Characterisation of the metabolome of ocular tissues and post-mortem changes in the rat retina

Time-dependent post-mortem biochemical changes have been demonstrated in donor cornea and vitreous, but there have been no published studies to date that objectively measure post-mortem changes in the retinal metabolome over time. The aim of the study was firstly, to investigate post-mortem, time-dependent changes in the rat retinal metabolome and secondly, to compare the metabolite composition of healthy rat ocular tissues. To study post-mortem changes in the rat retinal metabolome, globes were enucleated and stored at 4oC and sampled at 0, 2, 4, 8, 24 and 48 hours post-mortem. To study the metabolite composition of rat ocular tissues, eyes were dissected immediately after culling to isolate the cornea, lens, vitreous and retina, prior to storing at –80oC. Tissue extracts were subjected to Gas Chromatograph Mass Spectrometry (GC-MS) and Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS). Generally, the metabolic composition of the retina was stable for 8 hours post-mortem when eyes were stored at 4oC, but showed increasing changes thereafter. However, some more rapid changes were observed such as increases in TCA cycle metabolites after 2 hours post-mortem, whereas some metabolites such as fatty acids only showed decreases in concentration from 24 hours. A total of 42 metabolites were identified across the ocular tissues by GC-MS (MSI level 1) and 2782 metabolites were annotated by UHPLC-MS (MSI level 2) according to MSI reporting standards. Many of the metabolites detected were common to all of the tissues but some metabolites showed partitioning between different ocular structures with 655, 297, 93 and 13 metabolites being uniquely detected in the retina, lens, cornea and vitreous respectively. Only a small percentage (1.6%) of metabolites found in the vitreous were exclusively found in the retina and not other tissues. In conclusion, mass spectrometry-based techniques have been used for the first time to compare the metabolic composition of different ocular tissues. The metabolite composition of the retina of eyes kept at 4oC post-mortem is mostly stable for at least 8 hours.