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Yong Hwan Park, Edwin J Ostrin, Sangbae Kim, Rui Chen, Benjamin J Frankfort; Transcriptomic profiling of retinal ganglion cells following microbead induced intraocular pressure elevation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5159.
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© ARVO (1962-2015); The Authors (2016-present)
Intraocular pressure (IOP) is the major risk factor for glaucoma, however very little is understood on how elevated IOP modulates gene expression in retinal ganglion cells (RGCs) in glaucoma. In this study, RNA sequencing is used to examine the initial transcriptomic changes that occur in adult mouse RGCs after IOP elevation level to two distinct levels.
Elevation of IOP was induced by injection of polystyrene microbeads into the anterior chamber of left of eyes of both male and female wild-type C57BL/6J (6 weeks old). IOPs were observed for 2 weeks. Bead injected eyes with an average IOP increase of either 1-4mmHg or >4mmHg were divided into mild IOP and moderate IOP groups, respectively. Saline-injected eyes served as controls with IOP change < ± 1mmHg. RGCs were purified utilizing an established 4-step immunopanning technique with an antibody against Thy1.2. Total RNAs were isolated using a TRIzol/spin column-based nucleic acid extraction kit. Samples having RIN values >7 were utilized. For each treatment group, 100pg of total RNA per retina were pooled together and then split into triplicate samples, and total cDNA amplified with the SMART-Seq v4. Gene expression changes were analyzed with the HiSeq2500 at 20 million paired-end reads per sample.
2 weeks after injection, the average difference in IOPs were elevated significantly (p<0.0001) in both the mild IOP (2.65 ± 0.3 mmHg, n=12) and moderate IOP (6.96 ± 0.76mmHg, n=6) groups when compared to the control group (0.02 ± 0.13mmHg, n=12). Isolation of RGCs yielded 45,246 ± 2,654 cells per retina of which 88.1 ± 4.2% stained positive for the RGC marker, RBPMS. The number of upregulated genes was 86 and 204, and the number of downregulated genes was 223 and 200 in the mild and moderate IOP groups, respectively.
Total adult RGC RNA was successfully isolated and sequenced following mild and moderate IOP elevation via the microbead technique. Dissimilarities in the number of genes with expression changes in the two pressure groups suggest that IOP level may elicit differential responses. Future analysis will identify specific pathways and upstream modulators that will determine elevated IOP’s contribution to RGC abnormality in glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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