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East, E.; Golding, J.P. and Phillips, J.B.
(2007).
Abstract
Traumatic injuries to the spinal cord and dorsal root entry zone (DREZ) are debilitating and often lead to paralysis and loss of sensation for the patient. At the cellular and molecular level, the repair site microenvironment is inhibitory for axon growth due to formation of a glial scar. A common finding of current strategies aimed at bridging CNS lesions, in particular recent tissue engineering approaches using fibronectin (Phillips et al., Biomaterials 2004), is that whilst axons readily enter and traverse the bridging graft, they are less likely to exit the graft and reconnect with their targets. The aim of this work was to develop a cell culture model to investigate reactive gliosis following damage to the spinal cord.
Astrocytes in the CNS under physiological conditions express low levels of GFAP, but following trauma exhibit a reactive phenotype characterised by GFAP up-regulation. Primary glial cell cultures were analysed in 2D monolayers and 3D collagen gels for GFAP expression. In 2D cultures approximately twice the number of cells were positive for GFAP compared to those cultured in 3D collagen gels. As 3D astrocyte cultures more closely modelled the in vivo situation, we used this model to investigate the response of astrocytes to cells found at the inhibitory interface following damage. The preliminary model involved placing dorsal root ganglia (DRG) explants into the centre of astrocyte gels. Classification of astrocyte morphology revealed significantly more ramified cells in DRG-adjacent regions (6 fold higher), than in control areas away from the DRG. A more advanced model was then developed in which dissociated DRGs were labelled with CellTrackerTM, seeded onto astrocyte-populated collagen gels and maintained in culture for 5 days. Astrocytes near the DRG interface showed marked GFAP up-regulation and adopted a reactive morphology which was observed up to 1mm away. Intensity of GFAP fluorescence at this interface was 3 fold higher than that seen away from the interface or in controls (astrocyte only gels).
Astrocytes in 3D culture exhibit a lower basal level of reactivity than cells grown in monolayer. This model provides a useful tool for investigating triggers of reactive gliosis, as demonstrated by the response observed to cells found at the inhibitory interfaces formed following damage to the spinal cord and could be used as a way to improve existing therapies and develop new strategies aimed at CNS repair.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)
