Treatment of early age-related macular degeneration in a cell culture model with femtosecond laser pulses

Abstract

This thesis work focuses on obtaining preliminary results demonstrating the isolated femtosecond laser ablation of sub-RPE drusen-like deposits. Age-related macular degeneration (AMD) is an age-onset degenerative condition of the retina that can lead to legal blindness. The early stages of the condition are characterized by the development of hemi-spherical cellular debris accumulations, known as drusen, under the retina. This project has two specific aims: demonstrating the laser ablation of AMD related sub-RPE deposits and assembling a prototype for a clinical therapeutic beam delivery system. An articulating arm is fitted with silver mirrors for 800 nm and coupled to a slit lamp ophthalmic device for femtosecond laser delivery to the retina. A novel cell culture model using the immortalized cell line ARPE-19 is developed. The cell culture model is evaluated by confocal and transmission electron microscopy for the presence of sub-RPE drusen-like deposits. The samples are stained for two known drusen components, apolipoprotein E and cholesterol, to verify the artificial drusen-like deposits share a similar biochemical composition to drusen in vivo. Sub-RPE drusen-like deposits in the ARPE-19 model were targeted with 10 femtosecond pulse trains, 1.9-4.2 nJ/pulse, centered at 800 nm wavelength, from a Ti:Sapphire laser. Fluorescence microscopy was used to qualitatively confirm successful deposit ablation. After laser ablation more fluorescent dye was added to the dish and monitored to see if the areas targeted by the laser regain fluorescence. Although areas of the sample did regain fluorescence, the targeted deposit accumulation never regained fluorescence. Thus, eliminating the possibility of laser photobleaching being responsible for deposit removal as opposed to plasma mediated breakdown.