Abstract
Purpose :
Glaucoma is a retinal neurodegenerative disease and is a leading cause of blindness. Neurotrophic factor deprivation due to axonopathy has been implicated in glaucoma pathogenesis. Our previous studies have shown that secreted neurotrophic protein Neuritin-1 (NRN1) could substantially reduce retinal ganglion cell (RGC) damage after optic nerve injury. Thus, we propose to promote RGC survival by the exogenous addition of NRN1 using our novel ex vivo perfused pressurized Translaminar Autonomous System (TAS) that can simulate glaucoma pathogenesis.
Methods :
Human glaucomatous and non-glaucomatous donor eyes were obtained from eye banks according to the Declaration of Helsinki. Retinal quality was assessed by gene expression and electroretinograms (ERG) prior to culture. Levels of NRN1 and THY-1 were evaluated via immunostaining and qPCR (N=8). To recapitulate the in vivo pressure stimulus, human posterior cups (N=4) were cultured in the TAS model at normal (16 mmHg, N=4) and high IOP (30 mmHg, N = 4) for 7 days with and without hNRN1 (200ng/mL). RGC survival was evaluated by assessing markers of RGC (THY-1, RBPMS), gliosis (GFAP, IBA-1), apoptosis (CASP3) and ECM deposition (COLIV, FN) by qPCR and western blotting. Retinal function was assessed by ex-vivo ERG. To find the NRN1 receptor, the pressurized eyes were treated with Insulin (10nm) and Insulin receptor (IR) inhibitor (200nm) in combination with NRN1 and downstream effects were assessed as aforementioned.
Results :
Donor retinas had measurable retinal gene expression and electrical activity even after 48 hours of death. Significant reduction of NRN1 (p<0.05, N=4) and THY-1 was observed in glaucomatous retinas. In contrast to control eyes, significant decrease in gliosis (GFAP, p<0.05, N=4, IBA-1, p<0.05, N=4) with reduced ECM (COLIV, FN, p<0.05, N=4) was found in NRN1-treated eyes. Notably, at high IOP, NRN1 significantly reduced apoptosis (p<0.05, N=4) and increased RGC survival (RBPMS, THY-1, p<0.05, N=4). Also, measurable retinal functional activity was observed with NRN1 treatment. The blockage of IR, enhanced gliosis and apoptosis suggest NRN1 activation through IR.
Conclusions :
Our study validated that hNRN1 promote RGC survival and improves retinal functions under degenerative conditions. This study substantiates human NRN1 as a novel therapeutic target to rescue RGC from glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.