Oxygen and HIF1α-dependent SDF1 expression in primary astrocytes.

In the naturally hypoxic in utero fetal environment of preterm infants, oxygen and oxygen-sensitive signaling pathways play an important role in brain development, with hypoxia-inducible factor-1α (HIF1α) being an important regulator. Early exposure to nonphysiological high oxygen concentrations by birth in room can induce HIF1α degradation and may affect neuronal and glial development. This involves the dysregulation of astroglial maturation and function, which in turn might contribute to oxygen-induced brain injury. In this study, we investigated the effects of early high oxygen exposure on astroglial maturation and, specifically, on astroglial stromal cell-derived factor 1 (SDF1) expression in vivo and in vitro. In our neonatal mouse model of hyperoxia preterm birth brain injury in vivo, high oxygen exposure affected astroglial development and cortical SDF1 expression. These results were further supported by reduced Sdf1 expression, impaired proliferation, decreased total cell number, and altered expression of astroglial markers in astrocytes in primary cultures grown under high oxygen conditions. Moreover, to mimic the naturally hypoxic in utero fetal environment, astroglial Sdf1 expression was increased after low oxygen exposure in vitro, which appears to be regulated by HIF1α activity. Additionally, the knockdown of Hif1α revealed HIF1α-dependent Sdf1 expression in vitro. Our results indicate HIF1α and oxygen-dependent chemokine expression in primary astrocytes and highlight the importance of oxygen conditions for brain development.

Neonatal Oxidative Stress Impairs Cortical Synapse Formation and GABA Homeostasis in Parvalbumin-Expressing Interneurons.

Neonatal brain injury is often caused by preterm birth. Brain development is vulnerable to increased environmental stress, including oxidative stress challenges. Due to a premature change of the fetal living environment from low oxygen in utero into postnatal high-oxygen room air conditions ex utero, the immature preterm brain is exposed to a relative hyperoxia, which can induce oxidative stress and impair neuronal cell development. To simulate the drastic increase of oxygen exposure in the immature brain, 5-day-old C57BL/6 mice were exposed to hyperoxia (80% oxygen) for 48 hours or kept in room air (normoxia, 21% oxygen) and mice were analyzed for maturational alterations of cortical GABAergic interneurons. As a result, oxidative stress was indicated by elevated tyrosine nitration of proteins. We found perturbation of perineuronal net formation in line with decreased density of parvalbumin-expressing (PVALB) cortical interneurons in hyperoxic mice. Moreover, maturational deficits of cortical PVALB+ interneurons were obtained by decreased glutamate decarboxylase 67 (GAD67) protein expression in Western blot analysis and lower gamma-aminobutyric acid (GABA) fluorescence intensity in immunostaining. Hyperoxia-induced oxidative stress affected cortical synaptogenesis by decreasing synapsin 1, synapsin 2, and synaptophysin expression. Developmental delay of synaptic marker expression was demonstrated together with decreased PI3K-signaling as a pathway being involved in synaptogenesis. These results elucidate that neonatal oxidative stress caused by increased oxygen exposure can lead to GABAergic interneuron damage which may serve as an explanation for the high incidence of psychiatric and behavioral alterations found in preterm infants.

A unique cerebellar pattern of microglia activation in a mouse model of encephalopathy of prematurity.

Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Using a mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of pro-inflammatory IL-1ß, we sought to uncover causes of cerebellar damage. In this model, IL-1ß is administered between postnatal day (P) 1 to day 5, a timing equivalent to the last trimester for brain development in humans. Structural MRI analysis revealed that systemic IL-1ß treatment induced specific reductions in gray and white matter volumes of the mouse cerebellar lobules I and II (5% false discovery rate [FDR]) from P15 onwards. Preceding these MRI-detectable cerebellar volume changes, we observed damage to oligodendroglia, with reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) at P10 and P15. Increased density of IBA1+ cerebellar microglia were observed both at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebellums and cerebrums revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but cerebellar microglia displayed a unique type I interferon signaling dysregulation. Collectively, these data suggest that perinatal inflammation driven by systemic IL-1ß leads to specific cerebellar volume deficits, which likely reflect oligodendrocyte pathology downstream of microglial activation. Further studies are now required to confirm the potential of protective strategies aimed at preventing sustained type I interferon signaling driven by cerebellar microglia as an important therapeutic target.

Postnatal myelination of the immature rat cingulum is regulated by GABAB receptor activity.

