Intravital microscopy of corneal inflammation after burn injury

Abstract

Abstract: : Purpose: In response to injury or infection, inflammatory cells migrate into the cornea. We investigated the movement of bone marrow–derived cells in the murine cornea in response to burn injury. Methods: C57BL/6 GFP chimeric mice were generated by transplantation of bone marrow cells from C57BL/6–TgN(ACTBEGFP) donors into lethally irradiated recipients and used for experiments five months later. At baseline and at 2, 5, and 8h after silver nitrate burn injury of the central cornea, GFP⁺ cells in the corneal periphery and at the wound edge were recorded by timelapse fluorescence intravital microscopy (n=10). Video sequences (3 frames/min for 30 min) were analyzed for the density and movement of GFP⁺ cells. Results: The entire cornea was populated with GFP⁺ cells, the density of which was lowest in the center (36 cells/mm²) and increased toward the periphery. All GFP⁺ cells of uninjured corneas exhibited some motion such as extension and retraction of cellular processes, but little to no migration from one location to another. These cells were generally large and dendritiform in appearance. Following injury, a sharp increase in the number of smaller round or oblong GFP⁺ cells appeared, first in the periphery and later at the wound edge. At 2h post–injury, cells in the periphery were associated with significantly increased velocity compared to cell velocity in uninjured corneas (9.4±1.0 and 5.5±0.8 µm/min, respectively, P=0.004). Interestingly, movement of GFP⁺ cells at the wound edge was similar to that in uninjured corneas (4.3±0.7 and 6.0±1.0 µm/min, respectively, P=0.781). GFP⁺ cells in the periphery and at the wound edge traveled even more rapidly at 5h (22.0±1.9 and 18.5±1.1 µm/min, respectively) and at 8h (13.8±1.6 and 18.7±1.7 µm/min, respectively). Analysis of individual cell paths indicated that the overall progression of cells from the corneal periphery toward the center was non–linear in time and space; cell migration was often punctuated by pauses and/or changes in travel direction. Conclusions: Bone marrow–derived cells recruited in response to burn injury reach the central corneal wound in a few hours, but do so in a circuitous fashion. Few, if any, bone–marrow derived cells in the central cornea at the time of injury move to the wound.

Keywords: inflammation • cornea: basic science • imaging/image analysis: non–clinical