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Pierre-Serge Launay, David Godefroy, José-Alain Sahel, William Rostène, Christophe Baudouin, Stephane Melik Parsadaniantz, Annabelle Reaux-le Goazigo; Combined clearing method, retrograde tracer and lightsheet microscopy to map corneal neurons in a whole mouse trigeminal ganglion. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3375.
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© ARVO (1962-2015); The Authors (2016-present)
Clearing and imaging intact transparent organs has recently provided new way to image whole tissues. Corneal innervation is supplied by primary sensory neurons located in the ophthalmic branch of the trigeminal ganglion (TG). The distribution of corneal neurons in the TG has been previously studied with classical histological method using tissue sections. Here we used a new developed 3-dimensional imaging of solvent cleared organ (3DISCO) procedure to achieve imaging of unsectioned mouse TG by optical clearing and light-sheet laser-scanning ultramicroscopy to report the somatotopy of mouse corneal neurons.
Adult male C57BL/6 mice were anesthetized with Ketamine/Xylazine. After mechanical removing of the corneal epithelium, retrograde tracer (Alexa 555-conjugated cholera toxin subunit B) was carefully placed on the left mouse cornea for 10min. Two days after the CTB application, animals were perfused with 0.9% saline followed by 4% paraformaldehyde. After fixation, TGs were disseted out and were cleared by 3DISCO method. Large serial imaging on whole TG was performed by using light-sheet ultramicroscopic technology. Image analysis was performed by Imaris software.
We combined optical clearing, light-sheet laser-scanning microscopy, fluorescent retrograde tracer and Imaris sofware. We reported the optimized steps for easily and rapidly clearing a mouse TG. The procedure gave excellent results and took less than 3 hours. In a second set of experiments, CTB 555-conjugated was placed on cornea to retrograde-labeled corneal neurons. Two days later, TGs were cleared by 3DISCO and serial imaging was performed by light sheet ultramicroscopic technology. We provided a 3D reconstitution of corneal afferent neurons. We reported that trigeminal neurons supplying corneal innervation exhibit a specific limited dorsolateral localization within the TG. By using an automated counting, we found an average of 78 ± 6 and 80 ± 3 retrograde labeled corneal neurons within the intact mouse TG in two separate experimental series.
We proposed an optimized 3DISCO clearing method combined with ultramicroscopy technology to precisely map the 3D organization of corneal neurons within intact mouse TG. This method, which is rapid and reliable, could be suitable to evaluate corneal nerve alteration in preclinical animal model of dry eye disease.
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