Modulation of Clinically Significant Genetic Loci for Glaucoma in a Murine Retinal Explant Model

Abstract

Purpose: Retinal explants have been used as a relatively high throughput model for investigation of pathogenesis and potential neuroprotective therapies in glaucoma. Here we describe modulation of clinically significant genetic loci from Genome Wide Association Studies in glaucoma in a murine retinal explant model.

Methods: Mouse (C57BL/6) retinal explants were created and incubated at 37 degrees for between 1 and 7 days. A minimum of 3 eyes were used per experiment with the contralateral eyes used as internal control. Ganglion cell survival was quantified histologically using anti-BIII tubulin antibody in wholemount retinas. This was compared with in situ staining with anti-RNA-binding protein with multiple splicing (RBPMS) antibody. DAPI was used as a comparator stain. mRNA expression was measured by rtPCR in separate experiments performed under the same conditions. A caspase activity assay was used to assess apoptosis (Caspase-Glo, Promega). DMSO was used as a vehicle control. Explants were treated with a pan-caspase inhibitor (Z-VAD-FMK), IL1β or irbesartan.

Results: Application of Z-VAD-FMK increased cell survival by approximately 1.5 fold at day 4 (peak effect) when assessed histologically compared to control. RBPMS staining diminished with time with almost none by day 4.This suggests that BIII tubulin stains cellular structure which is maintained, whereas RBPMS stain is lost more quickly as RNA binding proteins are destroyed earlier in the apoptosis pathway. Peak differentiation of caspase activity was at day 1-2 with no significant differentiation at day 4. By establishing an appropriate time window for mRNA expression, we were able to investigate modulation of two GWAS significant genetic loci, CDKN2B and ABCA1 with drugs known to affect cell survival in this model. Histologically, irbesartan almost doubled cell survival and IL1β reduced cell survival by approximately 50%. CDKN2B mRNA expression increased approximately 1.7 fold at day 1 by application of irbesartan compared to day 1 vehicle controls. Likewise ABCA1 mRNA was increased approximately four fold when Z-VAD-FMK was applied but not modulated at all when IL1β was applied.

Conclusions: These findings suggest that a murine ex vivo model can be a relatively rapid way to investigate modulation of clinically relevant genes for glaucoma and further investigate these findings in vivo utilising C57BL/6 transgenic mice.