Novel biomarkers of preterm brain injury from blood transcriptome in sheep model of intrauterine asphyxia.

BACKGROUND: Infants born preterm have a higher incidence of neurological deficits. A key step in finding effective treatments is to identify biomarkers that reliably predict outcome. METHODS: Following umbilical cord occlusion (UCO) in pregnant sheep, whole fetal blood RNA was sequenced pre- and post-UCO, brain injury outcome was determined by battery of neuropathology scoring and the transcriptome signature correlated to the degree of brain injury. Additionally, we developed a novel analytical procedure to deduce cell blood composition over time. RESULTS: Sixty-one genes were identified with significant altered expression after UCO. In pre-UCO blood, the level of three mRNAs (Trex2, Znf280b, novel miRNA) and in post-UCO, four mRNAs (Fam184a, Angptl2, novel lincRNA and an unknown protein-coding gene) were associated to brain injury (FDR < 0.01). Several of these mRNAs are related to inflammation and angiogenesis. Pathway analysis highlighted genes playing a role in perinatal death and growth failure. Results also indicate that several leukocyte populations undergo significant changes after UCO. CONCLUSION: We have used a whole transcriptomic approach to uncover novel biomarkers in fetal blood that correlate to neuropathology in the preterm sheep brain. The current data forms a basis for future studies to investigate mechanisms of these mRNAs in the injury progression. IMPACT: Trend analysis of genes following asphyxia reveal a group of genes associated with perinatal death and growth failure. Several pre-asphyxia transcripts were associated to brain injury severity suggesting genomic susceptibility to injury. Several post-asphyxia transcripts were correlated to brain injury severity, thus, serve as potential novel biomarkers of injury outcome. Successfully adaptation of cell profiling algorithms suggests significant changes in blood cell composition following asphyxia.

Assessment of radial glia in the frontal lobe of fetuses with Down syndrome.

Down syndrome (DS) occurs with triplication of human chromosome 21 and is associated with deviations in cortical development evidenced by simplified gyral appearance and reduced cortical surface area. Radial glia are neuronal and glial progenitors that also create a scaffolding structure essential for migrating neurons to reach cortical targets and therefore play a critical role in cortical development. The aim of this study was to characterise radial glial expression pattern and morphology in the frontal lobe of the developing human fetal brain with DS and age-matched controls. Secondly, we investigated whether microstructural information from in vivo magnetic resonance imaging (MRI) could reflect histological findings from human brain tissue samples. Immunohistochemistry was performed on paraffin-embedded human post-mortem brain tissue from nine fetuses and neonates with DS (15-39 gestational weeks (GW)) and nine euploid age-matched brains (18-39 GW). Radial glia markers CRYAB, HOPX, SOX2, GFAP and Vimentin were assessed in the Ventricular Zone, Subventricular Zone and Intermediate Zone. In vivo diffusion MRI was used to assess microstructure in these regions in one DS (21 GW) and one control (22 GW) fetal brain. We found a significant reduction in radial glial progenitor SOX2 and subtle deviations in radial glia expression (GFAP and Vimentin) prior to 24 GW in DS. In vivo, fetal MRI demonstrates underlying radial projections consistent with immunohistopathology. Radial glial alterations may contribute to the subsequent simplified gyral patterns and decreased cortical volumes observed in the DS brain. Recent advances in fetal MRI acquisition and analysis could provide non-invasive imaging-based biomarkers of early developmental deviations.

Interneuron Development Is Disrupted in Preterm Brains With Diffuse White Matter Injury: Observations in Mouse and Human.

