Abstract
Purpose :
The aqueous humor (AH) outflow pathway through the trabecular meshwork (TM) and Schlemm’s canal (SC) is known to have regions of both high and low flow. Increased areas of low-flow regions increase risk for elevated IOP. The mechanism of how high and low-flow regions are developed and regulated is unknown. CGRP positive C-fibers are the major type of sensory neurons innervating the TM and SC. Upon activation afferent neurons secrete CGRP locally, and CGRP receptors are known to be expressed in TM and SC cells. It has previously been shown in other cell types that CGRP can modulate ECM protein expression and remodeling as well as enhance phagocytosis, both important aspects of TM function and IOP regulation. The goal of this project was to determine whether CGRP positive neurites innervating the TM and SC are involved in formation of high and low-flow regions.
Methods :
High and low-flow regions were determined in 9-month-old wild-type C57BL/6J mice using yellow-green fluorescent tracer latex beads (0.02 µm). Beads were injected intracamerally with a 35G needle at a rate 4 nL/s using a syringe pump controller for 45 min. Anterior segment flat mounts were co-labeled with antibodies against CGRP (afferent neurons), CALCRL (CGRP receptor), and PECAM1 (SC endothelium), and imaged by confocal microscopy to acquire tiled Z-stacks of entire outflow area. Density of CGRP neurites in each flow region was determined by volumetric analysis in ImageJ. Primary human TM cells in culture were exposed to fluorescently labeled beads with or without CGRP (0.1 nM-5mM) for 24 hours and processed for immunocytochemistry. Phagocytosed beads were imaged and quantified using ImageJ analysis.
Results :
Both high and low-flow regions were identified in C57BL/6J mice, with significantly more CGRP positive neurites per square area of high-flow regions compared to low-flow regions (p<0.05, n=10 eyes). The CGRP receptor, CALCRL, is expressed in both TM and SC cells and there is significantly more CALCRL expression in high-flow regions compared to low-flow regions (p<0.01, n=5 eyes). CGRP significantly increased phagocytosis in primary TM cells in culture compared to untreated controls (p<0.01), while Substance P treated cells had no significant differences in phagocytosis.
Conclusions :
These data suggest innervation of CGRP positive C-fibers in the TM and SC may influence the development and regulation of high and low-flow regions of aqueous humor outflow.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.