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.

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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.

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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

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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 ...... 38 2.6.4 Optimisation ...... 39 2.6.5 Evaluation in burned tissue ...... 41 2.7 Stain assessment ...... 42 2.8 Chapter 2 Discussion ...... 43 Chapter 3: Burn Wound Progression Analysis ...... 47 3.1 Introduction ...... 47 3.2 Burn model selection ...... 47 3.3 Burn Assessment Methodology ...... 50 3.3.1 Burn Depth ...... 50 3.3.2 Development of Burn Damage Rubric ...... 52 3.4 Results ...... 56 3.4.1 Burn Depth ...... 56 3.4.2 Burn Intensity ...... 57 3.4.3 Burn Severity Correlation ...... 63 3.5 Chapter 3 Discussion ...... 63 Chapter 4: Discussion and Conclusion ...... 69 4.1 Discussion ...... 69 4.2 Conclusion ...... 75 Bibliography ...... 77 Appendices ...... 85

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List of Figures

Figure 1.1 A) Burn depth classifications which lead to differing healing outcomes from Johnson (2). B) The different zones of a burn injury which explain their healing capacity, as described by Jackson's model (3), adapted from Johnson, 2018 (2)...... 1 Figure 1.2 Schematic of pathological processes following burn injury resulting in burn wound progression. From (52)...... 9 Figure 1.3 Burned sections data representing burn treatment group, burn conditions and timepoints. The number of burns represented in each group are shown, n equals number of sections for each group...... 13 Figure 2.1 Burned section data representing burn treatment group, burn conditions and timepoints for Chapter 2 stain optimisations. The number of burns represented in each group are shown, n represents the number of sections in each group...... 16 Figure 2.2 Optimised haematoxylin and eosin stain on normal, unburned porcine skin. A) Low magnification of general morphology of skin, clear distinction between purple epidermis and pink dermis (Scale: 200μm); B) High magnification image of cell nuclei within the skin stained dark purple within a light pink dermis (Scale: 50μm); C) Healthy hair follicle consisting of an intact internal keratin layer surrounded by healthy, intact keratinocytes (Scale: 50μm); D) Sweat glands located in deep dermis, with intact surrounding cells (Scale: 100μm); E) Small blood vessel in deep dermis (Scale 50μm); F) Small arteriole with clear lumen (Scale: 50μm) ...... 18 Figure 2.3 Reported burn induced damage markers of injury identified with haematoxylin and eosin staining of porcine skin. A) Epidermal loss of adherence from dermis. Clear separation identified between pink dermis and purple epidermal layer (Scale: 50μm); B) Flattening of dermal-epidermal junction with elongated string bean nuclei present in the basal epidermal layer, indicated with arrows (Scale: 200μm); C) Low magnification of collagen denaturation within the dermis (Scale: 50μm); D) High magnification of collagen denaturation, identified by bundles of collagen fibres and irregular fissures between collagen fibres (Scale: 200μm); E) Low magnification of inflammatory band in dermis (Scale: 50μm); F) High magnification of inflammatory band, individual dark inflammatory cells identified by arrows infiltrated within the dermis (Scale: 200μm); G) Hair follicle damage identified by spacing between the border and necrotic cells indicated by the arrow (Scale: 200μm); H) Blocked blood vessels indicated by arrows and free red blood cells found within the dermis, stained a clear bright red. (Scale: 200μm)...... 20 Figure 2.4 Verhoeff’s Van Gieson stain optimisation on normal, unburned porcine skin. Staining was optimised primarily through modifying the duration of differentiation and section thickness. Epidermis and elastin

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appearance indicated optimal differentiation duration, while collagen appearance and clarity of stain was observed to determine optimal section thickness. Optimal staining was observed by a lightly stained epidermis with visible keratinocytes, well-defined collagen fibres and clear individual elastin fibres in dermis and arterioles. The desired stain appearance is presented in H and I. Differentiation durations: A) 20s; B) 30s; C) 30s; D) 60s; E) 90s; F) 90s; G) 45s; H) 90s; I) 90s. Section thickness: A-F) 5μm; G-I) 3μm. C, F, I) Indicate internal elastic lamina of arterioles in the adjacent section. Arrows indicate elastin fibres, stained black. (Scale: 50μm) ...... 23 Figure 2.5 Burn induced injuries visualised with Verhoeff’s Van Gieson staining of burned porcine skin. Collagen changes are identified in A- D. A, B) Altered structure of dermal collagen, with inconsistent spacing between collagen fibres. C, D) Change in collagen colour from fuchsia to yellow located superficial to a band of inflammatory cells within the dermis. E) Low magnification of inflammatory band appears as grey background between collagen fibres; F) High magnification of inflammatory band, individual cell nuclei stained black are infiltrating the dermis. A, C, E) Scale: 50μm; B, D, F) Scale: 200μm. G) Elastin fibres in superficial dermis appear long and thin, similar to elastin fibres in normal skin; H) Elastin fibres in deep dermis are short and thin, appearing fragmented and clustered (Scale: 50μm). Generally, elastin fibres seem unchanged between burned and normal skin, however in some instances, elastin fibres in the deep dermis appear shortened and thicker as shown in H...... 24 Figure 2.6 Gomori Trichrome Optimisation using either the Gibson protocol (A, B, C, F) or Sigma Aldrich protocol (D, E) on normal, unburned porcine skin. To optimise the Gibson protocol, the post-fixing step was omitted or included for 60 minutes and charged slides were used. A) No post-fixing, uncharged slide, acetic acid dip; B) 60 minutes post-fixing, uncharged slide, acetic acid dip; C) 60 minutes post- fixing, charged slide, acetic acid rinse. Optimal staining as indicated in (C), with collagen fibres stained light blue, and blood vessels, muscle and other dermal structures stained red. This was achieved by using charged slides which enabled thorough acetic acid rinsing of Gomori trichrome solution (Scale: 200μm). To optimise the Sigma Aldrich protocol, the haematoxylin was differentiated, and the section was carefully handled during the acetic acid stage. D) Protocol was followed directly; E) Protocol was altered with the addition of haematoxylin differentiation and an additional acetic acid rinse. The inclusion of a haematoxylin differentiation stage allows cell nuclei to be more clearly distinguished from the surrounding structures. The addition of an acetic acid rinse allowed for a less intense collagen stain to better visualise other structures, optimal staining is not achieved due to variance in epidermal staining and fissure formation within the dermal tissue (Scale: 200μm). F) High magnification of collagen fibres with optimal staining using Gibson protocol, individual fibres clearly stained blue with fragments of red staining (Scale 50μm)...... 28

