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Micah Guthrie, Christian Osswald, Jennifer Kang Mieler; Direct In-Vivo Measurement of Nitric Oxide in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2506. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Nitric oxide (NO) concentration depth profiles were recorded in the in-vivo rat retina using direct measurements from an NO-sensing electrode under control conditions and nitric oxide synthase (NOS) inhibition.
NO concentration depth profiles were recorded with double-barreled microelectrodes (10 µm tip size). One barrel contained a silver chloride wire that recorded electroretinograms (ERGs) referenced to a subdermal electrode, and the other barrel contained a Nafion®-coated carbon fiber that measured NO amperometrically at +0.9 V. Recordings were made from a dark-adapted in-vivo rat eyecup preparation in which the cornea and lens were removed. The retina was penetrated with the electrode recording ERGs to determine location of the electrode tip inside the retina. The electrode was then withdrawn (1.5 µm/sec) while recording NO current to create a NO retinal depth profile. The profiles were expressed in terms of % depth, with 100% indicating the choroid and 0% indicating the vitreoretinal interface. Control NO profiles were compared to profiles recorded after the administration of the NOS inhibitor L-NG-Nitroarginine methyl ester (L-NAME, 10 mM vitreous concentration) to verify the contribution of NO to the current recorded.
Control NO profiles (n=3) had an average of 2.42 ± 0.48 µM NO at 100% retinal depth, which gradually decreased to an average of 1.22 ± 0.14 µM at 0% depth. There were spikes of NO in the ganglion and amacrine cell layers on the order of 200 to 400 nM. NO profiles after L-NAME administration (n=3) had an average of 0.79 ± 0.20 µM at 100% retinal depth gradually decreasing to an average of 0.28 ± 0.06 µM at 0% depth. No spikes of NO were seen in the ganglion or amacrine cell layers after L-NAME. Control and L-NAME NO measurements were statistically different at both 100% depth (t-test, p=0.035) and 0% depth (p=0.004).
The measurements showed a clear NO gradient through the retina, with NO being highest in the outer retina near the choroid and lowest at the retinal surface. The high NO levels near the photoreceptors and the NO spikes near the ganglion and amacrine cell layers correspond with previously suggested sources of NO production in the retina. The change in NO levels between the control and L-NAME measurements indicate that the electrodes could be used to detect changes in in-vivo NO levels in disease models.
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