Impact of neonatal noninvasive resuscitation strategies on lung mechanics, tracheal pressure, and tidal volume in preterm lambs.

This study investigated the relationship between three respiratory support approaches on lung volume recruitment during the first 2 h of postnatal life in preterm lambs. We estimated changes in lung aeration, measuring respiratory resistance and reactance by oscillometry at 5 Hz. We also measured intratracheal pressure in subsets of lambs. The first main finding is that sustained inflation (SI) applied noninvasively (Mask SI; n = 7) or invasively [endotracheal tube (ETT) SI; n = 6] led to similar rapid lung volume recruitment (∼6 min). In contrast, Mask continuous positive airway pressure (CPAP) without SI (n = 6) resuscitation took longer (∼30-45 min) to reach similar lung volume recruitment. The second main finding is that, in the first 15 min of postnatal life, the Mask CPAP without SI group closed their larynx during custom ventilator-driven expiration, leading to intratracheal positive end-expiratory pressure of ∼17 cmH2O (instead of 8 cmH2O provided by the ventilator). In contrast, the Mask SI group used the larynx to limit inspiratory pressure to ∼26 cmH2O (instead of 30 cmH2O provided by the ventilator). These different responses affected tidal volume, being larger in the Mask CPAP without SI group [8.4 mL/kg; 6.7-9.3 interquartile range (IQR)] compared to the Mask SI (5.0 mL/kg; 4.4-5.2 IQR) and ETT SI groups (3.3 mL/kg; 2.6-3.7 IQR). Distinct physiological responses suggest that spontaneous respiratory activity of the larynx of preterm lambs at birth can uncouple pressure applied by the ventilator to that applied to the lung, leading to unpredictable lung pressure and tidal volume delivery independently from the ventilator settings.NEW & NOTEWORTHY We compared invasive and noninvasive resuscitation on lambs at birth, including or not sustained inflation (SI). Lung volume recruitment was faster in those receiving SI. During noninvasive resuscitation, larynx modulation reduced tracheal pressure from that applied to the mask in lambs receiving SI, while it led to increased auto-positive end-expiratory pressure and very large tidal volumes in lambs not receiving SI. Our results highlight the need for individualizing pressures and monitoring tidal volumes during resuscitation at birth.

Mesenchymal stromal cell extracellular vesicles improve lung development in mechanically ventilated preterm lambs.

Novel therapies are needed for bronchopulmonary dysplasia (BPD) because no effective treatment exists. Mesenchymal stromal cell extracellular vesicles (MSC-sEVs) have therapeutic efficacy in a mouse pup neonatal hyperoxia BPD model. We tested the hypothesis that MSC-sEVs will improve lung functional and structural development in mechanically ventilated preterm lambs. Preterm lambs (∼129 days; equivalent to human lung development at ∼28 wk gestation) were exposed to antenatal steroids, surfactant, caffeine, and supported by mechanical ventilation for 6-7 days. Lambs were randomized to blinded treatment with either MSC-sEVs (human bone marrow MSC-derived; 2 × 1011 particles iv; n = 8; 4 F/4 M) or vehicle control (saline iv; 4 F/4 M) at 6 and 78 h post delivery. Physiological targets were pulse oximetry O2 saturation 90-94% ([Formula: see text] 60-90 mmHg), [Formula: see text] 45-60 mmHg (pH 7.25-7.35), and tidal volume 5-7 mL/kg. MSC-sEVs-treated preterm lambs tolerated enteral feedings compared with vehicle control preterm lambs. Differences in weight patterns were statistically significant. Respiratory severity score, oxygenation index, A-a gradient, distal airspace wall thickness, and smooth muscle thickness around terminal bronchioles and pulmonary arterioles were significantly lower for the MSC-sEVs group. S/F ratio, radial alveolar count, secondary septal volume density, alveolar capillary surface density, and protein abundance of VEGF-R2 were significantly higher for the MSC-sEVs group. MSC-sEVs improved respiratory system physiology and alveolar formation in mechanically ventilated preterm lambs. MSC-sEVs may be an effective and safe therapy for appropriate functional and structural development of the lung in preterm infants who require mechanical ventilation and are at risk of developing BPD.NEW & NOTEWORTHY This study focused on potential treatment of preterm infants at risk of developing bronchopulmonary dysplasia (BPD), for which no effective treatment exists. We tested treatment of mechanically ventilated preterm lambs with human mesenchymal stromal cell extracellular vesicles (MSC-sEVs). The results show improved respiratory gas exchange and parenchymal growth of capillaries and epithelium that are necessary for alveolar formation. Our study provides new mechanistic insight into potential efficacy of MSC-sEVs for preterm infants at risk of developing BPD.

Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr < 0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS < 0.0001) and increased levels of CD45+ leukocytes (PLPS < 0.0001) and MPO+ (PLPS < 0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr = 0.045) and MPO+ cells (PCr = 0.012) in the lungs and reduced thiol oxidation in plasma (PCr < 0.01) and lung tissue (PCr = 0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.

Pilot dose-ranging of rhIGF-1/rhIGFBP-3 in a preterm lamb model of evolving bronchopulmonary dysplasia.

BACKGROUND: Low levels of insulin-like growth factor-1 (IGF-1) protein in preterm human infants are associated with bronchopulmonary dysplasia (BPD). We used our preterm lamb model of BPD to determine (1) dosage of recombinant human (rh) IGF-1 bound to binding protein-3 (IGFBP-3) to reach infant physiologic plasma levels; and (2) whether repletion of plasma IGF-1 improves pulmonary and cardiovascular outcomes. METHODS: Group 1: normal, unventilated lambs from 128 days gestation through postnatal age 5 months defined normal plasma levels of IGF-1. Group 2: continuous infusion of rhIGF-1/rhIGFBP-3 (0.5, 1.5, or 4.5 mg/kg/day; n = 2) for 3 days in mechanically ventilated (MV) preterm lambs determined that 1.5 mg/kg/day dosage attained physiologic plasma IGF-1 concentration of ~125 ng/mL, which was infused in four more MV preterm lambs. RESULTS: Group 1: plasma IGF-1 protein increased from ~75 ng/mL at 128 days gestation to ~220 ng/L at 5 months. Group 2: pilot study of the optimal dosage (1.5 mg/kg/day rhIGF-1/rhIGFBP-3) in six MV preterm lambs significantly improved some pulmonary and cardiovascular outcomes (p < 0.1) compared to six MV preterm controls. RhIGF-1/rhIGFBP-3 was not toxic to the liver, kidneys, or lungs. CONCLUSIONS: Three days of continuous iv infusion of rhIGF-1/rhIGFBP-3 at 1.5 mg/kg/day improved some pulmonary and cardiovascular outcomes without toxicity. IMPACT: Preterm birth is associated with rapid decreases in serum or plasma IGF-1 protein level. This decline adversely impacts the growth and development of the lung and cardiovascular system. For this pilot study, continuous infusion of optimal dosage of rhIGF-1/rhIGFBP-3 (1.5 mg/kg/day) to maintain physiologic plasma IGF-1 level of ~125 ng/mL during mechanical ventilation for 3 days statistically improved some structural and biochemical outcomes related to the alveolar formation that would favor improved gas exchange compared to vehicle-control. We conclude that 3 days of continuous iv infusion of rhIGF-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.

Early extubation to noninvasive respiratory support of former preterm lambs improves long-term respiratory outcomes.

Invasive mechanical ventilation (IMV) and exposure to oxygen-rich gas during early postnatal life are contributing factors for long-term pulmonary morbidities faced by survivors of preterm birth and bronchopulmonary dysplasia. The duration of IMV that leads to long-term pulmonary morbidities is unknown. We compared two durations of IMV (3 h vs. 6 days) during the first 6-7 days of postnatal life in preterm lambs to test the hypothesis that minimizing the duration of IMV will improve long-term respiratory system mechanics and structural outcomes later in life. Moderately preterm (∼85% gestation) lambs were supported by IMV for either 3 h or 6 days before weaning from all respiratory support to become former preterm lambs. Respiratory system mechanics and airway reactivity were assessed monthly from 1 to 6 mo of chronological postnatal age by the forced oscillation technique. Quantitative morphological measurements were made for smooth muscle accumulation around terminal bronchioles and indices of alveolar formation. Minimizing IMV to 3 h led to significantly better (P < 0.05) baseline respiratory system mechanics and less reactivity to methacholine in the first 3 mo of chronological age (2 mo corrected age), significantly less (P < 0.05) accumulation of smooth muscle around peripheral resistance airways (terminal bronchioles), and significantly better (P < 0.05) alveolarization at the end of 5 mo corrected age compared with continuous IMV for 6 days. We conclude that limiting the duration of IMV following preterm birth of fetal lambs leads to better respiratory system mechanics and structural outcomes later in life.

Hippocampal epigenetic and insulin-like growth factor alterations in noninvasive versus invasive mechanical ventilation in preterm lambs.

BACKGROUND: The brain of chronically ventilated preterm human infants is vulnerable to collateral damage during invasive mechanical ventilation (IMV). Damage is manifest, in part, by learning and memory impairments, which are hippocampal functions. A molecular regulator of hippocampal development is insulin-like growth factor 1 (IGF1). A gentler ventilation strategy is noninvasive respiratory support (NRS). We tested the hypotheses that NRS leads to greater levels of IGF1 messenger RNA (mRNA) variants and distinct epigenetic profile along the IGF1 gene locus in the hippocampus compared to IMV. METHODS: Preterm lambs were managed by NRS or IMV for 3 or 21 days. Isolated hippocampi were analyzed for IGF1 mRNA levels and splice variants for promoter 1 (P1), P2, and IGF1A and 1B, DNA methylation in P1 region, and histone covalent modifications along the gene locus. RESULTS: NRS had significantly greater levels of IGF1 P1 (predominant transcript), and 1A and 1B mRNA variants compared to IMV at 3 or 21 days. NRS also led to more DNA methylation and greater occupancy of activating mark H3K4 trimethylation (H3K4me3), repressive mark H3K27me3, and elongation mark H3K36me3 compared to IMV. CONCLUSIONS: NRS leads to distinct IGF1 mRNA variant levels and epigenetic profile in the hippocampus compared to IMV. IMPACT: Our study shows that 3 or 21 days of NRS of preterm lambs leads to distinct IGF1 mRNA variant levels and epigenetic profile in the hippocampus compared to IMV. Preterm infant studies suggest that NRS leads to better neurodevelopmental outcomes later in life versus IMV. Also, duration of IMV is directly related to hippocampal damage; however, molecular players remain unknown. NRS, as a gentler mode of respiratory management of preterm neonates, may reduce damage to the immature hippocampus through an epigenetic mechanism.

Former-preterm lambs have persistent alveolar simplification at 2 and 5 months corrected postnatal age.

Preterm birth and mechanical ventilation (MV) frequently lead to bronchopulmonary dysplasia, the histopathological hallmark of which is alveolar simplification. How developmental immaturity and ongoing injury, repair, and remodeling impact completion of alveolar formation later in life is not known, in part because of lack of suitable animal models. We report a new model, using former-preterm lambs, to test the hypothesis that they will have persistent alveolar simplification later in life. Moderately preterm lambs (~85% gestation) were supported by MV for ~6 days before being transitioned from all respiratory support to become former-preterm lambs. Results are compared with term control lambs that were not ventilated, and between males (M) and females (F). Alveolar simplification was quantified morphometrically and stereologically at 2 mo (4 M, 4 F) or 5 mo (4 M, 6 F) corrected postnatal age (cPNA) compared with unventilated, age-matched term control lambs (4 M, 4 F per control group). These postnatal ages in sheep are equivalent to human postnatal ages of 1-2 yr and ~6 yr, respectively. Multivariable linear regression results showed that former-preterm lambs at 2 or 5 mo cPNA had significantly thicker distal airspace walls ( P < 0.001 and P < 0.009, respectively), lower volume density of secondary septa ( P < 0.007 and P < 0.001, respectively), and lower radial alveolar count ( P < 0.003 and P < 0.020, respectively) compared with term control lambs. Sex-specific differences were not detected. We conclude that moderate preterm birth and MV for ~6 days impedes completion of alveolarization in former-preterm lambs. This new model provides the opportunity to identify underlying pathogenic mechanisms that may reveal treatment approaches.

Preterm birth and ventilation decrease surface density of glomerular capillaries in lambs, regardless of postnatal respiratory support mode.

BACKGROUND: Prematurity is often complicated by respiratory support, including invasive mechanical ventilation (IMV) and noninvasive support (NIS). Compared with IMV, NIS reduces injury to the lung and brain. Prematurity may also disrupt glomerular architecture. Whether NIS differentially affects glomerular architecture is incompletely understood. We hypothesized that IMV would lead to greater disruption of glomerular architecture than NIS. METHODS: This is a secondary analysis of kidneys from moderately preterm lambs delivered at ~131 d gestation (term ~150 d) that had antenatal steroid exposure and surfactant treatment before resuscitation by IMV. At ~3 h of age, half of the lambs were switched to NIS. Support was for 3 d or 21 d. Structural indices of glomerular architecture were quantified. RESULTS: The number of glomerular generations was unaffected by moderate preterm birth and respiratory support, either IMV or NIS. At 3 d and 21 d of IMV or NIS, glomerular capillary surface density was not different. Glomerular capillary surface density was significantly lower in the inner and outer cortex compared with unventilated gestation age-matched or postnatal age-matched reference lambs. CONCLUSION: Moderate preterm birth and invasive or noninvasive respiratory support decreases glomerular capillarization in the lamb kidney. This adverse effect on glomerular development may contribute to increased risk for adult-onset hypertension and renal dysfunction.

Alveolar formation is dysregulated by restricted nutrition but not excess sedation in preterm lambs managed by noninvasive support.

BACKGROUND: Preterm birth and respiratory support with invasive mechanical ventilation frequently leads to bronchopulmonary dysplasia (BPD). A hallmark feature of BPD is alveolar simplification. For our preterm lamb model of BPD, invasive mechanical ventilation is associated with postnatal feeding intolerance (reduced nutrition) and sedation. In contrast, preterm lambs managed by noninvasive support (NIS) have normal alveolar formation, appropriate postnatal nutrition, and require little sedation. We used the latter, positive-outcome group to discriminate the contribution of reduced nutrition vs. sedation on alveolar simplification. We hypothesized that, restricted nutrition, but not sedation with pentobarbital, contributes to impaired indices of alveolar formation in preterm lambs managed by NIS. METHODS: Preterm lambs managed by NIS for 21d were randomized into three groups: NIS control, NIS plus restricted nutrition, and NIS plus excess sedation with pentobarbital. We quantified morphological and biochemical indices of alveolar formation, as well as mesenchymal cell apoptosis and proliferation. RESULTS: Restricted nutrition impaired morphological and biochemical indices of alveolar formation, and reduced mesenchymal cell apoptosis and proliferation. Excess sedation with pentobarbital did not alter these indices, although mesenchymal cell apoptosis was less. CONCLUSION: Our results demonstrate that restricted nutrition, but not excess sedation, contributes to impaired alveolar formation during the evolution of BPD in chronically ventilated preterm lambs.

High-frequency nasal ventilation for 21 d maintains gas exchange with lower respiratory pressures and promotes alveolarization in preterm lambs.

BACKGROUND: Short-term high-frequency nasal ventilation (HFNV) of preterm neonates provides acceptable gas exchange compared to endotracheal intubation and intermittent mandatory ventilation (IMV). Whether long-term HFNV will provide acceptable gas exchange is unknown. We hypothesized that HFNV for up to 21 d would lead to acceptable gas exchange at lower inspired oxygen (O2) levels and airway pressures compared to intubation and IMV. METHODS: Preterm lambs were exposed to antenatal steroids and treated with perinatal surfactant and postnatal caffeine. Lambs were intubated and resuscitated by IMV. At ~3 h of age, half of the lambs were switched to noninvasive HFNV. Support was for 3 or 21 d. By design, Pao2 and Paco2 were not different between groups. RESULTS: At 3 d (n = 5) and 21 d (n = 4) of HFNV, fractional inspired O2 (FiO2), peak inspiratory pressure (PIP), mean airway, intratracheal, and positive end-expiratory pressures, oxygenation index, and alveolar-arterial gradient were significantly lower than matched periods of intubation and IMV. Pao2/FiO2 ratio was significantly higher at 3 and 21 d of HFNV compared to matched intubation and IMV. HFNV led to better alveolarization at 3 and 21 d. CONCLUSION: Long-term HFNV provides acceptable gas exchange at lower inspired O2 levels and respiratory pressures compared to intubation and IMV.

Nucleated Red Blood Cell Emergence-Time in Newborn Lambs Following a Dose of Darbepoetin Alfa.

