Developmental changes of parameters for astrogliosis during cultivation of purified cerebral astrocytes from newborn rats.
Astrogliosis is a common phenomenon seen in most neuropathological changes of the central nervous system. Several in vitro models have been used to study the mechanisms and conditions for the induction of astrogliosis, however many do not take into account that the metabolic and structural characteristics of astrocytes change with time in culture. Thus, it appears difficult to attribute changes of, e.g., GFAP to the normal change in vitro as opposed to additional changes due to an astrogliotic reaction. The present study was therefore undertaken to characterize these developmental changes in purified astroglial secondary cultures during cultivation to provide a basis for further investigations of astrogliosis in vitro. During 6 weeks of cultivation (3-43 days) GFAP (ELISA) increased much more (22-fold) than the cell number (2.5-fold) and the total protein (3.5-fold). The GFAP/protein ratio increased during the first 4 weeks of cultivation and reached a plateau thereafter, which was accompanied by a significant increase of GFAP mRNA (Northern blot). At the ultrastructural level (transmission electron microscopy) gliofilaments in the perinuclear region as well as in the cell processes of 4-day-old astrocytes showed a dispersed pattern, whereas an accumulation of gliofilaments was found in 39-day-old cells, which formed large aggregated bundles localized mostly in the cell processes. Our results show that in vitro astrocytes undergo developmental changes in their accumulation of GFAP and intermediate filaments which reach a stable steady state after 4 weeks in culture. These 'normal' developmental changes will have to be taken into account, when experiments with variations of the level of GFAP are performed. Stable culture conditions for experimentation appear to be present after 4 weeks in culture.
Röhl
Claudia
C
Department of Anatomy, University of Kiel, Olshausenstr 40, D-24098 Kiel, Germany. claudia.roehl@gmx.net
Held-Feindt
Janka
J
Sievers
Jobst
J
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000502:physiology* / Q000648:ultrastructure; D015152:Blotting, Northern; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D002531:Cerebellum / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism*; D005911:Gliosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008854:Microscopy, Electron; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger