Abstract
Purpose :
Our rNAION closely resembles clinical NAION with early intraocular ON edema, a cellular inflammatory response, and isolated retinal ganglion cell (RGC) and ON loss. Like clinical NAION, rNAION can show variable in severity. This variability can require end-analysis of a considerable number of animals. We report our evaluation of variables associated with rNAION induction and present early biomarker for stroke severity, using intravenous fluorescein (IVFA) and indocyanine green angiography (ICGA), that can directly visualize early ON edema, and enable estimation of stroke severity.
Methods :
We used male Sprague Dawley rats. Heart rate, body temperature and hematocrit were measured prior to induction. rNAION was induced as previously described. 3-5hr post-induction, slit lamp observations were performed and animals intravenously injected with 0.1ml of 10% solution of sodium fluorescein (fluorescite; Alcon) and ICG (5mg/ml). Eyes were imaged using Heidelberg SD-OCT and ONH cross-section diameter was measured. En face ONH fluorescence was evaluated at 12 minutes post-injection using bluepeak (488nm) and ICGA channels (804nm). The intensity of the fluorescence were analized on imagJ. 30 days post induction, retinae were immunostained with Brn-3a antibody and Brn-3a positive RGCs stereologically counted.
Results :
Animals had a mean rectal temperature of 37.2±0.3 mean heart rate of 287/minute ± 45 and mean hematocrit (Hct) of 45.6. There was a low correlation of RGC loss with temp, HR or HCT. No apparent vessel or fundus changes were seen at 2 hours on slit lamp observation. A pale disc with slightly hazy disc border was seen at 5 hours post induction in some severe strokes. Cross-sectional ONH diameter revealed no apparent change at early times post induction; however IVFA and ICG revealed significant leakage at 3-5 hours. En face fluorescent imaging intensity was strongly correlated with later RGC loss.
Conclusions :
Fluorescent imaging can detect vessel permeability changes in the early phase of the rNAION and the degree of leakage strongly correlates with the later RGC loss, confirming its use as an early stroke biomarker. Identifying animals with similar degrees of damage prior to assignment into treatment groups may significantly improve the experimental rigor of neuroprotection agent studies.
This is a 2020 ARVO Annual Meeting abstract.