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C. K. Leung, J. D. Lindsey, Q. Liu, D. U. Bartsch, R. N. Weinreb; Longitudinal in vivo Imaging of Retinal Ganglion Cells Degeneration After Optic Nerve Crush in a Transgenic Mice Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2880.
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Expression of Thy-1 by retinal ganglion cells (RGCs) is down-regulated following optic nerve crush and prior to RGC death. The present study investigated whether fluorescence loss in RGCs of transgenic mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) could be followed in vivo using a modified confocal scanning laser ophthalmoscope.
Six Thy1-CFP mice aged 3-6 months were imaged with a blue-light confocal scanning laser ophthalmoscope (bCSLO, modified Heidelberg Engineering HRA1 with 460 nM excitation and 490 nm detection) before and weekly for 3 weeks after optic nerve crush in one randomly selected eye. Sham procedure was performed in the opposite eye. The mice were held steady by an assistant during the imaging and no anesthesia was required. Corresponding retinal areas before and after optic nerve crush were compared and the fluorescent ganglion cell layer neurons were counted manually.
Fluorescent points corresponding to CFP-expressing ganglion cell layer neurons were discernable with the bCSLO (Figure 1a). A consistent and progressive loss of fluorescent points was found after optic nerve crush (Figure 1b-d) with 34.6+/-8.9%, 16.0+/-4.2% and 11.6+/-6.0% of the fluorescent cells remaining 1, 2, and 3 weeks after the crush, respectively (n=6). No change in the fluorescent cell density was found in the opposite eyes in which the sham procedures were performed.
The present results demonstrate that the bCSLO can readily image fluorescent ganglion cell layer neurons and provide a non-invasive approach to monitor the progressive loss of CFP expression in RGCs of transgenic Thy1-CFP mice. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator of the functional integrity of RGCs and offer a new model to study RGC degeneration in glaucoma and to study the effect of neuroprotective agents.
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