title: Quantification of total and perfused blood vessels in murine skin autografts using a fluorescent double-labeling technique.
abstract: BACKGROUND
  Two theories exist regarding the revascularization of skin autografts: direct anastomosis between graft vessels and bed vessels, and ingrowth of bed vessels (angiogenesis) into the graft. This study characterizes revascularization, spatially and chronologically, in a murine skin autograft model using a double-labeling technique.
  
  
  METHODS
  Full-thickness (1 cm2) skin grafts were performed on adult male C57/Bl6 mice. After 48 hours, 60 hours, 3 days, 5 days, and 14 days (n = 3 mice per time point) terminal intracardiac perfusion with a fluorescein/dextran dye demonstrated vascular filling of graft blood vessels. Fluorescence immunohistochemistry of CD31+ endothelial cells allowed counting of total vessels and fluorescein perfusion quantification of patent vessels in the lateral graft area, central graft area, graft bed, and wound margins.
  
  
  RESULTS
  Initial filling of graft vessels was seen after 48 hours. This included vessels in the papillary dermis of the graft, and there was no significant difference in the percentage of filled vessels in the deep dermis of the graft compared with the papillary dermis of the graft. A rapid increase in vessel filling was seen between 48 and 60 hours in all areas of the graft. Vessel filling occurred mainly in the central area of the graft rather than in the lateral areas.
  
  
  CONCLUSIONS
  The short time course of vessel filling indicates that the initial onset of revascularization is attributable to early anastomoses between graft and bed vessels, mainly in the central area of the graft. These findings have implications for both autograft revascularization in a clinical setting and in the development of tissue-engineered skin substitutes.
authors.affiliation: UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Siobhán
authors.initials: S
authors.lastname: O'Ceallaigh
authors.forename: Sarah Elizabeth
authors.initials: SE
authors.lastname: Herrick
authors.forename: Joanne Elizabeth
authors.initials: JE
authors.lastname: Bluff
authors.forename: Duncan Angus
authors.initials: DA
authors.lastname: McGrouther
authors.forename: Mark W J
authors.initials: MW
authors.lastname: Ferguson
mesh_terms: D000714:Anastomosis, Surgical; D000818:Animals; D020405:Dermis / Q000098:blood supply; D004730:Endothelium, Vascular / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D018919:Neovascularization, Physiologic; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D012867:Skin / Q000098:blood supply*; D016038:Skin Transplantation / Q000502:physiology*; D013997:Time Factors; D020858:Tissue and Organ Harvesting; D014182:Transplantation, Autologous
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1