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Figure 2.7 Burn induced injuries visualised with Gomori’s Trichrome staining using optimised Gibson protocol on burned porcine skin. A) Low magnification of collagen within the dermis (Scale 50μm); B) High magnification of collagen, with damage indicated by darker blue stained bundled fibres (Scale: 200μm); C) Epidermal loss of adherence from the basal layer observed within red stained epidermis (Scale: 200μm) ; D) Clustering of red blood cells within a blood vessel, appearing as a blocked vessel indicated by the arrow (Scale: 200μm); E) Low magnification of inflammatory band located within the dermis (Scale: 50μm); F) High magnification of inflammatory band, showing individual inflammatory cell infiltration into the dermis surrounded by red background staining (Scale: 200μm); G) Change of collagen colour from blue to red, located superficial to an inflammatory band, due to altered, burned collagen structure (Scale: 50μm); H) Web-like structures located in the deep dermis stained red, their appearance is consistent with neutrophil extracellular traps, previously identified in literature (Scale: 200μm)...... 30 Figure 2.8 Optimising Martius Scarlet Blue stain in porcine skin, compared to porcine placenta. A) Porcine placenta positive control (Scale: 200μm) Optimal staining is shown with yellow red blood cells, red fibrin and blue collagen.; B) Burned skin exhibiting red fibrin deposits, indicated by the arrow (Scale: 50μm); C) Unburned skin, superficial dermis; D) Unburned skin, deep dermis (Scale: 200μm). Optimal staining within the skin can be observed as individual red blood cells within the vessels, clear collagen fibres and fibrin deposits adjacent to blood vessels...... 34 Figure 2.9 Burn induced injuries visualised with Martius Scarlet Blue staining. A, B) Change in collagen structure indicated by irregular spacing between collagen fibres. Arrows indicate free red blood cells within the dermis, stained yellow (Scale: 50μm); C) Collagen colour changed from blue to bright red (arrows) superficial to inflammatory cells; D) Completely blocked blood vessel indicated by an arrow, with red fibrin deposit staining adjacent to the vessel (Scale: 200μm); E) Low magnification image of inflammatory band (Scale: 50μm); F) High magnification of inflammatory band, showing individual inflammatory cell nuclei stained black and arrows indicating red fibrin deposits in the dermis (Scale: 200μm); G,H) Web-like structures located in the deep dermis and adipose tissue, stained red. Webs are consistent in appearance to neutrophil extracellular traps reported in literature (69, 74) (Scale: 200μm)...... 36 Figure 2.10 Fibrinogen immunofluorescence staining optimisation. Porcine skin and porcine placenta controls were stained with a rabbit anti- human fibrinogen primary antibody (ab34269, Abcam, Cambridge, United Kingdom) and a goat anti-rabbit secondary antibody tagged with Alexa Fluor 488 (A11008, Invitrogen, Carlsbad, California, USA) to identify fibrin and fibrinogen deposits. A) No antigen retrieval, primary antibody (1:100) overnight, secondary antibody (1:500) for 1 hour; B) Antigen retrieval using 0.05% trypsin, primary (1:100) overnight, secondary (1:500) 1 hour; C) Heat antigen retrieval

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by microwaving for 20 minutes, primary (1:100) overnight, secondary (1:500) 1 hour; D) Heat antigen retrieval, primary (1:100) 1 hour, secondary (1:500) 1 hour; E) Heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour; F) Heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour; G, H) Porcine placenta positive controls, heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour. Arrows indicate positive fibrinogen/fibrin staining in the sections (Scale: 50μm)...... 40 Figure 3.1 Burn depth information data for the sections chosen for this study, based on data previously calculated by Christine Andrews (68). Each line represents a specific group chosen for analysis, including progression (pink) and non-progression (green) burns, and burns which are of low temperature – long duration (circles or triangles) and burns which are high temperature – short duration (squares or diamonds), dotted lines represent criteria thresholds for each group, progression (<75% at 1 hour and ൒75% at 72 hours and non- progression (between 25% and 75% over 72 hours). A) Combined burn depth, points are represented as the mean ± standard deviation, with n=3 replicates for each condition; B-C) Individual burn data for progression burns; D-E) Individual burn data for non-progression burns...... 48 Figure 3.2 Burned sections data representing burn treatment group, burn conditions and timepoints. The number of burns represented in each group are shown, n equals number of sections for each group...... 49 Figure 3.3 Screen captures of the burn depth area measurements, calculated in CaseViewer. Area of structural tissue injury is defined by the green line; the total area of the section is defined by the blue line. Burn depth was calculated as injury area/total area. Scale: 200μm...... 51 Figure 3.4 Screen captures of two techniques utilised to draw quadrants in burn intensity assessments. Lines were measured and drawn at depths 25, 50, and 75%, forming four quadrants across the section. A) Quadrant lines are straight horizontal lines, depths measured at left and right borders; B) Quadrant lines are made up of two lines joined at the median, depths measured at left border, right border, and centre line...... 54 Figure 3.5 Burn depth results for burned sections analysed in Chapter 3. Non- progression burns (green) and progression burns (pink) plotted on as mean ± standard deviation across 1 hour, 24 hours and 72 hours post- burn with 6 replicates for each point. Dotted lines represent criteria thresholds for each group, progression (<75% at 1 hour and ൒75% at 72 hours and non-progression (between 25% and 75% over 72 hours)...... 56 Figure 3.6 Intensity scores for epidermal burn damage markers, of non- progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total epidermal damage, calculated as the average of all the individual marker intensity scores (string bean nuclei, shrunken nuclei and loss of adherence) at each timepoint; B) Intensity score for elongated string bean nuclei in the epidermis; C) Intensity score for shrunken nuclei; D) Intensity score for loss of adherence of the epidermis from the

A Histological Analysis of Burn Wound Progression ix

dermis. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each timepoint. Statistical analysis demonstrated no statistically significant differences in epidermal burn damage markers...... 58 Figure 3.7 Intensity scores for blood vessel burn damage markers of non- progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total blood vessel damage, calculated as the average of all the individual marker intensity scores (partially blocked vessels, blocked vessels, shrunken nuclei and structural damage at each time point; B) Intensity score for partially blocked vessels; C) Intensity score for blocked vessels; D) Intensity score for shrunken nuclei; D) Intensity score for structural damage. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant differences assessed using Mann-Whitney test between timepoints and treatment group indicated as * = p<0.05, ** = p<0.01...... 59 Figure 3.8 Intensity scores for hair follicle burn damage markers, of non- progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total hair follicle damage, calculated as the average of all the individual marker intensity scores (elongated string bean nuclei, structural damage and shrunken nuclei) at each timepoint; B) Intensity score for elongated string bean nuclei; C) Intensity score for structural damage; D) Intensity score for shrunken nuclei. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections (for all except 1- hour NP, 1-hour P and 72-hour P, where n=5) at each time point. Significant differences assessed using Mann-Whitney test between treatment groups indicated as * = p<0.05...... 60 Figure 3.9 Intensity scores for glandular burn damage markers, of non- progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total glandular damage, calculated as the average of all the individual marker intensity scores (elongated string bean nuclei, structural damage and shrunken nuclei) at each timepoint; B) Intensity score for elongated string bean nuclei; C) Intensity score for structural damage; D) Intensity score for shrunken nuclei. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant differences assessed using Mann-Whitney test between treatment groups indicated as * = p<0.05, ** = p<0.01...... 61 Figure 3.10 Intensity scores of red blood cell presence in dermis as burn damage markers, non-progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Overall red blood cells in dermis, calculated as the average of all the forms of extravasated red blood cell intensity scores (nearby blood vessels and free red blood cells) at each timepoint; B) Red blood cells nearby blood vessels; C) Intensity score for free red blood cells in dermis. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant

