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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.
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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.
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Oxigênio , Retinopatia da Prematuridade , Animais , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Camundongos , Anticorpos Monoclonais Humanizados/farmacologia , Camundongos Endogâmicos C57BL , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Hiperóxia/patologia , Hiperóxia/complicações , Retina/patologia , Retina/metabolismo , Retina/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Camundongos Transgênicos , Neovascularização Retiniana/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Microglia/patologia , Microglia/metabolismo , Microglia/efeitos dos fármacosRESUMO
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.
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Corioamnionite , Nascimento Prematuro , Recém-Nascido , Feminino , Lactente , Animais , Humanos , Gravidez , Proteínas Hedgehog , Macaca mulatta , Escherichia coli , Recém-Nascido Prematuro , Cerebelo , RNA Nuclear PequenoRESUMO
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.
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Lesões Encefálicas , Hiperóxia , Animais , Humanos , Recém-Nascido , Camundongos , Animais Recém-Nascidos , Técnicas de Inativação de Genes , Hipocampo , Hiperóxia/complicações , Recém-Nascido Prematuro , Camundongos Knockout , Proteínas de Ligação a Fosfato , Proteínas Citotóxicas Formadoras de PorosRESUMO
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.
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Betametasona , Maturidade dos Órgãos Fetais , Animais , Betametasona/farmacologia , Feminino , Glucocorticoides/farmacologia , Humanos , Pulmão , Gravidez , Cuidado Pré-Natal , Ovinos , Esteroides/farmacologiaRESUMO
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.
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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.
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Glucocorticoides , Sistema Hipotálamo-Hipofisário , Animais , Betametasona/análogos & derivados , Betametasona/farmacologia , Peso ao Nascer , Feminino , Glucocorticoides/uso terapêutico , Pulmão/metabolismo , Sistema Hipófise-Suprarrenal , Gravidez , OvinosRESUMO
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.
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Hérnias Diafragmáticas Congênitas , Animais , DNA/metabolismo , Modelos Animais de Doenças , Feto , Hérnias Diafragmáticas Congênitas/metabolismo , Pulmão , Modelos Anatômicos , Éteres Fenílicos/toxicidade , Ratos , Ratos Sprague-Dawley , Teratogênicos/metabolismo , Teratogênicos/farmacologiaRESUMO
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.
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Encéfalo/patologia , Vesículas Extracelulares/patologia , Piroptose/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Animais , Animais Recém-Nascidos , Caspase 1/sangue , Exossomos/patologia , Feminino , Mediadores da Inflamação/metabolismo , Interleucina-1beta/sangue , Masculino , Proteínas de Ligação a Fosfato/sangue , Proteínas Citotóxicas Formadoras de Poros/sangue , Gravidez , Ratos , Ratos Sprague-Dawley , Respiração Artificial , Transdução de SinaisRESUMO
We review the history of antenatal corticosteroid therapy (ACS) and present recent experimental data to demonstrate that this, one of the pillars of perinatal care, has been inadequately evaluated to minimize fetal exposure to these powerful medications. There have been concerns since 1972 that fetal exposures to ACS convey risk. However, this developmental modulator, with its multiple widespread biologic effects, has not been evaluated for drug choice, dose, or duration of treatment, despite over 30 randomized trials. The treatment used in the United States is two intramuscular doses of a mixture of 6 mg betamethasone phosphate (Beta P) and 6 mg betamethasone acetate (Beta Ac). To optimize outcomes with ACS, the goal should be to minimize fetal drug exposure. We have determined that the minimum exposure needed for fetal lung maturation in sheep, monkeys, and humans (based on published cord blood corticosteroid concentrations) is about 1 ng/ml for a 48-h continuous exposure, far lower than the concentration reached by the current dosing. Because the slowly released Beta Ac results in prolonged fetal exposure, a drug containing Beta Ac is not ideal for ACS use. IMPACT: Using sheep and monkey models, we have defined the minimum corticosteroid exposure for a fetal lung maturation. These results should generate new clinical trials of antenatal corticosteroids (ACS) at much lower fetal exposures to ACS, possibly given orally, with fewer risks for the fetus.
