Advanced imaging of aqueous outflow pathways in models of congenital glaucoma due to LTBP2 mutation

Abstract

Purpose : Primary congenital glaucoma (PCG) is a major cause of childhood blindness. Causal mutations in LTBP2 have been identified in human PCG and in feline congenital glaucoma (FCG). Although impaired aqueous humor outflow results in intraocular pressure (IOP) elevation, mechanisms for glaucoma associated with LTBP2 mutations remain unknown. Limitations of conventional histology have hampered characterization of morphology of highly complex three dimensional aqueous outflow pathways. To address these limitations, the eyes of cats homozygous for LTBP2 mutation and ltbp2^-/- knock-out mice were evaluated using light sheet fluorescence microscopy (LSFM).

Methods : A total of 6 eyes from cats with FCG (3 12-15 week old and 3 adult) and 6 age-matched control eyes, as well as 3 adult ltbp2 ^-/-, DBA/2J, DBA/2J-Gpnmb+ and wt eyes were bleached, immunofluorescence (IF) labeled for vascular markers, tissue-cleared with ethyl cinnamate and imaged by LSFM. Feline anterior segments were dissected in 4 quadrants (superior, inferior, nasal and temporal). The nature and quantity of the post-trabecular meshwork aqueous outflow vessels including Schlemm’s canal in mice/angular aqueous plexus in cats, collector channels and intrascleral venous plexus, were evaluated using Imaris v9 with Surface Module followed by signal segmentation.

Results : Mouse eyes and feline anterior segments were effectively cleared by this method, enabling visualization of the complex, 3D network of aqueous outflow vessels by IF labeling and LSFM. 3D image analysis identified collapse of intrascleral vessels in FCG cats with both chronic IOP elevation and within just a few weeks of mild IOP elevation, compared to age-matched controls.

Conclusions : We demonstrate that LSFM is a powerful tool for evaluation of the distal aqueous outflow pathways. Initial results suggest that LTBP2 mutation may affect the morphology of the post-trabecular aqueous humor outflow pathways in both murine and feline eyes. Studies are ongoing to delineate precise mechanisms responsible for glaucoma in these animal models.

This is a 2020 ARVO Annual Meeting abstract.