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I. Soto–Reyes, T.H. López–Roca, R.E. Blanco; FGF–2 Application to the Optic Nerve After Axotomy Transiently Increases nNOS Activity in the Retina and Tectum . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4002.
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Purpose: Nitric oxide (NO) is a free radical gas produced by the enzyme nitric oxide synthase (NOS) that acts as a signal messenger in the nervous system. The expression of neuronal NOS is correlated with periods of axonal growth and synapse formation. We have shown that fibroblast growth factor–2 (FGF–2) increases growth associated protein–43 in retinal ganglion cells (RGCs) and tectum after axotomy. The objective of this study is to determine the changes in nNOS expression in the retina and tectum of the adult frog Rana pipiens after optic nerve axotomy and the application of FGF–2. Methods: Frog optic nerves were cut and immediately treated with FGF–2 or saline solution (PBS). After 1, 4, 6 and 12 weeks the retinas and tecta of experimental and control animals were dissected and processed for immunohistochemistry. Retinal and tectal sections were labeled with antibodies against nNOS and reacted for NADPH–diaphorase. Quantitative changes of nNOS in retina and tectum were measured by Western blots. An antibody against neurofilament (160kDa) was used as a marker for regenerating axons arriving at the tectum. Results: In the control retina nNOS immunoreactivity was found in Müller cells, in the inner plexiform layer (IPL), in cells of the ganglion cell layer (GCL) and in some cells of the inner nuclear layer (INL). Axotomy decreased the amount of nNOS protein detected in the retina. FGF–2 application to the injured optic nerve increased nNOS immunoreactivity in the GCL at 4 and 12 weeks after axotomy. In the tectum nNOS immunoreactivity was found in some neurons from layers 2, 4, 6 and 8, and in the nerve processes of layer 3, 5, 7, and the retinorecipent layer 9 (laminae A–G). At 6 weeks after axotomy nNOS immunoreactivity was considerably decreased in tectal layer 9, however a significant increase in nNOS expression and NADPH–d activity in the tectum was observed in the FGF–2–treated nerves. Western blot analysis of the tecta at this stage confirmed this result. Neurofilament staining of the tecta of animals treated with FGF–2 showed a significant increase in the number of regenerating axons that were found arriving at the tectum when compared with the axotomized animals treated with PBS. Conclusions: Axotomy decreases nNOS and NADPH–d reactivity in axonal processes in the tectum, particularly retinorecipient layer 9. Application of FGF–2 after axotomy increases the nNOS in both retina and tectum. Regenerating axons reach the tectum earlier in FGF–2 treated animals and coincide with the increase in nNOS expression.
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