Synopsis

Keywords: Deuterium, Aging, Human eye

In the absence of a blood supply the lens operates an internal microcirculation to deliver nutrients, remove metabolic wastes, and controll the lens volume, which together to maintain the optical properties of the lens. While this microcirculation is generated by circulating fluxes of ions and water, visualising water flow throughout the whole lens and especially into the central of lens in real time has proven challenging. To address this, we developed and optimised new deuterium oxide (D₂O) MRI protocols to image water flow within multiple organ-cultured bovine lenses that can be routinely performed using 3T clinical MRI.

Purpose

The lens is an avascular organ in the eye. It operates a microcirculation to uptake nutrients and export waste using water as a carrier¹. As a negative imaging tracer, deuterium oxide (D₂O) can track the water flow in real time with MRI². In this study, we aim to establish and optimise protocols to visualise the regulation of water movement through the lens using deuterium oxide (D₂O) contrast imaging.

Methods

Lenses were extracted from bovine eyes obtained from a local abattoir, and up to 9 lenses were placed into a customised MRI-compatible sample holder (Fig.1) that contained isotonic artificial aqueous humour (AAH). At the start of the experiment (time = 0), the AAH bathing medium was replaced with either isotonic (290 mOsm/L), hypotonic (210 mOsm/L) or hypertonic (410 mOsm/L) D2O/AAH solutions to cause osmotic water fluxes or isotonic + ouabain (1mM) to inhibit the microcirculation³. Scans were performed by a 3T MRI (VIDA, Simens) equipped with a 16-channel hand/wrist coil. A proton-density (PD) - weighted image was acquired every 15 minutes using a turbo spin echo (TSE) sequence with FOV = 180×180 mm, matrix size = 512×512, TE = 8.70ms, TR = 2000ms, slice thickness = 3mm, and four averages. Scans were performed every 15 minutes for up to 2 hours to capture the change in signal intensity caused by the penetration of D2O into the lens under different conditions. To extract the rate of change in signal intensity, an ROI was drawn in the lens core to average the signal change in that region of the lens. The average signal of the core from different perturbations was normalised to its time 0 signal. The D₂O flow rate into the lens core is calculated as the slope of the linear fitting of the normalised signal.

Results

The usage of D₂O in the bathing medium caused a progressive reduction in signal intensity as the D₂O penetrated the lens and replaced H2O (Fig. 2A). This signal reduction was more apparent in the outer cortex of the lens, which has a higher water content than the central lens core, however, quantification of change in signal intensity in the core over time (Fig. 2B) showed that D₂O did penetrate the lens core, as predicted by the microcirculation system at a rate of 1.70 ± 0.3 × 10-3 mins ^-1. Furthermore, this rate of delivery of D₂O to the lens core could be increased (2.27 ± 0.3 × 10-3 mins^-1) or decreased (1.3 ± 0.5× 10-3 mins ^-1) by exposing the lens to hypotonic or hypertonic stress respectively (Fig. 2C), while under isotonic conditions ouabain also decreased the delivery of D₂O (1.5 ± 0.6 × 10-3 mins ^-1) to the lens.

Discussion

Our use of D2O-MRI has shown that water fluxes are convected to the core of the lens as predicted by the microcirculation. The up-regulation of this microcirculation system has been proposed to be the target of developing novel anti-cataract therapies designed to enhance the delivery of anti-oxidants to the lens core, the lens region affected in age-related nuclear cataract¹. Our new protocols thus offer the potential to identify and test therapies designed to enhance the delivery of such anti-cataract therapies.

Acknowledgements

New Zealand Health Research Council Programme grant to fund this project.