Real-time in-vivo imaging of retinal cell apoptosis after laser exposure
Steffen Schmitz-Valckenberg 1*,
Li Guo 2,
Annelie Maass 2,
William Cheung 2,
Anthony Vugler 3,
Stephen E. Moss 4,
Peter MG Munro 5,
Frederick W. Fitzke 1,
and
M. Francesca Cordeiro 2
1 Visual Science, UCL Institute of Ophthalmology, London, United Kingdom
2 Glaucoma & Retinal Neurodegeneration Research Group, UCL Institute of Ophthalmology, London, United Kingdom
3 Cellular Therapy, UCL Institute of Ophthalmology, London, United Kingdom
4 Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
5 Research Inf. Core Services, UCL Institute of Ophthalmology, London, United Kingdom
* To whom correspondence should be addressed. E-mail: s.schmitz-valckenberg{at}ucl.ac.uk.
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Abstract |
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Purpose: To investigate whether DARC (Detection of Apoptosing Retinal Cells) could detect cells undergoing apoptosis in a laser model of retinal damage.
Methods: Laser lesions were placed with a frequency-doubled Nd:YAG-laser on the retina in 34 eyes of anaesthetised dark agouti rats. Lesion size and laser-induced retinal elevation were analysed using in-vivo reflectance imaging. Development of retinal cell apoptosis was assessed using intravitreal fluorescent-labeled annexin 5 in-vivo with DARC technology from baseline until 90 minutes after laser application. Histological 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 both lesion size (p < 0.0001) and retinal elevation (p < 0.0001) were demonstrated. Histology confirmed the presence of apoptosing retinal cells both in the inner nuclear and ganglion cell layers.
Conclusion: 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.
Key Words:
laser, apoptosis, scanning laser ophthalmoscopy, in-vivo imaging, annexin 5
