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Phillips, James; East, Emma and Golding, Jon
(2008).
Abstract
Damage to the CNS causes reactive gliosis and the formation of a glial scar, one of the principal impediments to neuronal repair. Astrocytes are key cells in the formation of the glial scar and they respond to damage stimuli by up-regulating specific proteins, releasing cytokines, becoming hypertrophic and ramified and forming a dense meshwork of processes. The glial scar has been well characterised in animal models but there is a need for effective cell culture models in which reactive gliosis can be continuously monitored in a controlled environment. The aim of this study was to develop a cell culture system in which the progression of reactive gliosis in astrocytes could be monitored with a view to using the model to inform the development of therapeutic strategies for improving repair of spinal cord injuries.
Primary rat astrocytes maintained in 3-dimensional (3D) type-1 collagen gels showed a significantly lower level of reactivity than 2D cultures as determined by immunofluorescence detection of GFAP, vimentin, aquaporin-4, CSPGs and S100β. The 3D cells were therefore reminiscent of astrocytes in the undamaged CNS, and could be used as a baseline for investigating induction and progression of reactive gliosis. In order to verify this system as a useful model, 3D astrocyte cultures were either stimulated continually with TGFβ1 or maintained in control media for 15 days. Immunofluorescence detection and image analysis were used to quantify changes in GFAP, vimentin, aquaporin-4 and CSPG in response to TGFβ1-stimulation. There was a clear increase in detection of this panel of reactivity markers with time in culture in the astrocytes exposed to TGFβ1 whilst control cultures showed little change. For further validation of this system as a useful model that permits detailed analyses, protein and RNA were extracted and used for Western blotting and RT-PCR respectively, and levels of IL-6, a cytokine released by reactive astrocytes, were monitored using ELISA.
The results from this study indicate that astrocytes in 3D collagen gels remain in a relatively non-reactive state compared to those in 2D cultures, but when stimulated with TGFβ1 they become reactive. Therefore this 3D cell culture system provides a useful model in which astrocytes can be maintained with a non-reactive phenotype and then can be stimulated to undergo reactive gliosis in a manner that closely resembles the behaviour of astrocytes in vivo. Because a wide range of biochemical, molecular and microscopic techniques can be used to monitor these cellular responses in real-time, this model will provide a powerful test-bed for the rational design of improved therapeutic interventions to treat CNS damage.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)
