Rat Cerebral White Matter Research Articles

Altered Oligodendroglial and Neuroglial Gene Expression in Adult Rat Cerebral White Matter Following Short- and Long-Term Ethanol Exposures and Abbreviated Abstinence

Ethanol-mediated white matter degeneration is associated with impaired oligodendrocyte functions linked to myelin maintenance. This study examines ethanol-induced time course-dependent shifts in myelin gene expression in a chronic+binge exposure model and assesses therapeutic responses to short-term abstinence. Six-week-old male Long Evans rats were fed isocaloric liquid diets with 0% or 26% ethanol (caloric content) for 3 or 8 weeks. During the last 2 weeks of ethanol feeding, the rats were binged with 2 g/kg ethanol by intraperitoneal injection, 3x/week. In a subgroup pair-fed for 6 weeks, ethanol was tapered over 2 days and then withdrawn for 12 days. Gene expression in temporal lobe white matter was measured by qRT-PCR analysis. Chronic+binge ethanol exposures caused short-term, long-term, or step-wise changes in neuroglial gene expression such that: 1) NCAM1 mRNA levels were significantly elevated after 3 weeks (P=0.04); 2) PLP was reduced after 8 weeks (P=0.003); and 3) MAG (P=0.008) and RTN4 (P=0.02) progressively declined from 3 to 8 weeks of exposure. Abstinence did not reverse long-term ethanol effects on PLP, MAG or RTN4. Instead, it significantly reduced RPAIN (P=0.002) and Olig2 (P=0.02) relative to responses after 8 weeks of continuous ethanol exposure. In conclusion, chronic+binge ethanol exposures mainly inhibited mature myelin-associated gene expression. Short-term abstinence failed to abrogate those adverse effects of ethanol. Instead, the corresponding reductions in RPAIN and Olig2 expression suggest that during the acute phase of withdrawal or abstinence, molecular indices of white matter degeneration may worsen and that additional therapeutic measures may be required for optimum recovery.

Strain-Specific Differences in Perinatal Rodent Oligodendrocyte Lineage Progression and Its Correlation with Human

Progress in the development of rat models of human periventricular white matter injury (WMI) has been hampered by uncertainty about the developmental window in different rodent strains that coincides with cerebral white matter development in human premature infants. To define strain-specific differences in rat cerebral white matter maturation, we analyzed oligodendrocyte (OL) lineage maturation between postnatal days (P)2 and P14 in three widely studied strains of rat: Sprague-Dawley, Long-Evans and Wistar (W). We previously reported that late OL progenitors (preOL) are the major vulnerable cell type in human periventricular WMI. Strain-specific differences in preOL maturation were found at P2, such that the W rat had the highest percentage and density of preOL relative to the other strains. Overall, at P2, the state of OL maturation was similar to preterm human cerebral white matter. However, by P5, all three strains displayed a similar magnitude and extent of OL maturation that persisted with progressive myelination between P7 and P14. PreOL were the predominant OL lineage stage present in the cerebral cortex through P14, and thus OL lineage maturation occurred latter than in white matter. The hippocampus also displayed a later onset of preOL maturation in all three strains, such that OL lineage maturation and early myelination was not observed to occur until about P14. This timing of preOL maturation in rat cortical gray matter coincided with a similar timing in human cerebral cortex, where preOL also predominated until at least 8 months after full-term birth. These studies support that strain-specific differences in OL lineage immaturity were present in the early perinatal period at about P2, and they define a narrow window of preterm equivalence with human that diminishes by P5. Later developmental onset of preOL maturation in both cerebral cortex and hippocampus coincides with an extended window of potential vulnerability of the OL lineage to hypoxia-ischemia in these gray matter regions.

Persistent roles of signal transduction of platelet-derived growth factor B in genesis, growth, and anaplastic transformation of gliomas in an in-vivo serial transplantation model

We previously reported that retrovirally transduced platelet-derived growth factor-B (PDGFB) in glial progenitors of the rat cerebral white matter, subventricular zone, or brain stem induced malignant brain tumors closely resembling human glioblastoma (GBM). While human GBMs may progress over the period of several months to a few years, prospective, long-term in-vivo observation of histological changes of the tumor tissues is not feasible in these models, because the animals undergo rapid tumor progression and mortality within approximately 1 month. We thus performed successive, long-term in-vivo transplantation of the PDGFB-induced tumor cells into the rat cerebrum. Primary retroviral transduction of PDGFB in the glial progenitors of the rat basal ganglia induced malignant glioma resembling human GBM or anaplastic oligodendroglioma (AOL) consisting of relatively monomorphous tumor cells expressing markers for the oligodendrocyte lineage. In the course of long-term successive transplantation, tumor cells presented pleomorphism as well as focal GFAP expression. This suggests that secondary chromosomal aberration and dysregulation of gene expression following accelerated cell cycle by PDGFB stimulation would induce morphological and immunophenotypic changes in tumor cells. Furthermore, while the primary tumors contained only a minor fraction of proviral GFP-expressing or hemagglutinin-expressing cells, most tumor cells came to express these proviral genes in the course of serial transplantation suggesting a persistent role of PDGFB-expressing cells in maintenance and growth of the tumors. This model would be useful for investigation of the long-term effects of PDGFB stimulation in glioma tissues on anaplastic evolution.

