Abstract
Purpose: :
The presence of distinct populations of antigen-presenting cells (APCs) in the normal cornea has been firmly established. Their functional characterization has, thus far, mainly relied on the analysis of ex vivo studies. There remains a clear need to investigate the behavior of APCs in the context of the intact cornea in real-time. The purpose of this study was to dissect the migratory properties of these cells, by developing an intravital multi-photon microscopy (MP-IVM) model, which allows for the visualization of single cells deep within the entire cornea over time.
Methods: :
Localization and trafficking properties of APCs in live, anesthetized mice, were studied by MP-IVM during steady state and inflammation. Time-lapse MP-IVM (30-60 minute duration) of the cornea was applied to transgenic mice expressing yellow fluorescent protein (YFP) for CD11c (dendritic cells [DCs]) or enhanced green FP (EGFP) for MHC class II (mature DCs) at different time-points. High-performance 4D imaging software (Volocity) was used to create 3D movies, track and quantitate cell movement.
Results: :
MP-IVM studies of the full-thickness normal cornea demonstrated that DCs were sparsely distributed centrally and more dense in the periphery (p<0.01). Epithelial DCs had dendrites, with unusual terminal bulbs, extending anteriorly into the surface epithelium. Epithelial and stromal APCs were clearly distinguished with aid of second harmonic generation. MP-IVM demonstrated APCs with continuous sampling motions in steady state, while APCs generally did not migrate laterally. During inflammation, increased numbers of APCs were demonstrated, exhibiting extreme morphological changes. An increase in lateral and vertical migration (p<0.01), increased velocity (p<0.01), track length (p<0.001), and meandering index (total displacement/path length, p<0.05) were shown in inflammation, particularly in stromal subpopulations. Interestingly, MHC-II mature APCs were more motile than steady-state APCs, interacting with each other.
Conclusions: :
These studies are the first to demonstrate long-term migratory kinetics of corneal APCs in steady state and inflammation through high-resolution intravital multi-photon microscopy. Our model of high-resolution corneal MP-IVM is a powerful tool to study the migration and cell interactions of APCs and other immune cells in physiological settings and disease models.
Keywords: inflammation • imaging/image analysis: non-clinical • cornea: basic science