Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases

We report neuropathological, biochemical and molecular studies on two patients with childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome, a leukodystrophy recently defined according to clinical and radiological criteria. Both had severe cavitating orthochromatic leukodystrophy without atrophy, predominating in hemispheric white matter, whereas U-fibers, internal capsule, corpus callosum, anterior commissure and cerebellar white matter were relatively spared. The severity of white matter lesions contrasted with the rarity of myelin breakdown products and astroglial and microglial reactions. In the white matter, there was an increase in a homogeneous cell population with the morphological features of oligodendrocytes, in many instances presenting an abundant cytoplasm like myelination glia. These cells were negative for glial fibrillary acidic protein and antibodies PGM1 and MIB1. Some were positive for myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, but the majority were positive for human 2′-3′ cyclic nucleotide 3′ phosphodiesterase and all were positive for carbonic anhydrase II, confirming that they are oligodendrocytes. Myelin protein and lipid content were reduced. The PLP gene, analyzed in one case, was not mutated or duplicated. The increased number of oligodendrocytes without mitotic activity suggests an intrinsic oligodendroglial defect or an abnormal interaction with axons or other glial cells. This neuropathological study supports the notion that CACH syndrome constitutes a specific entity. Received: 23 February 1998 / Revised: 12 August 1998, 20 October 1998 / Accepted: 21 October 1998     

Glial Cell Lineages in the Rat Cerebral Cortex

I have traced the fates of glial cell progenitors in the rat cerebral cortex marked with a recombinant retrovirus throughout most of the period of corticogenesis, from embryonic (E) day 14 to postnatal (P) day 14. Discrete clusters of clonally related glia were examined in serially cut sections, and their phenotypes identified using reliable light and electron microscopic criteria. Analysis of a large number of clones marked with retrovirus at various stages of embryonic life contained, with very few exceptions, either all astrocytes or all oligodendrocytes. This observation suggests that the ventricular zone contains separate progenitor cells for the two glial cell types. Oligodendrocyte clones were rarely seen in the cortices injected with retrovirus at the early stages of corticogenesis (E14-E16), suggesting that there is a very small number of oligodendrocyte progenitors in the ventricular zone at these early stages. Their frequency increased significantly at later embryonic ages. At these later stages, ventricular zone cells also give rise to progenitor cells that make up the subventricular zone in early postnatal life. Injections of retrovirus in this proliferative zone shortly after birth resulted in the generation of labeled astrocyte and oligodendrocyte clones in the cortical gray and white matter, with the astrocyte clones being in the majority. Injections at increasingly later stages resulted in the presence, predominantly in the white matter of both hemispheres and in the corpus callosum, of progressively more oligodendrocyte clones and fewer astrocyte clones. Injections at P14 generated only oligodendrocyte clones in the white matter of both hemispheres. A small number of clusters (<10%) generated after subventricular zone injections contained both astrocytes and oligodendrocytes, suggesting that single subventricular zone cells can differentiate into both glial cell types.     

Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor

Demyelinating diseases are characterized by an extensive loss of oligodendrocytes and myelin sheaths from axolemma. These neurological disorders are a common cause of disability in young adults, but so far, there is no effective treatment against them. It has been suggested that neural stem cells (NSCs) may play an important role in brain repair therapies. NSCs in the adult subventricular zone (SVZ), also known as Type-B cells, are multipotential cells that can self-renew and give rise to neurons and glia. Recent findings have shown that cells derived from SVZ Type-B cells actively respond to epidermal-growth-factor (EGF) stimulation becoming highly migratory and proliferative. Interestingly, a subpopulation of these EGF-activated cells expresses markers of oligodendrocyte precursor cells (OPCs). When EGF administration is removed, SVZ-derived OPCs differentiate into myelinating and pre-myelinating oligodendrocytes in the white matter tracts of corpus callosum, fimbria fornix and striatum. In the presence of a demyelinating lesion, OPCs derived from EGF-stimulated SVZ progenitors contribute to myelin repair. Given their high migratory potential and their ability to differentiate into myelin-forming cells, SVZ NSCs represent an important endogenous source of OPCs for preserving the oligodendrocyte population in the white matter and for the repair of demyelinating injuries.     

Amyloid beta peptide-induced corpus callosum damage and glial activation in vivo

The effects of stereotaxic injection of amyloid beta-peptide (Abeta1-42) into rat brain to induce white matter damage have been studied. Administration of 1 nmol Abeta1-42 into corpus callosum resulted in considerable damage to axons as evidenced by the loss of neurofilament-immunoreactive (NF-ir) fibers 6 h and 3 and 7 days post-injection. Significant damage was also evident to myelin (using Luxol fast blue myelin staining) and oligodendrocytes (using CC1 immunocytochemistry); in the latter case marked caspase-3 immunoreactivity was evident in oligodendrocytes. Additionally, the numbers of GFAP-ir astrocytes and OX-42/OX-6-ir microglia were markedly increased following Abeta1-42 injection. These results suggest that Abeta plays an important pathophysiological role in white matter damage and that inflammatory responses may contribute to Abeta-induced demyelination and oligodendrocyte injury in corpus callosum. Loss of function of cells in corpus callosum could provide a potential new model for the study of white matter damage in Alzheimer's disease.     

Evidence for neuronal regulation of oligodendrocyte development: Cellular localization of platelet-derived growth factor α receptor and A-chain mRNA during cerebral cortex development in the rat

Oligodendrocyte responses in vitro to platelet-derived growth factor (PDGF) include proliferation, survival, migration, and changes in cell morphology and molecular expression. Studies of mixed glial cultures established that astrocytes secrete PDGF; thus astrocytes are considered to be key regulators of oligodendrocyte development in vitro. We previously demonstrated PDGF α receptor mRNA expression by oligodendrocyte progenitors and preoligodendrocytes during postnatal development of rat cerebral cortex. In the present study, we have mapped the spatial and temporal expression of PDGF A-chain ligand mRNA and α receptor mRNA to determine if the cell-cell interactions that form the basis for PDGF regulation of oligodendrocyte development in vitro are also present in vivo. By in situ hybridization (ISH) we demonstrate that at embryonic day 17 (E17) cells expressing receptor mRNA (PDGFRα+) are initially in the subventricular zone, at a distance from cells expressing ligand mRNA (PDGF+) in the cortical plate. By E20 PDGFRα+ cells are found throughout the corpus callosum and cortical gray matter. PDGF+ cells are restricted to the cortical plate prenatally and only appeared in the corpus callosum postnatally. Combined immunocytochemistry and ISH demonstrated the PDGF+ cells colocalized with neurofilament, but not with GFAP. These data establish that PDGF is expressed by neurons during PDGFRα+ oligodendrocyte progenitor migration from the subventricular zone to the corpus callosum and gray matter. Furthermore, neurons continue to express PDGF during the generation and differentiation of appropriate numbers of oligodendrocytes needed to myelinate axons as the nervous system matures. © 1996 Wiley-Liss, Inc.     

Migrating and myelinating potential of subventricular zone neural progenitor cells in white matter tracts of the adult rodent brain

Adult neural stem cells in the subventricular zone (SVZ) produce neuronal progenitors that migrate along the rostral migratory stream (RMS) and generate olfactory interneurons. Here, we evaluate the migratory potential of SVZ cells outside the RMS and their capacity to generate oligodendrocytes in the adult brain. We show that SVZ cells migrate long distances when grafted into white matter tracts such as the cingulum (Ci) and corpus callosum (CC). Furthermore, 22 days postinjection, most present morphologic and phenotypic characteristics of cells committed to the oligodendrocyte lineage. Cells grafted in shiverer CC and Ci become MBP-positive oligodendrocytes, abundantly myelinating these white matter tracts. Type A progenitors are involved in this myelinating process. Altogether, this study reveals the migrating and myelinating potential of SVZ cells in a new environmental context. Therefore, SVZ cells stand as interesting candidates for the development of novel therapeutic strategies for demyelinating diseases.     

Ontogeny of glycerol phosphate dehydrogenase-positive oligodendrocytes in rat brain. Impaired differentiation of oligodendrocytes in the myelin deficient mutant rat.

The ontogeny of oligodendrocytes in the myelin deficient (md) rat mutant and in control rats was explored immunohistochemically using an antiserum against the oligodendrocyte specific enzyme, glycerol phosphate dehydrogenase (GPDH), and the avidin-biotin complex technique. In control rats, GPDH was demonstrated to be expressed relatively early in oligodendrocyte differentiation, prior to either myelin basic protein or proteolipid protein expression. With development, oligodendrocytes containing GPDH increased in number, apparent staining intensity, cell soma area and process elaboration. Fewer GPDH+oligodendrocytes were observed in the brain of mutant rats than in unaffected littermates at all developmental ages, and major developmental increases in oligodendrocyte density were delayed. The density of GPDH+oligodendrocytes was reduced by about 40% in both the corpus callosum and in the cingulate cortex of P22-25 and mutants compared with control rats. The oligodendrocyte cell soma area was not influenced by the md condition, and increased 2-fold with development in rats of both genotypes. The area of coronal sections occupied by the corpus callosum increased about 2.5-fold with development, and was 30% smaller in mutant rats late in their lifespan than in unaffected littermates. The reductions in oligodendrocyte density reported here are of insufficient magnitude to fully account for biochemically measured reductions in oligodendrocyte gene expression accompanying the md trait, indicating that gene expression per oligodendrocyte is also impaired. Cell counts in control rats also revealed that oligodendrocytes are overproduced during development. Cell density and the total number of corpus callosum GPDH+oligodendrocytes per section were maximal at P22-25 and then decreased to adult values. These results suggest that glial cells, like neurons, may be generated in excessive numbers, and some subsequently die, as a normal concomitant of development.     

Myelin protein expression in the myelin-deficient rat brain and cultured oligodendrocytes

The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10-20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0-2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.     

Myelin loss and oligodendrocyte pathology in white matter tracts following traumatic brain injury in the rat

Axonal injury is an important contributor to the behavioral deficits observed following traumatic brain injury (TBI). Additionally, loss of myelin and/or oligodendrocytes can negatively influence signal transduction and axon integrity. Apoptotic oligodendrocytes, changes in the oligodendrocyte progenitor cell (OPC) population and loss of myelin were evaluated at 2, 7 and 21 days following TBI. We used the central fluid percussion injury model (n = 18 and three controls) and the lateral fluid percussion injury model (n = 15 and three controls). The external capsule, fimbriae and corpus callosum were analysed. With Luxol Fast Blue and RIP staining, myelin loss was observed in both models, in all evaluated regions and at all post‐injury time points, as compared with sham‐injured controls (P ≤ 0.05). Accumulation of β‐amyloid precursor protein was observed in white matter tracts in both models in areas with preserved and reduced myelin staining. White matter microglial/macrophage activation, evaluated by isolectin B4 immunostaining, was marked at the early time points. In contrast, the glial scar, evaluated by glial fibrillary acidic protein staining, showed its highest intensity 21 days post‐injury in both models. The number of apoptotic oligodendrocytes, detected by CC1/caspase‐3 co‐labeling, was increased in both models in all evaluated regions. Finally, the numbers of OPCs, evaluated with the markers Tcf4 and Olig2, were increased from day 2 (Olig2) or day 7 (Tcf4) post‐injury (P ≤ 0.05). Our results indicate that TBI induces oligodendrocyte apoptosis and widespread myelin loss, followed by a concomitant increase in the number of OPCs. Prevention of myelin loss and oligodendrocyte death may represent novel therapeutic targets for TBI.     

Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3-4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpy(msd) (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild-type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.     

Rosmarinic acid ameliorates hypoxia/ischemia induced cognitive deficits and promotes remyelination

Rosmarinic acid,a common ester extracted from Rosemary,Perilla frutescens,and Salvia miltiorrhiza Bunge,has been shown to have protective effects against various diseases.This is an investigation into whether rosmarinic acid can also affect the changes of white matter fibers and cognitive deficits caused by hypoxic injury.The right common carotid artery of 3-day-old rats was ligated for 2 hours.The rats were then prewarmed in a plastic container with holes in the lid,which was placed in 37°C water bath for 30 minutes.Afterwards,the rats were exposed to an atmosphere with 8% O2 and 92% N2 for 30 minutes to establish the perinatal hypoxia/ischemia injury models.The rat models were intraperitoneally injected with rosmarinic acid 20 mg/kg for 5 consecutive days.At 22 days after birth,rosmarinic acid was found to improve motor,anxiety,learning and spatial memory impairments induced by hypoxia/ischemia injury.Furthermore,rosmarinic acid promoted the proliferation of oligodendrocyte progenitor cells in the subventricular zone.After hypoxia/ischemia injury,rosmarinic acid reversed to some extent the downregulation of myelin basic protein and the loss of myelin sheath in the corpus callosum of white matter structure.Rosmarinic acid partially slowed down the expression of oligodendrocyte marker Olig2 and myelin basic protein and the increase of oligodendrocyte apoptosis marker inhibitors of DNA binding 2.These data indicate that rosmarinic acid ameliorated the cognitive dysfunction after perinatal hypoxia/ischemia injury by improving remyelination in corpus callosum.This study was approved by the Animal Experimental Ethics Committee of Xuzhou Medical University,China (approval No.20161636721) on September 16,2017.     

Lentiviral transduction of murine oligodendrocytes in vivo

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP). For all constructs, intracerebral injections to lateral corpus callosum resulted in widespread GFP expression in forebrain white matter glial cells. Intense cellular GFP fluorescence was observed within 3 days after injection and lasted for at least 28 days. The CMV promoter directed GFP expression in multiple glial cell types, whereas the MBP promoter targeted GFP specifically to oligodendrocytes. Expression of the membrane-targeted Lck-eGFP construct distinctly labeled individual myelinating processes of oligodendrocytes. Lentiviral constructs expressing eGFP or Lck-eGFP under the MBP promoter provide excellent visualization of oligodendrocyte morphology in intact white matter, and may prove valuable for delivering additional genes of interest to oligodendrocytes in vivo.     

Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder

Alcohol exposure during central nervous system (CNS) development can lead to fetal alcohol spectrum disorder (FASD). Human imaging studies have revealed significant white matter (WM) abnormalities linked to cognitive impairment in children with FASD; however, the underlying mechanisms remain unknown. Here, we evaluated both the acute and long‐term impacts of alcohol exposure on oligodendrocyte number and WM integrity in a third trimester‐equivalent mouse model of FASD, in which mouse pups were exposed to alcohol during the first 2 weeks of postnatal development. Our results demonstrate a 58% decrease in the number of mature oligodendrocytes (OLs) and a 75% decrease in the number of proliferating oligodendrocyte progenitor cells (OPCs) within the corpus callosum of alcohol‐exposed mice at postnatal day 16 (P16). Interestingly, neither mature OLs nor OPCs derived from the postnatal subventricular zone (SVZ) were numerically affected by alcohol exposure, indicating heterogeneity in susceptibility based on OL ontogenetic origin. Although mature OL and proliferating OPC numbers recovered by postnatal day 50 (P50), abnormalities in myelin protein expression and microstructure within the corpus callosum of alcohol‐exposed subjects persisted, as assessed by western immunoblotting of myelin basic protein (MBP; decreased expression) and MRI diffusion tensor imaging (DTI; decreased fractional anisotropy). These results indicate that third trimester‐equivalent alcohol exposure leads to an acute, albeit recoverable, decrease in OL lineage cell numbers, accompanied by enduring WM injury. Additionally, our finding of heterogeneity in alcohol susceptibility based on the developmental origin of OLs may have therapeutic implications in FASD and other disorders of WM development.     

Alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor CXCR2

Oligodendrocyte precursor cell (OPC) proliferation and migration are critical for the development of myelin in the central nervous system (CNS). Previous studies showed that localized expression of the chemokine CXCL1 signals through the receptor CXCR2 to inhibit the migration and enhance the proliferation of spinal cord OPCs during development. Here, we report structural and functional alterations in the adult CNS of Cxcr2-/- mice. In Cxcr2-/- adult mice, we observed regional alterations in the density of oligodendrocyte lineage cells in Cxcr2-/- adult mice, with decreases in the cortex and anterior commissure but increases in the corpus callosum and spinal cord. An increase in the density and arborization of spinal cord NG2 positive cells was also observed in Cxcr2-/- adult mice. Compared with wild-type (WT) littermates, Cxcr2-/- mice exhibited a significant decrease in spinal cord white matter area, reduced thickness of myelin sheaths, and a slowing in the rate of central conduction of spinally elicited evoked potentials without significant changes in axonal caliber or number. Biochemical analyses showed decreased levels of myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). In vitro studies showed reduced numbers of differentiated oligodendrocytes in Cxcr2-/- spinal cord cultures. Together, these findings indicate that the chemokine receptor CXCR2 is important for the development and maintenance of the oligodendrocyte lineage, myelination, and white matter in the vertebrate CNS.     

Lineage tracing reveals dynamic changes in oligodendrocyte precursor cells following cuprizone‐induced demyelination

The regeneration of oligodendrocytes is a crucial step in recovery from demyelination, as surviving oligodendrocytes exhibit limited structural plasticity and rarely form additional myelin sheaths. New oligodendrocytes arise through the differentiation of platelet‐derived growth factor receptor α (PDGFRα) expressing oligodendrocyte progenitor cells (OPCs) that are widely distributed throughout the CNS. Although there has been detailed investigation of the behavior of these progenitors in white matter, recent studies suggest that disease burden in multiple sclerosis (MS) is more strongly correlated with gray matter atrophy. The timing and efficiency of remyelination in gray matter is distinct from white matter, but the dynamics of OPCs that contribute to these differences have not been defined. Here, we used in vivo genetic fate tracing to determine the behavior of OPCs in gray and white matter regions in response to cuprizone‐induced demyelination. Our studies indicate that the temporal dynamics of OPC differentiation varies significantly between white and gray matter. While OPCs rapidly repopulate the corpus callosum and mature into CC1 expressing mature oligodendrocytes, OPC differentiation in the cingulate cortex and hippocampus occurs much more slowly, resulting in a delay in remyelination relative to the corpus callosum. The protracted maturation of OPCs in gray matter may contribute to greater axonal pathology and disease burden in MS.     

Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help in the development of strategies to promote remyelination. In the present study, we examined the ability of the precursor cells of the adult mouse subventricular zone (SVZ) to differentiate into remyelinating oligodendrocytes. After lysolecithin-induced demyelination of the corpus callosum, progenitors of the rostral SVZ (SVZa) and the rostral migratory pathway (RMS), expressing the embryonic polysialylated form of the neural cell adhesion molecule (PSA-NCAM), increased progressively with a maximal expansion occurring after 2 weeks. This observation correlated with an increase in the proliferation activity of the neural progenitors located in the SVZa and RMS. Moreover, polysialic acid (PSA)-NCAM-immunoreactive cells arizing from the SVZa were detected in the lesioned corpus callosum and within the lesion. Tracing of the constitutively cycling cells of the adult SVZ and RMS with 3H-thymidine labelling showed their migration toward the lesion and their differentiation into oligodendrocytes and astrocytes but not neurons. These data indicate that, in addition to the resident population of quiescent oligodendrocyte progenitors of the adult CNS, neural precursors from the adult SVZ constitute a source of oligodendrocytes for myelin repair.     

Toll-like receptor 4 participates in the myelin disruptions associated with chronic alcohol abuse

