Bioluminescence as a novel method of monitoring retinal ganglion cell death associated with glaucoma

Objectives Glaucoma is a leading causes of blindness in the United States and worldwide. Retinal ganglion cells that ultimately become the optic nerve are damaged as a result of the increased pressures associated with glaucoma. Convention animal models of glaucoma, although robust, require sacrificing large groups of animals as glaucoma progresses, thus limiting the ability to longitudinally follow ganglion cell loss. We hypothesize that using Bioluminescent Imaging (BLI) with a luciferin tagged adeno-associated virus Type 2 (AAV2-GFP-luc) to infect the retinal ganglion cells will allow longitudinal assessment of retinal ganglion cell damage induced via optic nerve crush. This technique will provide an opportunity to develop a novel animal model of glaucoma that will greatly reduce the number of animals used in future studies and be of great benefit to the glaucoma research community.

Methods 10 adult Sprague Dawley male rats were allowed to acclimate ~1 week. Animals underwent intravitreal injections of 2x10^12 AAV2-GFP-luc dual construct while under isoflurane anesthesia. Animals were recovered, transferred to BSL 2 housing, and after 4 weeks animals returned for baseline luciferin imaging. Animals were anesthetized via isoflurane anesthesia, dosed with of 150 mg/kg d-luciferin SQ, and dynamically imaged using the IVIS SpectrumCT with an exposure time of ~2 min and a frame duration of ~2 min for a total of 20 images. Two days following baseline imaging, animals underwent optic nerve crushes to induce retinal ganglion cell death in one of the two eyes, and sham-crush in the contralateral eye, and were returned to their home cage for post-operative recovery. Dynamic BLI was repeated at time points 1, 2, 4, 6, and 8 week post nerve crush/sham. Data was analyzed using both area under the curve and peak analysis after each time point, and animals were euthanized via isoflurane overdose and enucleated for further histological analysis.

Results Results of dynamic BLI imaging revealed an initial increase in ocular emission during the AAV2 infective phase which platitude by week 4 post transfection. Post optic nerve crush, ocular emission decrease with time, while sham operated eyes remained largely the same through 8 week period when analyzed via both peak and area under the curve methods. Histological analyses were consistent with the dynamic BLI data.

Conclusions These results indicate that the optic nerve crush procedure produced sufficient ganglion cell death as indicated by the decrease ocular emsission, and that BLI can be used as a minimally invasive way to quantitate retinal ganglion cell damage as it relates to glaucoma.