Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
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DOI：
10.1167/iovs.10-6101
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发表时间：
2011-05-01
影响因子：
4.4
通讯作者：
Lagali, Neil
中科院分区：
文献类型：
--
作者：
Peebo, Beatrice Bourghardt;Fagerholm, Per;Lagali, Neil
目的.阐明炎症性角膜血管生成中导致新毛细血管发芽的时间顺序。在Wistar大鼠中通过角膜缝线放置诱导血管生成。通过延时体内共聚焦显微镜检查炎症区域长达7天。在第6和12小时以及第1、2、4和7天，切除角膜，用CD 31、CD 34、CD 45、CD 11b、CD 11 c、Ki-M2 R、NG 2和α-SMA的一抗进行平板免疫荧光。从第0天到第4天，量化单个角膜缘血管的体内外渗和扩张特征。从诱导后数小时开始，在第1天达到峰值，CD 45（+）CD 11b（+）髓样细胞从角膜缘血管外渗，并在基质内形成无内皮通道，通往炎症刺激物。角膜缘血管直径在第2 ~ 3天增加3倍，血管芽出现，并在不到1天后转化为灌注的毛细血管芽。纺锤形CD 11b（+）骨髓系细胞的亚群，而不是树突状细胞或成熟的巨噬细胞，似乎直接促进进一步的毛细血管芽生长。这些细胞整合到芽尖附近的血管内皮中，共表达内皮标记物CD 31。芽苗的灌注特征与供血血管不同，芽苗顶端多为开口状。时间推移在体内角膜共焦显微镜可用于跟踪角膜血管生成的时间序列的事件。该技术揭示了髓样细胞在炎症性角膜环境中促进血管发芽的潜在作用。（Invest Ophthalmol维斯科学。2011;52：3060-3068）DOI：10.1167/iovs.10-6101
PURPOSE. To elucidate the temporal sequence of events leading to new capillary sprouting in inflammatory corneal angiogenesis.METHODS. Angiogenesis was induced by corneal suture placement in Wistar rats. The inflamed region was examined by time-lapse in vivo confocal microscopy for up to 7 days. At 6 and 12 hours and 1, 2, 4, and 7 days, corneas were excised for flat mount immunofluorescence with primary antibodies for CD31, CD34, CD45, CD11b, CD11c, Ki-M2R, NG2, and alpha-SMA. From days 0 to 4, the in vivo extravasation and expansion characteristics of single limbal vessels were quantified.RESULTS. Starting hours after induction and peaking at day 1, CD45(+)CD11b(+) myeloid cells extravasated from limbal vessels and formed endothelium-free tunnels within the stroma en route to the inflammatory stimulus. Limbal vessel diameter tripled on days 2 to 3 as vascular buds emerged and transformed into perfused capillary sprouts less than 1 day later. A subset of spindle-shaped CD11b(+) myeloid-lineage cells, but not dendritic cells or mature macrophages, appeared to directly facilitate further capillary sprout growth. These cells incorporated into vascular endothelium near the sprout tip, co-expressing endothelial marker CD31. Sprouts had perfusion characteristics distinct from feeder vessels and many sprout tips were open-ended.CONCLUSIONS. Time-lapse in vivo corneal confocal microscopy can be used to track a temporal sequence of events in corneal angiogenesis. The technique has revealed potential roles for myeloid cells in promoting vessel sprouting in an inflammatory corneal setting. (Invest Ophthalmol Vis Sci. 2011;52:3060-3068) DOI:10.1167/iovs.10-6101