Environmental enhancement of growth factor-mediated oligodendrocyte precursor proliferation

The timing of oligodendrogenesis depends on the specific location within the central nervous system, suggesting the local environment influences oligodendrocyte precursor proliferation. Spinal cord and optic nerve oligodendrocyte precursors both proliferate in response to platelet-derived growth factor (PDGF). Here we show that neurotrophin-3 (NT-3) enhanced PDGF-induced proliferation of optic nerve oligodendrocyte precursors, and these cells were labeled by an anti-trkC antibody. By contrast, NT-3 did not enhance PDGF-induced proliferation of spinal cord oligodendrocyte precursors, and these cells were not labeled by an anti-trkC antibody. Furthermore, PDGF-induced oligodendrocyte precursor proliferation was greater in spinal cord than in optic nerve cultures. The difference in NT-3 response between spinal cord and optic nerve oligodendrocyte precursors appears cell intrinsic, while the enhanced PDGF-induced proliferation of spinal cord oligodendrocyte precursors appears environmentally regulated. The spinal cord PDGF proliferation-enhancing activity may provide a mechanism to allow temporal and spatial regulation of gliogenesis.     

Induction of metallothionein in astrocytes and microglia in the spinal cord from the myelin-deficient jimpy mouse

Jimpy is a shortened life-span murine mutant whose genetic disorder results in severe pathological alterations in the CNS, including hypomyelination, oligodendrocyte death and strong astroglial and microglial reaction. The knowledge of metallothionein (MT) regulation in the CNS and especially of MT presence in specific glial cell types under pathological conditions is scarce. In the present study, immunocytochemical detection of MT-I + II has been performed in spinal cord sections from 10-12- and 20-22-day-old jimpy and normal animals. The identification of MT-positive glial cells was achieved through double labeling combining MT immunocytochemistry and selective markers for oligodendrocytes, astrocytes and microglia. MT was found in glial cells and was present in the spinal cord of jimpy and normal mice at both ages, but there were remarkable differences in MT expression and in the nature of MT-positive glial cells depending on the type of mouse. The number of MT-positive cells was higher in jimpy than in normal spinal cords. This was apparent in all spinal cord areas, although it was more pronounced in white than in the gray matter and at 20-22 days than at 10-12 days. The mean number of MT-positive glia in the jimpy white matter was 1.9-fold (10-12 days) and 2.4-fold (20-22 days) higher than in the normal one. Astrocytes were the only parenchymal glial cells that were positively identified as MT-producing cells in normal animals. Interestingly, MT in the jimpy spinal cord was localized not only in astrocytes but also in microglial cells. The occurrence of MT induction in relation to reactive astrocytes and microglia, and its role in neuropathological conditions is discussed.     

Efficacy and pharmacokinetics of a new intrarectal quinine formulation in children with Plasmodium falciparum malaria

The O-2A progenitor cell, which serves as a stem cell for the myelinating oligodendrocyte, has been implicated as a major target for radiation-induced spinal cord injury. In an attempt to increase the number of O-2A cells in the spinal cord, we applied an ex vivo gene therapy procedure for delivering platelet derived growth factor (PDGF). Recombinant fibroblasts expressing PDGF A chain were injected into the cisterna magna of adult rats, which resulted in cell seeding of the subarachnoid space of the cervical spinal cord. The number of O-2A progenitors in the cervical spinal cord was then assessed with an in vitro clonogenic assay. O-2A cells were found to be increased 8 days after recombinant cell injection, and they remained elevated up to at least 14 days. Analysis of O-2A colonies indicated that the implantation of PDGF-expressing cells increased the number of O-2A progenitors without affecting their in vitro proliferation potential or differentiation capacity. These data suggest that implantation of PDGF-expressing cells in the subarachnoid space of the cervical spinal cord may influence a stem cell population critical to the repair of demyelinated lesions.     

