Intrauterine inflammation and postnatal intravenous dopamine alter the neurovascular unit in preterm newborn lambs.

BACKGROUND: Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses. METHODS: At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPSDA, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTLDA, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit. RESULTS: LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS. CONCLUSION: LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

Creatine supplementation reduces the cerebral oxidative and metabolic stress responses to acute in utero hypoxia in the late-gestation fetal sheep.

Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real-time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days' gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg-1  h-1 ) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from -24 to 72 h post-UCO and analysed for percentage change of hydroxyl radicals (• OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein and lipid oxidation were assessed at post-mortem 72 h post-UCO. Prior to UCO, creatine treatment reduced pyruvate and glycerol concentrations in the microdialysate outflow. Creatine treatment reduced interstitial cerebral • OH outflow 0 to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO presented with reduced levels of hypoxaemia ( PO2${P_{{{\rm{O}}_{\rm{2}}}}}$ and SO2${S_{{{\rm{O}}_{\rm{2}}}}}$ ) during UCO which associated with reduced interstitial cerebral pyruvate, lactate and • OH accumulation. No effects of creatine treatment on immunohistochemical markers of oxidative stress were found. In conclusion, fetal creatine treatment decreased cerebral outflow of • OH and was associated with an improvement in cerebral bioenergetics following acute hypoxia. KEY POINTS: Fetal hypoxia can cause persistent metabolic and oxidative stress responses that disturb energy homeostasis in the brain. Creatine in its phosphorylated form is an endogenous phosphagen; therefore, supplementation is a proposed prophylactic treatment for fetal hypoxia. Fetal sheep instrumented with a cerebral microdialysis probe were continuously infused with or without creatine-monohydrate for 10 days before induction of 10 min umbilical cord occlusion (UCO; 131 days' gestation). Cerebral interstitial fluid was collected up to 72 h following UCO. Prior to UCO, fetal creatine supplementation reduced interstitial cerebral pyruvate and glycerol concentrations. Fetal creatine supplementation reduced cerebral hydroxyl radical efflux up to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO and reduced levels of systemic hypoxaemia during UCO were associated with reduced cerebral interstitial pyruvate, lactate and • OH following UCO. Creatine supplementation leads to some improvements in cerebral bioenergetics following in utero acute hypoxia.

Genetic and microstructural differences in the cortical plate of gyri and sulci during gyrification in fetal sheep.

Gyrification of the cerebral cortex is a developmentally important process, but the mechanisms that drive cortical folding are not fully known. Theories propose that changes within the cortical plate (CP) cause gyrification, yet differences between the CP below gyri and sulci have not been investigated. Here we report genetic and microstructural differences in the CP below gyri and sulci assessed before (at 70 days of gestational age [GA] 70), during (GA 90), and after (GA 110) gyrification in fetal sheep. The areal density of BDNF, CDK5, and NeuroD6 immunopositive cells were increased, and HDAC5 and MeCP2 mRNA levels were decreased in the CP below gyri compared with sulci during gyrification, but not before. Only the areal density of BDNF-immunopositive cells remained increased after gyrification. MAP2 immunoreactivity and neurite outgrowth were also increased in the CP below gyri compared with sulci at GA 90, and this was associated with microstructural changes assessed via diffusion tensor imaging and neurite orientation dispersion and density imaging at GA 98. Differential neurite outgrowth may therefore explain the localized changes in CP architecture that result in gyrification.

Dobutamine treatment reduces inflammation in the preterm fetal sheep brain exposed to acute hypoxia.

BACKGROUND: Impaired cerebral autoregulation in preterm infants makes circulatory management important to avoid cerebral hypoxic-ischemic injury. Dobutamine is frequently used as inotropic treatment in preterm neonates, but its effects on the brain exposed to cerebral hypoxia are unknown. We hypothesized that dobutamine would protect the immature brain from cerebral hypoxic injury. METHODS: In preterm (0.6 gestation) fetal sheep, dobutamine (Dob, 10 µg/kg/min) or saline (Sal) was infused intravenously for 74 h. Two hours after the beginning of the infusion, umbilical cord occlusion (UCO) was performed to produce fetal asphyxia (Sal+UCO: n = 9, Dob+UCO: n = 7), or sham occlusion (Sal+sham: n = 7, Dob+sham: n = 6) was performed. Brains were collected 72 h later for neuropathology. RESULTS: Dobutamine did not induce cerebral changes in the sham UCO group. UCO increased apoptosis and microglia density in white matter, hippocampus, and caudate nucleus, and astrocyte density in the caudate nucleus. Dobutamine commenced before UCO reduced microglia infiltration in the white matter, and microglial and astrocyte density in the caudate. CONCLUSION: In preterm hypoxia-induced brain injury, dobutamine decreases neuroinflammation in the white matter and caudate, and reduces astrogliosis in the caudate. Early administration of dobutamine in preterm infants for cardiovascular stabilization appears safe and may be neuroprotective against unforeseeable cerebral hypoxic injury.

