Abstract
Purpose :
To develop a peripapillary retinal neve fiber layer (pRNFL) thickness measurement to assess glaucomatous optic neuropathy in an animal model as it is used as a clinical imaging biomarker for optic neuropathies including glaucoma. Optical coherence tomography (OCT), the method for pRNFL thickness measurement in clinics, has constraints for application in animal models due to technical and optical limits.
Methods :
Rat RGC axons were anterogradely labeled with 6µl of 0.5% CTB (Cholera toxin subunit B, Alexa Fluor™ 594 conjugate) per eye by intravitreal (IVT) injection, then ONT (optic nerve transection) was performed unilaterally (OS eye) using blunt dissection method with a complete transection of optic nerve, 48 hours post CTB injection. Both eyes were collected on day 3, 5 and 7 post-ONT surgery and retinae were dissected and fixed for flat-mount analyses. Confocal images of RGC axons were acquired for all retinal flat mounts using a LSM900, Airyscan 2.0 microscope and 10x objective. Z-stack images of RGC axons were obtained for each quadrant of the retinal flat-mount. pRNFL thickness was measured by the total number of Z-stack slices and its fixed interval (0.42µm):
(pRNFL) thickness =Total number of Z-stack slices (with visible axon fibers) X Z- stack interval
Brn-3a immunostaining is used for RGC density evaluation in retina flat-mount post ONT.
Results :
pRNFL thickness in ONT eye was reduced significantly (P value was 0.04, 0.0002, and 0.006) on Day 3, 5, and 7 post ONT compared to the pRNFL thickness in control eye with reduction of 14± 4.0%, 22± 4.8% and 41± 5.3% respectively.
Time-dependent RGC density loss was observed in another group of rat retinae post ONT; Day 3: 33.6 ± 7.5%; day 7: 55.2 ± 9.9% and day 14: 83.1 ± 4.4% (p<0.005).
Conclusions :
Measurement of the pRNFL thickness with confocal imaging provides a novel approach to estimate the pRNFL thickness in ONT rat model with direct measurement of visible axon fibers. We found that the thickness of RNFL gradually becomes thinner in a time-dependent manner post ONT, which is consistent with RGC cell density loss. pRNFL thickness measurement with confocal imaging may serve as an additional readout to evaluate the RGC damage and glaucoma progression.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.