Alcohol abuse and alcoholism can cause brain damage, loss of white matter, myelin fiber disruption, and even neuronal injury. The underlying mechanisms of these alterations remain elusive. We have shown that chronic ethanol intake, by activating glial toll-like receptor 4 (TLR4) receptors, triggers the production of inflammatory mediators and can cause brain damage. Because neuroinflammation may be associated with demyelination and neuronal damage, we evaluate whether the ethanol-induced TLR4-dependent proinflammatory environment in the brain could be involved in the myelin disruptions observed in alcoholics. Using brains from wild-type (WT) and TLR4 knockout (KO, TLR4(-/-) ) mice, we demonstrate that chronic ethanol treatment downregulated proteins involved in myelination [proteolipid protein (PLP), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein, 2,3-cyclic-nucleotide-3-phosphodiesterase, and myelin-associated glycoprotein], while increased chondroitin sulfate proteoglycan NG2 (NG2)-proteoglycan in several brain regions of ethanol-treated WT mice. The immunohistochemistry analysis also revealed that ethanol-treatment-altered myelin morphology reduced the number of MBP-positive fibers and caused oligodendrocyte death, as demonstrated by an increase in caspase-3-positive oligodendrocytes. The in vivo imaging system further confirmed that chronic ethanol intake markedly reduced the PLP in WT mice. Most myelin alterations were not observed in brains from ethanol-treated TLR4(-/-) mice. Electron microscopy studies revealed that although 41-47% of axons showed myelin sheath disarrangements in the cerebral cortex and corpus callosum of WT ethanol-treated mice, respectively, small focal fiber disruptions were noticed in these brain areas of ethanol-treated TLR4(-/-) mice. In summary, the present results suggest that ethanol-induced neuroinflammation might be involved in myelin disruptions and white matter loss observed in human alcoholics.     

Effects of intrauterine inflammation on developing rat brain

Damage to the white matter in the brain during development can lead to cerebral palsy (CP), a heterogeneous group of clinical syndromes that results in life-long disorders of movement and posture. Periventricular leukomalacia (PVL) is a pathological process within the white matter characterized by oligodendrocyte loss and is associated with the development of CP. Clinically, CP and PVL are associated with intrauterine infection and inflammation, but mechanisms involved are not well understood. We developed a model of intrauterine inflammation in Lewis and Fischer 344 rats to study the effects of intrauterine inflammation on developing glia. Pregnant rats were intracervically injected with lipopolysaccharide (LPS) at 15 days of gestation (E15) and a dose of LPS that caused low fetal mortality was determined. At E20, treated fetuses had increased TUNEL(+) nuclei and tumor necrosis factor (TNF)-alpha-immunoreactive areas within the brains. In a second series of animals allowed to survive until postnatal day 21 (PND 21), immunostaining was performed against several glial markers. Staining for the oligodendrocyte-specific proteins 2', 3'-cyclic nucleotide phosphodiesterase (CNP) and myelin proteolipid protein (PLP) was decreased in treated pups compared to shams within the corpus callosum, a white matter structure used as a representative area of developing white matter. Treated pups had activated astrocytes lining cerebral blood vessels, as observed by glial fibrillary acidic protein (GFAP) staining, while sham pups did not. Activated microglia were not detected using OX42 as a cell marker. Our model of intrauterine inflammation causes increased TUNEL and TNF-alpha staining early after injury, suggesting increased apoptotic cell death, possibly by cytokine-related mechanisms.     

Upregulation of Kv 1.4 protein and gene expression after chronic spinal cord injury.

After spinal cord injury (SCI), white matter tracts are characterized by demyelination and increased sensitivity to the K(+) channel blocker 4-aminopyridine (4-AP). These effects appear to contribute to neurological impairment after SCI, although the molecular changes in K(+) channel subunit expression remain poorly understood. We examined changes in gene expression of the 4-AP-sensitive voltage-gated K(+) channel Kv 1.4 after chronic SCI in the rat. Quantitative immunoblotting showed that Kv 1.4 protein was significantly increased at 6 weeks, but not at 1 week, after SCI in spinal cord white matter. Kv 1.4 was localized to astrocytes, oligodendrocytes, and oligodendrocyte progenitor cells but not to axons in both the normal and the injured spinal cord white matter. Because glial cells proliferate after SCI, we used immunogold electron microscopy to quantify Kv 1.4 protein in individual glial cells and found a sixfold increase of Kv 1.4 in cells of the oligodendrocyte lineage after chronic injury. Finally, quantitative in situ hybridization showed that Kv 1.4 mRNA was significantly upregulated in spinal cord white matter, but not gray matter, after SCI. In summary, we show that Kv 1.4 is expressed in glial cells and not in axons in the rat spinal cord white matter and that its expression is markedly increased in cells of the oligodendrocyte lineage after chronic SCI. Given that K(+) channels play a role in glial cell proliferation, cells exhibiting changes in Kv 1.4 expression may represent proliferating oligodendroglia in the chronically injured spinal cord.