The use of transplanted glial cells to reconstruct glial environments in the CNS

Transplantation studies have demonstrated that glia-depleted areas of the CNS can be reconstituted by the introduction of cultured cells. Thus, the influx of Schwann cells into glia-free areas of demyelination in the spinal cord can be prevented by the combined introduction of astrocytes and cells of the O-2A lineage. Although Schwann cell invasion of areas of demyelination is associated with destruction of astrocytes, the transplantation of rat tissue culture astrocytes ("type-1") alone cannot suppress this invasion, indicating a role for cells of the O-2A lineage in reconstruction of glial environments. By transplanting different glial cell preparations and manipulating lesions so as to prevent meningeal cell and Schwann cell proliferation it is possible to demonstrate that the behaviour of tissue culture astrocytes ("type-1") and astrocytes derived from O-2A progenitor cells ("type-2") is different. In the presence of meningeal cells, tissue culture astrocytes clump together to form cords of cells. In contrast, type-2 astrocytes spread throughout glia-free areas in a manner unaffected by the presence of meningeal cells or Schwann cells. Thus, progenitor-derived astrocytes show a greater ability to fill glia-free areas than tissue culture astrocytes. Similarly, when introduced into infarcted white matter in the spinal cord, progenitor-derived astrocytes fill the malacic area more effectively than tissue culture astrocytes, although axons do not regenerate into the reconstituted area.     

Axonal and nonneuronal cell responses to spinal cord injury in mice lacking glial fibrillary acidic protein

We have examined the regeneration of corticospinal tract fibers and expression of various extracellular matrix (ECM) molecules and intermediate filaments [vimentin and glial fibrillary acidic protein (GFAP)] after dorsal hemisection of the spinal cord of adult GFAP-null and wild-type littermate control mice. The expression of these molecules was also examined in the uninjured spinal cord. There was no increase in axon sprouting or long distance regeneration in GFAP-/- mice compared to the wild type. In the uninjured spinal cord (i) GFAP was expressed in the wild type but not the mutant mice, while vimentin was expressed in astrocytes in the white matter of both types of mice; (ii) laminin and fibronectin immunoreactivity was localized to blood vessels and meninges; (iii) tenascin and chondroitin sulfate proteoglycan (CSPG) labeling was detected in astrocytes and the nodes of Ranvier in the white matter; and (iv) in addition, CSPG labeling which was generally less intense in the gray matter of mutant mice. Ten days after hemisection there was a large increase in vimentin+ cells at the lesion site in both groups of mice. These include astrocytes as well as meningeal cells that migrate into the wound. The center of these lesions was filled by laminin+/fibronectin+ cells. Discrete strands of tenascin-like immunoreactivity were seen in the core of the lesion and lining its walls. Marked increases in CSPG labeling was observed in the CNS parenchyma on either side of the lesion. These results indicate that the absence of GFAP in reactive astrocytes does not alter axonal sprouting or regeneration. In addition, except for CSPG, the expression of various ECM molecules appears unaltered in GFAP-/- mice.     

The relationship between serum gamma-glutamyl transpeptidase levels and hypertension: common in drinkers and nondrinkers

Lesions in CNS white matter involving loss of glial cells with concurrent destruction of the glia limitans lead to widespread remyelination of CNS axons by Schwann cells. Previous studies have demonstrated that this situation can be changed by transplanting cultured CNS glial cells into lesions early on in the repair process. In this study we have transplanted cultured astrocytes into the sub-arachnoid space above such a lesion in order to (1) influence the normal repair process by transplant-assisted reconstruction of the glia limitans, and (2) explore the potential of a minimally invasive route for introducing cells to white matter lesions. In some cases, it proved possible to influence normal repair by transplanting cells via the sub-arachnoid route, although the results were inconsistent. However, the experiment permitted observations to be made on the migration of transplanted astrocytes across the surface of and within the spinal cord.     

Calcium binding protein calcyphosine in dog central astrocytes and ependymal cells and in peripheral neurons

Calcyphosine is a calcium binding protein discovered in the dog thyroid in 1979. Calcyphosine mRNA and immunoreactivity were detected using Western and Northern blotting in the cerebral cortex, cerebral white matter and cerebellum. Using immunohistochemistry and in situ hybridization, both are present in ependymal cells, choroid plexus cells and several types of astrocytes of the subependymal cerebral layer, the cerebellar Bergmann layer, the retinal ganglion cell layer, the optic nerve and the posterior pituitary. Both are also present in neurons of nasal olfactory mucosa, enteric Auerbach and Meissner plexuses, orthosympathic and spinal cord ganglia as well as in endocrine cells of neural crest origin in the adrenal medulla. Calcyphosine immunoreactive astrocytes were also present mainly in hemispheric cerebral gray and white matter, hemispheric subcortical structures, brain stem and spinal cord. These results show that calcyphosine is a characteristic calcium binding protein of astrocytes and ependymal cells in the central nervous system and of neurons in the peripheral nervous system. This is of interest in view of the importance of calcium regulation in these cells, and since calcyphosine a calcium binding protein phosphorylated by cAMP dependent process, may be an intermediate between cAMP and inositol phosphate cascades.     

Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats

Following spinal cord contusion in the rat, apoptosis has been observed in the white matter for long distances remote from the center of the lesion and is primarily associated with degenerating fiber tracts. We have previously reported that many of the apoptotic cells are oligodendrocytes. Here we show that the oligodendrocyte death is maximal at 8 days postinjury and suggest that loss of oligodendrocytes may result in demyelination of axons that have survived the initial trauma. There are two mechanisms that may account for the observed oligodendrocyte apoptosis. The apoptotic cell death may result from the loss of trophic support after axonal degeneration or it may be the consequence of microglial activation. The hypothesis that oligodendrocyte apoptosis is secondary to microglial activation is supported by our observations of microglia with an activated morphology in the same regions as apoptosis and apparent contact between some of the apoptotic oligodendrocytes and microglial processes. In addition to oligodendrocyte apoptosis, a subpopulation of microglia appears to be susceptible to apoptotic cell death as well, as evidenced by the presence of apoptotic bodies in OX42 immunopositive profiles. Thus, the population of apoptotic cells following spinal cord contusion is comprised of oligodendrocytes and putative phagocytic microglia or macrophages. Given the delayed time course of oligodendrocyte death, the apoptotic death of oligodendrocytes may be amenable to pharmacological intervention with subsequent improvement in functional recovery.     

Notochord is essential for oligodendrocyte development in Xenopus spinal cord

Oligodendrocyte precursors originate in the ventral ventricular zone of the developing spinal cord. To examine whether the notochord is essential for the development of oligodendrocytes in Xenopus spinal cord the notochord was prevented from forming, ablated, or transplanted during early stages of development. Differentiated oligodendrocytes did not appear in spinal cord regions lacking a notochord in animals in which notochord failed to develop after UV irradiation at the one-cell stage. Similarly, differentiated oligodendrocytes were not detected in the spinal cord adjacent to the site of segmental notochord ablation at embryonic or larval stages. Transplantation of an additional notochord dorsal to the spinal cord induced the premature appearance of differentiated oligodendrocytes in adjacent lateral and dorsal spinal cord white matter. These results indicate that the development of Xenopus spinal cord oligodendrocytes is dependent on local influences from the notochord and suggest that the notochord is essential for oligodendrocyte development in Xenopus spinal cord.     

Identification of macrophage migration inhibitory factor (MIF) in rat spinal cord and its kinetics on experimental spinal cord injury