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Corticosteroides/administração & dosagem , Maturidade dos Órgãos Fetais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nascimento Prematuro/tratamento farmacológico , Cuidado Pré-Natal , Corticosteroides/efeitos adversos , Corticosteroides/farmacocinética , Animais , Composição de Medicamentos , Cálculos da Dosagem de Medicamento , Feminino , Idade Gestacional , Humanos , Pulmão/crescimento & desenvolvimento , Modelos Biológicos , Gravidez , Nascimento Prematuro/diagnóstico , Nascimento Prematuro/fisiopatologia , Medição de Risco , Fatores de RiscoRESUMO
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.
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Budesonida/farmacologia , Lesão Pulmonar/tratamento farmacológico , Pulmão/efeitos dos fármacos , Surfactantes Pulmonares/farmacologia , Respiração Artificial/efeitos adversos , Animais , Animais Recém-Nascidos/metabolismo , Citocinas/metabolismo , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Respiração com Pressão Positiva/métodos , Gravidez , Nascimento Prematuro/metabolismo , RNA Mensageiro/metabolismo , Respiração/efeitos dos fármacos , Ovinos , Volume de Ventilação Pulmonar/efeitos dos fármacosRESUMO
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.
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Betametasona/análogos & derivados , Maturidade dos Órgãos Fetais/efeitos dos fármacos , Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Animais , Aquaporina 1/efeitos dos fármacos , Aquaporina 1/genética , Aquaporina 5/efeitos dos fármacos , Aquaporina 5/genética , Betametasona/sangue , Betametasona/farmacologia , Gasometria , Dióxido de Carbono , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Feminino , Maturidade dos Órgãos Fetais/genética , Glucocorticoides/sangue , Pulmão/metabolismo , Pulmão/fisiopatologia , Complacência Pulmonar/efeitos dos fármacos , Espectrometria de Massas , Troca Materno-Fetal , Pressão Parcial , Assistência Perinatal , Reação em Cadeia da Polimerase , Gravidez , Nascimento Prematuro , Cuidado Pré-Natal , Proteína A Associada a Surfactante Pulmonar/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/efeitos dos fármacos , Proteína B Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/efeitos dos fármacos , Proteína C Associada a Surfactante Pulmonar/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Distribuição Aleatória , Respiração Artificial , OvinosRESUMO
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 â¼48 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.
Assuntos
Betametasona/análogos & derivados , Parto Obstétrico , Maturidade dos Órgãos Fetais/efeitos dos fármacos , Feto/efeitos dos fármacos , Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Animais , Betametasona/farmacologia , Idade Gestacional , Infusões Intravenosas , Injeções Intramusculares , Pulmão/embriologia , Cuidado Pré-Natal , Ovinos , Fatores de TempoRESUMO
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.
Assuntos
Produtos Biológicos/administração & dosagem , Displasia Broncopulmonar/prevenção & controle , Budesonida/administração & dosagem , Glucocorticoides/administração & dosagem , Pulmão/efeitos dos fármacos , Fosfolipídeos/administração & dosagem , Surfactantes Pulmonares/administração & dosagem , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/fisiopatologia , Budesonida/farmacocinética , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Idade Gestacional , Glucocorticoides/farmacocinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Nascimento Prematuro , Respiração Artificial , Carneiro Doméstico , Distribuição TecidualRESUMO
Mechanical ventilation with normal tidal volumes (VT) causes lung and systemic inflammation in preterm sheep. Mechanical ventilation is associated with bronchopulmonary dysplasia (BPD) in preterm infants, and the addition of budesonide to surfactant decreases BPD in clinical trials. Budesonide with surfactant will decrease the lung injury from mechanical ventilation for 24 h in preterm sheep. Lambs at 126 ± 1 day gestational age were delivered and randomized to either: 1) surfactant (200 mg/kg) or 2) surfactant mixed with budesonide (0.25 mg/kg) before mechanical ventilation with VT of 7-8 ml/kg for 2, 6, or 24 h (n = 6 or 7/group). Lung physiology and budesonide levels in the plasma and the lung were measured. Lung tissue, bronchoalveolar lavage fluid (BALF), liver, and brain tissues were evaluated for indicators of injury. High initial budesonide plasma levels of 170 ng/ml decreased to 3 ng/ml at 24 h. Lung tissue budesonide levels were less than 1% of initial dose by 24 h. Although physiological variables were generally similar, budesonide-exposed lambs required lower mean airway pressures, had higher hyperoxia responses, and had more stable blood pressures. Budesonide decreased proinflammatory mRNA in the lung, liver, and brain. Budesonide also decreased total protein and proinflammatory cytokines in BALF, and decreased inducible nitric oxide synthase activation at 24 h. In ventilated preterm lambs, most of the budesonide left the lung within 24 h. The addition of budesonide to surfactant improved physiology, decreased markers of lung injury, and decreased systemic responses in liver and brain.
