Combination of metallic impregnation and autoradiography of brain sections

Summary After labelling with ¹⁴C-thymidine, frozen sections or paraffin sections of the brain of adult mice or rats were first stained by metallic impregnation and then coated with chrome alum gelatine and with an emulsion layer of about 10 m. On the autoradiographs ¹⁴C-tracks are readily recognized above labelled astrocytes or oligodendrocytes, and these can be well discriminated, if the sections are processed by the silver carbonate method of Rio-Hortega. In contrast, no labelling is obtained, if the gold chloride sublimate method of Cajal is applied.     

Constitutive expression of interleukin-1beta (IL-1beta) in rat oligodendrocytes

The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1beta mRNA, the type I IL-1beta receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1beta probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1beta probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1beta mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1beta. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1beta. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1beta is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.     

Immunohistochemical localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in adult bovine cerebrum and cerebellum

We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

All astrocytes are not created equal—the role of astroglia in brain injury

In two recent papers published in Nature Neuroscience and Cell Stem Cells, Magdalena Götz and colleagues shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behavior of astrocytes to chronic and acute brain injury.Astrocytes have important roles in the brain, for example by regulating neurotransmitter clearance, controlling the formation and maintenance of synapses, and by contributing to the blood–brain barrier (BBB; for a review see [1]). In addition, astrocytes respond to acute and chronic injury by hypertrophy and induced proliferation. Notably, astrocytes in the mammalian brain represent a highly heterogeneous population and the exact cellular identity of the astrocytic response in the damaged brain remains largely unknown (for a review see [2]). Thus, live-imaging and single-cell studies are required to unravel the complexity of astrocyte behaviour and distinguish between the good and the bad effects of astrocytic activation on brain function and tissue homeostasis in response to acute and chronic injury.It is thought that astrocytes respond to injury through hypertrophy of cell bodies and processes, upregulation of the intermediate filaments GFAP and vimentin, extension of processes, proliferation and gradual overlapping of astrocytic domains (for a review see [3]). Interestingly, it is known that although some aspects of the astrocyte response to injury can be detrimental—such as the formation of a glial scar—it can also be beneficial by limiting the invasion of immune cells into the brain parenchyma [4,5,6]. However, our understanding of the response of astrocytes to injury assumes a global homogeneous response, and an unawareness of the more complex and diverse in vivo situation. Two papers from the group of Magdalena Götz, published in Nature Neuroscience and Cell Stem Cell, begin to unmask the heterogeneity of the astrocyte response to injury through in vivo live imaging after brain injury and by using multiple lesion models and comparing their effects on astroglial behaviour and properties within the injured brain.In the first study, Bardehle et al used in vivo two-photon laser-scanning microscopy to monitor individual astrocytes for up to 28 days after a stab wound to the somatosensory cortex [7]. To visualize single cells, astrocytes were labelled using different lines: GLAST^CreERT2/eGFP or Confetti reporter, labelling 60–80% of all astrocytes; Aldh1l1-eGFP mice, labelling all astrocytes; and hGFAP-eGFP mice, labelling only those astrocytes with the highest GFAP expression. The authors found that most GFP⁺ astrocytes maintained their morphology after injury and that only subsets showed signs of hypertrophy and polarization towards the injury site. Interestingly, only a small population of astrocytes divided, all of which had their somata apposed to blood vessels (juxtavascular) and depended on proper functioning of the small RhoGTPase Cdc42 for their proliferative response. Strikingly, none of the labelled astrocytes migrated towards the lesion site, suggesting that the increase in GFAP reactivity often seen at the site of injury is not due to astrocyte migration, but rather is due to increased GFAP expression through hypertrophy, an increased number of proliferative cells and the upregulation of GFAP in cells that might not express detectable levels of GFAP before injury. Notably, migration of other glial cells (microglia and NG2⁺ glia) to the injury site was observed, suggesting that the migratory properties in response to injury in the brain might not be general to all glia. Thus, the contribution of activated astrocytes to the formation of a glial scar in the brain following injury might be limited and need to be reconsidered. In addition, the location of proliferating astroglial cells at juxtavascular positions, and their limited movement, suggest that these proliferating astrocytes might be a subset that is responsible for the ‘beneficial'' astrocytic response to injury by tightening the BBB, preventing the invasion of cells into the lesioned brain parenchyma. Thus, observing the glial response after brain injury in real time within their in vivo environment identified a highly selective and cell-specific astrocyte response, challenging previously held concepts of astroglial migration and massive astrocyte proliferation after injury.In the next study, Sirko et al analysed how the