Temporal changes in microvessel leakiness during wound healing discriminated by in vivo fluorescence recovery after photobleaching

Regeneration of injured tissue is a dynamic process, critically dependent on the formation of new blood vessels and restructuring of the nascent plexus. Endothelial barrier function, a functional correlate of vascular restructuring and maturation, was quantified via intravital microscopic analysis of 150 kDa FITC-dextran-perfused blood vessels within discrete wounds created in the panniculus carnosus (PC) muscle of dorsal skinfold chamber (DSC) preparations in mice. Time to recovery of half-peak fluorescence intensity (t(1/2)) within individual vessel segments in three functional regions of the wound (pre-existing vessels, angiogenic plexus and blind-ended vessels (BEVs)) was quantified using in vivo fluorescence recovery after photo-bleaching (FRAP) and linear regression analysis of recovery profiles. Plasma flux across the walls of new vessel segments, particularly BEVs, was greater than that of pre-existing vessels at days 5-7 after injury (P < 0.05). TNP-470 reduced the permeability of BEVs at the leading edge of the advancing vascular plexus as measured by the decrease in luminal t(1/2) (P < 0.05), confirming the utility of FRAP as a quantitative measure of endothelial barrier function. Furthermore, these data are suggestive of a role for TNP-470 in selection for less leaky vascular segments within healing wounds. Increased FITC-dextran leakage was observed from pre-existing vessels after treatment with TNP-470 (P < 0.05), consistent with induction of transient vascular damage, although the significance of this finding is unclear. Using in vivo FRAP this study demonstrates the relationship between temporal changes in microvascular macromolecular flux and the morphology of maturing vascular segments. This combination of techniques may be useful to assess the therapeutic potential of angiogenic agents in restoring pre-injury levels of endothelial barrier function, following the establishment of a functional vascular plexus such as in models of wounding or tumour development.