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A primary culture technique of adult retina for regeneration studies on adult CNS neurons

Nature Protocols volume 2, pages 131–140 (2007)Cite this article

Abstract

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5–7 days. Mere preparation of a single retina should be completed within 20 min.

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Figure 1: Scheme on access to the ON in vivo and ON axotomy.
Figure 2: Timeline for pre-preparations and retinal culture, as well as for RGC labeling, growth stimulation and molecular expression analysis.
Figure 3: Scheme on retinal extraction from the eye cup.
Figure 4: Selective steps of technical progression of the culture setting.
Figure 5: Examples of a retinal culture assay.
Figure 6: Examples for tracing techniques of cultured RGCs.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), by the Bundesministerium für Bildung und Forschung (BMBF), the IZKF and by the Ernst Abbe Foundation.

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Authors and Affiliations

  1. Department of Neurology, Neuroregeneration Laboratory, University of Jena Medical School, Erlanger Allee 101, Jena, D-07747, Germany

    Alexandra Kretz, Julia K Marticke, Christian Schmeer & Stefan Isenmann

  2. Department of Neurology, University of Tübingen Medical School, Hoppe-Seyler-Strasse 3, Tübingen, D-72076, Germany

    Caroline J Happold

  3. Department of Neurology, HELIOS Klinik Wuppertal, and University Witten/Herdecke,

    Stefan Isenmann

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  1. Alexandra Kretz
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Correspondence to Alexandra Kretz.

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Kretz, A., Marticke, J., Happold, C. et al. A primary culture technique of adult retina for regeneration studies on adult CNS neurons. Nat Protoc 2, 131–140 (2007). https://doi.org/10.1038/nprot.2007.12

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