Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI) are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor NDI, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in inter-organ communication in diverse pathological processes. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of alveolar macrophage (AM) markers, CD11b, CD11c, and CD206, and ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction inspired oxygen (FiO2) therapy (HO2, ≥30%) had increased levels of AM-derived EV-ASC compared with infants on lower FiO2 (LO2, <30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain crosstalk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Continuous but not pulsed low-dose fetal betamethasone exposures extend the durability of antenatal steroid therapy.

Antenatal steroid (ANS) therapy is the standard care for women at imminent risk of preterm labor. Despite extensive and long-standing use, 40%-50% of babies exposed antenatally to steroids do not derive benefit; remaining undelivered 7 days or more after ANS treatment is associated with a lack of treatment benefit and increased risk of harm. We used a pregnant sheep model to evaluate the impact of continuous versus pulsed ANS treatments on fetal lung maturation at an extended, 8-day treatment to delivery interval. Continuous low-dose ANS treatments for more than 72 h in duration improved fetal lung maturation at 8 days after treatment initiation. If fetal ANS exposure was interrupted, the beneficial ANS effect was lost. Truncated treatments, including that simulating the current clinical treatment regimen, did not improve lung function. Variable fetal lung maturation was correlated to the amount of saturated phosphatidylcholine present in the lung fluid. These data demonstrate that 1) the durability of ANS therapy may be enhanced by employing an extended, low-dose treatment regimen by reducing total dose and 2) interrupting the continuity of fetal exposure by allowing it to fall below a minimal threshold was associated with comparably poor functional maturation of the preterm ovine lung.

The complex challenge of antenatal steroid therapy nonresponsiveness.

Antenatal steroid therapy is standard care for women at imminent risk of preterm delivery. When deliveries occur within 7 days of treatment, antenatal steroid therapy reduces the risk of neonatal death and improves preterm outcomes by exerting diverse developmental effects on the fetal organs, in particular the preterm lung and cardiovascular system. There is, however, sizable variability in antenatal steroid treatment efficacy, and an important percentage of fetuses exposed to antenatal steroid therapy do not respond sufficiently to derive benefit. Respiratory distress syndrome, for example, is a central metric of clinical trials to assess antenatal steroid outcomes. In the present analysis, we addressed the concept of antenatal steroid nonresponsiveness, and defined a failed or suboptimal response to antenatal steroids as death or a diagnosis of respiratory distress syndrome following treatment. For deliveries at 24 to 35 weeks' gestation, the number needed to treat to prevent 1 case of respiratory distress syndrome was 19 (95% confidence interval, 14-28). Reflecting gestation-dependent risk, for deliveries at >34 weeks' gestation the number needed to treat was 55 (95% confidence interval, 30-304), whereas for elective surgical deliveries at term this number was 106 (95% confidence interval, 61-421). We reviewed data from clinical and animal studies investigating antenatal steroid therapy to highlight the significant incidence of antenatal steroid therapy nonresponsiveness (ie, residual mortality or respiratory distress syndrome after treatment), and the potential mechanisms underpinning this outcome variability. The origins of this variability may be related to both the manner in which the therapy is applied (ie, the treatment regimen itself) and factors specific to the individual (ie, genetic variation, stress, infection). The primary aims of this review were: (1) to emphasize to the obstetrical and neonatal communities the extent of antenatal steroid response variability and its potential impact; (2) to propose approaches by which antenatal steroid therapy may be better applied to improve overall benefit; and (3) to stimulate further research toward the empirical optimization of this important antenatal therapy.

Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights when administered to pregnant sheep in combination with betamethasone acetate.

