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I. Kawaguchi, T. Higashide, S. Ohkubo, H. Takeda, K. Sugiyama; In vivo Imaging and Quantitative Evaluation of Rat Retinal Nerve Fiber Layer and Retinal Ganglion Cells Using a Scanning Laser Ophthalmoscope . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1249.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to determine whether a scanning laser ophthalmoscope (SLO) is useful for in vivo imaging and quantitative evaluation of retinal nerve fiber layer (RNFL) and retinal ganglion cells (RGCs) using an optic nerve crush model.
Following experiments were conducted on Brown Norway rats anesthetized by an intraperitoneal injection of sodium pentobarbital. RGCs were retrogradely labeled bilaterally with a fluorescent dye, 4–(4–(dihexadecylamino)styryl)–N–methylpyridinium iodine. Two months later, the optic nerve of the right eye was crushed intraorbitally with a clip for 30 seconds. The left eye served as the untreated control. Fundus images of both eyes were recorded by SLO using an argon blue laser (488 nm) before and 1, 2, and 4 weeks after the optic nerve crush. The focused plane was sequentially moved by changing the refractive values in the SLO setting. The range of refractive values (ΔF) in which RNFL reflex was clearly observed was subjectively determined in a masked fashion. Fluorescent RGCs were imaged using optical filter sets for fluorescein angiography. A retinal area 0.5 to 1.5 mm from the center of the optic disc, where cells were intensely stained and were well–focused in SLO images throughout the experimental period, was selected for each eye and was used for cell counting. To examine the reliability of SLO for RNFL thickness measurement and RGC counting, histological analyses were also performed at baseline and 1, 2, 4 weeks after the optic nerve crush using different sets of rats.
The ΔF value before and 1, 2, and 4 weeks after the optic nerve crush was 7.1±0.7, 7.0±1.0, 3.6±0.5 and 1.6±0.5 D, respectively (n=6). ΔF was unchanged in the control eyes during the experimental period. The ΔF value correlated significantly with the histologically determined RNFL thickness (Spearman's rank–order correlation coefficient, r = 0.86, P < 0.001, n=4 for each time point). The relative density of fluorescent cells in SLO images 1, 2, and 4 weeks after the optic nerve crush, with those at baseline being 100%, were 68.4±14.4, 35.2±4.2, 25.4±5.7% (n=6). Cell counts by SLO were not significantly different from those by retinal flatmounts (n=6 for each time point).
SLO is a useful and valuable tool for in vivo imaging and quantitative evaluation of rat RNFL and RGCs.
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