Preterm brain injury, occurring in approximately 30% of infants born <32 weeks gestational age, is associated with an increased risk of neurodevelopmental disorders, such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). The mechanism of gray matter injury in preterm born children is unclear and likely to be multifactorial; however, inflammation, a high predictor of poor outcome in preterm infants, has been associated with disrupted interneuron maturation in a number of animal models. Interneurons are important for regulating normal brain development, and disruption in interneuron development, and the downstream effects of this, has been implicated in the etiology of neurodevelopmental disorders. Here, we utilize postmortem tissue from human preterm cases with or without diffuse white matter injury (WMI; PMA range: 23+2 to 28+1 for non-WMI group, 26+6 to 30+0 for WMI group, p = 0.002) and a model of inflammation-induced preterm diffuse white matter injury (i.p. IL-1ß, b.d., 10 µg/kg/injection in male CD1 mice from P1-5). Data from human preterm infants show deficits in interneuron numbers in the cortex and delayed growth of neuronal arbors at this early stage of development. In the mouse, significant reduction in the number of parvalbumin-positive interneurons was observed from postnatal day (P) 10. This decrease in parvalbumin neuron number was largely rectified by P40, though there was a significantly smaller number of parvalbumin positive cells associated with perineuronal nets in the upper cortical layers. Together, these data suggest that inflammation in the preterm brain may be a contributor to injury of specific interneuron in the cortical gray matter. This may represent a potential target for postnatal therapy to reduce the incidence and/or severity of neurodevelopmental disorders in preterm infants.

Dysmaturation of Somatostatin Interneurons Following Umbilical Cord Occlusion in Preterm Fetal Sheep.

INTRODUCTION: Cerebral white matter injury is the most common neuropathology observed in preterm infants. However, there is increasing evidence that gray matter development also contributes to neurodevelopmental abnormalities. Fetal cerebral ischemia can lead to both neuronal and non-neuronal structural-functional abnormalities, but less is known about the specific effects on interneurons. OBJECTIVE: In this study we used a well-established animal model of fetal asphyxia in preterm fetal sheep to study neuropathological outcome. We used comprehensive stereological methods to investigate the total number of oligodendrocytes, neurons and somatostatin (STT) positive interneurons as well as 3D morphological analysis of STT cells 14 days following umbilical cord occlusion (UCO) in fetal sheep. MATERIALS AND METHODS: Induction of asphyxia was performed by 25 min of complete UCO in five preterm fetal sheep (98-100 days gestational age). Seven, non-occluded twins served as controls. Quantification of the number of neurons (NeuN), STT interneurons and oligodendrocytes (Olig2, CNPase) was performed on fetal brain regions by applying optical fractionator method. A 3D morphological analysis of STT interneurons was performed using IMARIS software. RESULTS: The number of Olig2, NeuN, and STT positive cells were reduced in IGWM, caudate and putamen in UCO animals compared to controls. There were also fewer STT interneurons in the ventral part of the hippocampus, the subiculum and the entorhinal cortex in UCO group, while other parts of cortex were virtually unaffected (p > 0.05). Morphologically, STT positive interneurons showed a markedly immature structure, with shorter dendritic length and fewer dendritic branches in cortex, caudate, putamen, and subiculum in the UCO group compared with control group (p < 0.05). CONCLUSION: The significant reduction in the total number of neurons and oligodendrocytes in several brain regions confirm previous studies showing susceptibility of both neuronal and non-neuronal cells following fetal asphyxia. However, in the cerebral cortex significant dysmaturation of STT positive neurons occurred in the absence of cell loss. This suggests an abnormal maturation pattern of GABAergic interneurons in the cerebral cortex, which might contribute to neurodevelopmental impairment in preterm infants and could implicate a novel target for neuroprotective therapies.

Post-mortem Characterisation of a Case With an ACTG1 Variant, Agenesis of the Corpus Callosum and Neuronal Heterotopia.

