Effect of Postmortem Time on Gene Expression Profile of Perfused Human Fellow Eyes

Abstract

Purpose: : Arrival of fresh postmortem human eyes to a laboratory is subjected to uncontrollable factors (e.g. time of death, immediate transportation, geographic location) which are believed to hamper reliable results. We investigate whether outflow facility (C), morphology and microarray profiles of fellow eyes (identical genetic background) are affected by postmortem time.

Methods: : A pair of eyes from a 76 year old Caucasian female was received at 8h postmortem in wet ice. The OD was immediately dissected and placed in the perfusion system (early). The fellow eye, OS was stored in original container at 4ºC and perfused at 32h (late). Each eye was perfused for 3 days. At the end of the experiment, a wedge was fixed for morphology and the remaining trabecular meshwork (TM) dissected for RNA. Gene expression analysis was done on U133 Plus2.0 Affymetrix GeneChips (54,678 gene probes). Comparative heat maps of normalized expression signals, overlapping Venn Maps, and enrichment of GO functional categories were obtained with the GeneSpring 7.3.1 software.

Results: : On the first overlapping perfusing day (day 2 OD, day 1 OS) C values were 0.16 and 0.17 µl/min/mmHg respectively. On the second one (day 3 OD, day 2 OS), the values were 0.14 for the OD and 0.19 for OS. Light and electron microscopy of OS showed a full structured TM. Overall gene expression between the two eyes was remarkably similar. The number of Present Genes was 21,611 in OD compared to 22,390 genes in OS with 90% overlapping (Venn map). The Absent Genes list contained 19,902 (OD) and 19,154 (OS) overlapping 85%. Specifically, the most expressed nonribosomal genes in each eye were also similar. Prostaglandin D2 synthase (PTGDS) was the 4^th most expressed in OD and the 3^th in OS; Myocilin (MYOC) was the 9^nd and 7^th highest and Matrix GLA protein (MGP) the 18^th and 4^th. Heat maps of both eyes on genes lists relevant to TM physiology (ECM, oxidative stress, cytoskeleton) very almost equal. There were 13 GO molecular function categories with p≤0.05, 12 of them overlapping. RNA binding (GO 3723) was the most significant category in both eyes with p ≤3.8E-59, 7.8% genes in OD and p ≤2.3E-64, 7.8% genes in OS. Catalytic activity (GO 3824) was the 8^th ranked in OD (p ≤3.7E-9, 36.9% genes) and the 5^th in OS (p ≤3.0E-16, 37.3% genes). Chaperone regulator activity category (GO 30188) was the 12^th ranked in both eyes with p ≤0.009, 0.1% genes in OD and p ≤0.01, 1.1% genes in OS.

Conclusions: : There is a very high physiological, morphological and molecular similarity between eyes perfused at 8h and 32h postmortem. These results validate the use of longer postmortem time as well as report for the first time the comparative expression profile of two eyes from the same individual.

Keywords: trabecular meshwork • gene microarray • gene/expression