Abstract
Purpose :
Characterization of the blood supply to the rat optic nerve (ON) using techniques such as vascular casting and histology have provided insight into the potential contribution of ON angioarchitecture to glaucoma pathophysiology. The purpose of this study was to determine the utility of LSFM to volume image the vasculature supplying the ON in the rat eye.
Methods :
Freshly enucleated Brown Norway rat eyes (n=3) were fixed for 16 hours in 4% PFA after removing the cornea and lens. The eyes were added to 10% hydrogen peroxide for 3 days for pigment bleaching. Following 4 days of permeabilization with dimethyl sulfoxide, blocking was performed with goat serum. Immunolabeling of the vascular endothelium was achieved with 1:150 primary anti-CD31 antibodies. The specimen was then incubated with a secondary antibody (1:1000) and cleared with increasing concentrations of tetrahydrofuran while in a desiccator cabinet. Final clearing was performed with dibenzyl ether for refractory index matching (fig 1). Samples were then mounted with the ON oriented upward in a Miltenyi Biotec LSFM system. Using a 4x objective, laser sheet width set to 30%, numerical aperture of 3.98 μm, and exposure time of 30 ms, the posterior globe was imaged at a step size of 1 μm. The 3D datasets were exported to Imaris for further analysis.
Results :
Representative images of the posterior vasculature are seen in figure 2. Our LSFM protocol resulted in the visualization of the major arterioles to the eye, including the long posterior ciliary arteries (LPCA), the central retina artery (CRA) and anastomoses contributing to the circle of Zinn-Haller (fig 2a). The microvasculature within the ONH was less consistently visualized but can be seen in fig 2c.
Conclusions :
LSFM successfully visualized vessels of the posterior rat globe. The ability to incorporate additional immunolabeling, such as amyloid precursor protein (to assess axonal transport blockade) and hypoxic markers to colocalize glaucomatous damage with vascular anatomy makes it an intriguing technique to understand the role ischemia plays in the pathophysiology of glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.