Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

Abstract

Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models.

Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry.

Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes.

Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury.