Impact of hypoxia-ischemia and dopamine treatment on dopamine receptor binding density in the preterm fetal sheep brain.

Dopamine is often used to treat hypotension in preterm infants who are at risk of hypoxic-ischemic (HI) brain injury due to cerebral hypoperfusion and impaired autoregulation. There is evidence that systemically administered dopamine crosses the preterm blood-brain barrier. However, the effects of exogenous dopamine and cerebral HI on dopaminergic signaling in the immature brain are unknown. We determined the effect of HI and dopamine on D1 and D2 receptor binding and expressions of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the striatum of the preterm fetal sheep. Fetal sheep (99 days of gestation, term = 147days) were unoperated controls (n = 6) or exposed to severe HI using umbilical cord occlusion and saline infusion (UCO + saline, n = 8) or to HI with dopamine infusion (UCO + dopamine, 10 µg/kg/min, n = 7) for 74 h. D1 and D2 receptor densities were measured by autoradiography in vitro. DAT, TH, and cell death were measured using immunohistochemistry. HI resulted in cell death in the caudate nucleus and putamen, and dopamine infusion started before HI did not exacerbate or ameliorate these effects. HI led to reduced D1 and D2 receptor densities in the caudate nucleus and reduction in DAT protein expression in the caudate and putamen. Fetal brains exposed to dopamine in addition to HI were not different from those exposed to HI alone in these changes in dopaminergic parameters. We conclude that dopamine infusion does not alter the striatal cell death or the reductions in D1 and D2 receptor densities and DAT protein expression induced by HI in the preterm brain.NEW & NOTEWORTHY This is the first study on the effects of hypoxia-ischemia and dopamine treatment on the dopaminergic pathway in the preterm brain. In the striatum of fetal sheep (equivalent to ∼26-28 wk of human gestation), we demonstrate that hypoxia-ischemia leads to cell death, reduces D1 and D2 receptors, and reduces dopamine transporter. Intravenous dopamine infusion at clinical dosage used in preterm human infants does not alter the striatal cell death, D1 and D2 receptor density levels, and DAT protein expressions after hypoxia-ischemia in the preterm brain.

Description of a method for inducing fetal growth restriction in the spiny mouse.

Intrauterine or fetal growth restriction (IUGR) is a major complication of pregnancy and leads to significant perinatal morbidities and mortality. Typically, induction of IUGR in animals involves the complete occlusion or ablation of vessels to the uterus or placenta, acutely impairing blood flow and fetal growth, usually with high fetal loss. We aimed to produce a model of reduced fetal growth in the spiny mouse with minimal fetal loss. At 27 days gestational age (term is 38-39 days), a piece of silastic tubing was placed around the left uterine artery to prevent the further increase of uterine blood flow with advancing gestation to induce IUGR (occluded). Controls were generated from sham surgeries without placement of the tubing. Dams were humanely euthanized at 37 days gestational age and all fetuses and placentas were weighed and collected. Of the 17 dams that underwent surgery, 15 carried their pregnancies to 37 days gestational age and 95% of fetuses survived to this time. The difference in fetal body weight between occluded and control was ~21% for fetuses in the left uterus side: there were no differences for fetuses in the right uterus side. Offspring from the occluded group had significantly lower brain, liver, lung, kidney and carcass weights compared with shams. Preventing the gestation-related increase of uterine blood flow induced significant growth restriction in the fetal spiny mouse, with minimal fetal loss. This technique could be readily adapted for other small animal.

Effects of combined IUGR and prenatal stress on the development of the hippocampus in a fetal guinea pig model.

Intrauterine growth restriction (IUGR) and maternal stress during pregnancy are two compromises that negatively impact neurodevelopment and increase the risk of developing later life neuropsychiatric disorders such as schizophrenia, depression and behavioural disorders. Neurosteroids, particularly allopregnanolone, are important in protecting the developing brain and promoting many essential neurodevelopmental processes. Individually, IUGR and prenatal stress (PS) reduce myelination and neurogenesis within affected fetal brains, however less information is available on the combined effects of these two disorders on the term fetal brain. This study aimed to investigate how IUGR and PS impairs the neurosteroid pathway when combined using a guinea pig model, and how these then disrupt the neurodevelopment of the fetus. Uterine artery blood flow restriction was performed at GA30-35 to induce growth restriction, whilst PS was induced by exposure of the dam to a strobe light during gestation commencing GA40 and repeated every 5 days. Exposure in this model caused reductions in hippocampal CA1 MBP immunostaining of male fetuses in both IUGR alone and IUGR+PS paradigms but only by IUGR in the subcortical white mater, compared with control males. Plasma allopregnanolone was reduced by both stressors irrespective of sex, whereas GFAP or MAP2 expression were not affected by either stressor. Female neurodevelopment, as assessed by these markers, was unimpeded by these compromises. The addition of prenatal stress did not further compound these deficits.

