Yu Q, Kent C R, Tytell M
Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1010, USA.
Mol Vis. 2001 Mar 7;7:48-56.
To evaluate the uptake by the rat retina of an intravitreally injected mixture of the constitutive and inducible forms of the 70 kD heat shock protein (Hsc/Hsp70) and test its potential to protect photoreceptors from light damage.
Hsc/Hsp70 and actin (control protein) were labeled with fluorescein (referred to as fl-Hsc/Hsp70 and fl-actin). The labeled proteins were microinjected intravitreally into the normal or light damaged rat eye and each eye collected at three intervals after the injections. Retinal uptake of Hsc/Hsp70 or actin was studied in frozen sections using epifluorescence microscopy and in western blots of retinal homogenates using an anti-fluorescein antibody. Additionally, the cytoprotective effects of Hsc/Hsp70 were tested in rats that first were exposed to bright light (170 ft-c) for 24 h and then given an intravitreal injection of the protein immediately thereafter. Ten days later, photoreceptor damage was evaluated by measuring the area of the outer nuclear layer at fixed locations along the circumference of the retina.
The fluorescein-labeled proteins were detected in the retina one h after administration and were retained there for more than 6 h. They were diffusely distributed, primarily in the nerve fiber layer, ganglion cell layer, and plexiform layers. Fl-Hsc/Hsp70 was also found in the outer nuclear layer (ONL) at 6 h after injection. At 24 h post-injection, the proteins were undetectable by epifluorescence microscopy of retinal sections, but could still be detected in western blots of retinal homogenates. The pattern of protein uptake was similar in light-damaged retinas. Ten days after light damage, the retinas in those eyes that received injections of Hsc/Hsp70 had greater ONL areas compared to either the light-damaged retinas of uninjected eyes or those that had received actin. The difference was statistically significant (p<0.05).
Intravitreally injected Hsc/Hsp70 is taken up by retinal cells and, when administered after an acute injury like light damage, increased the number of surviving photoreceptors.
评估玻璃体腔内注射组成型和诱导型70 kD热休克蛋白（Hsc/Hsp70）混合物后大鼠视网膜对其摄取情况，并测试其保护光感受器免受光损伤的潜力。
用荧光素标记Hsc/Hsp70和肌动蛋白（对照蛋白）（分别称为fl-Hsc/Hsp70和fl-肌动蛋白）。将标记的蛋白质经玻璃体腔微量注射到正常或光损伤的大鼠眼内，并在注射后三个时间点采集每只眼睛。使用落射荧光显微镜在冰冻切片中研究视网膜对Hsc/Hsp70或肌动蛋白的摄取情况，并使用抗荧光素抗体在视网膜匀浆的蛋白质印迹中进行研究。此外，在首先暴露于强光（170英尺烛光）24小时、然后立即经玻璃体腔注射该蛋白质的大鼠中测试Hsc/Hsp70的细胞保护作用。十天后，通过测量视网膜圆周上固定位置的外核层面积来评估光感受器损伤情况。
给药1小时后在视网膜中检测到荧光素标记的蛋白质，并且在那里保留超过6小时。它们呈弥漫性分布，主要在神经纤维层、神经节细胞层和神经丛层。注射后6小时在Hsc/Hsp70外核层（ONL）中也发现了fl-Hsc/Hsp70。注射后24小时，视网膜切片的落射荧光显微镜检查未检测到蛋白质，但仍可在视网膜匀浆的蛋白质印迹中检测到。在光损伤的视网膜中蛋白质摄取模式相似。光损伤十天后，与未注射的光损伤视网膜或接受肌动蛋白注射的视网膜相比，接受Hsc/Hsp70注射的眼睛的视网膜具有更大的ONL面积。差异具有统计学意义（p<0.05）。
经玻璃体腔注射的Hsc/Hsp70被视网膜细胞摄取，并且在像光损伤这样的急性损伤后给药时，可增加存活光感受器的数量。
