Yaylali V, Ohta T, Kaufman S C, Maitchouk D Y, Beuerman R W
Lions Eye Research Laboratories, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans, USA.
Cornea. 1998 Nov;17(6):646-53. doi: 10.1097/00003226-199811000-00013.
To investigate the cellular dynamics of vessel formation during corneal neovascularization in the living eye by confocal microscopy.
Corneal neovascularization was initiated by placing a 7-0 silk suture through the corneal stroma 3 mm from the limbus at the 12 o'clock position in both eyes of 10 New Zealand white rabbits. The corneas were examined for vessel ingrowth at intervals from 1 to 15 days after suture placement using a tandem scanning confocal microscope with a 20X water immersion objective, as well as a slit-lamp biomicroscope. Changes in the limbal vessels were recorded on videotape for later analysis. As early vessel growth appeared to be associated with corneal nerves, the total number of sprouts and the number of sprouts along nerves were counted in confocal images, and the results analyzed for statistical significance. Vessel growth and the structural relationship between vascular buds and the deep stromal nerves were examined by light and transmission electron microscopy.
The early events of cell migration from the limbal microvessels were found to be associated with the deep stromal nerves; although this association was easily visualized by confocal microscopy, it could not be documented by slit-lamp biomicroscopy. By 18 h after suture placement, the limbal vessels were dilated and the first vascular buds appeared as short, pointed, or flat-topped protrusions from the deep limbal capillaries. By 96 h, the capillary buds had increased in density and had begun to form lumens. Movement of red blood cells was established between 72 and 80 h after the first signs of bud formation, at the same time that cells of immune origin were seen. Confocal microscopy revealed and transmission electron microscopy verified that new bud formation began with the formation of vascular tubes by endothelial migration along the deep stromal nerves. The total number of sprouts and the number of sprouts associated with stromal nerves were similar on days 1 and 2 but differed on days 3-7, suggesting an association between sprouts and nerves in the early stages of neovascularization.
Using real-time white light confocal microscopy, we were able, for the first time, to observe the process of corneal neovascularization in the living eye, from the earliest stages within hours after initiation to 2 weeks. The deep stromal nerves appear to serve as a focus for the growth of new vessels, by attracting and supporting vessel growth and/or by providing a potential space for movement of the endothelial cells. Confocal microscopy may provide a new approach to achieving a better understanding of the mechanisms involved in corneal neovascularization.
通过共聚焦显微镜研究活体眼中角膜新生血管形成过程中的血管生成细胞动力学。
在10只新西兰白兔的双眼12点位置距角膜缘3mm处穿过角膜基质放置一根7-0丝线，引发角膜新生血管形成。在缝线放置后1至15天内，使用配备20倍水浸物镜的串联扫描共聚焦显微镜以及裂隙灯生物显微镜对角膜进行血管长入情况检查。记录角膜缘血管的变化并录制到录像带上以供后续分析。由于早期血管生长似乎与角膜神经有关，在共聚焦图像中计数新芽总数以及沿神经的新芽数量，并对结果进行统计学显著性分析。通过光学显微镜和透射电子显微镜检查血管生长以及血管芽与深层基质神经之间的结构关系。
发现从角膜缘微血管迁移的细胞早期事件与深层基质神经有关；尽管这种关联通过共聚焦显微镜很容易观察到，但裂隙灯生物显微镜无法记录。缝线放置后18小时，角膜缘血管扩张，第一批血管芽表现为从深层角膜缘毛细血管伸出的短而尖或平顶的突起。到96小时时，毛细血管芽密度增加并开始形成管腔。在芽形成的最初迹象出现后72至80小时之间，红细胞开始移动，与此同时可见免疫源性细胞。共聚焦显微镜显示并经透射电子显微镜证实，新的芽形成始于内皮细胞沿深层基质神经迁移形成血管管。新芽总数以及与基质神经相关的新芽数量在第1天和第2天相似，但在第3至7天有所不同，表明在新生血管形成的早期阶段新芽与神经之间存在关联。
使用实时白光共聚焦显微镜，我们首次能够观察活体眼中角膜新生血管形成的过程，从开始后数小时的最早阶段到2周。深层基质神经似乎通过吸引和支持血管生长和/或通过为内皮细胞提供潜在的移动空间，成为新血管生长的焦点。共聚焦显微镜可能为更好地理解角膜新生血管形成所涉及的机制提供一种新方法。
