Abstract
Abstract: :
Purpose: New methods are needs for monitoring the chemical composition in the vitreous to better understand retinal processes and disease. A low-flow push-pull perfusion method is described to sample from the vitreous of rats. Rats with normal and moderately elevated intraocular pressure were examined with low-flow push-pull perfusion. Methods: A low-flow push-pull probe is constructed of concentric fused silica capillaries and is placed into the central vitreous of a rat by insertion through a 29-gauge needle guide that is inserted through the sclera and conjuctiva. Saline is infused through the outer capillary (160/100 µm outer diamater/inner diameter) to the tip of the probe at 20-60 nL/min with a syringe pump. Using a vacuum pump and regulators, 500 nL fluid samples are withdrawn through a interior capillary (90/20 µm outer diamater/inner diameter) for subsequent analysis. Glutamate and aspartate are derivatized with primary-amine reactive, fluorescein isothiocyanate prior to separation by capillary electrophoresis and detection by laser-induced fluorescence. The composition of glutamate and aspartate was determined with Long-Evans rats that had normal and long-term, moderately elevated intraocular pressure. Intraocular pressure was raised by reducing episcleral blood flow. Results: Perfusates were collected from both the anterior and posterior chambers after visual confirmation of push-pull probe placement. Sample collection from vitreous proceeds without interruption when there was an excess infusion rate of 10 nL/min. In vitro collection efficiencies of fluorescein dye were determined to be 62.45 ± 2.29% in free solution (N=3) and 51.7 ± 7.6% in a viscolastic solution of hyaluronic acid (N=3). Over the course of 1.5 hour of sampling from the aqueous or vitreous glutamate levels varied by an relative average of <10%. The average normal vitreal glutamate concentration measured in perfusates was found to be 2.94 ± 1.92 µM (N=4). Surprisingly, the measured glutamate concentration of experimental rat eyes with reduced episcleral flow for 6 weeks generated a glutamate concentration of 2.15 ± 0.61 µM (N=2). Conclusions: The results of this study demonstrate a minimally invasive method for following chemical composition of the rat vitreous and suggest that glutamate levels do not change with long-term, moderately elevated intraocular pressure in the rat.
Keywords: vitreous • excitatory neurotransmitters • retina: neurochemistry