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Abstract #66690 Published in IGR 17-4
Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy
Kojima S; Inoue T; Kikuta J; Furuya M; Koga A; Fujimoto T; Ueta M; Kinoshita S; Ishii M; Tanihara HInvestigative Ophthalmology and Visual Science 2016; 57: 1207-1212
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.
Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan 2Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.
Full articleClassification:
5.1 Rodent (Part of: 5 Experimental glaucoma; animal models)
12.8.2 With tube implant or other drainage devices (Part of: 12 Surgical treatment > 12.8 Filtering surgery)
