Developmental regulation of group I metabotropic glutamate receptors in the premature brain and their protective role in a rodent model of periventricular leukomalacia

Cerebral white matter injury in premature infants, known as periventricular leukomalacia (PVL), is common after hypoxia-ischemia (HI). While ionotropic glutamate receptors (iGluRs) can mediate immature white matter injury, we have previously shown that excitotoxic injury to premyelinating oligodendrocytes (preOLs) in vitro can be attenuated by group I metabotropic glutamate receptor (mGluR) agonists. Thus, we evaluated mGluR expression in developing white matter in rat and human brain, and tested the protective efficacy of a central nervous system (CNS)-penetrating mGluR agonist on injury to developing oligodendrocytes (OLs) in vivo. Group I mGluRs (mGluR1 and mGluR5) were strongly expressed on OLs in neonatal rodent cerebral white matter throughout normal development, with highest expression early in development on preOLs. Specifically at P6, mGluR1 and mGLuR5 were most highly expressed on GalC-positive OLs compared to neurons, axons, astrocytes and microglia. Systemic administration of (1S,3R) 1-aminocyclopentane-trans-1,3,-dicarboxylic acid (ACPD) significantly attenuated the loss of myelin basic protein in the white matter following HI in P6 rats. Assessment of postmortem human tissue showed both mGluR1 and mGluR5 localized on immature OLs in white matter throughout development, with mGluR5 highest in the preterm period. These data indicate group I mGluRs are highly expressed on OLs during the peak period of vulnerability to HI and modulation of mGluRs is protective in a rodent model of PVL. Group I mGluRs may represent important therapeutic targets for protection from HI-mediated white matter injury.     

Transient presence and functional interaction of endogenous GABA and GABAA receptors in developing rat optic nerve.

GABA (gamma-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABAA receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABAA receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABAA receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.     

Functional metabotropic glutamate receptors are expressed in oligodendrocyte progenitor cells

We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent oligodendroglial progenitor cells (OPC) and rat brain oligodendrocytes. Our RT-PCR analysis detected mRNAs for mGluR3 and mGluR5 isoforms in OPCs. Although neurons express both mGluR5a and mGluR5b splice variants, only mGluR5a was identified in OPCs. Antibodies to mGluR2/3 and mGluR5 detected the corresponding receptor proteins in immunoblots of OPC membrane fractions. Furthermore, immunocytochemical analysis identified mGluR5 in oligodendrocyte marker O4-positive OPCs. The expression of mGluR5 was also demonstrated in oligodendrocyte marker (O4 and O1) positive cells in white matter of postnatal 4- and 7-day-old rat brain sections using immunofluorescent double labelling and confocal microscopy. The mGluR5 receptor function was assessed in CG-4 OPCs with fura-2 microfluorometry. Application of the mGluR1/5 specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced calcium oscillations, which were inhibited by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). The DHPG induced calcium oscillations required Ca2+ release from intracellular stores. In OPCs the group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) decreased forskolin-stimulated cAMP synthesis, indicating the presence of functional mGluR3. The newly identified mGluR3 and mGluR5a may be involved in the differentiation of oligodendrocytes, myelination and the development of white matter damage.     

Postnatal Changes in Cathepsin D in Rat Neural Tissue

Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl-¹⁴C]-methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.     

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.     

Improvement of Hypoxia–Ischemia-Induced White Matter Injury in Immature Rat Brain by Ethyl Pyruvate

Ethyl pyruvate (EP) has been reported to be neuroprotective in several models of brain injury, yet its influence on periventricular leukomalacia still remains elusive. Here we investigated whether repeated administration of EP could protect against white matter injury after hypoxia–ischemia (HI) (right common carotid artery ligation and 6 % O₂ for 60 min) in post-natal 3 day rat pups. EP was injected (50 mg/kg, intraperitoneally) 10 min, 1 and 24 h after HI insult. Treatment with EP significantly reduced HI-induced ventricular enlargement, loss of developing oligodendrocytes, and hypomyelination. We further demonstrated a marked inhibitory effect of EP on inflammatory responses, as indicated by the decreased number of activated microglia and astrocytes and the reduced release of proinflammatory cytokines. Moreover, EP down-regulated the expression of cleaved caspase-3 and Bax, and up-regulated Bcl-2 expression after HI exposure. In conclusion, our results demonstrated that EP was able to provide potent protection on white matter injury through blocking the cerebral inflammatory responses and modulating the apoptotic death program of oligodendrocytes, indicating a potential neuroprotective agent in neonatal brain injury.     

Activation of group I metabotropic glutamate receptors reduces neuronal apoptosis but increases necrotic cell death in vitro

Glutamate released during acute CNS insults acts at metabotropic glutamate receptors (mGluR), including group I mGluR. Blockade of group I mGluR during in vitro neuronal trauma provides neuroprotection, whereas activation exacerbates such injury. However, the effects of group I mGluR agonists or antagonists have been primarily studied in in vitro models characterized by necrotic cell death. We examined the role of group I mGluR in the modulation of neuronal injury induced during oxygen-glucose deprivation (OGD), a well-studied model of necrosis, and by application of two well established pro-apoptotic agents: staurosporine and etoposide. Inhibition of group I mGluR attenuated necrosis induced by OGD, whereas selective activation of group I mGluR exacerbated such injury. In contrast, activation of group I mGluR, including selective activation of mGluR5, significantly attenuated apoptotic cell death induced by both staurosporine and etoposide. This effect was completely reversed by co-application of a group I mGluR antagonist. Thus, group I mGluR appear to exhibit opposite effects on necrotic and apoptotic neuronal cell death. Our findings suggest that activation of mGluR1 exacerbates neuronal necrosis whereas both mGluR1 and mGluR5 play a role in attenuation of neuronal apoptosis.     

Glial progenitors of the neonatal subventricular zone differentiate asynchronously, leading to spatial dispersion of glial clones and to the persistence of immature glia in the adult mammalian CNS

The subventricular zone (SVZ) of the developing mammalian forebrain gives rise to astrocytes and oligodendrocytes in the neocortex and white matter, and neurons in the olfactory bulb in perinatal life. We have examined the developmental fates and spatial distributions of the descendants of single SVZ cells by infecting them in vivo at postnatal day 0-1 (P0-1) with a retroviral "library". In most cases, individual SVZ cells gave rise to either oligodendrocytes or astrocytes, but some generated both types of glia. Members of glial clones can disperse widely through the gray and white matter. Progenitors continued to divide after stopping migration, generating clusters of related cells. However, the progeny of a single SVZ cell does not differentiate synchronously: individual clones contained both mature and less mature glia after short or long intervals. For example, progenitors that settled in the white matter generated three types of clonal oligodendrocyte clusters: those composed of only myelinating oligodendrocytes, of both myelinating oligodendrocytes and non-myelinating oligodendrocytes, or of only non-myelinating cells of the oligodendrocyte lineage. Thus, some progenitors do not fully differentiate, but remain immature and may continue to cycle well into adult life.     

Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

A key aim of therapy for multiple sclerosis (MS) is to promote the regeneration of oligodendrocytes and remyelination in the central nervous system (CNS). The present study provides evidence that the vitamin K-dependent protein growth arrest specific 6 (Gas6) promotes such repair in in vitro cultures of mouse optic nerve and cerebellum. We first determined expression of Gas6 and TAM (Tyro3, Axl, Mer) receptors in the mouse CNS, with all three TAM receptors increasing in expression through postnatal development, reaching maximal levels in the adult. Treatment of cultured mouse optic nerves with Gas6 resulted in significant increases in oligodendrocyte numbers as well as expression of myelin basic protein (MBP). Gas6 stimulation also resulted in activation of STAT3 in optic nerves as well as downregulation of multiple genes involved in MS development, including matrix metalloproteinase-9 (MMP9), which may decrease the integrity of the blood–brain barrier and is found upregulated in MS lesions. The cytoprotective effects of Gas6 were examined in in vitro mouse cerebellar slice cultures, where lysolecithin was used to induce demyelination. Cotreatment of cerebellar slices with Gas6 significantly attenuated demyelination as determined by MBP immunostaining, and Gas6 activated Tyro3 receptor through its phosphorylation. In conclusion, these results demonstrate that Gas6/TAM signaling stimulates the generation of oligodendrocytes and increased myelin production via Tyro3 receptor in the adult CNS, including repair after demyelinating injury. Furthermore, the effects of Gas6 on STAT3 signaling and matrix MMP9 downregulation indicate potential glial cell repair and immunoregulatory roles for Gas6, indicating that Gas6-TAM signaling could be a potential therapeutic target in MS and other neuropathologies.     

