Monoclonal antibodies to P epitope of chordin detect type II astrocytes in glial cultures

Abstract

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/

Dependence of antitumor effect of hormonal cytostatic cortifen on expression of glucocorticoid receptors in brain tumor cells

“…Cultured rat glioblastoma 101/8 (GB) and anaplastic astrocytoma 15-47 (AA) were obtained from collection of Institute of Human Morphology [6] and inoculated intracranially [5].…”