Blazynski C, Cohen A I
Exp Eye Res. 1984 Mar;38(3):279-90. doi: 10.1016/0014-4835(84)90166-0.
To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.
为了评估药物制剂对特定视网膜层代谢物水平的影响，我们设计了一种装置，该装置能够对外翻眼杯（玻璃体表面暴露）或分离的视网膜（光感受器表面暴露）进行超灌注，记录视网膜电图（ERG），并以允许获取视网膜冷冻切线切片的构型进行快速冷冻。随后可以对冻干切片的特定层样品进行称重，并通过洛瑞法（Lowry和Passonneau，1972年）进行生化处理，或者可以从称重样品中提取环核苷酸并进行测定。为了测试该装置的实用性，我们将超灌注兔视网膜特定层中测得的鸟苷3'，5'-单磷酸（cGMP）和腺苷3'，5'-单磷酸（cAMP）水平与从光适应和暗适应兔眼中取出并快速冷冻的视网膜层所报道的浓度进行了比较。对于大多数层，超灌注制剂和全眼制剂中环核苷酸的浓度相似。在实验应用中，我们用一种通过螯合使其游离钙水平急剧降低的生理盐水对暗适应的眼杯和视网膜进行超灌注，并观察到cGMP水平仅在那些包含光感受器部分的视网膜层中升高。当将乙二醇双（β-氨基乙醚）N，N'-四乙酸（EGTA）（3 mM）应用于分离视网膜的光感受器表面时，在10分钟内cGMP增加了三倍。然而，当用钙螯合培养基对外翻眼杯的玻璃体表面进行10分钟的超灌注时，cGMP增加三倍需要20 mM EGTA，3 mM EGTA无效。在另一项研究中，眼杯的玻璃体表面或视网膜的光感受器表面用含有3 mM 3-异丁基-1-甲基黄嘌呤（IBMX）的生理盐水进行超灌注，以改变视网膜cAMP水平。在这两种情况下，内核层和内网状层中cAMP增加了八倍，而外核层和外网状层中增加了三到四倍。这种药物还导致色素上皮中cAMP大幅增加。