Cytoplasmic Actin Gamma 1 (ACTG1) gene variant are autosomal dominant and can cause CNS anomalies (Baraitser Winter Malformation Syndrome; BWMS). ACTG1 anomalies in offspring include agenesis of the corpus callosum (ACC) and neuronal heterotopia which are ectopic nodules of nerve cells that failed to migrate appropriately. Subcortical and periventricular neuronal heterotopia have been described previously in association with ACC. In this case report, we investigated a neonatal brain with an ACTG1 gene variant and a phenotype of ACC, and neuronal heterotopia (ACC-H) which was diagnosed on antenatal MR imaging and was consistent with band heterotopia seen on post-mortem brain images. Histologically clusters of neurons were seen in both the subcortical and periventricular white matter (PVWM) brain region that coincided with impaired abnormalities in glial formation. Immunohistochemistry was performed on paraffin-embedded brain tissue blocks from this case with ACTG1 variant and an age-matched control. Using tissue sections from the frontal lobe, we examined the distribution of neuronal cells (HuC/HuD, calretinin, and parvalbumin), growth cone (drebrin), and synaptic proteins (synaptophysin and SNAP-25). Additionally, we investigated how the ACTG1 variant altered astroglia (nestin, GFAP, vimentin); oligodendroglia (OLIG2) and microglia (Iba-1) in the corpus callosum, cortex, caudal ganglionic eminence, and PVWM. As predicted in the ACTG1 variant case, we found a lack of midline radial glia and glutamatergic fibers. We also found disturbances in the cortical region, in glial cells and a lack of extracellular matrix components in the ACTG1 variant. The caudal ganglionic eminence and the PVWM regions in the ACTG1 variant lacked several cellular components that were identified in a control case. Within the neuronal heterotopia, we found evidence of glutamatergic and GABAergic neurons with apparent synaptic connections. The data presented from this case study with BWMS with variants in the ACTG1 gene provides insight as to the composition of neuronal heterotopia, and how disturbances of important migratory signals may dramatically affect ongoing brain development.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury.

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer's sector of the Ammon's horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

Myelination induction by a histamine H3 receptor antagonist in a mouse model of preterm white matter injury.

Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1ß (IL-1ß) injections. We initiated GSK247246 treatment (i.p at 7 mg/kg or 20 mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7 mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.

γδ T Cells Contribute to Injury in the Developing Brain.

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αßT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.

Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia.

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets.

Does Caspase-6 Have a Role in Perinatal Brain Injury?

Apoptotic mechanisms are centre stage for the development of injury in the immature brain, and caspases have been shown to play a pivotal role during brain development and in response to injury. The inhibition of caspases using broad-spectrum agents such as Q-VD-OPh is neuroprotective in the immature brain. Caspase-6, an effector caspase, has been widely researched in neurodevelopmental disorders and found to be important following adult stroke, but its function in the neonatal brain has yet to be detailed. Furthermore, caspases may be important in microglial activation; microglia are required for optimal brain development and following injury, and their close involvement during neuronal cell death suggests that apoptotic cues such as caspase activation may be important in microglial activation. Therefore, in this study we aimed to investigate the possible apoptotic and non-apoptotic functions caspase-6 may have in the immature brain in response to hypoxia-ischaemia. We examined whether caspases are involved in microglial activation. We assessed cleaved caspase-6 expression following hypoxia-ischaemia and conducted primary microglial cultures to assess whether the broad-spectrum inhibitor Q-VD-OPh or caspase-6 gene deletion affected lipopolysaccharide (LPS)-mediated microglial activation and phenotype. We observed cleaved caspase-6 expression to be low but present in the cell body and cell processes in both a human case of white matter injury and 72 h following hypoxia-ischaemia in the rat. Gene deletion of caspase-6 did not affect the outcome of brain injury following mild (50 min) or severe (60 min) hypoxia-ischaemia. Interestingly, we did note that cleaved caspase-6 was co-localised with microglia that were not of apoptotic morphology. We observed that mRNA of a number of caspases was modulated by low-dose LPS stimulation of primary microglia. Q-VD-OPh treatment and caspase-6 gene deletion did not affect microglial activation but modified slightly the M2b phenotype response by changing the time course of SOCS3 expression after LPS administration. Our results suggest that the impact of active caspase-6 in the developing brain is subtle, and we believe there are predominantly other caspases (caspase-2, -3, -8, -9) that are essential for the cell death processes in the immature brain.

Cellular mechanisms of toll-like receptor-3 activation in the thalamus are associated with white matter injury in the developing brain.

