Fate of developing astrocytes in the optic nerve of the myelin-deficient rat

There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelindeficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media. J. Comp. Neurol. 378:105–116, 1997. © 1997 Wiley-Liss, Inc.     

A distinct set of synaptic-like microvesicles in atroglial cells contain VGLUT3

There is increasing evidence for vesicular release of glutamate from astrocytes. We have previously demonstrated existence of VGLUT1 on astrocytic synaptic-like microvesicles (SMLVs) in several brain regions indicating a role in astroglial glutamate release. As VGLUT3 is prominently expressed in non-neuronal cells, this prompted us to investigate whether VGLUT3 is also involved in astroglial release of glutamate. Confocal microscopic investigations revealed that astrocytes in the hippocampus and the frontal cortex, as well as Bergmann glia in the cerebellum were labeled for VGLUT3. Immunogold cytochemistry showed that VGLUT3 gold particles were located over SMLVs in perisynaptic astrocytic and Bergmann glial processes. The specificity of the VGLUT3 immunoreactivity was demonstrated by abolished VGLUT3 labeling in astroglia in VGLUT3 knock-out mice. Double immunogold labeling showed that astrocytic processes contained labeling for VGLUT3 and VGLUT1, but the antibodies labeled separate subpopulations of vesicles in the processes. The ratio of gold particle densities between glial processes and nerve terminals were higher for VGLUT3 than for VGLUT1, suggesting that VGLUT3 is particularly abundant in astrocytic processes. Thus, our data show that VGLUT3 localizes to a distinct set of SMLVs in perisynaptic astroglial processes and suggest that VGLUT3 is important for glutamate release from astrocytes.     

Increased 14-3-3 immunoreactivity in glial elements in patients with multiple sclerosis

14-3-3 proteins have been shown to be increased in the cerebrospinal fluid from patients with several kinds of neurological diseases, including multiple sclerosis (MS). To investigate whether 14-3-3 proteins are closely related to the pathogenesis of MS, we performed immunohistochemical studies for 14-3-3 in autopsied brains from ten patients with MS, five patients with progressive multifocal leukoencephalopathy (PML), and seven normal control subjects. Formalin-fixed, paraffin-embedded sections from all cases were immunostained with a specific anti-14-3-3 antibody, and some sections from the MS cases were double-immunostained with antibodies raised against 14-3-3 and glial markers. In the normal control brains, 14-3-3 immunoreactivity was localized mainly in the neuronal somata and processes, and some glial cells showed only weak immunoreactivity. In the plaque lesions from the MS cases, the astrocytes and oligodendrocytes were intensely immunostained, and strong immunoreactivity was also found in some microglia and macrophages, most of which were located in the perivascular areas. In the PML brains, a similar immunolabeling pattern was observed in the demyelinated lesions, in which the astrocytes and oligodendrocytes exhibited dense 14-3-3 immunoreactivity. Our results suggest that 14-3-3 may be up-regulated in the glial cells, especially in astrocytes and oligodendrocytes, in patients with MS or PML. The exaggerated 14-3-3 accumulation in these glial elements may be associated with the pathogenesis of both demyelinating disorders.     

Muscarinic acetylcholine receptor-expression in astrocytes in the cortex of young and aged rats

The present report describes the cellular and subcellular distribution pattern of immunoreactivity to M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein, in astrocytes in the cerebral cortex of young and aged rats. Most M35-positive astrocytes were localized in the superficial layers of the cortex and part of the corpus callosum. At the ultrastructural level, immunoprecipitates were localized in the Golgi complexes, but the nucleus, rough endoplasmic reticulum, mitochondria, and microfilaments were generally free of labeling. Labeling was also present associated to the cell membrane, although without the characteristic immunoreactive postsynaptic membrane thickening found in neuronal structures. In aging rats of 30-34 months, the number of M35-labeled astrocytes doubled, whereas the neuronal staining slightly decreased in the same region in half of the animals studied. Fluorescent double-labeling for M35 and GFAP, an astrocytic microfilament protein, revealed that all M35-positive glial cells express GFAP and, conversely, that almost all GFAP glial cells were M35-immunostained. Based on the high incidence of coexpression of mAChRs and GFAP, both proteins may be functionally linked to each other. Rough semiquantitative estimates revealed that in young adult rats the GFAP/M35-immunoreactive astrocytes made up approximately one fifth of all cortical astrocytes. An important aspect of the presently demonstrated immunoreactivity of astroglia to mAChR proteins is its labeling in situ instead of in tissue culture. This finding may further support investigation, e.g., on anatomical relations of astroglia with neuronal and vascular elements, and its reactivity in experimental conditions. © 1993 Wiley-Liss, Inc.     

Reactive astroglia-neuron relationships in the human cerebellar cortex: A quantitative, morphological and immunocytochemical study in Creutzfeldt-Jakob disease

In order to investigate the role of neuron-glia interactions in the response of astroglial to a non-invasive cerebellar cortex injury, we have used two cases of the ataxic form of Creutzfeldt-Jakob disease (CJD) with distinct neuronal loss and diffuse astrogliosis. The quantitative study showed no changes in cell density of either Purkinje or Bergmann glial cells in CJ-1, whereas in the more affected CJ-2 a loss of Purkinje cells and an increase of Bergmann glial cells was found. The granular layer in both CJD cases showed a similar loss of granule cells (about 60% ) in parallel with the significant increase in GFAP+ reactive astrocytes. GFAP immunostaining revealed greater reactivity of Bergmann glia in CJ-2 than in CJ-1, as indicated by the thicker glial processes and the higher optical density. Granular layer reactive astrocytes were regularly spaced. In both CJD cases there was strict preservation of the spatial arrangement of all astroglial subtypes—Fañanas cells, Bergmann glia and granular layer astrocytes. Reactive Fañanas and Bergmann glial cells and microglia/macrophages expressed vimentin, while only a few vimentin+ reactive astrocytes were detected in the granular layer. Karyometric analysis showed that the increase in nuclear volume in reactive astrloglia was directly related with the level of glial hypertrophy. The number of nucleoli per nuclear section was constant in astroglial cells of human controls and CJD, suggesting an absence of polyploidy in reactive astroglia. Ultrastructural analysis revealed junctional complexes formed by the association of macula adherens and gap junctions. In the molecular layer numerous vacant dendritic spines were ensheathed by lamellar processes of reactive Bergmann glia. Our results suggest that quantitative (neuron/astroglia ratio) and qualitative changes in the interaction of neurons with their region-specific astroglial partners play a central role in the astroglial response pattern to the pathogenic agent of CJD.     

Evidence for a role for apoptosis in central pontine myelinolysis

We have performed an immunocytochemical study of autopsy material from five cases of central pontine myelinolysis (CPM) and five age and sex-matched control subjects with the aim of exploring the possible involvement of apoptotic mechanisms in oligodendrocytes in this condition. We searched for immunoreactivity of glial cells for the apoptotic-related markers death receptor (DR) 3, Bax and Bak, the anti-apoptotic marker Bcl-2 and the cell cycle marker Ki-67. The latter marker was studied because it is known that entry of cells into the cell division cycle can trigger apoptosis if it does not result in mitosis. In CPM there was marked up-regulation of expression of the myelination-related enzyme carbonic anhydrase isoenzyme II and also markedly increased expression of Ki-67 in nuclei of glial cells of all types but particularly those of cells morphologically resembling oligodendrocytes. Despite this, there was no clear increase in density of such cells in CPM. The pro-apoptotic markers Bax, Bak and DR3 were all modestly increased in glial cell cytoplasm in CPM, while there was no change in expression of Bcl-2, which was only sparsely detected in glial cells both in controls and cases of CPM. The ratio of pro- to anti-apoptotic factors would appear from this evidence to have altered in favour of apoptosis in glial cells, most of which had the appearance of oligodendrocytes and many of which expressed Ki-67. We interpret this preliminary study as favouring apoptosis as a mechanism of oligodendrocytic death in CPM. Further study of this mechanism in CPM may lead to identification of factors that could reduce or prevent this rare but serious and often fatal disease.     

