April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Visualization of Corneal Antigen-Presenting Cell Migration by Multi-Photon Intravital Microscopy
Author Affiliations & Notes
  • P. Hamrah
    Cornea/Ophthalmology, Harvard Med Sch/MA Eye Ear Infirm, Boston, Massachusetts
    Immune Disease Institute/Harvard Medical School, Boston, Massachusetts
  • D. Mantopoulos
    Cornea/Ophthalmology, Harvard Med Sch/MA Eye Ear Infirm, Boston, Massachusetts
  • A. Turhan
    Cornea/Ophthalmology, Harvard Med Sch/MA Eye Ear Infirm, Boston, Massachusetts
  • L. Zheng
    Immune Disease Institute/Harvard Medical School, Boston, Massachusetts
  • S. Massberg
    Immune Disease Institute/Harvard Medical School, Boston, Massachusetts
  • U. H. von Andrian
    Immune Disease Institute/Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  P. Hamrah, None; D. Mantopoulos, None; A. Turhan, None; L. Zheng, None; S. Massberg, None; U.H. von Andrian, None.
  • Footnotes
    Support  NEI K12-EY016335, New England Corneal Transplant Research Fund, Falk Medical Research Trust
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3436. doi:
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      P. Hamrah, D. Mantopoulos, A. Turhan, L. Zheng, S. Massberg, U. H. von Andrian; Visualization of Corneal Antigen-Presenting Cell Migration by Multi-Photon Intravital Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3436.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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