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Title: Longitudinal in vivo characaterization of the role of angiogenesis during wound healing
Author: Machado, Maria João Coelho
ISNI:       0000 0004 2720 4484
Awarding Body: University of Ulster
Current Institution: Ulster University
Date of Award: 2010
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Transforming chronic wounds into wounds that close by secondary intention (through the formation of normal granulation tissue) is one of the goals of manipulating wound healing induced angiogenesis. In this thesis, the contribution of new blood vessel formation in the process of closure of a small heat induced wound in the panniculus carnosus is examined. Wound healing in skeletal muscle is a temporally restricted process. A detailed ultrastructural analysis of individual burn wounds in the panniculus carnosus revealed that by day 3 after injury, inflammatory polymorphonuclear cells were the predominant cell type. Angiogenic sprouts were apparent in histological sections from days 5 to 7 post injury, when numerous fibroblasts and regenerating myofibres were also observed. Progressive maturation of the myotubes took place from day 9 post-wounding onwards but, persistent necrosis and fibrosis in BALB/c mice was still apparent by day 15 post-injury. Angiogenesis is a highly dynamic process, in which plexus geometry and blood vessel permeability vary both temporally and spatially. Thus, intravital microscopy was used to image wound revascularization within the dorsal skinfold chamber in situ. Wound area was measured 3, 6 and 9 days after injury and stereological principles used to systematically measure vascular sprouting and functional vessel density at fixed 250~m distance increments towards the wound centre. The wounded panniculus carnosus began to revascularise by day 3, when vasodilation of the surviving vessel segments was noted even in more distal areas of the plexus. The steepest decline in wound area occurred between days 3 and 6, when numerous blind-ended vessels, in addition to tortuous and disorganized newly developed vascular plexi were observed. Complete wound closure did not occur by day 9 but a more organized, albeit less dense, plexus spanned most of the previous avascular area. In blind-ending vessels (BEVs), which develop at the leading edge of the growing vascular plexus, luminal content correlated with internal surface area, as described by our group (Guerreiro-Lucas et al., 2008). Additionally, in situ Fluorescence Recovery after Photobleaching (FRAP) in three different functional areas of the remodelling plexus (i.e., BEV s, angiogenic plexus and pre-existing vessels), quantitated the £lux of £Iuorescently labelled plasma into the vessels and across the vessel wall, highlighting different levels of maturity in these vessel types. Recovery of fluorescence intensity in the interstitial space adjacent to plasma-filled BEV s was significantly slower than close to £lowing vessels. Luminal recovery became faster from days 5 to 7 post injury. In mice bearing skeletal muscle wounds, administration of a potent angio- inhibitory agent (TNP-470) delayed wound closure and microvascular ingrowth into healing wounds. The diameter of pre-existing vessels and leakage of FITC- dextran increased in animals treated with TNP-470. The combined results from this study reveal that sprouting was inhibited but the newly-formed vessels were more closely aligned with the axis of myofibres, leading to a "normalized" vasculature. The coupling of in situ temporal imaging of angiogenesis during wound healing with stereological analysis of vascular morphology represents unique longitudinal data that can be used to inform mathematical models of wound healing-induced angiogenesis. Furthermore, perturbing the development of these vascular plexi (e.g. with TNP-470), provides a method of experimental validation of the modelling predictions arising from in silica simulations and establishes a new experimental paradigm.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available