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E. J. Lee, G. D. Scott, J. T. Rosenbaum, S. R. Planck; Inhibition of Neutrophil Migration in the Injured Murine Cornea by Mmp-2/9 Blockade. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2388.
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Matrix metalloproteinases-2 and -9 (gelatinases A and B, MMP-2/9) are produced by leukocytes to degrade extracellular matrix components and may facilitate cell migration in the inflamed cornea. We examined the role of MMP-2/9 in neutrophil trafficking using a synthetic inhibitor or the antibiotic doxycycline, which also inhibits MMP activity and synthesis.
Mice expressing enhanced green fluorescent protein (eGFP) in the lysozyme M locus were used to visualize neutrophils following silver nitrate injury to the central cornea. At 5 h after injury, eyes received: 1) PBS, 2) MMP-2/9 inhibitor III (MMPi, 6 µg in 6 µl)(Calbiochem), or 3) doxycycline hyclate (60 µg in 6 µl). Doses were split between temporal subconjunctival and central corneal stroma injections.Corneas were dissected for ex vivo examination 1 h post-treatment to determine eGFP+ cell density in sectors adjacent to and opposite the injection site (n=4/group). Cell migration was recorded by in vivo videomicroscopy just before and at 1 h after treatment. Randomly selected cells were tracked and analyzed.
Asymmetric distribution of neutrophils in MMPi- and doxycycline-treated, but not PBS-treated, corneas suggests a local effect on neutrophil migration. In the corneal sector adjacent to the subconjunctival injection there were significantly fewer cells in doxycycline-treated corneas in both the periphery (within 100 µm internal to limbal vessel)(p=0.047) and mid-periphery (~600 µm internal to limbal vessel)(p=0.048) when compared to PBS-treated controls. There was a similar decrease in cells in MMPi-treated corneas in the mid-periphery (p=0.048), but not in the periphery (p=0.886). Cells near the inhibitor injection site exhibited decreased speed compared to PBS controls. Before treatment, eGFP+ cells in the cornea (113 cells, 12 corneas) traveled at a lateral speed of 6.6±0.4 µm/min (median±SEM). Treatment with MMPi reduced speeds to 3.6±0.9 µm/min (p=0.019 vs. pre-treatment) and with doxycycline to 2.4±0.9 µm/min (p=0.002) compared to 7.9±1.0 µm/min with PBS (p=0.731 vs. pre-treatment).
Treatment with either MMP-2/9 inhibitor or doxycycline reduced neutrophil migration through tissue stroma. Additionally, doxycycline treatment affected neutrophil entry into the corneal periphery.
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