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