BACKGROUND: Antenatal corticosteroid therapy is a standard of care for women at imminent risk of preterm labor. However, the optimal (maximum benefit and minimal risk of side effects) antenatal corticosteroid dosing strategy remains unclear. Although conveying overall benefit when given to the right patient at the right time, antenatal corticosteroid treatment efficacy is highly variable and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high maternal-fetal betamethasone concentrations it generates are redundant for fetal lung maturation. OBJECTIVE: Using an established sheep model of prematurity and postnatal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of low-dosage treatment with betamethasone acetate only against a standard dosage of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynecologists for women at risk of imminent preterm delivery between 24 0/7 and 35 6/7 weeks' gestation. STUDY DESIGN: Ewes carrying a single fetus at 122±1 days' gestation (term=150 days) were randomized to receive either (1) maternal intramuscular injections of sterile saline (the saline negative control group, n=12), (2) 2 maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate+betamethasone acetate administered at 24-hour dosing intervals (the betamethasone phosphate+betamethasone acetate group, n=12); or (3) 2 maternal intramuscular injections of 0.125 mg/kg betamethasone acetate administered at 24-hour dosing intervals (the betamethasone acetate group, n=11). The fetuses were surgically delivered 48 hours after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. The fetuses were euthanized after ventilation, and the lungs were collected for analysis using quantitative polymerase chain reaction and Western blot assays. Fetal plasma adrenocorticotropic hormone levels were measured in the cord blood samples taken at delivery. RESULTS: Preterm lambs were defined as either antenatal corticosteroid treatment responders or nonresponders using an arbitrary cutoff, being a PaCO2 level at 30 minutes of ventilation being more extreme than 2 standard deviations from the mean value of the normally distributed saline control group values. Compared with the animals in the saline control group, the animals in the antenatal corticosteroid treatment groups showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (messenger RNA and surfactant protein assays) consistent with functional maturation. However, the betamethasone acetate group had a significantly higher treatment response rate than the betamethasone phosphate+betamethasone acetate group. These physiological results were strongly correlated to the amount of surfactant protein A. Birthweight was lower in the betamethasone phosphate+betamethasone acetate group and the fetal hypothalamic-pituitary-adrenal axis was suppressed to a greater extent in the betamethasone phosphate+betamethasone acetate group. CONCLUSION: Low-dosage antenatal corticosteroid therapy solely employing betamethasone acetate was sufficient for fetal lung maturation. The elevated maternal-fetal betamethasone concentrations associated with the coadministration of betamethasone phosphate did not in addition improve lung maturation but were associated with greater fetal hypothalamic-pituitary-adrenal axis suppression, a lower antenatal corticosteroid treatment response rate, and lower birthweight-outcomes not desirable in a clinical setting. These data warranted a clinical investigation of sustained low-dosage antenatal corticosteroid treatments that avoid high maternal-fetal betamethasone exposures.

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats.

BACKGROUND: Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS: Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS: DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS: This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. IMPACT: There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Surfactant plus budesonide decreases lung and systemic responses to injurious ventilation in preterm sheep.

Mechanical ventilation from birth with normal tidal volumes (VT) causes lung injury and systemic responses in preterm sheep. The addition of budesonide to surfactant therapy decreases these injury markers. Budesonide and surfactant will decrease the injury from injurious VT ventilation in preterm sheep. Lambs at 126 ± 1 day gestational age were ventilated from birth with either: 1) Normal VT [surfactant 200 mg/kg before ventilation, positive end expiratory pressure (PEEP) 5 cmH2O, VT 8 mL/kg] or 2) Injury VT (high pressure, 100% oxygen, no PEEP) for 15 min, then further randomized to surfactant + saline or surfactant + 0.25 mg/kg budesonide with Normal VT for 6 h. Lung function and lung, liver, and brain tissues were evaluated for indicators of injury. Injury VT + saline caused significant injury and systemic responses, and Injury VT + budesonide improved lung physiology. Budesonide decreased lung inflammation and decreased pro-inflammatory cytokine mRNA in the lung, liver, and brain to levels similar to Normal VT + saline. Budesonide was present in plasma within 15 min of treatment in both ventilation groups, and less than 5% of the budesonide remained in the lung at 6 h. mRNA sequencing of liver and periventricular white matter demonstrated multiple pathways altered by both Injury VT and budesonide and the combination exposure. In lambs receiving Injury VT, the addition of budesonide to surfactant improved lung physiology and decreased pro-inflammatory cytokine responses in the lung, liver, and brain to levels similar to lambs receiving Normal VT.

Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.

BACKGROUND: Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. OBJECTIVE: We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. STUDY DESIGN: Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days' gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. RESULTS: One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas- and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. CONCLUSION: This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling.

The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation.

