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J. S. Pritchett, J. S. Pulido, S. A. Shippy; In vivo Characterization of Nitric Oxide Metabolites and Nitric Oxide Synthases in the Rat Posterior Chamber Using Low-Flow Push-Pull Perfusion. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5370.
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© ARVO (1962-2015); The Authors (2016-present)
Nitric oxide (NO) plays an important role in several homeostatic processes in the eye. Measurement of the enzymatic activity that leads to NO production would be beneficial for understanding its functions in normal and diseased retinas. In this study, an in vivo sampling tool was used to deliver nitric oxide synthase (NOS) inhibitors to identify potential sources of NO in the rat retina.
Low-flow push-pull perfusion (LFPPP) was utilized to deliver selective and non-selective NOS inhibitors directly to various regions located in the vitreous cavity or on the vitreoretinal interface (VRI). Sampling probes were positioned along the VRI over the optic nerve head (ONH) or 1-2 mm peripheral to the ONH (PONH) and in the middle vitreous (MV). Physiological saline was infused to establish basal NO metabolite levels then the infusion capillary was transferred to an infusion syringe containing one of the NOS inhibitors. The inhibitors used were a non-selective NOS inhibitor (N-nitro-L-arginine methyl ester; L-NAME), a nNOS selective inhibitor (7-nitroindazole; 7NI), and an eNOS selective inhibitor (N-iminoethyl-L-ornithine; L-NIO). After infusion of the inhibitor for 30 minutes, the infusion capillary was returned to the infusion syringe containing the physiological saline solution. Collected perfusates were analyzed by capillary electrophoresis (CE) coupled with UV detection for the NO metabolite content.
The results show significantly (P<0.05) different nitrate levels at each of the sampling sites. Basal levels at the ONH (n=5), MV (n=5), and PONH (n=5) were 10.25 ± 2.75, 16.38 ± 2.83,and 27.53 ± 4.17 micromolar, respectively. The effect of the inhibitors on the presence of nitrate demonstrates an isoform specific production that is comparable to ex vivo, immunohistochemical studies. Switching the infusion line at these low 50 nL flow rates does not affect the retinal tissue under the tip and does not affect the ability to collect perfusates with this method. Control sampling in the MV demonstrates no effect of drug infusion.
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