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K. T. Galster, K. Garman, D. Choi, S. R. Planck, J. T. Rosenbaum; Inhibition of Neutrophil Migration in the Murine Eye Revealed by Intravital Microscopy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2648.
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Injection of endotoxin (LPS) into a murine eye induces a local neutrophil infiltration. In this study we sought to determine if intravital time-lapse fluorescence videomicroscopy would reveal alterations in the extravascular movement of these neutrophils resulting from treatment with two antagonists: cytochalasin D which should inhibit actin-based movement and pertussis toxin which should interfere with chemokine signaling via G-protein coupled receptors.
Female, 5-6 week old BALB/c mice were injected intravitreally with LPS (E. coli 055:B5). Rhodamine (0.27 mg) was injected i.p. 10 min prior to each video recording. Initial videos were recorded for 1 hr starting 4 hrs post LPS treatment. For the cytochalasin D experiments, mice received 4 µg LPS at 0 time and 101 ng cytochalasin D at 5.5 hrs (2 µl intravitreally, n=3). Video recordings were initiated immediately after treatment, and cell migration was tracked for 1 to 2 hours. For the pertussis toxin experiments, mice received 250 ng LPS at 0 time and 160 ng Bordetella pertussis toxin at 5.5 hrs. (200 µl i.v., n=2); cell tracking started 1 hr later. After image stabilization to compensate for tissue movement, at least 8 cells were randomly chosen by masked observers in each video and their XY coordinates determined for each video frame. A mixed effects statistical model was used to test the differences in speeds between two groups of each data set while mice were treated as a random factor.
Locally injected cytochalasin D, markedly reduced cell migration (p=0.003, control 9.5±1.8 µm/min, cytochalasin D 4.5±0.6 µm/min) but did not kill cells as evidenced by continued movement. Surprisingly pertussis toxin had no substantial effect on migration although the variance of velocity may be increased.
Intravital time-lapse videomicroscopy is feasible for recording the movement of neutrophils in extravascular iris tissue and analysis of these recording is capable of revealing differences in migratory behavior due to the action of pharmacologic agents. The minimal effect of pertussis toxin requires further study to determine if it failed to block G-protein coupled chemokine receptor signaling or if alternative pathways were active.
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