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Sachi Kojima, Toshihiro Inoue, Junichi Kikuta, Masayuki Furuya, Ayaka Koga, Tomokazu Fujimoto, Mayumi Ueta, Shigeru Kinoshita, Masaru Ishii, Hidenobu, Tanihara; Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy. Invest. Ophthalmol. Vis. Sci. 2016;57(3):1207-1212. doi: 10.1167/iovs.15-18507.
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To visualize intravital immune cell dynamics in the subconjunctival tissue during the wound-healing process using multiphoton microscopy.
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.
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).
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.
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