myelin-basic-protein and Hypoxia-Ischemia--Brain

This work was conducted in the frames of a multicenter clinical trial. The aim was to study efficacy of cytoflavin (infusion solution) in the prevention and treatment of posthypoxic CNS lesions in premature newborns. The study included 120 premature newborns (gestation period 28-36 weeks) who was born in severe distress and needed the intensive therapy after primary reanimation measures. Cytoflavin was prescribed in the first 2-4 h after the delivery to 61 newborns. The control group included 59 newborns who did not receive the drug. To assess treatment efficacy, the determination of some plasma neurospecific proteins (GFAP, NSE, MBP) was carried out along with standard clinical/instrumental and laboratory monitoring. The results revealed the marked cerebroprotective effect of cytoflavin. The significantly higher rate of normalization of KOC, pO2, PCO2 and elimination of lactate acidosis that led to the reduction of severity and frequency of ischemic and hemorrhagic CNS lesions as well as lower levels of plasma neurospecific proteins were seen in the main group compared to the control one.

Topics: Drug Combinations; Female; Fetal Hypoxia; Flavin Mononucleotide; Glial Fibrillary Acidic Protein; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Inosine Diphosphate; Intensive Care Units, Neonatal; Intensive Care, Neonatal; Male; Myelin Basic Protein; Niacinamide; Resuscitation; Succinates; Treatment Outcome

Multiple randomized controlled trials have shown that hypothermia is a safe and effective treatment for neonatal moderate or severe hypoxic-ischemic encephalopathy (HIE). The neuroprotective mechanisms of hypothermia need further study. The aim of this study was to investigate the effect of hypothermia on the serum levels of myelin basic protein (MBP) and tumor necrosis factor-α (TNF-α) as well as neurodevelopmental outcomes in neonatal HIE.. Eighty-five neonates with moderate-to-severe HIE were divided into a hypothermia group (. After 3 days of treatment, serum levels of MBP and TNF-α in the control group were not significantly different from levels before treatment (. Hypothermia can reduce serum levels of MBP and TNF-α in neonates with HIE. Inhibiting the release of TNF-α may be one of the mechanisms by which hypothermia protects the myelin sheath.. · Hypothermia can reduce serum levels of MBP and TNF-α in neonatal HIE.. · Hypothermia improves neurodevelopmental outcomes and reduces the rate of neurodevelopmental impairment.. · Hypothermia is a feasible and effective treatment for neonates with moderate or severe HIE..

Topics: Humans; Hypothermia; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Infant; Infant, Newborn; Myelin Basic Protein; Tumor Necrosis Factor-alpha

As the interest in the neuroprotective possibilities of docosahexaenoic acid (DHA) for brain injury has grown in the recent years, we aimed to investigate the long-term effects of this fatty acid in an experimental model of perinatal hypoxia-ischemia in rats. To this end, motor activity, aspects of learning, and memory function and anxiety, as well as corticofugal connections visualized by using tracer injections, were evaluated at adulthood. We found that in the hours immediately following the insult, DHA maintained mitochondrial inner membrane integrity and transmembrane potential, as well as the integrity of synaptic processes. Seven days later, morphological damage at the level of the middle hippocampus was reduced, since neurons and myelin were preserved and the astroglial reactive response and microglial activation were seen to be diminished. At adulthood, the behavioral tests revealed that treated animals presented better long-term working memory and less anxiety than non-treated hypoxic-ischemic animals, while no difference was found in the spontaneous locomotor activity. Interestingly, hypoxic-ischemic injury caused alterations in the anterograde corticofugal neuronal connections which were not so evident in rats treated with DHA. Thus, our results indicate that DHA treatment can lead to long-lasting neuroprotective effects in this experimental model of neonatal hypoxia-ischemic brain injury, not only by mitigating axonal changes but also by enhancing cognitive performance at adulthood.

Topics: Animals; Animals, Newborn; Axons; Behavior, Animal; Brain; Cognitive Dysfunction; Docosahexaenoic Acids; Glial Fibrillary Acidic Protein; Gliosis; Hippocampus; Hypoxia-Ischemia, Brain; Membrane Potential, Mitochondrial; Microglia; Mitochondria; Motor Activity; Myelin Basic Protein; Rats, Sprague-Dawley; Spatial Memory

Topics: Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Estetrol; Hippocampus; Hydrogen Peroxide; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Neurons; Neuroprotective Agents; Oxidative Stress; Pilot Projects; Rats; Rats, Sprague-Dawley

Hypoxic-ischemic (HI) injury to the developing brain remains a major cause of morbidity. To date, few therapeutic strategies could provide complete neuroprotection. Erythropoietin (EPO) has been shown to be beneficial in several models of neonatal HI. This study examines the effect of treatment with erythropoietin on postnatal day 2 (P2) rats introduced with HI injury.. Rats at P2 were randomized into four groups: sham, bilateral carotid artery occlusion (BCAO), BCAO + early EPO, and BCAO + late EPO groups. Pups in each group were injected with either saline or EPO (5000U/kg) intraperitoneally once at immediately (early) or 48h (late) after HI induction. Body weight was assessed at P2 before and day 7 after HI. Mortality Rate was assessed at 24h, 48h and 72h after HI and brain water content was assessed at 72h. Brain weight and expression of myelin basic protein (MBP) were assessed at day 7 and day 14. At day 31 to 35 following HI insult, neurological behavior function was assessed via Morris water maze (MWM) test.. HI cause significant higher mortality in male than in female (P=0.0445). Among the surviving animal, HI affect significantly the body growth, brain growth, MBP expression, and neurological behavior. EPO treatments at both early and late time points significantly benefit the rats in injury recovery, in which they promoted weight gains, reduced brain edema, as well as improved spatial learning ability and memory.. We demonstrated a single dose of EPO at 5000U/kg immediately or 48h after HI injury had significant benefit for the P2 rats in injury recovery, and there was no adverse effect associated with either EPO treatment.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Body Weight; Brain Edema; Developmental Disabilities; Disease Models, Animal; Erythropoietin; Hypoxia-Ischemia, Brain; Maze Learning; Myelin Basic Protein; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies.

Topics: Animals; Animals, Newborn; Apoptosis; Axons; Brain Injuries; Calcium-Binding Proteins; Calpain; Caspases; Enzyme Activation; Erythropoietin; Female; Hypoxia-Ischemia, Brain; Membrane Proteins; Myelin Basic Protein; Rats, Sprague-Dawley

OLIG1 is an oligodendrocyte (OL) transcription factor, which can contribute to the proliferation and differentiation of OLs, and the maturation of myelin. The aim of this study was to clarify the role of OLIG1 in neonatal Sprague Dawley rats with periventricular leukomalacia (PVL), induced by hypoxia‑ischemia (HI). Newborn rats in the HI group were subjected to ligation of the right carotid artery, followed by 8% oxygen delivery for 2 h, while rats in the normoxia group were only subjected to isolation of the right carotid artery, without exposure to hypoxia. Samples of brain tissue from rats in both groups were collected at 1, 3, 7, 14 and 21 days. In the HI group, observation by transmission electron microscopy (TEM) revealed OLs with a damaged nuclear membrane, cellular atrophy, deformation and necrosis, and cells in myelin with a high number of small vacuoles. A double‑label immunofluorescence assay revealed the translocation of OLIG1 from the cytoplasm to the nucleus, while western blot and reverse transcription‑quantitative polymerase chain reaction assays showed that there is a significant decrease, followed by an increase, in the gene and protein expression levels of OLIG1 and myelin basic protein (MBP). Despite the increase at the late stages of HI, the final levels of these proteins remained lower than the corresponding levels in the normoxia group. In conclusion, the decreased protein expression of OLIG1 following HI plays an important role in inhibiting the development and maturation of OLs and myelin. Although OLIG1 may, via its nuclear translocation, promote the growth and development of myelin to a certain extent, this factor fails to fully repair injured myelin.

Topics: Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Brain; Female; Gene Expression; Hypoxia-Ischemia, Brain; Immunohistochemistry; Leukomalacia, Periventricular; Male; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Oligodendroglia; Rats; RNA, Messenger

Hypomyelination is the major cause of neurodevelopmental deficits that are associated with perinatal white matter injury. Chondroitin sulfate proteoglycans (CSPGs) are known to exert inhibitory effects on the migration and differentiation of oligodendrocytes (OLs). However, few studies describe the roles of CSPGs in myelination by OLs and the cognitive dysfunction that follows perinatal white matter injury. Here, we examined the alterations in the expression of CSPGs and their functional impact on the maturation of OLs and myelination in a neonatal rat model of hypoxic-ischemic (HI) brain injury. Three-day-old Sprague-Dawley rats underwent a right common carotid artery ligation and were exposed to hypoxia (6% oxygen for 2.5h). Rats were given chondroitinase ABC (cABC) via an intracerebroventricular injection to digest CSPGs. Animals were sacrificed at 7, 14, 28 and 56days after HI injury and the accompanying surgical procedure. We found that the expression of CSPGs was significantly up-regulated in the cortical regions surrounding the white matter after HI injury. cABC successfully degraded CSPGs in the rats that received cABC. Immunostaining showed decreased expression of the pre-oligodendrocyte marker O4 in the cingulum, external capsule and corpus callosum in HI+cABC rats compared to HI rats. However HI+cABC rats exhibited greater maturation of OLs than did HI rats, with increased expression of O1 and myelin basic protein in the white matter. Furthermore, using electron microscopy, we demonstrated that myelin formation was enhanced in HI+cABC rats, which had an increased number of myelinated axons and decreased G-ratios of myelin compared to HI rats. Finally, HI+cABC rats performed better in the Morris water maze task than HI rats, which indicates an improvement in cognitive ability. Our results suggest that CSPGs inhibit both the maturation of OLs and the process of myelination after neonatal HI brain injury. The data also raise the possibility that modifying CSPGs may repair this type of lesion associated with demyelination.

Topics: Animals; Animals, Newborn; Cell Differentiation; Chondroitin Sulfate Proteoglycans; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; Rats, Sprague-Dawley; White Matter

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.

Topics: Animals; Animals, Newborn; Astrocytes; Bacterial Proteins; Blood-Brain Barrier; Cell Count; Cells, Cultured; Cerebral Cortex; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Integrin beta1; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Myelin Basic Protein; Phosphopyruvate Hydratase; Platelet Endothelial Cell Adhesion Molecule-1; Vimentin

For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined.. HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo.. At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment.. Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

Topics: Animals; Animals, Newborn; Behavior, Animal; Biomarkers; Brain; Cells, Cultured; Cognition; Disease Models, Animal; Hypoxia-Ischemia, Brain; Mesenchymal Stem Cell Transplantation; Mice, Inbred C57BL; Microtubule-Associated Proteins; Motor Activity; Myelin Basic Protein; Recognition, Psychology; Recovery of Function; Risk Assessment; Time Factors

To study the effects of caffeine citrate on myelin basic protein (MBP) expression in the cerebral white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the related mechanism.. Forty-eight seven-day-old Sprague-Dawley neonatal rats were randomly assigned to 3 groups: sham operation (n=16), HIBD (n=16) and HIBD+caffeine citrate (n=16). The rats in the HIBD and HIBD+caffeine citrate groups were subjected to left common carotid artery ligation, and then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen for 2 hours to induce HIBD. The rats in the sham operation group were only subjected to a sham operation, without the left common carotid artery ligation or hypoxia exposure. Caffeine citrate (20 mg/kg) was injected intraperitoneally before hypoxia ischemia (HI) and immediately, 24 hours, 48 hours and 72 hours after HI. The other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time points. On postnatal day 12, the expression of MBP in the left subcortical white matter was detected by immunohistochemistry, and the levels of adenosine A1 receptor mRNA and A2a receptor mRNA in the left brain were detected by real-time PCR.. The expression of MBP in the left subcortical white matter in the HIBD group was lower than in the sham operation group (P<0.05). The MBP expression in the HIBD+caffeine citrate group was significantly higher than in the HIBD group, but was still lower than the sham operation group (P<0.05). Real-time PCR showed that the adenosine A1 receptor mRNA expression was significantly higher in the HIBD group than in the sham operation group, and it was significantly lower in the HIBD+caffeine citrate group than in the HIBD group (P<0.05).. Caffeine citrate can improve brain white matter damage following HIBD in neonatal rats and the protection mechanism might be related with the down-regulation of adenosine A1 receptor expression.

Topics: Animals; Animals, Newborn; Caffeine; Citrates; Female; Hypoxia-Ischemia, Brain; Male; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptor, Adenosine A2A; RNA, Messenger; White Matter

Despite advances in neonatal care, hypoxic-ischemic brain injury is still a serious clinical problem, which is responsible for many cases of perinatal mortality, cerebral palsy, motor impairment and cognitive deficits. Resveratrol, a natural polyphenol with important anti-oxidant and anti-inflammatory properties, is present in grapevines, peanuts and pomegranates. The aim of the present work was to evaluate the possible neuroprotective effect of resveratrol when administered before or immediately after a hypoxic-ischemic brain event in neonatal rats by analyzing brain damage, the mitochondrial status and long-term cognitive impairment. Our results indicate that pretreatment with resveratrol protects against brain damage, reducing infarct volume, preserving myelination and minimizing the astroglial reactive response. Moreover its neuroprotective effect was found to be long lasting, as behavioral outcomes were significantly improved at adulthood. We speculate that one of the mechanisms for this neuroprotection may be related to the maintenance of the mitochondrial inner membrane integrity and potential, and to the reduction of reactive oxygen species. Curiously, none of these protective features was observed when resveratrol was administered immediately after hypoxia-ischemia.

Topics: Animals; Animals, Newborn; Astrocytes; Behavior, Animal; Brain; Brain Injuries; Cognition Disorders; Disease Models, Animal; Female; Hypoxia-Ischemia, Brain; Male; Membrane Potential, Mitochondrial; Mitochondria; Myelin Basic Protein; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Resveratrol; Stilbenes

Myelination failure is associated with perinatal cerebral hypoxia-ischemia (PHI) induced brain injury in premature infants. How to efficiently promote remyelination is crucial for improving cognitive deficits caused by brain injury. Here, we demonstrated that quercetin (Que), a kind of flavonoids, significantly improved cognitive deficits and the behavior of PHI-rat in Morris water maze and open field tasks. After administration of Que to PHI-rat, the number of neogenetic Olig2⁺ oligodendrocyte progenitor cells (OPCs) was evidently increased in the subventricular zone. Additionally, in corpus callosum (CC), the expression of MBP (myelin basic protein) was increased, and the myelin sheaths reached normal level at 30 days with more compact while less damaged myelin sheaths and more mature oligodendrocytes (OLs) repopulating the CC compared with PHI groups. In a word, our findings indicated that Que could remarkably improve both cognition performance and myelination in the context of PHI-induced brain injury by promoting the proliferation of OPCs and strengthening survival of OLs in vivo.

Topics: Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Cell Proliferation; Cognition Disorders; Corpus Callosum; Exploratory Behavior; Gene Expression; Hippocampus; Hypoxia-Ischemia, Brain; Locomotion; Maze Learning; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Neural Stem Cells; Neuroprotective Agents; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Quercetin; Rats

Periventricular leukomalacia (PVL) is a major form of brain injury among preterm infants, which is characterized by extensive loss and dysfunction of premyelinating oligodendrocytes (pre-OLs) induced by hypoxia-ischemia (HI). Therapeutic hypothermia, which is a standard treatment for term infants with HI encephalopathy, is not indicated for preterm infants because its safety and effect have not been established. Here we investigate the effectiveness and mechanism of hypothermia for the inhibition of pre-OLs damage in PVL. For in vivo studies, 6-day-old rats underwent left carotid artery ligation, followed by exposure to 6% oxygen for 1 hr under hypothermic or normothermic conditions. The loss of myelin basic protein (MBP) was inhibited by hypothermia. For in vitro studies, primary pre-OLs cultures were subjected to oxygen-glucose deprivation (OGD) under normothermic or hypothermic conditions, and dorsal root ganglion neurons were subsequently added. Hypothermia inhibited apoptosis of pre-OLs, and, despite specific downregulation of 21.5- and 17-kDa MBP mRNA expression during hypothermia, recovery of the expression after OGD was superior compared with normothermia. OGD caused disarrangement of MBP distribution, decreased the levels of phosphorylated 21.5-kDa MBP, and disturbed the capacity to contact with neurons, all of which were restored by hypothermia. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 during and after OGD significantly reduced the protective effects of hypothermia on apoptosis and myelination, respectively. These data suggest that phosphorylated exon 2-containing (21.5- and possibly 17-kDa) MBP isoforms may play critical roles in myelination and that hypothermia attenuates apoptosis and preserves the contact between OLs and neurons via ERK1/2 phosphorylation.

