Neuroprotection, the Role of Iron in Photoreceptor Toxicity Induced by Subretinal Hemorrhage

Abstract

Purpose:: Sub-retinal hemorrhage (SRH) resulting from choroidal neovascularization is a common finding in patients with acute visual loss from exudative age-related macular degeneration (AMD) and several other retinal disorders. A neuroprotective therapy against short-term hemorrhage-induced retinal toxicity holds the potential to complement and enhance current anti-vascular endothelial growth factor therapies in AMD. In this phase of the project, our aims are: 1) to elucidate the mechanism of retinal cell death; 2) clarify the role of intracellular iron and iron transporters in retinal toxicity, and identify potential therapeutic targets in this pathway; and 3) to test potential neuroprotective therapies for their ability to slow or prevent photoreceptor loss during the acute crisis of a subretinal hemorrhage.

Methods:: Autologous whole blood will be obtained from anesthetized rabbits via the ear vein, for immediate subretinal blood injection. Blood alone or along with either IVTA, deferoxamine or SIH will be injected (approximately 100uL) into the subretinal space to create a bullous retinal elevation involving the central retina. At chosen intervals, rabbits will be euthanized and enucleated, and eyes will be fixed and embedded in paraffin or cyrosectioned. Retinal sections will be made through the region of subretinal blood and multiple sections analyzed and averaged in each eye. Counts of cells exhibiting degenerative morphology will be made to identify all retinal layers affected. Perl’s stain will be used to stain for iron. TUNEL staining will be used to identify cells undergoing specific apoptotic cell death, in the photoreceptor layer as well as other layers.

Results:: Histologic data indicates that iron from the subretinal blood cells migrating into the photoreceptors (PR) is a mechanism of retinal neuronal cell toxicity. There is a nuclear locus of iron accumulation, which precedes photoreceptor loss. Macrophages were found in the PR layers and the RPE. In addition, we have found that apoptosis, or programmed cell death, is initiated within 48 hours of exposure to subretinal blood. Furthermore, we have established the timing and histologic characteristics of retinal damage following injection of autologous blood into the subretinal space.

Conclusions:: Iron accumulates in the outer segments, with a gradient with more iron in the outer segments near the subretinal blood. These findings suggest that that this photoreceptor iron accumulation may contribute to retinal degeneration following SRH.

Keywords: age-related macular degeneration