Meredith A. Kelleher

Sex-dependent effect of a low neurosteroid environment and intrauterine growth restriction on foetal guinea pig brain development

Progesterone and its neuroactive metabolite, allopregnanolone, are present in high concentrations during pregnancy, but drop significantly following birth. Allopregnanolone influences foetal arousal and enhances cognitive and behavioural recovery following traumatic brain injury. Inhibition of allopregnanolone synthesis increases cell death in foetal animal brains with experimental hypoxia. We hypothesised that complications during pregnancy, such as early or preterm loss of placental steroids and intrauterine growth restriction (IUGR), would disrupt the foetal neurosteroid system, contributing to poor neurodevelopmental outcomes. This study aimed to investigate the effects of chronic inhibition of allopregnanolone synthesis before term and IUGR on developmental processes in the foetal brain. Guinea pig foetuses were experimentally growth restricted at mid-gestation and treated with finasteride, an inhibitor of allopregnanolone synthesis. Finasteride treatment reduced foetal brain allopregnanolone concentrations by up to 75% and was associated with a reduction in myelin basic protein (MBP) (P = 0.001) and an increase in glial fibrillary acidic protein expression in the subcortical white matter brain region (P < 0.001). IUGR resulted in decreased MBP expression (P < 0.01) and was associated with a reduction in the expression of steroidogenic enzyme 5α-reductase (5αR) type 2 in the foetal brain (P = 0.061). Brain levels of 5αR1 were higher in male foetuses (P = 0.008). Both IUGR and reduced foetal brain concentrations of allopregnanolone were associated with altered expression of myelination and glial cell markers within the developing foetal brain. The potential role of neurosteroids in protecting and regulating neurodevelopmental processes in the foetal brain may provide new directions for treatment of neurodevelopmental disorders in infants who are exposed to perinatal insults and pathologies.

Changes in Neuroactive Steroid Concentrations After Preterm Delivery in the Guinea Pig

Background: Preterm birth is a major cause of neurodevelopmental disorders. Allopregnanolone, a key metabolite of progesterone, has neuroprotective and developmental effects in the brain. The objectives of this study were to measure the neuroactive steroid concentrations following preterm delivery in a neonatal guinea pig model and assess the potential for postnatal progesterone replacement therapy to affect neuroactive steroid brain and plasma concentrations in preterm neonates. Methods: Preterm (62-63 days) and term (69 days) guinea pig pups were delivered by cesarean section and tissue was collected at 24 hours. Plasma progesterone, cortisol, allopregnanolone, and brain allopregnanolone concentrations were measured by immunoassay. Brain 5α-reductase (5αR) expression was determined by Western blot. Neurodevelopmental maturity of preterm neonates was assessed by immunohistochemistry staining for myelination, glial cells, and neurons. Results: Brain allopregnanolone concentrations were significantly reduced after birth in both preterm and term neonates. Postnatal progesterone treatment in preterm neonates increased brain and plasma allopregnanolone concentrations. Preterm neonates had reduced myelination, low birth weight, and high mortality compared to term neonates. Brain 5αR expression was also significantly reduced in neonates compared to fetal expression. Conclusions: Delivery results in a loss of neuroactive steroid concentrations resulting in a premature reduction in brain allopregnanolone in preterm neonates. Postnatal progesterone therapy reestablished neuroactive steroid levels in preterm brains, a finding that has implications for postnatal growth following preterm birth that occurs at a time of neurodevelopmental immaturity.

The guinea pig as an animal model for studying perinatal changes in microvascular function

Introduction:Microvascular dysfunction, characterized by inappropriate vasodilatation and high blood flow in the peripheral microcirculation, is linked to physiologic instability and poor outcome in neonates. Specifically, preterm neonates have significantly higher levels of baseline microvascular blood flow than term neonates at 24 h postnatal age. Because of similarities between human and guinea pig endocrine profiles and maturity at birth, we hypothesized that preterm guinea pig neonates would provide a suitable model for studying the mechanisms underlying transitional microvascular function.Results:Guinea pigs that were delivered preterm showed immaturity and had markedly reduced viability. Baseline microvascular blood flow was significantly higher in preterm animals than in term animals. No effect of intrauterine growth restriction or birth weight on baseline microvascular blood flow was observed in either preterm or term animals.Discussion:These results are consistent with recent clinical findings and support the use of the guinea pig as a suitable model for future studies of the mechanisms underlying perinatal microvascular behavior.Methods:Guinea pigs were delivered either prematurely or at term. Laser Doppler flowmetry was used to study microvascular blood flow at 23 h postnatal age.

Zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.Zika virus infection during pregnancy can result in birth defects, but underlying pathogenesis at the maternal-fetal interface is unclear. Here, the authors use non-invasive in vivo imaging of Zika-infected rhesus macaques and show that infection results in abnormal oxygen transport across the placenta.

Effect of postnatal progesterone therapy following preterm birth on neurosteroid concentrations and cerebellar myelination in guinea pigs

Allopregnanolone protects the fetal brain and promotes normal development including myelination. Preterm birth results in the early separation of the infant from the placenta and consequently a decline in blood and brain allopregnanolone concentrations. Progesterone therapy may increase allopregnanolone and lead to improved oligodendrocyte maturation. The objectives of this study were to examine the efficacy of progesterone replacement in augmenting allopregnanolone concentrations during the postnatal period and to assess the effect on cerebellar myelination - a region with significant postnatal development. Preterm guinea pig neonates delivered at 62 days of gestation by caesarean section received daily s.c. injections of vehicle (2-Hydroxypropyl-β-cyclodextrin) or progesterone (16 mg/kg) for 8 days until term-equivalent age (TEA). Term delivered controls (PND1) received vehicle. Neonatal condition/wellbeing was scored, and salivary progesterone was sampled over the postnatal period. Brain and plasma allopregnanolone concentrations were measured by radioimmunoassay; cortisol and progesterone concentrations were determined by enzyme immunoassay; and myelin basic protein (MBP), proteolipid protein (PLP), oligodendroctye transcription factor 2 (OLIG2) and platelet-derived growth factor receptor-α (PDGFRα) were quantified by immunohistochemistry and western blot. Brain allopregnanolone concentrations were increased in progesterone-treated neonates. Plasma progesterone and cortisol concentrations were elevated in progesterone-treated male neonates. Progesterone treatment decreased MBP and PLP in lobule X of the cerebellum and total cerebellar OLIG2 and PDGFRα in males but not females at TEA compared with term animals. We conclude that progesterone treatment increases brain allopregnanolone concentrations, but also increases cortisol levels in males, which may disrupt developmental processes. Consideration should be given to the use of non-metabolizable neurosteroid agonists.

Loss of neurosteroid-mediated protection following stress during fetal life.

Elevated levels of neurosteroids during late gestation protect the fetal brain from hypoxia/ischaemia and promote neurodevelopment. Suppression of allopregnanolone production during pregnancy leads to the onset of seizure-like activity and potentiates hypoxia-induced brain injury. Markers of myelination are reduced and astrocyte activation is increased. The placenta has a key role in maintaining allopregnanolone concentrations in the fetal circulation and brain during gestation and levels decline markedly after both normal and preterm birth. This leads to the preterm neonate developing in a neurosteroid deficient environment between delivery and term equivalence. The expression of 5α-reductases is also lower in the fetus prior to term. These deficiencies in neurosteroid exposure may contribute to the increase in incidence of the adverse patterns of behaviour seen in children that are born preterm. Repeated exposure to glucocorticoid stimulation suppresses 5α-reductase expression and allopregnanolone levels in the fetus and results in reduced myelination. Both fetal growth restriction and prenatal maternal stress lead to increased cortisol concentrations in the maternal and fetal circulation. Prenatal stress results in reduced expression of key GABAA receptor subunits that normally heighten neurosteroid sensitivity. These stressors also result in altered placental allopregnanolone metabolism pathways. These findings suggest that reduced neurosteroid production and action in the perinatal period may contribute to some of the adverse neurodevelopmental and behavioural outcomes that result from these pregnancy compromises. Studies examining perinatal steroid supplementation therapy with non-metabolisable neurosteroid analogues to improve these outcomes are warranted.

Mechanisms leading to increased risk of preterm birth in growth-restricted guinea pig pregnancies.

Intrauterine growth restriction (IUGR) is a risk factor for preterm labor; however, the mechanisms of the relationship remain unknown. Prostaglandin (PG), key stimulants of labor, availability is regulated by the synthetic enzymes, prostaglandin endoperoxidases 1 and 2 (PTGS1 and 2), and the metabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (HPGD). We hypothesized that IUGR increases susceptibility to preterm labor due to the changing balance of synthetic and metabolizing enzymes and hence greater PG availability. We have tested this hypothesis using a surgically induced IUGR model in guinea pigs, which results in significantly shorter gestation. Myometrium, amnion, chorion, and placentas were collected from sham operated or IUGR pregnancies, and PTGS1 and HPGD protein expression were quantified throughout late gestation (>62 days) and labor. The PTGS1 expression was significantly upregulated in the myometrium of IUGR animals, and chorionic HPGD expression was markedly decreased (P < .01 and P < .001, respectively). These findings suggest a shift in the balance of PG production over metabolism in IUGR pregnancies leads to a greater susceptibility to preterm birth.

Antenatal prevention of cerebral palsy and childhood disability: is the impossible possible?

This review covers our current knowledge of the causes of perinatal brain injury leading to cerebral palsy‐like outcomes, and argues that much of this brain damage is preventable. We review the experimental evidence that there are treatments that can be safely administered to women in late pregnancy that decrease the likelihood and extent of perinatal brain damage that occurs because of acute and severe hypoxia that arises during some births, and the additional impact of chronic fetal hypoxia, infection, inflammation, growth restriction and preterm birth. We discuss the types of interventions required to ameliorate or even prevent apoptotic and necrotic cell death, and the vulnerability of all the major cell types in the brain (neurons, astrocytes, oligodendrocytes, microglia, cerebral vasculature) to hypoxia/ischaemia, and whether a pan‐protective treatment given to the mother before birth is a realistic prospect.

Increased placental T cell trafficking results in adverse neurobehavioral outcomes in offspring exposed to sub-chronic maternal inflammation

Interleukin-1 beta (IL-1β) is a cytokine mediator of perinatal brain injury. The effect of sub-chronic systemic IL-1β exposure in perinatal and offspring outcomes is unclear. The aim of this study was to examine the effects of maternal IL-1β exposure on pregnancy and offspring outcomes. At E15, CD1 dams were allocated to receive intraperitoneal injection of phosphate buffered saline or mouse recombinant IL-1β (1 mcg) for four consecutive days. We analyzed pup survivaland neurobehavioral status. At E18, placental H&E staining and fetal brain Nissl staining was performed. Placental gene expression was analyzed by qPCR and T cell infiltration was analyzed by flow cytometry. Effects of inflammation on feto-placental blood flow were analyzed by Doppler ultrasonography. IL-1β decreased pup survival (P < .0001) and adversely affected offspring performance on neurodevelopmental tests (P < .05). Placentas of exposed dams exhibited significant thinning of maternal and fetal sides, and fetal brain exhibited cortical thinning. Placental qPCR analysis revealed significant upregulation of NFκB2 (P = .0021) and CXCL11 (P = .0401). While maternal IL-1β exposure did not affect feto-placental blood flow, placental flow cytometry showed an increase in placental infiltration of CD4+ T cells at 24 h post-injection (hpi, P < .0001) and CD8+ T cells at 72 hpi (P = .0217). Maternal sub-chronic, systemic inflammation with IL-1β decreased pup survival and played a key role in perinatal brain injury. The mechanisms behind these outcomes may involve immune system activation and alterations in placental T cell trafficking.

Effect of Inhibition of 5α Reduced Steroid Synthesis on Apoptotic Brain Cell Death and Neurosteroidogenic Enzyme Expression in the Fetal and Neonatal Guinea Pig

Background: We have developed a sheep model of intrauterine ureaplasma infection. We aimed to examine the capability of ureaplasmas in the amniotic fluid to infect the fetus and alter fetal development...