Abstract
Purpose :
The retraction of retinal ganglion cell (RGC) dendrites is among the earliest pathological changes during glaucoma. We recently demonstrated that insulin, administered after arbor retraction, promoted remarkable RGC dendrite and synapse regeneration. Here, we asked the following questions: 1) Is the reduction of high intraocular pressure (IOP) sufficient to promote dendrite regeneration? 2) What are the signalling components downstream of insulin that stimulates RGC dendrite regeneration in glaucoma?
Methods :
Thy1-YFP mice, which allow visualization of RGC dendritic arbors, received an intracameral injection of magnetic microbeads to induce ocular hypertension. Daily topical application of brinzolamide, a carbonic anhydrase inhibitor, was used to reduce IOP. RGC dendrites were imaged with confocal microscopy and 3D reconstructed using Imaris software. To study molecular mechanisms, RGCs were isolated by Fluorescence-Activated Cell Sorting from insulin- or vehicle-treated glaucomatous retinas and sham-operated controls, followed by transcriptome analysis (RNA-seq).
Results :
Brinzolamide reduced IOP in vehicle-treated controls (sham: 10.7±0.3 mmHg, brinzolamide: 11.3±0.4 mmHg, vehicle: 20.5±0.8 mmHg, n=12 mice/group, Student’s t-test, p<0.001). Analysis of RGC dendrites showed that IOP lowering was not sufficient to promote dendritic regrowth. Total RGC dendritic length increased in insulin-treated glaucomatous eyes (4,515±149 µm) to values similar to those found in controls (4,566±233 µm), but not in brinzolamide-treated eyes (2,534±164 µm) or vehicle (2,981±169 µm) (n=6 mice/group, ANOVA, p<0.001). RNA-seq analysis of insulin- and vehicle-treated glaucomatous retinas identified key regulatory pathways that might be implicated in insulin-induced RGC dendrite regeneration including the mTOR pathway, glycolysis, fatty acid metabolism, DNA repair, and myc-targets (FDR<0.05).
Conclusions :
IOP reduction alone is not sufficient to promote RGC dendrite regeneration, suggesting that endogenous insulin or related factors cannot fulfill the role of exogenous insulin. Importantly, several molecular pathways are activated during insulin-mediated regeneration. These findings support a critical role for insulin administration to restore RGC connections and retinal function and identify new pathways that might reveal novel therapeutic targets for glaucoma.
This is a 2020 ARVO Annual Meeting abstract.