A Histological Analysis of Burn Wound Progression x

differences assessed using Mann-Whitney test between timepoints indicated as ** = p<0.01...... 62 Figure 3.11 Correlation data of progression burns at 24 hours between depth score and total blood vessel damage intensity scores. Two-tailed nonparametric Spearman correlation was conducted with 95% confidence interval. * = p<0.05...... 63

A Histological Analysis of Burn Wound Progression xi

List of Tables

Table 1.1 Stains described in literature to identify thermally induced collagen denaturation...... 4 Table 1.2 Methods to identify thermally-induced cellular necrosis...... 6 Table 1.3: Methods to identify thermally induced cellular apoptosis in literature ...... 7 Table 1.4 Stains to identify thermally induced vascular pathologies in skin...... 10 Table 2.1 Burn condition information for porcine scald burns utilised in Chapter 2 for burn marker identification and stain evaluation. Burns included samples collected at one hour, 24 hours and 72 hours post- burn. LT+LD: low temperature – long duration, HT+SD: high temperature – short duration...... 16 Table 2.2 Gomori’s trichrome initial protocols ...... 26 Table 3.1 Burns selection information detailing animal number, burn conditions (temperature and duration) and progression criteria. LT+LD: low temperature/long duration, HT+SD: high temperature/short duration...... 50 Table 3.2 Burn injury rubric utilised to identify features and burn markers in burn intensity measurement...... 53 Table 3.3 Burn intensity scoring quadrants. Depth measured from superficial to deep epidermis, with quadrants indicating damage associated to burn classifications...... 54

A Histological Analysis of Burn Wound Progression xii

List of Abbreviations

Abbreviation Description BRANZ Burns Registry of Australia and New Zealand BV Blood vessel BWP Burn wound progression CC3a Cleaved Caspase 3a DDPT Deep dermal partial thickness et al. et alia (Latin for “and others”) EVG Elastic Van Gieson FFPE Formalin fixed; paraffin embedded GT Gomori’s Trichrome H&E Haematoxylin and Eosin HMGB1 High mobility group box 1 HPS Haematoxylin Phloxine Saffron Hx Haematoxylin IF Immunofluorescence IHC Immunohistochemistry LDH Lactate dehydrogenase LDI Laser doppler imaging MSB Martius Scarlet Blue MT Masson’s Trichrome NET Neutrophil extracellular trap RBC Red blood cell SPT Superficial partial thickness TdT Terminal deoxynucleotidyl transferase TUNEL TdT dUTP nick end labelling VG Van Gieson VVG Verhoeff’s Van Gieson RBC Red blood cell PTA Phosphotungstic acid

A Histological Analysis of Burn Wound Progression xiii Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

Signature: QUT Verified Signature

Date: 26-Apr-2021

A Histological Analysis of Burn Wound Progression xiv

Acknowledgements

Firstly, I would like to acknowledge and thank my supervisory team, for leading me and supporting me through a challenging two years. When first embarking on this journey, I did not know what to expect however, with your guidance and supervision I have learnt and accomplished so much. I would like to start by expressing my gratitude and appreciation to the Australian Government Research Training Program for granting me a scholarship to support myself personally throughout my MPhil.

I would like to acknowledge and thank my principal supervisor, Associate Professor Leila Cuttle. This entire project would not have been possible without your endless encouragement, guidance, and motivation throughout the past two years. From first inviting me to volunteer at the Brisbane ANZBA Conference in 2018, before I had even started, you ignited my passion for burns and research. You have provided me with countless opportunities I would have never experienced, and I cannot thank you enough. As we know, the year of 2020 was especially challenging, but you continued your support through countless Zoom meetings, phone calls and lengthy emails. It has been an absolute pleasure to be your student.

To Professor Damien Harkin, your impact on this project started before it even began! Without seeing your enthusiasm for histology during my undergrad, I would not have developed the curiosity and interest for histology I have today. Your guidance and insight during this project were invaluable and your skill in histological analysis has been immensely helpful. I appreciate all the continuous support in editing my drafts and in aiding me throughout my histological staining.

To Doctor Christine Andrews, I cannot thank you enough for your support and involvement during this project. Of course, this work could not have been possible without your initial studies. Your guidance through countless samples and depth data was immensely helpful in the fruition of this thesis. Your expertise and enthusiasm in burn depth assessment could not have been more valuable and I appreciate all your insights throughout this journey.

To the QUT staff at the IHBI Histology Facility, thank you for your support and aid in sectioning and especially imaging during the lockdown. Special thanks to Miranda Kay for the sectioning of my many, complicated samples. To Chris Cazier,

A Histological Analysis of Burn Wound Progression xv

thank you for giving up your time in aliquoting staining solutions and allowing me to use your lab and instruments at any time, not to mention my countless emails about stain details! To Zoe Dettrick from QUT Research Methods Group, I thank you for your cooperation in discussing my complicated project over Zoom and aiding me in any statistical analyses.

Finally, I would like to acknowledge and thank the people around me. Thank you to the Centre for Children’s Burns and Trauma Research (CCBTR) team, ANZBA 2019 was a blast and your enthusiasm in research is unmatched. To the Centre for Children’s Health Research (CCHR) team on Level 8, you made me look forward to lunches each day, and helped me keep my sanity with countless cakes throughout the years. To my friends and family, thank you for dealing with my ramblings of optimal burns first aid, correcting burn depth classifications, and essentially my entire thesis. I think its right to assume I have become a little passionate in this field. Your persistent support and encouragement throughout the past two years has been immeasurable could not be more appreciated.