BACKGROUND: Antenatal corticosteroids (ACS) are the standard of care for maturing the fetal lung and improving outcomes for preterm infants. Antenatal corticosteroid dosing remains nonoptimized, and there is little understanding of how different treatment-to-delivery intervals may affect treatment efficacy. The durability of a lung maturational response is important because the majority of women treated with antenatal corticosteroids do not deliver within the widely accepted 1- to 7-day window of treatment efficacy. OBJECTIVE: We used a sheep model to test the duration of fetal exposures for efficacy at delivery intervals from 1 to 10 days. MATERIALS AND METHODS: For infusion studies, ewes with single fetuses were randomized to receive an intravenous bolus and maintenance infusion of betamethasone phosphate to target 1-4 ng/mL fetal plasma betamethasone for 36 hours, with delivery at 2, 4 ,or 7 days posttreatment or sterile saline solution as control. Animals receiving the clinical treatment were randomised to receive either a single injection of 0.25 mg/kg with a 1:1 mixture of betamethasone phosphate + betamethasone acetate with delivery at either 1 or 7 days posttreatment, or 2 treatments of 0.25 mg/kg betamethasone phosphate + betamethasone acetate spaced at 24 hours (giving ∼48 hours of fetal steroid exposure) with delivery at 2, 5, 7, or 10 days posttreatment. Negative control animals were treated with saline solution. All lambs were delivered at 121 ± 3 days gestational age and ventilated for 30 minutes to assess lung function. RESULTS: Preterm lambs delivered at 1 or 2 days post-antenatal corticosteroid treatment had significant improvements in lung maturation for both intravenous and single-dose intramuscular treatments. After 2 days, the efficacy of 36-hour betamethasone phosphate infusions was lost. The single dose of 1:1 betamethasone phosphate + betamethasone acetate also was ineffective at 7 days. In contrast, animals treated with 2 doses had significant improvements in lung maturation at 2, 5, and 7 days, with treatment efficacy reduced by 10 days. CONCLUSION: In preterm lambs, the durability of antenatal corticosteroids treatment depends on the duration of fetal exposure and is independent of the intravenous or intramuscular maternal route of administration. For acute 24- to 48-hour posttreatment deliveries, a 24-hour fetal antenatal corticosteroids exposure was sufficient for lung maturation. A fetal exposure duration of at least 48 hours was necessary to maintain long-term treatment durability. A single-dose ACS treatment should be sufficient for women delivering within <48 hours of antenatal corticosteroids treatment.

Dose of budesonide with surfactant affects lung and systemic inflammation after normal and injurious ventilation in preterm lambs.

BACKGROUND: The addition of budesonide (Bud) 0.25 mg/kg to surfactant decreased the lung and systemic responses to mechanical ventilation in preterm sheep and the rates and severity of bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that lower budesonide concentrations in surfactant will decrease injury while decreasing systemic corticosteroid exposure. METHODS: Preterm lambs received either (1) protective tidal volume (VT) ventilation with surfactant from birth or (2) injurious VT ventilation for 15 min and then surfactant treatment. Lambs were further assigned to surfactant mixed with (i) Saline, (ii) Bud 0.25 mg/kg, (iii) Bud 0.1 mg/kg, or (iv) Bud 0.04 mg/kg. All lambs were then ventilated with protective VT for 6 h. RESULTS: Plasma Bud levels were proportional to the dose received and decreased throughout ventilation. In both protective and injurious VT ventilation, <4% of Bud remained in the lung at 6 h. Some of the improvements in physiology and markers of injury with Bud 0.25 mg/kg were also found with 0.1 mg/kg, whereas 0.04 mg/kg had only minimal effects. CONCLUSIONS: Lower doses of Bud were less effective at decreasing lung and systemic inflammation from mechanical ventilation. The plasma Bud levels were proportional to dose given and the majority left the lung.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

Oral antenatal corticosteroids evaluated in fetal sheep.

BACKGROUND: The use of antenatal corticosteroids (ACS) in low-resource environments is sporadic. Further, drug choice, dose, and route of ACS are not optimized. We report the pharmacokinetics and pharmacodynamics of oral dosing of ACS using a preterm sheep model. METHODS: We measured pharmacokinetics of oral betamethasone-phosphate (Beta-P) and dexamethasone-phosphate (Dex-P) using catheterized pregnant sheep. We compared fetal lung maturation responses of oral Beta-P and Dex-P to the standard treatment with 2 doses of the i.m. mixture of Beta-P and betamethasone-acetate at 2, 5, and 7 days after initiation of ACS. RESULTS: Oral Dex-P had lower bioavailability than Beta-P, giving a lower maximum maternal and fetal concentration. A single oral dose of 0.33 mg/kg of Beta-P was equivalent to the standard clinical treatment assessed at 2 days; 2 doses of 0.16 mg/kg of oral Beta-P were equivalent to the standard clinical treatment at 7 days as assessed by lung mechanics and gas exchange after preterm delivery and ventilation. In contrast, oral Dex-P was ineffective because of its decreased bioavailability. CONCLUSION: Using a sheep model, we demonstrate the use of pharmacokinetics to develop oral dosing strategies for ACS. Oral dosing is feasible and may facilitate access to ACS in low-resource environments.

Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep.

