A novel primary culture technique for adult retina allows for evaluation of CNS axon regeneration in rodents.
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.
Kretz
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Neuroregeneration Laboratory, Department of Neurology, University of Jena Medical School, Erlanger Allee 101, D-07747 Jena, Germany.
Hermening
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Isenmann
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D000818:Animals; D002450:Cell Communication / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D020439:Growth Cones / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D006133:Growth Substances / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D036421:Nerve Fibers, Unmyelinated / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009416:Nerve Regeneration / Q000187:drug effects / Q000235:genetics*; D016501:Neurites / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D012162:Retinal Degeneration / Q000235:genetics / Q000503:physiopathology; D014161:Transduction, Genetic / Q000379:methods; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D006133:Growth Substances; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein