Abstract
Purpose :
Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. We investigated the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA+/+) that constitutively expresses only FN containing the EDA isoform.
Methods :
Eyes (n=3/strain) were processed for electron microscopy, polymerized in EPON, ultrathin sections (80 nm) were cut and placed on formvar coated slot grids, and poststained with uranyl acetate and lead citrate. Consecutive images of the entire TM area spanning from anterior to posterior parts of Schlemm’s canal (SC) were collected at 2500x and montaged into a single image. ECM accumulation and basement membrane thickness were quantified by ImageJ analysis. TLR4 expression in ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols (n=3 eyes/strain). IOP was measured using a rebound tonometer and ON damage assessed by PPD stain (n=20-22 eyes/strain).
Results :
Ultrastructure analyses show the TM of B6.EDA+/+ mice have significantly increased accumulation of ECM between the TM beams with few empty spaces compared to C57BL/6J control mice (P<0.05). SC basement membrane is thicker and more continuous in B6.EDA+/+ mice compared to C57BL/6J control. No significant structural differences were detected in the TM of EDA null mice. The mRNA expression of TLR4, a known ligand of FN-EDA, was increased in the TM of B6.EDA+/+ mice compared to C57BL/6J control eyes (p<0.05). IOP was significantly higher in B6.EDA+/+mice compared to C57BL/6J control eyes (p<0.01) and significant ON damage was detected at one year of age (p<0.001). TLR4 mRNA is expressed in the ONH, and is present in mouse ganglion cell axons, microglia, and astrocytes. There was no significant difference in astrocyte density in B6.EDA+/+ mice compared to C57BL/6J control eyes; however, there was a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA+/+ mice compared to C57BL/6J control eyes (p<0.01).
Conclusions :
B6.EDA+/+ mice have increased accumulation of ECM in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, and damage to retina ganglion cell axons. These data implicate B6.EDA+/+ mice as a novel mouse model of glaucoma.
This is a 2021 ARVO Annual Meeting abstract.