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Chapter 1: Literature Review

1.1 BURN INJURIES

Traumatic burn injuries cause long-lasting psychological and physical complications for patients. Children with severe burns are especially affected, as their treatment and wound management is often ongoing throughout the childhood growth period and potentially continues into adulthood. The Burns Registry of Australia and New Zealand (BRANZ) reports that paediatric burns comprise approximately 30% of reported cases, with the most common causes of burn injury admissions being scalds with hot liquids (57%) and contact with hot objects (27%) (1).

Accurate assessment of burn depth is a critical factor in determining prognosis and treatment. Burns are currently classified into four depth classifications, each characterised by differences in healing time (re-epithelialisation) and outcomes (Figure 1.1 A). Superficial burns, which are limited to the epidermis, often heal within a week without scarring. Superficial partial thickness (SPT) burns extend into the upper dermis and may result in blistering, but typically re-epithelialise within two weeks without scarring. Deep-dermal partial thickness (DDPT) burns extend deep into the dermis, requiring longer than three weeks to re-epithelialise, and are likely to scar. Full thickness (FT) burns are defined by damage through the entire epidermis and dermis, these almost always require a surgical intervention and will scar (2).

A B

Figure 1.1 A) Burn depth classifications which lead to differing healing outcomes from Johnson (2). B) The different zones of a burn injury which explain their healing capacity, as described by Jackson's model (3), adapted from Johnson, 2018 (2).

Chapter 1: Literature Review 1

When defining the damage within a wound, burns are described as consisting of three concentric zones (from the centre outwards): zone of coagulation, zone of stasis and zone of hyperaemia (3) (Figure 1.1 B). These zones are observed irrespective of depth and describe the regenerative capacity of the tissue.

1.2 BURN WOUND PROGRESSION

Burn wound progression (BWP), also called “burn wound conversion” (4) or “secondary burn wound progression” (5), is defined as the process of further tissue damage occurring in burn wounds following the initial damage at the time of injury. The literature also describes BWP as the process by which superficial partial thickness burns convert to deep partial-thickness and full-thickness burns (6), or as “continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult” (7). It is apparent that the phrasing and definition of this phenomenon across literature is inconsistent, and to better understand BWP it would be valuable to develop an agreed definition. For the purposes of this study, the definition of BWP aligns mostly with Shupp et al.’s, i.e “continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult”, as it describes the occurance of damage occuring within the zone of stasis. However, this should not be limited to necrosis as damage may occur due to a variety of mechanisms.

It was initially considered that the progression of burn wounds mainly occurs due to changes within the zone of stasis, as described by Jackson’s 1953 model. The zone of stasis was originally defined as an area which develops complete cessation of blood flow (3). However, subsequent investigations by Jackson determined that after excising the zone of coagulation, if a skin graft is placed on the zone of stasis, it will survive, indicating that the zone of stasis does not necessarily progress to have complete cessation of blood flow (8). This was further supported by observations made using laser Doppler imaging (LDI). Burns found to take longer than 21 days to heal were associated with LDI blood flow of <140 perfusion units (PU), whereas burns that healed within 14 days were associated with >600 PU. The variances in blood flow for burns healing between 14 and 21 days (between 200 and 600 PU) suggests that these areas of neither minimal or maximal perfusion are indicative of the zone of stasis (9). Due to the variability of perfusion, and other factors, the zone of stasis is a region of the wound in which either BWP or burn wound healing is suspected to occur. Thus, this area of tissue is of most interest when investigating BWP.

Chapter 1: Literature Review 2

The occurrence of BWP is acknowledged both in the literature and by clinicians’ anecdotal observations of burn injuries. However, there is no consensus on the mechanisms that are responsible for this progression and is unclear which treatments are best for preventing BWP.

1.3 MECHANISMS OF BURN WOUND PROGRESSION AND THEIR MEASUREMENT

There are many proposed mechanisms responsible for BWP however, appropriately identifying these mechanisms are difficult due to the complexity of burn wounds, thus wound mechanisms may vary from burn to burn (10). Many therapies and interventions that target these mechanisms have been evaluated in attempts to reduce or prevent BWP (5, 7). Further research to investigate the pathophysiological mechanisms responsible for BWP is required. Current literature regards histological assessment of skin tissue biopsies as the ‘gold standard’ in assessing burn depth (11, 12). Various staining techniques can be employed depending on the target of interest, cost, time, and stain specificity. Assessment of burn depth is often examined as the deepest point of injury. Several histological features could potentially be used either alone or collectively as markers for assessment of BWP.

1.3.1 Collagen Denaturation Collagen denaturation has been observed in partial to full thickness burns as a direct product of thermal injury. Literature suggests this causes a difference in collagen stainability occurring due to loss of crystallinity or parallel alignment of native collagen molecules (13) and can be used as a marker for burn depth measurements. Although burn depth has been assessed by collagen denaturation, some evidence suggests the depth indicated occurs superficial to cell death, specifically endothelial and epithelial cellular necrosis (12), which can occur deeper in the tissue. This suggests that although collagen denaturation is thermally induced, the true depth of injury may develop due to other pathophysiological pathways in the skin. Several different stains have been used to observe collagen denaturation (Table 1.1).

Masson’s trichrome (MT), Gomori’s trichrome (GT) and Verhoeff’s Van Gieson (VVG) stains distinguish between normal and denatured collagen by exploiting the

Chapter 1: Literature Review 3

Table 1.1 Stains described in literature to identify thermally induced collagen denaturation. Stain Injury markers Reference Masson’s Denatured collagen stains red, contrasts with (12-16) Trichrome normal blue stain. Gomori’s Denatured collagen stains red, contrasts with (17) Trichrome normal blue stain. Picrosirius Red Birefringence of collagen, differentiating type I (18, 19) and type III. Verhoeff’s Van Stains elastic fibres black, and collagen fuchsia. (16, 20- Gieson Burned collagen stains dark blue, purple to black, 22) contrasts from normal fuchsia dermis. Haematoxylin Collagen appears thin, flattened, or absent, with (23-25) and Eosin inconsistent sized gaps between fibres. altered molecular structure of the denatured collagen. An early study that used human skin sections heated to denaturation point and stained with MT determined that collagen heated to denaturation failed to retain the Masson’s red (a mixture of Ponceau 2R and Acid Fuchsin) component of the stain (14). This confirmed the occurrence of the structural changes within the dermis and validated the stain’s capability to detect and distinguish this damage in skin. This research inspired the widespread use of trichrome dyes to analyse burn wounds. Trichromes primarily stain connective tissues and muscle and are used to observe muscle pathologies such as myocardial tissue damage (26). Although MT is widely utilised in burns research, the studies which also involved H&E stain analysis prioritised burn depth assessment from the H&E results (12). This suggests that MT may not be as sensitive to detecting collagen denaturation as described in literature (27, 28). GT is another method that has been used to study collagen fibres and structural changes in skin pathologies (29, 30). An artificial skin designed as a skin substitute for autografts in burn wounds, was stained with GT to observe and differentiate between mature and young collagen (31). However, the use of GT to observe specifically burn wounds is limited, as described in Table 1.1. This stain is commonly used for collagen assessment in non-burn wound analysis, its effectiveness in burn wounds deserves to be further investigated. VVG is a modification to the Van Gieson stain by introducing Verhoeff’s haematoxylin to stain elastic fibres, prior to staining of collagen (red) and background tissue elements (yellow) by the application of Van Gieson stain (a mixture of acid fuchsin and picric acid). Due to its demonstration of elastin, this stain is also described in the literature as Elastic Van Gieson (EVG) (12). The VVG stain has been used in burn models to

