myelin-basic-protein and Hypoxia

The objective of this study was to investigate changes in serum biomarkers of acute brain injury, including white matter and astrocyte injury during chronic foetal hypoxaemia. We have previously shown histopathological changes in myelination and neuronal density in fetuses with chronic foetal hypoxaemia at a level consistent with CHD.. Mid-gestation foetal sheep (110 ± 3 days gestation) were cannulated and attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile fluid environment mimicking the intrauterine environment. Fetuses were maintained with an oxygen delivery of 20-25 ml/kg/min (normoxemia) or 14-16 ml/kg/min (hypoxaemia). Myelin Basic Protein and Glial Fibrillary Acidic Protein serum levels in the two groups were assessed by ELISA at baseline and at 7, 14, and 21 days of support.. Based on overlapping 95% confidence intervals, there were no statistically significant differences in either Myelin Basic Protein or Glial Fibrillary Acidic Protein serum levels between the normoxemic and hypoxemic groups, at any time point. No statistically significant correlations were observed between oxygen delivery and levels of Myelin Basic Protein and Glial Fibrillary Acidic Protein.. Chronic foetal hypoxaemia during mid-gestation is not associated with elevated serum levels of acute white matter (Myelin Basic Protein) or astrocyte injury (Glial Fibrillary Acidic Protein), in this model. In conjunction with our previously reported findings, our data support the hypothesis that the brain dysmaturity with impaired myelination found in fetuses with chronic hypoxaemia is caused by disruption of normal developmental pathways rather than by direct cellular injury.

Topics: Animals; Biomarkers; Brain Injuries; Female; Fetus; Glial Fibrillary Acidic Protein; Humans; Hypoxia; Myelin Basic Protein; Oxygen; Pregnancy; Sheep

This study aims to evaluate the protective effects of progesterone on white matter injury and brain immaturity in neonatal rats with chronic hypoxia.. Three-day old Sprague-Dawley rats were randomly divided into 3 groups: (1) control (n = 48), rats were exposed to normoxia (fraction of inspired oxygen: 21% ± 0%); (2) chronic hypoxia (n = 48), rats were exposed to hypoxia (fraction of inspired oxygen: 10.5% ± 1.0%); and (3) progesterone (n = 48), rats were exposed to hypoxia and administrated with progesterone (8 mg/kg/d). Hematoxylin-eosin staining, immunohistochemistry, real-time quantitative polymerase chain reaction, and Western blot analyses were compared on postnatal day 14 in different groups. Motor skill and coordination abilities of rats were assessed via rotation experiments.. Increased brain weights (P < .05), narrowed ventricular sizes (P < .01), and rotarod experiment scores (P < .01) were better in the progesterone group than in the chronic hypoxia group. The number of mature oligodendrocytes and myelin basic protein expression increased in the progesterone group compared with the chronic hypoxia group (P < .01). The polarization of M1 microglia cells in the corpus callosum of chronic hypoxia-induced hypomyelination rats was significantly increased, whereas there were fewer M2 microglia cells. Conversely, progesterone therapy had an opposite effect and caused an increase in M2 microglia polarization versus a reduction in M1 microglia cells.. Progesterone could prevent white matter injury and improve brain maturation in a neonatal hypoxic rat model; this may be associated with inducing a switch from M1 to M2 in microglia.

Topics: Animals; Animals, Newborn; Behavior, Animal; Brain; Cell Plasticity; Chronic Disease; Disease Models, Animal; Female; Hypoxia; Leukoencephalopathies; Male; Microglia; Motor Activity; Myelin Basic Protein; Neuroprotective Agents; Oligodendroglia; Progesterone; Rats, Sprague-Dawley; White Matter

Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub-ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory-related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid-cervical transplantation of SVZ-derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non-GFP rats. Whole body plethysmography conducted at 4 and 8wks post-transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P=0.003) or hypercapnic respiratory challenge (P=0.019). Phrenic nerve output was assessed at 8wks post-transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P<0.001). Stereological analyses at 8wks post-injury indicated survival of ~50% of transplanted NPCs with ~90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P<0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ-derived NPCs can improve respiratory recovery following high cervical SCI.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Animals, Newborn; CD11b Antigen; Cervical Vertebrae; Disease Models, Animal; Female; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Hypoxia; Lateral Ventricles; Male; Myelin Basic Protein; Neural Stem Cells; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Recovery of Function; Respiration Disorders; Spinal Cord Injuries