Does maternal-fetal transfer of creatine occur in pregnant sheep?

Our aim was to determine the disposition of creatine in ovine pregnancy and whether creatine is transferred across the placenta from mother to fetus. Pregnant ewes received either 1) a continuous intravenous infusion of creatine monohydrate or saline from 122 to 131 days gestation, with maternal and fetal arterial blood and amniotic fluid samples collected daily for creatine analysis and fetal tissues collected at necropsy at 133 days for analysis of creatine content, or 2) a single systemic bolus injection of [13C]creatine monohydrate at 130 days of gestation, with maternal and fetal arterial blood, uterine vein blood, and amniotic fluid samples collected before and for 4 h after injection and analyzed for creatine, creatine isotopic enrichment, and guanidinoacetic acid (GAA; precursor of creatine) concentrations. Presence of the creatine transporter-1 (SLC6A8) and l-arginine:glycine amidinotransferase (AGAT; the enzyme synthesizing GAA) proteins were determined by Western blots of placental cotyledons. The 10-day creatine infusion increased maternal plasma creatine concentration three- to fourfold (P < 0.05) without significantly changing fetal arterial, amniotic fluid, fetal tissues, or placental creatine content. Maternal arterial 13C enrichment was increased (P < 0.05) after bolus [13C]creatine injection without change of fetal arterial 13C enrichment. SLC6A8 and AGAT proteins were identified in placental cotyledons, and GAA concentration was significantly higher in uterine vein than maternal artery plasma. Despite the presence of SLC6A8 protein in cotyledons, these results suggest that creatine is not transferred from mother to fetus in near-term sheep and that the ovine utero-placental unit releases GAA into the maternal circulation.

Intrauterine Growth Restriction Alters the Postnatal Development of the Rat Cerebellum.

Intrauterine growth restriction (IUGR) is a major cause of antenatal brain injury. We aimed to characterize cerebellar deficits following IUGR and to investigate the potential underlying cellular and molecular mechanisms. At embryonic day 18, pregnant rats underwent either sham surgery (controls; n = 23) or bilateral uterine vessel ligation to restrict blood flow to fetuses (IUGR; n = 20). Offspring were collected at postnatal day 2 (P2), P7, and P35. Body weights were reduced at P2, P7, and P35 in IUGR offspring (p < 0.05) compared with controls. At P7, the width of the external granule layer (EGL) was 30% greater in IUGR than control rats (p < 0.05); there was no difference in the width of the proliferative zone or in the density of Ki67-positive cells in the EGL. Bergmann glia were disorganized at P7 and P35 in IUGR pups, and by P35, there was a 10% decrease in Bergmann glial fiber density (p < 0.05) compared with controls. At P7, trophoblast antigen-2 (Trop2) mRNA and protein levels in the cerebellum were decreased by 88 and 40%, respectively, and astrotactin 1 mRNA levels were increased by 20% in the IUGR rats (p < 0.05) compared with controls; there was no difference in ASTN1 protein. The expressions of other factors known to regulate cerebellar development (astrotactin 2, brain-derived neurotrophic factor, erb-b2 receptor tyrosine kinase 4, neuregulin 1, sonic hedgehog and somatostatin) were not different between IUGR and control rats at P7 or P35. These data suggest that damage to the migratory scaffold (Bergmann glial fibers) and alterations in the genes that influence migration (Trop2 and Astn1) may underlie the deficits in postnatal cerebellar development following IUGR.

Excitatory and inhibitory neuron imbalance in the intrauterine growth restricted fetal guinea pig brain: Relevance to the developmental origins of schizophrenia and autism.

