Bioenergetic neuroprotection in experimental glaucoma

Abstract

Glaucoma refers to a group of ocular conditions united by a clinically characteristic intraocular pressure optic neuropathy and associated degeneration of the retinal ganglion cells (RGCs). It is a leading cause of blindness worldwide. Although the pathogenesis of the most common variant, primary open-angle glaucoma, remains poorly understood, there is considerable evidence that energy insufficiency plays a pathogenic role. Currently, the only proven strategy to treat glaucoma is intraocular pressure (IOP) reduction by medical, laser or surgical modalities. Whilst these IOP lowering therapies have been proven to retard glaucomatous progression, there is inter-individual responsiveness and a significant proportion of individuals progress to visual impairment. Additional neuroprotective strategies that augment IOP-lowering therapy would be highly clinically desirable and likely to reduce the burden of glaucomatous visual impairment at the individual and society levels. This thesis investigates bioenergetic neuroprotection in experimental glaucoma and specifically the effects of pyruvate supplementation in facilitating neuroprotection or recovery of compromised, but not dead, RGCs. The thesis consists of two related but independent sections. The first section describes a novel automated cell counting program, conceived to facilitate highly accurate automated counts of immunolabelled RGCs on retinal wholemounts. Quantifying RGCs on retinal wholemounts remains one of the key histopathological end points in pre-clinical glaucoma neuroprotection studies, yet manual or semi-automated methods are labour intensive, monotonous, time consuming and subject to inter and intra-observer variability. Limited software is available to expedite counting of immunolabelled RGCs on wholemounts. Their use is restricted due to cost constraints with purchasing licences for commercially available software, limited applicability to a variety of RGC / neuronal specific immunolabels, inability to distinguish cells in cluster, and heavy manual image pre-processing steps. We have overcome these barriers with our innovative software and validated its use against a variety of RGC specific immunolabels (Brn3a and RBPMS). Our results demonstrate excellent accuracy and a 40-fold reduction in time compared to manual counting. Ultimately this software promises to expedite data acquisition and statistical analysis in pre-clinical glaucoma neuroprotection research, potentially accelerating translation to clinical trials. The second section demonstrates the highly significant neuroprotective effects of oral pyruvate supplementation in our experimental rat model of glaucoma. Experimental glaucoma was induced unilaterally by laser photocoagulation of the trabecular meshwork and episcleral veins, in control and pyruvate-supplemented rats. At two weeks, the retina and optic nerves were processed for quantification of the number of surviving RGCs and axonal injury, respectively. The combined results clearly demonstrate RGC preservation, decreased axonal loss and degeneration, and attenuated microglial proliferation and phagocytic activity in the retinal nerve fibre layer and optic nerves of pyruvate-supplemented glaucomatous rats. Retinal cell cultures demonstrated that the presence of pyruvate counteracted the loss of both glia and neurons when subjected to either glucose deprivation or oxidative stress, suggesting that pyruvate supplementation has multimodal mechanisms of neuroprotection. These results unveil a potential new therapy for glaucoma with promise of translation into clinical trials.