Abstract
Purpose :
Transgenic mice that express various fluorophores in retinal ganglion cells (RGCs) have helped our understanding of glaucoma pathophysiology. However, the alternative approach of exogenous delivery of markers for both structural and functional integrity using adeno-associated viral (AAV) vectors has been of great interest. The aim of this study was to transfect RGCs to express a functional marker and characterize cellular functional loss in acute and chronic models of optic nerve injury as well as with aging.
Methods :
C57BL/6 mice received intravitreal injection of AAV2-GCaMP6s. In vivo imaging (CLSO/OCT; Spectralis Multiline; Heidelberg Engineering) was performed weekly for 8-weeks to analyze cellular labelling and ganglion cell complex (GCC) thickness. Mice were divided into 3 groups: optic nerve transection (ONT; 3-days), experimental glaucoma induced by an intracameral injection of a cross-linking hydrogel (EG; 8-weeks), or aging (AG). For EG, in vivo images and IOP measurements were obtained weekly. For AG, in vivo images were obtained weekly over the initial 8-weeks and every 6 months until 18 months post-injection. Calcium imaging was performed to quantify cellular function and immunohistochemistry (IHC) to quantify cellular density. Retinas were stained for RBPMS (a marker for RGCs), ChAT (a marker for cholinergic amacrine cells), and GFP (a marker for GCaMP). For EG and ONT experimental eyes were compared to untreated fellow eyes, while for AG, eyes were compared to 8-week mice.
Results :
GCaMP labelling was visible 1-week post-injection and increased over 6-weeks before it plateaued. In EG, IOP remained elevated over 8 weeks (mean (SD), 10.7 (0.6) vs 19.2 (2.8) mmHg at 8-weeks in control vs EG). In EG, in vivo fluorescence labelling and GCC thickness decreased 18% and 14% from baseline, respectively. Corresponding figures AG were 15% and 6%. Functional loss was detected in all experimental groups, compared to control of 33%, 37%, and 34% (ONT, EG and AG, respectively). Finally, IHC showed a 19%, 21%, and 12% loss of RBPMS+ cells compared to fellow eyes (ONT, EG, and AG, respectively).
Conclusions :
Functional loss was comparable between 3-day ONT, 8-week EG, and AG groups, despite being different models of RGC loss. This work provides evidence that exogenous functional fluorescent markers can be used to assess functional changes and that age is a vital determinant of loss of RGC function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.