Purchase this article with an account.
S.R. Kottegoda, K. Thongkhao–on, X. Zhao, J.S. Pulido, S. Shippy; New Approach to Study Diverse Neurochemicals of the Rat Vitreous by Low–flow Push–pull Perfusion . Invest. Ophthalmol. Vis. Sci. 2004;45(13):273.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Diverse chemicals are involved in neurosignaling in the retina during normal and anomalous conditions. The applicability of low–flow push–pull perfusion for in vivo collection from the vitreous was tested for 1) nitrate and nitrite – metabolites of nitric oxide 2) amino acids 3) peptides and proteins by matrix–assisted laser desorption ionization time–of–flight mass spectrometry (MALDI TOF MS). Methods: A concentric low–flow push–pull probe is made of fused–silica capillaries and placed in the rat vitreous close to upper retina through a 29–gauge needle guide. To sample the tissue at the tip of the probe, saline is infused through an outer capillary (170/100 µm) and perfusate is simultaneously collected through an interior withdrawing capillary (90/20 µm). Then, these small volume samples were assayed separately for nitrate/nitrite, amino acids or peptides by capillary electrophoresis (CE) with UV detection, CE with laser–induced fluorescence detection and MALDI TOF MS, respectively. Results:Vitreous samples can be collected for more than 2.5 hrs without any interruption and placement is confirmed by indirect ophthalmoscopy. Nitrates and nitrites are recovered from the vitreous and their basal levels were lower than the brain. A total of 13 amino acids are clearly resolved by capillary electrophoresis. Basal levels of some important neurochemicals including glutamate, aspartate, glycine, and D–serine were found to be 1.7 ± 0.5, 2.3 ± 0.5, 4.5 ± 1.9 and 2.0 ± 1.3 µM respectively. A number of peptides are recovered from the vitreous and their molecular weight ranged from 0.5 to 66kD. After a 20 min incubation of the vitreal perfusate with dynorphin A, a significant decrease is observed in the dynorphin A concentration. This implies the presence of proteases collected from the vitreous. Conclusions: The results of this study indicate the wide applicability of the low–flow push–pull perfusion for neurochemical analysis of the rat vitreous. The method provides recovery of nitrate, nitrite, amino acids, biogenic amines, peptides including retinal enzymes from the vitreous and will provide a more accurate neurochemical picture of the retina during normal function and in retinal diseases.
This PDF is available to Subscribers Only