Topics: Animals; Animals, Newborn; Apoptosis; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Ganglia, Spinal; Gene Expression Regulation; Glucose; Hypothermia, Induced; Hypoxia; Hypoxia-Ischemia, Brain; Leukoencephalopathies; Male; Myelin Basic Protein; Neurons; Oligodendroglia; Rats; Rats, Sprague-Dawley

Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month.. Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction.. Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001).. Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions.

Topics: Animals; Animals, Newborn; Axons; Brain; Calcium-Binding Proteins; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Female; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Inflammation; Leukoencephalopathies; Lipopolysaccharides; Microfilament Proteins; Myelin Basic Protein; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin

In this study, we investigated the effects of a bioactive high-affinity TrkB receptor agonist 7,8- dihydroxyflavone (7,8 DHF) on neonatal brain injury in female and male mice after hypoxia ischemia (HI). HI was induced by exposure of postnatal day 9 (P9) mice to 10% O2 for 50 minutes at 37°C after unilateral ligation of the left common carotid artery. Animals were randomly assigned to HI-vehicle control group [phosphate buffered saline (PBS), intraperitoneally (i.p.)] or HI + 7,8 DHF-treated groups (5 mg/kg in PBS, i.p at 10 min, 24 h, or with subsequent daily injections up to 7 days after HI). The HI-vehicle control mice exhibited neuronal degeneration in the ipsilateral hippocampus and cortex with increased Fluoro-Jade C positive staining and loss of microtubule associated protein 2 expression. In contrast, the 7,8 DHF-treated mice showed less hippocampal neurodegeneration and astrogliosis, with more profound effects in female than in male mice. Moreover, 7,8 DHF-treated mice improved motor learning and spatial learning at P30-60 compared to the HI-vehicle control mice. Diffusion tensor imaging of ex vivo brain tissues at P90 after HI revealed less reduction of fractional anisotropy values in the ipsilateral corpus callosum of 7,8 DHF-treated brains, which was accompanied with better preserved myelin basic protein expression and CA1 hippocampal structure. Taken together, these findings strongly suggest that TrkB agonist 7,8 DHF is protective against HI-mediated hippocampal neuronal death, white matter injury, and improves neurological function, with a more profound response in female than in male mice.

Topics: Aging; Animals; Cerebral Cortex; Corpus Callosum; Female; Flavones; Gliosis; Hippocampus; Hypoxia-Ischemia, Brain; Learning; Male; Mice; Microtubule-Associated Proteins; Myelin Basic Protein; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroimaging; Neuroprotective Agents; Receptor, trkB; Recovery of Function; Sex Characteristics

Hypothermia has been proposed as a treatment for reducing neuronal damage in the brain induced by hypoxic ischemia. In the developing brain, hypoxic ischemia-induced injury may give rise to cerebral palsy (CP). However, it is unknown whether hypothermia might affect the development of CP. The purpose of this study was to investigate whether hypothermia would have a protective effect on the brains of immature, 3-day old (P3) mice after a challenge of cerebral ischemia. Cerebral ischemia was induced in P3 mice with a right common carotid artery ligation followed by hypoxia (6% O2, 37°C) for 30 min. Immediately after hypoxic ischemia, mice were exposed to hypothermia (32°C) or normothermia (37°C) for 24 h. At 4 weeks of age, mouse motor development was tested in a behavioral test. Mice were sacrificed at P4, P7, and 5 weeks to examine brain morphology. The laminar structure of the cortex was examined with immunohistochemistry (Cux1/Ctip2); the number of neurons was counted; and the expression of myelin basic protein (MBP) was determined. The hypothermia treatment was associated with improved neurological outcomes in the behavioral test. In the normothermia group, histological analyses indicated reduced numbers of neurons, reduced cortical laminar thickness in the deep, ischemic cortical layers, and significant reduction in MBP expression in the ischemic cortex compared to the contralateral cortex. In the hypothermia group, no reductions were noted in deep cortical layer thickness and in MBP expression in the ischemic cortex compared to the contralateral cortex. At 24 h after the hypothermia treatment prevented the neuronal cell death that had predominantly occurred in the ischemic cortical deep layers with normothermia treatment. Our findings may provide a preclinical basis for testing hypothermal therapies in patients with CP induced by hypoxic ischemia in the preterm period.

Topics: Animals; Animals, Newborn; Brain; Cell Count; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Immunohistochemistry; Mice; Motor Activity; Myelin Basic Protein; Neurons

The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis.

Topics: Animals; Animals, Newborn; Antigens; Brain Injuries; Carotid Artery Diseases; Disease Models, Animal; Excitatory Amino Acid Antagonists; Functional Laterality; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Leukoencephalopathies; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Minocycline; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Polysaccharides; Proteoglycans; Quinoxalines; Receptors, AMPA; Synapses; Vesicular Glutamate Transport Protein 1; Vesicular Glutamate Transport Protein 2

To minimize as much as possible the neurological consequences from hypoxic-ischemic (HI) brain injury, neuroprotective strategies are urgently required. In this sense, there is growing interest in the neuroprotective potential of melatonin after perinatal asphyxia, due to its high efficacy, low toxicity and ready cross through the blood-brain barrier. Twenty six Wistar rats at postnatal day 7 were randomly assigned to: two hypoxic-ischemic groups: pups with the left common carotid artery ligated and then submitted to hypoxia (HI group) and animals that received a dose of 15 mg/kg melatonin just after the hypoxic-ischemic event and repeated twice with an interval of 24 hours (HI+MEL group). Pups without ischemia or hypoxia were used as controls (Sham group). Seven days after surgery, brains were collected and coronal sections Nissl-stained, TUNEL-labeled, or MBP- and GFAP-immunolabeled prior to determining brain infarct area, quantify surviving neurons and evaluate oligodendroglial injury and reactive astrogliosis. The number of surviving neurons showing a well preserved architecture in HI+MEL group was similar to that observed in the Sham group. Moreover, TUNEL-positive cells only appeared in the HI group. The ratio of left-to-right hemispheric MBP immunostaining showed a significant decrease in the HI group in comparison with Sham pups, which was restored after melatonin administration. Melatonin also reduced reactive gliosis. Thus, our results suggest that treatment with melatonin after neonatal hypoxia-ischemia led to a neuroprotective effect reducing cell death, white matter demyelination and reactive astrogliosis.

Topics: Animals; Animals, Newborn; Apoptosis; Biomarkers; Brain; Brain Infarction; Demyelinating Diseases; Disease Models, Animal; Glial Fibrillary Acidic Protein; Gliosis; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Leukoencephalopathies; Melatonin; Myelin Basic Protein; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Time Factors

Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.

Topics: 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; Administration, Intranasal; Age Factors; Animals; Animals, Newborn; Antigens; Apoproteins; Autophagy-Related Proteins; Brain Injuries; Bromodeoxyuridine; Caspase 3; Cell Death; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Colchicine; Corpus Callosum; Cytochalasin B; Female; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Hypoxia; Hypoxia-Ischemia, Brain; Intermediate Filament Proteins; Intracellular Signaling Peptides and Proteins; Lateral Ventricles; Male; Mice; Mice, Transgenic; Myelin Basic Protein; Nerve Fibers, Myelinated; Nerve Tissue Proteins; Nestin; Neurogenesis; Neuroprotective Agents; Oligodendroglia; Platelet-Derived Growth Factor; Proteoglycans; SOXB1 Transcription Factors; Time Factors; Transferrin

Chronic demyelination can result in axonopathy and is associated with human neurological conditions such as multiple sclerosis (MS) in adults and cerebral palsy in infants. In these disorders, myelin regeneration is inhibited by impaired differentiation of oligodendrocyte progenitors into myelin-producing oligodendrocytes. However, regulatory factors relevant in human myelin disorders and in myelin regeneration remain poorly understood. Here we have investigated the role of the transcription factor nuclear factor IA (NFIA) in oligodendrocyte progenitor differentiation during developmental and regenerative myelination.. NFIA expression patterns in human neonatal hypoxic-ischemic encephalopathy (HIE) and MS as well as developmental expression in mice were evaluated. Functional studies during remyelination were performed using a lysolecithin model, coupled with lentiviral misexpression of NFIA. The role of NFIA during oligodendrocyte lineage development was characterized using chick and mouse models and in vitro culture of oligodendrocyte progenitors. Biochemical mechanism of NFIA function was evaluated using chromatin immunoprecipitation and reporter assays.. NFIA is expressed in oligodendrocyte progenitors, but not differentiated oligodendrocytes during mouse embryonic development. Examination of NFIA expression in white matter lesions of human newborns with neonatal HIE, as well active MS lesions in adults, revealed that it is similarly expressed in oligodendrocyte progenitors and not oligodendrocytes. Functional studies indicate that NFIA is sufficient to suppress oligodendrocyte progenitor differentiation during adult remyelination and embryonic development through direct repression of myelin gene expression.. These studies suggest that NFIA participates in the control of oligodendrocyte progenitor differentiation and may contribute to the inhibition of remyelination in human myelin disorders.

Topics: Adenomatous Polyposis Coli Protein; Animals; Arabidopsis Proteins; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Chromatin Immunoprecipitation; Disease Models, Animal; DNA-Binding Proteins; Electroporation; Embryo, Mammalian; Gene Expression Regulation, Developmental; Homeodomain Proteins; Humans; Hypoxia-Ischemia, Brain; Infant; Infant, Newborn; Intramolecular Transferases; Leukoencephalopathies; Lysophosphatidylcholines; Mice; Mice, Transgenic; Multiple Sclerosis; Myelin Basic Protein; NFI Transcription Factors; Oligodendroglia; Spinal Cord; Stem Cells; Time Factors; Transcription Factors

There are mixed reports on the neuroprotective properties of erythropoietin (EPO) in animal models of birth asphyxia. We investigated the effect of EPO on short- and long-term outcome after neonatal hypoxic-ischemic (HI) brain injury in mice and compared the effect of two different dose regimens of EPO. Nine-day-old mice were subjected to HI, and EPO was injected i.p. at 0, 24, and 48 h after HI in a dose of either 5 or 20 kU/kg. Paw preference in the cylinder rearing test (CRT) was used as a measure of sensorimotor function. Only in female mice, administration of EPO at 5 kU/kg but not 20 kU/kg improved sensorimotor function, reduced striatum atrophy and hippocampal lesion volume, and enhanced myelin basic protein (MBP) staining as determined at 4 and 9 wk after HI. In addition, at 72 h after HI, more Ki 67 cells were found in the subventricular zone and dentate gyrus after EPO 5 kU/kg treatment, indicating an increase in progenitor cell proliferation. In conclusion, EPO improves sensorimotor function after neonatal HI and protects against striatum atrophy, hippocampus injury, and white matter loss. The protective effect of EPO is dose-dependent and only present in females.

Topics: Animals; Apoptosis; Cell Proliferation; Dose-Response Relationship, Drug; Erythropoietin; Female; Hippocampus; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Male; Mice; Myelin Basic Protein; Myelin Sheath; Neurogenesis; Psychomotor Performance; Sex Factors; Time Factors

Myelinated axons have a distinct protein architecture essential for action potential propagation, neuronal communication, and maintaining cognitive function. Damage to myelinated axons, associated with cerebral hypoperfusion, contributes to age-related cognitive decline. We sought to determine early alterations in the protein architecture of myelinated axons and potential mechanisms after hypoperfusion. Using a mouse model of hypoperfusion, we assessed changes in proteins critical to the maintenance of paranodes, nodes of Ranvier, axon-glial integrity, axons, and myelin by confocal laser scanning microscopy. As early as 3 d after hypoperfusion, the paranodal septate-like junctions were damaged. This was marked by a progressive reduction of paranodal Neurofascin signal and a loss of septate-like junctions. Concurrent with paranodal disruption, there was a significant increase in nodal length, identified by Nav1.6 staining, with hypoperfusion. Disruption of axon-glial integrity was also determined after hypoperfusion by changes in the spatial distribution of myelin-associated glycoprotein staining. These nodal/paranodal changes were more pronounced after 1 month of hypoperfusion. In contrast, the nodal anchoring proteins AnkyrinG and Neurofascin 186 were unchanged and there were no overt changes in axonal and myelin integrity with hypoperfusion. A microarray analysis of white matter samples indicated that there were significant alterations in 129 genes. Subsequent analysis indicated alterations in biological pathways, including inflammatory responses, cytokine-cytokine receptor interactions, blood vessel development, and cell proliferation processes. Our results demonstrate that hypoperfusion leads to a rapid disruption of key proteins critical to the stability of the axon-glial connection that is mediated by a diversity of molecular events.

Topics: Age Factors; Animals; Ankyrins; Axons; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Chronic Disease; Corpus Callosum; Disease Models, Animal; Electron Microscope Tomography; Gene Expression Profiling; Gene Expression Regulation; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Myelin Basic Protein; Myelin-Associated Glycoprotein; NAV1.6 Voltage-Gated Sodium Channel; Nerve Fibers, Myelinated; Nerve Growth Factors; Nerve Tissue Proteins; Neurofilament Proteins; Neuroglia; Neurons; Oligonucleotide Array Sequence Analysis; Optic Nerve; Ranvier's Nodes; Signal Transduction; Sodium Channels

Simvastatin, the most widely used cholesterol-lowering drug, has been reported to protect the adult brain from ischemia. Nevertheless, little is known about its action on developing brain after stroke. Although a few reports have found recently that simvastatin displays anti-inflammation and anti-apoptosis properties and improves the cognitive and morphological consequences in the neonatal rats after hypoxia-ischemia (HI) damage, to our best knowledge, there has been no study of the effect of it on myelin formation after neonatal brain damage. Therefore, we investigated whether simvastatin could promote the myelination of oligodendrocytes in the neonatal rats after HI and explored the possible role of microglial responses in this process.. Postnatal day 7 Sprague-Dawley rats were subjected to HI. White matter integrity and myelination were evaluated by the densitometry of myelin basic protein (MBP) immunostaining. OX-42 immunoreactivity and nissl staining were used for identifying microglial responses and the structure changes of white matter and adjacent gray matter after HI. Simvastatin was administrated prophylactically to rats.. HI induced serious hypomyelination especially in external and internal capsules 3 and 7 days after HI, accompanying with microglial activation remarkably. Simvastatin treatment greatly increased the densities of MBP immunostaining, inhibited microglial activation and reduced the numbers of pyknotic cells and neuronal loss.. The present study shows that simvastatin treatment in neonatal rats attenuates HI-induced developing oligodendrocytes injury and hypomyelination. Its anti-inflammatory properties via suppression of microglial activation are likely to contribute to this action. It provides experimental evidence to support the neuroprotective effects of statins in neonatal ischemic stroke.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Body Weight; CD11b Antigen; Demyelinating Diseases; Disease Models, Animal; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Simvastatin

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Mice; Mice, Transgenic; Myelin Basic Protein

Deferoxamine (DFO) and erythropoietin (EPO) have each been shown to provide neuroprotection in neonatal rodent models of brain injury. In view of the described anti-oxidative actions of DFO and the anti-apoptotic and anti-inflammatory effects of EPO, we hypothesized that the combination of DFO and EPO would increase neuroprotection after neonatal hypoxic-ischemic brain injury as compared to single DFO or EPO treatment. At postnatal day 7 rats underwent right common carotid artery occlusion followed by a 90-min exposure to 8% oxygen. Rats were treated intraperitoneally with DFO (200mg/kg), recombinant human EPO (1 kU/kg), a combination of DFO-EPO or vehicle at 0, 24 and 48 h after hypoxia-ischemia (HI) and were sacrificed at 72 h. DFO-EPO administration reduced the number of cleaved caspase 3-positive cells in the ipsilateral cerebral cortex. Early neuronal damage was assessed by staining for microtubuli-associated protein (MAP)-2. In our model 63+/-9% loss of ipsilateral MAP-2 was observed after HI, indicating extensive brain injury. DFO, EPO or DFO-EPO treatment did not improve neuronal integrity as defined by MAP-2. Cerebral white matter tracts were stained for myelin basic protein (MBP), a constituent of myelin. Hypoxia-ischemia strongly reduced MBP staining which suggests white matter damage. However, DFO, EPO and DFO-EPO treatment had no effect on the loss of MBP staining. Finally, HI-induced loss of striatal tyrosine hydroxylase staining was not attenuated by DFO, EPO or DFO-EPO. Although DFO-EPO treatment reduced the number of cleaved caspase 3(+) cells, treatment with DFO, EPO, or with the combination of DFO and EPO did not protect against gray or white matter damage in the experimental setting applied.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Antioxidants; Brain Infarction; Caspase 3; Cerebral Cortex; Cytoprotection; Deferoxamine; Disease Models, Animal; Drug Combinations; Drug Synergism; Erythropoietin; Hypoxia-Ischemia, Brain; Microtubule-Associated Proteins; Myelin Basic Protein; Nerve Degeneration; Nerve Fibers, Myelinated; Neurons; Rats; Rats, Wistar