Toll-like receptor-3 (TLR3) has been identified in a variety of intracellular structures (e.g. endosomes and endoplasmic reticulum); it detects viral molecular patterns and damage-associated molecular patterns. We hypothesized that, after white matter injury (WMI) has occurred, localization and activation of TLR3 are altered in gray matter structures in response to damage-associated molecular patterns and activated glia. Therefore, we investigated the subcellular localization of TLR3 and its downstream signaling pathway in postmortem brain sections from preterm infants with and without WMI (7 patients each). We assessed astroglia (glial fibrillary acidic protein-positive), microglia (ionized calcium-binding adaptor molecule-1-positive), and neuronal populations in 3 regions of the thalamus and in the posterior limb of the internal capsule and analyzed TLR3 messenger RNA and protein expression in the ventral lateral posterior thalamic region, an area associated with impaired motor function. We also assessed TLR3 colocalization with late endosomes (lysosome-associated membrane protein-1) and phagosomal compartments in this region. Glial fibrillary acidic protein, ionized calcium-binding adaptor molecule-1, and TLR3 immunoreactivity and messenger RNA expression were increased in cases with WMI compared with controls. In ventral lateral posterior neurons, TLR3 was colocalized with the endoplasmic reticulum and the autophagosome, suggesting that autophagy may be a stress response associated with WMI. Thus, alterations in TLR3 expression in WMI may be an underlying molecular mechanism associated with impaired development in preterm infants.

Microglia toxicity in preterm brain injury.

Microglia are the resident phagocytic cells of the central nervous system. During brain development they are also imperative for apoptosis of excessive neurons, synaptic pruning, phagocytosis of debris and maintaining brain homeostasis. Brain damage results in a fast and dynamic microglia reaction, which can influence the extent and distribution of subsequent neuronal dysfunction. As a consequence, microglia responses can promote tissue protection and repair following brain injury, or become detrimental for the tissue integrity and functionality. In this review, we will describe microglia responses in the human developing brain in association with injury, with particular focus on the preterm infant. We also explore microglia responses and mechanisms of microglia toxicity in animal models of preterm white matter injury and in vitro primary microglia cell culture experiments.

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction.

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long-term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.

Myo-inositol metabolism in appropriately grown and growth-restricted fetuses: a proton magnetic resonance spectroscopy study.

OBJECTIVE: Myo-inositol (Myo-ins) is a marker of neuroglial cells, being present in the astrocytes of brain tissue, but also functions as an osmolyte. Numbers of astrocytes are known to increase following injury to the brain. Growth-restricted fetuses are at increased risk of later neurodevelopmental impairments even in the absence of overt lesions and despite preserved/increased cerebral blood flow. This study aims to investigate brain Myo-ins metabolism in fetuses with intrauterine growth restriction (IUGR) and evidence of cerebral redistribution using magnetic resonance spectroscopy (MRS) at a short echo time. STUDY DESIGN: Biometry and Doppler assessment of blood flow was assessed using ultrasound in 28 fetuses with IUGR and 47 appropriately grown control subjects. MRI was used to exclude overt brain injury. Proton magnetic resonance spectroscopy of the fetal brain was then performed at an echo time of 42 ms to examine the Myo-ins:Choline (Cho), Myo-ins:Creatine (Cr) and Cho:Cr ratios. RESULTS: No alterations in brain Myo-ins:Cho, Myo-ins:Cr or Cho:Cr ratios were detected between appropriately grown and growth restricted fetuses. CONCLUSIONS: IUGR is not associated with a measureable difference in brain myo-inositol ratios. This may be due to the protective effects of preserved cerebral blood flow in growth restriction and comparable astrocyte numbers when compared to controls.

Microglia activation in the extremely preterm human brain.

BACKGROUND: The periventricular white matter (PVWM) of the immature preterm brain is selectively vulnerable to a spectrum of injury. Although essential for normal brain development, the presence of resident microglia may exacerbate PVWM injury. METHODS: We used immunohistochemistry to investigate microglia profile in human preterm noninjured control brains and in brains with evidence of germinal matrix hemorrhage/intraventricular hemorrhage (GMH/IVH), with median gestational age (GA) of 24.1 and 25.4 wk, respectively. RESULTS: The number of microglia in the PVWM was higher than the other brain regions in both the control and GMH/IVH groups. Microglial density increased further in the PVWM of GMH/IVH brains, regardless of hemorrhage severity and despite normal macroscopic and imaging appearances to the PVWM. This was due to an increase in activated Iba1/CD68- and not Iba/CD45-immunopositive microglia. However, there were very few CD68/Ki67 colocalized cells, suggesting that the source of this increase may be due to a quick transformation of CD45-immunopositive hematopoietic microglia into CD68-immunopositive microglia. There was also increased apoptosis in the PVWM of all cases of GMH/IVH, with axonal injury and increased tumor necrosis factor-α (TNF-α) expression evident in the most severe cases. CONCLUSION: Isolated GMH/IVH may influence ongoing brain development, with a significant role played by microglial activation.

