Colocalization of segmental outflow and endogenous eNOS activity in the conventional outflow pathway of mice

Abstract

 

Purpose

 

Aqueous humor filtration through the conventional outflow pathway is non-uniformly distributed or “segmental”, but it remains unclear what regulates segmental outflow patterns. We hypothesize that spatial variations in the expression of endothelial nitric oxide synthase (eNOS) within Schlemm’s canal (SC) cells may influence the segmental distribution of outflow in the trabecular meshwork (TM).

 

Methods

 

We used transgenic mice expressing GFP driven by the human eNOS promoter as a reporter lacking eNOS activity. Enucleated eyes from five eNOS-GFP mice on a C57BL/6 background (15-16 weeks old) were perfused with fluorescent nanoparticles (500nm) at 8mmHg for 45 mins, followed by 2 hours at either 8 or 16mmHg to decorate segmental outflow patterns in the TM. By confocal microscopy, the intensity of GFP expression in SC and the intensity of tracer within the TM were quantified in 12-16 regions of interest (200µm x 200µm each) per eye. ANCOVA was used for statistical analysis.

 

Results

 

When considering all eyes, there was a positive correlation between tracer intensity in the TM and GFP expression in SC (p<5×10^-5; N=10, η²=0.107). This relationship holds true when considering eyes perfused at only 8 or 16 mmHg as well (p=0.003 and p=0.004, respectively; N=5 each). However, when considering the effect of pressure on the correlation between GFP expression and tracer intensity, no statistical significance was detected (p=0.543).

 

Conclusions

 

These data suggest that endogenous eNOS expression in SC tends to coincide with segmental outflow patterns in the TM. This relationship could be attributed to greater hydrodynamic shear stress in high-flow regions that increases local eNOS expression in SC. Alternatively, elevated eNOS activity may promote local outflow through the TM. The latter implies that altered eNOS activity, as may occur in glaucoma, may disrupt normal outflow segmentation and contribute to elevated conventional outflow resistance.  

 

Figure1: Segmental outflow patterns in the TM of an enucleated eNOS-GFP (green) mouse eye perfused with 500nm fluorescent tracers (red).