Developing oligodendrocytes (pre-OLs) are highly vulnerable to hypoxic-ischemic injury and associated excitotoxicity and oxidative stress. 17beta-Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)-alpha and ER-beta are both expressed in OLs. We examined the effect of 17beta-estradiol on oxygen-glucose deprivation and oxidative stress-induced cell death in rat pre-OLs in vitro and on hypoxic-ischemic brain injury in vivo. Pre-OLs in culture were subjected to oxygen-glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17beta-estradiol. LDH release, the Alamar blue assay, and phase-contrast microscopy were used to assess cell viability. Hypoxic-ischemic injury was generated in 6-day-old rats (P6) by unilateral carotid ligation and hypoxia (6% O(2) for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 microg/kg 17beta-estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17beta-Estradiol produced significant protection against OGD-induced cell death in primary OLs (EC(50) = 1.3 +/- 0.46 x 10(-9) M) and against oxidative stress. Moreover, 17beta-estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic-ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia.

Topics: Animals; Animals, Newborn; Biomarkers; Brain; Cell Death; Cell Survival; Cells, Cultured; Estradiol; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroprotective Agents; Oligodendroglia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Stem Cells

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia-ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1-2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1-2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter. Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.

Topics: Animals; Brain; Cell Death; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Inflammation; Myelin Basic Protein; Random Allocation; Rats; Tumor Necrosis Factor-alpha; Tyrosine

Although periventricular white matter injury is a leading cause of major neurologic disability in premature infants, the relationship between myelination deficiency and long-term cognitive dysfunction is not well understood. The purpose of this study was to investigate oligodendrocytes myelination and long-term spatial cognitive function in rats with perinatal hypoxia-ischemia (HI). Postnatal day 3 (P3) rats were subjected to right carotid artery ligation followed by 2.5 h of hypoxia (6% oxygen). Brain injury during the early and late phases was evaluated by immunostaining at P6 (72 h after the injury) and P47. Spatial cognitive function was evaluated at P42 using the Morris Water Maze test followed by histologic evaluation. HI caused an increase in pre-oligodendrocytes, astrocytes, and microglia in the ipsilateral white matter 72 h after the insult compared to contralateral regions and sham-operated controls (both p<0.05). There were significant decreases in myelin basic protein (MBP)and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-labeled oligodendrocytes with glial fibrillary acidic protein (GFAP)-labeled glial scarring in the ipsilateral periventricular white matter at P47 compared to contralateral regions and sham-operated controls (all p<0.05). The rats with HI had spatial learning deficits in navigation trials (longer escape latency and swimming distance) and memory dysfunction in probe trials (fewer number of platform crossings and percentage of time in the target quadrant) compared with sham-operated controls (p<0.05). In this neonatal rat model of HI, myelination deficiency induced by activated astrocytes and microglia during the early phase with subsequent glial scarring was associated with long-term spatial learning and memory dysfunction.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Analysis of Variance; Animals; Animals, Newborn; Antigens, CD; Antigens, Differentiation, Myelomonocytic; CA1 Region, Hippocampal; Cell Count; Cognition Disorders; Demyelinating Diseases; Exploratory Behavior; Female; Gliosis; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Maze Learning; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Rats; Space Perception; Time Factors

The first 3 weeks of life is the peak time of oligodendrocytes development and also the critical period of cholesterol increasing dramatically in central nervous system in rats. Neonatal hypoxia-ischemia (HI) brain damage happening in this period may disturb the brain cholesterol balance as well as white matter development.. To test this hypothesis, postnatal day 7 (P7) Sprague-Dawley rats were subjected to HI insult. Cholesterol concentrations from brain and plasma were measured. White matter integrity was evaluated by densitometric analysis of myelin basic protein (MBP) immunostaining and electron microscopy. Brain TNF-alpha and IL-6 levels were also measured.. HI-induced brain cholesterol, but not the plasma cholesterol, levels decreased significantly during the first three days after HI compared with naïve and sham operated rats (p<0.05). Obvious hypomyelination was indicated by marked reductions in MBP immunostaining on both P10 and P14 (p<0.01) and less and thinner myelinated axons were detected on P21 by electron microscopy observation. High expressions of brain TNF-alpha and IL-6 12 h after HI (p<0.05) were also observed.. The present work provides evidence that HI insult destroyed brain cholesterol homeostasis, which might be important in the molecular pathology of hypoxic-ischemic white matter injury. Proinflammatory cytokines insulting oligodendrocytes, may cause cholesterol unbalance. Furthermore, specific therapeutic interventions to maintain brain cholesterol balance may be effective for the recovery of white matter function.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Brain; Cholesterol; Enzyme-Linked Immunosorbent Assay; Hypoxia-Ischemia, Brain; Interleukin-6; Microscopy, Electron, Transmission; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Hypoxia-ischemia (H/I) in the premature infant leads to white matter injury termed periventricular leukomalacia (PVL), the leading cause of subsequent neurological deficits. Glutamatergic excitotoxicity in white matter oligodendrocytes (OLs) mediated by cell surface glutamate receptors (GluRs) of the AMPA subtype has been demonstrated as one factor in this injury. Recently, it has been shown that rodent OLs also express functional NMDA GluRs (NMDARs), and overactivation of these receptors can mediate excitotoxic OL injury. Here we show that preterm human developing OLs express NMDARs during the PVL period of susceptibility, presenting a potential therapeutic target. The expression pattern mirrors that seen in the immature rat. Furthermore, the uncompetitive NMDAR antagonist memantine attenuates NMDA-evoked currents in developing OLs in situ in cerebral white matter of immature rats. Using an H/I rat model of white matter injury, we show in vivo that post-H/I treatment with memantine attenuates acute loss of the developing OL cell surface marker O1 and the mature OL marker MBP (myelin basic protein), and also prevents the long-term reduction in cerebral mantle thickness seen at postnatal day 21 in this model. These protective doses of memantine do not affect normal myelination or cortical growth. Together, these data suggest that NMDAR blockade with memantine may provide an effective pharmacological prevention of PVL in the premature infant.

Topics: Animals; Animals, Newborn; Antigens, Differentiation; Biomarkers; Brain; Disease Models, Animal; Excitatory Amino Acid Antagonists; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Male; Memantine; Myelin Basic Protein; Nerve Fibers, Myelinated; Oligodendroglia; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Wallerian Degeneration

Periventricular white matter injury in premature infants occurs following hypoxia/ischaemia and systemic infection, and results in hypomyelination, as well as neuromotor and cognitive deficits later in life. Inflammatory infiltrates are seen within human cerebral white matter from periventricular leucomalacia (PVL) cases.. In this study, we examine the time course of CD-68+ microglial cell responses relative to cell death within white matter following hypoxia/ischaemia in a rat model of PVL. We also tested the efficacy of the minocycline, an agent that suppresses microglial activation, in this model when administered as a post-insult treatment.. We show that preoligodendrocyte injury in the post-natal day 6 begins within 24 h and continues for 48-96 h after hypoxia/ischaemia, and that microglial responses occur primarily over the first 96 h following hypoxia/ischaemia. Minocycline treatment over this 96 h time window following the insult resulted in significant protection against white matter injury, and this effect was concomitant with a reduction in CD-68+ microglial cell numbers.. These results suggest that anti-inflammatory treatments may represent a useful strategy in the treatment of PVL, where clinical conditions would favour a post-insult treatment strategy.

Topics: Animals; Animals, Newborn; Cell Death; Disease Models, Animal; Humans; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Microglia; Minocycline; Myelin Basic Protein; Rats; Rats, Long-Evans; Tegmentum Mesencephali

Hypoxic-ischemic brain injury is regulated in part by neurotransmitter and chemokine signaling via G-protein-coupled receptors (GPCRs). GPCR-kinase 2 (GRK2) protects these receptors against overstimulation by inducing desensitization. Neonatal hypoxic-ischemic brain damage is preceded by a reduction in cerebral GRK2 expression. We determined the functional importance of GRK2 in hypoxic-ischemic brain damage. Nine-day-old wild-type and GRK2(+/-) mice with a approximately 50% reduction in GRK2 protein were exposed to unilateral carotid artery occlusion and hypoxia. In GRK2(+/-) animals, gray and white matter damage was aggravated at 3 weeks after hypoxia-ischemia. In addition, cerebral neutrophil infiltration was increased in GRK2(+/-) animals. Neutrophil depletion reduced brain damage, but neuronal loss was still more pronounced in GRK2(+/-) animals. Onset of neuronal loss was advanced in GRK2(+/-) animals regardless of neutrophil depletion. White matter injury was advanced in GRK2(+/-) animals and was not affected by neutrophil depletion. Activation/infiltration of microglia/macrophages was stronger in GRK2(+/-) brains but only occurred 24 h after hypoxia-ischemia and is therefore not the primary cause of increased damage. During hypoxia, cerebral blood flow was reduced to the same extent in both genotypes. In vitro, GRK2(+/-) hippocampal slices and cerebellar granular neurons were more sensitive to glutamate-induced death. We propose the novel concept that the kinase GRK2 regulates onset and magnitude of hypoxic-ischemic brain damage. Increased gray and white matter damage in GRK2(+/-) animals was not dependent on infiltrating neutrophils and occurred before microglia/macrophage activation was detected. Collectively, our data suggest that cerebral GRK2 has an important endogenous neuroprotective role in ischemic cerebral damage.

Topics: Animals; Animals, Newborn; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Brain Injuries; G-Protein-Coupled Receptor Kinase 2; Gene Expression Regulation, Developmental; Glutamic Acid; Hypoxia-Ischemia, Brain; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Neuroglia; Neurons; Neutrophil Infiltration; Peroxidase; Regional Blood Flow; Subcellular Fractions; Time Factors

To study the effect of intracerebral transplantation of bone marrow stromal cells (BMSCs) on brain white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD).. Thirty-four 7-day-old neonatal rats were randomly assigned to three groups: normal control (n=10), HIBD (n=12) and HIBD+BMSCs transplantation (n=12). The HIBD and the HIBD+BMSCs transplantation group rats were subjected to left carotid artery ligation, followed by hypoxia exposure for 2 hrs, in order to induce HIBD. The rats in the HIBD+BMSCs transplantation group received transplantation of BMSCs labeled nucleus with Hochest 33324 into the left hippocampus 24 hrs after HIBD induction. Myelin basic protein (MBP) expression in the left corpus callosum and the subcortical white matter and the number of oligodendrocyte precursors positively stained O4 in the left periventricular area and the subcortical white matter were detected by immunohistochemistry at ages of 45 days.. The labeled BMSCs survived and were found mainly in the left hemisphere 37 days after transplantation. The positive rate of O4 expressed by the transplanted BMSCs was 3.70+/-1.09%. More hypomyelination in the left corpus callosum and the subcortical white matter, and less number of O4 positive oligodendrocytes in the left periventricular area and the subcortical white matter were found in the HIBD group compared with the normal control group (P<0.01). The HIBD rats receiving BMSCs transplantation had increased O4 positive oligodendrocytes in the left periventricular area and the subcortical white matter and improved MBP immunoreactivity in the left corpus callosum and the subcortical white matter compared with the HIBD group (P<0.01).. Intracerebral transplantation of BMSCs can improve brain white matter damage in neonatal rats with HIBD.

Topics: Animals; Animals, Newborn; Antigens, Differentiation; Bone Marrow Cells; Brain; Hypoxia-Ischemia, Brain; Immunohistochemistry; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Stromal Cells

Inhibition of matrix metalloproteinase-9 (MMP-9) protects the adult brain after cerebral ischemia. However, the role of MMP-9 in the immature brain after hypoxia-ischemia (HI) is unknown. We exposed MMP-9(-/-) [MMP-9 knock-out (KO)] and wild-type (WT) mice to HI on postnatal day 9. HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (10% O2; 36 degrees C). Gelatin zymography showed that MMP-9 activity was transiently increased at 24 h after HI in the ipsilateral hemisphere and MMP-9-positive cells were colocalized with activated microglia. Seven days after 50 min of HI, cerebral tissue volume loss was reduced in MMP-9 KO (21.8 +/- 1.7 mm3; n = 22) compared with WT (32.3 +/- 2.1 mm3; n = 22; p < 0.001) pups, and loss of white-matter components was reduced in MMP-9 KO compared with WT pups (neurofilament: WT, 50.9 +/- 5.4%; KO, 18.4 +/- 3.1%; p < 0.0001; myelin basic protein: WT, 57.5 +/- 5.8%; KO, 23.2 +/- 3.5%; p = 0.0001). The neuropathological changes were associated with a delayed and diminished leakage of the blood-brain barrier (BBB) and a decrease in inflammation in MMP-9-deficient animals. In contrast, the neuroprotective effects after HI in MMP-9-deficient animals were not linked to either caspase-dependent (caspase-3 and cytochrome c) or caspase-independent (apoptosis-inducing factor) processes. This study demonstrates that excessive activation of MMP-9 is deleterious to the immature brain, which is associated with the degree of BBB leakage and inflammation. In contrast, apoptosis does not appear to be a major contributing factor.

Topics: Animals; Animals, Newborn; Apoptosis Inducing Factor; Blood-Brain Barrier; Brain; Brain Infarction; Caspase 3; Cell Death; Cytochromes c; Encephalitis; Gene Expression Regulation, Developmental; Hypoxia-Ischemia, Brain; Immunohistochemistry; Indoles; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Neurofilament Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Time Factors

A recent study has suggested that hyperbaric oxygen (HBO) therapy administered within 3 hrs following hypoxic-ischemic brain damage (HIBD) may alleviate brain white matter damage (WMD) in neonatal rats. However it is unclear whether a delayed HBO therapy (more than 3 hrs following HIBD) has neuroprotective effects in neonatal rats. This study aimed to explore the effect of HBO therapy administered at different time points following HIBD on WMD in neonatal rats.. The HIBD model was prepared according to the Rice-Vannucci procedure in 7-day-old Sprague-Dawley rats. HBO therapy was administered at 3, 6, 12, 24 or 72 hrs after HIBD, once daily for consecutive 7 days. T-maze test, the foot-fault test and the radial arm maze test were performed after 14 days of HIBD. Myelin basic protein (MBP) in the callositas and corpora striata was examined by immunohistochemical method 28 days after HIBD.. The rats receiving HBO therapy at 3, 6 and 12 hrs after HIBD performed significantly better in the T-maze test, the radial arm maze test and the foot-fault test than the untreated HIBD rats. There were no significant differences in the behavioral test results between the HBO-treated groups administered HBO at 24 and 72 hrs after HIBD and the untreated HIBD group. The MBP expression in the HBO-treated groups treated within 12 hrs after HIBD was significantly higher than that in the untreated HIBD group (P < 0.05). When the HBO therapeutic window was delayed to 24 hrs after HIBD, there were no significant differences in the MBP expression between the HBO-treated and the untreated HIBD groups.. HBO therapy administered within 12 hrs following HIBD can alleviate brain WMD in neonatal rats, but the efficacy of HBO therapy administered 24 hrs after HIBD does not appear to be satisfactory.

Topics: Animals; Animals, Newborn; Brain; Female; Hyperbaric Oxygenation; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Maze Learning; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Time Factors

We previously demonstrated that IGF-1 blocks glutamate-mediated death of late oligodendrocyte progenitors (OPs) by preventing Bax translocation, mitochondrial cytochrome c release and cleavage of caspases 9 and 3. Here, we demonstrate that IGF-1 prevents caspase 3 activation in late OPs when administered up to 16 h following exposure to glutamate. Moreover, late addition of IGF-1 to OPs previously exposed to toxic levels of glutamate promotes oligodendrocyte maturation as measured by myelin basic protein expression. We also demonstrate that intraventricularly administered IGF-1 retains OPs in the perinatal white matter after hypoxia-ischemia when given after insult. These results suggest that delayed administration of IGF-1 will rescue OPs in the immature white matter and promote myelination following hypoxia-ischemia.

