Immunoelectron microscopic characterization of glial intermediate filaments in human gliomas
Glial fibrillary acidic protein (GFAP) is found in varying amounts in the cytoplasm of most normal and neoplastic cells of astroglial origin. Though not glial specific, immunoelectron microscopy has shown that vimentin and GFAP are coexpressed as monomers of glial intermediate filaments. These structures display irreversible assembly and a slow metabolic turnover. Although currently applied as astroglial markers, these intermediate filament proteins may reflect the functional and developmental differentiation status of the cells in which they are expressed. Some authors have tried to apply these aspects as diagnostic parameters for grades of malignancy and anaplasia whilst other workers have indicated variable concentrations of GFAP in different astroglial cell types and entities. Different processing protocols, including the use of epoxy and acrylic resins, omission of osmium tetroxide and variations in concentration and incubation time of primary fixatives, were evaluated to find a compromise between antigen availability and acceptable ultrastructure. Thin sections were labelled on grid for GFAP (Dako A561) and vimentin (Dako M725) by means of the indirect immunogold method. For semi- quantification of relative antigen concentrations, a novel method was devised to calculate the labelling density, percentage heterogeneity of the particle distribution and the surface area investigated. This allowed expression of labelling results as a three figure unit. Standardized post-embedding immunoelectron microscopy was performed on 11 normal and neoplastic human tissue specimens. The tissue was exposed to conventional immersion fixation in glutaraldehyde and osmium tetroxide prior to modified embedding in LR White resin. The validity of these results was verified by correlation with conventional histopathological, immunohistochemical and clinical data obtained for each specimen. The presence of epoxy resin in thin sections was shown to reduce antigen availability to such an extent that very low to negative labelling was encountered. Acrylic LR White resin allowed more acceptable immunodetection, but at the cost of inferior ultrastructure and greater instability of thin sections in the electron beam. This masked the effects of glutaraldehyde fixation on the density of the tissuefixative matrix which included destruction of the vimentin and some GFAP associated epitopes. Although osmium tetroxide was required for acceptable ultrastructure, it reduced the labelling sensitivity by 20% and was responsible for premature curing of acrylic resin during impregnation of tissue. Despite superior resolution gained by electron microscopy and the advantage of semi-quantification of labeling results, the labelling sensitivity of this technique is lesser than that of light microscopical immunohistochemistry. Immunoelectron microscopy confirmed the association between GFAP and glial intermediate filaments in almost all the glial tumours studied, correlating well with GFAP expression in matching specimens demonstrated at light microscopical level. In the absence of intermediate filaments, no positivity for GFAP or vimentin was found in oligodendroglial components of mixed tumours. GFAP positivity in astrocytomas was demonstrated by between 17 and 126 particles / µm2, whilst lower figures were obtained for the glioblastoma (PD = 8) and some of the mixed gliomas (Pd = 6). Rosenthal fibres showed both peripheral and central positive labelling for GFAP, thus providing more evidence for their hypothetical degenerative, astroglial nature. The meningioma studied, was GFAP negative, but produced low density positivity for vimentin. Coexpression of GFAP and vimentin was demonstrated in an astroblastoma and degenerative infant brain tissue, thus supporting the presence of both these proteins development of glial structures. Although sites of likely glial intermediate filament synthesis were found, the antigen availability for vimentin was too low to allow a reliable assessment of specific vimentin localization and determination of the GFAP : vimentin ratio in individual intermediate filaments and/or astroglial fibres. Variations in particle densities (PD) which demonstrated GFAP in the various astroglial entities studied, were considered to be a result of variable technical and tissue processing factors rather than truly significant differences in expression of GFAP in individual intermediate filaments. This lead to the conclusion that the GFAP concentration / glial intermediate filament area is likely to be constant for mature glial intermediate filaments and therefore cannot be used to distinguish between different astroglial cells or entities. Whether each cell has a different number of glial intermediate filaments, has not been established satisfactorily. Following complementary conventional immunohistochemistry and careful orientation of biopsy material, the procedure can be applied to suitable specimens for the electron microscopical localization of high concentrations of aldehyde resistant, cytoplasmic antigens.