BACKGROUND: Among spinal cord injuries, secondary injury is considered to be a "reversible" process and seems to be a key target for the treatment of spinal cord injury. Recently, macrophage migration inhibitory factor (MIF) has been reevaluated as being one of the most important cytokines which act during wound healing, proliferation and differentiation of cells. However, the expression of MIF in the spinal cord has not been investigated yet. PURPOSE: The purpose of this paper is to demonstrate the MIF expression in normal rat spinal cord and to evaluate the kinetics of MIF after spinal cord injury. MATERIALS & METHODS: Female Wistar (280-320 g) rats were studied. Spinal cord injury was made by the clip compression method at the level of C7/Th1 (56 g, For 1 min.). The expression of MIF was examined by immunohistochemistry and northern blot analysis. MIF content in the cerebrospinal fluid (CSF) was measured by enzyme-linked immunosorbent assays (ELISA). Furthermore, to examine the MIF function on neuronal cell, cell proliferation assay (MTS assay) was carried out using PC12, pheochromocytoma cell line, and LN444, glioblastoma cell line, in the presence of anti-MIF monoclonal antibody. RESULTS: MIF stain was positive in normal rat spinal cord white matter. The expression of MIF decreased between 1 hour and 6 hours after injury. It was found to have re-appeared 24 hours after injury. The kinetics of MIF mRNA expression showed reverse-correlation with those of the MIF positive stain. MIF content in CSF was found to be elevated soon after injury. MTS assay suggested that MIF had some proliferative function on neuronal cells. CONCLUSION: MIF exists in the rat white matter. And it's immediately released into the CSF and then re-synthesized 24-hr after injury. MIF shows a cell proliferative function on neuronal cells. These results suggest that MIF plays an important role for secondary spinal cord injury.     

Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes

Microglial reactivity associated with induction of MHC class II (HLA-DR) antigen is a sensitive indicator for pathological events in the CNS. To assess the response of glial cells after lesions of supraspinal descending tracts, HLA-DR, CD68 and GFAP were studied immunohistochemically on spinal cord tissue of 5 patients who died after unilateral infarction of the middle cerebral artery territory, and 5 control cases. In patients who died shortly after a stroke (4-14 days) increased HLA-DR-immunoreactivity (HLA-DR-IR) could be observed in the intermediate grey matter and in the ventral horn. The CD68-IR was much less intense. After longer survival times (5 weeks to 4 months). HLA-DR-IR in the grey matter was clearly lower than that observed in the spinal cord of short survival times, but very abundant in the dorsolateral funiculus, specifically within the corticospinal tract. In white matter areas, CD68-IR was almost identical to the HLA-DR-IR. Within the grey matter, CD68-IR was similar to the control tissue. A moderate increase of GFAP-positive astrocytes could be seen only in the grey matter after longer survival times. It seems probable, that the dynamics of HLA-DR-positive microglia reflect the early phagocytosis of presynaptic terminals by microglia in target regions of descending fibre tracts. In the white matter, the removal of degenerating axons by phagocytosing microglia expressing HLA-DR and CD68 antigens is a slower process which occurs over a period of months.     

Density-dependent feedback inhibition of oligodendrocyte precursor expansion

The myelin sheath in the vertebrate CNS is formed by oligodendrocytes. The number of oligodendrocytes in a mature axon tract must be sufficient to myelinate all appropriate axons. How the number of oligodendrocytes is matched to axonal requirements and whether such matching involves axon-oligodendrocyte signaling or intrinsic oligodendrocyte self-regulation are not clear. Using a combination of in vitro analyses, we demonstrate that oligodendrocyte precursors closely regulate their numbers through interactions between adjacent precursors. In low-density rat spinal cord cultures, the number of oligodendrocyte lineage cells increases rapidly. The addition of large numbers of oligodendrocyte precursors substantially reduces precursor expansion and results in a normalization of oligodendrocyte lineage cell numbers in the cultures over time. Thus, the number of oligodendrocyte lineage cells that develop appears dependent on the density of oligodendrocyte lineage cells. This normalization of cell number is reflected in assays of clonal potential and proliferation. For example, precursors gave rise to fewer progeny and proliferated less at high density. Reduced precursor expansion at high density was not attributable to the depletion of growth factors. Cocultures of high and low densities did not inhibit precursor expansion in low-density cultures, suggesting the requirement for local cell-cell interactions. The inhibition of precursor expansion was cell-type-specific and dependent on the presence of oligodendrocyte lineage cells. We propose that this density-dependent feedback inhibition of oligodendrocyte precursor expansion may play a primary role in regulating the number of oligodendrocytes in the developing spinal cord.     

Failure of spinal cord oligodendrocyte development in mice lacking neuregulin

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/-) in a temporal sequence of developmental events that replicates that observed in vivo. However, in spinal cord explants derived from mice lacking neuregulin (NRG -/-), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG -/- mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG -/- explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.     

Role of group I metabotropic glutamate receptors in traumatic spinal cord white matter injury

Metabotropic glutamate receptors (mGluRs) participate in glutamate neural transmission, but their role in the pathophysiology of spinal cord injury (SCI) has not been explored. Accordingly, we examined the role of group I mGluRs, which are linked to phospholipase C, in mediating SCI using an in vitro model. A dorsal column segment was isolated from the spinal cord of adult rats, maintained in vitro, and injured by compression for 15 sec with a clip having a 2 g closing force. Under control conditions after SCI, the compound action potential (CAP) amplitude was reduced to 69.1 +/- 5.4% of baseline. Blockade of group I mGluR receptors with MCPG, 4CPG, or AIDA resulted in improved recovery of CAP amplitude (82.2 +/- 2.0%, 86.2 +/- 3.9%, and 86.0 +/- 2.5% of baseline, respectively). The group I/II agonist trans-ACPD and selective group I agonist DHPG exacerbated the posttraumatic reduction of CAP amplitude. The phospholipase C inhibitor U-73122 improved recovery of CAP amplitude after traumatic spinal cord axonal injury. Western blotting and immunocytochemistry demonstrated the presence of mGluR1alpha-immunopositive astrocytes and the absence of mGluR5 in spinal cord white matter. These studies are consistent with the hypothesis that activation of group I mGluR receptors after SCI exacerbates posttraumatic axonal injury through a phospholipase C dependent mechanism. The presence of mGluR1alpha labeling on astrocytes suggests a role for these cells in the pathophysiology of SCI. Additional studies in vivo, are required to further clarify the role of mGluRs in acute traumatic SCI.     

Blood-spinal cord barrier function and morphometry after single doses of x-rays in rat spinal cord

PURPOSE: The effects of irradiation on blood-spinal cord barrier (BSCB) function and ultrastructure were evaluated using a rat spinal cord model. METHODS AND MATERIALS: Rats received a single dose of 25 Gy to the cervical spinal cord (C2-T2). At various times following irradiation and before the onset of paralysis, BSCB function was assessed using horseradish peroxidase (HRP) as a vascular tracer, and barrier-related structural changes in the capillaries were evaluated using morphometric techniques. RESULTS: Focal extravasation of HRP was seen at 93 days after irradiation, and extensive extravasation was apparent by 114 days in white matter, but not in gray matter. At 93 days, pathologic changes apparent by light microscopy were very minor in the white matter of the irradiated segment. By 107 days, myelin beading, Wallerian degeneration, edema, and histiocytes were apparent in white matter, and these features became increasingly prominent over the following weeks. No noteworthy changes were seen in gray matter at these times. Electron microscopic examination showed that, during the first 93 days following irradiation, more than half of the endothelial cells in white matter had disappeared (p < 0.05). In terms of the putative vascular pores, no abnormalities in endothelial junctions (the presumed small pore) were found, but there was an increase in the density of endothelial vesicles (a putative form of the large pore) in irradiated white matter (p < 0.001), but not in gray matter. Pericytes, thought to act as a second line of defence in the blood-brain barrier, increased in size but not in number in the irradiated white matter of the spinal cord. CONCLUSION: We suggest that radiation damage to endothelial cells, which form the BSCB prior to the onset of neurological deficit, may play an important role in the pathogenesis of white matter necrosis.     

