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Natalia Karagianni, Victor H. Guaiquil, John T. Pena, Mark I. Rosenblatt; Antero- and Retrograde Labeling of Corneal Nerves in Vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1794. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To visualize the corneal nerves and their neuron cell bodies in vivo via anterograde labeling using DiI and GFP tagged lentiviral vector injections and retrograde labeling using FM1-43FX a lipophilic styryl dye.
6 to 8 week-old C57BL/6 mice were used. For anterograde labeling, animals were deeply anesthetized and immobilized within a stereotactic frame. A 35g microinjection needle was passed to stereotactic coordinates to target the ophthalmic branch (V1) of the trigeminal ganglion (TG). Either DiI or plenti6.3-mGFP lentiviral vectors were microinjected into the (TG). Animals were sacrificed at day 4 (for DiI injected animals) and day 15 (for plenti6.3mGFP lentiviral vector injected animals) and the TG was exposed and imaged by fluorescent microscopy. Corneal wholemounts of these treated animals were imaged to localize the fluorescence stained nerves. For retrograde labeling, a pledget saturated with 5 mM FM1-43 was placed on the center of the cornea for 30 min. 4 days post surgery TGs were harvested and dissociated. Neuronal cells from the TGs were cultured and imaged by fluorescent microscopy.
The stereotactic surgery technique successfully allowed labeling of the targeted TG with DiI and plenti6.3-mGFP lentiviral vectors. No staining was found in the non-injected contralateral TG or surrounding tissue. Corneal nerves also stained positive in corneal wholemounts. The staining localized preferentially at the periphery of the cornea. We found that 2-5 % of the TG neuronal cells cultured from FM1-43 treated mice stained positive for the dye.
Antero- and retrograde corneal nerve labeling in vivo was achieved. These techniques may improve our ability to study basic and translational aspects of corneal neurobiology.
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