Download Use of Intravital Multi-Photon Microscopy to Study In Vivo Migratory

Use of Intravital Multi-Photon Microscopy to
Study In Vivo Migratory Kinetics of Corneal
Bone Marrow-Derived Cells
Pedram Hamrah, M.D., Dimosthenis Mantopoulos, MD;
Lixin Zheng, MD; Ulrich von Andrian, MD, PhD
Massachusetts Eye & Ear Infirmary, Department of Ophthalmology & Immune
Disease Institute, Harvard Medical School
Financial Disclosure: The authors have no financial disclosure related to this project
Support: NEI K12-EY016335, New England Corneal Transplant Research Fund, Falk
Medical Research Trust
Antigen-presenting Cells
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Sentinels of the immune system
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Dendritic cells, macrophages and B cells
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Dendritic Cells and macrophages are the
professional antigen-presenting cells
(APC) of the cornea
Implicated in corneal transplantation and
allergic immunity, microbial keratitis, and
dry eye disease
Corneal, unlike limbal, APC are universally
MHC class II-negative and immature in the epithelium and stroma
Limbus
Green = Class II
(Maturation marker)
Limbus
Red = CD45
(Bone marrow marker)
Green = Class II
Periphery
Center
Red = CD11c
(DC marker)
Red = CD11c
Green = CD80
Green = CD80
(B7 costimulatory
marker)
Hamrah et al., Novel Characterization of MHC class II-negative Population of Resident Corneal Langerhans
cell-type Dendritic Cells. Invest Ophthalmol Vis Sci, 2002; 43:639-646
Hamrah et al., The Corneal Stroma is Endowed with Significant Numbers of Resident Dendritic Cells.
Invest Ophthalmol Vis Sci, 2003; 44:581-589
Hamrah and Dana, Immune homeostasis of the eye: Antigen Presenting Cells in the Eye and Ocular
Surface. Encyclopedia of the Eye. Elsevier. In press
Advantages of Multi-Photon
Microscopy

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Deeper imaging into scattering specimens
Reduced out of plane photobleaching and
photodamage in optically thick specimens
Access to nonlinear signals other than
fluorescence such as second harmonic
scattering
Purpose and Methods
The purpose of this study was to dissect
migratory properties of antigen presenting cells
by novel multi-photon intravital microscopy.
Multi-photon intravital microscopy (MPM) of the
cornea was applied to investigate localization
and trafficking properties of corneal APCs in
transgenic mice in steady state and inflammation
in vivo.
CD11c-eYFP
MHC class II-eGFP
Day 5 Inflammation
MHC class II-eGFP
Day 5 inflammation
Normal
Inflamed
Results



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Intravital MPM studies of the normal cornea demonstrated
that APCs were sparsely distributed centrally and more
dense in the periphery
Epithelial and stromal APCs were distinguished by second
harmonic generation that visualizes stromal collagen
While APCs demonstrated continuous sampling motions in
steady state, cells generally did not migrate laterally
During inflammation, increased numbers of APCs were
demonstrated, exhibiting extreme morphological changes
An increase in lateral and vertical migration was shown
particularly in stromal subpopulations
Conclusions


Our 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.
Collectively, these models allow for dissecting molecular
regulation of APC recruitment to, and migration in the
cornea.