Neurodevelopmental disorders such as schizophrenia and autism are thought to involve an imbalance of excitatory and inhibitory signaling in the brain. Intrauterine growth restriction (IUGR) is a risk factor for these disorders, with IUGR onset occurring during critical periods of neurodevelopment. The aim of this study was to determine the impact of IUGR on excitatory and inhibitory neurons of the fetal neocortex and hippocampus. Fetal brains (n = 2) were first collected from an unoperated pregnant guinea pig at mid-gestation (32 days of gestation [dg]; term ∼67 dg) to visualize excitatory (Ctip2) and inhibitory (calretinin [CR] and somatostatin [SST]) neurons via immunohistochemistry. Chronic placental insufficiency (CPI) was then induced via radial artery ablation at 30 dg in another cohort of pregnant guinea pigs (n = 8) to generate IUGR fetuses (52 dg; n = 8); control fetuses (52 dg; n = 7) were from sham surgeries with no radial artery ablation. At 32 dg, Ctip2- and CR-immunoreactive (IR) cells had populated the cerebral cortex, whereas SST-IR cells had not, suggesting these neurons were yet to complete migration. At 52 dg, in IUGR versus control fetuses, there was a reduction in SST-IR cell density in the cerebral cortex (p = .0175) and hilus of the dentate gyrus (p = .0035) but not the striatum (p > .05). There was no difference between groups in the density of Ctip2-IR (cortex) or CR-IR (cortex, hippocampus) neurons (p > 0.05). Thus, we propose that an imbalance in inhibitory (SST-IR) and excitatory (Ctip2-IR) neurons in the IUGR fetal guinea pig brain could lead to excitatory/inhibitory dysfunction commonly seen in neurodevelopmental disorders such as autism and schizophrenia.

Creatine in the fetal brain: A regional investigation of acute global hypoxia and creatine supplementation in a translational fetal sheep model.

Background: Creatine supplementation during pregnancy is a promising prophylactic treatment for perinatal hypoxic brain injury. Previously, in near-term sheep we have shown that fetal creatine supplementation reduces cerebral metabolic and oxidative stress induced by acute global hypoxia. This study investigated the effects of acute hypoxia with or without fetal creatine supplementation on neuropathology in multiple brain regions. Methods: Near-term fetal sheep were administered continuous intravenous infusion of either creatine (6 mg kg-1 h-1) or isovolumetric saline from 122 to 134 days gestational age (dGA; term is approx. 145 dGA). At 131 dGA, global hypoxia was induced by a 10 min umbilical cord occlusion (UCO). Fetuses were then recovered for 72 h at which time (134 dGA) cerebral tissue was collected for either RT-qPCR or immunohistochemistry analyses. Results: UCO resulted in mild injury to the cortical gray matter, thalamus and hippocampus, with increased cell death and astrogliosis and downregulation of genes involved in regulating injury responses, vasculature development and mitochondrial integrity. Creatine supplementation reduced astrogliosis within the corpus callosum but did not ameliorate any other gene expression or histopathological changes induced by hypoxia. Of importance, effects of creatine supplementation on gene expression irrespective of hypoxia, including increased expression of anti-apoptotic (BCL-2) and pro-inflammatory (e.g., MPO, TNFa, IL-6, IL-1ß) genes, particularly in the gray matter, hippocampus, and striatum were identified. Creatine treatment also effected oligodendrocyte maturation and myelination in white matter regions. Conclusion: While supplementation did not rescue mild neuropathology caused by UCO, creatine did result in gene expression changes that may influence in utero cerebral development.

The Effects of In Utero Fetal Hypoxia and Creatine Treatment on Mitochondrial Function in the Late Gestation Fetal Sheep Brain.

