Microglia were successfully cultured from human brain tissue from normal and neurologically diseased cases obtained 3.5-10 hours postmortem. Final cell preparations were more than 99% pure as judged by latex bead phagocytosis, expression of microglial phenotypic markers, and absence of astrocytic markers. The expression of complement genes C1qB, C3, and C4 as well as genes for interleukin-(IL-)1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)alpha, IL-1 receptor antagonist, and transforming growth factor beta, but not inducible nitric oxide synthase, by these cells was detected by polymerase chain reaction (PCR) analysis. The pattern of gene expression was evaluated following stimulation of the cells with lipopolysaccharide, phorbol myristate acetate, gamma interferon, and beta amyloid peptide. There was considerable variation in gene response to these activating agents. However, it was of interest that beta-amyloid peptide (1-40) increased the expression of IL-1 beta mRNA in these cells. The number of cases in this study was too small to permit evaluation of microglial response according to the disease state, but the results demonstrate the potential for such studies in the future.

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.

The proportion of cultured rat oligodendrocytes (OL) that extended processes of over three soma diameter in length is dependent on the age of the animals from which the brains were derived; up to 70% of neonatal OL attained this criterion within 3 days, and this proportion progressively decreased with advancing ages of the animals (1, 3, and 6 months). The lower extent of process formation from older rat OL could be augmented, and indeed to equal neonatal levels, by treatment of cells with phorbol esters that stimulate protein kinase C: 4 beta-phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA). Enhancement of process formation by PDB and PMA was also observed for cultured adult human and bovine OL. For adult OL from all three species, a phorbol ester that binds but that does not activate protein kinase C, 4 alpha-phorbol-12,13-didecanoate, did not result in enhancement of process formation. Selectively to biologically active phorbol esters was shown by the inability of a wide range of growth factors to promote process extension. Immunohistochemical analyses indicate that the type III isozyme of protein kinase C predominates in cultured OL; the apparent intensity of immunoreactive PKC was not different between controls or cultures treated for 12 days with PDB, suggesting that the persistent presence of PDB might not have down-regulated the enzyme, in contrast to other cell types. We propose that stimulation of protein kinase C is critical to the triggering of process formation by cultured OL in vitro.

The effects of phorbol esters have been attributed to the activation of the enzyme protein kinase C. While much has been described for the actions of phorbol esters on neurons and synaptic transmission, sparse data exist on the effects of phorbol esters on oligodendrocytes, the cells that make and maintain myelin in the central nervous system. In this report, we show that 10 and 100 nM of a phorbol ester, 4 beta-phorbol-12,13-dibutyrate, extensively enhanced process formation by cultured bovine oligodendrocytes. This effect was blocked by two inhibitors of protein kinase C, sodium heparin and polymixin B. We propose the hypothesis that activation of protein kinase C is an important process that leads to the differentiation of oligodendrocytes and the formation of myelin in vivo.