pCH is an important risk factor for brain injury and long-term morbidity in children, occurring during the developmental stages of neurogenesis, neuronal migration, and myelination. We show that a rodent model of pCH results in an early decrease in mature myelin. Although pCH does increase progenitor oligodendrocytes in the developing brain, BrdU labeling revealed a loss in dividing progenitor oligodendrocytes, indicating a defect in mature cell replacement and myelinogenesis. Mice continued to exhibited hypomyelination, concomitant with long-term impairment of motor function, weeks after cessation of pCH. The implication of a novel neuroimmunologic interplay, pCH also induced a significant egress of infiltrating CD4 T cells into the developing brain. This pCH-mediated neuroinflammation included oligodendrocyte-directed autoimmunity, with an increase in peripheral myelin-specific CD4 T cells. Thus, both the loss of available, mature, myelin-producing glial cells and an active increase in autoreactive, myelin-specific CD4 T cell infiltration into pCH brains may contribute to early pCH-induced hypomyelination in the developing CNS. The elucidation of potential mechanisms of hypoxia-driven autoimmunity will expand our understanding of the neuroimmune axis during perinatal CNS disease states that may contribute to long-term functional disability.

Topics: Animals; Autoimmunity; Behavior, Animal; CD4-Positive T-Lymphocytes; Cerebral Cortex; Disease Models, Animal; Female; Hypoxia; Inflammation; Mice; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neuroglia; Pregnancy; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets

Very low birth weight (VLBW) premature infants experience numerous, often self-limited non-bradycardic episodes of intermittent hypoxemia (IH). We hypothesized that these episodes of IH affect postnatal white matter (WM) development causing hypomyelination and neurological handicap in the absence of cellular degeneration. Based on clinical data from ten VLBW neonates; a severity, daily duration and frequency of non-bradycardic IH episodes were reproduced in neonatal mice. Changes in heart rate and cerebral blood flow during IH were recorded. A short-term and long-term neurofunctional performance, cerebral content of myelin basic protein (MBP), 2'3' cyclic-nucleotide 3-phosphodiesterase (CNPase), electron microscopy of axonal myelination and the extent of cellular degeneration were examined. Neonatal mice exposed to IH exhibited no signs of cellular degeneration, yet demonstrated significantly poorer olfactory discrimination, wire holding, beam and bridge crossing, and walking-initiation tests performance compared to controls. In adulthood, IH-mice demonstrated no alteration in navigational memory. However, sensorimotor performance on rota-rod, wire-holding and beam tests was significantly worse compared to naive littermates. Both short- and long-term neurofunctional deficits were coupled with decreased MBP, CNPase content and poorer axonal myelination compared to controls. In neonatal mice mild, non-ischemic IH stress, mimicking that in VLBW preterm infants, replicates a key phenotype of non-cystic WM injury: permanent hypomyelination and sensorimotor deficits. Because this phenotype has developed in the absence of cellular degeneration, our data suggest that cellular mechanisms of WM injury induced by mild IH differ from that of cystic periventricular leukomalacia where the loss of myelin-producing cells and axons is the major mechanism of injury.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Animals, Newborn; Caspase 3; Cerebrovascular Circulation; Disease Models, Animal; Gene Expression Regulation, Developmental; Heart Rate; Hypoxia; Leukoencephalopathies; Mice; Mice, Inbred C57BL; Muscle Strength; Myelin Basic Protein; Nerve Fibers, Myelinated; Nervous System Diseases; Oxygen; Psychomotor Performance; Statistics, Nonparametric; Time Factors

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

The NG2-positive cells are the oligodendrocyte precursors, which, when terminally differentiated, are capable of myelinating the central nervous system. There is however an ever-growing list of evidences that NG2 cells actually possess an intrinsic neurogenic potential and they are capable of neuronal differentiation in response to environmental stimuli. To address the question, we have established a model of an indirect co-culture system of the freshly isolated rat neonatal NG2 cells and organotypic slices derived from two distinct CNS regions (hippocampus and spinal cord) to mimic the nervous tissue microenviroment. The cell differentiation in microenvironment of OGD-injured hippocampal slices has been studied as well. The molecular analysis of selected trophic factors has been performed to determine the patterns of their expression. Indeed, the comparison of the cell commitment and development in various microenvironments has pointed to significant dissimilarities. First of all, the medium being continuously conditioned by the hippocampal slices efficiently promoted neurogenesis. The effect has been significantly abolished in co-cultures with the injured tissue. The less pronounced susceptibility to adopting neuronal phenotype and the considerable slowdown of oligodendroglial development was observed in the co-cultures with the spinal cord slices. The role of BDNF in oligodendroglial progenitor commitment and development has been investigated proving that it is one of the key players in the examined processes. The specificity of the instructive clues cocktail might module the fate choice of mobilized endogenous or transplanted cells, which should be taken into consideration while planning neurorepair strategies.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antibodies; Brain-Derived Neurotrophic Factor; Cell Count; Cell Differentiation; Coculture Techniques; Gene Expression Regulation; Glucose; Hippocampus; Hypoxia; Myelin Basic Protein; Nerve Tissue Proteins; O Antigens; Oligodendroglia; Organ Culture Techniques; Rats; Rats, Wistar; Spinal Cord

Ischemic brain injury is widely modeled in vitro with paradigms of oxygen-glucose deprivation (OGD), which leads to cell death. The prevention and attenuation of brain injury by the tetracycline antibiotic minocycline has been attributed largely to suppression of microglial activation, but its benefits in oligodendrocyte cells have not been well characterized. Using primary cultures of rat oligodendroglial precursor cells (OPC) exposed to OGD, we investigated the direct effects of minocycline on the survival, proliferation, and maturation of oligodendroglial lineage cells. OGD for 2 hr caused a decrease in the total number of OPC and the amount of proliferating progenitors by 50%, which was attenuated by inclusion of minocycline. The reduced numbers of immature oligodendroglial cells at 72 hr and of mature oligodendrocytes at 120 hr after OGD were partially restored by minocycline. In OPC, OGD caused an increase of reactive oxygen species (ROS) and production of TUNEL-positive cell numbers, which was abolished by minocycline. Minocycline preferentially increased the expression of superoxide dismutase under OGD but not in control OPC. Minocycline also prevented the OGD-induced downregulation of the transcription factors Sox10 and Olig2 and of myelin-specific genes 2'3' cyclic nucleotide phosphodiesterase (CNP) and myelin basic protein (MBP) in response to OGD. These studies demonstrate direct protective actions of minocycline on oligodendroglial-lineage cells, suggesting potential benefit in white matter injury involving OGD.

Topics: Analysis of Variance; Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; Embryo, Mammalian; Embryonic Stem Cells; Female; Gangliosides; Gene Expression Regulation; Glucose; Hypoxia; Ki-67 Antigen; Minocycline; Myelin Basic Protein; O Antigens; Oligodendroglia; Oxidative Stress; Pregnancy; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles; Time Factors; Transcription 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

Inflammation in the periventricular white matter (PWM) of hypoxic neonatal brain causes myelination disturbances. In this connection, macrophage colony-stimulating factor (M-CSF) has been reported to regulate release of proinflammatory cytokines that may be linked to PWM damage. We sought to determine if M-CSF derived from amoeboid microglial cells (AMC) would promote proinflammatory cytokine production by astrocytes in the PWM following hypoxic exposure, and, if so, whether it is associated with axon degeneration and myelination disturbances. In 1-day hypoxic rats, expression of M-CSF was upregulated in AMC. This was coupled with increased expression of CSF-1 receptor, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in astrocytes, and TNF-receptor 1 and IL-receptor 1 on the axons. Neurofilament-200 immunopositive axons and myelin basic protein immunopositive processes appeared to undergo disruption in 14-days hypoxic rats. By electron microscopy, some axons showed degenerative changes affecting the microtubules and myelin sheath. Primary cultured microglial cells subjected to hypoxia showed enhanced release of M-CSF. Remarkably, primary cultured astrocytes treated with conditioned-medium derived from hypoxic microglia or M-CSF exhibited increased production of TNF-alpha and IL-1beta. Our results suggest that AMC-derived M-CSF promotes astrocytes to generate proinflammatory cytokines, which may be involved in axonal damage following a hypoxic insult.

Topics: Animals; Animals, Newborn; Brain; Cells, Cultured; Cerebral Ventricles; Chemotactic Factors; Cytokines; Enzyme-Linked Immunosorbent Assay; Glial Fibrillary Acidic Protein; Hypoxia; Microglia; Myelin Basic Protein; Nerve Fibers, Myelinated; Neurofilament Proteins; Rats; Rats, Wistar; RNA, Messenger; Time Factors

The aim of the present study is to evaluate the effect of reduced fetal oxygen supply on cerebral white matter in the adult offspring and further assess its susceptibility to postnatal hypoxia and high-fat diet. Based on a 3 x 2 full factorial design consisting of three factors of maternal hypoxia, postnatal high-fat diet, and postnatal hypoxia, the ultrastructure of myelin, axon and capillaries were observed, and the expression of myelin basic protein (MBP), neurofilament-H+L(NF-H+L), and glial fibrillary acidic protein (GFAP) was analyzed in periventricular white matter of 16-month-old offspring. Demyelination, injured axon and damaged microvasculars were observed in maternal hypoxia offspring. The main effect of maternal hypoxia lead to decreased expression of MBP or NF-H+L, and increased expression of GFAP (all P<0.05). Moreover, there was positive three-way interaction among maternal hypoxia, high-fat diet and postnatal hypoxia on MBP, NF-H+L or GFAP expression (all P<0.05). In summary, our results indicated that maternal hypoxia during pregnancy in rats lead to changes of periventricular white matter in adult offspring, including demyelination, damaged axon and proliferated astroglia. This effect was amplified by high-fat diet and postnatal hypoxia.