Myelination of axons in the neonatal brain is a highly complex process primarily achieved by oligodendroglial cells (OLs). OLs express receptors for γ-aminobutyric acid (GABA) which is released from cortical interneurons on a basal level, while glial cells can be a source of GABA, too. We investigated GABA-induced oligodendroglial maturation, proliferation, apoptosis, and myelin production after pharmacological inhibition of GABAA and GABAB in the neonatal rat brain. Daily injections of the reverse GABAA receptor agonist (DMCM) and the GABAB receptor antagonist (CGP35348) were performed from postnatal day 6 (P6) to P11. MBP expression was examined by Western blots and immunohistochemistry. Furthermore, we determined the number of CC1+ OLIG2+ and CNP+ OLIG2+ cells to assess maturation, the number of PCNA+ OLIG2+ oligodendrocytes to assess proliferation, the number of oligodendrocyte precursor cells (PDGFRα+ OLIG2+ ), and apoptosis of OLs (CASP3A+ OLIG2+ ) as well as apoptotic cells in total (CASP3A+ DAPI+ ) at P11 and P15. In addition, we analyzed the expression Pdgfrα and CNP. MBP expression was significantly reduced after CGP treatment at P15. In the same animal group, CNP expression and CNP+ OLIG2+ cells decreased temporarily at P11. At P15, the proliferation of PCNA+ OLIG2+ cells and the number of PDGFRα+ OLIG2+ cells increased after GABAB receptor antagonization whereas no significant differences were visible in the Pdgfrα gene expression. No changes in apoptotic cell death were observed. CGP treatment induced a transient maturational delay at P11 and deficits in myelin expression at P15 with increased oligodendroglial proliferation. Our in vivo study indicates GABAB receptor activity as a potential modulator of oligodendroglial development.

Paracetamol (Acetaminophen) and the Developing Brain.

Paracetamol is commonly used to treat fever and pain in pregnant women, but there are growing concerns that this may cause attention deficit hyperactivity disorder and autism spectrum disorder in the offspring. A growing number of epidemiological studies suggests that relative risks for these disorders increase by an average of about 25% following intrauterine paracetamol exposure. The data analyzed point to a dose-effect relationship but cannot fully account for unmeasured confounders, notably indication and genetic transmission. Only few experimental investigations have addressed this issue. Altered behavior has been demonstrated in offspring of paracetamol-gavaged pregnant rats, and paracetamol given at or prior to day 10 of life to newborn mice resulted in altered locomotor activity in response to a novel home environment in adulthood and blunted the analgesic effect of paracetamol given to adult animals. The molecular mechanisms that might mediate these effects are unknown. Paracetamol has diverse pharmacologic actions. It reduces prostaglandin formation via competitive inhibition of the peroxidase moiety of prostaglandin H2 synthase, while its metabolite N-arachidonoyl-phenolamine activates transient vanilloid-subtype 1 receptors and interferes with cannabinoid receptor signaling. The metabolite N-acetyl-p-benzo-quinone-imine, which is pivotal for liver damage after overdosing, exerts oxidative stress and depletes glutathione in the brain already at dosages below the hepatic toxicity threshold. Given the widespread use of paracetamol during pregnancy and the lack of safe alternatives, its impact on the developing brain deserves further investigation.

Reduction of cortical parvalbumin-expressing GABAergic interneurons in a rodent hyperoxia model of preterm birth brain injury with deficits in social behavior and cognition.

The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the underlying mechanisms are not known. In a translational hyperoxia model, exposing mice pups at P5 to 80% oxygen for 48 h to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin-expressing interneurons until adulthood. Developmental delay of cortical myelin was observed, together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning and attention. These results demonstrate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate that an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.

GABAB Receptor-Mediated Impairment of Intermediate Progenitor Maturation During Postnatal Hippocampal Neurogenesis of Newborn Rats.

