Intravital Imaging of the Cellular Dynamics of LysM-Positive Cells in a Corneal Suture Mouse Model

Abstract

Purpose: Intravital imaging is a technique used to analyze cellular and molecular dynamics stereoscopically and time-dependently in an in vivo animal model. In LysM-eGFP mice [gene-targeted mice expressing enhanced green fluorescent protein (EGFP) under the control of the endogenous lysozyme M promoter], myeloid granulocyte cells are labeled with GFP in vivo, and the resulting endogenous neutrophils are brightly labeled. The purpose of this present study was to analyze the dynamics of LysM-positive granulocytes (neutrophils) in vivo in a LysM-eGFP corneal suture mouse model using intravital imaging.

Methods: A 1-mm 10-1 nylon suture was surgically inserted through all layers in the center of the cornea of a LysM-eGFP mouse. Dynamics of the LysM-positive granulocytes were then analyzed in vivo using intravital imaging.

Results: In the unsutured normal cornea (0 hours) of the LysM-eGFP mouse model, LysM-positive granulocytes were present in the corneal stroma of only the limbal region. At 2 hours after insertion of the corneal suture, LysM-positive granulocytes migrated from the limbal capillary loop to the cornea and conjunctiva, and reached the corneal suture at 6 hours after insertion. The number of LysM-positive granulocytes then increased and peaked at 48 hours after insertion of the suture. At 72 hours after insertion, infiltration of the LysM-positive granulocytes continued, yet decreased.

Conclusions: The findings of this study show that neutrophils migrated from the limbal capillary loop and infiltrated to the corneal suture. Through the use of intravital imaging, the cellular dynamics that underlie various physiological and pathological conditions of the cornea were able to be elucidated.

Keywords: 557 inflammation • 551 imaging/image analysis: non-clinical • 484 cornea: stroma and keratocytes