Monitoring of in vivo manipulation of nitric oxide synthases at the rat retina using the push–pull perfusion sampling and capillary electrophoresis

• In vivo sampling with inhibitor infusion to assay of nitric oxide synthase activity.
• Nitric oxide metabolite, nitrate, is significantly decreased with inhibitor infusions.
• Nitrate levels decrease at peripheral to, but not directly over optic nerve head.
• Sub-type selective inhibitor infusions display unique nitrate decrease responses.

Proteins play a variety of functional roles in tissues that underlie tissue health. The measurement of protein function is important to both understand normal and dysfunctional tissue states. Low-flow push–pull perfusion sampling (LFPS) has been used to collect submicroliter volumes of extracellular fluid which are well suited to capillary electrophoresis for compositional quantitative analysis. In this study, LFPS is used to deliver pharmacological agents to the in vivo retinal tissues at the probe sampling tip during sampling to measure protein function. Two native nitric oxide synthase enzymes were pharmacologically inhibited and the enzyme product NO metabolite, nitrate, was determined with capillary electrophoresis from the perfusates. LFPS delivered inhibitors including the non-selective N(G)-nitro-Larginine methyl ester (L-NAME), the nNOS selective 7-nitroindazole (7-NI), and eNOS N5-(1-imioethyl)-L-ornithine, dihydrochloride (L-NIO) were perfused to the sampling region either directly over a rat retina optic nerve head or 1-mm peripheral to the ONH. At the PONH, 65, 55 and 60% of baseline nitrate levels, respectively, were observed with inhibitor infusion. These are statistically significant (P < 0.05) compared to saline drug infusion. However, infusion of the inhibitors to the ONH did lead to significant nitrate concentration decreases. This data suggests that the endogenous enzymes, nNOS and eNOS, are both spatially and functionally localized to the PONH at the in vivo rat retina.