Rat Retina-RPE-Choroid Preparation: Electrophysiological Responses

大鼠视网膜-RPE-脉络膜制备：电生理反应

• 批准号: 7594094
• 负责人: sheldon s miller
• 金额: $ 17.31万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594094
• 关键词: Apical; Barium; Bathing; Calcium; Cells; Chloride Channels; Choroid; Elevation; In Vitro; Membrane; Membrane Potentials; Microelectrodes; Mutation; Neural Retina; Perfusion; Physiology; Pigments; Potassium; Potassium Channel; Preparation; Rattus; Resistance; Retina; Retinal; Retinal Photoreceptors; Side; Structure of retinal pigment epithelium; Thinking; Vision; Vitelliform macular dystrophy; apical membrane; basolateral membrane; in vitro Model; in vivo; monolayer; research study; response; thymidine 5' -triphosphate

The RPE is a monolayer of pigmented cells that interact with retinal photoreceptors and helps maintain visual function. We have developed a new preparation to study RPE physiology in rat that includes the neural retina. The retina-retinal pigment epithelium-choroid (R-RPE-Ch) preparation was mounted in an Ussing chamber which allowed independent perfusion of retinal and choroidal baths. The intact monolayer had a mean (+/- SD) transepithelial potential of 4.3 +/- 1.0 mV and a total transepithelial resistance of 152 +/- 37 ohm*cm-squared (n =10). As in other mammalian RPE preparations, we found barium-sensitive potassium channels at the apical and basolateral membranes. Intracellular microelectrodes at the basolateral membrane showed that step increases of basal potassium concentration depolarized the basolateral membrane 8 mV (n=2) and were blocked by barium. Decreasing apical potassium concentration mimics light-onset and hyperpolarizes the apical membrane; increasing apical potassium concentration depolarized the apical membrane by 6 mV (n=3). The potassium concentration responses were blocked by barium. Elevation of calcium concentration in the basal bath 10 fold, from 1.8 to 18 mMincreased TTP by 0.6 mV(p<0.001.; n=5). This increase was inhibited by 50% (p<0.01; n=3) when pretreated with DIDS (1mM) on the basolateral side. The R-RPE-Ch preparation was developed to study light-induced changes in RPE physiology, and provides an in-vitro model of in-vivo function. It can be used to analyze light-evoked alterations in RPE physiology that occur in Bests disease, which is thought to result from a mutation in basolateral membrane calcium-activated chloride channels.

RPE是单层色素细胞，与视网膜光感受器相互作用，有助于维持视觉功能。我们已经开发了一种新的制剂来研究包括神经视网膜在内的大鼠RPE生理学。 将视网膜-视网膜色素上皮-脉络膜（R-RPE-Ch）制备物安装在允许视网膜和脉络膜浴独立灌注的Ussing室中。完整单层的平均（+/- SD）跨上皮电位为4.3 +/- 1.0 mV，总跨上皮电阻为152 +/- 37 ohm* cm-2（n =10）。 在其他哺乳动物的视网膜色素上皮制剂，我们发现钡敏感的钾通道的顶端和基底外侧膜。基底外侧膜细胞内微电极显示，基础钾离子浓度的阶跃增加使基底外侧膜去极化8 mV（n=2），并被钡离子阻断。 降低顶端钾浓度模拟光发作和超极化顶端膜;增加顶端钾浓度去极化顶端膜6 mV（n=3）。 钾浓度反应被钡阻断。基础浴中钙浓度升高10倍，从1.8到18 mM，TTP增加0.6 mV（p<0.001; n=5）。当在基底外侧用DIDS（1 mM）预处理时，这种增加被抑制50%（p<0.01; n=3）。R-RPE-Ch制剂被开发用于研究光诱导的RPE生理学变化，并提供体内功能的体外模型。它可用于分析Bests病中发生的RPE生理学中的光诱发改变，该改变被认为是由基底外侧膜钙激活氯离子通道中的突变引起的。