astroglial response varies between different types of acute or more chronic brain injury [8]. To this end the authors used four different models of injury: MCAo lesion (invasive), stab wound (invasive), APPPS1 mutation (non-invasive) and ectopic p25 activation in neurons (non-invasive). They analysed comparative data for reactive gliosis and induction of stem cell properties in activated astroglia found after brain injury (Figure 1). Interestingly, the two non-invasive, chronic lesion models induced the least response from astrocytes, with astrocytes undergoing hypertrophy but having low levels of proliferation and virtually no neurosphere-forming capacity, indicating that chronic injury in these models does not enhance astrocyte proliferation or acquisition of stem cell properties. In contrast, a much larger astrocytic response occurred in the invasive models, in which astrocytes not only underwent hypertrophy but also had a relatively high proliferative rate and formed multipotent and self-renewing neurospheres in vitro. The authors then showed that Sonic hedgehog (SHH) levels increased dramatically, but only in invasive models, and that SHH levels correlated with in vivo astrocyte proliferation rates and in vitro stem cell potential between injury conditions. By using pharmacological and genetic gain- and loss-of-function strategies, SHH signalling could indeed be identified as a crucial mediator of injury-induced acquisition of stem cell properties in astrocytes. Thus, Sirko et al identified substantial differences with respect to glial response between chronic and acute injury models and identified a molecular pathway (SHH) that at least partly accounts for enhanced astroglial response in invasive injury models.Open in a separate windowFigure 1Glial cell response, stem cell potential and extracellular Sonic hedgehog (SHH) levels vary depending on the type of brain injury. Astrocytes (yellow), NG2⁺ glial cells (blue) and microglia (red) reside in the uninjured intact brain, in which only NG2⁺ cells usually proliferate. When this tissue is studied in vitro to measure its stem cell potential, virtually no neurospheres are formed. After different types of injury, however, morphological and proliferative changes occur to all cells and their in vitro stem cell potential can be reactivated. In six-month-old APPPS1 mice, all glial cells change their morphology, with astrocytic and NG2⁺ hypertrophy of cell body and processes, and hypertrophy and reduction of processes in microglia. While few astrocytes proliferate, large amounts of proliferation ocurrs in both NG2⁺ glia and microglia. This tissue in vitro can form a few spheres that are self-renewing and multipotent, generating astrocytes, neurons and oligodendrocytes. In a model of neuronal death (CK/p25; overexpressing p25 in the postnatal forebrain), astrocytes and microglia change their morphology as described above. Astrocytes and NG2⁺ glia do not have any increase in proliferation rates, whereas microglia proliferate greatly. This tissue has little stem cell potential and makes only a few primary multipotent spheres. Finally, in the more invasive stab wound injury to the cortex, all glial cells become morphologically reactive, and astrocytes, NG2⁺ glia and microglia all proliferate in response. This tissue has the largest stem cell potential, capable of making both primary and secondary spheres with multipotent progeny. In each situation, the levels of SHH (green) can be correlated with the proliferation rates of astrocytes and in vitro stem cell potential, such that only in stab wound injury are SHH levels significantly upregulated. APPPS1, co-expresses mutated amyloid precursor protein 1 and mutated presenilin 1; NG2⁺, neuron-glial antigen 2.The two papers by the Götz group shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behaviour of astrocytes to chronic and acute brain injury. Surprisingly, only subsets of astrocytes proliferate or polarize, and none of them migrate towards the lesion. The juxtavascular position of proliferating astrocytes suggests that these cells might have access to the increase in SHH after invasive injury, which can regulate their division. However, it is not clear whether this proliferation is through their de-differentiation and acquisition of neural stem cell potential, or whether it is a result of a mature astrocyte division. That the astrocyte progeny remains with the original cell at the juxtavascular location suggests that they might be acting in a positive way to limit the migration of invading immune cells into the brain. Further studies on whether the increase in juxtavascular, astroglial proliferation affects the BBB permeability or decreases the number of invading cells will be important to understand this effect. If it turns out that enhanced astroglial proliferation might be generally beneficial for the injured brain, it is also tempting to speculate that for other brain injuries where the proliferation rates and SHH levels are reduced, enhanced glial proliferation in close proximity to blood vessels might help to reduce tissue damage and to improve regeneration and repair. Thus, SHH could represent a future therapeutic target to activate glial proliferation in the context of non-invasive, chronic brain injury. In any case, the acquisition of stem cell properties allowing astrocytes to form neurospheres in vitro is not directly tied to the in vivo use of these stem cell properties (for a review, see [9]). Whether the de-differentiation of astrocytes and proliferation of stem cells in vivo is beneficial or detrimental remains unclear. However, the new data have set the cellular framework for future studies to understand injury-induced astroglial stem cell characteristics in vivo and whether this in vitro potential might be unleashed for regenerative strategies in vivo.     