The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration

Spinal cord oligodendrocytes originate in the ventricular zone and subsequently migrate to white matter, stop, proliferate, and differentiate. Here we demonstrate a role for the chemokine CXCL1 and its receptor CXCR2 in patterning the developing spinal cord. Signaling through CXCR2, CXCL1 inhibited oligodendrocyte precursor migration. The migrational arrest was rapid, reversible, concentration dependent, and reflected enhanced cell/substrate interactions. White matter expression of CXCL1 was temporo-spatially regulated. Developing CXCR2 null spinal cords contained reduced oligodendrocytes, abnormally concentrated at the periphery. In slice preparations, CXCL1 inhibited embryonic oligodendrocyte precursor migration, and widespread dispersal of postnatal precursors occurred in the absence of CXCR2 signaling. These data suggest that population of presumptive white matter by oligodendrocyte precursors is dependent on localized expression of CXCL1.     

Distribution of mGluR1α and SMI 311 immunoreactive Lugaro cells in the kitten cerebellum

The Lugaro cell is a feedback interneuron of the cerebellar cortex, recognizable by its characteristic morphology. Postnatal neuronal migration to the cortex has been described for several cerebellar interneurons. Since in our previous studies we observed Lugaro-like cells (LCs) in the white matter (WM) and internal granular layer (IGL) of the cerebellum of young cats, we assumed that a proportion of these cells migrate also postnatally to their destination. In the present study using and immunostaining for the metabotropic glutamate receptor mGluR1α and neurofilament protein SMI 311 the number and spatial distribution of LCs at different postnatal days were investigated. We found that the number and distribution of both mGluR1a-immunoreactive (ir) and of SMI 311-ir LCs changed with age in the developing cerebellar cortex of kittens: developing LCs express mGluR1α already in the newborn, while expression of SMI 311-ir in LCs appears only about a week later. At postnatal day 1 (P1) relatively few mGluR1-ir LCs were detected in the WM and at the border of WM and IGL. Later, their number increased sharply until P15 (6–7 fold) and decreased continuously between P15 and P135. SMI 311-ir LCs were not present at P1 and even at P8 only a few were observed in the WM or in infraganglionic positions. Their number increased gradually (12–14 fold) until adulthood when their number was stabilized at 8.000–10.000/cerebellum. At the same time the number of probably ectopic SMI 311-ir LCs decreased with age: at P22 about one third of them was found in “ectopic” position, whereas in the adult cat only about 10–12% of LCs's was either in the WM or scattered in the whole depth of the granular layer. These results suggest that: (1) most LCs appear in the cerebellar cortex postnatally; and (2) postnatal migration and incorporation of LCs to the cortex is a much longer process than previously expected, occurring even after the cytoarchitectonic built-up (about P65–P70 in cat) of the cerebellum.     

Myelination of prospective large fibres in chicken ventral funiculus

In mammals, the oligodendrocyte population includes morphological and molecular varieties. We reported previously that an antiserum against the T4-O molecule labels a subgroup of oligodendrocytes related to large myelinated axons in adult chicken white matter. We also reported that T4-O immunoreactive cells first appear in the developing ventral funiculus (VF) at embryonic day (E)15, subsequently increasing rapidly in number. Relevant fine structural data for comparison are not available in the literature. This prompted the present morphological analysis of developing and mature VF white matter in the chicken. The first axon-oligodendrocyte connections were seen at E10 and formation of compact myelin had started at E12. Between E12 and E15 the first myelinating oligodendrocytes attained a Schwann cell-like morphology. At hatching (E21) 60% of all VF axons were myelinated and in the adult this proportion had increased to 85%. The semilunar or polygonal oligodendrocytes associated with adult myelinated axons contained many organelles indicating a vivid metabolic activity. Domeshaped outbulgings with gap junction-like connections to astrocytic profiles were frequent. Oligodendrocytes surrounded by large myelinated axons and those surrounded by small myelinated axons were cytologically similar. But, thick and thin myelin sheaths had dissimilar periodicities and Marchi-positive myelinoid bodies occurred preferentially in relation to large myelinated axons. We conclude that early oligodendrocytes contact axons and form myelin well before the first expression of T4-O and that emergence of a T4-O immunoreactivity coincides in time with development of a Type IV phenotype. Our data also show that oligodendrocytes associated with thick axons are cytologically similar to cells related to thin axons. In addition, the development of chicken VF white matter was found to be similar to the development of mammalian white matter, except for the rapid time course.     

Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system

Four monoclonal antibodies are characterized that have been obtained from a fusion of mouse myeloma P3-NS1/1-Ag4-1 with spleen cells from BALB/c mice immunized with white matter from bovine corpus callosum. The corresponding antigens (O antigens) are designated O1, O2, O3, and O4. The localization of these antigens was investigated by indirect immunofluorescence in cultures of early postnatal mouse cerebellum, cerebrum, spinal cord, optic nerve, and retina. When tested on live cultures none of the O antibodies reacted with the surface of astrocytes, neurons, or fibroblasts, however, all are positive on the surface of oligodendrocytes. The identity of these cells was determined by double-immunolabeling experiments with indpendent cell-type-specific antigenic markers (glial fibrillary acidic protein, tetanus toxin receptors, fibronectin, and galactocerebroside). Antigen O1 is exclusively expressed on galactocerebroside-positive cells, whereas O2, O3, and O4 are expressed on additional cells that are negative for any of the markers tested. None of the O antigens is expressed on the surface of cultured retinal cells. In fresh-frozen sections of adult mouse cerebellum all O antigens are detectable in white matter tracts and in vesicular structures of the granular layer. O2 and O3 antigens are in addition detectable in GFA protein-positive radial fibers in the molecular layer. In fixed cerebellar cultures, where intracellular antigens are accessible, O1, O2, and O3 antibodies label astrocytes in a GFA protein-like pattern. O antigens are expressed in mouse, rat, chicken, and human central nervous systems. O antibodies belong to the IgM immunoglobulin subclass and have been used in complement-dependent cytotoxic elimination of cerebellar oligodendrocytes in culture. At limiting antibody dilutions all processes of oligodendrocytes are preferably lysed over cell bodies.     

Thiamine attenuates hypoxia-induced cell death in cultured neonatal rat cardiomyocytes

Previous studies have demonstrated that thiamine (vitamin B1) has a cytoprotective effect against ischemic damage to the heart, and that heat shock protein 70 (Hsp70) is capable of protecting cardiac cells from lethal ischemia/hypoxia. We show here that thiamine has a cytoprotective effect on cultured neonatal rat cardiomyocytes under hypoxic insult, and also protects the cardiomyocytes against hypoxia-induced apoptosis; caspase-3 activation, PARP cleavage and DNA fragmentation are all inhibited. Moreover, it increases the level of Hsp70 protein in the cardiomyocytes even under prolonged hypoxic stress and its effects on hypoxia-induced cardiac cell death are antagonized by an Hsp70 inhibitor. These results suggest that the cytoprotective effect of thiamine in cardiomyocytes under hypoxic stress is due to its ability to induce Hsp70.     

High-affinity GABA uptake and GABA-metabolizing enzymes in pig forebrain white matter: a quantitative study

GABA receptor activation in central nervous white matter may be protective during white matter hypoxia in the adult, and it may modify axonal conduction, especially in the developing brain. GABA uptake is important for the shaping of the GABA signal, but quantitative data are lacking for GABA uptake and GABA-metabolizing enzymes in central nervous white matter. We report that high-affinity uptake of GABA in adult pig corpus callosum, fimbria, subcortical pyramidal tracts, and occipital white matter is approximately 20% of that in temporal cortex gray matter. Tiagabine (0.1 microM), an antiepileptic drug that specifically inhibits the GAT-1 GABA transporter inhibited GABA uptake 50% in temporal cortex and 60-68% in white structures. This finding indicates that GAT-1 is an important GABA transporter in white matter and suggests that white matter GABA uptake is inhibited during tiagabine therapy. GABA transaminase activity in white structures was approximately 20% of neocortical values. Glutamate decarboxylase (GAD) activity in white structures was only 4% of that in neocortex (7-12 pmol/mg tissue x min(-1) versus approximately 200 pmol/mg tissue x min(-1)). Since white matter activity of citrate synthase of the tricarboxylic acid cycle was approximately 25% of neocortical values ( approximately 0.4 nmol/mg tissue x min(-1) versus approximately 1.5 nmol/mg tissue x min(-1)), the low GAD activity suggests a slower metabolic turnover of GABA in white than in gray matter.     

Degradation of myelin basic protein by a membrane-associated metalloprotease: Neural distribution of the enzyme

A metalloprotease activity associated with myelin membrane preparations degrades myelin basic protein (MBP), generating a characteristic fragment designated peptide C (MBP 74-170). Using an immunoblotting assay, peptide C-generating activity was detected in mammalian, avian, reptilian, and amphibian brains. The activity was present in rat brain as early as postnatal day 1 and also in adult rat peripheral nerve. Immunohistochemistry with a monoclonal antibody to the purified enzyme revealed that the metalloprotease was present in oligodendrocytes of optic nerve, of both white and grey matter of spinal cord, and also in the cytoplasm of both myelinating and non-myelinating Schwann cells of peripheral nerve.Special issue dedicated to Dr. Alan N. Davison     

TGF?1 Stimulates the Over-Production of White Matter Astrocytes from Precursors of the “Brain Marrow” in a Rodent Model of Neonatal Encephalopathy

Background

In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.

