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author:("araur, Edwin")
1.  Ischemia Induced Neuroinflammation is Associated with Disrupted Development of Oligodendrocyte Progenitors in a Model of Periventricular Leukomalacia 
Developmental neuroscience  2013;35(0):182-196.
Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time-window can produce long-lasting defects in oligodendroglial maturation, myelination-deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy.
Here, we report that permanent unilateral carotid ligation on postnatal day 5 (P5) in CD-1 mice induces an inflammatory response, as defined by microglial activation and recruitment, as well as significant changes in cytokine expression (increased IL-1b, IL-6, TGF-b1, and TNF-a) following ischemia. Transient reduction in counts of oligodendrocyte progenitor cells (OPCs) at 24 and 48 hours post-ischemia, a shift in OPC cell size and morphology towards the more immature form, as well as likely migration of OPCs were found. These OPC changes were topographically associated with areas showing microglial activation, and OPC counts negatively correlated with increased microglial staining.
The presented data shows a striking neuroinflammatory response in an ischemia-induced model of PVL, associated with oligodendroglial injury. Future studies modulating the neuroinflammatory response in this model, may contribute to a better understanding of the interaction between microglia and OPCs in PVL and open opportunities for future therapies.
doi:10.1159/000346682
PMCID: PMC3764456  PMID: 23445614
Infants; inflammation; ischemia; microglia; neonatal; oligodendrocyte progenitor; white matter
2.  In vivo magnetization transfer MRI shows dysmyelination in an ischemic mouse model of periventricular leukomalacia 
Periventricular leukomalacia, PVL, is the leading cause of cerebral palsy in prematurely born infants, and therefore more effective interventions are required. The objective of this study was to develop an ischemic injury model of PVL in mice and to determine the feasibility of in vivo magnetization transfer (MT) magnetic resonance imaging (MRI) as a potential monitoring tool for the evaluation of disease severity and experimental therapeutics. Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal day 5 (P5); at P60, in vivo T2-weighted (T2w) and MT-MRI were performed and correlated with postmortem histopathology. In vivo T2w MRI showed thinning of the right corpus callosum, but no significant changes in hippocampal and hemispheric volumes. Magnetization transfer MRI revealed significant white matter abnormalities in the bilateral corpus callosum and internal capsule. These quantitative MT-MRI changes correlated highly with postmortem findings of reduced myelin basic protein in bilateral white matter tracts. Ventriculomegaly and persistent astrogliosis were observed on the ligated side, along with evidence of axonopathy and fewer oligodendrocytes in the corpus callosum. We present an ischemia-induced mouse model of PVL, which has pathologic abnormalities resembling autopsy reports in infants with PVL. We further validate in vivo MRI techniques as quantitative monitoring tools that highly correlate with postmortem histopathology.
doi:10.1038/jcbfm.2011.68
PMCID: PMC3208153  PMID: 21540870
brain ischemia; glial cells; MRI; perinatal hypoxia; white matter disease
3.  Derivation of Glial Restricted Precursors from E13 mice 
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
doi:10.3791/3462
PMCID: PMC3399460  PMID: 22760029
Neuroscience;  Issue 64;  Physiology;  Medicine;  periventricular leukomalacia;  oligodendrocytes;  glial restricted precursors;  spinal cord;  cell culture
4.  Probing Cellular Protein Complexes via Single Molecule Pull-down 
Nature  2011;473(7348):484-488.
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.
doi:10.1038/nature10016
PMCID: PMC3103084  PMID: 21614075

Results 1-4 (4)