Liane Bologa

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.

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.

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.

Parvalbumin, a Neuronal Protein in Brain Cell Cultures

Abstract: 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 Ca + ‐binding protein parvalbumin (M., 12,000). In embryonic cultures a large proportion of cells was intensely stained by anti‐ parvalbumin. In double‐labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron‐specific enolase, the enolase‐con‐ taining 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 newbornmice. 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.

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.

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.

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.

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.

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.