Antenatal glucocorticoids reduce growth in appropriately grown and growth-restricted ovine fetuses in a sex-specific manner.

Antenatal glucocorticoids are administered to mature the fetal lungs before preterm birth. Glucocorticoids also have non-pulmonary effects, including reducing fetal body and brain growth. The present study examined whether glucocorticoid administration has a sex-specific effect on growth in appropriately grown (control) and intrauterine growth-restricted (IUGR) fetal sheep. IUGR was induced at 0.7 gestation in fetal sheep by single umbilical artery ligation. On Days 5 and 6 after surgery, IUGR or control fetuses were exposed to the synthetic glucocorticoid betamethasone (BM; 11.4mg) or saline via intramuscular maternal administration. On Day 7, a postmortem was conducted to determine fetal sex and weight. Compared with control fetuses, the birthweight of male and female IUGR fetuses was significantly reduced (by 18.5±4.4% (P=0.002) and 21.7±6.0% (P=0.001), respectively). Maternal administration of BM significantly reduced bodyweight in both control and IUGR fetuses (by 11.3±2.8% and 20.5±3.6% in control male and female fetuses, respectively; and by 22.9±3.1% and 38.3±3.4% in IUGR male and female fetuses, respectively; P<0.001 for all, versus control+saline) fetuses. In control and IUGR animals the degree of growth restriction was greater in females than males (P<0.05) following administration of BM. These data suggest that antenatal glucocorticoids reduce fetal growth in a sex-specific manner, with females more growth restricted than males.

Genetic inhibition of caspase-2 reduces hypoxic-ischemic and excitotoxic neonatal brain injury.

OBJECTIVE: Perinatal brain injury is a major cause of neurodevelopmental handicaps. Multiple pathways of oxidant stress, inflammation, and excitotoxicity lead to cell damage and death, including caspase-dependent apoptosis. Caspase-2 (Casp2; Nedd-2, Ich-1) is a developmentally regulated initiator caspase, which poorly cleaves other caspases but can initiate mitochondrial outer membrane permeabilization. We have investigated if Casp2 could mediate perinatal ischemic brain damage. METHODS: Casp2 expression in human neonatal brains and developmental patterns in rats and mice were evaluated. Casp2-deficient (Casp2(-/-)), wild-type (WT), and heterozygous (Casp2(+/-)) newborn C57BL/6 mice were subjected to hypoxia-ischemia (unilateral carotid occlusion + exposure to 10% oxygen for 50 minutes) or intracerebral injection of the excitotoxic N-methyl-D-aspartate-receptor agonist ibotenate. In addition, Casp2 specific siRNAs were preinjected into the brain of WT newborn mice 24 hours before ibotenate treatment. Brain tissues were examined by immunohistochemical staining (cresyl violet, MAP2, NF68, Casp2, Casp3) and Western blotting. Lesion volumes and injury in the cortical plates and white matter were quantified together with activated Casp3. RESULTS: Casp2 is highly expressed in the neonatal brain. Casp2-deficient mice subjected to hypoxia-ischemia at postnatal day 9 present significantly lower cerebral infarction, reduced white matter injury, and reduced Casp3 activation in the thalamus and hippocampus. Both Casp2(-/-) mice and siRNA-administered WT mice conferred reduction of gray and white matter injury after excitotoxic insult at postnatal day 5. Casp3 activation was also found reduced in Casp2-deficient mice subjected to excitotoxicity. INTERPRETATION: These data suggest for the first time a role of Casp2 in neonatal brain damage.

