Effects of Hypoxic Preconditioning and Vascular Endothelial Growth Factor on the Survival of Isolated Primary Retinal Ganglion Cells.

Hyoung Won Bae,Sang Yeop Lee,Ah Reum Hwang,Chan Yun Kim,Gong Je Seong,Wungrak Choi

doi:10.3390/biom11030391

Abstract

This study aimed to investigate the effect of hypoxic preconditioning (HPC) on primary retinal ganglion cell (RGC) survival and the associated mechanism, including the role of vascular endothelial growth factor (VEGF). Retinas were separated from the enucleated eyeballs of Sprague–Dawley rats on postnatal days 1–4. RGCs were harvested using an immunopanning-magnetic separation system and maintained for 24 h in a defined medium. Hypoxic damage (0.3% O2) was inflicted on the cells using a CO₂ chamber. Anti-VEGF antibody (bevacizumab) was administered to RGCs exposed to hypoxic conditions, and RGC survival rate was compared to that of non-anti-VEGF antibody-treated RGCs. HPC lasting 4 h significantly increased RGC survival rate. In the RGCs exposed to hypoxic conditions for 4 h, VEGF mRNA and protein levels were significantly increased. Treatment with high dose bevacizumab (>1 mg/mL) countered HPC-mediated RGC survival. Protein kinase B and focal adhesion kinase levels were significantly increased in 4-h hypoxia-treated RGCs. HPC showed beneficial effects on primary RGC survival. However, only specifically controlled exposure to hypoxic conditions rendered neuroprotective effects. Strong inhibition of VEGF inhibited HPC-mediated RGC survival. These results indicate that VEGF may play an essential role in promoting cell survival under hypoxic conditions.

Highlights

Accepted: 4 March 2021Glaucoma is one of the leading causes of blindness worldwide, with the estimated number of patients exceeding 64 million [1,2]
The response of retinal ganglion cell (RGC) exposed to hypoxic conditions for various lengths of time was measured to identify the sublethal hypoxic preconditioning (HPC)
Following treatment in DMEM/F-12 for 24 h, the harvested RGCs were exposed to hypoxic conditions by incubation in a CO2 chamber for 2, 4, 6, 12, and 24 h; the cells were counted (Figure 1A)

Summary

Introduction

Glaucoma is one of the leading causes of blindness worldwide, with the estimated number of patients exceeding 64 million [1,2]. It is a chronic disease that requires lifetime treatment upon diagnosis. Treatment begins with medication to lower the intraocular pressure (IOP) to halt the progression of the disease. The disease continues to progress in certain patients despite satisfactory decrease in IOP [3,4]. Studies have searched for additional treatment strategies. Based on the ability of the cells to adapt to stressful conditions, preconditioning-induced, neuroprotection-based approaches to prevent or slow the progressive loss of the retinal ganglion cells (RGCs) have received considerable attention [5]

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