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S. Shippy, S.R. Kottegoda, K. Thonghkhao–on, J. Pulido; Nanoliter Sampling of the Rat Vitreoretinal Interface in vivo . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3959.
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Purpose:Chemical signaling plays important roles in retinal neurotransmission, however, there are relatively few tools to follow chemical changes at the in vivo retina. This study presents a sampling tool for describing the extracellular chemical composition at specific sites of the vitreoretinal interface (VRI) of the rat. Methods: Low–flow push–pull perfusion is used to collect samples of the rat vitreous and VRI. Concentric style probes are made of 90–micron outer– and 20–micron inner diameter fused–silica capillary inserted into170–micron outer– and 100–micron inner–diameter capillary. Probes are inserted into the posterior chamber through a 29–gauge needle and physiological saline is infused and withdrawn at 50 nL/min flow rates. Collected perfusates are fluorescently derivatized and analyzed for amino acids by capillary electrophoresis with laser induced fluorescence. Probe tip placement at the VRI is guided by indirect ophthalmoscopy and confirmed by infusion of L–trans–pyrrolidine–2,4–dicarboxylic acid (PDC) a glutamate transport protein blocker. Results: More than 11 individual amino acids were recovered and analyzed from rat vitreous and VRI. In vivo sample collection was performed more than 2.5–hrs and amino acid basal levels were stabilized after 40–45 min. Statistically significant, elevated glutamate levels were observed in relation to PDC infusion. The glutamate elevation was significantly higher at sampling regions more distant from (>450%) rather than directly over the optic nerve head (>200%). Conclusions:The results of this study indicate the applicability of low–flow push–pull perfusion for neurochemical analysis of the in vivo VRI of the rat. The site selectivity for pharmacological manipulation of central versus peripheral locations on the retina provides new in vivo information not available by other methods. The application of this tool to animal models may provide an improved understanding of the neurochemical basis of disease and identify novel potential alternatives for treatment.
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