BACKGROUND: Nucleated red blood cells (NRBC) are very uncommon in the blood of children and adults, but small numbers are not rare in neonates on the day of birth. Elevated NRBC counts in neonates can be seen following erythropoietin dosing. Limited studies in human neonates suggest the time-interval between erythropoietin dosing and the first appearance of NRBC in the blood (the "NRBC emergence-time") is in excess of 24 hours. METHODS: We made serial blood counts (Sysmex veterinary analyzer) on ten newborn lambs; five were dosed with darbepoetin (10 µg/kg), and five were dosed with a vehicle-control to assess the NRBC emergence time under relatively controlled laboratory conditions. RESULTS: The first appearance of NRBC was at 24 h (2757 ± 3210 NRBC/µL vs. 0/µL in controls). Peak was 48-72 h (16,758 ± 8434/µL vs. 0/µL in controls), followed by fewer at 96 hours (7823 ± 7114/µL vs. 0/µL in controls). Similarly, reticulocytes peaked at 48-72 h (113,094 ± 3210/µL vs. 10,790 ± 5449/µL in controls), with no changes in platelets or leukocytes. CONCLUSION: The NRBC emergence time in newborn lambs is similar to reports from newborn humans. By extrapolation, if a neonate has a high NRBC at birth, the erythropoietic stimulus likely occurred within the interval 24 to perhaps 96+ hours prior to birth.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Impact of fetal treatments for congenital diaphragmatic hernia on lung development.

The extent of lung hypoplasia impacts the survival and severity of morbidities associated with congenital diaphragmatic hernia (CDH). The alveoli of CDH infants and in experimental models of CDH have thickened septa with fewer type II pneumocytes and capillaries. Fetal treatments of CDH-risk preterm birth. Therefore, treatments must aim to balance the need for increased gas exchange surface area with the restoration of pulmonary epithelial type II cells and the long-term respiratory and neurodevelopmental consequences of prematurity. Achievement of sufficient lung development in utero for successful postnatal transition requires adequate intra-thoracic space for lung growth, maintenance of sufficient volume and appropriate composition of fetal lung fluid, regular fetal breathing movements, appropriate gas exchange area, and ample surfactant production. The review aims to examine the rationale for current and future therapeutic strategies to improve postnatal outcomes of infants with CDH.

IUGR decreases elastin mRNA expression in the developing rat lung and alters elastin content and lung compliance in the mature rat lung.

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.

Mechanism of reduced lung injury by high-frequency nasal ventilation in a preterm lamb model of neonatal chronic lung disease.

The mechanism underlying the potentially beneficial effects of the "gentler" modes of ventilation on chronic lung disease (CLD) of the premature infant is not known. We have previously demonstrated that alveolar parathyroid hormone-related protein-peroxisome proliferator-activated receptorγ (PTHrP-PPARγ) signaling is critically important in alveolar formation, and this signaling pathway is disrupted in hyperoxia- and/or volutrauma-induced neonatal rat lung injury. Whether the same paradigm is also applicable to CLD, resulting from prolonged intermittent mandatory ventilation (IMV), and whether differential effects of the mode of ventilation on the PTHrP-PPARγ signaling pathway explain the potential benefits of the "gentler" modes of ventilation are not known. Using a well-established preterm lamb model of neonatal CLD, we tested the hypothesis that ventilatory support using high-frequency nasal ventilation (HFNV) promotes alveolar PTHrP-PPARγ signaling, whereas IMV inhibits it. Preterm lambs managed by HFNV or IMV for 21 d following preterm delivery at 132-d gestation were studied by Western hybridization and immunofluorescence labeling for key markers of alveolar homeostasis and injury/repair. In lambs managed by IMV, the abundance of key homeostatic alveolar epithelial-mesenchymal markers was reduced, whereas it was significantly increased in the HFNV group, providing a potential molecular mechanism by which "gentler" modes of ventilation reduce neonatal CLD.

Preterm Birth With Neonatal Interventions Accelerates Collagen Deposition in the Left Ventricle of Lambs Without Affecting Cardiomyocyte Development.

