Abstract

PurposeElevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated.MethodsA chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot.ResultsExposure of HORCs to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD.ConclusionsDirectly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.

Highlights

  • Glaucoma is a group of optic neuropathies leading to progressive loss of visual field due to the degeneration of retinal ganglion cells (RGCs) in the inner retina and loss of their axons in the optic nerve [1]

  • Exposure of Human organotypic retinal cultures (HORCs) to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, Lactate dehydrogenase (LDH) release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls

  • oxygen glucose deprivation (OGD) increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/ min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD

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Summary

Introduction

Glaucoma is a group of optic neuropathies leading to progressive loss of visual field due to the degeneration of retinal ganglion cells (RGCs) in the inner retina and loss of their axons in the optic nerve [1]. Vision loss caused by glaucoma is irreversible. Glaucoma is the second most common cause of world blindness after cataract [2] and the most common cause of irreversible blindness. IOP above the normal range of 11 to 21mmHg has been shown to increase the likelihood of developing glaucoma with higher pressures leading to a progressive worsening of vision [4,5,6,7]. Fundamental questions remain, as to the mechanism by which elevated IOP causes degeneration of the RGCs and subsequent loss of vision in glaucoma [8]

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