Distribution of astroglia in glomeruli of the rat main olfactory bulb: Exclusion from the sensory subcompartment of neuropil

During an entire lifetime, sensory axons of regenerating olfactory receptor neurons can enter glomeruli in the olfactory bulb and establish synaptic junctions with central neurons. The role played by astrocytes in this unique permissiveness is still unclear. Glomerular astrocytes have been identified by immunocytochemistry for glial fibrillary acidic protein and S100 proteins at the light and electron microscopic levels. The latter labeling included submicroscopic lamellar and filopodial extensions of astroglial processes. Cell bodies and processes accumulate along the border between juxtaglomerular walls and glomerular neuropil. Within glomeruli, a network of astroglial processes encloses mesh-like neuropil zones devoid of astroglia. Electron microscopy confirmed the division into subcompartments of glomerular neuropil: 1) The “sensory-synaptic subcompartment” includes all sensory axon terminals and terminal dendritic branches receiving sensory input, whereas astroglia are excluded; 2) in the “central-synaptic subcompartment,” astroglial processes are intermingled with other neuropil components: dendrites of relay cells and interneurons, dendrodendritic synapses, centrifugal (cholinergic and serotonergic) axons, their axodendritic synapses, and blood vessels. Unevenly distributed astroglial processes in this subcompartment are attached to vascular basal laminae, stem dendrites, and subpopulations of dendrodendritic synapses, especially those colocalized with centrifugal projections (“triadic synapses”). Astroglia-free parts of the “central” subcompartment contain segments of dendrites and subpopulations of dendrodendritic synapses. Because of the subdivision of the glomerular neuropil into portions with and without glial components, glia do not completely demarcate the border between the “sensory” and the “central” subcompartments. Interdigitation between the subcompartments varies among glomeruli and even within a single glomerulus. The mesh width of astroglial networks covaries with numerical relations between sensory and dendrodendritic synapses. This distribution pattern of astrocytes suggests that these glial cells monitor brain-derived effects on olfactory glomerular neuropil rather than olfactory input and that astroglial processes are (re-)arranged accordingly. J. Comp. Neurol. 388:191–210, 1997. © 1997 Wiley-Liss, Inc.     

Uptake of [3H]GABA by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study

Uptake of [3H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [3H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galactocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [3H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by beta-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [3H]beta-alanine, which labeled only astrocytes. Oligodendroglial [3H]GABA uptake was Na+-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

Heterogeneity of astrocytes in human optic nerve head

Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuro pathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16–25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 1 astrocytes were present in the mature prelaminar and laminar regions. Glial celia expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and fINK-1/N-CAM and were identified as type lB astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GaIC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for a type lB astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment. © 1995 Wiley-Liss Inc.     

Schwann cell and oligodendrocyte remyelination in lysolecithin-induced lesions in irradiated rat spinal cord

Localised irradiation of adult rat spinal cord was achieved by implanting for 2 weeks a 192Ir pin alongside vertebral segments in the thoraco-lumbar region of the spinal column. Following removal of the implant, lysolecithin (LPC) was injected directly into the dorsal columns in order to induce demyelination in the most intensely irradiated segments of spinal cord. Eight weeks after LPC injection, remyelination was much less extensive in dorsal columns which absorbed more than 40Gy than in LPC lesions in less intensely irradiated spinal cords or in unirradiated animals. No oligodendrocytes, few astrocyte processes and little myelin debris lay among the demyelinated axons. However, capillary vessels were surrounded by astroglial end-feet so that the glial-limiting membrane remained intact in the demyelinated regions. There were some oligodendrocyte remyelinated fibres around the edges of the demyelinated zones but none among the naked axons. Schwann cells, which probably migrated into the lesions from the proximal segments of the dorsal roots, provided some fibres with myelin sheaths. These remyelinated fibres abutted demyelinated axons without an intervening glial limiting membrane or astrocyte process. Oligodendrocytes may fail to migrate into the demyelinated regions because of the scarcity of astrocyte processes. A possible explanation for the limited Schwann cell remyelination may be that the presence of astroglial end-feet around capillaries deprived Schwann cells of ready access to the demyelinated regions.     

