Jinghua Chai

Inflammation-induced preterm birth alters neuronal morphology in the mouse fetal brain

Adverse neurological outcome is a major cause of long‐term morbidity in ex‐preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non‐infectious model of preterm birth by administering RU486. Pro‐inflammatory cytokines (IL‐10, IL‐1β, IL‐6 and TNF‐α) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro‐inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation‐induced preterm birth, but not non‐infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule‐associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation‐induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology.

MAGNESIUM SULFATE REDUCES INFLAMMATION-ASSOCIATED BRAIN INJURY IN FETAL MICE

OBJECTIVE The purpose of this study was to investigate whether magnesium sulfate (MgSO(4)) prevents fetal brain injury in inflammation-associated preterm birth (PTB). STUDY DESIGN In a mouse model of PTB, mice exposed to lipopolysaccharide (LPS) or normal saline (NS) by intrauterine injection were randomized to intraperitoneal treatment with MgSO(4) or NS [corrected]. From the 4 treatment groups (NS + NS; LPS + NS; LPS + MgSO(4); and NS + MgSO(4)), fetal brains were collected for quantitative polymerase chain reaction studies and primary neuronal cultures. Messenger RNA expression of cytokines, cell death, and markers of neuronal and glial differentiation were assessed. Immunocytochemistry and confocal microscopy were performed. RESULTS There was no difference between the LPS + NS and LPS + MgSO(4) groups in the expression of proinflammatory cytokines, cell death markers, and markers of prooligodendrocyte and astrocyte development (P > .05 for all). Neuronal cultures from the LPS + NS group demonstrated morphologic changes; this neuronal injury was prevented by MgSO(4) (P < .001). CONCLUSION Amelioration of neuronal injury in inflammation-associated PTB may be a key mechanism by which MgSO(4) prevents cerebral palsy.

Preterm and Term Cervical Ripening in CD1 Mice (Mus musculus): Similar or Divergent Molecular Mechanisms?

Premature cervical ripening is believed to contribute to preterm birth (PTB). Preterm cervical ripening may be due to an aberrant regulation in timing of the same processes that occur at term, or may result from unique molecular mechanisms. Using mouse models of PTB, this study sought to investigate if the molecular mechanisms that govern cervical ripening were similar between preterm and term. Lipopolysaccharide (LPS) is infused into the uterine horn to create a mouse model of inflammation-induced PTB. For a noninfectious model of PTB, RU486 was administered. Both models result in delivery of pups in 8–24 h. Cervical tissues were collected from these models, as well as throughout gestation. Cervical tissues from E15 (preterm), E15 LPS (preterm inflammation), and E18.5 (term) were used for microarray analysis (n = 18). Additional experiments using gestational time course specimens were performed to confirm microarray results. Specific gene pathways were differentially expressed between the groups. Genes involved in immunity and inflammation were increased in the cervix in inflammation-induced PTB; term labor was not associated with differential expression of immune pathways. Cytokine expression was not increased in cervices during term labor, but was increased in the pospartum period. Epithelial cell differentiation pathway was significantly altered in term, but not preterm, labor. Activation of immune pathways may be sufficient for cervial ripening, but does not appear necessary. Differential expression of the epithelial cell differentiation pathway appears necessary in the process of cervical repair. Our results indicate that the molecular mechanisms governing preterm and term cervical ripening are distinctly different.

Beyond white matter damage: fetal neuronal injury in a mouse model of preterm birth

OBJECTIVE The purpose of this study was to elucidate possible mechanisms of fetal neuronal injury in inflammation-induced preterm birth. STUDY DESIGN With the use of a mouse model of preterm birth, the following primary cultures were prepared from fetal brains: (1) control neurons (CNs), (2) lipopolysaccharide-exposed neurons (LNs), (3) control coculture (CCC) that consisted of neurons and glia, and (4) lipopolysaccharide-exposed coculture (LCC) that consisted of lipopolysaccharide-exposed neurons and glia. CNs and LNs were treated with culture media from CN, LN, CCC, and LCC after 24 hours in vitro. Immunocytochemistry was performed for culture characterization and neuronal morphologic evidence. Quantitative polymerase chain reaction was performed for neuronal differentiation marker, microtubule-associated protein 2, and for cell death mediators, caspases 1, 3, and 9. RESULTS Lipopolysaccharide exposure in vivo did not influence neuronal or glial content in cocultures but decreased the expression of microtubule-associated protein 2 in LNs. Media from LNs and LCCs induced morphologic changes in control neurons that were comparable with LNs. The neuronal damage caused by in vivo exposure (LNs) could not be reversed by media from control groups. CONCLUSION Lipopolysaccharide-induced preterm birth may be responsible for irreversible neuronal injury.

