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Originally published In Press as doi:10.1167/iovs.07-1335 on February 15, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:2773-2780.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1335
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Real-Time In Vivo Imaging of Retinal Cell Apoptosis after Laser Exposure

Steffen Schmitz-Valckenberg,1 Li Guo,1 Annelie Maass,1 William Cheung,1 Anthony Vugler,1 Stephen E. Moss,1 Peter M. G. Munro,1 Frederick W. Fitzke,1 and M. Francesca Cordeiro1,2

1From the Institute of Ophthalmology, University College London, London, United Kingdom; and 2Western Eye Hospital, London, United Kingdom.

PURPOSE. To investigate whether the detection of apoptosing retinal cells (DARC) could detect cells undergoing apoptosis in a laser model of retinal damage.

METHODS. Laser lesions were placed, with the use of a frequency-doubled Nd:YAG laser, on the retina in 34 eyes of anesthetized Dark Agouti rats. Lesion size and laser-induced retinal elevation were analyzed using in vivo reflectance imaging. Development of retinal cell apoptosis was assessed using intravitreal fluorescence-labeled annexin 5 in vivo with DARC technology from baseline until 90 minutes after laser application. Histologic analysis of retinal flat mounts and cross-sections was performed.

RESULTS. The lateral and anteroposterior depth extension of the zone of laser damage was significantly larger for higher exposure settings. A strong diffuse signal, concentrated at the outer retina, was seen with DARC for low exposures (<300 ms and <300 mW). In comparison, higher exposures (>300 ms and >300 mW) resulted in detectable hyperfluorescent spots, mainly at the level of the inner retinal layers. Dose-dependent effects on spot density and positive correlation of spot density between lesion size (P < 0.0001) and retinal elevation (P < 0.0001) were demonstrated. Histology confirmed the presence of apoptosing retinal cells in the inner nuclear and the ganglion cell layers.

CONCLUSIONS. This is the first time that DARC has been used to determine apoptotic effects in the inner nuclear layer. The ability to monitor changes spatially and temporally in vivo promises to be a major advance in the real-time assessment of retinal diseases and treatment effects.






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