The neurotransmitter GABA and its receptors assume essential functions during fetal and postnatal brain development. The last trimester of a human pregnancy and early postnatal life involves a vulnerable period of brain development. In the second half of gestation, there is a developmental shift from depolarizing to hyperpolarizing in the GABAergic system, which might be disturbed by preterm birth. Alterations of the postnatal GABA shift are associated with several neurodevelopmental disorders. In this in vivo study, we investigated neurogenesis in the dentate gyrus (DG) in response to daily administration of pharmacological GABAA (DMCM) and GABAB (CGP 35348) receptor inhibitors to newborn rats. Six-day-old Wistar rats (P6) were daily injected (i.p.) to postnatal day 11 (P11) with DMCM, CGP 35348, or vehicle to determine the effects of both antagonists on postnatal neurogenesis. Due to GABAB receptor blockade by CGP 35348, immunohistochemistry revealed a decrease in the number of NeuroD1 positive intermediate progenitor cells and a reduction of proliferative Nestin-positive neuronal stem cells at the DG. The impairment of hippocampal neurogenesis at this stage of differentiation is in line with a significantly decreased RNA expression of the transcription factors Pax6, Ascl1, and NeuroD1. Interestingly, the number of NeuN-positive postmitotic neurons was not affected by GABAB receptor blockade, although strictly associated transcription factors for postmitotic neurons, Tbr1, Prox1, and NeuroD2, displayed reduced expression levels, suggesting impairment by GABAB receptor antagonization at this stage of neurogenesis. Antagonization of GABAB receptors decreased the expression of neurotrophins (BDNF, NT-3, and NGF). In contrast to the GABAB receptor blockade, the GABAA receptor antagonization revealed no significant changes in cell counts, but an increased transcriptional expression of Tbr1 and Tbr2. We conclude that GABAergic signaling via the metabotropic GABAB receptor is crucial for hippocampal neurogenesis at the time of rapid brain growth and of the postnatal GABA shift. Differentiation and proliferation of intermediate progenitor cells are dependent on GABA. These insights become more pertinent in preterm infants whose developing brains are prematurely exposed to spostnatal stress and predisposed to poor neurodevelopmental disorders, possibly as sequelae of early disruption in GABAergic signaling.

In vitro P38MAPK inhibition in aged astrocytes decreases reactive astrocytes, inflammation and increases nutritive capacity after oxygen-glucose deprivation.

Proper astroglial functioning is essential for the development and survival of neurons and oligodendroglia under physiologic and pathological circumstances. Indeed, malfunctioning of astrocytes represents an important factor contributing to brain injury. However, the molecular pathways of this astroglial dysfunction are poorly defined. In this work we show that aging itself can drastically perturb astrocyte viability with an increase of inflammation, cell death and astrogliosis. Moreover, we demonstrate that oxygen glucose deprivation (OGD) has a higher impact on nutritive loss in aged astrocytes compared to young ones, whereas aged astrocytes have a higher activity of the anti-oxidant systems. P38MAPK signaling has been identified to be upregulated in neurons, astrocytes and microglia after ischemic stroke. By using a pharmacological p38α specific inhibitor (PH-797804), we show that p38MAPK pathway has an important role in aged astrocytes for inflammatory and oxidative stress responses with the subsequent cell death that occurs after OGD.

Glial Factors Regulating White Matter Development and Pathologies of the Cerebellum.

The cerebellum is a brain region that undergoes extremely dynamic growth during perinatal and postnatal development which is regulated by the proper interaction between glial cells and neurons with a complex concert of growth factors, chemokines, cytokines, neurotransmitters and transcriptions factors. The relevance of cerebellar functions for not only motor performance but also for cognition, emotion, memory and attention is increasingly being recognized and acknowledged. Since perturbed circuitry of cerebro-cerebellar trajectories can play a role in many central nervous system pathologies and thereby contribute to neurological symptoms in distinct neurodevelopmental and neurodegenerative diseases, is it the aim with this mini-review to highlight the pathways of glia-glia interplay being involved. The designs of future treatment strategies may hence be targeted to molecular pathways also playing a role in development and disease of the cerebellum.

Antioxidative effects of caffeine in a hyperoxia-based rat model of bronchopulmonary dysplasia.

BACKGROUND: While additional oxygen supply is often required for the survival of very premature infants in intensive care, this also brings an increasing risk of progressive lung diseases and poor long-term lung outcomes. Caffeine is administered to neonates in neonatal intensive care for the prevention and treatment of apneas and has been shown to reduce BPD incidence and the need for mechanical ventilation, although it is still unclear whether this is due to a direct pulmonary action via antagonism of adenosine receptors and/or an indirect action. This experimental study aims to investigate the action of caffeine on the oxidative stress response in pulmonary tissue in a hyperoxia-based model of bronchopulmonary dysplasia in newborn rats. METHODS: Newborn Wistar rats were exposed to 21% or 80% oxygen for 3 (P3) or 5 (P5) postnatal days with or without recovery on room air until postnatal day 15 (P15) and treated with vehicle or caffeine (10 mg/kg) every 48 h beginning on the day of birth. The lung tissue of the rat pups was examined for oxidative stress response at P3 and P5 immediately after oxygen exposure or after recovery in ambient air (P15) by immunohistological staining and analysis of lung homogenates by ELISA and qPCR. RESULTS: Lungs of newborn rats, corresponding to the saccular stage of lung development and to the human lung developmental stage of preterms, showed increased rates of total glutathione and hydrogen peroxide, oxidative damage to DNA and lipids, and induction of second-phase mediators of antioxidative stress response (superoxide dismutase, heme oxygenase-1, and the Nrf2/Keap1 system) in response to hyperoxia. Caffeine reduced oxidative DNA damage and had a protective interference with the oxidative stress response. CONCLUSION: In addition to the pharmacological antagonism of adenosine receptors, caffeine appears to be a potent antioxidant and modulates the hyperoxia-induced pulmonary oxidative stress response and thus protective properties in the BPD-associated animal model. Free-radical-induced damage caused by oxidative stress seems to be a biological mechanism progress of newborn diseases. New aspects of antioxidative therapeutic strategies to passivate oxidative stress-related injury should be in focus of further investigations.

