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Intravitreal (ITV) drug delivery is a new cornerstone for retinal therapeutics. Yet, predicting the disposition of formulations in the human eye remains a major translational hurdle. A prominent, but poorly understood, issue in pre-clinical ITV toxicity studies is unintended particle movements to the anterior chamber (AC). These particles can accumulate in the AC to dangerously raise intraocular pressure. Yet, anatomical differences, and the inability to obtain equivalent human data, make investigating this issue extremely challenging. We have developed an organotypic perfusion strategy to re-establish intraocular fluid flow, while maintaining homeostatic pressure and pH. Here, we used this approach with suitably sized microbeads to profile anterior and posterior ITV particle movements in live versus perfused porcine eyes, and in human donor eyes. Small-molecule suspensions were then tested with the system after exhibiting differing behaviours in vivo. Aggregate particle size is supported as an important determinant of particle movements in the human eye, and we note these data are consistent with a poroelastic model of bidirectional vitreous transport. Together, this approach uses ocular fluid dynamics to permit, to our knowledge, the first direct comparisons between particle behaviours from human ITV injections and animal models, with potential to speed pre-clinical development of retinal therapeutics.

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This review describes how the morphology and distribution of the mitochondria of the epithelium and the superficial fibre layers of the lens were studied using confocal scanning laser microscopy. This research was correlated with an effort to use the optical properties of the intact lens in culture as a proxy for the cornea in measuring ocular toxicity. In turn, this work led to the confocal study of the in vitro and then the in vivo cornea and their possible use in using confocal microscopy to evaluate the effect of various treatments on the integrity of the surface of the eye. Finally, confocal examination of the mitochondria of the lens has provided an avenue to the study of mitochondrial dynamics.

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Pre-screening of cosmetic ingredients is vital for consumer safety. Previous in vivo techniques, such as the Draize test, have proved to be unreliable in predicting ocular irritancy and therefore there is a need for alternate testing methodologies. One such test is the scanning laser in vitro assay system which quantifies irritancy based on the focusing ability of the cultured bovine lens. In combination with confocal microscopy, a more thorough documentation of ocular irritancy can be achieved. This study investigates the response of cultured bovine lenses over time to butyl, methyl and propyl parabens, which are common antimicrobial agents found in cosmetic and ophthalmic products. The focusing ability of the lens was measured with an automated laser scanner over a period of 96 h. At 120 h post-treatment, the lenses were analysed by using a confocal laser scanning microscope to determine the characteristics of nuclei, and the morphology and distribution of mitochondria within the lenses. Irritancy to the three parabens was investigated at both an optical and cellular level. Each of the parabens was tested at 0.002% and 0.2%, where the 0.2% butyl paraben was found to be the most irritating.

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In order to elucidate the correlation between lens optical function and metabolic function, in vitro bovine lens optical quality and mitochondrial integrity was measured following treatment with carbonyl cyanide m-chlorophenylhydrazone (the mitochondrial depolarizing agent, CCCP). The results indicate that in vitro exposure to CCCP resulted in concentration and time-dependent loss of sharp focus. The concentrations tested included 65.0, 32.5, 16.25 and 8.125 microm CCCP. Lenses treated with two lower concentrations show recovery from damage at the 24-h scan point. In lenses treated with 65 microM CCCP, mitochondria in lens epithelial and superficial cortical fibre cells appeared short and swollen. The results of this study indicate that lens optical function and mitochondrial integrity are closely correlated.

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PURPOSE: This study is part of an effort to clarify mitochondrial distribution in the lens in order to better understand lens metabolic function. This study of the rat lens involves: 1) Using confocal microscopy, Rhodamine-123 and Calcium Green fluorescent dyes, to characterise the distribution of mitochondria and calcium in whole rat lenses of different ages in epithelial and superficial cortical fibre cells approaching sutures and 2) Using a scanning laser system to measure the optical quality at the sutures. METHODS: Lenses of rats from age 1 week to 22 months were pre-incubated for 24 hrs in 1.5 ml medium 199 (M199). Those exhibiting damage, as evaluated by protein leakage or visual opacities, were discarded. Lenses were labelled with 50 microg/ml Calcium Green for 45 min and/or 14 microM Rhodamine-123 for 25 min and embedded in 1% agarose in M199 for inverted laser scanning confocal microscopy with a 40 x water immersion lens. The lens optical properties were determined with a scanning laser system. RESULTS: Lens focal length variability significantly increased at the sutures of 13 month-old lenses, the only age investigated. An absence of both mitochondria and calcium was observed at the sutures in rat lenses of all ages. Elongated (up to 108 mm) mitochondria were present in superficial cortical fibre cells approaching the sutures of 16 month-old lenses. Calcium Green fluorescent staining was seen closer to the border of the suture, where mitochondria were absent. Along the axis, 1 week-old lenses showed a mitochondria free zone (MFZ) starting 177 microm below the lens surface, whereas in 22 month-old lenses the MFZ started only 29 microm below the surface. In the equatorial fibre cells, mitochondria were seen to a depth of 220 microm. CONCLUSIONS: Optical quality near and at the suture decreased in 13 month-old lenses despite the reduction in light scattering that should be associated with absence of mitochondria at the sutures. This suggests that mitochondrial loss in superficial cortical fibre cells may originate at the sutures and may compensate for loss of optical quality at the sutures.

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The effect of several antioxidants and cysteine-elevating precursor drugs (prodrugs) was tested on lens damage occurring after in vitro exposure to low levels of 60Co-gamma-irradiation, to simulate in vitro the exposure to radiation in vivo of (1) astronauts (2) jet crews (3) military radiation accident personnel. Tocopherol (100 microM), ascorbic acid (1 mM), R-alpha-lipoic acid (1 mM), and taurine (0.5 mM) protected against radiation-associated protein leakage. MTCA and ribocysteine protected lenses against opacification, LDH and protein leakage, indicating that antioxidants and prodrugs of cysteine appear to offer protection against lens damage caused by low level radiation.

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