Role of C/EBP homologous protein (CHOP) in the survival of retinal ganglion cells after retinal ischemia/reperfusion injury

Abstract

Purpose: Retinal ischemia/reperfusion (I/R) causes apoptotic death of retinal ganglion cells (RGC). CHOP is a pro-apoptotic protein and a unfolded protein response (UPR) marker that plays a role in ER-stress mediated apoptotic cell death. The purpose of this study was to investigate the role of CHOP in mouse RGC survival following retinal I/R injury.

Methods: Retinal I/R was induced in adult C57BL/6J (WT) and CHOP-/- mice by cannulation of the anterior chamber of the left eye with a needle connected to a reservoir of saline. Intraocular pressure was increased to 120 mmHg for 60 min, after which the needle was withdrawn to restore retinal circulation. Uninjured right eyes served as controls. Expression of CHOP protein and other UPR markers (p-eIF2α and BiP) in WT mice post-I/R was studied using Western blot and immunohistochemistry. To compare RGC survival between WT and CHOP-/- mice, retinal flat mount staining with RGC marker, Brn3a was performed. Scotopic threshold response electroretinography (STR-ERG) was performed at 0.03 mcd.s/m² light intensity to evaluate retinal function.

Results: CHOP protein was up-regulated by 30 % in I/R injured eyes (1.30 ± 0.11 arbitrary units (a.u.)) compared to control eyes (1 ± 0.07 a.u.) in WT mice three days after I/R injury (p < 0.05). Protein levels of p-eIF2α and BiP also increased by 19% (I/R: 1.19 ± 0.15 a.u., Control: 1 ± 0.06 a.u.) and 11% (I/R: 1.11 ± 0.02 a.u., Control: 1 ± 0.03 a.u.) respectively (both p < 0.05). Co-localization of CHOP and Brn3a confirmed the up-regulation of CHOP specifically in the RGCs. In the uninjured control eyes, CHOP knockout did not affect baseline RGC density or STR-ERG amplitude. I/R injury decreased RGC densities and STR-ERG amplitudes in both WT and CHOP-/- mice. However, survival of RGCs in I/R-injured CHOP-/- mouse eyes (3337.1 ± 316.4 RGC/mm²) was 48% higher (p < 0.05) than that of I/R-injured WT mouse eyes (2248.7 ± 225.9 RGC/mm²) three days after I/R injury. STR-ERG amplitudes were 83 % higher in CHOP-/- I/R eyes (18.6 ± 1.1 μV) compared to WT I/R eyes (10.1 ± 0.9 μV) (p < 0.05).

Conclusions: Absence of CHOP partially protects against the loss of RGCs and reduction in retinal function (STR-ERG) after I/R injury. These results indicate that CHOP and thus ER-stress play an important role in RGC apoptosis in retinal I/R injury.

Keywords: 688 retina • 615 neuroprotection • 531 ganglion cells