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Ushasree Pattamatta, Kieran Benjamin Garbutcheon Singh, krishna tumuluri, duri kim, Andrew JR White; A method for the production of rabbit retinal explants to be utilised for neuronal apoptotic models. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2565. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To study retinal apoptosis in a human ex vivo retinal explant model, it is ideal to have the tissue as close to post mortem as possible. A number of patients, on the basis of clinical indication, require either enucleation or evisceration with the tissue often discarded. The retinal tissue from these eyes should be viable for retinal explant studies. This study uses a rabbit model to develop the technique that can be further developed to reliably obtain tissue in a timely fashion and can then be used to produce human retinal explants.Methods:
New Zealand White Rabbit explants were created after enucleating whole eye specimens immediately postmortem from an unrelated study. An oculoplastic surgeon then used a Freer's dissector to produce an evisceration specimen. These specimens were either used to make retinal explants immediately or stored in Gibco Hibernate A medium for two days before making explants. Explants prepared immediately and those made from the specimens stored in hibernate were cultured at an air/medium interface on membrane inserts for 4 days ex-vivo. These explants were treated either with Z-VAD-FMK (a pan-caspase inhibitor; 100 μM) or vehicle. Retinal cell density was estimated by 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI) using immunohistochemistry.
It was found that explants could be viably produced through the method used. This was confirmed by staining with a ganglion cell specific transcription marker (RBPMS). Further, Z-VAD-FMK reduced the amount of retinal ganglion cell death in the rabbit retinal explant models as previously shown in our mouse model. Retinal explants treated with Z-VAD-FMK demonstrated a two fold (p<0.05) increase in number of retinal cells (8520±60 cells/mm2) on day 4 compared to the control (3260±60 cells/mm2; Vehicle alone). There was no difference in the cell counts between the explants prepared immediately and explants made from hibernate medium (p<0.05).Conclusi
This study indicates that the method used to produce the rabbit retinal explants was reliable and reproducible. The use of hibernate media before culturing the rabbit retinal explants was successful and provides a foundation for human retinal explant studies using a similar techniques as a model for studying ganglion cell specific apoptosis and neuroprotective therapies. Positive results could lead to rapid and likely successful clinical trials.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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