A Method for Preparing Primary Retinal Cell Cultures for Evaluating the Neuroprotective and Neuritogenic Effect of Factors on Axotomized Mature CNS Neurons

Veselin Grozdanov1, Adrienne Müller1, Vetrivel Sengottuvel1, Marco Leibinger1, Dietmar Fischer1

1 Department of Experimental Neurology, University of Ulm, Ulm, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.22
DOI:  10.1002/0471142301.ns0322s53
Online Posting Date:  October, 2010
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Retinal ganglion cells (RGCs) are central nervous system neurons with a very limited ability for axon regeneration. This unit details a cell culture technique, which can be used to functionally screen factors/compounds for their neuritogenic and neuroprotective effects on RGCs. In this protocol, the retina is isolated, digested in a papain solution, and after trituration, the RGCs are cultured. The neuritogenic effect of applied factors/compounds on RGCs in the medium is functionally determined by measuring the average neurite length of βIII‐tubulin‐positive RGCs in culture after 3 days. This protocol takes 3 to 7 days to perform depending on the application to complete, and is suitable to reliably test pharmacological and genetic approaches for their axon growth‐promoting and neuroprotective potential on mature RGCs. Curr. Protoc. Neurosci. 53:3.22.1‐3.22.10. © 2010 by John Wiley & Sons, Inc.

Keywords: axon regeneration; retinal cultures; retinal ganglion cell; neuroprotection

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Poly‐D‐lysine solution (see recipe)
  • Laminin solution (see recipe), optional
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 70011‐036) or Hank's buffered salt solution (HBSS)
  • Papain (30 to 35 U/mg; Worthington)
  • Dulbecco's modified Eagle medium (DMEM; Invitrogen, cat. no. 41966‐029)
  • L‐cysteine (0.3 µg/ml; Sigma)
  • Adult Sprague‐Dawley rats (180 to 220 g; Charles River) or adult C57BL/6 mice (20 to 23 g; Charles River)
  • 70% ethanol
  • Culture medium (see recipe)
  • Drugs of interest or other factors (e.g., CNTF)
  • Paraformaldehyde (PFA) solution (see recipe)
  • Methanol
  • Blocking solution (see recipe)
  • βIII‐tubulin antibody (TUJ‐1, 1:2000; Convance)
  • Anti–mouse IgG antibody (1:1000; Alexa Fluor 488; Invitrogen)
  • 4‐well tissue culture dish (Nunc), sterile
  • 37°C CO 2 incubator
  • 37°C water bath
  • Sartorius Minisart sterile syringe filters (0.22 µm; Sartorius)
  • Vannas microscissors (Hermle)
  • Microforceps (Dumont, cat. no. 5; Hermle)
  • 15‐ and 50‐ml Falcon tubes (Sarstedt)
  • 5‐, 10‐, and 25‐ml serological pipets
  • 5‐ or 10‐ml Pasteur pipets
  • Centrifuge, suitable for cell centrifugation
  • 40‐µm cell strainer (Falcon)
  • Inverse fluorescent‐microscope, equipped with camera (Carl Zeiss)
  • Processing software (AxioVision, Carl Zeiss)
NOTE: Protocols using live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Literature Cited

Literature Cited
   Berkelaar, M., Clarke, D.B., Wang, Y.C., Bray, G.M., and Aguayo, A.J. 1994. Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats. J. Neurosci. 14:4368‐4374.
   Berry, M., Ahmed, Z., Lorber, B., Douglas, M., and Logan, A. 2008. Regeneration of axons in the visual system. Restor. Neurol. Neurosci. 26:147‐174.
   Fischer, D., Pavlidis, M., and Thanos, S. 2000. Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture. Invest. Ophthalmol. Vis. Sci. 41:3943‐3954.
   Fischer, D., Heiduschka, P., and Thanos, S. 2001. Lens‐injury‐stimulated axonal regeneration throughout the optic pathway of adult rats. Exp. Neurol. 172:257‐272.
   Fischer, D., He, Z., and Benowitz, L.I. 2004a. Counteracting the Nogo receptor enhances optic nerve regeneration if retinal ganglion cells are in an active growth state. J. Neurosci. 24:1646‐1651.
   Fischer, D., Petkova, V., Thanos, S., and Benowitz, L.I. 2004b. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J. Neurosci. 24:8726‐8740.
   Fischer, D., Hauk, T.G., Muller, A., and Thanos, S. 2008. Crystallins of the beta/gamma‐superfamily mimic the effects of lens injury and promote axon regeneration. Mol. Cell. Neurosci. 37:471‐479.
   Fournier, A.E., Takizawa, B.T., and Strittmatter, S.M. 2003. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J. Neurosci. 23:1416‐1423.
   Gallagher, S. 2010. Digital image processing and analysis with ImageJ. Curr. Protoc. Essen. Lab. Tech. 3:A.3C.1‐A.3C.24.
   GrandPre, T., Li, S., and Strittmatter, S.M. 2002. Nogo‐66 receptor antagonist peptide promotes axonal regeneration. Nature 417:547‐551.
   Hauk, T., Müller, A., Lee, J., Schwendener, R., and Fischer, D. 2008. Neuroprotective and axon growth promoting effects of intraocular inflammation do not depend on oncomodulin or the presence of large numbers of activated macrophages. Exp. Neurol. 209:469‐482.
   Hauk, T.G., Leibinger, M., Muller, A., Andreadaki, N., Knippschild, U., and Fischer, D. 2009. Stimulation of axon regeneration in the mature optic nerve by intravitreal application of the Toll‐like receptor 2 agonist Pam3Cys. Invest. Ophthalmol. Vis. Sci. 51:459‐464.
   Leaver, S.G., Cui, Q., Bernard, O., and Harvey, A.R. 2006a. Cooperative effects of bcl‐2 and AAV‐mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice. Eur. J. Neurosci. 24:3323‐3332.
   Leaver, S.G., Cui, Q., Plant, G.W., Arulpragasam, A., Hisheh, S., Verhaagen, J., and Harvey, A.R. 2006b. AAV‐mediated expression of CNTF promotes long‐term survival and regeneration of adult rat retinal ganglion cells. Gene Ther. 13:1328‐1341.
   Leibinger, M., Muller, A., Andreadaki, A., Hauk, T.G., Kirsch, M., and Fischer, D. 2009. Neuroprotective and axon growth‐promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor. J. Neurosci. 29:14334‐14341.
   Leon, S., Yin, Y., Nguyen, J., Irwin, N., and Benowitz, L.I. 2000. Lens injury stimulates axon regeneration in the mature rat optic nerve. J. Neurosci. 20:4615‐4626.
   Lorber, B., Berry, M., Douglas, M.R., Nakazawa, T., and Logan, A. 2009. Activated retinal glia promote neurite outgrowth of retinal ganglion cells via apolipoprotein E. J. Neurosci. Res. 87:2645‐2652.
   Muller, A., Hauk, T.G., and Fischer, D. 2007. Astrocyte‐derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain 130:3308‐3320.
   Muller, A, Hauk, TG, Leibinger, M, Marienfeld, R, and Fischer, D. 2009. Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF. Mol. Cell. Neurosci. 41:233‐246.
   Rudge, J.S. and Silver, J. 1990. Inhibition of neurite outgrowth on astroglial scars in vitro. J. Neurosci. 10:3594‐3603.
   Yin, Y., Cui, Q., Li, Y., Irwin, N., Fischer, D., Harvey, A.R., and Benowitz, L.I. 2003. Macrophage‐derived factors stimulate optic nerve regeneration. J. Neurosci. 23:2284‐2293.
   Yiu, G. and He, Z. 2006. Glial inhibition of CNS axon regeneration. Nat. Rev. Neurosci. 7:617‐627.
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