Topics: Animals; Astrocytes; Axons; Cerebral Ventricles; Dietary Fats; Female; Glial Fibrillary Acidic Protein; Hypoxia; Maternal Exposure; Maternal-Fetal Exchange; Myelin Basic Protein; Neurofilament Proteins; Pregnancy; Rats; Rats, Sprague-Dawley

Oligodendroglia play an important role in axonal conduction in the CNS and are sensitive to oxidative toxicity induced by glutamate in the absence of ionotropic glutamate receptors. In this study, oligodendrocyte signalling cascades were examined, in response to glutamate-induced oxidative injury and to excitotoxicity. Rat cortical oligodendrocytes, differentiated in culture, were highly vulnerable to glutamate-induced cell death. Competitive inhibition of cystine uptake and increased oxidative stress appeared responsible for this death, and caused an accumulation of intracellular peroxides as well as chromatin fragmentation and condensation. Glutamate receptor subtype agonists (quisqualate, ibotenate) known to inhibit cystine uptake were cytotoxic, but not NMDA itself; moreover, glutamate receptor antagonists were not protective. Oligodendrocytes were also vulnerable to overactivation of glutamate receptors, as kainic acid and AMPA proved to be toxic. AMPA toxicity required the presence of cyclothiazide, suggesting rapid desensitization of AMPA receptors. Glutamate-induced oxidative stress and kainate/AMPA receptor stimulation activated the mitogen-activated protein kinase (MAP kinase) pathway, as well as the transcription factor ELK. However, MAP kinase kinase inhibitors only protected against injury from glutamate-induced oxidative stress. Oligodendrocytes were sensitive to oxygen-glucose deprivation injury as well, in a MAP kinase dependent fashion. Glutamate toxicity may conceivably be operative in neuropathological conditions that disrupt neuronal/oligodendrocyte interactions in axons, e.g. multiple sclerosis and ischaemia-reperfusion injury.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Animals, Newborn; Annexin A5; Blotting, Western; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Chromatin Assembly and Disassembly; Cystine; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Fluoresceins; G(M1) Ganglioside; Glial Fibrillary Acidic Protein; Glucose; Glutamic Acid; Hypoxia; Immunohistochemistry; Indoles; JNK Mitogen-Activated Protein Kinases; Kainic Acid; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Myelin Proteins; Myelin-Associated Glycoprotein; Nogo Proteins; Oligodendroglia; Oligopeptides; Peroxides; Phosphorylation; Quinoxalines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Cell Surface; Signal Transduction; Time Factors

Our aim was to test the hypothesis that the fetal brainstem is relatively spared, compared to other brain regions, from hypoxia-induced damage. We have used established experimental models of acute and chronic intrauterine compromise in sheep to mimic conditions that can arise in human pregnancy. The acute insult was 12 h of placental insufficiency induced by restricted utero-placental blood flow at 90 days of gestation (term approximately 147 days). Five weeks after this insult (n=7 fetuses) there was no overt damage to the brainstem nor were there alterations to the blood vessel morphology, volume of the medulla or of medullary nuclei compared to controls (n=8). This regimen is known to have significant effects on the forebrain and cerebellum. The chronic insult was induced in five fetuses via embolisation of the umbilico-placental circulation from 120 to 140 days of gestation. An additional three fetuses were found to be spontaneously hypoxemic (SH) immediately after surgery. At 140 days, in brainstems of all chronically hypoxemic fetuses compared to controls (n=8), there was an increase (P<0.05) in the percentage of neuropil occupied by blood vessels and abnormal myelin in the most severely SH fetus but no other morphological or neurochemical alterations. This regimen is known to cause marked damage to the cerebral hemispheres and to a lesser extent to the cerebellum. We suggest that the absence of marked structural or neurochemical alterations in the brainstem is most likely due to the maintenance of oxygen delivery to the brainstem during fetal hypoxemia.

