Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy.
Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy.
复制标题
使用多光子显微镜观察结膜手术后的活体免疫细胞动态。
DOI：
10.1167/iovs.15-18507
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发表时间：
2016
影响因子：
4.4
通讯作者：
Tanihara H.
中科院分区：
文献类型：
--
作者：
Kojima S;Inoue T;Kikuta J;Furuya M;Koga A;Fujimoto T;Ueta M;Kinoshita S;Ishii M;Tanihara H.
目的：可视化活体免疫细胞的动态在结膜下组织在伤口愈合过程中，使用多光子microscopic.Methods：基因靶向小鼠表达增强绿色荧光蛋白的内源性溶菌酶M启动子（LysM-eGFP小鼠）的控制下，用异氟烷麻醉，并通过10-0尼龙结膜缝合损伤。通过静脉内注射70 kDa罗丹明缀合的葡聚糖使血管可视化。使用多光子显微镜，在损伤前20分钟和损伤后0.5、3、6和72小时每分钟获取结膜下组织的三维图像。原始图像数据进行处理的四维图像和分析的数量和速度的LysM-eGFP-阳性cells using Imaris software.Results：活体LysM-eGFP-阳性细胞和红色标记的血管被成功地可视化使用多光子显微镜。结膜和巩膜胶原纤维被检测为二次谐波产生信号，其被着色为蓝色。与无损伤的小鼠相比，结膜手术后结膜下组织中LysM-eGFP阳性细胞的数量以时间依赖性的方式增加。结膜下组织细胞运动速度在术后3 h显著增加（5.9±3.2 μm/min; P< 0.0001），并持续到伤后72 h（5.9±3.3 μm/min）。这项技术可能是一个有用的工具，以表征各种眼损伤后，如青光眼手术的伤口愈合过程的分子机制。
Purpose: To visualize intravital immune cell dynamics in the subconjunctival tissue during the wound-healing process using multiphoton microscopy.Methods: Gene-targeted mice expressing enhanced green fluorescent protein under the control of the endogenous lysozyme M promoter (LysM-eGFP mice) were anesthetized with isoflurane, and injured by a 10-0 nylon conjunctival suture. Vessels were visualized by intravenous injection of 70 kDa rhodamine-conjugated dextran. Using a multiphoton microscope, the three-dimensional images of the subconjunctival tissue were acquired every minute for 20 minutes before and 0.5, 3, 6, and 72 hours after injury. Raw imaging data were processed for four-dimensional images and analyzed for the number and the velocity of the LysM-eGFP–positive cells using Imaris software.Results: The intravital LysM-eGFP–positive cells and the red-labeled vessels were successfully visualized using a multiphoton microscope. The conjunctival and scleral collagen fibers were detected as secondary harmonic generation signals, which were colored blue. Compared with mice without injury, the number of LysM-eGFP–positive cells in the subconjunctival tissue after conjunctival surgery increased in a time-dependent manner. The cell velocities significantly increased until 3 hours after surgery (5.9±3.2 μm/min; P< 0.0001) and the elevated level was sustained until 72 hours after injury (5.9±3.3 μm/min).Conclusion: This is the first report to visualize and evaluate intravital cellular dynamics during inflammation in the subconjunctival tissue using multiphoton microscopy. This technique may be a useful tool to characterize the molecular mechanisms of the wound-healing process after various ocular injuries, such as glaucoma surgery.