Subplate in the developing cortex of mouse and human.

The subplate is a largely transient zone containing precocious neurons involved in several key steps of cortical development. The majority of subplate neurons form a compact layer in mouse, but are dispersed throughout a much larger zone in the human. In rodent, subplate neurons are among the earliest born neocortical cells, whereas in primate, neurons are added to the subplate throughout cortical neurogenesis. Magnetic resonance imaging and histochemical studies show that the human subplate grows in size until the end of the second trimester. Previous microarray experiments in mice have shown several genes that are specifically expressed in the subplate layer of the rodent dorsal cortex. Here we examined the human subplate for some of these markers. In the human dorsal cortex, connective tissue growth factor-positive neurons can be seen in the ventricular zone at 15-22 postconceptional weeks (PCW) (most at 17 PCW) and are present in the subplate at 22 PCW. The nuclear receptor-related 1 protein is mostly expressed in the subplate in the dorsal cortex, but also in lower layer 6 in the lateral and perirhinal cortex, and can be detected from 12 PCW. Our results suggest that connective tissue growth factor- and nuclear receptor-related 1-positive cells are two distinct cell populations of the human subplate. Furthermore, our microarray analysis in rodent suggested that subplate neurons produce plasma proteins. Here we demonstrate that the human subplate also expresses α2zinc-binding globulin and Alpha-2-Heremans-Schmid glycoprotein/human fetuin. In addition, the established subplate neuron marker neuropeptide Y is expressed superficially, whereas potassium/chloride co-transporter (KCC2)-positive neurons are localized in the deep subplate at 16 PCW. These observations imply that the human subplate shares gene expression patterns with rodent, but is more compartmentalized into superficial and deep sublayers. This increased complexity of the human subplate may contribute to differential vulnerability in response to hypoxia/ischaemia across the depth of the cortex. Combining knowledge of cell-type specific subplate gene expression with modern imaging methods will enable a better understanding of neuropathologies involving the subplate.

Magnetic resonance imaging of white matter diseases of prematurity.

Periventricular leucomalacia (PVL) and parenchymal venous infarction complicating germinal matrix/intraventricular haemorrhage have long been recognised as the two significant white matter diseases responsible for the majority of cases of cerebral palsy in survivors of preterm birth. However, more recent studies using magnetic resonance imaging to assess the preterm brain have documented two new appearances, adding to the spectrum of white matter disease of prematurity: punctate white matter lesions, and diffuse excessive high signal intensity (DEHSI). These appear to be more common than PVL but less significant in terms of their impact on individual neurodevelopment. They may, however, be associated with later cognitive and behavioural disorders known to be common following preterm birth. It remains unclear whether PVL, punctate lesions, and DEHSI represent a continuum of disorders occurring as a result of a similar injurious process to the developing white matter. This review discusses the role of MR imaging in investigating these three disorders in terms of aetiology, pathology, and outcome.

Cardiovascular responses to maternal betamethasone administration in the intrauterine growth-restricted ovine fetus.

OBJECTIVE: The objective was to characterize the effect of glucocorticoid treatment on fetal organ blood flow and regional cerebral blood flow in normally grown fetuses and fetuses with intrauterine fetal growth restriction (IUGR). STUDY DESIGN: Studies were undertaken in both control and IUGR fetal sheep; growth restriction was induced by ligation of 1 umbilical artery. Fetuses received colored microspheres for organ blood flow calculations before and after 2 maternal betamethasone injections (BM1 and BM2). RESULTS: Following BM1, cardiac output was significantly decreased in the control fetuses and blood flow to the heart and placenta was unchanged, whereas total cerebral blood flow was significantly decreased (P<.001), consistent with cerebral vasoconstriction. In the fetuses with IUGR, the cardiac output was significantly increased at +33 hours relative to BM1, and blood flow was increased in all organs; notably, there was a 2-fold increase in cerebral blood flow (P=.03). CONCLUSION: The cardiovascular response of the fetus with IUGR to glucocorticoids is profoundly different from the control fetuses, which may induce both short- and long-term injury.