Year

2004

Degree Name

Doctor of Philosophy

Department

Department of Chemistry

Abstract

Age-related nuclear cataract is a major cause of blindness and is thought to result from oxidation of key cellular omponents. Molecular oxygen (O2) is a possible oxidant in this process. The aim of this thesis was to investigate the regulation of tissue O2 levels in the ocular lens. We mapped the distribution of O2 within isolated lenses using a fluorescent optode. All lenses examined (bovine, rabbit and human) showed U-shaped Po2 profiles across both the optic and equatorial axes resulting in core hypoxia. When equilibrated in physiological conditions (Po2 environment of 36mmHg), the lens nucleus Po2 was 1.6 ± 0.5 mmHg (n=6), 12.5 ± 2.5 mmHg (n=6), and 13.8 ± 7.6 mmHg (n=24 pairs) in the bovine, rabbit and human lens, respectively.

We examined a possible role of Po2 gradients on the development of the embryonic chicken lens. The lens contains two populations of fibre cells, differentiating fibers (DF) in the outer cortex that contain a full complement of organelles (including mitochondria), and mature fibers (MF) in the lens core that do not. Incubating chicken embryos in hyperoxic conditions resulted in larger lenses and an increase in the depth of the DF population compared to lenses from embryos raised in normoxia. This is consistent with the hypothesis that hypoxia triggers the elimination of organelles from the lens.

The consumption of O2 by the lens was region-dependent. Both respirometric measurements (performed on dissected regions of the bovine lens) and a diffusion/consumption mathematical model showed that approximately 90% of O2 consumption occurred in the cortex, the remainder occurring in the core which contains only MF. The distribution of mitochondria in the DF layer was mapped using 2-photon or confocal microscopy on living lenses treated with mitochondria-specific dyes (rhodamine 123 or Mito Tracker) and in fixed tissue by immunofluorecence using an antibody to cytochrome c-oxidase. The depth of the mitochondria-containing cell layer varied in different species, being 500-760 μm in the bovine lens and 50-100 μm in the human. The Po2 gradients in the lens extended beyond the boundaries of the mitochondria-containing DF layer, consistent with a role for non-mitochondrial O2 consumption (albeit a minor one) in the MF region in the intact lens. We therefore examined both mitochondrial and nonmitochondrial processes which may be responsible for modulating lens O2 levels. Mitochondria-inhibition studies showed that the contribution of oxidative phosphorylation to bovine lens QO2 was approximately 90%. Interestingly, mitochondria-inhibiting drugs and age had an insignificant effect on human lens QO2. Ascorbate, found in high levels in the lens, was investigated as an O2 consumer. Bovine and human lens core O2 consumption could be induced by metals and inhibited by the metal-chelator DETAPAC, characteristics which are consistent with ascorbate-dependent O2 consumption. We found no evidence for a role for a trans-plasma membrane oxido-reductase system or photo-oxidation as nonmitochondrial O2 consumers.

The movement of O2 through the lens tissue was studied by decreasing lens QO2. Sub-physiological temperatures resulted in a marked decrease in lens QO2 and a concomitant flooding of the lens core with O2. The rapid rise in lens Po2 at lower temperatures may be of clinical relevance. During vitrectomy, cool, O2-rich solutions are infused into the eye. We hypothesise that rising lens core O2 levels during vitrectomy are the cause of the strikingly high incidence of post-vitrectomy nuclear cataract. We have initiated a clinical study to examine whether the use of warmed solutions during surgery can decrease the incidence of post-vitrectomy cataract.

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