Real-time PCR and immunocytochemical study of chondroitin sulfate proteoglycans after scratch wounding in cultured astrocytes

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 counter­part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sulfate 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 phosphacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent signal for phosphacan and neurocan was more intense in astrocytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differentially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suitable for investigation of astrocyte-derived response to injury.

Keywords

• reactive astrocytes
• chondroitin sulfate proteoglycans
• in vitro model
• scratch-wound assay
• gene expression