Cerebral haemodynamic response to somatosensory stimulation in near-term fetal sheep.

KEY POINTS: Cerebral haemodynamic response to neural stimulation has been extensively investigated in animal and clinical studies, in both adult and paediatric populations, but little is known about cerebral haemodynamic functional response in the fetal brain. The present study describes the cerebral haemodynamic response measured by near-infrared spectroscopy to somatosensory stimulation in fetal sheep. The cerebral haemodynamic response in the fetal sheep brain changes from a positive (increase in oxyhaemoglobin (oxyHb)) response pattern to a negative or biphasic response pattern when the duration of somatosensory stimulation is increased, probably due to cerebral vasoconstriction with prolonged stimulations. In contrast to adult studies, we have found that changes in fetal cerebral blood flow and oxyHb are positively increased in response to somatosensory stimulation during hypercapnia. We propose this is related to reduced vascular resistance and recruitment of cerebral vasculature in the fetal brain during hypercapnia. ABSTRACT: Functional hyperaemia induced by a localised increase in neuronal activity has been suggested to occur in the fetal brain owing to a positive blood oxygen level-dependent (BOLD) signal recorded by functional magnetic resonance imaging following acoustic stimulation. To study the effect of somatosensory input on local cerebral perfusion we used near-infrared spectroscopy (NIRS) in anaesthetised, partially exteriorised fetal sheep where the median nerve was stimulated with trains of pulses (2 ms, 3.3 Hz) for durations of 1.8, 4.8 and 7.8 s. Signal averaging of cerebral NIRS responses to 20 stimulus trains repeated every 60 s revealed that a short duration of stimulation (1.8 s) increased oxyhaemoglobin in the contralateral cortex consistent with a positive functional response, whereas longer durations of stimulation (4.8, 7.8 s) produced more variable oxyhaemoglobin responses including positive, negative and biphasic patterns of change. Mean arterial blood pressure and cerebral perfusion as monitored by laser Doppler flowmetry always showed small, but coincident increases following median nerve stimulation regardless of the type of response detected by the NIRS in the contralateral cortex. Hypercapnia significantly increased the baseline total haemoglobin and deoxyhaemoglobin, and in 7 of 8 fetal sheep positively increased the changes in contralateral total haemoglobin and oxyhaemoglobin in response to the 7.8 s stimulus train, compared to the response recorded during normocapnia. These results show that activity-driven changes in cerebral perfusion and oxygen delivery are present in the fetal brain, and persist even during periods of hypercapnia-induced cerebral vasodilatation.

Maternal creatine in pregnancy: a retrospective cohort study.

OBJECTIVE: To estimate creatine concentrations in maternal plasma and urine, and establish relationships with maternal characteristics, diet and fetal growth. DESIGN: Retrospective cohort study. SETTING: Lyell McEwin Hospital, Adelaide, Australia. POPULATION: A biobank of plasma and urine samples collected at 13, 18, 30 and 36 weeks' gestation from 287 pregnant women from a prospective cohort of asthmatic and non-asthmatic women. METHODS: Creatine was measured by enzymatic analysis. Change in creatine over pregnancy was assessed using the Friedman test. Linear mixed models regression was used to determine associations between maternal factors and diet with creatine across pregnancy and between creatine with indices of fetal growth at birth. MAIN OUTCOME MEASURES: Maternal creatine concentrations, associations between maternal factors and creatine and between creatine and fetal growth parameters. RESULTS: Maternal smoking, body mass index, asthma and socio-economic status were positively and parity negatively associated with maternal plasma and/or urine creatine. Maternal urine creatine concentration was positively associated with birthweight centile and birth length. After adjustment, each µmol/l increase in maternal urinary creatine was associated with a 1.23 (95% CI 0.44-2.02) unit increase in birthweight centile and a 0.11-cm (95% CI 0.03-0.2) increase in birth length. CONCLUSIONS: Maternal factors and fetal growth measures are associated with maternal plasma and urine creatine concentrations. TWEETABLE ABSTRACT: Maternal creatine is altered by pregnancy; fetal growth measures are associated with maternal creatine concentrations.

