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J.M. Nickerson, S.M. Wolchok, V.T. Ciavatta, J.H. Boatright; Delivery of dyes into intact mouse eyes by electrical fields . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4761.
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Purpose: We recently transferred small charged dyes and naked DNA across human sclera (Mol Vis 2003; 9:569–78) by an electric field. Linear DNAs from 50 bp to 12 kb and a 3 kb plasmid passed through the sclera. Microgram amounts were delivered in a few minutes. The latter tests were conducted in vitro in an electrophoresis apparatus on squares of cleaned sclera. Here, we fabricated a new apparatus to test whether dyes and DNAs penetrate quickly into intact mouse globes by an electric field. Methods: Freshly harvested mouse eyes were stained with 1% rose bengal or other stains, destained repeatedly with PBS, and extraocular muscles and fat removed by dissection. The eye was positioned on a U–shaped lower reservoir and an upper reservoir, plugged with agarose, was placed in contact with the globe. Xylene cyanol was added on top of the agarose plug. Platinum electrodes and 1x TAE buffer completed a circuit to a power supply. The eye was exposed to an electric field of 3 V/cm for up to 3 h, embedded in OCT, sectioned on a cryostat, and observed by brightfield microscopy. Results: Digital time–lapse photography during electrophoresis revealed that most of the xylene cyanol associated with the globe. Cryostat sections showed xylene cyanol inside the globe, across the entire sclera, and at least as far inside as the RPE. Conclusions:The new apparatus was designed to reduce analysis complications by minimizing external electrophoretic flow around the eye via a water–tight seal. Rose bengal (1% in PBS) was more effective than fluorescein or trypan blue in identifying extraocular materials and aiding dissection. Experiments with intact mouse globes show the passage of dye through the sclera, but required the prior removal of extraocular muscles, adherent materials, and fat deposits that blocked direct contact of dye with the external surface of the sclera. Electric fields are promising as a noninvasive DNA delivery tool, where gene therapy can be accomplished by small or large RNAs or DNAs. The intact globe of the mouse could be challenged electrophoretically in this new apparatus to measure the bulk transit of materials inside the eye. None of the internal structures of the eye served as an insulating barrier that might have blocked flow of dye in the electric field.
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