Abstract
Purpose :
The two most common optic neuropathies in adults, glaucoma and non-arteritic anterior ischemic optic neuropathy have uncertain etiologies. This gap in knowledge will be explored with imaging methods to visualize and digitize the path and diameters of long stretches of peri-optic nerve arteries. These digitized data will inform development of software models to analyze biomechanical and other factors that might affect optic nerve head perfusion.
Methods :
After exposure of six en bloc human orbits, the take-off of the ophthalmic arteries were cannulated with an angiocath to inject CM-Dil dye (ThermoFisher: C7001) in situ and the orbits were fixed in 4% paraformaldehyde. Orbit dissection under a stereomicroscope to remove the extraocular muscles and peripheral orbital fat left the length of the optic nerve and surrounding blood vessels and fat intact. The orbits then underwent tissue clearing per a modified ClearEye protocol (Darche, 2023): dehydration in serial phosphate-buffered saline (PBS)/methanol (50%, 80%, and 100%) solutions for 2h each, and 20% Quadrol in PBS for 7 days. In one case, melanin of the retinal pigment epithelium was bleached with 10% hydrogen peroxide in methanol for 10 days. Delipidation was achieved following placement in dichloromethane (exchanged every 2-3 days) until the specimens ceased to float. The samples were transferred to ethyl cinnamate (replaced after 24 hours) for refractive index matching. A custom-built MESOSPIM light sheet fluorescence microscope (LSFM) with a 561nm excitation laser and associated emission filter sets (Voigt, 2019) captured 3D images of the injected peri-neural blood vessel.
Results :
In our initial sample, we observed a longitudinal stretch (3-4 mm) of peri-neural vessels using LSFM, but excessive background fluorescence and incomplete optic nerve impaired complete visualization. For our second sample, prolonged bleaching removed the CM-Dil dye signal. Our methods continue to evolve to achieve our goals.
Conclusions :
Tissue clearing combined with en-block imaging methods such as light sheet microscopy is an alternative to traditional histology. Despite the major advantage of producing 3D volumes without the need for time-consuming serial sectioning and reconstruction, the conditions for vessel labeling and tissue clearing still require optimization for each step for the human eye.
This abstract was presented at the 2024 ARVO Imaging in the Eye Conference, held in Seattle, WA, May 4, 2024.