Oligodendrocyte degeneration and recovery after focal cerebral ischemia

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Diminished calcium homeostasis and increased susceptibility to excitotoxicity of JS 3/16 progenitor cells after differentiation to oligodendroglia

JS 3/16, derived from passaged oligodendroglial cultures prepared from rat cerebral white matter, differentiate from progenitors (OP) into complex process-bearing, galactocerebroside-positive but myelin basic protein-negative immature oligodendrocyte-like cells (ImO) after withdrawal of trophic factors. We found that JS 3/16 ImO are markedly more susceptible than OP to cell death after sustained α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-GluR) activation. This excitotoxicity is preceded by loss of intracellular Ca2+ homeostasis, which is more marked in ImO than OP. We identified three factors likely to contribute to the diminished Ca2+ homeostatic capacity of ImO. First, signal intensities of immunoreactive GluR2, GluR3, and GluR4 AMPA-GluR subunits are increased 1.3- to 2.2-fold in ImO over OP without comparable changes in RNA editing and alternative splicing. Second, transcriptional levels of genes encoding Na+-Ca2+ exchanger proteins and a plasma membrane ATPase (PMCA1), which are necessary for Ca2+ extrusion across the plasma membrane, are lower in ImO than in OP. Third, ImO have more depolarized basal mitochondrial membrane potential (ΔΨ) than OP, and ΔΨ collapses within 15 min after onset of AMPA-GluR activation in almost all ImO, but not in the majority of OP. This ΔΨ collapse limits the capacity of ImO mitochondria to buffer the rise in intracellular Ca2+ caused by AMPA-GluR activation. The JS 3/16 line provides a valuable system for analysis of intracellular Ca2+ homeostasis and AMPA-GluR-mediated excitotoxicity in the oligodendroglial lineage. GLIA 31:165–180, 2000. © 2000 Wiley-Liss, Inc.

Diminished calcium homeostasis and increased susceptibility to excitotoxicity of JS 3/16 progenitor cells after differentiation to oligodendroglia

JS 3/16, derived from passaged oligodendroglial cultures prepared from rat cerebral white matter, differentiate from progenitors (OP) into complex process-bearing, galactocerebroside-positive but myelin basic protein-negative immature oligodendrocyte-like cells (ImO) after withdrawal of trophic factors. We found that JS 3/16 ImO are markedly more susceptible than OP to cell death after sustained alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-GluR) activation. This excitotoxicity is preceded by loss of intracellular Ca(2+) homeostasis, which is more marked in ImO than OP. We identified three factors likely to contribute to the diminished Ca(2+) homeostatic capacity of ImO. First, signal intensities of immunoreactive GluR2, GluR3, and GluR4 AMPA-GluR subunits are increased 1.3- to 2.2-fold in ImO over OP without comparable changes in RNA editing and alternative splicing. Second, transcriptional levels of genes encoding Na(+)-Ca(2+) exchanger proteins and a plasma membrane ATPase (PMCA1), which are necessary for Ca(2+) extrusion across the plasma membrane, are lower in ImO than in OP. Third, ImO have more depolarized basal mitochondrial membrane potential (Delta Psi) than OP, and Delta Psi collapses within 15 min after onset of AMPA-GluR activation in almost all ImO, but not in the majority of OP. This Delta Psi collapse limits the capacity of ImO mitochondria to buffer the rise in intracellular Ca(2+) caused by AMPA-GluR activation. The JS 3/16 line provides a valuable system for analysis of intracellular Ca(2+) homeostasis and AMPA-GluR-mediated excitotoxicity in the oligodendroglial lineage.

Partial purification of a novel mitogen for oligodendroglia

A protein with a MWapp of 50-70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS-polyacrylamide gel, this protein ("oligodendroglial trophic factor," OTF) elicited half-maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet-derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage.

