A HISTOLOGICAL ANALYSIS OF BURN WOUND PROGRESSION Raymond Thai Quan Bachelor of Biomedical Science Submitted in fulfilment of the requirements for the degree of Master of Philosophy School of Biomedical Sciences Faculty of Health Queensland University of Technology 2021 Keywords Anti-Fibrinogen, burn depth, burn marker, burn wound conversion, burn wound progression, damage marker, fibrin, formalin fixed; paraffin embedded, haematoxylin and eosin, histology, immunofluorescence, immunohistochemistry, MSB fibrin stain, pathology, porcine burn model, scald, skin, structural damage A Histological Analysis of Burn Wound Progression i Abstract Burn wound progression is the phenomenon in which burns progress in depth following the initial injury. This can cause superficial partial burns, which would normally heal within two weeks, to progress into deep-dermal partial thickness or full thickness burns, resulting in delayed healing and poorer long-term outcomes. In the literature, histological burn wound analysis involves the detection of multiple burn damage markers including collagen denaturation, cellular necrosis, cellular apoptosis, and vascular pathologies. A variety of histochemical and immunohistochemical stains have been utilised to detect these markers. In this study, Haematoxylin and eosin (H&E), Verhoeff’s Van Gieson (VVG), Gomori’s Trichrome (GT), Martius Scarlet Blue (MSB) and a fibrinogen antibody were optimised and evaluated for their ability to detect burn markers within a porcine scald burn model. H&E was effective for observing cellular necrosis, collagen denaturation and vascular pathologies. Collagen denaturation was also detected by VVG and GT with the additional detection of elastin fibres and vascular pathologies, respectively. MSB staining provided detection of collagen denaturation and vascular pathologies with the additional detection of fibrin deposition. Immunostaining for fibrinogen validated the fibrin staining in MSB. The analysis of burn wound progression was conducted by assessing a porcine burn wound model and comparing burns that progressed (n=6) within 72 hours to burns that did not progress (n=6). Sample replicates for each burn were obtained at one hour, 24 hours and 72 hours post-burn and comprised of high temperature, short duration and low temperature, long duration burns. A novel burn rubric for H&E stained tissue was developed to measure burn intensity, allowing for comprehensive analysis of the pathologies present in the entire section. Blood vessel occlusion was found to play a significant role in the progression of burns, with non-progression burns having higher intensity scores of completely occluded vessels at one hour post-burn. Increased intensity of partially occluded blood vessels was observed at 72 hours post- burn in burns that progressed. The difference in intensity of blood vessel damage between progression burns and non-progression burns demonstrates the complexity of assessing the mechanisms responsible for burn wound progression. A Histological Analysis of Burn Wound Progression ii The assessment of burns requires a multifaceted approach in determining the mechanisms involved in burn wound progression. This study developed a rubric to advance the current methods of burn assessment and found that vascular pathologies are a key marker in examining burn wounds and are of importance when assessing burn wound progression. A Histological Analysis of Burn Wound Progression iii Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii Table of Contents .................................................................................................................... iv List of Figures ......................................................................................................................... vi List of Tables .......................................................................................................................... xii List of Abbreviations ............................................................................................................. xiii Statement of Original Authorship ......................................................................................... xiv Acknowledgements ................................................................................................................ xv Chapter 1: Literature Review ............................................................................. 1 1.1 Burn Injuries .................................................................................................................. 1 1.2 Burn Wound Progression ............................................................................................... 2 1.3 Mechanisms of Burn Wound Progression and their measurement ................................ 3 1.3.1 Collagen Denaturation ............................................................................ 3 1.3.2 Cellular Necrosis .................................................................................... 5 1.3.3 Cellular Apoptosis .................................................................................. 6 1.3.4 Vascular Pathologies .............................................................................. 7 1.4 Burn Wound Model ..................................................................................................... 10 1.5 Summary and Gaps in Knowledge ............................................................................... 11 1.5.1 Summary .............................................................................................. 11 1.5.2 Gaps in Knowledge .............................................................................. 12 1.6 Thesis Outline .............................................................................................................. 13 1.7 Hypothesis and Aims ................................................................................................... 14 Chapter 2: Selection and Optimisation of Staining Methods......................... 15 2.1 Introduction .................................................................................................................. 15 2.2 Haematoxylin and Eosin .............................................................................................. 16 2.2.1 Introduction .......................................................................................... 16 2.2.2 Reagents ............................................................................................... 17 2.2.3 Initial Protocol ...................................................................................... 17 2.2.4 Optimisation ......................................................................................... 18 2.2.5 Evaluation in burned tissue .................................................................. 19 2.3 Verhoeff’s Van Gieson ................................................................................................ 21 2.3.1 Introduction .......................................................................................... 21 2.3.2 Reagents ............................................................................................... 21 2.3.3 Initial Protocol ...................................................................................... 21 2.3.4 Optimisation ......................................................................................... 22 A Histological Analysis of Burn Wound Progression iv 2.3.5 Evaluation in burned tissue .................................................................. 23 2.4 Gomori’s Trichrome .....................................................................................................25 2.4.1 Introduction .......................................................................................... 25 2.4.2 Reagents ............................................................................................... 26 2.4.3 Initial Protocol ...................................................................................... 26 2.4.4 Optimisation ......................................................................................... 26 2.4.5 Evaluation in burned tissue .................................................................. 29 2.5 Martius Scarlet Blue .....................................................................................................32 2.5.1 Introduction .......................................................................................... 32 2.5.2 Reagents ............................................................................................... 32 2.5.3 Protocol ................................................................................................ 32 2.5.4 Optimisation ......................................................................................... 33 2.5.5 Evaluation in burned tissue .................................................................. 35 2.6 Immunohistochemistry .................................................................................................37 2.6.1 Introduction .......................................................................................... 37 2.6.2 Reagents ............................................................................................... 37 2.6.3 Protocol