视网膜静脉阻塞后视网膜对大分子物质通透性的影响

目的 观察病理状态下视网膜对大分子物质直径的通透极限变化以及视网膜不同亚层对于大分子物质的通透性差异。 方法 光动力（PDT）方法制作小型猪视网膜分支静脉阻塞(BRVO)模型模拟视网膜水肿状态，2 d后处死取出双眼视网膜，另外未造模眼作为对照，使 用自行设计的膜通透装置进行视网膜通透性研究。使用相对分子质量为376.0×10.3的羧基荧光素和相对分子质量分别为4.4×10.3、9.3×10.3、19.6×10.3、38.9×10.3、71.2×10.3、150.0×10.3的异硫氰酸荧光黄标记葡聚糖（FITC-葡聚糖），将其溶于RPMI 1640 溶液，与视网膜内界 膜面或光感受器面相接触，不同时间段使用荧光分光光度计对通过视网膜的FITC-葡聚糖溶 液进行浓度检测，计算后得出视网膜对大分子物质通透的极限直径；将视网膜进行冰冻切片和荧光观察，观察视网膜不同亚层的荧光分布，了解视网膜不同层对大分子物质的阻挡能力 。 结果 相对分子质量为4.4×10.3的FITC-葡聚糖接触到视网膜内侧面时，内核层阻挡了绝大部分FITC-葡聚糖；相对分子质量为19.6×10.3～71.2×10 .3 的FITC-葡聚糖接触到视网膜内侧面时，神经纤维层和内丛状层阻挡了绝大部分FITC-葡聚糖；相对分子质量150.0×10.3的FITC-葡聚糖接触到视网膜内侧面时，神经纤维层阻挡了绝大部分FITC-葡聚糖；而相对分子质量4.4×10.3～150.0×10.3 的FITC-葡聚糖接触到视网膜外侧面时，外核层阻挡了绝大部分FITC-葡聚糖。无论是接触视网膜内侧面还是外侧面，羧基荧光素能通过全层视网膜，主要聚集在内核层和外核层。BRVO后视网膜内层发生水肿，出现囊样空腔，失去了屏障作用，和正常视网膜相比，大分子物质通透极限由（6.5±0.39）nm增加到（6.18±0.54 ）nm（t=4.143, P=0.0001）。 结论 BRVO 后视网膜内层屏障功能破坏，大分子物质通透极限增大，但很有限。外核层是决定视网膜通透性的最主要屏障，视网膜外层遭受损伤后 会明显影响大分子物质的通透极限。 （中华眼底病杂志，2008，24：197-201）

Effects of retina on macromolecules diffusion after retinal vein occlusion

Objective To observe the change of diffusion upper limit of macromolecules through pathological retina and the difference between the layers of retina. Methods Retinal edema was emulated by establishing branch retinal vein occlusion(RVO)model in miniature pig eyes under photodynamic method.Two days later,the retinas of both eyeballs were peeled off.The diffusion test apparatus was designed by ourselves.FITC-dextrans of various molecular weights(4.4,9.3,19.6,38.9,71.2 and 150 kDa)and Carboxyfluorescein(376 Da)were dissolved in RPMI-1640 solutions and diffused through inner or outer surface of retina.The rate of transretinal diffusion was determined with a spectrophotometer.Theoretical maximum size of molecule(MSM)was calculated by extrapolating the trend-linear relationship with the diffusion rate.In separate experiments to determine the sites of barrier tO diffusion,FITC-dextrans were applied to either the inner or outer retinal surface,processed as frozen sections.and viewed with a fluorescence microscope. Results FITC-dextrans applying tO inner retinal surface,4.4 kDa dextrans were largely blocked by inner nuclear layer(INL);19.6-71.2 kDa dextrans were blocked by the nerve fiber layer(NFL)and inner plexiform layer;150 kDa dextrans were blocked by NFL.FITC-dextrans applying to outer retinal surface,most dextrans with various molecular weights were blocked before outer nuclear layer(ONL).No matter applying to the inner or outer surface,Carboxyfluorescein can diffuse through the whole retina and aggregate at INL and ONL.After RVO,the inner part of retina became edema and cystoid,loosing the barrier function.Compared with the normal retina,the MSM in RVO tissues increased(6.5±0.39nm Vs 6.18±0.54nm,t=4.143,P=0.0001). Conclusions After RVO,the barrier function of inner part of retinal is destroyed and the upper limit of diffusion macromolecule size increased.which is nevertheless limited.ONL acts as bottle-neck barriers to diffusion,if the outer part of retina is damaged,the change of the diffusion upper limit will be prominent.