Oligodendrocyte maturation and myelin gene expression in PDGF-treated cultures from rat cerebral white matter

Myelination in the CNS is accompanied by the differentiation of oligodendrocytes as well as the coordinate expression of a group of myelin-specific genes, including those encoding proteolipid protein and myelin basic protein. In order to compare the timing of the onset of myelin gene expression with the known sequence of oligodendrocyte maturation, we analyzed cerebral white matter cultures grown in the presence of platelet-derived growth factor for expression of the mRNAs encoding these myelin proteins, as well as for the numbers of oligodendrocytes and their precursors. Platelet-derived growth factor treatment increased the rate of oligodendrocyte precursor cell proliferation and the number of mature oligodendrocytes. Platelet-derived growth factor also produced a significant increase in oligodendrocyte precursors prior to an increase in their proliferation rate, suggesting that platelet-derived growth factor may also have an effect on oligodendrocyte precursor survival. Furthermore, steady-state levels of proteolipid protein and myelin basic protein mRNAs increased within 24 of the addition of platelet-derived growth factor, before any significant change in the numbers of oligodendrocytes or their precursors, demonstrating that platelet-derived growth factor also regulates myelin gene expression. At later times after platelet-derived growth factor addition, however, when the number of oligodendrocytes and their precursors was rapidly increasing, the increase in proteolipid protein and myelin basic protein mRNA levels was proportionally much greater than the increase in oligodendroglial lineage cells, suggesting that platelet-derived growth factor also increased the number of proteolipid protein and myelin basic protein transcripts per cell; this interpretation was confirmed by in situ hybridization analysis. Finally, by examining the co-expression of galactocerebroside using the epitopes recognized by the Ranscht monoclonal antibody and proteolipid protein mRNA in individual cells by a combination of in situ hybridization and immunohistochemistry, we demonstrated that oligodendrocytes express proteolipid protein and myelin basic protein mRNA. Oligodendrocyte maturation, as measured by surface galactocerebroside expression, is thus contemporaneous with the activation of myelin-specific gene expression.

Differential regional and cellular distribution of β-amyloid precursor protein messenger RNAs containing and lacking the kunitz protease inhibitor domain in the brain of human, rat and mouse

The β-amyloid precursor protein is the precursor of the main component of senile plaques (the β-amyloid peptide or β/A4) found in the brain of aged humans and, in higher amounts, in the brain of Alzheimer's disease and Down's syndrome subjects. Four different forms of β-amyloid precursor protein messenger RNAs have been described in humans and rodents: β-amyloid precursor protein 695, β-amyloid precursor protein 714, β-amyloid precursor protein 751 and β-amyloid precursor protein 770 messenger RNAs (numbers corresponding to the number of encoded amino acids). The two latter forms are characterized by containing in their sequence a region with high homology to the Kunitz family of serine protease inhibitors. We have used oligonucleotide probes to study the distribution of the different messenger RNAs encoding each of the four β-amyloid precursor proteins by in situ hybridization histochemistry in human, rat and mouse brain. We found that β-amyloid precursor protein 695, β-amyloid precursor protein 714 and β-amyloid precursor protein 751 messenger RNAs were widely distributed in the human, rat and mouse brain and that their distribution was roughly similar in most brain areas in these three species. The distribution of β-amyloid precursor protein 770 messenger RNA was not so wide and differed among the three species studied, β-amyloid precursor protein 751 and 770 messenger RNAs were the only forms present at significant levels in rodent choroid plexus and meninges, while β-amyloid precursor protein messenger RNA isoforms containing and lacking the Kunitz domain were detected in the human choroid plexus. We also observed that the relative levels of β-amyloid precursor protein 751 and 770 messenger RNAs in the rat cerebral white matter as well as in the mouse and human striatum were higher than those of the β-amyloid precursor protein messenger RNAs lacking the Kunitz domain. While the most abundant β-amyloid precursor protein messenger RNAs in the brain of all three species under study were, in descending order, β-amyloid precursor protein 695 and β-amyloid precursor protein 751 messenger RNAs, the least abundant form was not the same for all species: in human it was β-amyloid precursor protein 714 messenger RNA and in rat and mouse brain it was β-amyloid precursor protein 770 messenger RNA. Our results show differences both inter- and intraspecies of the relative abundance and distribution of four β-amyloid precursor protein messenger RNAs in rat, mouse and human brain. The non-coincidence of the distribution of β-amyloid precursor protein messenger RNA isoforms suggests different physiological functions for the β-amyloid precursor proteins. Interspecies differences should be considered when comparing results from animal and human studies.

Rat brain microvessel extracellular matrix modulates the phenotype of cultured rat type 1 astroglia

Astroglia from immature rat cerebral white matter which were plated on the insoluble extracellular matrix (ECM) secreted by rat cerebral microvessel endothelial cells (RCMEC) and maintained in a defined medium were induced to become stellate and to express glutamine synthetase. These effects were not elicited by RCMEC-conditioned medium. ECM secreted by rat pleural mesothelium elicited a lesser proportion of stellate astroglia and did not induce glutamine synthetase.