Methodology/Principal Finding

For these studies we used the Vannucci H/I rat model where P6 rats are subjected to unilateral common carotid ligation followed by 75 min of systemic hypoxia. Retroviral lineage tracing studies combined with morphological and immunohistochemical analyses revealed the preferential generation of SVZ-derived white matter astrocytes instead of oligodendrocytes post hypoxia/ischemia. Microarray and QRT-PCR analyses of the damaged SVZ showed increased expression of several cytokines and receptors that are known to promote astrocyte differentiation, such as EGF, LIF and TGFß signaling components. Using gliospheres to model the neonatal SVZ, we evaluated the effects of these cytokines on signal transduction pathways regulating astrocyte generation, proliferation and differentiation. These studies demonstrated that combinations of EGF, LIF and TGFß1 reconstituted the increased astrogliogenesis. TGFß1-induced Smad 2/3 phosphorylation and the combination of EGF, LIF and TGFß1 synergistically increased STAT3 phosphorylation over single or double cytokine combinations. Pharmacologically inhibiting ALK5 signaling in vitro antagonized the TGFß1-induced increase in astrocyte generation and antagonizing ALK5 signaling in vivo similarly inhibited astrogliogenesis within the SVZ during recovery from H/I.

Conclusion/Significance

Altogether, these data indicate that there is aberrant specification of glial precursors within the neonatal SVZ during recovery from neonatal H/I that is a consequence of altered cytokine signaling. Our studies further suggest that antagonizing the ALK5 receptor will restore the normal pattern of cell differentiation after injury to the immature brain.     

Acute lipopolysaccharide-mediated injury in neonatal white matter glia: role of TNF-alpha, IL-1beta, and calcium

Bacterial infection is implicated in the selective CNS white matter injury associated with cerebral palsy, a common birth disorder. Exposure to the bacterial endotoxin LPS produced death of white matter glial cells in isolated neonatal rat optic nerve (RON) (a model white matter tract), over a 180-min time course. A delayed intracellular Ca(2+) concentration ([Ca(2+)](i)) rise preceded cell death and both events were prevented by removing extracellular Ca(2+). The cytokines TNF-alpha or IL-1beta, but not IL-6, mimicked the cytotoxic effect of LPS, whereas blocking either TNF-alpha with a neutralizing Ab or IL-1 with recombinant antagonist prevented LPS cytotoxicity. Ultrastructural examination showed wide-scale oligodendroglial cell death in LPS-treated rat optic nerves, with preservation of astrocytes and axons. Fluorescently conjugated LPS revealed LPS binding on microglia and astrocytes in neonatal white and gray matter. Astrocyte binding predominated, and was particularly intense around blood vessels. LPS can therefore bind directly to developing white matter astrocytes and microglia to evoke rapid cell death in neighboring oligodendroglia via a calcium- and cytokine-mediated pathway. In addition to direct toxicity, LPS increased the degree of acute cell death evoked by ischemia in a calcium-dependent manner.     

Activation of epidermal growth factor receptors directs astrocytes to organize in a network surrounding axons in the developing rat optic nerve

In postnatal developing optic nerves, astrocytes organize their processes in a cribriform network to group axons into bundles. In neonatal rat optic nerves in vivo, the active form of EGFR tyrosine kinase is abundantly present when the organization of astrocytes and axons is most actively occurring. Blocking activity of EGFR tyrosine kinase during the development of rat optic nerves in vivo inhibits astrocytes from extending fine processes to surround axons. In vitro, postnatal optic nerve astrocytes, stimulated by EGF, organize into cribriform structures which look remarkably like the in vivo structure of astrocytes in the optic nerve. In addition, when astrocytes are co-cultured with neonatal rat retinal explants in the presence of EGF, astrocytes that are adjacent to the retinal explants, re-organize to an astrocyte-free zone into which neurites grow out from the retinal tissue. We hypothesize that in the developing optic nerve, EGFR activity directs the formation of a histo-architectural structure of astrocytes which surrounds axons and provides a permissive environment for axon development.     

Hypoxia during pregnancy in rats leads to the changes of the cerebral white matter in adult offspring

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 × 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.