title: Time-lapse in vivo imaging of corneal angiogenesis: the role of inflammatory cells in capillary sprouting.
abstract: OBJECTIVE
  To elucidate the temporal sequence of events leading to new capillary sprouting in inflammatory corneal angiogenesis.
  
  
  METHODS
  Angiogenesis was induced by corneal suture placement in Wistar rats. The inflamed region was examined by time-lapse in vivo confocal microscopy for up to 7 days. At 6 and 12 hours and 1, 2, 4, and 7 days, corneas were excised for flat mount immunofluorescence with primary antibodies for CD31, CD34, CD45, CD11b, CD11c, Ki-M2R, NG2, and α-SMA. From days 0 to 4, the in vivo extravasation and expansion characteristics of single limbal vessels were quantified.
  
  
  RESULTS
  Starting hours after induction and peaking at day 1, CD45(+)CD11b(+) myeloid cells extravasated from limbal vessels and formed endothelium-free tunnels within the stroma en route to the inflammatory stimulus. Limbal vessel diameter tripled on days 2 to 3 as vascular buds emerged and transformed into perfused capillary sprouts less than 1 day later. A subset of spindle-shaped CD11b(+) myeloid-lineage cells, but not dendritic cells or mature macrophages, appeared to directly facilitate further capillary sprout growth. These cells incorporated into vascular endothelium near the sprout tip, co-expressing endothelial marker CD31. Sprouts had perfusion characteristics distinct from feeder vessels and many sprout tips were open-ended.
  
  
  CONCLUSIONS
  Time-lapse in vivo corneal confocal microscopy can be used to track a temporal sequence of events in corneal angiogenesis. The technique has revealed potential roles for myeloid cells in promoting vessel sprouting in an inflammatory corneal setting.
authors.affiliation: Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Bourghardt Peebo
authors.forename: Per
authors.initials: P
authors.lastname: Fagerholm
authors.forename: Catharina
authors.initials: C
authors.lastname: Traneus-Röckert
authors.forename: Neil
authors.initials: N
authors.lastname: Lagali
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000941:Antigens / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002196:Capillaries / Q000473:pathology; D002452:Cell Count; D016510:Corneal Neovascularization / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D016850:Limbus Corneae / Q000098:blood supply; D008297:Male; D018613:Microscopy, Confocal; D022423:Myeloid Cells / Q000378:metabolism / Q000473:pathology*; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D059008:Time-Lapse Imaging; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000199:Actins; D000941:Antigens; D015703:Antigens, CD; D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D018836:Inflammation Mediators; D011509:Proteoglycans; D000099216:Chondroitin Sulfate Proteoglycan 4; C498862:Acta2 protein, rat