In vitro studies on the comparative sensitivities of cells of the central nervous system to diphtheria toxin

We have used tissue culture techniques and cell type-specific antibodies to compare the sensitivity of the various cell types of the white matter tracts of the rat central nervous system to in vitro exposure to diphtheria toxin (DTx). We have found that oligodendrocytes and Type 2 astrocytes (Raff et al. 1983a), which at least in the rat optic nerve, appear to be derived from a single bipotential progenitor cell (Raff et al. 1983b), are both more susceptible to DTx than are either Type 1 astrocytes or spinal neurones. The loss of oligodendrocytes and Type 2 astrocytes caused by exposure to DTx in vitro appeared to be irreversible. Even when cultures were maintained for a month following initial treatment with DTx, these glial populations were not reestablished, suggesting that precursors for these macroglial cell types were as sensitive to the effects of DTx as were the oligodendrocytes and Type 2 astrocytes themselves. Our results are discussed in the light of the failure of diphtheritic lesions to remyelinate in vivo.     

Flow microfluorometry detects IgM autoantibody to oligodendrocytes in multiple sclerosis

Freshly isolated canine oligodendrocytes were reacted by indirect membrane immunofluorescence with 44 sera from patients with multiple sclerosis (MS). Using analysis by flow microfluorometry (FMF), we found significant IgM antibody binding to the surfaces of oligodendrocytes in the MS sera. The fluorescence intensity of cell surface reactions for MS sera (F.I. > 40 = 37.2±21.34%) was significantly different (P < 0.001) to that for 53 sera from normal subjects (10.0±8.97%), 15 sera from patients with Murray Valley encephalitis (6.18±5.3%), 22 with brain tumours (6.28±5.3%), 25 with SLE (13.42±3.04%). Cell surface binding was and 7 miscellaneous neurological disorders (6.87±3.045). Cell surface binding was restricted to oligodendrocytes and was absorbed out by oligodendrocytes but not by liver cells or kidney cells. Oligodendrocytes were identified by conventional histology, phase-contrast optics, electron microscopy (EM) and by cell surface reactions with anti-galactocerebroside, a specific immunocytological marker for oligodendrocytes. A cell sort of the single 0° FMF scatter peak followed by EM examination confirmed that the reactive cells consisted exclusively of oligodendrocytes. Viability of oligodendrocytes before and after the staining reactions, was>80% as assessed by trypan blue and fluorescein diacetate exclusion. The possibility that immune reactions mediated by the surface-reactive antibody with readily accessible cell surface autoantigens in vivo may contribute to the loss of oligodendrocytes and demyelination in MS is raised.     

Suppression of remyelination in the CNS by X-irradiation

Summary Rat and cat spinal cords were exposed to 2000, 3000 or 4000 rads of x-irradiation prior to producing an area of primary demyelination in the dorsal columns by the injection of lysolecithin. In animals irradiated with 4000 rads no remyelination by either Schwann cells or oligodendrocytes occurred. With 2000 rads both types of remyelination occurred, but when compared to unirradiated controls, central remyelination was less extensive, while Schwann cells remyelinated a greater percentage of axons. With 3000 rads the results were variable, some animals responded similarly to those in the 4000 rad group, whereas others responded as the 2000 rad animals.Oligodendrocytes were found among the persistently demyelinated axons in the 4000 rad animals and their processes were associated with, but only rarely formed a myelin sheath round, the demyelinated axons. It was concluded that irradiation damage to local cells was responsible for the inhibition of remyelination but it could not be determined if this was due to its effect on the oligodendrocytes alone. The origin of the oligodendrocytes found among the demyelinated axons is discussed in this context.     

Concentrations of glutamate released following spinal cord injury kill oligodendrocytes in the spinal cord

We investigated in vivo in rats whether sufficient glutamate is released following spinal cord injury (SCI) to kill oligodendrocytes. Microdialysis sampling was used to establish the level of glutamate released (550 +/- 80 microM) in the white matter during SCI. This glutamate concentration was administered into the spinal cords of other rats and the densities of oligodendrocytes remaining 24 and 72 h later determined by counting cells immunostained with the oligodendrocyte marker CC-1. Administration of ACSF, 4.0 mM glutamate (estimated resulting tissue exposure 500 microM) and 10.0 mM glutamate by microdialysis reduced oligodendrocyte density 22%, 57%, and 74%, respectively, relative to normal at 24 h post-exposure. Therefore, sufficient glutamate is released following SCI to damage white matter. Oligodendrocyte densities near the fiber track were not significantly different at 72 h from 24 h post-exposure, so most glutamate-induced oligodendrocyte death occurs within 24 h after exposure. Injecting the AMPA/kainate receptor blocker NBQX into the spinal cord during glutamate administration reduced the glutamate-induced decrease in oligodendrocyte density, evidence for AMPA/kainate receptor involvement in glutamate-induced oligodendrocyte death. This work directly demonstrates in vivo that following SCI glutamate reaches concentrations toxic to white matter and that AMPA/kainate receptors mediate this glutamate toxicity to oligodendrocytes.     