Topics: Animals; Animals, Newborn; Apoptosis; Caspase 3; Cells, Cultured; Cytoprotection; Drug Administration Schedule; Glutamic Acid; Hypoxia-Ischemia, Brain; Insulin-Like Growth Factor I; Myelin Basic Protein; Nerve Degeneration; Nerve Fibers, Myelinated; Nerve Growth Factors; Neurotoxins; Oligodendroglia; Rats; Rats, Sprague-Dawley; Rats, Wistar; Stem Cells; Time Factors; Treatment Outcome

This study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD.. Seven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining.. The BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage.. The HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

Topics: Animals; Animals, Newborn; Bromodeoxyuridine; Female; Hyperbaric Oxygenation; Hypoxia-Ischemia, Brain; Immunohistochemistry; Intermediate Filament Proteins; Male; Mice; Myelin Basic Protein; Nerve Tissue Proteins; Nestin; Neurons; Rats; Rats, Sprague-Dawley; Stem Cells

Hypoxic ischemic (HI) injury in neonates may have devastating, long-term consequences. Recently completed clinical trials in HI neonates indicate that hypothermia within 6 h of birth results in modest improvement in the combined outcome of death or severe disability. The aim of this study was to investigate the effects of combining hypothermia and N-acetylcysteine (NAC) on brain injury, neonatal reflexes and myelination after neonatal HI. Seven-day-old rats were subjected to right common carotid artery ligation and hypoxia (8% oxygen) for 2 h. Systemic hypothermia (30 + 0.5 degrees C) was induced immediately after the period of HI and was maintained for 2 h. NAC (50 mg/kg) was administered by intraperitoneal injection daily until sacrifice. Brain infarct volumes were significantly reduced at 48 h post-HI in the hypothermia plus NAC group (21.5 +/- 3.84 mm3) compared with vehicle (240.85 +/- 4.08 mm3). Neonatal reflexes were also significantly improved by combination therapy at days 1 and 7. There was a significant loss of right hemispheric brain volume in the untreated group at 2 and 4 wk after HI insult. Brain volumes were preserved in hypothermia plus NAC group and were not significantly different when compared with the sham group. Similarly, increased myelin expression was seen in brain sections from hypothermia plus NAC group, when stained for Luxol Fast Blue (LFB), Myelin Basic Protein (MBP) and Proteolipid protein (PLP). These results indicate that hypothermia plus NAC combination therapy improves infarct volume, myelin expression and functional outcomes after focal HI injury.

Topics: Acetylcysteine; Animals; Animals, Newborn; Combined Modality Therapy; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Myelin Proteolipid Protein; Rats; Rats, Sprague-Dawley

The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1-day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real-time RT-PCR, western blotting and immunohistochemistry. Up-regulated mRNA and protein expression of HIF-1alpha, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron-microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia.

Topics: Animals; Animals, Newborn; Blotting, Western; Brain; Colorimetry; Corpus Callosum; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Immunoenzyme Techniques; Immunohistochemistry; Lateral Ventricles; Microscopy, Electron; Myelin Basic Protein; Myelin Sheath; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vascular Endothelial Growth Factor A

Inflicted traumatic brain injury (iTBI) involves a combination of mechanical trauma and hypoxemia. Serum biomarker concentrations may provide objective information about their relative importance to the pathophysiology of iTBI. We compared the time course of neuron-specific enolase (NSE), S100B and myelin basic protein after pediatric hypoxic-ischemic brain injury, iTBI and noninflicted TBI (nTBI). The time to reach peak concentrations of all three biomarkers was shorter after nTBI. Initial and peak S100B, initial and peak myelin basic protein and peak NSE concentrations were no different between the three groups. Initial NSE concentration was highest after nTBI. These results suggest that the biochemical response of the brain to iTBI is distinct from the response to nTBI and shares temporal similarities with hypoxic-ischemic brain injury. This may have important implications for the treatment and prognosis of children with iTBI.

Topics: Age Factors; Aging; Biomarkers; Brain; Brain Injuries; Child; Child, Preschool; Diagnosis, Differential; Female; Humans; Hypoxia-Ischemia, Brain; Infant; Infant, Newborn; Male; Myelin Basic Protein; Nerve Growth Factors; Nerve Tissue Proteins; Phosphopyruvate Hydratase; Predictive Value of Tests; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Time Factors; Up-Regulation

Inflammation is likely to be important in the pathophysiology of white matter damage in the immature brain. In order to investigate the involvement of interleukin (IL)-18, we subjected 9-day-old IL-18-deficient and wild-type (WT) mice to hypoxia-ischemia (HI) (unilateral carotid ligation and exposure to 10% oxygen) and white matter injury was evaluated after 3 days by immunostaining for myelin basic protein (MBP) and neurofilament (NF). The immunoreactivity of MBP was significantly higher by 92, 49 and 21%, respectively, in subcortical white matter, striatum and thalamus in IL-18-deficient mice versus WT mice following HI. Similarly, there was a more pronounced immunoreactivity of NF by 78% in the subcortical white matter in IL-18 KO versus WT mice. IL-18 was expressed by astrocytes and microglia, whereas the IL-18 receptor was mainly found in astrocytes localized in and around the subventricular white matter. Taken together, these results indicate that release of IL-18 may play an important role in the development of white matter injury in the neonatal brain.

Topics: Animals; Animals, Newborn; Astrocytes; Female; Hypoxia-Ischemia, Brain; Immunohistochemistry; Interleukin-18; Male; Mice; Mice, Knockout; Microglia; Myelin Basic Protein

Ischemia/reperfusion injury to the developing brain is a major cause of neurologic abnormalities in preterm infants. To investigate the underlying mechanisms, we modified a previously described rat model of unilateral uterine-artery ligation on the 17th embryonic day (E17). Growth retardation was taken as an index of in utero ischemia, and pups born with a birth weight more than 2 standard deviations below that of controls were compared with the same-litter, normal-growth control pups born from the nonligated horn. Prenatal ischemia probably associated with hypoxia and followed by reperfusion at birth induced white matter damage at a developmental stage corresponding to extreme prematurity in humans. On P0 (day of birth), growth-retarded pups exhibited lesions in the cingular white matter and internal capsule with increased counts of activated microglial cells for 2 weeks compared with controls. Astrogliosis was detected in the injured white matter. On P3, increased apoptotic cell death was seen in O4-positive preoligodendrocytes, which were abnormally scarce on P7. Defective myelination, as assessed by myelin-binding-protein labeling, was detected until adulthood. The diffuse white matter damage in growth-retarded rats replicated the main features of white matter damage in human preterm infants.

Topics: Animals; Animals, Newborn; Brain Diseases; Caspase 3; Caspases; CD11b Antigen; Cell Count; Cell Death; Disease Models, Animal; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Glucose Transporter Type 2; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Male; Myelin Basic Protein; Neovascularization, Pathologic; O Antigens; Plant Lectins; Pregnancy; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric

White matter of the neonatal brain is highly sensitive to hypoxic-ischemic insult. The susceptibility of premature oligodendrocytes (OLs) to free radicals (FRs) produced during hypoxia-ischemia (HI) has been proposed as one of the mechanisms involved. To test this hypothesis, and to further investigate if the FR scavenger alpha-phenyl-N-tert-butyl-nitrone (PBN) attenuates hypoxic-ischemic white matter damage (WMD), postnatal day 4 (P4) SD rats were subjected to bilateral common carotid artery ligation (BCAL), followed by 8% oxygen exposure for 20 min. Pathological changes were evaluated on P6 and P9, 2 and 5 days after the HI insult. HI caused severe WMD including rarefaction, necrosis and cavity formation in the corpus callosum, external and internal capsule areas. OL injury was evidenced by degeneration of O4 positive OLs on P6. Disrupted myelination was verified by decreased immunostaining of myelin basic protein (MBP) on P9. Axonal injury was demonstrated by increased amyloid precursor protein (APP) immunostaining on both P6 and P9. Two lipid peroxidation end products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed a one-fold elevation within 1-24 h following HI. 4-HNE immunostaining was found to specifically localize in the white matter area. Furthermore, pyknotic O4+ OLs were double-labeled with 4-HNE. These findings suggest that FRs are involved in the pathogenesis of neonatal WMD. PBN (100 mg/kg, i.p.) treatment alleviated the pathological changes of WMD following HI. It improved the survival of O4 positive OLs, attenuated hypomyelination and reduced axonal damage. PBN treatment also decreased the brain concentration of MDA/4-HNE and positive 4-HNE staining in the white matter area. These findings indicate that in the current WMD model, PBN protects both OLs and axons, the two main components in the white matter, from neonatal HI insult. FR scavenging appears to be the primary mechanism underlying its neuroprotective effect.

Topics: Age Factors; Aldehydes; Amyloid beta-Peptides; Analysis of Variance; Animals; Animals, Newborn; Antigens, Differentiation; Brain; Cell Count; Cell Survival; Cyclic N-Oxides; Free Radical Scavengers; Hypoxia-Ischemia, Brain; Immunohistochemistry; Infarction, Middle Cerebral Artery; Malondialdehyde; Myelin Basic Protein; Nitrogen Oxides; Oligodendroglia; Rats; Staining and Labeling; Time Factors

Neonatal hypoxic-ischemic (HI) white matter injury is a major contributor to chronic neurological dysfunction. Immature oligodendrocytes (OLGs) are highly vulnerable to HI injury. As little is known about in vivo OLG repair mechanisms in neonates, we studied whether new OLGs are generated after HI injury in P7 rats. Rats received daily BrdU injections at P12-14 or P21-22 and sacrificed at P14 to study the level of cell proliferation or at P35 to permit dividing OLG precursors to differentiate. In P14 HI-injured animals, the number of BrdU+ cells in the injured hemisphere is consistently greater than controls. At P35, sections were double-labeled for BrdU and markers for OLGs, astrocytes, and microglia. Double-labeled BrdU+/myelin basic protein+ and BrdU+/carbonic anhydrase+ OLGs are abundant in the injured striatum, corpus callosum, and the infarct core. Quantitative studies show four times as many OLGs are generated from P21-35 in HI corpora callosa than controls. Surprisingly, the infarct core contains many newly generated OLGs in addition to hypertrophied astrocytes and activated microglia. These glia and non-CNS cells may stimulate OLG progenitor proliferation or induce their migration. At P35, astrogliosis and microgliosis are dramatic ipsilaterally but only a few microglia and some astrocytes are BrdU+. This finding indicates microglial and astrocytic hyperplasia occurs shortly after HI but before the P21 BrdU injections. Although the neonatal brain undergoes massive cell death and atrophy the first week after injury, it retains the potential to generate new OLGs up to 4 weeks after injury within and surrounding the infarct.

Topics: Animals; Animals, Newborn; Astrocytes; Atrophy; Biomarkers; Bromodeoxyuridine; Carbonic Acid; Cell Count; Cell Death; Cell Differentiation; Cell Division; Cerebral Infarction; Disease Models, Animal; Gliosis; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Nerve Fibers, Myelinated; Oligodendroglia; Rats; Rats, Sprague-Dawley; Stem Cells

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brain's angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.

Topics: Analysis of Variance; Animals; Brain; Carotid Artery Diseases; Carotid Artery, Common; CD11b Antigen; Cell Count; Constriction; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Microscopy, Electron; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Rats; Rats, Wistar

The purpose of this study was to determine the immunoreactivity of selected structural proteins in the preterm and near-term ovine fetal brain and the response to intermittent umbilical cord occlusion as a measure of altered cellular growth. The intermediate filament proteins nestin, vimentin, and glial fibrillary acidic protein was used as markers for astroglial maturation and astrogliosis, and myelin basic protein as a marker for oligodendrocytes and myelin formation.. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days; umbilical cord occlusion was performed in the experimental group by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then killed, and the fetal brain was perfusion fixed and processed for immunohistologic examination of the gray and white matter. Immunoreactivity was quantified with an image analysis system and expressed as the fractional area positive stain for each protein.. In both preterm and near-term animal groups, umbilical cord occlusion caused a large decline in arterial Po(2) (to approximately 7 mm Hg), a modest decline in pH (to approximately 7.30), and a modest rise in Pco(2) (to approximately 61 mm Hg; all P <.01), with a return to control values after the occluder release and no cumulative acidosis over each day of study. Vimentin and glial fibrillary acidic protein immunoreactivity showed reciprocal changes, with vimentin decreased and glial fibrillary acidic protein increased in both the gray and white matter of the control group from 0.75 to 0.90 of gestation, which can be attributed to the transition of radial glia into mature astrocytes. Myelin basic protein immunoreactivity increased approximately 3-fold in the white matter of the control group with advancing gestation, which likely reflected active oligodendrocyte differentiation and increased myelination at this time of development. Intermittent umbilical cord occlusion over 4 days resulted in an approximately 60% decrease in nestin, vimentin, and glial fibrillary acidic protein immunoreactivity, which was qualitatively similar for both the gray and white matter and likely indicative of altered protein synthesis and/or degradation, but only in the preterm group and with no change in myelin basic protein immunoreactivity.. There is considerable change in the immunoreactivity of structural proteins within the ovine fetal brain over the latter part of gestation and consistent with a high rate of protein turnover, as previously reported. Intermittent umbilical cord occlusion as studied with minimal evidence for necrotic cell injury appears capable of altering selected protein synthesis/degradation, more so in younger animals when protein turnover is higher, which might then impact on the brain's development.

Topics: Animals; Brain; Constriction; Female; Fetus; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Immunohistochemistry; Intermediate Filament Proteins; Labor, Obstetric; Myelin Basic Protein; Nerve Tissue Proteins; Nestin; Pregnancy; Sheep; Umbilical Cord; Vimentin

Postresuscitation cerebral hypothermia is consistently neuroprotective in experimental preparations; however, its effects on white matter injury are poorly understood. Using a model of reversible cerebral ischemia in unanesthetized near-term fetal sheep, we examined the effects of cerebral hypothermia (fetal extradural temperature reduced from 39.4 +/- 0.1 degrees C to between 30 and 33 degrees C), induced at different times after reperfusion and continued for 72 hours after ischemia, on injury in the parasagittal white matter 5 days after ischemia. Cooling started within 90 minutes of reperfusion was associated with a significant increase in bioactive oligodendrocytes in the intragyral white matter compared with sham cooling (41 +/- 20 vs 18 +/- 11 per field, P < 0.05), increased myelin basic protein density and reduced expression of activated caspase-3 (14 +/- 12 vs 91 +/- 51, P < 0.05). Reactive microglia were profoundly suppressed compared with sham cooling (4 +/- 6 vs 38 +/- 18 per field, P < 0.05) with no effect on numbers of astrocytes. When cooling was delayed until 5.5 hours after reperfusion there was no significant effect on loss of oligodendrocytes (24 +/- 12 per field). In conclusion, hypothermia can effectively protect white matter after ischemia, but only if initiated early after the insult. Protection was closely associated with reduced expression of both activated caspase-3 and of reactive microglia.

Topics: Animals; Caspase 3; Caspases; Cerebral Cortex; Demyelinating Diseases; Enzyme Activation; Female; Fetus; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Hybridization; Microglia; Myelin Basic Protein; Myelin Proteolipid Protein; Oligodendroglia; Pregnancy; Proliferating Cell Nuclear Antigen; Reperfusion Injury; RNA, Messenger; Sheep; Time Factors

Distinctive cerebral lesions with disruptions to the developing white matter are found in very low birth weight (VLBW) infants. Although hypoxia-ischemia (HI) is a causal pathway, the pathogenesis of cerebral white matter injury in the VLBW infant is not fully understood. Pertinent murine models would facilitate the investigation of the processes leading to these cerebral lesions and enable the evaluation of therapeutic strategies. Postnatal d 3 (P3) rats are at a stage of cortical oligodendroglial maturation and axonal outgrowth similar to very preterm infants. Our aim was to characterize the effects of a focal hypoxic-ischemic injury at P3 on subsequent cerebral development. Three groups of P3 Wistar rats were investigated: group I underwent right carotid ligation followed by 6% hypoxia for 30 min (HI), group 2 had carotid ligation only, and group 3 had no intervention. At P21, in the HI group, the right cortical area was reduced compared with controls (p < 0.01). There were no significant alterations in the size of the dorsal hippocampus, striatum, and thalamus. The cortical myelinated area was reduced in the HI animals compared with controls (p < 0.01). There was a corresponding loss of myelinated axons extending up into the cortex, with deep cortical neuronal and axonal architecture markedly disrupted. Glial fibrillary acidic protein immunohistology showed a reactive gliosis in the deep parietal cortex (p < 0.01). Moderate HI injury in the immature rat brain compromised cortical growth and led to a selective alteration of cortical myelinated axons with persistent gliosis. These alterations induced at P3 by unilateral HI share neuropathological similarities with the diffuse white matter lesions found in VLBW infants.

Topics: Age Factors; Animals; Cerebral Cortex; Coloring Agents; Glial Fibrillary Acidic Protein; Gliosis; Hypoxia-Ischemia, Brain; Myelin Basic Protein; Myelin Sheath; Rats; Rats, Wistar; Rosaniline Dyes; Staining and Labeling