Dopamine treatment during acute hypoxia is neuroprotective in the developing sheep brain.

Dopamine is often used to treat hypotension in preterm infants; these infants are at risk of developing brain injury due to impaired autoregulation and cerebral hypoperfusion. However the effects of dopamine on the immature brain under conditions of cerebral hypoxia are not known. We hypothesized that pretreatment with dopamine would protect the immature brain from injury caused by cerebral hypoxia. Preterm fetal sheep were used to determine the effects of intravenous dopamine on hypoxia-induced brain injury. In 16 pregnant sheep at 90days of gestation (0.6 of term, term=147days) catheters were implanted aseptically into the fetal carotid artery and jugular vein; an inflatable occluder was placed loosely around the umbilical cord for later induction of fetal hypoxemia. At 5days after surgery, dopamine (10µg/kg/min, n=7 fetuses) or saline (n=9 fetuses) was infused for 74h. Two hours after commencing the dopamine/saline infusion, we induced umbilical cord occlusion (UCO) for up to 25min to produce fetal asphyxia. Fetuses were allowed to recover, and brains were collected 72h later for assessment of neuropathology. Un-operated twin fetuses were used as age-matched non-UCO controls (n=8). In UCO+saline fetuses, microglial and apoptotic cell density in the subcortical and periventricular white matter, caudate nucleus and hippocampus was greater than that in age-matched controls; oxidative stress was elevated in the subcortical and periventricular white matter and caudate nucleus compared to that in age-matched controls. In UCO+dopamine fetuses microglial density and oxidative stress in the cerebral white matter and caudate nucleus were not different to that of age-matched controls. Apoptotic cell death was decreased in the cerebral white matter of UCO+dopamine brains, relative to UCO+saline brains. We conclude that pretreatment with dopamine does not exacerbate hypoxia-induced injury in the immature brain and may be neuroprotective because it led to decreased apoptosis, oxidative stress and neuroinflammation in the cerebral white matter and decreased neuroinflammation in the caudate nucleus.

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-ß-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.

Preterm birth affects GABAA receptor subunit mRNA levels during the foetal-to-neonatal transition in guinea pigs.

Modulation of gamma-aminobutyric acid A (GABAA) receptor signalling by the neurosteroid allopregnanolone has a major role in late gestation neurodevelopment. The objective of this study was to characterize the mRNA levels of GABAA receptor subunits (α4, α5, α6 and δ) that are key to neurosteroid binding in the brain, following preterm birth. Myelination, measured by the myelin basic protein immunostaining, was used to assess maturity of the preterm brains. Foetal guinea pig brains were obtained at 62 days' gestational age (GA, preterm) or at term (69 days). Neonates were delivered by caesarean section, at 62 days GA and term, and maintained until tissue collection at 24 h of age. Subunit mRNA levels were quantified by RT-PCR in the hippocampus and cerebellum of foetal and neonatal brains. Levels of the α6 and δ subunits were markedly lower in the cerebellum of preterm guinea pigs compared with term animals. Importantly, there was an increase in mRNA levels of these subunits during the foetal-to-neonatal transition at term, which was not seen following preterm birth. Myelination was lower in preterm neonatal brains, consistent with marked immaturity. Salivary cortisol concentrations, measured by EIA, were also higher for the preterm neonates, suggesting greater stress. We conclude that there is an adaptive increase in the levels of mRNA of the key GABAA receptor subunits involved in neurosteroid action after term birth, which may compensate for declining allopregnanolone levels. The lower levels of these subunits in preterm neonates may heighten the adverse effect of the premature decline in neurosteroid exposure.

Treatment of pregnant spiny mice at mid gestation with a synthetic glucocorticoid has sex-dependent effects on placental glycogen stores.

INTRODUCTION: Elevated maternal glucocorticoids during human pregnancy suppress fetal growth, more so if the fetus is male. The synthetic glucocorticoid dexamethasone (DEX) is known to affect placental glucose transport, but whether this also affects placental glycogen stores has not been investigated. METHOD: We examined the short and long term consequences of a single, 60 h exposure to DEX at mid gestation on the glycogen pathway in the placenta of the spiny mouse, with a focus on identifying sex-dependent differences in expression of genes involved in glycogen cell formation (PCDH12), and regulation of glycogen synthesis (GSK3B, GYS1, GBE1, FOXO1, UGP2). RESULTS: Placentas from female fetuses had increased amounts of glycogen on day 25 of gestation (term is 39 days) as identified by positive Periodic acid Schiff (PAS) reaction staining. DEX administration initially reduced expression of GSK3B, GYS1, GBE1, FOXO1, UGP2 in both male and female placentas, but reduced histologically detectable glycogen storage in placentas of female fetuses only. The DEX-induced reduction in expression of GSK3B and UGP2 persisted until day 37 of gestation, an effect that was significantly greater in the male placenta. DISCUSSION/CONCLUSION: We conclude that constitutive placental glycogen storage is regulated in pregnancy in a sex-dependant manner, and that glucocorticoids such as DEX induce sex-dependent changes in glycogen storage. Placental glycogen metabolism and its response to glucocorticoids may contribute to the different sensitivities of male and female fetuses to the effects of maternal illness and stress in utero.

Sexually dimorphic placental development throughout gestation in the spiny mouse (Acomys cahirinus).

OBJECTIVES: It has been hypothesized that male fetuses down regulate placental growth during periods of accelerated fetal growth. We aimed to investigate this, and determine whether sexual dimorphism was apparent in the spiny mouse placenta. We hypothesized that expression of fetal growth promoters would be higher in placentas of males, whereas genes involved in placental structural development would be more highly expressed in placentas of females. METHODS: Spiny mouse dams, a precocial rodent with an in utero endocrine milieu dissimilar from other rodents, but akin to humans, were sacrificed at gestational ages 15-37 (term = 39 days). Placentas were collected and processed for histology or qPCR analysis of selected genes (GCM1, MAP2K1, SLC2A1, NR3C1, IGF1, IGF1R). RESULTS: Fetal and placental weights were similar for both sexes. Placentas of female fetuses had less spongy zone (P(SEX) < 0.0001), and more labyrinth (P(SEX) < 0.0001) than males. Early placenta and labyrinth expression of SLC2A1 was higher in males than females (P(SEX) < 0.05). Labyrinthine IGF1R remained constant until term in the female, compared with male where expression increased until term. Peak MAP2K1 expression occurred earlier in the male placenta than the female. Spongy zone SLC2A1 remained constant until term in the female, compared with male where expression increased until term. CONCLUSIONS: The spiny mouse is a species that exhibits sexually dimorphic placental development. We suggest that these sex differences in placental gene expression and structure may underlie or compound the male vulnerability to a sub-optimal in utero environment.

Antioxidant therapies: a potential role in perinatal medicine.

Pregnancies complicated by impaired placentation, acute severe reductions in oxygen supply to the fetus, or intrauterine infection are associated with oxidative stress to the mother and developing baby. Such oxidative stress is characterized as an upregulation in the production of oxidative or nitrative free radicals and a concomitant decrease in the availability of antioxidant species, thereby creating a state of fetoplacental oxidative imbalance. Recently, there has been a good deal of interest in the potential for the use of antioxidant therapies in the perinatal period to protect the fetus, particularly the developing brain, against oxidative stress in complications of pregnancy and birth. This review will examine why the immature brain is particularly susceptible to oxidative imbalance and will provide discussion on antioxidant treatments currently receiving attention in the adult and perinatal literature - allopurinol, melatonin, α-lipoic acid, and vitamins C and E. In addition, we aim to address the interaction between oxidative stress and the fetal inflammatory response, an interaction that may be vital when proposing antioxidant or other neuroprotective strategies.

Alcohol exposure during late gestation: multiple developmental outcomes in sheep.

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life.

Effects of birth asphyxia on neonatal hippocampal structure and function in the spiny mouse.

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term=39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP(3)R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.

A maternal diet supplemented with creatine from mid-pregnancy protects the newborn spiny mouse brain from birth hypoxia.

The creatine-phosphocreatine shuttle is essential for the maintenance of cellular ATP, particularly under hypoxic conditions when respiration may become anaerobic. Using a model of intrapartum hypoxia in the precocial spiny mouse (Acomys cahirinus), the present study assessed the potential for maternal creatine supplementation during pregnancy to protect the developing brain from the effects of birth hypoxia. On day 38 of gestation (term is 39 days), the pregnant uterus was isolated and placed in a saline bath for 7.5 min, inducing global hypoxia. The pups were then removed, resuscitated, and cross-fostered to a nursing dam. Control offspring were delivered by caesarean section and recovered immediately after release from the uterus. At 24 h after birth hypoxia, the brains of offspring from dams fed a normal diet showed significant increases in lipid peroxidation as measured by the amount of malondialdehyde. In the cortical subplate, thalamus and piriform cortex there were significant increases in cellular expression of the pro-apoptotic protein BAX, cytoplasmic cytochrome c and caspase-3. When pregnant dams were fed the creatine supplemented diet, the increase in malondialdehyde, BAX, cytochrome c and caspase 3 were almost completely prevented, such that they were not different from control (caesarean-delivered) neonates. This study provides evidence that the neuroprotective capacity of creatine in the hypoxic perinatal brain involves abrogation of lipid peroxidation and apoptosis, possibly through the maintenance of mitochondrial function. Further investigation into these mechanisms of protection, and the long-term development and behavioural outcomes of such neonates is warranted.

