilator. Toxicity potential of NO however remains a great concern. The postulated injurious mechanisms include NO-induced activation of matrix metalloproteinases (MMP) with consequent breakdown of the alveolar extracellular matrix. Endothelin-1 (ET-1) is reportedly a marker of acute pulmonary injury. 8-Isoprostanes are lipid peroxidation products of oxyradical-induced cell membrane injury. We hypothesized that lung lavage fluid from animals exposed to hyperoxia and NO will show increased laminin and hydroxyproline contents as early markers of alveolar capillary basement membrane and matrix breakdown. Additionally, we reasoned that lung lavage fluid ET-1 and 8-isoprostane concentration will correlate with laminin and hydroxyproline concentration. To test this hypothesis, we designed the following study. Twenty-eight newborn piglets were randomized into four exposure groups to breathe for five days in a 200-liter exposure chamber. The groups were (i) room air (RA), (ii) RA+50 ppm NO, (RA+NO), (iii) hyperoxia(FiO2≥0.96) (O2), and (iv) hyperoxia + 50 ppm NO(O2+NO). Lung lavage fluid was obtained and analyzed by ELISA to quantitate laminin, ET-1, and 8-isoprostane concentrations. Lavage fluid hydroxyproline was measured by a colorimetric method. Mean hydroxyproline levels were about twenty fold higher in the O2 and O2 + NO group as compared to other groups. ET-1 concentration was higher in the O2 + NO group than in any other group (P

Pulmonary production of NO increases in adult animals exposed to endotoxin. Neonatal endotoxemia is usually accompanied by pulmonary hypertension. The existence of pulmonary hypertension in the presence of increased NO production is inconsistent with NO's role as a vasodilator. We examined pulmonary NO production in neonatal endotoxemia by measuring exhaled NO and by comparing plasma NO metabolite concentration in paired pulmonary artery and pulmonary vein samples. Ten to fourteen day old piglets were anesthetized, tracheostomized and instrumented with pulmonary artery and systemic arterial catheters. Each piglet received 20 mcg/kg E. Coli lipopolysaccharide infused over 30 minutes. PVR, SVR, exhaled NO and paired PA and PV plasma NO metabolites were measured at 60 minute intervals. One hundred twenty minutes after endotoxin infusion was completed, the piglets were randomized to receive either a nitric oxide synthase inhibitor (L-NAME or aminoguanidine), the substrate for NO production (L-Arginine) or saline control. PVR increased significantly from baseline and was sustained throughout the 240 minute experimental period with no difference among groups after randomization. There was no change in peak exhaled NO over time in the control group or in those receiving L-NAME or aminoguanidine. Exhaled NO increased significantly in the two hours following administration of L-Arginine (4.41 ± 2.6 to 8.1± 3.8 ppb, p < 0.01). There was no difference in plasma NO metabolites over time within groups or between groups. We conclude that neonatal endotoxemia does not acutely increase pulmonary production of NO. We speculate that an inability to increase NO production may play a role in pulmonary hypertension associated with neonatal sepsis.

Hyperoxia causes an increase in lung water, whereas NO, with both antioxidant and oxidant properties, could ameliorate or exacerbate oxidant-induced lung injury. The objective of this study was to assess the individual and combined effects of these gases on lung water and respiratory function. Mixed strain piglets (11-21 days old) were exposed to room air (RA)(n=6); RA plus 50 ppm NO (RA/NO) (n=8); hyperoxia > 0.96 FiO2 (O2) (n=6); or O2 plus 50 ppm NO(O2/NO) (n=6) for a maximum of 5 d, or to the onset of marked respiratory distress. At the end of the exposure, animals were anesthetized, had blood drawn from the right atrium for serum protein measurement, and were then sacrificed. Pleural fluid was quantitated and the lungs were removed en bloc and weighed. The right lung underwent bronchoalveolar lavage with 175 ml of saline for protein measurement. A 10-gram lung sample from the left upper lobe was used to measure extravascular lung water (EVLW). Animals exposed to RA or RA/NO appeared normal after 5 days, whereas the O2 group, sacrificed at 113 ± 10 hrs, and the O2/NO group, sacrificed at 102 ± 17 hrs, had severe respiratory distress. RA animals, with or without NO, had no pleural fluid, but the O2 and O2/NO pigs had pleural effusions of 15.8 ± 31.7 ml and 45.5± 54.4 ml, respectively, not different from each other. In the RA and RA/NO groups, wet lung weight was not different (56.3 ± 12.2 g and 50.0± 10.9 g, respectively), nor was the EVLW/dry lung (4.3 ± 0.2 ml/g and 4.5 ± 0.2 ml/g). However, these measures were significantly greater (p

Endothelin-1 (ET-1) is a potent, endothelial-derived vasoconstricting agent which may be an important modulator of perinatal vascular tone. The development of techniques to assay plasma ET-1 levels provides an opportunity to investigate the role of ET-1 in a variety of perinatal disorders. To date, however, these assays have revealed widely varying results, which may be due to inherent differences in purification/detection methods. Normally, ET-1 is close to zero, but increased levels in cord blood have been reported. Most of the reports of ET-1 levels are based on a radioimmunoassay (RIA) technique. There are three antigen-antibody assays available. We compared the ET-1 levels in cord blood of 8 full term, healthy neonates using: 1] an RIA kit (Peninsula Laboratories, Belmont, CA), which uses rabbit anti-ET-1 serum specific for the peptide; 2] an EIA kit (Cayman Chemical, Ann Harbor, MI), which uses acetylcholinesterase (AChE) as substrate; and 3] an EIA kit (R&D Systems, Minneapolis, MN) which uses an antibody to ET-1 conjugated to horseradish peroxidase (HRP). The purification methods also differ between the three methods. Each sample was divided into three 1.0 ml plasma aliquots, and each aliquot was analyzed in duplicate according to the manufacturer's specifications for each kit. Duplicate aliquots are averaged and shown in pg ET-1/ml plasma. In spite of almost identical x ± SD, the variance between the assays with the most comparable results (RIA/HRP) was 34.3± 27.8% (range = 3-76.2%) (n=8). Plasma ET-1 measurements may have prognostic value, but interpretation of immunoassays of ET-1 is limited, due to differences in purification procedures, antibody specificity, sensitivity of the assay and available sample size. Table

The interaction in the pulmonary circulation of two endogenously produced vasoactive substances, the vasodilator nitric oxide (NO) and the complex-acting peptide endothelin-1 (ET-1), may help determine both the severity of and response to treatment of pulmonary hypertension in neonates. To examine potential interactions, we investigated the effects of ET-1 infusions with and without inhaled NO. We studied ten, 14-21 day old, anesthetized, mechanically ventilated, piglets. We first determined if inhalation of NO altered pulmonary vascular resistance (PVR) in these normal animals breathing ambient air. Results: Table The percent decline in PVR with NO was variable, ranging from 0% to a 45% decrease. Percent decrease in PVR with NO was not correlated with the initial pulmonary artery pressure or with pulmonary blood flow. Next, we investigated the effects of concomitant administration of inhaled NO (60 ppm) during randomly administered low (0.1 μg/kg) or high (0.25 μg/kg) dose infusion of ET-1. Results (x ± SD): Table The acute increase in PVR with ET-1 was not moderated by inhaled NO when expressed as either absolute or percent increase in PVR. The increase in systemic vascular resistance with ET-1 was proportionately greater (mean increase of 50% above baseline with low ET-1 and 100% with high ET-1 by 5 min) than the increase in PVR. We conclude that, while breathing ambient air, PVR can be reduced in young animals by inhaled NO. We also conclude that ET-1-induced increases in PVR are not diminished by inhaled NO. ET-1 and inhaled NO may act at different sites in the pulmonary vasculature.

The contribution to neonatal pulmonary injury by reactive oxygen species has been difficult to assess. The recent discovery of a novel class of prostaglandins, the isoprostanes, may help as these substances appear to be generated in response to lipid peroxidation. Isoprostanes are noncylooxygenase derived and are produced in vivo. They may contribute to pulmonary pathophysiology by their vasoconstrictive properties. We determined if 8-epi-PGF2α, a member of the isoprostane family, would be detectable in the urine or tracheal aspirates of infants ≥ 37 weeks GA with pulmonary disorders treated with assisted ventilation and FiO2 of 1.0 and in control infants without pulmonary disorders, breathing room air. Isoprostane levels were measured by enzyme immune assay (EIA). Urine levels were normalized by the urine creatinine and tracheal aspirate levels by free Secretory Component (fSC) of IgA, also measured by EIA. Results are as follows: Table The 24 and 48 hr tracheal aspirate values in sick infants were not different. When sick and control groups combined data were compared, there was a significant difference (p