Oxidative Stress and the Lens: Characterising a Hyperbaric Oxygen model of Lens Ageing

Abstract

Age-related nuclear (ARN) cataracts are a leading cause of blindness, and appear to be an inevitable consequence of ageing. They occur when proteins precipitate, which is the conclusion of a long process that involves antioxidant depletion, metabolite transport disruption, and shifts in the pattern of internal refractive index distribution. These changes occur in vivo along with natural lens growth, which itself causes changes in lens size, shape, deformability, and refractive power. Exposure to sources of oxidative stress promotes ARN cataract, yet at age 80 >30% of Americans are non-cataractous. This would suggest that the pro-cataractous pathophysiological changes may be confounded by, yet distinct from, the normal ageing process. In an effort to identify specific events that occur throughout the process of cataractogenesis, I took the hyperbaric oxygen (HBO) model of in vitro oxidative stress and used complementary imaging techniques to spatially resolve the effects of oxidative stress, independent of ageing. I use imaging mass spectrometry (IMS) to map lens metabolites, and define compartments in the outer cortex, inner cortex, and nucleus based solely on measurements of the metabolome in each pixel. I map glutathione metabolism and its regional differences, as well as the effects of oxidative stress on the distribution of this key antioxidant. I present methods for IMS of lens α, β, and γ crystallins, and present images of their distribution in the normal bovine lens and in the HBO model. IMS of intact α crystallins was achieved with isotopic resolution. No effect of HBO on α crystallin primary structure was seen. HBO-induced mass shifts to specific β/γ crystallins consistent with glutathionylation are mapped to the outer cortex. T2-mapping MRI enabled the calculation of refractive index and the visualisation of lens GRIN. This revealed an HBO-induced drop in refractive index in the lens nucleus. In silico ray tracing demonstrated the effect this had on vision. T1-mapping MRI, which measures a signal proportional to free water, revealed an increase in free water in the nucleus that closely matches the HBO-induced shift in lens GRIN. I then track the transport of a small MRI tracer molecule by the lens microcirculation. In the control lens this was transported from the outer cortex to the nucleus, bypassing the inner cortex in accordance with microcirculation theory. However in the HBO treatment group, diffusion into the outer cortex was slower, and delivery to the nucleus was absent. Ultimately, these experiments show an HBO-induced increase in nuclear free water that is predicted to have significant effects on lens optics. In the absence of protein modifications in the nucleus, changes in protein tertiary structure are predicted to cause a release of proteinbound water as free water. This is expected to occur as a result of a decrease in pressure, which is consistent with the microcirculation being impaired by HBO. These observations share key features with the in vivo ageing lens, namely a hyperopic shift in refractive power, and a functional barrier to glutathione passage interior to the outer cortex. I show these results follow oxidative stress exposure and demonstrate causal relationships. Future directions will involve testing mechanistic relationships, and developing interventions to preserve key physiological features in the face of oxidative stress, and pathological lens ageing.