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A. J. Leedom, J. LeDue, K. Gronert; Modulation of Leukocyte Dynamics in the Cornea: Lipid Autacoid Function Revealed by in vivo Two Photon Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5684.
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© ARVO (1962-2015); The Authors (2016-present)
A hallmark of the protective bioactions of lipid autacoids is regulation of leukocyte trafficking. The dynamic activity of leukocytes in vivo during inflammation is poorly understood. Experimental approaches have been mostly limited to analyses of fixed/isolated tissues or cells, hence, we set out to develop an in vivo approach to study leukocyte behavior in the cornea using two photon microscopy.
A silk suture was placed in corneas of GFP+ mice for 7 d, triggering chronic inflammation. Mice were treated with selected protective lipid autacoids (100 ng tid) or saline for 7 d, anesthetized and imaged in vivo for 30 min using a two-photon microscope (TPM). This TPM uses a near-infrared Ti:Al2O3 laser as the excitation source. Epithelial cells were visualized by excitation of NADPH fluorescence at 740 nm while GFP+ cells were excited at 920 nm. Fluorescence was sorted into separate detectors by dichroic mirrors at 495 nm (NADPH) and 515 nm (GFP). High sensitivity GaAsP non-descanned detectors provided the high speed and signal to noise ratio necessary for in vivo imaging. Data was analyzed using software for cell tracking (Imaris). For ex vivo imaging, enucleated eyes were placed in line with the optical axis of the microscope objective.
By exciting fluorescence of GFP and NADPH sequentially at 920 and 740 nm, we located in 3D leukocytes and corneal epithelial cells, revealing an 18 fold increase in the number of GFP+ cells in the inflamed eye. This method was used in vivo to analyze leukocyte activity in space and time (velocity and position) in corneas. These experiments revealed highly dynamic activity of leukocytes in injured corneas. Topical treatment with protective lipid mediators (LXA4 and RvD1), markedly and differentially regulated this dynamic leukocyte behavior.
This study provides the first data on the dynamic in vivo behavior of leukocytes in chronically injured corneas. More importantly, it provides novel insights into to mechanisms of protective lipid autacoids and leukocyte function. In vivo TPM imaging provides a new dimension for studying the largely unexplored behavior of leukocyte.
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