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R.L. Bernardos, L.K. Barthel, P.A. Raymond; Localized Retinal Regeneration Following Thermal Lesions in Adult Zebrafish . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5327.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Adult teleost fish have the ability to generate new neurons in their retinas to replace those destroyed during injury. Recent data suggest a role for the Notch signaling pathway in activation of the regenerative response in zebrafish heart and fin tissue. The goal of the experiments presented here was to examine the role of Notch signaling in retinal regeneration. Methods: We developed a new method to produce a local thermal lesion in the neural retina in juvenile/adult zebrafish. Zebrafish, ≥2 months of age, were anesthetized in 0.16% MS–222 and then positioned on wet paper towels in a plastic petri plate on the stage of a dissecting microscope. A glass pasture pipette that had been flamed to seal the tip was mounted in a micromanipulator and used to torque the zebrafish’s eye away from the eye socket, exposing the dorsal surface. A second micromanipulator held a 0.3 mm copper wire attached to the end of a 15–watt soldering iron. The copper wire was advanced towards the eye until it just dimpled the outer surface of the sclera and was left in place for 5 to 10 seconds. The fish were revived in water from their fish habitat. Lesioned and control retinas were sectioned and assayed by in situ hybridization and immunocytochemisty to assess changes in notch expression, mitotic activity and neurogenesis. Results: The thermal lesion method resulted in reproducible damage to the neural retina. The depth of the lesion could be varied by changing the duration of the burn. A 5–second application of heat selectively destroyed the photoreceptor layer in a circular area of approximately 300 µm diameter, leaving the inner retinal layers largely intact. Zebrafish undergoing this procedure had a 100% survival rate and regenerated retinal neurons, including cone photoreceptors, within about 4 weeks. Proliferation of cells in the inner nuclear layer was enhanced and Müller glia showed a gliosis response locally in the region of the lesion between 2 and 8 days post–lesion (dpl). Notch1b and notch3 expression was upregulated in the region of the lesion at 3 dpl. Conclusions: The thermal lesion paradigm proved to be a useful and reproducible method to study regeneration in the zebrafish retina. The upregulation of notch1b and notch3 expression is consistent with a role for Notch signaling in retinal regeneration, and further studies are underway to elucidate Notch’s involvement in the regenerative response.
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