Assuntos
Budesonida , Pulmão , Pneumonia , Surfactantes Pulmonares , Respiração Artificial , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Budesonida/farmacocinética , Budesonida/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Inflamação/terapia , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Pneumonia/metabolismo , Pneumonia/patologia , Pneumonia/fisiopatologia , Pneumonia/terapia , Surfactantes Pulmonares/farmacocinética , Surfactantes Pulmonares/farmacologia , OvinosRESUMO
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.
Assuntos
Pulmão/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Proteína B Associada a Surfactante Pulmonar/análise , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/análise , Proteína C Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/análise , Surfactantes Pulmonares/metabolismo , Animais , Animais Recém-Nascidos , Pulmão/efeitos dos fármacos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/farmacologia , Fosfatidilcolinas/farmacologia , Proteína B Associada a Surfactante Pulmonar/farmacologia , Proteína C Associada a Surfactante Pulmonar/farmacologia , Surfactantes Pulmonares/farmacologia , Ovinos , Distribuição TecidualRESUMO
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.
Assuntos
Betametasona/análogos & derivados , Dexametasona/análogos & derivados , Glucocorticoides/administração & dosagem , Ovinos/embriologia , Administração Oral , Animais , Betametasona/administração & dosagem , Betametasona/farmacocinética , Disponibilidade Biológica , Dexametasona/administração & dosagem , Dexametasona/farmacocinética , Feminino , Glucocorticoides/farmacocinética , Pulmão/crescimento & desenvolvimento , GravidezRESUMO
Mechanical ventilation causes lung injury and systemic inflammatory responses in preterm sheep and is associated with bronchopulmonary dysplasia (BPD) in preterm infants. Budesonide added to surfactant decreased BPD by 20% in infants. We wanted to determine the effects of budesonide and surfactant on injury from high tidal volume (VT) ventilation in preterm lambs. Ewes at 125 ± 1 days gestational age had fetal surgery to expose fetal head and chest with placental circulation intact. Lambs were randomized to 1) mechanical ventilation with escalating VT to target 15 ml/kg by 15 min or 2) continuous positive airway pressure (CPAP) of 5 cmH2O. After the 15-min intervention, lambs were given surfactant 100 mg/kg with saline, budesonide 0.25 mg/kg, or budesonide 1 mg/kg. The fetuses were returned to the uterus for 24 h and then delivered and ventilated for 30 min to assess lung function. Budesonide levels were low in lung and plasma. CPAP groups had improved oxygenation, ventilation, and decreased injury markers compared with fetal VT lambs. Budesonide improved ventilation in CPAP lambs. Budesonide decreased lung weights and lung liquid and increased lung compliance and surfactant protein mRNA. Budesonide decreased proinflammatory and acute-phase responses in lung. Airway thickness increased in animals not receiving budesonide. Systemically, budesonide decreased monocyte chemoattractant protein-1 mRNA and preserved glycogen in liver. Results with 0.25 and 1 mg/kg budesonide were similar. We concluded that budesonide with surfactant matured the preterm lung and decreased the liver responses but did not improve lung function after high VT injury in fetal sheep.
