Extract from the brain of young mice produced weak cytoproliferative effect on cultured glial cells, while brain extract from old mice 3-4-and 30-60-fold stimulated glial proliferation in primary and passaged cell cultures, respectively. Key Words: cell culture; gliosis; brain aging; prion diseasesHuman and animal prion diseases (PD) are characterized by typical histopathological changes involving only the brain and, sometimes, spinal cord. They are characterized by neuronal loss, spongiform changes of the white and/or grey matter of the brain and spinal cord, formation of amyloid plagues, and gliosis. These changes considerably differ in various PD. Thus, amyloid plagues are observed in 9 and 70% patients with Creutzfeldt-Jacob disease (CJD) and kuru. respectively. However, gliosis is typical of all diseases including lethal hereditary insomnia, which is seldom characterized by spongiosis, but induces neuronal loss and astrogliosis in the mediodorsal and anterioventral thalamic nuclei [ 10].For a long time much attention was focused on the mechanism of PD-induced lesions in the central nervous system (CNS), plague origin, chemical composition, and topography, as well as topography of spongiform regions, mechanisms of neurodegeneration, and correlations between CNS damage and mutations in PRNP gene. At the same time, it was assumed that the initial histopathological changes are neuronal loss followed by spongiosis and accumulation of amyloid. This sequence culminates in glial reaction replacing defects (reactive gliosis) caused by progressive neuronal death [ 1]. Recently, the interest of many researchers was attracted to the role of glia in this multistage process of CNS damage by infectious (and other) proteins. In the late 80s, immunocytochemical and ultrastructural studies showed that, on one hand, glial cells are involved in phagocytosis of amyloid fibrils during AIzheimer disease (AD) [11,15], while on the other hand, they participate in the formation of amyloid fibrils during AD and CJD [9,14]. These data were confirmed by the discovery of activated microglia involved in the accumulation of amyloid during experimental scrapie m mice [2]. At the same time, variable and important role of microglia in the formation of amyloid plagues during PD was demonstrated. It was suggested that variability is specific for this nosologic form and realized on the level of mRNA synthesis for scrapie-associated amyloid protein precursor [7]. These data were supplemented by studies on the mechanisms of neuronal death. It was shown that synthetic peptide 106-126 homologous to amyloid protein isolated from the brain of patient died from Gerstmann--Straussler--Scheinker syndrome (GSSS) caused apoptotic death of neurons and pronounced proliferation of glial cells in culture [13]. Moreover. it was found that peptide 106-126 promotes in vitro formation of amyloid fibrils and is toxic for cultured neurons only in the presence of microglia responding to this peptide by enhanced generation of oxidative radicals [3].
The expression of chordin P-epitope in dissociated cell culture of rat brain is studied by indirect immunofluorescence. Specific fuorescence is confined to compact bipolar and stellate cells on a layer of P-negative spread cells. Granular cells of the cerebellum, spinal neurons, neurons, and Schwann cells are P-negative. Double staining with monoclonal and polyclonal antibodies to glial fibriUar acid protein (an astrocyte marker) shows that this protein is expressed by P-positive stellate cells and P-negative cells of the underlying monolayer. Thus, monoclonal antibodies to P-epitope detect type II astrocytes. Key Words: neuroglia development; chordin P-epitope; CNS culture; astrocytesIn 1984, a new acid glycopmtein was isolated from the sturgeon chord, characterized, and termed chordin [5,12]. Monoclonal antibodies (MAb) to the repeated site of chordin molecule (P-epitope) were obtained [13]. Further studies showed that P-epitope is also present in the nervous system of all vertebrates. Therefore, the proteins of the central nervous system (CNS) carrying P-epitope were called neurochordins [12]. Immunocytochemically detected expression of P-epitope in other organs and tissues was either not typical of all systematic groups or was transitory, occurring only during some stages of ontogenesis [1]. P-epitope was found in almost all parts of the brain and spinal cord of sterlet and triton larvae and chicken and human embryos. Its expression was higher in the white matter and nerve fiber bundles [1,2].Tissue distribution of P-epitope in vertebrates suggests its potential significance as a neurospecific marker. The specific cellular localization of proteins carrying P-epitope so far remains unclear due to the complex structure of the nervous tissue. This study is an attempt to localize neurochordin with the use of tissue and cell cultures as simple systems. For this purpose primary dissociated glial cultures of the forebrain, cerebellum, and spine as well as organotypical cultures of the sympathetic and spinal ganglia were studied by immunocytochemical methods. MATERIALS AND METHODSDissociated glial cultures were prepared as described elsewhere [10]. Cerebral hemispheres were isolated under sterile conditions from newborn (days 1-2) rats and put in serum-free culture medium to remove the membranes. Then the brain was minced and suspended by pipetting. The resultant suspension was successively filtered (230 and 140 ~t pore diameter). The filtrate was layered onto slides covered with 0.01% polylysine. Primary cultures were grown in Eagle's medium with and without 2 mM glutamine, 20% fetal calf serum, 50 U/ml penicillin, and 50 ~tg/
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