In vitro hormonal regulation of astrocyte proliferation.

Previously, we showed that purified myelin basic protein (MBP) induces a two-fold increase in the proliferation rate of astrocytes in culture. This observation allowed us to hipothesize that MBP-induced astroglial proliferation might be one of the causes of astrogliosis and astroglial scar formation in case of in vivo myelin breakdown. Also, we observed that dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEA-S) reduce the amount of 3H-Thymidine incorporated by cultured astrocytes. In the present study, we investigated by a combined 3H-Thymidine autoradiography/immunocytochemistry technique whether the mitogenic effect that MBP exerts upon astrocytes in vitro can be prevented by DHEA and DHEA-S. Results showed that a treatment of the cells with MBP only induces approximately a two-fold increase in the number of silver grains overlaying the nuclei of astrocytes. In cultures treated with both MBP and DHEA there was a 2.5-3 fold reduction in the number of silver grains, while DHEA-S provoked a 3-4 fold reduction. These results allow us to speculate that DHEA and DHEA-S could modulate the astrogliosis that usually accompanies myelin breakdown.

Theiler's virus-associated antigens on the surfaces of cultured glial cells.

Infection of the central nervous system by Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, produces chronic demyelinating disease in susceptible mice. In this immunoelectron microscopic study of TMEV infection of neonatal mouse brain cells in culture, TMEV antigen was found on the surfaces of infected oligodendrocytes and astrocytes by labeling with hyperimmune serum from TMEV-infected mice or with rabbit antiserum to purified inactivated DA strain TMEV. Brain-derived macrophages had no TMEV-specific antigen on their surfaces and were not able to maintain productive TMEV infection, even though TMEV antigen was present in the cytoplasm. The presence of TMEV antigens on the surfaces of oligodendrocytes (myelin-producing cells) was unexpected because picornaviruses are nonenveloped viruses and do not bud from cell surfaces. The finding is consistent with the hypothesis that demyelination follows damage of infected oligodendrocytes by immune cells or immunoglobulins that recognize surface virus antigen.

Expression of glial fibrillary acidic protein by differentiated astrocytes is regulated by serum antagonistic factors.

We report here that, in culture, the expression of glial fibrillary acidic protein (GFAP) by astrocytes, as well as their shape (flat-polygonal vs. stellate) can be regulated by 4 serum antagonistic factors. Three of these factors are stimulatory, while the fourth exerts an inhibitory effect upon these astrocytic properties. As suggested by temperature and trypsin treatments, the inhibitory factor is a polypeptide or a protein of 15-35 kDa. The stimulatory factors are smaller: two of them have a mol. wt. between 0.2 and 5 kDa; the third is smaller than 0.2 kDa. Treatments with chloroform/methanol, ammonium sulfate, neuraminidase, and papain, indicate that at least one glycolipid and one glycoprotein are involved. We speculate that, during development, cells from the astrocytic line could be susceptible selectively to one or another of these factors, which would explain their great plasticity.

Serum contains inducers and repressors of oligodendrocyte differentiation.

An important stage in oligodendrocyte development is the expression of galactocerebroside (GC), the major glycolipid in myelin. Although oligodendrocyte cell lineage and differentiation in vitro have been the object of many studies, to date there is sparse information on the regulation of GC expression in oligodendrocytes already committed to be positive for GC. We report here that GC expression in these cells is controlled by three serum factors. Two of these, possibly a lipoprotein and a mucoprotein, increase GC levels, whereas the third, probably a glycoprotein, exerts an inhibitory effect. The developmental increase of GC in postnatal rat brain cerebral cultures and its induction by serum factors are reversible phenomena. The isolation of the GC-regulatory factors would allow experimental manipulation of impaired GC expression by differentiated oligodendrocytes.

Serum antibodies to central nervous system antigens: an analysis of their relation with different human neurologic disorders.

In a previous paper we have presented a double ligand enzyme linked immunosorbent assay (ELISA) technique suitable for the detection of human antibodies to different brain antigens. In the present study, we have applied this technique to the analysis of 100 neurologically affected patients with regard to both a list of clinical parameters and the presence in their sera of nervous tissue specific antibodies, in an attempt to highlight the meaning of such antibodies in different neurologic disorder. We show that the presence of these antibodies cannot be used for elucidation of pathogenesis or for diagnostic purposes, but can be used as a prognostic index.

Buffers and H2O2 reduce neuronal death and/or enhance differentiation of neurons and astrocytes in dissociated mouse brain cultures.