A Mouse Model of Term Chorioamnionitis: Unraveling Causes of Adverse Neurological Outcomes

Maternal fever and/or chorioamnionitis at term are associated with an increased prevalence of adverse neurobehavioral outcomes in exposed offspring. Since the mechanisms of such injury are currently unknown, the objectives of this study were to elucidate whether intrauterine inflammation at term results in fetal brain injury. Specifically, we assessed brain injury by investigating the cytokine response, white matter damage, and neuronal injury and viability. A mouse model of intrauterine inflammation at term was utilized by injecting lipopolysaccharide (LPS), or normal saline into uterine horn. Compared to saline-exposed, LPS-exposed fetal brains had significantly increased IL-1β and IL-6 messenger RNA (mRNA) expression (P < .05 for both) and IL-6 protein levels by enzyme-linked immunosorbent assay (ELISA; P < 0.05). Fetal neurons were affected by the intrauterine and fetal brain inflammation, as demonstrated by significantly decreased microtubule-associated protein 2 (MAP2) mRNA expression and a decrease in immunocytochemical staining (a marker of neuronal cytoskeleton development; P < .05), altered neuronal morphology (P < 0.05), and delayed neurotoxicity (P < .05). These fetal neuronal changes occurred without overt changes in white matter damage markers. Marker of astrocyte development and astrogliosis (glial fibrillary acidic protein [GFAP]) did not show an increase; pro-oligodendrocyte marker (PLP1/DM20) was not significantly changed (P > .05). These studies may provide a critical mechanism for the observed long-term adverse neurobehavioral outcomes after exposure to chorioamnionitis at term.

TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth

In this study, we sought to assess how essential activation of toll-like receptor 4 (TLR-4) is to fetal brain injury from intrauterine inflammation. Both wild-type and TLR-4 mutant fetal central nervous system cells were exposed to inflammation using lipopolysaccharide in vivo or in vitro. Inflammation could not induce neuronal injury in the absence of glial cells, in either wild-type or TLR-4 mutant neurons. However, injured neurons could induce injury in other neurons regardless of TLR-4 competency. Our results indicate that initiation of neuronal injury is a TLR-4-dependent event, while propagation is a TLR-4-independent event.

Maternal mortality from systemic illness: unraveling the contribution of the immune response.

OBJECTIVE Maternal morbidity and/or mortality (MM) is increased in pyelonephritis and influenza. Alterations in the immune response could account for the increase MM. We sought to determine whether the immune response is functionally different during pregnant and nonpregnant (NP) states. STUDY DESIGN Mouse model of systemic and localized inflammation was used. Maternal serum was assessed for expression of T-helper cell type 1 and 2 cytokines. Maternal spleens were harvested for immunohistochemistry. RESULTS Systemic administration of lipopolysaccharides resulted in no mortality to NP mice compared with 88% in preterm and 100% in term mice. A potent cytokine response was present in both NP and pregnancy. Systemic inflammation in pregnancy results in increased CD8 and CD11c expression in spleens. CONCLUSION Differences in cytokine response to systemic inflammation is unlikley to modulate the increased MM during pregnancy. Altered T-cell and dendritic cell responses in pregnancy may be responsible for the increase in MM.

The negative regulators of the host immune response: an unexplored pathway in preterm birth

OBJECTIVE Toll-like receptors (TLRs) are essential mediators of host immunity. TLR activation must be tightly regulated to prevent an exaggerated immune response from devastating the host. These studies assessed the expression of negative regulators (interleukin receptor-associated kinase [IRAK]-3, IRAK-1, Fas-associated protein with death domain) during pregnancy and in preterm birth (PTB). STUDY DESIGN Tissues (uterine, cervix, placenta, and spleens) from the following experimental groups were harvested: (1) nonpregnant mice, (2) pregnant mice across gestation, (3) murine model of PTB, and (4) pregnant mice exposed to medroxyprogesterone acetate (MPA). RESULTS Negative regulators are differentially expressed in the uterus during pregnancy. In the setting of PTB, IRAK-3 is significantly increased in the uterus and cervix but not the placenta. In maternal spleens, IRAK-3 and IRAK-1 are increased in response to intrauterine inflammation. MPA can increase IRAK expression in cervical tissues. CONCLUSION Negative regulators of the maternal immune response may play an important role in protecting pregnancies from an exaggerated inflammatory response.