Oxygen and Glucose Deprivation on Human Müller Cells (MIO-M1) and Human Organotypic Retinal Cultures (HORCs) in Relation to Glaucoma

Purpose: The purpose of this research was to investigate the effect of glaucoma-related insults, specifically oxygen and/or glucose deprivation (OGD) on the survival and genes expression of human Müller cells (MIO-M1), and retinal ganglion cells (RGCs) using the human organotypic retinal culture (HORC) model. Methods: MIO-M1 cells and HORCs were exposed to different levels of OGD using a custom-built chamber to control oxygen levels. Cell survival was evaluated using MTS and LDH assays while RGC death in HORCs was investigated using NeuN immunohistochemistry and TUNEL-labelling. Expression of genes of interest was assessed using QRT-PCR. Results: Reduced levels of oxygen and glucose (1.11mMglucose/4%O2) caused proliferation of MIO-M1 cells. Full deprivation of glucose caused cell death, but full hypoxia did not affect survival. In HORCs, glucose deprivation and OGD, but not oxygen deprivation alone, caused loss of RGCs. Different levels of OGD regulated expression of genes associated with angiogenesis, glial activation, excitotoxicity and neuroprotection in MIO-M1 cells and HORCs. VEGF expression significantly increased in MIO-M1 cells and HORCs treated with full OGD, and VEGF protein was secreted under reduced levels (1.11mMglucose/4%O2). Secretion of VEGF in MIO-M1 cells and HORCs was also increased under conditions of raised glucose. The PKCβ inhibitor LY333531 decreased VEGF secretion under conditions of raised glucose and hypoxia. Co-culture of MIO-M1 cells resulted in more damage/apoptosis to HORCs and reduced RGCs survival. Conclusions: Use MIO-M1 cells and the HORC model were effective in studying the effect of OGD in relation to glaucoma. Glucose rather than oxygen was the key survival factor for RGCs and MIO-M1 cells. The secreted factors by Müller cells could have protective and detrimental effects on RGC survival. Investigation of mechanisms using these models may be of benefit in development of potential therapeutic interventions for retinal neurodegenerative diseases including glaucoma.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2015

Author dc:creator

• Aldarwesh, Amal

Language dc:language

en