Assuntos
Displasia Broncopulmonar , Budesonida , Feto , Nascimento Prematuro/terapia , Surfactantes Pulmonares , Animais , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Displasia Broncopulmonar/terapia , Budesonida/farmacocinética , Budesonida/farmacologia , Feminino , Feto/metabolismo , Feto/patologia , Feto/fisiopatologia , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Gravidez , Nascimento Prematuro/metabolismo , Nascimento Prematuro/patologia , Nascimento Prematuro/fisiopatologia , Surfactantes Pulmonares/farmacocinética , Surfactantes Pulmonares/farmacologia , Respiração Artificial/efeitos adversos , Respiração Artificial/métodos , OvinosRESUMO
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.
Assuntos
Betametasona/administração & dosagem , Maturidade dos Órgãos Fetais/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Pulmão/efeitos dos fármacos , Subfamília A de Transportador de Cassetes de Ligação de ATP/genética , Subfamília A de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Betametasona/análogos & derivados , Betametasona/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Glucocorticoides/farmacocinética , Modelos Animais , Gravidez , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , RNA Mensageiro/metabolismo , OvinosRESUMO
BACKGROUND: Antenatal corticosteroids are among the most important and widely used interventions to improve outcomes for preterm infants. Antenatal corticosteroid dosing regimens remain unoptimized and without maternal weight-adjusted dosing. We, and others, have hypothesized that, once a low concentration of maternofetal steroid exposure is achieved and maintained, the duration of the steroid exposure determines treatment efficacy. Using a sheep model of pregnancy, we tested the relationship among steroid dose, duration of exposure, and treatment efficacy. OBJECTIVE: The study was conducted to investigate the relative importance of duration and magnitude of fetal corticosteroid exposure to mature the preterm fetal ovine lung. STUDY DESIGN: Ewes with single fetuses at 120 days gestation received an intravenous bolus (loading dose) followed by a maintenance infusion of betamethasone phosphate to target 12-hour fetal plasma betamethasone concentrations of (1) 20 ng/mL, (2) 10 ng/mL, or (3) 2 ng/mL. In a subsequent experiment, fetal plasma betamethasone concentrations were targeted at 2 ng/mL for 26 hours. Negative control animals received sterile saline solution. Positive control animals received 2 intramuscular injections of 0.25 mg/kg Celestone Chronodose (betamethasone phosphate + betamethasone acetate) spaced at 24 hours. Preterm lambs were delivered surgically and ventilated 48 hours after treatment commenced. Maternal and fetal plasma betamethasone concentrations were confirmed by mass spectrometry in a parallel study of chronically catheterized, corticosteroid-treated ewes and fetuses. RESULTS: The loading and maintenance doses were achieved and maintained the desired fetal plasma betamethasone concentrations of approximately 20, 10, and 2 ng/mL for 12 hours. Compared with the 12-hour infusion-treated animals, lambs from the positive control (2 intramuscular doses of 0.25 mg/kg Celestone Chronodose) group had the greatest functional lung maturation (compliance, gas exchange, arterial pH) and molecular evidence of maturation (glucocorticoid receptor signaling activation), despite having maximum fetal plasma betamethasone concentrations 2.5 times lower than animals in the 20 ng/mL betamethasone infusion group. Lambs from the 12-hour 2-ng/mL betamethasone infusion group had little functional lung maturation. In contrast, lambs from the 26-hour 2-ng/mL betamethasone infusion group had functional lung maturation equivalent to lambs from the positive control group. CONCLUSION: In preterm lambs that were exposed to antenatal corticosteroids, high maternofetal plasma betamethasone concentrations did not correlate with improved lung maturation. The largest and most consistent improvements in lung maturation were in animals that were exposed to either the clinical course of Celestone Chronodose or a low-dose betamethasone phosphate infusion to achieve a fetal plasma betamethasone concentration of approximately 2 ng/mL for 26 hours. The duration of low-concentration maternofetal steroid exposure, not total dose or peak drug exposure, is a key determinant for antenatal corticosteroids efficacy. These findings underscore the need to develop an optimized steroid dosing regimen that may improve both the efficacy and safety of antenatal corticosteroids therapy.