Near-term acute hypoxia in utero can result in significant fetal brain injury, with some brain regions more vulnerable than others. As mitochondrial dysfunction is an underlying feature of the injury cascade following hypoxia, this study is aimed at characterizing mitochondrial function at a region-specific level in the near-term fetal brain after a period of acute hypoxia. We hypothesized that regional differences in mitochondrial function would be evident, and that prophylactic creatine treatment would mitigate mitochondrial dysfunction following hypoxia; thereby reducing fetal brain injury. Pregnant Border-Leicester/Merino ewes with singleton fetuses were surgically instrumented at 118 days of gestation (dGa; term is ~145 dGA). A continuous infusion of either creatine (n = 15; 6 mg/kg/h) or isovolumetric saline (n = 16; 1.5 ml/kg/h) was administered to the fetuses from 121 dGa. After 10 days of infusion, a subset of fetuses (8 saline-, 7 creatine-treated) were subjected to 10 minutes of umbilical cord occlusion (UCO) to induce a mild global fetal hypoxia. At 72 hours after UCO, the fetal brain was collected for high-resolution mitochondrial respirometry and molecular and histological analyses. The results show that the transient UCO-induced acute hypoxia impaired mitochondrial function in the hippocampus and the periventricular white matter and increased the incidence of cell death in the hippocampus. Creatine treatment did not rectify the changes in mitochondrial respiration associated with hypoxia, but there was a negative relationship between cell death and creatine content following treatment. Irrespective of UCO, creatine increased the proportion of cytochrome c bound to the inner mitochondrial membrane, upregulated the mRNA expression of the antiapoptotic gene Bcl2, and of PCG1-α, a driver of mitogenesis, in the hippocampus. We conclude that creatine treatment prior to brief, acute hypoxia does not fundamentally modify mitochondrial respiratory function, but may improve mitochondrial structural integrity and potentially increase mitogenesis and activity of antiapoptotic pathways.

The physiological effects of creatine supplementation in fetal sheep before, during, and after umbilical cord occlusion and global hypoxia.

The aim of this study was to investigate the effects of direct creatine infusion on fetal systemic metabolic and cardiovascular responses to mild acute in utero hypoxia. Pregnant ewes (n = 28) were surgically instrumented at 118 days gestation (dGa). A constant intravenous infusion of creatine (6 mg·kg-1·h-1) or isovolumetric saline (1.5 mL·h-1) began at 121 dGa. After 10 days, fetuses were subjected to 10-min umbilical cord occlusion (UCO) to induce mild global hypoxia (saline-UCO, n = 8; creatine-UCO, n = 7) or sham UCO (saline-control, n = 6; creatine-control, n = 7). Cardiovascular, arterial blood gases and metabolites, and plasma creatine were monitored before, during, and then for 72 h following the UCO. Total creatine content in discrete fetal brain regions was also measured. Fetal creatine infusion increased plasma concentrations fivefold but had no significant effects on any measurement pre-UCO. Creatine did not alter fetal physiology during the UCO or in the early recovery stage, up to 24 h after UCO. During the late recovery stage, 24-72 h after UCO, there was a significant reduction in the arterial oxygen pressure and saturation in creatine fetuses (PUCO × TREATMENT = 0.02 and 0.04, respectively). At 72 h after UCO, significant creatine loading was detected in cortical gray matter, hippocampus, thalamus, and striatum (PTREATMENT = 0.01-0.001). In the striatum, the UCO itself increased total creatine content (PUCO = 0.019). Overall, fetal creatine supplementation may alter oxygen flux following an acute hypoxic insult. Increasing total creatine content in the striatum may also be a fetal adaptation to acute oxygen deprivation.NEW & NOTEWORTHY Direct fetal creatine supplementation increased plasma and cerebral creatine concentrations but did not alter fetal body weight, basal cardiovascular output, or blood chemistry. Creatine-treated fetuses displayed changes to arterial oxygenation 24-72 h after acute global hypoxia. An increase in striatum total creatine levels following UCO was also noted and suggests that increasing creatine tissue availability may be an adaptive response against the effects of hypoxia.

Evaluation of 3K3A-Activated Protein C to Treat Neonatal Hypoxic Ischemic Brain Injury in the Spiny Mouse.

