Use of Intravital Multi-Photon Microscopy to Study In Vivo Migratory Kinetics of Corneal Bone Marrow-Derived Cells

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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
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Antigen-presenting Cells

• Sentinels of the immune system
• Dendritic cells, macrophages and B cells
• 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

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Corneal, unlike limbal, APC are universally MHC
class II-negative and immature in the epithelium
and stroma
Limbus Red CD45 (Bone marrow
marker) Green Class II
Limbus Green Class II (Maturation marker)
Periphery Red CD11c (DC marker) Green CD80
(B7 costimulatory marker)
Center Red CD11c Green CD80
Hamrah et al., Novel Characterization of MHC
class II-negative Population of Resident Corneal
Langerhans cell-type Dendritic Cells. Invest
Ophthalmol Vis Sci, 2002 43639-646 Hamrah et
al., The Corneal Stroma is Endowed with
Significant Numbers of Resident Dendritic Cells.
Invest Ophthalmol Vis Sci, 2003 44581-589
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Hamrah and Dana, Immune homeostasis of the eye
Antigen Presenting Cells in the Eye and Ocular
Surface. Encyclopedia of the Eye. Elsevier. In
press
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Advantages of Multi-Photon Microscopy

• 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

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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.

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CD11c-eYFP
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MHC class II-eGFP Day 5 Inflammation
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MHC class II-eGFP Day 5 inflammation
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Normal
Inflamed
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Results

• 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

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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.