NFκB-dependent increase of kynurenine pathway activity in human placenta: inhibition by sulfasalazine.

Catabolism of tryptophan via the kynurenine pathway is up-regulated in the human placenta by infection, resulting in the release of pro-inflammatory and neuroactive metabolites into the fetal circulation. In this study we determined if activation of NFκB is involved in the inflammation-induced increase of kynurenine pathway activity in the human placenta. Placentae obtained after elective caesarian section at 37-40 weeks gestation (n=8), and explants (35-40 mg) prepared from terminal villi were incubated under standard conditions in the presence of 10 µg/ml LPS for 24 or 48 h; duplicates of each explant were incubated either with or without 5mM sulfasalazine added to the medium. Expression of mRNAs for key kynurenine-forming enzymes, indoleamine 2,3-dioxygrenase (IDO) and tryptophan 2,3-doalxygenase (TDO) and the inflammatory cytokines TNFα and IL6 was studied by RT-PCR. Kynurenine output by explants was measured in samples in the incubation medium by absorbance at 363nm after separation from other metabolites using an HPLC technique. Expression of IDO, TDO, TNFα and IL6 mRNAs was increased with LPS treatment, a response mitigated by the presence of sulfasalazine (P<0.01, P<0.01, P=0.03 &P=0.04). Kynurenine output into the culture medium increased with LPS treatment but this was also prevented by sulfasalazine at 24h (mean ± SEM; 412.1 ± 40 vs. 147.7 ± 48.9 nM/mg, P=0.01) and 48 h (636 ± 39.1 vs. 135.5 ± 29.8 nM/mg, P=0.001, respectively). Sulfasalazine inhibited the LPS induction of both the kynurenine pathway and pro-inflammatory cytokines in the placenta, implicating NFκB in the LPS effect. Direct measurement of NFκB activity showed that sulfasalazine decreased NFκB activation under both control and LPS-treated conditions. These observations show that kynurenine pathway activity in the human placenta is increased by a NFκB dependent pathway, and suggests a new therapeutic strategy for the management of pregnancies with in utero infection.

The effect of betamethasone treatment on neuroactive steroid synthesis in a foetal Guinea pig model of growth restriction.

There are ongoing concerns that antenatal corticosteroids, which are administered to women at high risk of delivering preterm to reduce the incidence of respiratory distress syndrome, have adverse effects on foetal brain development and subsequent effects on behaviour and learning, when administered as repeated courses. The present study aimed to examine whether repeated betamethasone treatment alters the expression of the key-rate limiting enzyme, 5alpha-reductase, in the synthetic pathway of the potent neuroactive steroid allopregnanolone in the brain and placenta and whether this effect is potentiated in growth restricted foetuses. To investigate this, pregnant guinea pigs carrying either control (sham surgery) or growth-restricted foetuses were treated with vehicle or betamethasone (1 mg/kg/day) for 4 days prior to sacrifice (65d). Placental insufficiency was induced by the ablation of uterine artery branches supplying each placenta at mid gestation, resulting in foetal growth restriction characterised by 'brain sparing'. Real-time reverse transcriptase polymerase chain reaction was used to determine relative 5alpha-reductase type 1 and 2 mRNA expression in the placenta and brain. Immunohistochemistry was used to examine the glial fibrillary acidic protein (GFAP) expression in the subcortical white matter, CA1 and dentate regions of the hippocampus. 5alpha-reductase type 2 mRNA expression in the brain was markedly reduced by betamethasone treatment in male foetuses compared to vehicle-treated controls but not in female foetuses. In addition, 5alpha-reductase type 1 expression in the brain was increased by growth restriction and/or betamethasone treatment in female foetuses but expression in males foetuses did not increase. 5alpha-reductase type 2 expression in the placenta was markedly reduced by betamethasone treatment compared to vehicle-treated control. Intrauterine growth restriction and betamethasone treatment reduced GFAP expression in the CA1 region of the hippocampus in the brains of male but not female foetuses. These data indicate that betamethasone treatment suppresses placental expression and has sexually dimorphic effects on expression of neuroactive steroid synthetic enzymes in the brain. These actions may lead to adverse effects on the developing brain, particularly in male foetuses, such as the observed effects on GFAP expression.

Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain.

The neurosteroid allopregnanolone (AP) is a GABAergic agonist that suppresses central nervous system (CNS) activity in the adult brain, and by reducing excitotoxicity is considered to be neuroprotective. A role for neurosteroids in the developing brain, particularly in late gestation, is still debated. The aim of this study was to investigate effects on proliferation and cell death in the brain of late gestation fetal sheep after inhibition of AP synthesis using finasteride, a 5alpha-reductase type 2 (5alpha-R2) inhibitor. Catheters were implanted in fetal sheep at approximately 125 days of gestation. At 3-4 days postsurgery, fetuses received infusions of either finasteride (20 mg/kg/h; n=5), the AP analogue alfaxalone (5 mg/kg/h; n=5), or finasteride and alfaxalone together (n=5). Brains were obtained at 24 h after infusion to determine cell death (apoptotic or necrotic) and cell proliferation in the hippocampus and cerebellum, areas known to be susceptible to excitotoxic damage. Finasteride treatment significantly increased apoptosis (activated caspase-3 expression) in hippocampal CA3 and CA1, and cerebellar molecular and granular layers, an effect abolished by co-infusion of alfaxalone and finasteride. Double-label immunohistochemistry showed that both neurons and astrocytes were caspase-3 positive. Finasteride treatment also increased the number of dead (pyknotic) cells in the hippocampus and cerebellum (Purkinje cells), but not when finasteride+alfaxalone was infused. Cell proliferation (Ki-67-immunoreactivity) increased after finasteride treatment; double-labeling showed the majority of Ki-67-positive cells were astrocytes. Thus, steroids such as AP appear to influence the constitutive rate of apoptosis and proliferation in the hippocampus and cerebellum of the fetal brain, and suggest an important role for neurosteroids in the development of the brain.

High-performance computing for Monte Carlo radiotherapy calculations.

We report on the RTGrid project, which investigates approaches for using high-performance computing infrastructures, such as the grid, in order to reduce the turnaround time of Monte Carlo (MC) simulation-based radiotherapy treatment planning. The main aim of this project is to render accurate dose calculations using MC simulations clinically feasible. To this end, we have successfully implemented and deployed the RTGrid distributed simulation framework for MC dose calculations. In this paper, we present the main experimental findings.

Inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in the late gestation fetal sheep.

Allopregnanolone (AP) is a potent GABAergic agonist that suppresses CNS activity, seizure threshold, and excitotoxicity in the adult brain. AP is present in the fetal sheep brain and increases rapidly after asphyxial insult due to increased 5alpha-reductase type-2 (5alphaR-2) expression. The aim of this study was to use finasteride to suppress fetal neurosteroid synthesis, and then determine the effect on brain injury, particularly in the hippocampus, of asphyxia induced in utero by brief occlusion of the umbilical cord. Catheters and an inflatable umbilical cord cuff were implanted in fetal sheep at approximately 125 days gestation. Five days later the fetuses received either finasteride (20 mg/kg/h) or vehicle (40% hydroxypropyl-beta-cyclodextrin) for 2 h. The umbilical cord was occluded (UCO) for 5 min at 30 min after starting the infusion. The fetal brain was obtained 24 h later for examination of activated caspase-3 expression as an index of apoptosis, and to measure AP content. Finasteride treatment alone significantly reduced AP content and increased the number of caspase-3 positive cells in the hippocampus, cerebellum, and the subcallosal bundle, indicating that AP modulates the normal rate of apoptosis in the developing brain. UCO in vehicle and finasteride-treated fetuses produced a similar, marked decrease in O2 saturation (5.8+/-0.6%), but after finasteride treatment UCO caused a significantly greater increase in the number of caspase-3 positive cells in the hippocampal cornu ammonis 3 (CA3) (57.3+/-1.6%) compared with the vehicle-treated fetuses. Thus, 5alpha-reduced steroids such as AP may be protective in reducing cell death following acute fetal asphyxia. Perturbation of normal fetal neurosteroid levels in late gestation (e.g. due to preterm birth, or maternal synthetic steroid treatment to induce fetal lung maturation) could adversely affect brain development and increase its vulnerability to injury.