An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes.
An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes.
复制标题
DOI：
10.1167/iovs.16-19481
复制
发表时间：
2016-10-01
影响因子：
4.4
通讯作者：
John SW
中科院分区：
文献类型：
--
作者：
Kizhatil K;Chlebowski A;Tolman NG;Freeburg NF;Ryan MM;Shaw NN;Kokini AD;Marchant JK;John SW
控制房水（AQH）流出和眼压的分子机制需要进一步定义。鼠标是一个强大的系统，用于表征 AQH 流出的机械基础。为了加强小鼠的流出研究，我们开发了一种基于人类前房灌注培养系统的灌注系统。我们的鼠标系统允许以前不切实际的实验。我们设计了一种计算机控制的、基于泵的灌注系统，该系统具有用于安装整个解剖小鼠眼睛的平台（除去晶状体和虹膜，〜45％的引流组织被灌注）。我们测试了系统监测流出的能力，并测试了流出升高药物 Y27632（一种 rho 相关蛋白激酶 (ROCK) 抑制剂）的效果。最后，我们通过确定候选基因 Nos3 和 Cav1 的缺陷是否改变流出来测试系统检测遗传决定的流出减少的能力。使用我们的系统，发现 C57BL/6J 小鼠眼睛的流出设施 (C) 范围在 7.7 至 10.4 nl/分钟/mm Hg 之间（针对整个眼睛进行校正）。我们的系统很容易检测到 Y27632 诱导的 C 值增加了 74.4%。NOS3 抑制剂 L-NG-硝基精氨酸甲酯 (L-NAME) 和 Nos3 无效突变分别使 C 值降低了 28.3% 和 35.8%。同样，在 Cav1 中，空眼 C 降低了 47.8%。我们设计了一种独特的灌注系统，可以准确测量 C 的变化。然后，我们使用该系统证明 NOS3 和 CAV1 是控制流出机制的关键组成部分。
The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments. We engineered a computer-controlled, pump-based perfusion system with a platform for mounting whole dissected mouse eyes (minus lens and iris, ∼45% of drainage tissue is perfused). We tested the system's ability to monitor outflow and tested the effects of the outflow-elevating drug, Y27632, a rho-associated protein kinase (ROCK) inhibitor. Finally, we tested the system's ability to detect genetically determined decreases in outflow by determining if deficiency of the candidate genes Nos3 and Cav1 alter outflow. Using our system, the outflow facility (C) of C57BL/6J mouse eyes was found to range between 7.7 and 10.4 nl/minutes/mm Hg (corrected for whole eye). Our system readily detected a 74.4% Y27632-induced increase in C. The NOS3 inhibitor L-NG-nitroarginine methyl ester (L-NAME) and a Nos3 null mutation reduced C by 28.3% and 35.8%, respectively. Similarly, in Cav1 null eyes C was reduced by 47.8%. We engineered a unique perfusion system that can accurately measure changes in C. We then used the system to show that NOS3 and CAV1 are key components of mechanism(s) controlling outflow.