Abstract
Purpose :
Following optic nerve crush (ONC), retinal ganglion cell (RGC) apoptosis occurs within 7 days in wild-type C57BL/6 mice and never occurs in Bax-/- mice. Understanding how BAX dosage affects when apoptosis occurs is critical for developing an anti-BAX therapeutic to prevent loss of RGCs in optic neuropathy. To that end, we tested the hypothesis that RGCs in Bax+/- mice would experience a delay cell death following ONC.
Methods :
RGC loss in Bax+/- mice was assessed by cell counts of DAPI stained whole-mounts at various times from 3-12 weeks following ONC. Cell loss was measured as the fraction of cells in the damaged (OS) eye relative to the contralateral (OD) eye. The JNK-JUN pathway has been associated with activating BAX in RGCs following ONC. To explore if this pathway was continuously activated post crush in Bax+/- mice, we used immunofluorescence to assess phospho-c-JUN accumulation in the nuclei of RGCs. Bax+/- mice underwent ONC and were collected 1, 3 or 7 weeks later. Retinas were fixed in 4% PFA, sucrose cryoprotected and then eye cups were cryosectioned and stained for the RGC transcription factor, BRN3A, and for an end product of JNK pathway activation, phospho-c-JUN.
Results :
After ONC, Bax+/- retinas began to exhibit statistically significant cell loss at 8 weeks and by 12 weeks exhibited only a 12% loss of cells. Following ONC, phospho-c-JUN co-localized with BRN3A in 83%, 55% and 68% of BRN3A positive cells at 1, 3 and 7 weeks respectively, indicating that this pathway was persistently activated. We are currently interrogating the apoptotic activation mechanism months after ONC by viral reintroduction of an exogenous BAX protein into the RGCs of Bax-/- mice.
Conclusions :
BAX deficiency delays cell loss following ONC. Despite this, the apoptosis associated JNK pathway appeared to remain activate in RGCs for months after acute injury. Together, these findings demonstrate that a BAX therapeutic likely needs to be permanent to prevent the loss of damaged RGCs.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.