Peter Mathew MacFarlane

Background: Continuous positive airway pressure (CPAP) is a primary non-invasive respiratory support modality for preterm infants. While there are benefits to CPAP, evidence suggests it may contribute to long-term wheezing and airway hyperreactivity (AHR) in preterm infants. Most preterm neonates also receive caffeine in addition to CPAP, which is associated with decreased rates of BPD. In a mouse model, we investigated whether caffeine administration during neonatal CPAP prevents long-term AHR. Methods: Neonatal mice were fitted with a custom-made mask and received CPAP (6 cmH ₂ 0) for 3 h/day for the first 7 postnatal days. Mice also received a subcutaneous injection of caffeine (10 mg/kg/day) or saline (control mice) immediately prior to each CPAP session. Two weeks after treatment, mice were assessed for AHR and parameters of lung remodeling. Results: CPAP resulted in long-term AHR (vs control mice), which was associated with increased airway αSM-actin expression, epithelial and alveolar septal wall thickening, and decreased radial alveolar counts. Co-administration of caffeine prevented CPAP effects. Conclusion: Early caffeine administration protected against the long-term effects of CPAP on AHR and lung remodeling. These data demonstrate that neonatal caffeine benefits long-term respiratory function after CPAP administration by a direct effect on lung and airway development. Impact: Neonatal CPAP may have long-term adverse consequences to the immature lung. This study demonstrates that caffeine attenuates the adverse effects of CPAP on lung development and airway remodeling. The results enhance our understanding of the complex effects that caffeine can have on lung development. CPAP may be a contributor to wheezing disorders of former preterm infants, but can be prevented by caffeine therapy. This study raises the clinical significance of CPAP effects on lung development and how caffeine could be optimized to help mitigate its adverse consequences.

Mechanical forces associated with positive-pressure respiratory support used in the intensive care of preterm infants can have both positive and inadvertent consequences for lung development. Positive pressure support improves pulmonary gas exchange, limits atelectasis, and improves systemic oxygenation, but the injurious effects of invasive modalities, such as mechanical ventilation, on lung development are also well known. However, there are major gaps in our understanding of the adverse effects of less invasive forms of ventilation. Here, continuous positive airway pressure (CPAP) is one of the most common respiratory support modalities, representing the least invasive of the positive pressure spectrum. A major limitation in studying human airway development is the lack of age-appropriate models and the ability to administer CPAP to small neonatal animals. We resolved this problem by developing a mouse model of neonatal CPAP delivered daily to awake, un-anesthetized newborn mice -- a model that utilizes clinically relevant CPAP levels at a stage of mouse lung development that corresponds with preterm infants when they are likely to receive CPAP in the ICU. Here, we demonstrate our CPAP model, which includes a convenient, custom-designed system that can deliver CPAP non-invasively to the un-anesthetized newborn with easily adjustable levels. We also provide a demonstration of the precision cut lung slice method for the measurement of airway hyperreactivity ex vivo. The PCLS is described here because it provides a convenient functional readout of the pulmonary system, which can be technically challenging in small (neonatal) animal models, and also has wide applicability to other models unrelated to CPAP. We show data demonstrating that CPAP elicits airway hyperreactivity, which may have important clinical implications for the choice of respiratory support modality in the intensive care of preterm infants.

Mechanical forces associated with positive-pressure respiratory support used in the intensive care of preterm infants can have both positive and inadvertent consequences for lung development. Positive pressure support improves pulmonary gas exchange, limits atelectasis, and improves systemic oxygenation, but the injurious effects of invasive modalities, such as mechanical ventilation, on lung development are also well known. However, there are major gaps in our understanding of the adverse effects of less invasive forms of ventilation. Here, continuous positive airway pressure (CPAP) is one of the most common respiratory support modalities, representing the least invasive of the positive pressure spectrum. A major limitation in studying human airway development is the lack of age-appropriate models and the ability to administer CPAP to small neonatal animals. We resolved this problem by developing a mouse model of neonatal CPAP delivered daily to awake, un-anesthetized newborn mice -- a model that utilizes clinically relevant CPAP levels at a stage of mouse lung development that corresponds with preterm infants when they are likely to receive CPAP in the ICU. Here, we demonstrate our CPAP model, which includes a convenient, custom-designed system that can deliver CPAP non-invasively to the un-anesthetized newborn with easily adjustable levels. We also provide a demonstration of the precision cut lung slice method for the measurement of airway hyperreactivity ex vivo. The PCLS is described here because it provides a convenient functional readout of the pulmonary system, which can be technically challenging in small (neonatal) animal models, and also has wide applicability to other models unrelated to CPAP. We show data demonstrating that CPAP elicits airway hyperreactivity, which may have important clinical implications for the choice of respiratory support modality in the intensive care of preterm infants.

