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A. Kanamori, M.-M. Catrinescu, M. Traistaru, R. Beaubien, L. A. Levin; In vivo Imaging of Retinal Ganglion Cell Axons Within the Nerve Fiber Layer. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3199.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve injury causes the loss of retinal ganglion cells (RGCs) and their axons. The reduction in RGC counts over time in axonal injury is well studied, but the correlation with the time course of anterograde and retrograde axonal degeneration is less clear. We longitudinally observed retinal nerve fiber bundles (RNFB) stained with a chloromethyl derivative of fluorescein diacetate (CMFDA) in the living rat after optic nerve injury.
The right optic nerves of Long-Evans rats were transected within the meninges by a transconjunctival approach, sparing the retinal circulation. Three days after transection CMFDA was intravitreously injected to a vitreous concentration of 60 µM. Confocal scanning laser imaging was performed daily with a Heidelberg Retina Angiograph 2 (HRA2) using the fluorescein channel and images exported to ImageJ. Mean fluorescence intensity and number of CMFDA bundles were calculated. A separate group of animals underwent retrograde RGC labeling by overlaying the superior colliculi with Alexa Fluor 488-dextran.
RNFB lost 87 ± 15% of their fluorescence by 7 days after injection and 10 days after optic nerve transection, compared with 42 ± 26 % in untransected eyes (p = 0.005, n = 6). Similarly, the number of labeled RNFB decreased by 86 ± 13 % after optic nerve transection, vs. 49 ± 22 % in untransected eyes (p = 0.004; n = 6). To distinguish increased leakage of CMFDA as a result of injury from decreased number of CMFDA-positive axons, another group of animals were injected with CMFDA at 2 and 3 weeks after transection. There were far fewer axon bundles labeled, compared to the number labeled at three days after transection, consistent with a decrease in the number of axons and not just CMFDA leakage. The number of retrograde labeled RGC bodies and CMFDA bundles on HRA2 decreased rapidly between 5 and 7 days after transection.
Intravitreal CMFDA can be used to longitudinally monitor RGC axons within the retinal nerve fiber layer in vivo.
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