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Dynamic changes of cytoskeletal proteins in primary astrocyte cultures post scratch injury

Authors Leung, Doreen Siu Yi
Issue Date 1994
Summary Irrespective to the types of CNS injury, astrocytes are activated and become gliotic. Glial fibrillary acidic protein (GFAP) is one of the specific markers for astrocytes. Increment in GFAP is well documented in astrogliosis, the scar formation process by astrocytes in response to injury. Astrogliosis is also characterised by hyperplasia, hypertrophy and rearrangement of the cytoskeletons. The response of astrocytes to injury is so vigorous and fast that astrocytic scar formed can have harmful effect in neuronal regeneration and axonal remyelination. As GFAP and other cytoskeleton (including microtubules, actin and vimentin) are important in the change of morphology of reactive astrocytes, it is important to investigate the changes of these proteins in injured astrocytes. In this study, an in vitro scratch-wound model was used to study mechanical injury. A plastic pipette tip was used to scratch a confluent primary culture of astrocytes which was prepared from rat cerebral cortex. Confocal Scanning Laser Microscopy (CSLM) and Western Blotting techniques were used as tools to examine the morphological and biochemical changes of actin, GFAP, vimentin and β-tubulin. These proteins: were visualized by immunocytochemical staining and quantified by densitometer. The changes of cytoskeletal proteins in the astrocytes along the scratched zone and the distant inner non scratch region were compared at 8 time points (0,6 and 12 hours, day 1,2,3,5 and 9). Western Blotting quantified the total pooled responses of both scratched and non scratched astrocytes of the whole culture whereas CSLM was able to detect the regional differences of astrocvtes along and distant from the scratch injury. With analysis, the detected responses of the four cytoskeletal proteins to scratch injury were different. GFAP of the scratched astrocytes had an intense initial elevation, then decreased gradually and increased at a later stage. Microtubules as reflected by Western blotting remained at a constant level throughout the whole time course with only slight fluctuations. Actin had a significant decrease of about 50% at day 1 and 20% at day 2. Vimentin had a significant decrease of about 15% at day 1 and 50% increase at day 9. For regional comparison, all astrocytes along the scratch showed higher and dramatic changes in the intensity of staining of GFAP, vimentin, actin and β-tubulin. No changes were observed in the non scratched astrocytes in the inner region except vimentin which showed changes in both scratched and non scratched region. Staining for oncogene proteins for heat shock, c-myc and n-myc demonstrated intense immunofluorescence in astrocytes localized along the scratch edge. These proteins are good markers for injured cells. These observations concluded that CSLM and Western Blotting are a good combination in studying the changes in cytoskeletal proteins in injured astrocytes. The cytoskeletal proteins, GFAP, vimentin, β-tubulin and actin, altered differently in astrocytes in response to injury suggested that GFAP may not be the only cytoskeletal protein involved in astrogliosis, other cytoskeietal proteins such as vimentin, actin and microtubules may act as aan associate role and provide an architectural support for GFAP to act on and/or may act directly during astrogliosis. The correlation of their activity during healing processes require further studies.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 1994
Language English
Format Thesis
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