Biphasic Response in Optic Nerve Head (ONH) Gene Expression with Sustained Downregulation in Axonal Messages Following 8-hours’ Controlled Elevation of Intraocular Pressure (CEI)

Abstract

Purpose : We previously reported that a large number of ONH gene expression changes occur immediately after a CEI in rats (2016 ARVO E-Abstract 2536). Here we specifically examine the evolution of gene expression changes in messages localized within ONH axons and optic nerve injury up to 10-days following CEI.

Methods : Anesthetized rats were exposed unilaterally to an 8-hour CEI at 60 mm Hg. ONH RNA was extracted and gene expression compared between naïve (n=8) and ONHs harvested 0-hr, and 1-, 2-, 3-, 7-, and 10-days post-CEI using RNA-seq (n=6/group). Sequenced data from each ONH were aligned to the Ensembl rat genome using STAR and the Limma-Voom workflow was utilized for differential expression analysis. Optic nerves from these eyes were graded for injury on a scale from 1 (normal) to 5 (>50% of axons degenerating). DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Expression levels of genes in axonally annotated clusters were correlated with injury grades.

Results : The number of significant genes (1354 genes) peaked immediately at the end of the CEI (0-hr), followed by a lull (<4 significant genes per time point) at 1- and 2-days post CEI. Gene activity increased again at 3-days (136 genes) and persisted at 7- (78 genes) and 10- (339 genes) days. Nerve injury compared to naïve was not detected 3-days post-CEI (grade = 1.01 ± 0.01), but was increasingly apparent at 7- (1.72 ± 0.22; P = .0002) and 10-days (1.95 ± 0.42; <15% of axons degenerating; P = .005). Axonal messages at 0-Hr were decreased to ~57% of naïve values, and some declined to <40% by 10-days. 21 axonal messages showed decreased expression as early as 3-days. By 7-days, expression levels of 5 of these genes in individual ONHs correlated negatively with their injury grade (all had r² > 0.7 and P ≤.04). By 10-days, expression levels for all of these genes remained <50% of naïve.

Conclusions : A burst of ONH gene expression immediately after CEI is followed by an unexpected lull, and then a second increase in significant regulation. This includes sustained decrease in axonally associated mRNA that exceeded the proportion of axon degeneration observed. This may indicate: (1) impaired retinal ganglion cell function, (2) an altered axonal and ONH microenvironment and (3) compromised axonal resilience to future IOP insult.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.