Uptake [H]GABA by oligodendrocytes in dissociated brain cell culture: A combined autoradiographic and immunocytochemical study

Uptake of [³H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [³H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galatocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [³H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by β-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [³H]β-alanine, which labeled only astrocytes. Oligodendroglial [³H]GABA uptake was Na⁺-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

Early pathological changes in progressive multifocal leukoencephalopathy: a report of two asymptomatic cases occurring prior to the AIDS epidemic

Serial sections of formalin-fixed, paraffinembedded blocks from two asymptomatic, non-AIDS cases of progressive multifocal leukoencephalopathy (PML) were stained with a double-label immunocytochemical method for detection of glial fibrillary acidic protein and JC virus (JCV) capsid proteins and with luxol fast blue/hematoxylin-eosin. In case 1 small, rounded lesions of about 1-mm diameter were seen within a restricted area in the posterior part of the superior frontal gyrus of both cerebral hemispheres, suggesting an early manifestation of the disease. Fully developed demyelinated lesions of the classical type with JCV-infected oligodendrocytes appeared in the white matter and along its border with the cortex. Lesswell-developed lesions, believed to be precursors to the fully developed ones, were seen in the gray and white matter. Of special interest were areas which contained small collections of enlarged, glial fibrillary acidic protein (GFAP)-positive astrocytes without capsid antigen and which seemed to lack destruction of myelin as judged from the appearance of matching serial sections stained for myelin. Large lesions in the brain of case 2 showed the well-known features of advanced PML. The close relation between some astrocytes and oligodendrocytes with viral antigen raises the possibility of early intercellular passage of virus. Vacuolation, seen within or near lesions in both cases, has previously been noted in the CNS infected by HIV, but not in PML. It is suggested that PML, a disease of both oligodendrocytes and astrocytes, may actually begin in astroglial cells which, under the influence of a restricted JCV infection, become reactive, express GFAP and pass on virus to the more highly susceptible oligodendrocytes with which they are in contact.Supported in part by a grant N.S.07596 from the National Institute of Neurological Disorders and Stroke. The work was carried out in the Laboratory of Experimental Neurophathology, NINDS, and in the Department of Pathology II, Karolinska Institute, Stockholm     

Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study

Central nervous system (CNS) tissue was studied by immunocytochemistry and electron microscopy from three cases of multiple sclerosis (MS) in which evidence of ongoing myelin breakdown could be documented. The study focussed upon the role of glial cells in the pathogenesis of demyelination. In acute MS, demyelination involved the vesicular dissolution of myelin from intact axons and a paucity of fibrillary astrogliosis. Foamy macrophages, many of them probably derived from transformed and recently proliferated microglia, contained recognizable myelin debris and lipid droplets and were abundant throughout the lesions. These cells formed the major phagocytic population and stained positively for class II major histocompatibility complex antigens (HLA-DR; Ia). In acute MS lesions, rounded astrocytes were encountered which possessed membrane-bound compartments enclosing phagocytosed fragments of myelin basic protein-positive debris. Despite the superficial resemblance of these cells to foamy macrophages, the presence of intermediate filaments, glycogen granules and diffuse glial fibrillary acidic protein positivity supported an astroglial identity. Astrocyte processes were involved in myelin removal and invested recently demyelinated axons. Hypertrophic fibrous astrocytes were common in chronic active lesions, were capable of myelin degradation and on occasion, contained myelin debris attached to clathrin-coated pits. These astrocytes were sometimes Ia+. Oligodendrocytes were depleted from the center of active lesions but were numerous at the lesion margin, suggesting survival and proliferation. They stained positively for myelin-associated glycoprotein, a marker for immature oligodendrocytes. However, they were invariably Ia-. The findings confirm and further support a role for the astrocyte as both an antigen presenting cell and a phagocyte in the CNS during MS.     