Glial cell responses, complement, and clusterin in the central nervous system following dorsal root transection

We have examined the glial cell response, the possible expression of compounds associated with the complement cascade, including the putative complement inhibitor clusterin, and their cellular association during Wallerian degeneration in the central nervous system. Examination of the proliferation pattern revealed an overall greater mitotic activity after rhizotomy, an exclusive involvement of microglia in this proliferation after peripheral nerve injury, but, in addition, a small fraction of proliferating astrocytes after rhizotomy. Immunostaining with the phagocytic cell marker ED1 gradually became very prominent after rhizotomy, possibly reflecting a response to the extensive nerve fiber disintegration. Lumbar dorsal rhizotomy did not induce endogenous immunoglobulin G (IgG) deposition or complement expression in the spinal cord dorsal horn, dorsal funiculus, or gracile nucleus. This is in marked contrast to the situation after peripheral nerve injury, which appears to activate the entire complement cascade in the vicinity of the central sensory processes. Clusterin, a multifunctional protein with complement inhibitory effects, was markedly upregulated in the dorsal funiculus in astrocytes. In addition, there was an intense induction of clusterin expression in the degenerating white matter in oligodendrocytes, possibly reflecting a degeneration process in these cells. The findings suggest that 1) complement expression by microglial cells is intimately associated with IgG deposition; 2) axotomized neuronal perikarya, but not degenerating central fibers, undergo changes which induce such deposition; and 3) clusterin is not related to complement expression following neuronal injury but participates in regulating the state of oligodendrocytes during Wallerian degeneration.     

Glucocorticoid receptors and actions in the spinal cord of the Wobbler mouse, a model for neurodegenerative diseases

We have studied glucocorticoid receptors (GR) and actions in the spinal cord of the Wobbler mouse, a model for amyotrophic lateral sclerosis and infantile spinal muscular atrophy. Basal and stress levels of circulating corticosterone (CORT) were increased in Wobbler mice. Single point binding assays showed that cytosolic type II GR in the spinal cord of Wobbler mice of both sexes were slightly reduced compared with normal littermates. Saturation analysis further demonstrated a non-significant reduction in Bmax with increased Kd. In the hippocampus, however, we found down-regulation of GR, a probable response to increased CORT levels. We also found that the basal activity of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, was higher in Wobbler mice than in control animals. Both groups showed a two-fold stimulation of ODC activity after treatment with dexamethasone (DEX). Additionally, Wobbler mice presented with an intense proliferation of astrocytes immunoreactive (ir) for glial fibrillary acidic protein (GFAP) in grey and white matter of the spinal cord. The enhanced GFAP-ir was attenuated after four days of treatment with a corticosterone (CORT) pellet implant, producing a pharmacological increase in peripheral circulating CORT. Taking into consideration the content of GR and the changes in ODC activity and GFAP-ir brought about by glucocorticoids, we suggest that Wobbler mice are hormone responsive. Further elucidation of glucocorticoid effects in this model may be relevant for understanding the possible use of hormones in human neurodegenerative diseases.     

Alterations in temporal/spatial distribution of GFAP- and vimentin-positive astrocytes after spinal cord contusion with the New York University spinal cord injury device

Astrocytes become reactive as a result of various types of lesions and upregulate 2 intermediate filaments, glial fibrillary acidic protein (GFAP), and the developmentally regulated protein vimentin. Young female Sprague-Dawley rats were subjected to a spinal cord contusion at segment T10 using the New York University injury device. Animals were killed at 1, 2, 7, 14, and 30 days postinjury. Horizontal spinal cord sections spanning segments T7-T13 were assessed with antibodies to both intermediate filament proteins. The number of gray matter GFAP-positive astrocytes increased by 2 days postinjury, with segments adjacent (proximal) to the injury site showing greater responses than areas several segments away (distal). By 30 days following injury, astroglial cell numbers returned to normal levels. Vimentin-positive astrocytes also showed a graded proximal/distal response by 2 days following injury. Proximal regions remained significantly higher at 30 days following injury than control animals. Rostral/caudal changes were also evident, with regions caudal to the injury showing significantly higher numbers of vimentin positive astrocytes than those rostral, indicating that gray matter areas caudal to spinal cord injury may undergo more stress following spinal cord injury.