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Natl. LCA model mice. Moreover, the expression levels of DIO2 and were significantly higher in the diseased retinas, suggesting locally elevated TH signaling. We show that targeting DIOs protects cones, and intracellular inhibition of TH components locally in the retina may represent a novel strategy for retinal degeneration management.Yang, F., Ma, H., Belcher, J., Butler, M. R., Redmond, T. M., Boye, S. L., Hauswirth, W. W., Ding, X.-Q. Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration. mice, a model of the pediatric blinding disease Lebers congenital amaurosis (LCA), and mice with defect in mouse line was generated as has been described (15). The mouse line was obtained from The Jackson Laboratory (Bar Harbor, ME, USA). VD3-D6 The mouse line was provided by Dr. Anand Swaroop (National Eye Institute, National Institutes of Health, Bethesda, MD, USA) (16). The (17) and (18) lines were generated by cross-mating. All mice were maintained under cyclic 12-h lightCdark conditions. Cage illumination was 7 foot-candles (fc) during the light cycle. All animal maintenance and experiments were approved by the local Institutional Animal Care and Use Committee (University of Oklahoma Health Sciences Center) and conformed to the guidelines on the care and use of animals adopted by the Society for Neuroscience and the Association for Research in Vision and Ophthalmology (Rockville, MD, USA). Rabbit antibodies against mouse M-opsin and cone arrestin (CAR) were provided by Dr. Cheryl Craft (University of Southern California, Los STMN1 Angeles, CA, USA). Rabbit anti-S-opsin antibody was provided by Dr. Muna Naash (University of Houston, Houston, TX, USA). Mouse anti-rhodopsin antibody 1D4 was provided by Dr. Robert Molday (The University of British Columbia, Vancouver, BC, VD3-D6 Canada). Biotinylated peanut agglutinin (PNA) was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Rabbit anti-DIO2 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-DIO3 antibody was procured from Abcam (Cambridge, MD, USA). Mouse anti–actin was obtained from Abcam. Horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD, USA). Fluorescent goat anti-rabbit and goat anti-mouse antibodies and streptavidin-Cy3 were procured from Thermo Fisher Scientific (Waltham, MA, USA). Iopanoic acid (IOP) was purchased from TCI Chemicals America (Portland, OR, USA). T4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospholipon 90G was provided by Dr. Bruce Baretz (Lipoid LLC, Newark, NJ, USA). All other reagents were purchased from Sigma-Aldrich, Bio-Rad Laboratories (Hercules, CA, USA), and Thermo Fisher Scientific. Construction of AAV5-IRBP/GNAT2-DIO3 vectors The pCDM8-vector [in balanced salt solution (BSS); Alcon, Fort Worth, TX, USA] was injected intravitreally or subretinally into 1 eye of each mouse with a NanoFil microsyringe injector system with a 33-gauge blunt needle (Hamilton Co., Reno, NV, USA). The contralateral eye was injected with 1 l of vehicle. Only eyes with no apparent surgical complications were retained for further evaluation. Table 1 shows information including concentration/titer of the reagents delivered for ocular injection experiments. TABLE 1. ?Information for ocular delivery experiments and mice received injections at P5 and were analyzed for cone density at P25 and cone death at P15, respectively.mice received eye drops for 20 d, beginning on P5, and were analyzed for retinal function and cone density at the end of the experiment.AAV5-IRBP/Gnat2-mice received IOP eye drops or vehicle (25% phospholipon 90G in PBS) (10 l) 3/d for 20 d. Eyes were collected at the end of the experiments for evaluation of cone density and TUNEL+ cells. Eye preparation, immunofluorescence labeling, and confocal microscopy Mouse retinal whole mounts or cross-sections were prepared, and immunofluorescence labeling VD3-D6 using antibodies against cone markers was performed (23). For whole-mount preparations, eyes were enucleated, marked at the superior (dorsal) pole with a green dye, for dorsalCventral orientation, and fixed in 4% paraformaldehyde (Polysciences, Inc., Warrington, PA, USA) for 30 min at room temperature. For retinal cross-sections, mouse eyes were enucleated (the superior portion of the cornea was marked for orientation before enucleation) and fixed in Prefer (Anatech Ltd., Battle Creek, MI, USA) for 15 min at room temperature, and.