Glaucoma is the second leading cause of blindness, affecting at least 300,000 Canadians, and over 67,000,000 people worldwide. Retinal Ganglion cell (RGC) death is a major cause of visual impairment in optic neuropathies including glaucoma, AMD (Age Related Macular Degeneration), diabetic retinopathy, uveoretinitis and vitreo-retinopathy. Glaucoma is a disease in which the optic nerve and retinal ganglion cells are injured leading to peripheral vision loss and eventually blindness. Glaucoma is commonly characterized by an increase in intraocular pressure and is treated by ocular hypotensive drugs such as latanoprost. Despite the advent and therapeutic use of these drugs, vision loss as a result of RGC apoptosis and optic nerve atrophy continues, resulting in blindness.
Central nervous system (CNS) injury results in permanent functional loss due to the inability of adult CNS neurons to regenerate axons, and their susceptibility to programmed cell death (apoptosis). The unifying hallmark of visual diseases such as glaucoma and optic neuropathies is the death of Retinal Ganglion Cells (RGCs) of the eye. RGCs are CNS neurons that transmit visual information from the retina to the brain via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely cut (axotomized), transecting the axons of the entire RGC population. Optic nerve transection (FIG. 1) is a reproducible model of apoptotic neuronal cell death in the adult CNS (Bahr 2000; Koeberle and Bahr 2004; Magharious, D'Onofrio et al. 2011; Magharious, D'Onofrio et al. 2011). The optic nerve transection model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations. The diffusion of chemicals through the posterior chamber fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs (siRNAs), plasmids, or viral vectors to the cut end of the optic nerve (Garcia Valenzuela and Sharma 1998; Kugler, Klocker et al. 1999; Lingor, Koeberle et al. 2005; Koeberle and Schlichter 2010; Koeberle, Wang et al. 2010). This permits selective therapeutic targeting of RGCs without confounding effects on bystander neurons or surrounding glia. An additional benefit is the accuracy with which cell survival can be quantified after injury. The retina is a flat tissue and RGCs are located in the innermost layer, the ganglion cell layer. The survival of injured RGCs can be tracked by applying a fluorescent tracer (3% Fluorogold) to the cut end of the optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. Tracers applied by these methods are retrogradely transported back to the RGC cell bodies, labeling all of the RGCs in the retina. RGC survival is then quantified by fixing the eye, removing the retina, and flat-mounting the tissue (FIG. 2). In this preparation, the ganglion cell layer, which is a monolayer (one cell in thickness), is imaged in order to calculate the number of cells per unit area (cells/mm2) (FIG. 2). Optic nerve transection causes the apoptotic death of 90% of injured RGCs within 14 days postaxotomy (Villegas-Perez, Vidal-Sanz et al. 1988; Villegas-Perez, Vidal-Sanz et al. 1993; Berkelaar, Clarke et al. 1994; Peinado-Ramon, Salvador et al. 1996). RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate (FIG. 2). This provides a time window for experimental manipulations directed at developing therapeutics for CNS insults.
Stroke (ischemia) of the CNS can be studied via ophthalmic artery ligation. Ligature of the ophthalmic vessels (FIG. 3), without damaging the optic nerve, causes 50% RGC death within 14 days (Lafuente, Villegas-Perez et al. 2002). RGCs die by the process of apoptosis following axotomy, ischemia, and during the course of glaucoma (Berkelaar, Clarke et al. 1994; Quigley, Nickells et al. 1995; Bahr 2000; Lafuente, Villegas-Perez et al. 2002; Koeberle and Bahr 2004), hence these animal models provide a testing ground for therapeutics that can be directly applied in a clinical situation in order to prevent RGC death. Furthermore, the therapeutics developed via studies in these models are applicable to the treatment of insults in other regions of the CNS such as the brain or spinal cord, that include traumatic CNS injury, stroke, concussion, neurodegenerative diseases, and brain damage caused by tumours or surgical procedures.