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R McNulty, RJ W Truscott, S Bassnett; Lens and Vitreous pO2 Measured by Hypoxia Markers and Flourescent Optodes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2374.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: O2 is implicated in age related cataract formation. However, pO2 is difficult to measure in the lens using conventional O2 electrodes because the viscosity of the tissue impedes diffusion of gas to the O2-consuming electrode tip. Here we employ two novel techniques to determine pO2 in chicken and cow lens and vitreous. Methods: 1) Covalent binding to tissue by the hypoxia marker pimonidazole occurs only in hypoxic conditions (< 10 mmHg). Pimonidazole (0.35 mg) was injected into the chicken embryo eye. After 1 hour the lens was removed and processed for immunohistochemistry. Hypoxic regions were visualized using a pimonidazole antibody after antigen retrieval. 2) O2 tension behind the lens was determined in the chicken with an optode O2 sensor (OxyLab). This quantitative technique measures the fluorescence lifetime decay of a ruthenium-based sensor at the tip of an optic fiber. Fluorescence lifetime measurements are insensitive to viscosity and do not consume O2. 3) We also used the optode to measure pO2 in the larger bovine lens in vitro. Lenses were placed in Ringer's solution gassed with 20 mmHg O2 to simulate the intra-ocular environment. The optode was passed through the lens along the optic and equatorial axes. Results: 1) Bound pimonidazole was detected throughout the embryonic chicken lens at E10 and E17 indicating that the tissue was hypoxic. 2) Optode measurements showed a pO2 of <5 mmHg immediately behind the lens. In the mid-vitreous the pO2 was approximately 12 mmHg. 3) A standing pO2 gradient was detected within the bovine lens where pO2 fell to 5 mmHg within 1 mm of the surface and thereafter remained constant. Brief heat treatment (60C) eliminated this gradient. Conclusion: These data confirm that the lens is hypoxic in vivo. Furthermore, our in vitro measurements suggest that pO2 in the lens core is significantly lower than in the periphery. The techniques described here should prove useful in measuring pO2 within the human lens.
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