title: Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Sachi
authors.initials: S
authors.lastname: Kojima
authors.affiliation: Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Inoue
authors.affiliation: Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan 3WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan 4Japan Science and Technology Agency (JST) and Core Re.
authors.forename: Junichi
authors.initials: J
authors.lastname: Kikuta
authors.affiliation: Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan 5Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Furuya
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Koga
authors.affiliation: Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Ueta
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan 3WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan 4Japan Science and Technology Agency (JST) and Core Re.
authors.forename: Masaru
authors.initials: M
authors.lastname: Ishii
authors.affiliation: Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Tanihara
mesh_terms: D000818:Animals; D002465:Cell Movement; D003228:Conjunctiva / Q000473:pathology / Q000601:surgery*; D004195:Disease Models, Animal; D005131:Eye Injuries / Q000276:immunology* / Q000473:pathology; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000494:pharmacology; D021621:Imaging, Three-Dimensional; D007111:Immunity, Cellular; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D011184:Postoperative Period; D014822:Vitreous Body / Q000276:immunology / Q000473:pathology*
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins