Role of the Chondroitin Sulfate Proteoglycan, Neurocan, in Inhibition of Sensory Neurite Regeneration

In the adult mammalian brain and spinal cord, neuronal injury results in failed neurite regeneration, in part due to the up-regulation of chondroitin sulfate proteoglycans (CSPGs). CSPGs are molecules consisting of a protein core with covalently bound glycosaminoglycans (GAGS), specifically, chondroitin sulfate side-chains. The majority of CSPGs produced after injury originate from reactive astrocytes found in the glial scar surrounding the injury site. Although this milieu is very complex and involves more than just CSPGs, axonal regrowth may be improved if the expression of specific, highly inhibitory CSPGs produced after injury were attenuated selectively. Neurocan is one type of CSPG that is upregulated after injury and inhibits neurite regeneration. The over-arching goal of this study is to focus on the response and growth of sensory neurons in the presence of astrocytes that express neurocan, in relation to those astrocytes in which neurocan has been “knocked down” by shRNA transfection. In order to perform this analysis, the conditions needed for the co-culture experiment of chick astrocytes and chick neurons were optimized. A series of preliminary tests were performed on chick astrocytes, including a test to monitor the upregulation of CSPGs after treatment with Transforming Growth Factor-β (TGF-β), previously shown to mimic injury in an In vitro setting in rat astrocytes (G. Curinga et. al, 2008), and a test to demonstrate that chick astrocytes express Glial Fibrillary Acidic Protein (GFAP) in culture. Through these optimizations, co-culture analysis will be used to evaluate neuronal responses to neurocan. Other techniques used were tissue culture, immunofluorescence, and neurite outgrowth analyses