Neonatal hypoxic ischemic encephalopathy (HIE) resulting from intrapartum asphyxia is a global problem that causes severe disabilities and up to 1 million deaths annually. A variant form of activated protein C, 3K3A-APC, has cytoprotective properties that attenuate brain injury in models of adult stroke. In this study, we compared the ability of 3K3A-APC and APC (wild-type (wt)) to attenuate neonatal brain injury, using the spiny mouse (Acomys cahirinus) model of intrapartum asphyxia. Pups were delivered at 38 days of gestation (term = 39 days), with an intrapartum hypoxic insult of 7.5 min (intrapartum asphyxia cohort), or immediate removal from the uterus (control cohort). After 1 h, pups received a subcutaneous injection of 3K3A-APC or wild-type APC (wtAPC) at 7 mg/kg, or vehicle (saline). At 24 h of age, pups were killed and brain tissue was collected for measurement of inflammation and cell death using RT-qPCR and histopathology. Intrapartum asphyxia increased weight loss, inflammation, and apoptosis/necrosis in the newborn brain. 3K3A-APC administration maintained body weight and ameliorated an asphyxia-induced increase of TGFß1 messenger RNA expression in the cerebral cortex, immune cell aggregation in the corpus callosum, and cell death in the deep gray matter and hippocampus. In the cortex, 3K3A-APC appeared to exacerbate the immune response to the hypoxic ischemic insult. While wtAPC reduced cell death in the corpus callosum and hippocampus following intrapartum asphyxia, it increased markers of neuro-inflammation and cell death in control pups. These findings suggest 3K3A-APC administration may be a useful therapy to reduce cell death and neonatal brain injury associated with HIE.

Effects of hypoxia-ischemia and inotropes on expression of cardiac adrenoceptors in the preterm fetal sheep.

Preterm infants frequently suffer cardiovascular compromise, with hypotension and/or low systemic blood flow, leading to tissue hypoxia-ischemia (HI). Many preterm infants respond inadequately to inotropic treatments using adrenergic agonists such as dobutamine (DB) or dopamine (DA). This may be because of altered cardiac adrenoceptor expression because of tissue HI or prolonged exposure to adrenergic agonists. We assessed the effects of severe HI with and without DB/DA treatment on cardiac adrenoceptor expression in preterm fetal sheep. Fetal sheep (93-95 days) exposed to sham surgery or severe HI induced by umbilical cord occlusion received intravenous DB or saline for 74 h (HI + DB, HI, Sham + DB, Sham). The HI groups were also compared with fetal sheep exposed to HI and DA. Fetal hearts were collected to determine ß-adrenoceptor numbers using [125I]-cyanopindolol binding and mRNA expression of ß1-, ß2-, α1A-, α2A-, or α2B-adrenoceptors. The HI group had increased ß-adrenoceptor numbers compared with all other groups in all four heart chambers ( P < 0.05). This increase in ß-adrenoceptor numbers in the HI group was significantly reduced by DB infusion in all four heart chambers, but DA infusion in the HI group only reduced ß-adrenoceptor numbers in the left atria and ventricle. DB alone did not affect ß-adrenoceptor numbers in the sham animals. Changes in ß1-adrenoceptor mRNA levels trended to parallel the binding results. We conclude that HI upregulates preterm fetal cardiac ß-adrenoceptors, but prolonged exposure to adrenergic agonists downregulates adrenoceptors in the preterm heart exposed to HI and may underpin the frequent failure of inotropic therapy in preterm infants. NEW & NOTEWORTHY This is the first study, to our knowledge, on the effects of hypoxia-ischemia and adrenergic agonists on adrenoceptors in the preterm heart. In fetal sheep, we demonstrate that hypoxia-ischemia increases cardiac ß-adrenoceptor numbers. However, exposure to both hypoxia-ischemia and adrenergic agonists (dobutamine or dopamine) reduces the increase in ß-adrenoceptor numbers, which may underpin the inadequate response in human preterm infants to inotropic therapy using adrenergic agonists. Dobutamine alone does not affect the cardiac adrenoceptors in the sham animals.

Erythropoietin Protects Against Lipopolysaccharide-Induced Microgliosis and Abnormal Granule Cell Development in the Ovine Fetal Cerebellum.

