Abstract
Abstract: :
Purpose: (1) To study the kinetics of rhodopsin regeneration after a nearly complete bleach in mice with Met450 (C57BL/6) and Leu450 (Balb/c) variants of RPE65 protein. (2) To test effects of a ketamine/xylazine/urethane (25, 10 and 1000 mg/kg respectively) anesthesia, commonly used in ERG studies, on the kinetics of regeneration. Methods: Awake or anesthetized animals with fully dilated pupils were placed in an aluminum foil lined chamber with 2 white fluorescent tube lamps inside which provided nearly isotropic illumination. Alert animals were allowed to move freely; anesthetized mice were put on a heating plate and their temperature was maintained within 35–38oC throughout the experiment. Mice were illuminated at 5–7 klux for 10 min to bleach rhodopsin and allowed to regenerate in darkness for prescribed periods of time; they were then euthanized, the retinas removed through a corneal slit and the rhodopsin content measured by difference spectroscopy. Results: (1) In unanesthetized mice regeneration from the initial bleach level (>80% for both strains) proceeded linearly to regeneration of ∼ 70% of rhodopsin at the rate of 0.7% min–1 and 2.2% min–1 for C57BL/6 and Balb/c respectively. (2) anesthesia delayed regeneration by ∼ 30% in Balb/c and 60% in C57BL/6 animals. Control experiments demonstrated that the ketamine/xylazine/urethane cocktail, at a concentration corresponding to 25, 10 and 1000 mg/kg respectively, did not affect the rate of 11–cis retinal/opsin reconstitution in vitro. Conclusions: (1) A non–exponential, rate–limited process or processes determine the initial rate of rhodopsin regeneration after substantial bleaches in mice with both the Met450 and Leu450 variants of RPE65 protein. (2) Ketamine/xylazine/urethane anesthesia inhibits the rate of rhodopsin regeneration, but this effect may be strain–dependent.
Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium