BackgroundHypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI.MethodsPreterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis.ResultsGlobal HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function.ConclusionsOur data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.

Bronchopulmonary dysplasia is associated with chorioamnionitis and fetal lung inflammation. Ureaplasma species are the bacteria most frequently isolated from chorioamnionitis. Very chronic ureaplasma colonization of amniotic fluid causes low-grade lung inflammation and functional lung maturation in fetal sheep. Less is known about shorter exposures of the fetal lung. Therefore, we hypothesized that ureaplasmas would cause an acute inflammatory response that would alter lung development. Singleton ovine fetuses received intra-amniotic Ureaplasma parvum serovar 3 or control media at 110, 117, or 121 days and were delivered at 124 days gestational age (term = 150 days). Inflammation was assessed by 1) cell counts in bronchoalveolar lavage fluid (BALF), and 2) cytokine mRNA measurements, immunohistochemistry, and flow cytometry for inflammatory cells and elastin and α-smooth muscle actin (α-SMA) staining in lung tissue. Neutrophils were increased in BALF 3 days after exposure to ureaplasmas (P = 0.01). Myeloperoxidase-positive cells increased after 3 days (P = 0.03), and major histocompatibility complex (MHC) class II-positive cells increased after 14 days of ureaplasma exposure (P = 0.001). PU.1 (macrophage marker)- or CD3 (T lymphocyte marker)-positive cells were not induced by ureaplasmas. CD3-positive cells in the posterior mediastinal lymph node increased in ureaplasma-exposed animals at 3, 7, and 14 days (P = 0.002). Focal elastin depositions decreased in alveolar septa at 14 days (P = 0.002), whereas α-SMA increased in arteries and bronchioli. U. parvum induced a mild acute inflammatory response and changed elastin and α-SMA deposition in the lung, which may affect lung structure and subsequent development.

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different pro-inflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic injections of media (control) or Ureaplasma parvum serovar 3 either 7d or 70d before preterm delivery. Another group received an intraamniotic injection of E.coli lipo-polysaccharide (LPS) 2d prior to delivery. To test for interactions, intraamniotic U. parvum exposed animals were challenged with intraamniotic LPS and delivered 2d later. All animals were delivered at 124±1d gestation (Term=150d). Compared to the 2d LPS exposure group, the U. parvum 70d+LPS group had: 1) decreased lung pro and anti-inflammatory cytokine expression 2) fewer CD3+ T-lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7d did not change responses to a subsequent intraamniotic LPS challenge, and exposure to intraamniotic U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGFβ1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70d+LPS but not U. parvum 7d+LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of down-regulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

The proinflammatory stimulus of chorioamnionitis is commonly associated with preterm delivery. Women at risk of preterm delivery receive antenatal glucocorticoids to functionally mature the fetal lung. However, the effects of the combined exposures of chorioamnionitis and antenatal glucocorticoids on the fetus are poorly understood. Time-mated ewes with singleton fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) either preceding or following maternal intramuscular betamethasone 7 or 14 days before delivery, and the fetuses were delivered at 120 days gestational age (GA) (term = 150 days GA). Gestation matched controls received intra-amniotic and maternal intramuscular saline. Compared with saline controls, intra-amniotic LPS increased inflammatory cells in the bronchoalveolar lavage and myeloperoxidase, Toll-like receptor 2 and 4 mRNA, PU.1, CD3, and Foxp3-positive cells in the fetal lung. LPS-induced lung maturation measured as increased airway surfactant and improved lung gas volumes. Intra-amniotic LPS-induced inflammation persisted until 14 days after exposure. Betamethasone treatment alone induced modest lung maturation but, when administered before intra-amniotic LPS, suppressed lung inflammation. Interestingly, betamethasone treatment after LPS did not counteract inflammation but enhanced lung maturation. We conclude that the order of exposures of intra-amniotic LPS or maternal betamethasone had large effects on fetal lung inflammation and maturation.

Yearly more than 15 million babies are born premature (<37 weeks gestational age), accounting for more than 1 in 10 births worldwide. Lung injury caused by maternal chorioamnionitis or preeclampsia, postnatal ventilation, hyperoxia, or inflammation can lead to the development of bronchopulmonary dysplasia (BPD), one of the most common adverse outcomes in these preterm neonates. BPD patients have an arrest in alveolar and microvascular development and more frequently develop asthma and early-onset emphysema as they age. Understanding how the alveoli develop, and repair, and regenerate after injury is critical for the development of therapies, as unfortunately there is still no cure for BPD. In this review, we aim to provide an overview of emerging new concepts in the understanding of perinatal lung development and injury from a molecular and cellular point of view and how this is paving the way for new therapeutic options to prevent or treat BPD, as well as a reflection on current treatment procedures.