BACKGROUND: Adults born preterm (< 37 weeks' gestation) exhibit altered cardiac growth and are susceptible to cardiac dysfunction. Sheep studies have shown that moderate preterm birth results in maladaptive structural remodelling of the cardiac ventricles. The aim of this study was to examine ventricular structure in lambs born at a greater severity of preterm birth and ventilated postnatally. METHODS: Former-preterm lambs delivered at 128 days' gestation, and mechanically ventilated for a week after birth, were compared with unventilated lambs born at term (150 days' gestation), at 2 months (term: n = 10, former-preterm: n = 8), and 5 months (term: n = 9, former-preterm: n = 8) term-equivalent age. The right ventricle and left ventricle plus septum were analysed using immunohistochemistry, histology, and stereology. RESULTS: Cardiomyocyte number, cross-sectional area, proliferation, and apoptosis were not affected by preterm birth or age. Left ventricle plus septum interstitial collagen levels increased with age (P = 0.0015) and were exacerbated by preterm birth (P = 0.0006; 2 months term: 0.57% ± 0.07%, former-preterm: 1.44% ± 0.18%; 5 months term: 1.37% ± 0.25%, former-preterm: 2.15% ± 0.31%). Right ventricle interstitial collagen levels increased with age (P = 0.012) but were not affected by preterm birth. CONCLUSION: This study is the first to explore the effect of preterm birth combined with modern neonatal interventions on the ventricular myocardium in lambs. There was no adverse impact on cardiomyocyte growth in early postnatal life. Of concern, however, there was increased collagen deposition in the preterm hearts, which has the potential to induce cardiac dysfunction, especially if it becomes exaggerated with ageing.

INTRODUCTION: Les adultes nés avant terme (< 37 semaines de grossesse) montrent une altération de la croissance cardiaque et sont exposés à une dysfonction cardiaque. Les études sur les moutons ont montré que la prématurité modérée entraîne un remodelage structurel inadapté des ventricules du cœur. L'objectif de la présente étude était d'examiner la structure ventriculaire des agneaux grands prématurés et oxygénés après la naissance. MÉTHODES: Les agneaux anciens prématurés nés après 128 jours de gestation et sous respirateur durant une semaine ont été comparés aux agneaux nés à terme qui n'avaient pas été sous respirateur (150 jours de gestation) à l'âge du terme, soit deux mois (à terme : n = 10, anciens prématurés : n = 8) et cinq mois (à terme : n = 9, anciens prématurés : n = 8). Le ventricule droit et le ventricule gauche plus le septum ont été analysés par immunohistochimie, histologie et stéréologie. RÉSULTATS: Le nombre de cardiomyocytes, la surface en coupe transversale, la prolifération et l'apoptose n'étaient pas affectés par la naissance prématurée ou l'âge. Les concentrations interstitielles en collagène du ventricule gauche plus le septum augmentaient avec l'âge (P = 0,0015) et étaient exacerbées par la naissance prématurée (P = 0,0006; âge du terme, deux mois : [à terme : 0,57 % ± 0,07 %, anciens prématurés : 1,44 % ± 0,18 % ]; âge du terme, cinq mois : [à terme : 1,37 % ± 0,25 %, anciens prématurés : 2,15 % ± 0,31 %]). Les concentrations interstitielles en collagène du ventricule droit augmentaient avec l'âge (P = 0,012), mais n'étaient pas affectées par la naissance avant terme. CONCLUSION: Il s'agit de la première étude qui porte sur la combinaison des effets de la naissance avant terme aux interventions néonatales modernes sur le myocarde ventriculaire des agneaux. Aucune conséquence sur la croissance des cardiomyocytes dans la phase précoce de la vie postnatale n'a été observée. Toutefois, le dépôt accru de collagène dans le cœur des prématurés est préoccupant puisqu'il a le potentiel d'induire une dysfonction cardiaque, particulièrement s'il s'exacerbe avec le vieillissement.

Stretch-Induced Intimal Failure in Isolated Cerebral Arteries as a Function of Development.

Recent clinical studies have shown that traumatic brain injury is a significant risk factor for stroke. Motivated to better understand possible mechanisms of this association, we studied subfailure disruption of the intima in overstretched sheep cerebral arteries, as this has been implicated in the increased risk of stroke following blunt cerebrovascular injury. Middle cerebral arteries from four age groups (ranging from fetal to adult) were stretched axially to failure, and intimal disruption was captured with a video camera. All vessels demonstrated intimal disruption prior to catastrophic failure, with nearly all incurring disruption at stretch values well below those at ultimate stress (means of 1.56 and 1.73, respectively); the lowest stretch associated with intimal disruption was 1.29. The threshold of intimal failure was independent of age. Additional analysis showed that disruption included failure of both the endothelium and internal elastic lamina. Although our experiments were conducted at quasi-static rates, the results likely have important implications for vessel function following trauma. Future work should seek to identify subfailure disruption of the cerebrovasculature in head trauma.

Microarchitecture of the hearts in term and former-preterm lambs using diffusion tensor imaging.