Background/Objectives: Preterm infants are at risk of developing the chronic lung condition of bronchopulmonary dysplasia (BPD), with associated alveolar simplification and airway hyperreactivity. Inhibition of S-nitrosoglutathione (GSNO) reductase has been shown to rescue airway hyperreactivity in a murine model of BPD. Here, we investigate the effects of early treatment with N6022, a pharmacologic GSNO reductase inhibitor. Methods: Newborn C57BL/6 mice were exposed to either 21% (control) or 60% oxygen (BPD model) for 5 days after birth. Pups simultaneously received either subcutaneous saline or varying doses of N6022 for 5 days during hyperoxia exposure. Pups were then recovered in room air to 3 weeks postnatal age. H&E-stained lungs were analyzed for alveolar simplification and airway tethering. In vivo airway reactivity to inhaled methacholine was measured using a flexiVent system. In separate littermates, lungs were immediately harvested after 5 days of hyperoxia for protein quantification via automated capillary Westerns. Results: Alveolar simplification and decreased airway tethering were noted in the 60% + saline group. Pups treated with N6022 during hyperoxia displayed dose-dependent improvements in alveolar simplification and airway tethering. Similarly, hyperoxia-exposed pups had increased airway reactivity, as measured by elevated respiratory system resistance and elastance responses to methacholine. Treatment with 10 mg/kg/day N6022 during hyperoxia resulted in decreased resistance and elastance responses. TGF-β expressions were elevated in the 60% + saline group and attenuated in the 60% + N6022 groups. Conclusions: Early exposure to GSNO reductase inhibitors such as N6022 can prevent hyperoxia-induced alveolar simplification and airway hyperreactivity, with lasting effects even after cessation of treatment.

Caffeine is administered to preterm infants in neonatal intensive care units for prevention and treatment of apnea of prematurity. Although caffeine’s primary effect is to impact the respiratory drive of preterm infants, caffeine also has anti-inflammatory properties. This study investigated the role of caffeine on the inflammatory gene expression in THP-1 pre-monocytes exposed to lipopolysaccharide (LPS) in vitro, mimicking a clinical pro-inflammatory scenario. The effects of different physiologic dosages of caffeine administration post-LPS (treatment with caffeine) and pre-LPS (prophylaxis with caffeine) on pro-inflammatory gene expressions (TNF-α, NF-κB, IL-8, PPARγ) of the THP-1 cells were investigated. The post-LPS group showed a dose-dependent decrease in TNF-α at a caffeine concentration of 100 μM and NF-κB gene expression at 50 and 100 μM, with the implication that this is an optimal anti-inflammatory caffeine concentration range. Clinically, this would correspond to a serum caffeine level between 10 and 20 μg/mL, respectively. For the pre-LPS group, TNF-α and NF-κB gene expression decreased at all studied caffeine concentrations. These findings point to caffeine’s potential therapeutic capacity in regulating monocyte inflammatory responses to gram-negative infections in addition to regulating neuron response in the brainstem for preterm infants.

Introduction: Hyaluronan (HA) is a major component of the extracellular matrix. Increased pulmonary HA concentrations are associated with several respiratory disorders and is a pathophysiological feature of lung disease. We investigated whether elevated urine HA is a biomarker of an unfavorable 40-week respiratory outcome in preterm infants. Methods: Infants comprised a cohort of preterm neonates <31 weeks gestational age (GA) from the Prematurity-Related Ventilatory Control (Pre-Vent) multicenter study. HA was quantified in urine obtained at 1 week and 1 month of age. Respiratory status at 40 weeks post-menstrual age (PMA) was classified as unfavorable [either (1) deceased at or before 40 weeks PMA, (2) an inpatient on respiratory medication, O2 or other respiratory support at 40 weeks, or (3) discharged prior to 40 weeks on medications/O2/other respiratory support], or favorable (alive and previously discharged, or inpatient and off respiratory medications, off O2, and off other respiratory support at 40 weeks PMA). The association between urine HA and the unfavorable 40 week PMA outcome was examined using a multivariate logistic generalized estimation equation model. Results: Infants with higher HA at 1 week (but not 1 month) showed increased odds of unfavorable respiratory outcome at 40 weeks PMA (OR [95% CI] = 1.87 per 0.01 mg [1.27, 2.73]). Discussion and Conclusion: Neonatal urine screening for HA could identify infants at risk for death or need for respiratory support at term-corrected age (40 weeks PMA). The relationship between elevated HA at 1 week and an unfavorable 40 week outcome was stronger in infants with lower GA.

Background Hypoxemia is a physiological manifestation of immature respiratory control in preterm neonates, which is likely impacted by neurotransmitter imbalances. We investigated relationships between plasma levels of the neurotransmitter serotonin (5-HT), metabolites of tryptophan (TRP), and parameters of hypoxemia in preterm neonates. Methods TRP, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and kynurenic acid (KA) were analyzed in platelet-poor plasma at ~1 week and ~1 month of life from a prospective cohort of 168 preterm neonates <31 weeks gestational age (GA). Frequency of intermittent hypoxemia (IH) events and percent time hypoxemic (<80%) were analyzed in a 6 h window after the blood draw. Results At 1 week, infants with detectable plasma 5-HT had fewer IH events (OR (95% CI) = 0.52 (0.29, 0.31)) and less percent time <80% (OR (95% CI) = 0.54 (0.31, 0.95)) compared to infants with undetectable 5-HT. A similar relationship occurred at 1 month. At 1 week, infants with higher KA showed greater percent time <80% (OR (95% CI) = 1.90 (1.03, 3.50)). TRP, 5-HIAA or KA were not associated with IH frequency at either postnatal age. IH frequency and percent time <80% were positively associated with GA < 29 weeks. Conclusions Circulating neuromodulators 5-HT and KA might represent biomarkers of immature respiratory control contributing to hypoxemia in preterm neonates. Impact Hypoxemia events are frequent in preterm infants and are associated with poor outcomes. Mechanisms driving hypoxemia such as immature respiratory control may include central and peripheral imbalances in modulatory neurotransmitters. This study found associations between the plasma neuromodulators serotonin and kynurenic acid and parameters of hypoxemia in preterm neonates. Imbalances in plasma biomarkers affecting respiratory control may help identify neonates at risk of short- and long-term adverse outcomes.