Metrology of the zonular apparatus of human and non-human primates using fluorescent confocal microscopy: A pilot study

Abstract

 

Purpose

 

To characterize the zonular apparatus of human and non-human primate (NHP) eyes using indirect immunostaining and laser scanning confocal microscopy (LSCM).

 

Methods

 

The accommodative apparatus of 7 human (55-99 y/o), 9 cynomolgus (4.3-5.8 y/o), 6 baboons (4.0-4.6 y/o) and 1 rhesus (4 y/o) eyes were isolated to expose the lens, zonules, ciliary body, pars plicata, and pars plana. An indirect immunostaining protocol (Bassnett et al, IOVS 2013) was applied using goat-derived MAGP-1 primary antibody to target the microfibril-associated glycoprotein-1 (MAGP-1) molecules of the zonules followed by a fluorophore bound secondary antibody Alexa Fluor 488. DRAQ5 nuclear stain was used to visualize cells within the lens and ciliary processes. A LSCM (TCS SP5, Leica) was used to capture 5, 20, and 63x images that were processed (Advanced Fluorescence Lite, Leica). Individual volumetric images were merged manually (Adobe Photoshop Elements 8.0) to map the zonular pathway.

 

Results

 

The zonule arrangement along with the ciliary processes and lens could be observed in volumetric images revealing a vast network of fine fibers, some originating in the ciliary body and extending on and inserting into the lens capsule at a radial distance that is 5-10% of the total lens diameter. Extra-lenticular and lenticular attachment sites reveal fan-like projections and separation of individual fibers near the lens and ciliary processes. The images show a uniform distribution of zonules around the lens allowing for a circumferentially quasi-continuous transmission of radial forces during accommodation. Volumetric imaging analysis (in process) allows anterior (e.g. 99 y/o human: ~ 435 zonules and 4.5 y/o cynomolgus monkey: ~ 387 zonules) and posterior (e.g. average of two 4.7 y/o cynomolgus monkeys: ~ 418 zonules) zonule density assessment. Improvements are needed to identify the zonular attachment structure at the pars plana as it might affect the accommodation theory.

 

Conclusions

 

The zonular apparatus in human and NHP eyes can be successfully imaged from the lens equator to the pars plana. Zonule metrology (density, lengths, thicknesses) allows cross-species quantification of age-related changes as well as the morphological characterization of zonule transection (Nankivil et al, IOVS 2009) studies.

 

 

Green = zonules; Magenta = lens & ciliary body epithelial cells.

 

 

Keywords: 404 accommodation • 457 ciliary processes • 472 comparative anatomy