Diffusion tensor imaging (DTI) is an MRI technique that can be used to map cardiomyocyte tracts and estimate local cardiomyocyte and sheetlet orientation within the heart. DTI measures diffusion distances of water molecules within the myocardium, where water diffusion generally occurs more freely along the long axis of cardiomyocytes and within the extracellular matrix, but is restricted by cell membranes such that transverse diffusion is limited. DTI can be undertaken in fixed hearts and it allows the three-dimensional mapping of the cardiac microarchitecture, including cardiomyocyte organization, within the whole heart. The objective of this study was to use DTI to compare the cardiac microarchitecture and cardiomyocyte organization in archived fixed left ventricles of lambs that were born either preterm (n = 5) or at term (n = 7), at a postnatal timepoint equivalent to about 6 years of age in children. Although the findings support the feasibility of retrospective DTI scanning of fixed hearts, several hearts were excluded from DTI analysis because of poor scan quality, such as ghosting artifacts. The preliminary findings from viable DTI scans (n = 3/group) suggest that the extracellular compartment is altered and that there is an immature microstructural phenotype early in postnatal life in the LV of lambs born preterm. Our findings support a potential time-efficient imaging role for DTI in detecting abnormal changes in the microstructure of fixed hearts of former-preterm neonates, although further investigation into factors that affect scan quality is required.

Chronic lung disease in preterm lambs: effect of daily vitamin A treatment on alveolarization.

Neonatal chronic lung disease is characterized by failed formation of alveoli and capillaries, and excessive deposition of matrix elastin, which are linked to lengthy mechanical ventilation (MV) with O(2)-rich gas. Vitamin A supplementation has improved respiratory outcome of premature infants, but there is little information about the structural and molecular manifestations in the lung that occur with vitamin A treatment. We hypothesized that vitamin A supplementation during prolonged MV, without confounding by antenatal steroid treatment, would improve alveolar secondary septation, decrease thickness of the mesenchymal tissue cores between distal air space walls, and increase alveolar capillary growth. We further hypothesized that these structural advancements would be associated with modulated expression of tropoelastin and deposition of matrix elastin, phosphorylated Smad2 (pSmad2), cleaved caspase 3, proliferating cell nuclear antigen (PCNA), VEGF, VEGF-R2, and midkine in the parenchyma of the immature lung. Eight preterm lambs (125 days' gestation, term approximately 150 days) were managed by MV for 3 wk: four were treated with daily intramuscular Aquasol A (vitamin A), 5,000 IU/kg, starting at birth; four received vehicle alone. Postmortem lung assays included quantitative RT-PCR and in situ hybridization, immunoblot and immunohistochemistry, and morphometry and stereology. Daily vitamin A supplementation increased alveolar secondary septation, decreased thickness of the mesenchymal tissue cores between the distal air space walls, and increased alveolar capillary growth. Associated molecular changes were less tropoelastin mRNA expression, matrix elastin deposition, pSmad2, and PCNA protein localization in the mesenchymal tissue core of the distal air space walls. On the other hand, mRNA expression and protein abundance of VEGF, VEGF-R2, midkine, and cleaved caspase 3 were increased. We conclude that vitamin A treatment partially improves lung development in chronically ventilated preterm neonates by modulating expression of tropoelastin, deposition of elastin, and expression of vascular growth factors.

Electrostatic Filters to Reduce COVID-19 Spread in Bubble CPAP: An in vitro Study of Safety and Efficacy.

BACKGROUND: Bubble CPAP may be used in infants with suspected or confirmed COVID-19. Electrostatic filters may reduce cross infection. This study aims to determine if including a filter in the bubble CPAP circuit impacts stability of pressure delivery. METHODS: A new electrostatic filter was placed before (pre) or after (post) the bubble CPAP generator, or with no filter (control) in an in vitro study. Pressure was recorded at the nasal interface for 18 h (6 L/min; 7 cm H2O) on 3 occasions for each configuration. Filter failure was defined as pressure >9 cm H2O for 60 continuous minutes. The filter was weighed before and after each experiment. RESULTS: Mean (SD) time to reach the fail point was 257 (116) min and 525 (566) min for filter placement pre- and post-CPAP generator, respectively. Mean pressure was higher throughout in the pre-generator position compared to control. The filter weight was heavier at end study in the pre- compared to the post-generator position. CONCLUSIONS: Placement of the filter at the pre-generator position in a bubble CPAP circuit should be avoided due to unstable mean pressure. Filters are likely to become saturated with water over time. The post-generator position may accommodate a filter, but regular pressure monitoring and early replacement are required.