Hepatitis C Virus Neuroinvasion: Identification of Infected Cells

Hepatitis C virus (HCV) infection often is associated with cognitive dysfunction and depression. HCV sequences and replicative forms were detected in autopsy brain tissue and cerebrospinal fluid from infected patients, suggesting direct neuroinvasion. However, the phenotype of cells harboring HCV in brain remains unclear. We studied autopsy brain tissue from 12 HCV-infected patients, 6 of whom were coinfected with human immunodeficiency virus. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (CD68), oligodendrocytes (2′,3′-cyclic nucleotide 3′-phosphodiesterase), astrocytes (glial fibrillary acidic protein [GFAP]), and neurons (neuronal-specific nuclear protein); separated by laser capture microscopy (LCM); and tested for the presence of positive- and negative-strand HCV RNA. Sections also were stained with antibodies to viral nonstructural protein 3 (NS3), separated by LCM, and phenotyped by real-time PCR. Finally, sections were double stained with antibodies specific for the cell phenotype and HCV NS3. HCV RNA was detected in CD68-positive cells in eight patients, and negative-strand HCV RNA, which is a viral replicative form, was found in three of these patients. HCV RNA also was found in astrocytes from three patients, but negative-strand RNA was not detected in these cells. In double immunostaining, 83 to 95% of cells positive for HCV NS3 also were CD68 positive, while 4 to 29% were GFAP positive. NS3-positive cells were negative for neuron and oligodendrocyte phenotypic markers. In conclusion, HCV infects brain microglia/macrophages and, to a lesser extent, astrocytes. Our findings could explain the biological basis of neurocognitive abnormalities in HCV infection.     

Cell Reactions Following Acute Brain Injury: A Review

The proliferative behavior of glia following a cerebral stab wound in adult rats is reviewed. Proliferation was determined by both PCNA and [³H]thymidine labeling. Microglia were the first cells to divide and constituted the bulk of dividing cells. Both ramified and ameboid microglia divided. A smaller number of astrocytes entered the cell cycle a day later and were shown to derive from differentiated reactive cells. No differentiated oligodendroglia were labeled by thymidine, although a small number of dividing immature oligodendroglia could be detected in cultures of cells labeled in vivo. Recent studies of the properties of oligodendroglial precursors in brain repair mechanisms are discussed. The results so far support our conclusion that differentiated oligodendrocytes do not divide.     

S100 immunoreactive glial cells in the forebrain and midbrain of the lizard Gallotia galloti during ontogeny

We identified S100 immunoreactive cells in the brain of the lizard Gallotia galloti during ontogeny using immunohistochemical techniques for light and electron microscopy. In double labeling experiments with antibodies specific for S100A1 and S100B (anti-S100) and proliferative cell nuclear antigen (anti-PCNA), myelin basic protein (anti-MBP), phosphorylated neurofilaments (SMI-31), glial fibrillary acidic protein (anti-GFAP), or glutamine synthetase (anti-GS), we detected S100-like immunoreactivity in glial cells but never in neurons. Restricted areas of the ventricular zone were stained in the hypothalamus from E32 to postnatal stages, and in the telencephalon at E35, E36, and in adults. S100 immunoreactivity was observed predominantly in scattered PCNA-negative cells that increased in number from E35 to the adult stage in the myelinated tracts of the brain and had the appearance of oligodendrocytes. Quantitative analysis revealed that all of the S100-positive glial cells were GFAP-negative, whereas most of the S100-positive glial cells were GS-positive. Ultrastructurally, most of these S100-positive/GS-positive glial cells resembled oligodendrocytes of light and medium electron density. In adult lizards, a small subpopulation of astrocyte-like cells was also stained in the pretectum. We conclude that in the lizard S100 can be considered a marker of a subpopulation of oligodendrocytes rather than of astrocytes, as is the case in mammals. The S100-positive subpopulation of oligodendrocytes in the lizard could represent cells actively involved in the process of myelination during development and in the maintenance of myelin sheaths in the adult.     

Extracellular matrix of cultured glial cells: selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.     

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.Download video file.^{(159M, mp4)}     

Lectin binding by macroglial and microglial cells in the brains of young normal and myelin-deficient (md) mutant rats

To distinguish macroglia (oligodendrocytes and astrocytes) frommicroglia in the brain, ‘markers’ that have beenused in previous studies include carbonic anhydrase II (CAII)immunoreactivity for macroglia, and Ricinusand Bandeiraea(BS-I)lectins for microglia. However, in rats <1 week of age, manycells stained intensely with both anti-CAII and the labelledBS-I lectin. If some of the BS-I+/CAII+ cells were macroglia,and not microglia, BS-I should no longer be regarded as specificfor microglia. To confirm or rule out that possibility, lectinhistochemistry and double immunofluorescence staining were performedin tissue from the brains of normal young rats and from themicroglial cell-enriched brains of myelin-deficient mutant rats.BS-I+/ CAII+ cells were found and examined. The BS-I +/CAII-cellsresembled macrophages and microglia and did, indeed, differin sizes and shapes from the BS-I+/+cells. The BS-I+/CAII+ cellsappeared to represent CAII+ putative oligodendrocyte precursorsdescribed previously. Although less obvious, a lectin-bindingstructure was also observed in astrocytes. Lectins may cross-reactwith macroglial glycoproteins. For example, a glycoprotein foundin o ligodendrocytes and myelin, the myelin-associated glycoprotein(MAG), is related to the Ig superfamily and cell adhesion molecules.Therefore, it is cautioned that lectins and antibodies againstmembers of the latter families of proteins should be used ascell-type specific markers only if other parameters are alsoexamined. astrocytes carbonic anhydrase lectins microglia oligodendrocytes     

Morphological studies on neuroglia

Summary Autoradiographic studies showed that in the rat hippocampus microglia-like reactive cells (MRC) and astrocytes are capable of proliferation in response to kainic acid (KA)-induced lesions. A marked increase in the number of labeled MRC was observed during the first four days after the induction of the KA-lesion. A proliferative response of astrocytes occurred at two days after the KA-lesion. After the induction of a KA-lesion brain macrophages and oligodendrocytes were only slightly labeled with ³H-thymidine. It appears likely that MRC is the main cellular element responding to this type of lesion.This work was supported by a grant (No. 437002) from the Ministry of Education, Science and Culture, Japan     

The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.     

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Summary Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-streptavidin method, with either horseradish peroxidase or -galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Problems and experience in the light optical and histological presentation of glia cells]

1. 8 histological techniques and 13 modifications derived from those were tested on usefulness for the demonstration of glial cells in the adult rat brain. From these methods the impregnation techniques of Golgi-Kopsch, Valenzuela y Chacón and Rio del Hortega were modified according to a scheme of variance to find out the optimal variants. 2. The impregnation quality depends on the animal species, the animal age, the health of brains, the brain area, the balanced proportion of the treatment stages and the biochemical state of the glial cells. 3. The silver impregnation techniques are not so specific that only one glial type is stained, but one type prevails. The silver carbonate procedure according to Hortega allows to impregnate oligodendrocytes, microglial cells and astrocytes in frozen as well as in paraffin sections. The method of Golgi-Kopsch is more suited for oligodendrocytes and microglial cells than for astrocytes. Following the procedure of Valenzuela y Chacón especially astrocytes, but also microglial cells allow impregnation in both frozen and paraffin sections. 4. The different demonstration qualities of the proved methods call for critical examination of absolute measurements of cell size, length of processes and ramification density. 5. The presence of cell groups of different disposition towards impregnation within a glial type speaks for a biochemical inhomogeneity of the glial types.     

Inorganic mercury in human astrocytes,oligodendrocytes, corticomotoneurons and the locus ceruleus: implications for multiple sclerosis,neurodegenerative disorders and gliomas

Neurotoxic metals have been implicated in the pathogenesis of multiple sclerosis, neurodegenerative disorders and brain tumours but studies of the location of heavy metals in human brains are rare. In a man who injected himself with metallic mercury the cellular location of mercury in his brain was studied after 5 months of continuous exposure to inorganic mercury arising from metallic mercury deposits in his organs. Paraffin sections from the primary motor and sensory cortices and the locus ceruleus in the pons were stained with autometallography to detect inorganic mercury and combined with glial fibrillary acidic protein immunohistochemistry to identify astrocytes. Inorganic mercury was found in grey matter subpial, interlaminar, protoplasmic and varicose astrocytes, white matter fibrous astrocytes, grey but not white matter oligodendrocytes, corticomotoneurons and some locus ceruleus neurons. In summary, inorganic mercury is taken up by five types of human brain astrocytes, as well as by cortical oligodendrocytes, corticomotoneurons and locus ceruleus neurons. Mercury can induce oxidative stress, stimulate autoimmunity and damage DNA, mitochondria and lipid membranes, so its location in these CNS cells suggests it could play a role in the pathogenesis of multiple sclerosis, neurodegenerative conditions such as Alzheimer’s disease and amyotrophic lateral sclerosis, and glial tumours.     

A Brain-Specific Protein p25 Is Localized and Associated with Oligodendrocytes, Neuropil, and Fiber-Like Structures of the CA3 Hippocampal Region in the Rat Brain

Abstract: Developmental expression and cellular localization of a novel brain-specific 25-kDa protein (p25), a substrate of tau protein kinase II, were investigated in the rat brain using polyclonal antibodies raised against peptides synthesized based on the p25 amino acid sequence. By western immunoblotting, p25 was found to be expressed only slightly in the embryonic period; the expression increased from 11 days up to 5 weeks of age, and continued to increase gradually until 1–2 years of age. Immunohistochemistry revealed distinct staining of glial cells in most regions of the central nervous system in the adult rat brain. These positively immunostained cells were especially abundant in the white matter, such as the corpus callosum, cingulum, external capsule, and internal capsule. The glial cells were identified as oligodendrocytes, and the nuclei of the cells remained unstained. Whereas the neuropil in most parts of the brain was immunostained less intensely than glias, the neuropil in the first and second layers of the cerebral cortex and the dentate gyrus was relatively strongly stained. Fiber-like structures were also stained in the CA3 region of hippocampus.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Remyelination In Vitro Following Protein Kinase C Activator-Induced Demyelination

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2,3-cyclic nucleotide 3-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.