May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Particle-Mediated Gene Transfer in Retinal Explant Cultures as a Model for Studying Neurite Remodelling After Axotomy
Author Affiliations & Notes
  • P. Samsel
    Cardiff University, Cardiff, United Kingdom
    Optom & Vis Sciences,
  • H. Gutierrez
    Cardiff University, Cardiff, United Kingdom
    Biosciences,
  • J. T. Erichsen
    Cardiff University, Cardiff, United Kingdom
    Optom & Vis Sciences,
  • J. Albon
    Cardiff University, Cardiff, United Kingdom
    Optom & Vis Sciences,
  • A. M. Davies
    Cardiff University, Cardiff, United Kingdom
    Biosciences,
  • J. E. Morgan
    Cardiff University, Cardiff, United Kingdom
    Optom & Vis Sciences,
  • Footnotes
    Commercial Relationships  P. Samsel, None; H. Gutierrez, None; J.T. Erichsen, None; J. Albon, None; A.M. Davies, None; J.E. Morgan, None.
  • Footnotes
    Support  National Eye Research Centre, UK
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5904. doi:https://doi.org/
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      P. Samsel, H. Gutierrez, J. T. Erichsen, J. Albon, A. M. Davies, J. E. Morgan; Particle-Mediated Gene Transfer in Retinal Explant Cultures as a Model for Studying Neurite Remodelling After Axotomy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5904. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose:
 

Retinal explants have been used to study effects of inhibiting the apoptotic pathway on retinal ganglion cell death. However, it is poorly known how this can affect neurite regeneration and remodelling.Here we propose the use of biolistic transfection of explant cultures as a model to study the time course of neurite regeneration and remodelling in living retinal ganglion cells.

 
Methods:
 

Retinas were isolated from Brown Norwegian male rats 9-12 weeks of age and cultured on 0.4 µm Millicell culture plate inserts in Neurobasal-A medium at 37ºC in 5% CO2. A Helios gene gun system was used for particle-mediated transfer of a YFP-expressing plasmid to RGCs. Retinal explants were treated with a broad-spectrum, irreversible caspase inhibitor (Boc-D-FMK) to prevent apoptosis. The morphology of RGCs was observed by confocal microscopy.

 
Results:
 

Individual YFP-labelled RGCs were successfully kept alive for at least 7 days in explant cultures. YFP labelling allowed for the detailed morphology of the dendritic arbors and axons of RGCs to be analysed. RGCs in caspase inhibitor-supplemented explants displayed a vigorous regrowth of axons towards the optic disc compared with explants not treated with caspase inhibitors.

 
Conclusions:
 

Retinal explants, coupled with caspase inhibition, are a promising model for the analysis of dendritic and axonal plasticity following axotomy. This system, together with biolistic gene transfer, allows for the study of changes in neurite remodelling over time in the adult retina as well as the manipulation of signalling pathways controlling growth and survival.  

 
Keywords: ganglion cells • regeneration • retinal culture 
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