Chapter 1: Literature Review 4

assess the depth of burn by collagen denaturation, where staining differentiates between normal and denatured collagen. Hinshaw & Pearse developed a modified VVG that was consistent in differentiating between normal and burned tissues, where normal epidermis is grey and burned epidermis is yellow, with normal collagen staining yellow and burned collagen staining black or purple (20). This method was used to assess scald burns and confirmed FT burns when the entire epidermis and dermis stained black (22). An alternate method, in this instance VVG, was used to stain partial thickness contact burns. Collagen denaturation was reported to stain dark blue or purple to distinguish from pale red intact normal collagen (21). VVG shows promising results in observing collagen denaturation and its specificity for elastin staining may be advantageous in analysing burn wounds.

Sirius red (or picrosirius when combined with picric acid) staining has also been used to observe collagen changes in wounds by enhancing the natural birefringence of collagen. When observed under polarised light, type I and type III collagen glow red and green respectively, showing a contrast between the two types (32). This was observed in burned lamb skin, with burned skin presenting a higher ratio of type III collagen, however Cuttle et al. cautioned that these may be redeveloping fibres indicative of healing as opposed to denatured collagen (18). Human skin with Ehlers- Danlos syndrome has also been observed using picrosirius red, however differences between collagen types were unable to be distinguished (19).

1.3.2 Cellular Necrosis Cell viability in the zone of stasis is critical for determining the survivability of the tissue. Cell death as a result of burn injury may occur by either necrosis or apoptosis (7, 33). Cellular necrosis is commonly observed (12, 23, 34), however apoptosis is also observed as a result of thermal injury (35, 36). The contribution of these different cell death pathways is not well understood at different time points, or for different severity burns.

The necrotic dermal structures which are used as a criterion to determine burn depth are mostly consistent within current literature, and include endothelial cells, epithelial cells, hair follicles, interstitial cells and fibroblasts (23, 24). Evidence of endothelial cell injury has been shown to predict burn progression as early as one hour post-burn in porcine skin (37).

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Table 1.2 Methods to identify thermally-induced cellular necrosis. Method Cellular necrosis marker Literature Haematoxylin and Cells appear swollen, fragmented, (12, 22, 23, Eosin (H&E) pyknotic nuclei 25, 34, 38- 40) Lactate Viable cells with functional LDH convert (41, 42) dehydrogenase nitroblue tetrazolium substrate to an (LDH) substrate insoluble blue/purple formazan salt for detection – viable cells are stained, leaving dead cells unstained Vimentin antibody Vimentin is a cytoskeletal protein in (12, 43) mesenchymal cells, which becomes degraded in necrotic cells – viable mesenchymal cells are stained, necrotic cells unstained High mobility group HMBG1 is a binding protein released out (12, 44, 45) box 1 (HMGB1) of the nucleus into the cytoplasm during antibody necrosis - stains necrotic cells

Cellular necrosis in burns involves multiple structures including hair follicles and blood vessels (BV) (37). The H&E stain is commonly used to demonstrate general morphological changes including cellular necrosis. Alternatively, enzyme histochemistry (e.g. for lactate dehydrogenase or LDH) can be used to identify viable cells at time of tissue fixation (46). Immunohistochemistry (IHC) staining for vimentin and high mobility group box 1 (HMGB1) has also shown promise for assessing cellular necrosis (44, 45). Previously published work by Hirth et al indicates that in comparison to H&E, HPS and HMGB1 staining, burn depth assessment using vimentin antibodies is underestimated in the first 24 hours (12), but HMGB1 staining matches the burn depth assessment of H&E and HPS after 4 hours.

1.3.3 Cellular Apoptosis Apoptotic cell death in burns has been observed as early as 1997 in rat models through identifying nucleosomal DNA fragmentation (47). This was confirmed clinically in biopsies from human patients with DDPT burns using terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labelling (TUNEL) and Fas antigen expression. To identify the pathway of apoptosis within burns, Giles et al. applied a c-Jun inhibitor to FT burn wounds in mice. The transcription factor c-Jun undergoes phosphorylation in the extrinsic apoptotic pathway (35). This treatment reduced cell apoptosis and time to wound re-epithelialisation, confirming the detrimental effect of apoptosis in burn wounds.

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Table 1.3: Methods to identify thermally induced cellular apoptosis in literature Method Mechanism of apoptosis Literature Cleaved Caspase Detects processes in the (23, 44, 45) 3a (CC3a) extrinsic apoptotic pathway – antibody following activation of caspase-8, caspase 3a is cleaved to signal apoptosis. terminal TUNEL detects apoptosis (34, 49, 50) deoxynucleotidyl through the specific binding of transferase (TdT) TdT to fragmented DNA, DNA mediated dUTP fragmentation occurs during nick end labelling late stages of apoptosis (48). (TUNEL)

Apoptosis within burn wounds may occur immediately (within 30 minutes), intermediately (30 minutes to 4 hours) or be delayed (after 4 hours), as identified in UV radiation in skin (7). Heat-induced apoptosis has been observed up to three weeks following injury, with apoptosis decreasing over time (49). The extended timeframe with which apoptosis has been shown to occur makes it difficult to identify when it is most detrimental to BWP. Currently, apoptosis is detected in burn wounds by IHC staining of cleaved caspase 3a (CC3a) and TUNEL (37). These methods detect apoptosis at different stages, with CC3a detected earlier, so it would be more beneficial to use this method for early detection of cellular apoptosis.

Studies investigating cell death in burns have indicated that there is a secondary wave of necrosis within the first 24 hours of burn injury, occurring distinctly after the initial passive necrotic cell death but before evidence of thermally-induced apoptosis (44). This secondary onset of necrosis is suggested to be self-induced, similar to apoptosis. Thus, necroptosis describes the mechanism in which active cell signalling occurs to induce cell death however cells morphologically resemble necrotic cells (51). The occurrence of necroptosis in BWP was previously investigated through administrating a necroptosis inhibitor (Necrostatin-1), inhibiting the assembly of receptor-interacting protein kinases 1 (RIP-1) and 3 (RIP-3), which failed to reduce progression. This suggested either the inactivity of the inhibitor, or that necroptosis may have no role in BWP (33).

1.3.4 Vascular Pathologies Tissue viability in burn wounds is also highly dependent on the blood flow to the area as the cessation of blood flow leads to ischemic cell death. This is

Chapter 1: Literature Review 7

demonstrated by laser Doppler imaging of burn wounds, where burned areas of low perfusion correspond to deeper tissue damage (9). Observation of the pathological features in burn wounds related to blood flow may prove to be beneficial to assess and measure tissue death. The initial stimulus following wounding involves the aggregation of red blood cells within the injured tissue, which may lead to further pathologies that cause cellular necrosis or apoptosis (Figure 1.2) (52). However, this occlusion delays inflammatory infiltration which may aid in preventing harmful inflammatory mediators. BV occlusion has previously been reported to occur as early as one hour post-burn in porcine contact burn models of 100°C for 30 seconds and found to continue for up to 48 hours (53). At 24 hours post-burn, decreased blood flow is believed to result in the formation of microthrombi, and in combination with the initial BV occlusion, enable ischemic necrosis to progress deeper into the dermis (7, 53).

Fibrin and fibrinogen deposits play a role in thrombus formation and BV occlusion. Fibrinogen is an acute-phase protein which contains binding sites responsible for fibrin conversion, fibrin assembly, cross-linking and platelet interactions (54). During wound healing, fibrinogen located in plasma leaks into the wound to provide a provisional matrix at the site of injury, promoting fibroblast growth factor-2 (FGF-2) and vascular endothelial cell growth factor (VEGF) to bind (55). The assembly of fibrinogen at the site of injury aids in tissue repair. Further research shows that in addition to growth factors, endothelial cells infiltrate fibrin clots to initiate angiogenesis before fibroblasts degrade and replace the fibrin with collagen and extracellular matrix proteins (56). The resolution of the clot enables the initiation of the inflammatory response, as neutrophils and macrophages are then able to travel to the wound and migrate into the tissue. Following the inflammatory processes, tissue re-vascularisation and angiogenesis occurs, primarily between days 3 and 12 post-burn (57). Fibrinogen and fibrin production play a significant role in wound repair, however their influence in burns specifically, has not been well documented outside of fibrin sealants (58). The viability of the zone of stasis is highly dependent on blood perfusion levels within the skin, this suggests that observing fibrin within burn wounds may be advantageous in burn pathophysiology.

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Figure 1.2 Schematic of pathological processes following burn injury resulting in burn wound progression. From (52).

As endothelial cell injury has been shown to be consistently deeper than hair follicle necrosis and collagen denaturation (24), it is important to observe vascular markers in the zone of stasis. Distinct features of vascularity can be observed with histological staining to examine tissue perfusion and consequently burn depth. Characteristics that are commonly identified are vessel occlusion, thrombosis, and infiltration of inflammatory cells. The stains identified in literature to identify burn induced vascular pathologies are described in Table 1.4.

Masson’s trichrome stain was previously used in human partial thickness burns to detect blocked dermal vessels as a mass of red blood cells within the lumen (59). To observe perfusion markers, H&E staining is the most prominent method within the literature. To improve H&E staining, Hirth et al. used a haematoxylin phloxine saffron (HPS) stain, which has been reported to improve visualisation of collagen and BVs with the substitution of saffron and phloxine (12). The limited number of methods used outside of H&E may suggest that other stains do not surpass H&E for observing perfusion markers. Not only does H&E staining have a fast and simple protocol it also provides other comprehensive information. As mentioned above, H&E has been used to visualise collagen denaturation, blood perfusion pathologies and cell death. For these reasons, H&E is highly beneficial in observing general features of burn damage and should be of value in observing BWP.

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Table 1.4 Stains to identify thermally induced vascular pathologies in skin. Stain Injury markers Literature Masson’s Blocked dermal vessels (59) Trichrome Haematoxylin Vascular patency (normal endothelial cells and (12, 34, and Eosin (H&E) intact blood vessels), microthrombi, neutrophil 38, 39) and inflammatory cell infiltration, blood vessel occlusion Haematoxylin Blood vessel occlusion, microthrombi, (12) Phloxine Saffron infiltration of inflammatory cells (HPS) Martius Scarlet Young fibrin: orange red; older fibrin: light (60) Blue (MSB) blue.

The presence of fibrinogen and fibrin deposition within burn wounds has only previously been studied in the context of skin graft treatments. Fisseler-Eckhoff & Muller compared H&E, EVG and MSB staining of fibrin sealed transplants to detect fibrin, collagen and RBCs (60). They reported that MSB was effective for identifying fibrin and had the capacity to distinguish between young and old fibrin staining red orange and light blue, respectively. MSB may serve as robust stain, identifying vascular pathologies as well as connective tissues in skin.

1.4 BURN WOUND MODEL

The use of in vivo animal models in burns research has progressed our understanding of burn pathophysiology and wound healing. Unlike in vitro models, animal models allow for thorough analysis of burn pathophysiology and especially post-burn mechanisms, including vascular changes. Animal models are especially advantageous to human samples in histological analyses as the collection of biopsies will cause a secondary wound in patients.

To observe burn wounds and wound healing, it is beneficial to develop an animal model which best simulates human pathophysiology. Mouse and rat models in burns are not ideal. Although their skin structure is composed of the same dermal and epidermal layers as humans, their differences in skin elasticity and thickness are not ideal for modelling human wound healing (61). In addition to high elasticity, these animals also have a unique muscular layer, the panniculus carnosus, which influences wound healing, causing wound closure by contraction instead of reepithelialisation, like humans (62, 63).

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Porcine skin has shown to be a reliable burn model for its resemblance to human skin. Skin structure similarities include distinct skin layers of relative size; epidermis, dermis and subcutaneous fat (15). The physiology and pathophysiological response to wounding in porcine skin resembles human skin’s ability to reepithelialise and the time to reepithelialisation after partial thickness burns (64). Porcine burn models have been frequently researched and validated in literature with both contact and scald burns (15, 23, 24, 39, 65, 66). Although porcine skin is recognised as a valid model for human wound healing, limitations of this model exist. Porcine skin differs from human skin as it contains a relatively high elastin content and comparatively a less developed subepidermal plexus, which may alter healing outcomes (64).

In using a porcine burn model, the mechanism of burn is critical for burn assessment and should always be considered. Brans et al. (67) reports the differences between contact and scald burns as assessed in porcine burn models. Contact burns show clear demarcation between healthy and injured skin, whereas scald burns show intermingled pattern of injured skin where damage was observed in deep vascular plexus whilst superficial plexus was intact. Scald burns also took several days to establish, suggesting the occurrence of BWP. A systematic review carried out by Andrews and Cuttle (66) found that contact and scald burns of similar temperatures and burn durations differed in burn depth. The thermal conductivity between water and metals modifies the heat transfer to cause a burn wound, thus affecting the appearance and pathology of the wound.

1.5 SUMMARY AND GAPS IN KNOWLEDGE

1.5.1 Summary Burn wounds are a complex environment consisting of a variable zone of stasis, where multiple pathological mechanisms occur. This zone has the capacity to heal and sustain cellular structures within the skin, or to undergo BWP. Burn wound progression is defined as continued progression of injury following the initial insult. This progression is more severe when partial thickness burns convert to DDPT or FT burns, causing dramatically different healing times and outcomes. Burn wound progression is reported to take place within this zone due to the variability of blood flow into the tissue.

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Although the occurrence of BWP is not widely reported, burn depth studies have been conducted using various methods of models and analyses. The most common and supported method is histology, as it provides a snapshot of the processes and mechanisms within the injured tissue. Histochemical and IHC staining are effective in observing specific pathologies associated with burns. The most commonly reported features observed are collagen denaturation, cellular apoptosis, cellular necrosis, and vascular pathologies such as BV occlusions and thromboses. In observing burn markers utilised to assess burn depth, the mechanisms responsible for BWP can be investigated.

1.5.2 Gaps in Knowledge Burn wound progression is commonly acknowledged in the literature, whether it is described as “dynamic changes” (28, 38) or “secondary burn wound progression” (5). However, there is no consensus regarding the exact mechanisms involved in the progression of the wound. Whether these mechanisms alter with respect to time post- burn, severity or type of burn injury is unknown. The majority of current research around BWP investigates BV occlusion and endothelial cell death as major contributors (37, 52, 53). However, these studies use only contact burn models, and scald burns are still to be investigated, although they are the most common type of burn injuries experienced by children. Scald burns may continue to deepen over several days, indicating a period in which BWP may take place (67). The mechanisms associated with collagen denaturation, cellular necrosis, apoptosis, and vascular pathologies are known to occur during burns, but little research has been conducted into how these may interact, to contribute to BWP. As noted by Hirth et al., burn wound features that predict wound progression or lack of, would be invaluable in burns research, to develop more effective diagnoses and treatments (37).

In the literature, various stains have been utilised to identify different pathologies for wound and depth analysis. This variety in staining methods not only demonstrates the vast complexity of burn wounds but highlights the inability to optimise an effective stain and rubric for burn analyses. As BWP continues to be investigated, it would be beneficial to determine a consistent, reliable method to accurately analyse burns.

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1.6 THESIS OUTLINE

This thesis presents a histological analysis of the burn wounds with the view to gaining new insights into the potential mechanisms that determine BWP. This chapter provides an overview of the current literature about BWP and techniques that have been used to date to study it. The previously published research concerning BWP and its detection was critically reviewed to determine gaps in knowledge, to form project hypotheses. Chapter 2 describes optimisation methods of staining used, including the H&E, VVG, GT and MSB staining methods, as well as immunostaining for fibrin. Chapter 3 describes how the optimised stains were subsequently employed to evaluate burn damage, in which a rubric was developed to assess burn intensity with the view to developing histological markers for BWP. In this thesis, burns of different temperatures and durations, and progression vs non-progression burns were assessed. This allows for in-depth analysis of pathologies within the wound to cause BWP. A schematic illustrating the burn conditions and numbers of replicates examined is shown in Figure 1.3. The final chapter of the thesis provides a discussion and conclusions for the study, highlighting important discoveries made, limitations of the current work and proposed future directions.

Progression (6 burns, at 3 timepoints, n=18)

Low temperature – Long High temperature – Short duration (3 burns, n=9) duration (3 burns, n=9)

1 hour 24 hours 72 hours 1 hour 24 hours 72 hours (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3)

Non-Progression (6 burns, at 3 timepoints, n=18)

Low temperature – Long High temperature – Short duration (3 burns, n=9) duration (3 burns, n=9)

1 hour 24 hours 72 hours 1 hour 24 hours 72 hours (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3)

Figure 1.3 Burned sections data representing burn treatment group, burn conditions and timepoints. The number of burns represented in each group are shown, n equals number of sections for each group.

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1.7 HYPOTHESIS AND AIMS

Observation of burn damage markers at differing time points is vital for determining the occurrence of BWP. Many stains have been used previously to observe burn wounds, however in order to accurately observe BWP, appropriate stains must be chosen and optimised to investigate specific mechanisms. It is hypothesised that with optimal histological staining, mechanisms of BWP can be identified in the early stages after burn wound injury. This hypothesis was investigated via the following aims:

Aim 1: To choose stains of interest from literature and optimise their ability to identify burn damage markers.

Aim 2: To identify mechanisms of burn wound progression by:

a) Creating a rubric to assess burn damage markers, so that the intensity and type of burn damage can be detected in each tissue section.

b) Use the rubric to score progression and non-progression burn wound sections and identify any differences between them.

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Chapter 2: Selection and Optimisation of Staining Methods

2.1 INTRODUCTION

This chapter explains the process of optimising each stain protocol to then evaluate which stains are most suitable for identification of damage associated with BWP. In this chapter, the methods of the chosen stains were optimised to show skin structures clearly and effectively in normal tissue and markers of damage in burned tissue. Through critically analysing the literature regarding histological methods in detecting burn damage markers in skin, the chosen stains to evaluate were H&E, VVG, GT and MSB. In stain optimisation, methods were optimised to best stain porcine skin and highlight burn damage markers. Optimised stains were then compared to determine which stains would be selected for assessment based on their relevance and applicability for BWP.

In stain optimisation, an initial protocol was applied and evaluated. The initial protocol was then modified appropriately to improve its application to porcine skin. Once optimised, stains were applied to burned skin to observe its effectiveness for identifying burn damage markers. A selection of formalin fixed paraffin embedded (FFPE) porcine scald burns, and normal porcine skin was chosen to evaluate the stain in burn damage identification. Selected burn samples were collected post-burn at one hour, 24 hours and 72 hours with differing burn conditions as detailed in Figure 2.1 and Table 2.1. Porcine sections were obtained from previous work conducted by Dr. Christine Andrews, which assessed multiple burns at various burn temperatures and duration using a novel scald burn model (68). Ethics approval for this work was obtained from the University of Queensland Animal Ethics Committee (approval numbers: QCMRI/RCH/326/12/ QCMRI/NHMRC and QCMRI/446/15/QCHF). The porcine burn sections chosen for staining provided a wide variety of burn conditions at different time points, to assist with identifying burn damage markers. These burn conditions across multiple timepoints provided a range of different burn depths to assess. Figure 2.1 and Table 2.1 detail section selection and burn conditions.

