The Development of Human Organotypic Retinal Cultures (HORCs) to study Retinal Neurodegeneration

Nuwan Niyadurupola (1) , Peter Sidaway (2) , Andrew Osborne (3) , David C Broadway (4) , Julie Sanderson (5)

1 School of Pharmacy, University of East Anglia / Dept Ophthalmology, Norfolk and Norwich University H
2 Department of Ophthalmology, Norfolk and Norwich University Hospital; School of Pharmacy, University
3 UEA - University of East Anglia [Norwich]
4 Department of Ophthalmology, Norfolk and Norwich University Hospital
5 School of Pharmacy, University of East Anglia

Aims To develop human organotypic retinal cultures (HORCs) to study retinal ganglion cell (RGC) death in response to ischaemic and N-methyl-D-aspartate (NMDA) excitotoxic insults, both known to cause loss of RGCs and proposed as mechanisms involved in glaucomatous retinal neurodegeneration. Methods Human donor eyes were obtained within 24hr post mortem. The retina was isolated and explants cultured using two techniques. THY-1 mRNA (as assessed by real time quantitative PCR) and neuronal nuclei protein (NeuN) (as assessed by immunohistochemistry) were used as markers of human RGCs. Apoptosis was assessed by TUNEL labelling. Results The distribution of THY-1 mRNA and NeuN-labelling within the human retina was consistent with the expected distribution of RGCs. Gross morphology and retinal architecture remained stable over a 96hr culture period. THY-1 mRNA and NeuN-labelled RGC layer cells decreased over the culture period and there was an increase in TUNEL-labelling with time, but HORCs cultured in serum-free DMEM/HamF12 medium were useful for up to 48hr in culture. NMDA (10ìM) caused a reduction in THY-1 mRNA by 24hr and decreased the numbers of NeuN-labelled RGC layer neurones by 48hr, suggesting that the loss of THY-1 mRNA is a marker of RGC stress prior to RGC death. Simulated ischaemia (60 minute oxygen/glucose deprivation) caused a reduction at 24hr in both THY-1 mRNA and the numbers of NeuN-labelled neurones of HORCs. Conclusion HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes.

Mots clés: Retina; Degeneration; Experimental &#8211 laboratory

Nuwan Niyadurupola, Peter Sidaway, Andrew Osborne, David C Broadway, Julie Sanderson. The Development of Human Organotypic Retinal Cultures (HORCs) to study Retinal Neurodegeneration. British Journal of Ophthalmology, 2010, 95 (5), pp.720. ⟨10.1136/bjo.2010.181404⟩. ⟨hal-00601170⟩