Abstract
Purpose :
To investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals following a controlled elevation of IOP (CEI).
Methods :
An equal number of male and female rats underwent CEI for 8-hours, at pressures of 20 mmHg (mean rat IOP; CEI-20; N=11) and 50 mmHg (2.5x mean IOP; CEI-50; N=12). Naïve animals (n=12), receiving no anesthesia or surgical interventions, served as additional controls. Immediately after CEI, TM and ONH tissues were dissected and RNA isolated. Samples were processed with the Nanostring nCounter PanCancer Immune Profiling Panel, containing 770 genes, following the low input RNA protocol, and raw count data were uploaded to Rosalind® for differential expression analyses. For TM, three comparisons were made: “CEI-50 vs. CEI-20”, “CEI-50 vs. naïve”, and “CEI-20 vs. naïve”. For ONH, our previous RNA-seq study showed no significant gene responses in “CEI-20 vs. naïve”, so only “CEI-50 vs. naïve” ONH samples were compared. Gene functional assessment was evaluated with ShinyGO bioinformatics software.
Results :
Mean RNA concentration was 61 ng/TM and 60 ng/ONH. TM and ONH naïve samples clustered separately, indicating distinctive gene profiles. Naïve TM had 34 uniquely enriched genes, while the ONH had 33. For TM, 46 IOP-related genes were found to be significant in “CEI-50 vs. CEI-20” and “CEI-50 vs. naïve” groups, with ‘ECM space’ and ‘membrane raft’ identified as significant GO cellular components. Fifteen genes were common to both comparisons, which were related to the Notch (upregulated) and TGFβ (downregulated) KEGG pathways. For ONH, 22 significantly regulated genes were identified in the “CEI-50 vs. naïve” comparison and significantly affected biological process pathways were ‘response to cytokine’ and ‘immune response’.
Conclusions :
In response to CEI-50 for 8-hours, we identified IOP-related gene responses in both the TM and the ONH in the same animals. In the TM, Notch is a conserved pathway, which plays an important role in cell-cell communication and mechanotransduction. Downregulation of TGFβ pathway genes suggest that TM responses may be protective by preventing TGFβ-induced ECM synthesis. For ONH, the initial response to elevated IOP is also likely protective, with astrocytes likely playing a key role since they are the predominant glial type in this region.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.