Topics: Animals; Brain Stem; Cerebellum; Cerebral Cortex; Female; Fetus; Glial Fibrillary Acidic Protein; Hypoxia; Immunohistochemistry; Male; Myelin Basic Protein; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pregnancy; Pyramidal Tracts; Sheep; Synaptophysin

We tested the hypotheses that an episode of hypoxemia near mid-gestation in fetal sheep has long-term effects on brain development and that the extent and type of damage is related to the stage of development within a particular brain structure at the time of the hypoxemia. Fetal sheep (n = 8) were made hypoxemic at 90 +/- 2 days (term approximately 147 days) by restricting the maternal blood supply to the placenta for 12 hours (h) using a vascular clamp so as to reduce fetal arterial O2 saturation by 50%-60%. Fetuses were killed 35 days later and the brains analysed histologically and immunohistochemically. Age-matched fetuses (n = 8) were used as controls. Gross brain damage was observed in only 1 fetus, the most acidemic during the period of hypoxemia. There was a reduction of 12% (p < 0.05) in the cross-sectional area of the cerebral cortex in hypoxemic fetuses compared with controls. In lobule 6 of the cerebella of hypoxemic fetuses, significant reductions were seen in (a) the volume density of Purkinje cells (33%), (b) the width of the molecular layer (13%), (c) the area of the inner granule cell layer (13%), (d) the area of the white matter (18%), and (e) the total cross-sectional area (15%). There were also significant reductions in the area of arborization of Purkinje cell dendritic trees (50%), in the branching density (25%), and in the number of dendritic spines (31%). In the ventral hippocampi of hypoxemic fetuses, there was a 36% reduction (p < 0.05) in the volume density of CA1 pyramidal cells and a 50% increase (p < 0.05%) in the number of astrocytes. We conclude that an episode of hypoxemia near mid-gestation reduces neuronal numbers in the hippocampus and cerebellum and probably also in the cerebral cortex. The growth of neural processes in a particular region will be significantly retarded if the hypoxemia occurs at an early stage of the growth of neural processes (e.g. cerebellum) but not if development is well advanced at the time of the insult (e.g. hippocampus). Damage is sustained in the white matter of the cerebral hemispheres if the insult is particularly severe. Together, these deficits could affect neural connectivity and impair postnatal brain function.

Topics: Animals; Blood Pressure; Body Weight; Brain; Embryonic and Fetal Development; Female; Fetus; Gases; Gestational Age; Glial Fibrillary Acidic Protein; Hypoxia; Immunohistochemistry; Microtubule-Associated Proteins; Myelin Basic Protein; Organ Size; Pregnancy; Pregnancy Complications; Pregnancy, Animal; Sheep; Time Factors

We have previously established that 21-day-old postnatal rat oligodendrocytes, maintained in monolayer culture and subjected to 6 h of hypoxia, show reversible inhibition of synthesis of alpha-hydroxy fatty acid and myelin basic protein but a dramatic induction of a 22-kDa protein, suggesting that this is a good model to study the mechanism of CNS demyelination caused by hypoxic injury. We now report that hypoxia also dramatically inhibits the basal protein kinase C-mediated phosphorylation of myelin basic protein and myelin 2',3'-cyclic nucleotide phosphohydrolase by 80%, but that the inhibition of phosphorylation can be reversed by addition of a protein kinase C activator, phorbol 12-myristate 13-acetate. The mechanism of action appears to involve the uncoupling of signal transduction at a site before phospholipase C, because hypoxia did not affect protein kinase C activity or its translocation to the membrane fraction. The most potent activator of phospholipase C (as measured by inositol phosphate release) was carbachol (muscarinic M1 receptor agonist), followed by L-phenylephrine (alpha 1-adrenergic receptor agonist) in normal oligodendrocytes. Excitatory amino acids and histamine were ineffective. Hypoxia for 6 h completely inhibited both muscarinic and alpha 1-adrenergic receptor-mediated inositol monophosphate release but did not affect phospholipase D-coupled phosphatidylethanol production in response to carbachol. We therefore conclude from this and earlier work that early, reversible changes in oligodendrocyte metabolism result not simply from ATP depletion, but may specifically target GTP binding protein-mediated processes.

Topics: Animals; Hypoxia; Inositol Phosphates; Myelin Basic Protein; Oligodendroglia; Oligomycins; Phospholipase D; Phosphorylation; Protein Kinase C; Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Guanosine Triphosphate; Heat-Shock Proteins; Hypoxia; Molecular Weight; Myelin Basic Protein; Nerve Tissue Proteins; Oligodendroglia; Protein Binding; Rats; Rats, Sprague-Dawley; Time Factors