Establishment Of A Rat Model Of Anterior Ischemic Optic Neuropathy And Assessment Of Optic Nerve Damage

Objective1. To establish a rat model of anterior ischemic optic neuropathy (rAION).2. To qualitatively and quantitatively evaluate the time-course of optic nerve (ON) damage in rAION model.3. To investigate the neuroprotective effects of intravitreally administered nerve growth factor (NGF) on neuronal degeneration in rAION model.Methods1. rAION was induced by directly illuminating the ON of the right eye with532nm green laser, after intravenous infusion with the photosensitizing agent Rose Bengal (RB)(n=23). Sham laser treatment consisted of illuminating the ON region with a laser without RB injection(n=11); Rats in normal control group underwent no intervention (n=13). Effect of rAION on the appearance of ON and retina was observed with funduscope and fundus fluorescein angiography (FFA). Hematoxylin and eosin (H&E) staining and transmission electron microscopy were performed to assess rAION-indeced histologic changes in the retina and ON. After labeling RGCs by stereotactic injections of3%FluoroGold (FG) into the superior colliculus, the animals were sacrificed, and RGCs were counted on retinal flat mounts in a masked fashion.2. The rats were randomly divided into five groups:1-week (n=12),2-week (n=13),4-week (n=15),8-week (n=15) model group and the normal control group (n=10). Animals undergoing AION were sacrificed on7,14,28or56days post-injury. The density of RGCs was calculated by morphometric measurement with FG labeling. The RGC survival rate was defined as ratio of RGC density of right eye/left eye. Semithin sections of ON were stained with toluidine blue for evaluation of axonal degeneration.3. The rats were randomly divided into three groups:NGF treatment group (n=10), normal saline (NS) treatment group (n=10), and untreated group (n=8). Rats either received exogenous NGF (intravitreal injections,2μg/4μL) or NS12hours,4days, and10days after the modeling. Rats were euthanized at4weeks post-infarct. The number of surviving RGCs was measured by means of RGC staining using retrograde labeling of FG. Toluidine blue staining were performed in the semithin section of ON to identify axonal degeneration in NGF-treated rats.Results1. In the rAION-induced eyes, the optic disc was extremely swollen3days after photodynamic therapy. ON edema resolved on day7after induction. The focal filling defect of choroid and optic disc was observed during the early phase of FFA. H&E-stained longitudinal ON sections of rAION revealed peripapillary retinal detachment and vacuolar degeneration corresponded to swollen axons on day3after induction, and optic disc atrophy4weeks after the modeling. Ultrastructural study showed axonal edema and collapsed sheaths in the ischemic optic nerve3days after induction. Four weeks later, there were extensive axonal loss and severe glial scar, accompanied with a reduction of retinal nerve fiber layer thickness and RGC death in large numbers. Meanwhile, no obvious degenerative change was observed in the inner and outer nuclear layers. Retrograde labeling with FG revealed that rAION resulted in regional RGC loss.2. The density of labeled RGCs was2273±219cells/mm2in control group,2075±120cells/mm2in1-week group,1783±143cells/mm2in2-week group,1330±169cells/mm2in4-week group,869±301cells/mm2in8-week group, respectively; There were significant differences between all pairs of groups(P<0.05, respectively). The survival rate of RGCs was99.9%±3.4%,91.8%±2.9%,72.6%±5.7%,54.4%±7.0%,37.5%±13.0%, respectively; There were significant differences between all pairs of groups(P<0.05, respectively). During the2-week period closely after ischemic injury, RGCs death was a relatively slow process. Thereafter, the number of apoptotic RGCs rapidly increased, which lasted at least for6weeks. Toluidine blue staining of semithin sections of the ON revealed significant demyelination1week after ischemic damage. Progressive axonal degeneration existed for several weeks, which mainly affected the central region of the optic nerve with extensive reactive gliosis.3. The density of RGCs in NGF-treated rats was1447±70cells/mm2(P<0.05, compared with the NS-treated group,1298±97cells/mm2; P<0.05, compared with the untreated ischemic group,1313±153cells/mm2). Correspondingly, the survival rate of RGCs was significantly higher in the NGF-treated rats than in the NS-treated and the untreated groups(62.0%±2.8%vs.53.3%±5.1%,62.0%±2.8%vs.52.3%±7.9%; both P<0.05). There was obvious axonal loss and glial scar in NGF-treated rats, similar to what happened in untreated ischemic rats.Conclusions1. The findings of the rAION model resemble the histologic and physiologic defects seen in human nonarteritic anterior ischemic optic neuropathy. Therefore, rAION model in this study is appropriate for the following research.2. rAION results in later RGC death. The extended period of cell death after rAION induction is much more longer than previously recognized, when neuroprotective treatment may rescue damaged RGCs.3. Exogenous NGF has neuroprotective effect on rAION in our pilot study.