Developing novel imaging modalities to visualise the morphology of lens fibre cells in real time

Abstract

In diabetic patients dysfunction of the ability of the lens to regulate the volume of its fibre cells results in localised tissue damage in the outer cortex that manifests as increased light scattering and eventually cataract (Donaldson et al. 2009). Previous experiments to study the regulation of lens fibre cell volume generally utilized fixed lenses that were imaged at discrete time points. Using this method, it proved impossible to capture the time course of fibre cell volume regulation in the living lens. The initial aim of my PhD project was therefore to develop methods that allowed the dynamic changes in fibre cell volume to be visualised in real time in organ cultured lenses. To achieve this, I trialled a number of imaging modalities with little success. However, through a process of trial and error, I noticed that the clarity of lens cellular structure was improved when lenses were illuminated using off-angle light. Based on this fortuitous observation, I developed and optimised a new technique for imaging fibre cell structure in whole lenses, which I have termed Off-Angle-Light (OAL) microscopy. Using OAL, I recorded the dynamic changes to fibre cell morphology in both rat and bovine lenses that were exposed to osmotic challenge. Interestingly, these proof of principle experiments revealed a new phenomenon which changed the direction of my PhD project. I observed an unexpected shrinkage which slowly occurred in bovine lenses organ cultured under isotonic conditions, while no such phenomenon was observed in rat lenses. I hypothesised that the shrinkage in bovine lens was caused by the loss of zonular tension which occurred when the lens was first removed from the eye before being placed in organ culture. To test this hypothesis, I developed a simple dual-ring stretcher that enabled the in situ zonular tension to be maintained in organ cultured bovine lenses. I found for the first time that bovine lenses demonstrated both spatial and temporal changes to fibre cell morphology upon the cutting of the zonules to alter the tension applied to the lens. An immediate change in fibre cell morphology occurred in the deeper mature fibre cells, which I defined as Quick Adaption. This immediate change was followed by a Slow Remodelling of fibre cell morphology which was driven by the shrinkage of fibre cells in a localised zone in the outer cortex to restore the ordered morphology of the fibre cell columns in this area of the lens. In addition, I showed that this slow restoration of fibre cell order is potentially driven by changes to the actin cytoskeleton as an increase in F-actin labelling occurred in parallel to the fibre cell remodelling. Taken together, the new techniques I developed in this thesis have revealed that changing the tension applied to the lens alters fibre cell morphology, the actin cytoskeleton, and the overall geometry of the bovine lens. These techniques can now be used to open up new avenues of research to study the processes that actively regulate the maintenance of a highly ordered tissue architecture of the young, healthy lens and how the loss of this regulation manifests as presbyopia in middle age and cataract in the elderly.