The effect of mobile zinc on retinal ganglion cells death after glaucomatous optic nerve injury

Abstract

Purpose : Traditional therapies targeting RGCs failed to maintain the long-term cell survival or axon regeneration in glaucoma, which suggested unknown molecular mechanisms exist. Our previous research revealed that mobile zinc (Zn²⁺) increased rapidly in the early stage after injury directly to the optic nerve and mediated RGCs apoptosis (Li Y, et al. PNAS 2017). In addition, Zn²⁺ chelation enabled long survival of RGCs and axon regeneration. However, the effect of Zn²⁺ on RGCs death in the glaucoma model has not been fully elucidated.

Methods : 1. A mouse model of acute ocular hypertension (OH, 80 mmHg for 1 hour) was established, and the level and localization of Zn²⁺ in retina were monitored by autometallography (AMG) 6 hours post injury.
2. RGC-5 was cultured and treated with Zn²⁺in vitro. We studied the time-dependent effect of Zn²⁺ on RGCs apoptosis at 6h, 12h and 18h using flow cytometry labeled by Annexin V-FITC/PI.
3. The dose-dependent effect of Zn²⁺ (50, 100, 150 μM) on mitochondrial function was revealed by detecting fluorescenct level of JC-1, a fluorescence probe which reflects mitochondrial membrane potential with different color.
4. The oxygen-glucose deprivation for 3h and reperfusion for 6h (OGD/R model) was established on primary cultured mouse RGCs, and the effect of specific Zn²⁺ chelator ZX1 on RGCs apoptosis was detected by flow cytometry.

Results : 1. After OH, the level of Zn²⁺ increased rapidly (1.76±0.26-fold, P<0.05) in the inner-plexiform layer within 6 hours (Fig. 1).
2. 6 hours after Zn²⁺ treatment (150 μM) in vitro, significant death of RGC-5 was observed (15.32 ±0.32 %, P<0.05). The toxicity of Zn²⁺ increased in a time-dependent manner (Fig. 2A).
3. The mitochondrial membrane potential declined in accordance with Zn²⁺ concentration, and the toxicity of Zn²⁺ increased in a dose-dependent manner (Fig. 2B)
4. Zn²⁺ Chelator ZX1 (5 μM) significantly inhibited the apoptosis of RGCs in OGD/R model (P<0.0001, Fig. 2C).

Conclusions : Mobile Zn²⁺ increased rapidly in mouse retina after OH, leading to RGCs injury. Zn²⁺ chelators significantly enhanced the survival of RGCs. Apoptosis mediated by mitochondrial disfunction could be the mechanism underlying Zn²⁺ toxicity. This study provides a new and promising therapeutic target for glaucoma.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Zn²⁺-AMG staining in mouse retina at 6h after ocular hypertension.

 

Zn²⁺ induced cell death through mitochondrial disfunction in cultured retinal neurons.