Retinal Trilaminar Vascular Density in Healthy Mice

Abstract

Purpose : Mice are a popular model for retinal vascular study. Its vasculature is divided into a trilaminar network, but its characterization is scarce. Here, we quantify the 3D vascular density by measuring vessel path length at various eccentricities in healthy adult mice

Methods : We used 6 male mice on a C57BL6/J background (19-54 weeks). To quantify vessels, we used NG2DsRedBAC (JAX #:008241) mice that express fluorescent pericytes and smooth muscle cells to label retinal vasculature. One retina per mouse was dissected, flat mounted and imaged using a confocal microscope (Nikon ECLIPSE Ti2-e). We captured 15 high density cubes at 60X mag. typically 300 x 300 x 30 µm (0.1 µm step size) between 0.9 to 2.1 mm eccentricities. Vessels were manually traced with ImageJ’s SNT plugin and exported to MATLAB for analysis. We quantified vascular path length in each layer (Superficial (S), Intermediate (I), Deep (D)) and the diving connections (DC) between them (Fig. 1)

Results : On average, the density of retinal vessels was 74.82 mm of path length per square mm of retina (74.82 ± 14.47 mm/mm², mean ± SD). We found three clear plexuses of vessels that mainly avoided the retinal nuclear layers. Generally, vascular density increased with deeper stratifications. These were grouped as: S=17.55 ± 3.48 mm/mm², I=21.77 ± 8.36 mm/mm² and D=29.54 ± 5.64 mm/mm² (Fig.2). Unlike cortex, these plexuses were primarily lateral with 92.03% of vessels contained within them. The remaining vessels dive and interconnect the layers (DC=5.96 ± 4.03 mm/mm²). We found 2X more connections between the I-D layers (4.00 ± 3.40 mm/mm²) than the S-I layers (1.96 ± 1.21 mm/mm²). While age tended to decrease vascular path length, it was not significant (ANOVA, p=0.051). In the periphery, we found no eccentricity effect on vascular density (p=0.25). Combined, we estimated the total vascular path length of the mouse retinal circulation to be 1.12 ± 0.09 meters per retina

Conclusions : While the trilaminar structure of the mouse retina has been described, we now provide a metric of vascular density to these layers and diving connections. Generally, vessel density increases with progressively deeper stratifications. Our preparation maintains 3D structure for direct comparison to in vivo imaging. Further work will characterize association of vascular density with neurons and glia, to learn critical rules of retinal vascular patterning of the most popular animal model for biomedical research

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.