Disruption of astroglial interlaminar processes in Alzheimer's disease

A palisade of long, interlaminar astroglial processes in supragranular layers of the cerebral cortex is characteristic of adult individuals of anthropoid species. In the present study, this distinctive cytoarchitectonic feature was analyzed in tissue deriving from the neocortex of cases affected by Alzheimer's disease (n=14) and age-matched control cases (n=10). Samples of different cortical areas, and in particular prefrontal, temporal and striate fields, were analyzed. Astroglia was labeled by glial fibrillary acidic protein immunoreactivity, that allowed a clear distinction between the classical, stellate intralaminar astroglia and the interlaminar glial processes. The occurrence and relative density of neuritic plaques were ascertained in the same specimens with Bielchowsky staining. In most cortical regions of cases diagnosed as severe Alzheimer's disease by the donor institutions, interlaminar astroglia was found to be markedly altered or absent, and replaced by hypertrophic intralaminar astrocytes. Cases diagnosed as milder or uncertain Alzheimer's disease showed a less consistent involvement of the interlaminar glial palisade. Alterations of the interlaminar palisade in the cortex affected by Alzheimer's disease did not strictly correlate with the density of neuritic plaques in the examined specimens. The findings indicate that loss/severe disruption of the interlaminar palisade of astroglial processes is part of the array of neuropathological changes occurring in the cerebral cortex during Alzheimer's disease. In addition, our data indicate that different types of neocortical astrocytes (namely intralaminar and interlaminar astrocytes) respond differently to the pathobiology of Alzheimer's disease in the neocortex, inasmuch as interlaminar processes tend to disappear while intralaminar processes become reactive.     

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.     

S-100 immunoreactivity in rat brain glial cultures is associated with both astrocytes and oligodendrocytes

S-100 protein, a Ca²⁺-binding protein of the EFhand type, is most abundant in the brain, and is involved in cell differentiation and the molecular mechanisms underlying cytoskeletal organization. We have investigated the immunocytochemical localization of S-100 protein in rat brain glial cultures prepared from the cerebral hemispheres of newborn rats. In mixed glial cultures, containing astrocytes type I and II and oligodendrocytes at various stages of differentiation, S-100 immunoreactivity was detected in all three cell types. Double immunofluorescence analysis revealed that in astrocytes, S-100 immunoreactivity was mainly colocalized with glial fibrillary acidic protein (GFAP), while in oligodendrocytes a close association with microtubular structures was observed. For immunoblot analysis, highly enriched oligodendrocytes and astrocytes were separately cultured for another week, and their extracts were analyzed by immunoblotting. The immunoblots of the cell extracts of both cell types showed a single S-100-immunoreactive polypeptide with an apparent molecular weight of approximately 12,000 daltons. Thus, the data presented here demonstrate that S-100 protein is not confined to astrocytes but occurs also in oligodendrocytes of rat brain. The close association with the oligodendroglial cytoskeleton suggests that this protein could also play a regulatory role in the organization of microtubules in oligodendrocytes and hence may be involved in the formation and maintenance of the myelin-containing membrane sheets. © 1995 Wiley-Liss, Inc.     

INVASION OF SCHWANN CELLS INTO THE SPINAL CORD OF THE RAT FOLLOWING LOCAL INJECTIONS OF LYSOLECITHIN

NVASION OF SCHWANN CELLS INTO THE SPINAL CORD OF THE RAT FOLLOWING LOCAL INJECTIONS OF LYSOLECITHIN
Focal areas of primary demyelination were induced in the spinal cord of rats by local injection of lysolecithin. In addition to loss of myelin there was also loss of astrocytes and oligodendrocytes in some parts of the demyelinated area. All demyelinated axons were subsequently remyelinated either by oligodendrocytes or by Schwann cells. Schwann cells remyelinated axons in the centre of the lesion and beneath the pia; the remaining axons were remyelinated by oligodendrocytes. By three months after the lesion the two types of remyelinated fibres were always separated from each other by astrocytes covered by basement membrane. It was concluded that the glial limiting membrane (basement membrane-covered astrocytes) normally inhibits Schwann cell entry into the spinal cord. When this is destroyed they can enter, but when it becomes re-established their free migration into the neuropil is once again inhibited.     