Tissue of the mammalian central nervous system (CNS) undergoes complex and uncoordinated pathological responses upon injury. Efforts to develop pharmacological approaches to achieve functionally meaningful regeneration largely have been unsuccessful. Assuming that anoxia and drop in tissue H are initiating factors in most pathological sequences consequent to CNS injury, we studied the effects of increasing the buffering and oxygenating capacities of the medium on dissociated embryo brain cultures. The presence of H2O2 in the medium led to greatly enhanced neuronal survival and/or differentiation. Increased buffering capacity favored enhanced neurite outgrowth with remarkable elongation of fibers. A combination of the two gave a synergistic effect in which both of the above responses were seen. Both buffers and H2O2 enhanced astrocytic differentiation and extension of processes while reducing DNA synthesis. The results favor the view that attempts to encourage self-repair in CNS tissue or to enhance repair of CNS damage with potential therapeutic agents or procedures should be carried out in the context of a near optimal environment in which, at the least, pH and pO2 values are stably maintained within normal operational limits.

Effects of dehydroepiandrosterone and its sulfate on brain tissue in culture and on memory in mice.

Low concentrations of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) enhanced neuronal and glial survival and/or differentiation in dissociated cultures of 14-day mouse embryo brain. Posttrial intracisternal injection into the brains of mice undergoing active avoidance training alleviated amnesia and enhanced long-term memory. By minimizing degenerative changes in injured nerve tissue and facilitating plastic changes, DHEA and DHEAS may be of use in treatment of neurodegenerative and memory disorders in man.

Dehydroepiandrosterone and its sulfated derivative reduce neuronal death and enhance astrocytic differentiation in brain cell cultures.

Human studies of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) have shown age-related changes in serum levels of these two sex hormone precursors. The levels of both DHEA and DHEA-S are characterized by monotonic decreases after puberty in females and after 20-24 yr of age in males. Further studies have shown that DHEA and DHEA-S levels are significantly low or close to minimal at ages when the incidence of senile dementia of Alzheimer's type (SDAT) begins to increase. We propose that DHEA and DHEA-S play a significant role in normal function of neuronal cells and that supplementation with them may prevent neuronal loss and/or damage. In the present study, using methods of immunocytochemistry, autoradiography, and scanning electron microscopy, we show that a supplement of as little as 10(-8) M DHEA or DHEA-S greatly increases neuronal survival and differentiation and reduces astroglial proliferation rates in mouse brain cells in cultures. These results suggest that correcting the DHEA and the DHEA-S deficit may prevent and/or improve the SDAT condition in humans.

Theiler's virus in brain cell cultures: lysis of neurons and oligodendrocytes and persistence in astrocytes and macrophages.

The mechanisms of persistence and of demyelination in Theiler's virus (TV)-induced chronic neurologic disease (a murine model for multiple sclerosis) are, as yet, disputed. We investigated the tropism and persistence of TV in brain cell culture to better understand the pathogenesis of this disease. Using anti-genic markers to identify specific cells in culture, we have demonstrated that TV infects, lytically, neurons and oligodendrocytes and persistently astrocytes and macrophages. These results suggest that host cell factors play a key role in the mechanism of demyelination and the persistence of TV in the nervous system.

Regulation of myelin basic protein in oligodendrocytes by a soluble neuronal factor.

Myelin basic protein (MBP) is one of the most important myelin components. Based on our previous studies, we hypothesized that neurons might have regulatory effects on the production of MBP by oligodendrocytes, and we conducted studies designed to verify this hypothesis. Oligodendroglia-rich cultures from total brain of neonatal rats or mice and pure cultures of embryonic rats or chicks were prepared. Cultures of mouse fibroblasts and astrocytes were prepared as well. We show here that MBP production by oligodendrocytes was greatly enhanced by treatment with either pure neurons, rat neuronal conditioned medium, or chick neuronal conditioned medium, while chemically defined, hormonally supplemented medium or medium conditioned by astrocytes and fibroblasts had no effect on MBP expression. We conclude that the production of MBP by oligodendrocytes is regulated by a nonspecies specific soluble neuronal factor. The conservation of this phenomenon from avian to rodent species implies its critical role in myelination and suggests its potential application as a treatment in demyelination.

Myelin basic protein stimulates the proliferation of astrocytes: possible explanation for multiple sclerosis plaque formation.

