Unraveling the causes of regeneration failure in the adult injured CNS has remained a challenge in neurobiology. The notion that CNS neurons lose their regenerative potential during development has been challenged by the identification of several promoters of axon growth. Novel methods are required that allow to study and quantify interactions of molecular determinants, and to envisage future treatment applications. Here we report a novel, highly reproducible method for monitoring axonal regeneration of mature retinal ganglion cells (RGCs) in vitro. In contrast to earlier explantation methods, primary cultures derived from adult rodent retina are kept viable without growth factor supplements. Further, since intraretinal RGC axons remain unmyelinated, regeneration can be followed independently of non-permissive white matter compounds. Applying tracing techniques prior to retinal explantation, cell survival can be correlated to outgrowth activity on the single cell level. Following intervention with pharmacological, growth factor, or gene transfer treatments, retinal explants, and partially RGC neurites, can be processed for protein and gene expression analysis. This novel procedure will prove useful to get insight into complex cell survival and regeneration promoting cascades, and will complement in vivo strategies such as transgenic and knock out mouse models.
Copyright 2004 Elsevier B.V.