Transferrin: an early marker of oligodendrocytes in culture

In this study, the developmental pattern of transferrin expression, the iron transporting glycoprotein, was investigated morphologically and immunocytochemically in mixed glial cultures as well as pure cultures of mature oligodendrocytes, both derived from newborn rat brain. Double immunofluorescent labeling of pure oligodendrocyte cultures revealed that transferrin co-localizes with the oligodendroglial marker, myelin basic protein. During early development in mixed glial cultures, the presence of transferrin was detected at 3 days in vitro in small round process-bearing cells lying on top of astrocytes. These cells were galactocerebroside negative. However, at 7 days these process-bearing cells began to express galactocerebrosides and transferrin co-localized with the oligodendroglial marker. Transferrin did not co-localize with any neuronal or astroglial markers at any time. These results indicate that transferrin is an oligodendrocyte-specific marker which is expressed earlier than galactocerebroside.     

Localization of pore-forming subunit of the ATP-sensitive K(+)-channel,Kir6.2, in rat brain neurons and glial cells

Kir6.2, a subunit of the ATP-sensitive K(+) channel (K(ATP)), was localized in adult rat brain by immunohistochemistry and in situ hybridization. The Kir6.2 mRNA was widely expressed in most rat brain neuronal populations and nuclei examined, intensely in the mitral cell layer and tufted cells of the olfactory bulb, pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei and cuneate nuclei of the brain stem, moderately in the neocortex and cerebellar Purkinje cells, and weakly in the granular cell layer of the olfactory bulb and the granular layer of the cerebellum. In addition, glial cells also expressed the Kir6.2 gene weakly in the corpus callosum and cerebellar white matter. This wide localization of the gene was quite similar to that of Kir6.2 protein. Double stainings with anti-GFAP and anti-Kir6.2 antibodies were performed in this study. Glial cells showing immunoreactivity to both anti-Kir6.2 and anti-GFAP were confirmed to be astrocytes, and those showing only immunoreactivity to anti-Kir6.2 but not to anti-GFAP were presumed to be oligodendrocytes and confirmed by immunoelectron microscopy. Thus, it may be concluded that both oligodendrocytes and astrocytes contain Kir6.2. Under the electron microscope, we showed in vivo for the first time that the immunoreactive products were localized in the endoplasmic reticulum and Golgi apparatus as well as the plasma membranes of neurons and glial cells.     

Metallothionein I-II immunocytochemical reactivity in Binswanger's encephalopathy

Binswanger's disease is a subacute form of hypertensive encephalopathy characterized by patchy-confluent myelin loss of the deep hemispheric white matter, associated with marked regressive changes of the oligodendrocytes and variable astroglial reaction. To understand the distribution and the specificity of astrocyte pathology in Binswanger's disease we quantified reactive and degenerating astrocytes in different regions of the deep and subcortical white matter and of the cerebral cortex. Sections of frontal, temporal, parietal, and occipital lobes of 12 histologically proven cases of Binswanger's disease were immunostained with antibodies to glial fibrillary protein (GFAP) and to metallothionein I and II (MT-I-II), markers which specifically identify normal and reactive astrocytes. Control tissues were from 6 elderly patients without neurological diseases. The brains of Binswanger's disease were characterized by few and lightly immunostained astrocytes in the deep white matter, but normal and reactive astrocytes, strongly immunoreactive for GFAP and MT-I-II, were prominent in the subcortical white matter and the cerebral cortex. However, the relative distribution of GFAP positive and MT-I-II positive astrocytes was significantly different between the cerebral cortex and the subcortical white matter, the MT-I-II positive astrocytes being more frequent in the cerebral cortex, and the GFAP positive astrocytes in the subcortical white matter (p < 0.02). The GFAP and MT-I-II expressions in subsets of reactive astrocytes in the cortico-subcortical layers together with regressive astroglial changes in the deep white matter suggest that the dynamic plasticity of astroglia is topographically and biochemically differentiated in vascular dementia of Binswanger type.