Erythropoietin (EPO) ameliorates inflammation-induced injury in cerebral white matter (WM). However, effects of inflammation on the cerebellum and neuroprotective effects of EPO are unknown. Our aims were to determine: (i) whether lipopolysaccharide (LPS)-induced intrauterine inflammation causes injury to, and/or impairs development of the cerebellum; and (ii) whether recombinant human EPO (rhEPO) mitigates these changes. At 107 ± 1 days gestational age (DGA; ~0.7 of term), fetal sheep received LPS (~0.9 µg/kg; i.v.) or an equivalent volume of saline, followed 1 h later with 5000 IU/kg rhEPO (i.v.) or an equivalent volume of saline (i.v.). This generated the following experimental groups: control (saline + saline; n = 6), LPS (LPS + saline, n = 8) and LPS + rhEPO (n = 8). At necropsy (116 ± 1 DGA; ~0.8 of term) the brain was perfusion-fixed and stained histologically (H&E) and immunostained to identify granule cells (Neuronal Nuclei, NeuN), granule cell proliferation (Ki67), Bergmann glia (glial fibrillary acidic protein, GFAP), astrogliosis (GFAP) and microgliosis (Iba-1). In comparison to controls, LPS fetuses had an increased density of Iba-1-positive microglia (p < 0.005) in the lobular WM; rhEPO prevented this increase (p < 0.05). The thickness of both the proliferative (Ki67-positive) and post-mitotic zones (Ki67-negative) of the EGL were increased in LPS-exposed fetuses compared to controls (p < 0.05), but were not different between controls and LPS + rhEPO fetuses. LPS also increased (p < 0.001) the density of granule cells (NeuN-positive) in the internal granule layer (IGL); rhEPO prevented the increase (p < 0.01). There was no difference between groups in the areas of the vermis (total cross-section), molecular layer (ML), IGL or WM, the density of NeuN-positive granule cells in the ML, the linear density of Bergmann glial fibers, the areal density or somal area of the Purkinje cells, the areal coverage of GFAP-positive astrocytes in the lobular and deep WM, the density of Iba-1-positive microglia in the deep WM or the density of apopotic cells in the cerebellum. LPS-induced intrauterine inflammation caused microgliosis and abnormal development of granule cells. rhEPO ameliorated these changes, suggesting that it is neuroprotective against LPS-induced inflammatory effects in the cerebellum.

The anti-inflammatory agent N-acetyl cysteine exacerbates endotoxin-induced hypoxemia and hypotension and induces polycythemia in the ovine fetus.

BACKGROUND: Lipopolysaccharide (LPS) delivered acutely to the ovine fetus induces cerebral white matter injury and brain inflammation. N-acetyl cysteine (NAC) is potentially neuroprotective as it blocks the production of inflammatory cytokines and increases glutathione levels; however, it is unknown whether NAC affects the physiological status of the fetus already exposed to an inflammatory environment. OBJECTIVES: Our objective was to determine whether NAC influences the physiological effects of LPS exposure in the ovine fetus. METHODS: Catheterized fetal sheep underwent one of four treatments (saline, n = 6; LPS, n = 6; LPS + NAC, n = 6; NAC, n = 3) on 5 consecutive days from 95 days of gestation (term approximately 147 days). Fetal arterial pressure and heart rate were recorded and blood samples collected. RESULTS: LPS administration resulted in fetal hypoxemia and hypotension; simultaneous treatment with NAC exacerbated these effects and induced polycythemia. NAC treatment alone had no effect on the fetus. CONCLUSION: In the presence of LPS, NAC compromises fetal physiological status, suggesting that it may not be a suitable antenatal treatment for a fetus with evidence of inflammation.

Erythropoietin is neuroprotective in a preterm ovine model of endotoxin-induced brain injury.

Intrauterine infection and inflammation have been linked to preterm birth and brain damage. We hypothesized that recombinant human erythropoietin (rhEPO) would ameliorate brain damage in anovine model of fetal inflammation. At 107 +/- 1 day of gestational age (DGA), chronically catheterized fetal sheep received on 3 consecutive days 1) an intravenous bolus dose of lipopolysaccharide ([LPS] approximately 0.9 microg/kg; n = 8); 2) an intravenous bolus dose of LPS, followed at 1 hour by 5,000 IU/kg of rhEPO (LPS + rhEPO, n = 8); or 3) rhEPO (n = 5). Untreated fetuses (n = 8) served as controls. Fetal physiological parameters were monitored, and fetal brains and optic nerves were histologically examined at 116 +/- 1 DGA. Exposure to LPS, but not to rhEPO alone or saline, resulted in fetal hypoxemia, hypotension (p < 0.05), brain damage, including white matter injury, and reductions in numbers of myelinating oligodendrocytes in the corticospinal tract and myelinated axons in the optic nerve (p < 0.05 for both). Treatment of LPS-exposed fetuses with rhEPO did not alter the physiological effects of LPS but reduced brain injury and was beneficial to myelination in the corticospinal tract and the optic nerve. This is the first study in a long-gestation species to demonstrate the neuroprotective potential of rhEPO in reducing fetal brain and optic nerve injury after LPS exposure.