BACKGROUND: Antenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2-dose treatment with betamethasone-acetate+betamethasone-phosphate is more effective than 2 doses of betamethasone-phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone-acetate component induces similar lung maturation to betamethasone-phosphate+betamethasone-acetate with decreased dose and fetal exposure. OBJECTIVE: The purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone-acetate in preterm fetal sheep. STUDY DESIGN: Groups of 10 singleton-pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone-phosphate+betamethasone-acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone-acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure-volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone-phosphate 0.125 mg/kg, betamethasone-acetate 0.125 mg/kg, or betamethasone-acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours. RESULTS: All betamethasone-treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP-binding cassette subfamily A member 3, and aquaporin-5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone-acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone-acetate+betamethasone-phosphate. Betamethasone-acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone-phosphate 0.125 mg/kg. CONCLUSION: A single dose of betamethasone-acetate results in similar fetal lung maturation as the 2-dose clinical formulation of betamethasone-phosphate+betamethasone-acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone-acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus.

Antenatal dexamethasone vs. betamethasone dosing for lung maturation in fetal sheep.

BACKGROUND: Dexamethasone-phosphate (Dex-PO4) and the combination betamethasone-phosphate (Beta-PO4) + betamethasone-acetate (Beta-Ac) are the most used antenatal corticosteroids to promote fetal lung maturation. We compared fetal lung maturation induced by Beta-Ac+Beta-PO4, Dex-PO4, or Beta-PO4 alone. METHODS: Pregnant ewes received two intramuscular doses 24 h apart of 0.25 mg/kg/dose of Beta-Ac+Beta-PO4, Dex-PO4 or Beta-PO4; or 2 doses of 0.125 mg/kg/dose of Beta-PO4 at 6, 12, or 24 h intervals. Fetuses were delivered 48 h after the first dose and ventilated for 30 min. We assessed ventilatory variables, vital signs, and blood gas. After ventilation pressure-volume curves were measured and lungs were sampled for analysis. RESULTS: All treatments improved lung compliance and ventilation efficiency. Only Beta-Ac + Beta-PO4 required lower positive inspiratory pressure compared with control. Beta-Ac + Beta-PO4 and Beta-PO4 alone, but not Dex-PO4, increased the mRNA of surfactant proteins compared with control. Low-dose Beta-PO4 did not increase mRNA of surfactant proteins. There were no differences among Beta-PO4 treatment intervals. CONCLUSION: Beta-Ac + Beta-PO4 given as two doses 24 h apart was more effective in promoting fetal lung maturation than Dex-PO4 or Beta-PO4 alone, consistent with a prolonged exposure provided by the Beta-Ac + Beta-PO4. These results support the clinical use of combined Beta-Ac + Beta-PO4 preparations over phosphate corticosteroids alone for fetal lung maturation.

Intra-amniotic Ureaplasma parvum-Induced Maternal and Fetal Inflammation and Immune Responses in Rhesus Macaques.

BACKGROUND: Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized. METHODS: Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 107 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues. RESULTS: Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor. CONCLUSIONS: U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype.

Intra-amniotic LPS causes acute neuroinflammation in preterm rhesus macaques.

BACKGROUND: Chorioamnionitis is associated with an increased risk of brain injury in preterm neonates. Inflammatory changes in brain could underlie this injury. Here, we evaluated whether neuroinflammation is induced by chorioamnionitis in a clinically relevant model. METHODS: Rhesus macaque fetuses were exposed to either intra-amniotic (IA) saline, or IA lipopolysaccharide (LPS) (1 mg) 16 or 48 h prior to delivery at 130 days (85 % of gestation) (n = 4-5 animals/group). We measured cytokines in the cerebrospinal fluid (CSF), froze samples from the left brain for molecular analysis, and immersion fixed the right brain hemisphere for immunohistology. We analyzed the messenger RNA (mRNA) levels of the pro-inflammatory cytokines IL-1ß, CCL2, TNF-α, IL-6, IL-8, IL-10, and COX-2 in the periventricular white matter (PVWM), cortex, thalamus, hippocampus, and cerebellum by RT-qPCR. Brain injury was assessed by immunohistology for myelin basic protein (MBP), IBA1 (microglial marker), GFAP (astrocyte marker), OLIG2 (oligodendrocyte marker), NeuN (neuronal marker), CD3 (T cells), and CD14 (monocytes). Microglial proliferation was assessed by co-immunostaining for IBA1 and Ki67. Data were analyzed by ANOVA with Tukey's post-test. RESULTS: IA LPS increased mRNA expression of pro-inflammatory cytokines in the PVWM, thalamus, and cerebellum, increased IL-6 concentration in the CSF, and increased apoptosis in the periventricular area after 16 h. Microglial proliferation in the white matter was increased 48 h after IA LPS. CONCLUSIONS: LPS-induced chorioamnionitis caused neuroinflammation, microglial proliferation, and periventricular apoptosis in a clinically relevant model of chorioamnionitis in fetal rhesus macaques. These findings identify specific responses in the fetal brain and support the hypothesis that neuroinflammatory changes may mediate the adverse neurodevelopmental outcomes associated with chorioamnionitis.