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Substrate Inhibition of Tetrazolium Salt Reduction in Dark Adapted Retinae.

1965; SAGE Publishing; Volume: 118; Issue: 2 Linguagem: Inglês

10.3181/00379727-118-29885

1535-3702

W. L. Fowlks, Dallas E. Peterson,

Anesthesia and Neurotoxicity Research

Enoch(l) reported that the light adapted retina of rat, rabbit, and squirrel monkey reduced more nitroblue tetrazolium chloride (nitroBT) in simple buffered media using succinate as substrate than did the fellow dark adapted retina. The effect was enhanced when the tissue culture medium, TC-199 without phenol red, was used and the effect was reduced if ATP was added to the media. Sickle (2) has reported results which suggest that light stimulation of isolated frog retinae alters metabolism or metabolic pathways. Albino rabbits of either sex were dark adapted 2-18 hours. One eye was patched in the dark with a tight fitting metal cup rimmed with a soft rubber gasket before removal to the adapting light source, a 60 W tungsten bulb in a focusing reflector held about 12 inches from the eye. After 10 minutes exposure of the one eye to the adapting light the rabbit was beheaded. In the dark room both eyes were enucleated, and dissected, and the retinas excised, and placed in the incubation mixture. Low intensity, Wratten #29 filtered, light was used during these latter procedures. The incubations, a modification of Enoch's procedure(l), were carried out in 0.6 ml of media in the 25 mm dia. of a spot plate. The media used had substrate at 10-1-10-3 M, nitroBT at 0.75 mg/ml, 0.5 tris-chloride buf-er at pH 7.35-7.40, and enough NaCl to make the final media isotonic at 318-320 mOsmol. Incubation time was 10 minutes unless otherwise noted. The temperature during incubation was 21° ± 1°C. The reaction was stopped by addition of 1.0 ml of 5% TCA to the reaction mixture. The retinae were then transferred within ½ minute to 5% TCA. Judgments of the difference, if any, in amount of blue colored formazan deposited in the uninjured parts of the light adapted and dark adapted pair of retinae were made at this point.