In dissociated mouse brain cell cultures we frequently observed an association between myelin basic protein (MBP) positive oligodendrocytes and proliferating astrocytes. When MBP was added in a purified form to the culture medium, it greatly stimulated the proliferation of astrocytes, while other proteins tested did not. This finding allows us to speculate that the gliosis observed in demyelinating diseases or/and in central nervous system (CNS) injury would be due to the mitogenic effect exerted by MBP or its fragments when there is myelin breakdown.

Double ligand ELISA technique for the estimation of antibodies to brain tissue antigens in patients with neurological disorders.

A double ligand enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibodies against brain tissue antigens in the sera of patients with neurological diseases. The sera were tested on human white matter homogenate. The technique consists of successive incubations with the human serum to be tested, rabbit immunoglobulin G (IgG) to human immunoglobulins (Ig), alkaline phosphate-labeled protein A and alkaline phosphatase substrate. This procedure has the advantage of increased sensitivity compared to the classical ELISA. Application of this procedure to the sera of patients with neurological diseases showed that the unspecific binding is very low and the results are reliable. Moreover the test allows the detection of antibodies to chemically different antigenic structures that can occur in a variety of neurological diseases.

Cellular development and myelin production in primary cultures of embryonic mouse brain.

The development of cell cultures from embryonic mouse cerebral hemispheres has been followed in detail for periods up to 40 days in culture using a variety of approaches. Functionally well differentiated neurons (shown by receptor binding studies, immunocytochemistry and morphological examination) were found to be abundant early in culture and to form cell contacts with oligodendrocytes characterized both immunocytochemically and morphologically. Myelin-like membranes with the periodicity of classical myelin elaborated by oligodendrocytes were detected only after 30 days in culture when neurones were no longer present. These results are discussed with regard to possible mechanisms of initiation of myelin synthesis.

Parvalbumin, a neuronal protein in brain cell cultures.

Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000). In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.

Production and characterization of monoclonal antibodies to the myelin glycolipid sulfatide.

Sulfatide is enriched in the myelin sheath and accounts for 5% of the total lipids in this membrane. In the present work we describe the production and characterization of mouse monoclonal antibodies against sulfatide. The antibodies were detected and characterized in a previously described ELISA test system. The clone AIC3IA2 produced antibodies of the IgG3 class with high specificity for sulfatide. These antibodies showed almost no cross-reactivity with galactocerebroside or with any of the other lipids we tested. When used with the peroxidase antiperoxidase technique the antibodies stained a cell population either in fixed or unfixed brain cell cultures, indicating a surface localization of sulfatide in the respective cell population. In double-staining experiments the stained cell population was identified as myelin basic protein-positive oligodendrocytes.

Normal proliferation rate of galactocerebroside positive oligodendrocytes in brain cell cultures of the hypomyelinated mouse mutant jimpy.

Proliferation of oligodendrocytes from the jimpy (jp) hypomyelinated mouse mutant was studied in dissociated brain cell cultures. This was done by combining anti-galactocerebroside (GC) immunostaining (for identifying oligodendrocytes) with [3H]thymidine autoradiography (for identifying proliferating cells). Previously we showed that the expression of GC in culture by jp oligodendrocytes is not altered by the jp mutation. Present results show that in 7-, 14- and 21-day-old jp cultures oligodendrocytes proliferate at a rate similar to that of normal GC+ oligodendrocytes. This indicates that, in jp brain cell cultures, oligodendrocytes which are not affected by mutation in their capability to express GC are also unaffected with regard to their proliferation rate.

Development of immunologically identified brain cells in culture: quantitative aspects.

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.

Proliferation rate of oligodendrocytes in culture can be influenced by extrinsic factors.

We investigated whether in cultures of mechanically dissociated brain cells from newborn mice the reduction of the number of oligodendrocytes influences their proliferation rate. 14-day-old cultures were subjected to complement-dependent anti-galactocerebroside (GC) antibody-mediated cytotoxicity. The cytotoxic treatment completely destroyed oligodendrocytes. Thereafter, GC+ oligodendrocytes progressively reappeared. Their number was 20 and 66% compared to controls, 3 and 7 days after cytotoxicity, respectively. Proliferating oligodendrocytes were detected 3 and 7 days after cytotoxicity by combining the immunostaining for GC with 3H-thymidine autoradiography. The proliferation rate of oligodendrocytes in treated cultures was increased by 100 and 76% compared to controls, 3 and 7 days after cytotoxicity, respectively. These data suggest that the proliferation rate of oligodendrocytes can be influenced by extrinsic factors.

Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures.

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.