Canine distemper virus does not infect oligodendrocytes in vitro

Dissociated canine brain cell cultures were infected with virulent canine distemper virus (CDV). Double immunofluorescent labelling was done to simultaneously demonstrate viral antigen and specific glial cell markers. Virus containing oligodendrocytes were not found at any stage of the infection. A certain proportion of the infected cells were shown to be astrocytes. It was concluded that CDV has no obvious tropism for oligodendrocytes which could explain the mechanism of demyelination in distemper in vivo.     

Immunocytochemical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. II. Electron microscopic study

Following a former immunohistochemical study in the rat brain [Arluison, M., Quignon, M., Nguyen, P., Thorens, B., Leloup, C., Penicaud, L. Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. I. Immunohistochemical study. J. Chem. Neuroanat., in press], we have analyzed the ultrastructural localization of GLUT2 in representative and/or critical areas of the forebrain and hindbrain. In agreement with previous results, we observe few oligodendrocyte and astrocyte cell bodies discretely labeled for GLUT2 in large myelinated fibre bundles and most brain areas examined, whereas the reactive glial processes are more numerous and often localized in the vicinity of nerve terminals and/or dendrites or dendritic spines forming synaptic contacts. Only some of them appear closely bound to unlabeled nerve cell bodies and dendrites. Furthermore, the nerve cell bodies prominently immunostained for GLUT2 are scarce in the brain nuclei examined, whereas the labeled dendrites and dendritic spines are relatively numerous and frequently engaged in synaptic junctions. In conformity with the observation of GLUT2-immunoreactive rings at the periphery of numerous nerve cell bodies in various brain areas (see previous paper), we report here that some neuronal perikarya of the dorsal endopiriform nucleus/perirhinal cortex exhibit some patches of immunostaining just below the plasma membrane. However, the presence of many GLUT2-immunoreactive nerve terminals and/or astrocyte processes, some of them being occasionally attached to nerve cell bodies and dendrites, could also explain the pericellular labeling observed. The results here reported support the idea that GLUT2 may be expressed by some cerebral neurones possibly involved in glucose sensing, as previously discussed. However, it is also possible that this transporter participate in the regulation of neurotransmitter release and, perhaps, in the release of glucose by glial cells.     

Mechanisms of brain injury in newborn infants associated with the fetal inflammatory response syndrome

The fetal inflammatory response syndrome (FIRS) is characterized by umbilical cord inflammation and elevated fetal pro-inflammatory cytokines. Surviving neonates, especially very preterm infants, have increased rates of neonatal morbidity including neurodevelopmental impairment. The mechanism of brain injury in FIRS is complex and may involve “multiple hits.” Exposure to in utero inflammation initiates a cascade of the fetal immune response, where pro-inflammatory cytokines can cause direct injury to oligodendrocytes and neurons. Activation of microglia results in further injury to vulnerable pre-myelinating oligodendrocytes and influences the integrity of the fetal and newborn's blood-brain barrier, resulting in further exposure of the brain to developmental insults. Newborns exposed to FIRS are frequently exposed to additional perinatal and postnatal insults that can result in further brain injury. Future directions should include evaluations for new therapeutic interventions aimed at reducing brain injury by dampening FIRS, inhibition of microglial activation, and regeneration of immature oligodendrocytes.     

Traumatic brain injury in the neonate, child and adolescent human: an overview of pathology

In the middle of the last century it had been thought that a good recovery of function and behavior would occur after traumatic brain injury (TBI) in very young human beings. A recent major change in thinking states that early childhood TBI may result in a severe compromise of normal brain growth and development such that TBI, rather, may compromise later normal development resulting in a need for very long term patient care and management. The mechanisms of injury and pathology within the injured brain are reviewed and compared between when injury occurs at or close to the time of birth, in an infant, in a young child, in a child between ages 5 and 10, in young and older adolescents and in young adulthood. Our understanding of pathophysiological responses by cells of the human central nervous system has recently greatly increased but has really only served to illustrate the great complexity of interactions between different types of cell within the growing and developing CNS. The hypothesis is developed that the outcome for a very young patient differs with the relative state of development of injured cells at the locus of injury. And that the potential for either repair, re-instatement of normal cellular and organ function or for continued normal development is much reduced after an early brain insult (EBI) compared with TBI in a slightly older child or young adult patient. The advent of increasingly sophisticated non-invasive imaging technology has allowed assessment of the influence and time course of brain pathology both early and late after TBI. This has generated greater confidence on the part of clinicians in forecasting outcomes for an injured patient. But our increased understanding has still not allowed development of therapeutic strategies that might ameliorate the effect of an injury. It is suggested that an improved integration of major clinical and scientific effort needs to be made to appreciate the import of multiple interactions between cells forming the neurovascular unit in order to improve any potential for post-traumatic recovery after TBI in neonates and young children.     

Unravelling the T-cell-mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG34-56 peptide in incomplete adjuvant

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein (MOG(34-56) ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG(34-56) displayed high production of IL-17A and specific lysis of MOG(34-56) -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG(40-48) presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG(34-56,) comprised CD4(+) CD56(+) and CD4(+) CD8(+) CD56(+) T cells. Furthermore, phenotypical analysis showed that CD4(+) CD8(+) CD56(+) T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.