Transient Improvement of Cerebellar Oligodendroglial Development in a Neonatal Hyperoxia Model by PDGFA Treatment.

In preterm infants, the changes from fetal life to ex-utero conditions often coincide with reduced growth and white matter damage of the cerebellum. The premature increase in arterial oxygen tension caused by preterm birth may dysregulate cerebellar development. In a hyperoxia rat model of white matter damage to mimic a steep increase in oxygen levels by 24 h exposure to 80% O2 from postnatal day 6 (P6) to day 7, we analyzed growth factor (GF) synthesis of cerebellar astrocytes. Determination of GF production was performed in astrocytes after Magnetic-activated cell sorting (MACS) isolation from cerebelli after hyperoxia exposure ex vivo, and also in astroglial cultures. Oligodendrocyte progenitor cell (OPC) function was analyzed in cerebellar OPCs isolated by MACS after hyperoxia. Administration of PDGFA from P6 to P11, during hyperoxia and during 4 days recovery, was finally tested for protection of oligodendroglia and myelination. As a result, expression of the GFs Pdgfa, Fgf2, and Bdnf was diminished in cerebellar astrocytes in vitro and in vivo. Gene expression of Olig1, Olig2, Sox9, Sox10, and Cnp was reduced in OPCs in vivo. Nasal PDGFA application improved oligodendroglial proliferation after hyperoxia at P7. However, this treatment effect vanished until P9. Impaired MBP expression after hyperoxia was attenuated by PDGFA treatment until P11, but not beyond when PDGFA supply was stopped. In this study on neonatal cerebellar injury, it is documented for the first time that improvement of oligodendroglial proliferation and of myelination can be achieved by PDGFA treatment. However, the treatment benefit is not maintained long term.

Neonatal Hyperoxia Perturbs Neuronal Development in the Cerebellum.

Impaired postnatal brain development of preterm infants often results in neurological deficits. Besides pathologies of the forebrain, maldeveolopment of the cerebellum is increasingly recognized to contribute to psychomotor impairments of many former preterm infants. However, causes are poorly defined. We used a hyperoxia model to define neonatal damage in cerebellar granule cell precursors (GCPs) and in Purkinje cells (PCs) known to be essential for interaction with GCPs during development. We exposed newborn rats to 24 h 80% O2 from age P6 to P7 to identify postnatal and long-term damage in cerebellar GCPs at age P7 after hyperoxia and also after recovery in room air thereafter until P11 and P30. We determined proliferation and apoptosis of GCPs and immature neurons by immunohistochemistry, quantified neuronal damage by qPCR and Western blots for neuronal markers, and measured dendrite outgrowth of PCs by CALB1 immunostainings and by Sholl analysis of Golgi stainings. After hyperoxia, proliferation of PAX6+ GCPs was decreased at P7, while DCX + CASP3+ cells were increased at P11. Neuronal markers Pax6, Tbr2, and Prox1 were downregulated at P11 and P30. Neuronal damage was confirmed by reduced NeuN protein expression at P30. Sonic hedgehog (SHH) was significantly decreased at P7 and P11 after hyperoxia and coincided with lower CyclinD2 and Hes1 expression at P7. The granule cell injury was accompanied by hampered PC maturation with delayed dendrite formation and impaired branching. Neonatal injury induced by hyperoxia inhibits PC functioning and impairs granule cell development. As a conclusion, maldevelopment of the cerebellar neurons found in preterm infants could be caused by postnatal oxygen toxicity.

Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α.

The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes.

Neuroprotection by Caffeine in Hyperoxia-Induced Neonatal Brain Injury.

Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term "oxygen radical disease of prematurity". Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28-32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.

Oligodendroglial maldevelopment in the cerebellum after postnatal hyperoxia and its prevention by minocycline.

According to recent research, brain injury after premature birth often includes impaired growth of the cerebellum. However, causes of cerebellar injury in this population are poorly understood. In this study, we analyzed whether postnatal hyperoxia perturbs white matter development of the cerebellum, and whether cerebellar glial damage can be prevented by minocycline. We used a hyperoxia model in neonatal rats providing 24 h exposure to fourfold increased oxygen concentration (80% O2) from P6 to P7, followed by recovery in room air until P9, P11, P15, P30. Injections with minocycline were performed at the beginning and 12 h into hyperoxia exposure. Hyperoxia induced oxidative stress in the cerebellum at P7 as evidenced by increased nitrotyrosine concentrations. Numbers of proliferating, NG2+Ki67+ oligodendroglial precursor cells were decreased at P7 after hyperoxia and at P11 following recovery in room air. Numbers of mature, CC1+ oligodendrocytes were diminished in recovering hyperoxia rats, and myelin basic protein expression was still decreased at P30. Electron microscopy analysis of myelinated fibers at P30 revealed thinner myelin sheath after hyperoxia. Long-term injury of the cerebellum by neonatal hyperoxia was confirmed by reduced volumes in MRI measurements at P30. In response to 80% O2, expression of platelet-derived growth factor (PDGF)-A was largely reduced in cerebellar tissue and also in cultured cerebellar astrocytes. Treatment with minocycline during hyperoxia prevented oxidative stress, attenuated oligodendroglial injury, and improved astroglial PDGF-A levels. In conclusion, early hyperoxia causes white matter damage in the cerebellum with astroglial dysfunction being involved, and both can be prevented by treatment with minocycline. Neonatal exposure to hyperoxia causes hypomyelination of the cerebellum. Reduced astroglial growth factor production but not microglial inflammation seems to contribute to oligodendroglial damage, and minocycline rescues oligodendroglia development in the cerebellum after hyperoxia.

Minocycline protects the immature white matter against hyperoxia.

Poor neurological outcome in preterm infants is associated with periventricular white matter damage and hypomyelination, often caused by perinatal inflammation, hypoxia-ischemia, and hyperoxia. Minocycline has been demonstrated in animal models to protect the immature brain against inflammation and hypoxia-ischemia by microglial inhibition. Here we studied the effect of minocycline on white matter damage caused by hyperoxia. To mimic the 3- to 4-fold increase of oxygen tension caused by preterm birth, we have used the hyperoxia model in neonatal rats providing 24h exposure to 4-fold increased oxygen concentration (80% instead of 21% O2) from P6 to P7. We analyzed whether minocycline prevents activation of microglia and damage of oligodendroglial precursor cell development, and whether acute treatment of hyperoxia-exposed rats with minocycline improves long term white matter integrity. Minocycline administration during exposure to hyperoxia resulted in decreased apoptotic cell death and in improved proliferation and maturation of oligodendroglial precursor cells (OPC). Minocycline blocked changes in microglial morphology and IL-1ß release induced by hyperoxia. In primary microglial cell cultures, minocycline inhibited cytokine release while in mono-cultures of OPCs, it improved survival and proliferation. Long term impairment of white matter diffusivity in MRI/DTI in P30 and P60 animals after neonatal hyperoxia was attenuated by minocycline. Minocycline protects white matter development against oxygen toxicity through direct protection of oligodendroglia and by microglial inhibition. This study moreover demonstrates long term benefits of minocycline on white matter integrity.

Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations.

Gene regulation by transcription factors (TFs) determines developmental programs and cell identity. Consequently, mutations in TFs can lead to dramatic phenotypes in humans by disrupting gene regulation. To date, the molecular mechanisms that actually cause these phenotypes have been difficult to address experimentally. ChIP-seq, which couples chromatin immunoprecipitation with high-throughput sequencing, allows TF function to be investigated on a genome-wide scale, enabling new approaches for the investigation of gene regulation. Here, we present the application of ChIP-seq to explore the effect of missense mutations in TFs on their genome-wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The mutated glutamine (Q) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have lysine (K) at this position. Our results show that the mutation results in a shift in the binding profile of the mutant toward a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with another mutation, Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. The methodology described can be used to investigate a wide spectrum of TFs and mutations that have not previously been amenable to ChIP-seq experiments.