Petroll W M, Jester J V, Barry-Lane P, Cavanagh H D
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas 75235-9057, USA.
Invest Ophthalmol Vis Sci. 1995 Nov;36(12):2492-502.
To assess the relationships between cytoskeletal changes and apical-basal polarity during healing of mechanical scrape injuries in the cat corneal endothelium.
Ten cats (20 eyes) were used in this study. One mechanical scrape injury was created in the corneal endothelium of each eye using a blunt olive tip cannula. Tandem scanning confocal microscopy (TSCM) was performed at sequential time points after injury for in vivo assessment of cell morphology and wound healing rates. In two eyes, scanning electron microscopy was performed to allow verification of TSCM observations. Ten eyes were collected between 6 and 48 hours after wounding for in situ labeling of f-actin, ZO-1, or both.
Cat endothelial cell morphology observed using in vivo microscopy was identical to that shown using scanning electron microscopy. During healing, endothelial cells always remained attached to the endothelial sheet, although some showed extensions of lamellipodia into the open wound area. The in situ localization of f-actin also correlated with the TSCM in vivo wound morphology. Quantitative analysis showed that there was a decrease in the intensity of phalloidin-fluorescein isothiocyanate staining at the leading edge of the wound, suggesting a decrease in f-actin; a significant correlation was found between the relative intensity of f-actin staining and the distance from the wound margin (R = 0.98, P < 0.01). At 24 and 48 hours after injury, both ZO-1 and f-actin maintained an apical localization within cells immediately adjacent to the leading edge, despite the considerable distance of movement and dramatic decrease in the intensity of f-actin staining.
Overall, these data demonstrate that after scrape injury in the cat, endothelial cells exhibit a pattern of healing in which total intracellular f-actin is reduced, but normal cell connectivity and apical-basal polarity are maintained throughout.
评估猫角膜内皮机械刮伤愈合过程中细胞骨架变化与顶-基极性之间的关系。
本研究使用了10只猫（20只眼）。用钝头橄榄形插管在每只眼的角膜内皮造成一处机械刮伤。在损伤后的连续时间点进行串联扫描共聚焦显微镜检查（TSCM），以对细胞形态和伤口愈合率进行体内评估。对两只眼进行扫描电子显微镜检查，以验证TSCM的观察结果。在受伤后6至48小时收集10只眼，用于对f-肌动蛋白、紧密连接蛋白1（ZO-1）或两者进行原位标记。
使用体内显微镜观察到的猫内皮细胞形态与扫描电子显微镜显示的形态相同。在愈合过程中，内皮细胞始终附着在内皮层上，尽管有些细胞显示片状伪足延伸到开放伤口区域。f-肌动蛋白的原位定位也与TSCM体内伤口形态相关。定量分析表明，伤口前缘的鬼笔环肽-异硫氰酸荧光素染色强度降低，表明f-肌动蛋白减少；f-肌动蛋白染色的相对强度与距伤口边缘的距离之间存在显著相关性（R = 0.98，P < 0.01）。在损伤后24和48小时，尽管有相当大的移动距离且f-肌动蛋白染色强度显著降低，但ZO-1和f-肌动蛋白在紧邻前缘的细胞内仍保持顶端定位。
总体而言，这些数据表明，猫角膜内皮刮伤后，内皮细胞呈现出一种愈合模式，即细胞内总f-肌动蛋白减少，但整个过程中正常的细胞连接性和顶-基极性得以维持。
