The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes
The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes
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DOI：
10.1016/j.exer.2007.10.018
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发表时间：
2008-02-01
影响因子：
3.4
通讯作者：
Gong, Haiyan
中科院分区：
文献类型：
--
作者：
Lu, Zhaozeng;Overby, Darryl R.;Gong, Haiyan
rho激酶抑制剂Y-27632 （Y-27）影响肌动球蛋白细胞骨架网络，并已被证明可显著增加去核猪和兔眼以及猴子眼的流出设施(C)，但无明显毒性。这些反应背后的机制在很大程度上仍然未知。在这项研究中，我们研究了Y-27如何影响房水C、流出水的流体动力学模式以及内壁（IW）和导管旁结缔组织（JCT）的形态。12只牛眼在15mmhg下灌注含有5.5 mM葡萄糖的Dulbecco's PBS （DPBS）以建立稳定的基线c。7只眼前房交换并灌注含有50 LM Y-27的DPBS， 5只眼单独接受DPBS。然后将含有荧光微球（0.5 μ m, 0.002% v/v）的DPBS以固定体积灌注到眼睛中，以提供等量的示踪剂来标记水动力过滤模式。所有的眼睛都用卡诺夫斯基固定液灌注固定。在所有象限制作径向和额部切片，沿水丛（AP）的IW共聚焦图像。在>= 16张/眼的条件下测量IW的总长度（TL）和滤过长度（FL），计算平均有效滤过长度（PEFL = FL/TL）。用光镜和电镜对AP切片进行处理和检查。以>= 16显微图/眼为单位，测定IW的TL和JCT/IW分离长度（SL），并计算平均百分比分离长度（PSL = SL/TL）。经Y-27处理后，C从1.54 +/- 0.34 （SEM）增加到2.36 +/- 0.54 mu L/min / mmHg(58.2% +/- 18.9%)，而对照眼从1.67 +/- 0.41增加到1.71 +/- 0.39 mu L/min / mmHg (6.0 +/- 9.3%)， Y-27处理后与对照眼的百分比变化显著（p = 0.03）。对照眼的小梁网示踪剂呈节段性分布，倾向于聚集在收集器通道口附近，而y -27治疗眼的小梁网示踪剂沿IW分布更均匀，标记范围更广。y -27治疗组PEFL比对照组（18.2 +/- 4.5%;p < 0.001）大3倍（57.6 +/- 3.7%）。光镜检查显示，与对照眼相比，Y-27的IW和JCT明显膨胀，IW和JCT之间有明显的分离。y -27治疗组的PSL（59.3 +/- 3.6%）比对照组（20.8 +/- 2.0%,p < 0.001）大2.8倍。PEFL和PSL之间存在显著的正相关（p = 0.003），这表明随着JCT和IW之间的连通性减少，房水引流可用面积沿AP增加。我们的研究还证明了C和PSL之间存在显著的正相关（p = 0.01），这一发现与我们报道的牛眼“冲洗”效应期间发生的结果相同。我们的数据表明，y -27治疗后C和PEFL增加的结构相关性是JCT和IW之间的物理分离。y -27处理后C的增加可能与牛眼中发生的冲洗效应具有相似的机制。总的来说，这些发现支持了我们的假设，即JCT/IW连接性影响调节C的局部流出流体动力学，并表明针对JCT/IW连接性的策略是降低IOP的有希望的抗青光眼治疗方法。(c) 2007 Elsevier Ltd.版权所有。
Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 LM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 mu m; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in >= 16 images/eye, and the average percent effective filtration length (PEFL = FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in >= 16 micrographs/eye, and the average percent separation length (PSL = SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54 +/- 0.34 (SEM) to 2.36 +/- 0.54 mu L/min per mmHg (58.2 +/- 18.9%) while control eyes changed from 1.67 +/- 0.41 to 1.71 +/- 0.39 mu l/min per mmHg (6.0 +/- 9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p = 0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6 +/- 3.7%) compared to control eyes (18.2 +/- 4.5%; p < 0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3 +/- 3.6%) than in controls (20.8 +/- 2.0%; p < 0.001). A significant positive correlation was found between PEFL and PSL (p = 0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p = 0.01), a finding identical to what we reported to occur during the "washout" effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP. (c) 2007 Elsevier Ltd. All rights reserved.