Correlation of Retrograde Retinal Ganglion Cell Labeling and Optic Nerve Injury in a Rat Glaucoma Model

Abstract

Abstract: : Purpose: To correlate the loss of retrograde retinal ganglion cell (RGC) labeling with the amount of optic nerve degeneration due to intraocular pressure (IOP) elevation from aqueous outflow obstruction by episcleral vein sclerosis in rats. Methods: Brown Norway rats (N=13) received unilateral injections of hypertonic saline into episcleral veins to produce chronic IOP elevation. IOP was measured frequently by tonopen on awake animals prior to RGC labeling. At 3, 4 and 5 weeks post injection, RGCs were retrogradely labeled with Fluorogold from the superior colliculus. IOP was measured under anesthesia by tonopen 10 days later at the time of sacrifice. Automated counting was performed by applying standard imaging software on images of retinal flat mounts so that the total number of labeled RGCs could be determined. Optic nerves were graded by multiple, masked observers on a scale of 1 (normal) to 5 (entire nerve affected with at least 50% of axons degenerating). Results: Prior to RGC labeling, awake mean (±S.D.) tonopen IOPs and cumulative IOP doses (mm Hg-days) in the injected eye were 5.7±4 mm Hg and 164±104 mm Hg-days above the fellow eye. Final IOP for injected eyes was 17.3±10.7 mm Hg above the fellow eye. RGC counts in injected eyes were 12,352±26,601(including 7 retinas with no labeled RGCs) compared to 90,774±19,146 in fellow eyes. Mean nerve injury grades were 4.8±0.6 and 1.0±0.1 for injected and fellow eyes, respectively. RGC count was highly correlated to injury grade (r2=0.84), and also correlated to mean and cumulative IOP prior to labeling and to final IOP (r2=0.60, 0.60 and 0.57). The injected eye with the least optic nerve injury (grade 2.9) had a RGC count of 81,109. Decreased labeling in this retina was most striking in the paracentral superior region. Labeled RGCs in 4 of the remaining 5 elevated IOP retinas were primarily located in the inferior or inferior nasal regions. Conclusions: Following IOP elevation, grading of optic nerve injury by microscopic evaluation is highly correlated to total RGC count by retrograde fluorescent labeling, illustrating that both methods provide reliable quantitation of injury in this model. The regional distribution of labeled cells in experimental retinas suggests that RGCs in the inferior retina maybe more resistant to the effects of IOP than those located superiorly, corresponding to previous observations of regional localization of injury in the optic nerve head and optic nerve in this model.

Keywords: intraocular pressure • ganglion cells • animal model