Temporal changes in microvessel leakiness during wound healing discriminated byin vivofluorescence recovery after photobleaching

Non-technical summary Wound closure depends on vascular ingrowth into the injured area and this process has traditionally been studied in tissue sections of animals ex vivo. Following the creation of a wound within a surgically implanted dorsal window chamber, we captured micro- scopicimagesoftheingrowingvasculatureonsuccessivedaysafterinjury.Usingacombinationof surgical,confocalmicroscopicandmathematicaltechniqueswequantifiedthefluxofplasmainto and around vessels including: newly formed vessel sprouts, nascent flowing vascular segments and pre-existing vessels within the same wound. From these analyses we are able to discriminate: (1) vessels with differing maturity, (2) that vascular sprouts get progressively less leaky and (3) TNP-470 (an anti-angiogenic agent), reduces leakiness in sprouts with co-incident secondary effects on pre-existing vessels. These techniques can be used to assess both functional maturity and the effects of therapeutics on the vasculature of healing wounds. Abstract 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) preparationsinmice.Timetorecoveryofhalf-peakfluorescenceintensity(t1/2)withinindividual 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, particularlyBEVs, was greater than that of pre-existing vessels at days5-7 after injury (P