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(Investigative Ophthalmology and Visual Science. 2003;44:1993-1997.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0582
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Visualization of Cell Death In Vivo during Murine Endotoxin-Induced Uveitis

Peizeng Yang,1,2 Justine R. Smith,2 K. Sampath Damodar,2 Stephen R. Planck,2,3,4 and James T. Rosenbaum2,3,4

1From the Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People’s Republic of China; and the 2Casey Eye Institute and 3Departments of Medicine and 4Cell and Developmental Biology, Oregon Health & Science University, Portland Oregon.

PURPOSE. To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation.

METHODS. To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice. At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy. Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy. TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results.

RESULTS. Uveitis was induced in all the mice that received an endotoxin injection. The percentages of annexin V+ propidium- cells, annexin V+ propidium+ cells, and annexin V- propidium+ cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro. In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens. Both the number and the pattern of labeled cells changed dramatically over time. The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular.

CONCLUSIONS. A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V+ propidium-) and late apoptotic or necrotic (annexin V+ propidium+ and annexin V- propidium+) cells change over the course of EIU. A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU. This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.




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