Chapter 2: Selection and Optimisation of Staining Methods 15

Burn Conditions

High Temperature + Short Low Temperature + Long Duration Duration (6 burns, n=18) (2 burns, n=6)

1 hour 24 hour 72 hour 1 hour 24 hour 72 hour (n=6) (n=6) (n=6) (n=2) (n=2) (n=2)

Figure 2.1 Burned section data representing burn treatment group, burn conditions and timepoints for Chapter 2 stain optimisations. The number of burns represented in each group are shown, n represents the number of sections in each group.

Table 2.1 Burn condition information for porcine scald burns utilised in Chapter 2 for burn marker identification and stain evaluation. Burns included samples collected at one hour, 24 hours and 72 hours post-burn. LT+LD: low temperature – long duration, HT+SD: high temperature – short duration. Temperature (°C) Duration (seconds) Burn Conditions 55 600 LT+LD 55 300 LT+LD 55 120 LT+LD 55 60 LT+LD 60 60 LT+LD 60 30 LT+LD 85 5 HT+SD 90 5 HT+SD

2.2 Haematoxylin and Eosin

2.2.1 Introduction Haematoxylin (Hx) combined with eosin (H&E) is the routine stain used for studying tissue morphology. The amount of detail this stain produces for its cost and time is highly advantageous in histology work. The haematoxylin component stains negatively charged tissue components including chromatin found within cell nuclei. Nuclei are initially stained a deep red/burgundy colour, which is subsequently converted to a dark blue after alkaline treatment. The remaining tissue of components with a more basic nature (i.e. positively charged) including collagen fibers, are stained with the acidic dye, eosin, resulting in a bright red-pink stain. Hematoxylin may be applied either regressively or progressively. Regressive staining methods involve initial over-staining with hematoxylin followed by brief treatment with acidified alcohol until the required level of staining is achieved. In contrast, progressive methods

Chapter 2: Selection and Optimisation of Staining Methods 16

rely upon treatment with hematoxylin for a fixed period of time until the desired level of staining is achieved. A progressive staining method will be used in this study.

2.2.2 Reagents The reagents used for this stain in optimisation and final stages are as follows: Mayer’s Haematoxylin (MHS32, Sigma-Aldrich, St Louis, Missouri, USA), eosin (HT110132, Sigma-Aldrich, St Louis, Missouri, USA), graded ethanol (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (70%, 90%, 100%) xylene (Thermo Fisher

Scientific, Waltham, Massachusetts, USA), Scott’s water (0.35% NaHCO3 (Thermo

Fisher Scientific, Waltham, Massachusetts, USA), 2% MgSO4(Sigma-Aldrich, St Louis, Missouri, USA)), Depex neutral mounting medium (Chem-Supply, Gillman, South Australia, Australia).

2.2.3 Initial Protocol The initial stepwise protocol followed is described below.

1. Rehydrate sections – 2 rinses in xylene (3 minutes each) and 4 rinses in ethanol (1 minute each) in decreasing concentration (100%, 100%, 90%, 70%).

2. Place sections in water.

3. Stain with Mayer’s Haematoxylin for 5 minutes.

4. Rinse with running water.

5. Treat with Scott’s water for 30-60 seconds to convert hematoxylin to a blue colour (microscope check here to determine haematoxylin stain intensity).

6. Rinse with running water.

7. Quickly rinse sections in 90% ethanol.

8. Stain with eosin for 2 minutes.

9. Rinse in 90% ethanol.

10. Dehydrate sections – 2 rinses in 100% ethanol (1 minute each) and two rinses in xylene (3 minutes each).

11. Coverslip slides using Depex mounting medium. Leave to dry for at least 15 minutes.

Note: Routine H&E staining protocol was applied during this study.

Chapter 2: Selection and Optimisation of Staining Methods 17

2.2.4 Optimisation As this stain is commonly used in skin and burns to assess burn depth and markers of damage, no specific optimisation was required beyond confirming that five minutes incubation in Mayer’s haematoxylin was adequate to stain cell nuclei, with minimal to no staining within collagen. Normal, unburned skin was stained with routine H&E staining to confirm the appearance of healthy skin and its structures.

With optimal staining methods, the epidermal and dermal structures are stained clearly to identify relevant structures within the skin. In Figure 2.2 A-B, the structure of the epidermis, dermis and the dermal-epidermal junction is exhibited. Haematoxylin

Figure 2.2 Optimised haematoxylin and eosin stain on normal, unburned porcine skin. A) Low magnification of general morphology of skin, clear distinction between purple epidermis and pink dermis (Scale: 200μm); B) High magnification image of cell nuclei within the skin stained dark purple within a light pink dermis (Scale: 50μm); C) Healthy hair follicle consisting of an intact internal keratin layer surrounded by healthy, intact keratinocytes (Scale: 50μm); D) Sweat glands located in deep dermis, with intact surrounding cells (Scale: 100μm); E) Small blood vessel in deep dermis (Scale 50μm); F) Small arteriole with clear lumen (Scale: 50μm)

Chapter 2: Selection and Optimisation of Staining Methods 18

stains the skin cell nuclei dark purple, as seen in the high magnification image in Figure 2.2 B. Basal epidermal cell nuclei are circular in shape which differ from fibroblast nuclei in the dermis which are denser and more elliptical in shape. Dermal structures: hair follicles, glandular structures and BVs are easily identifiable in H&E staining, where healthy structures consist of clear and round cell nuclei, in and around the structures (Figure 2.2 C-F). Normal BVs retain a circular structure, consisting of a central open lumen. The routine H&E staining of normal porcine skin is a valid method to effectively identify epidermal and dermal structures as seen in Figure 2.2.

2.2.5 Evaluation in burned tissue H&E staining is the most commonly used stain in pathology, and its use in burns research is widely reported. The appearance of common burn injury markers described in the literature is shown in Figure 2.3. Burn induced epidermal injuries vary in appearance, the first instance is epidermal loss of adherence (Figure 2.3 A). The loss of adherence from the dermal layer is signified by white spacing between the epidermal and dermal layers, areas in which the spacing is substantial may be indicative of blister formation. The second instance is when the epidermis maintains contact with the dermis, in this case, injury is demonstrated by a change in cellular structure. Elongated string-bean nuclei are observed within the epidermis, as well as flattening of the dermal-epidermal junction (Figure 2.3 B).

In Figure 2.3 C-D, collagen denaturation is identified by the change in structural appearance and arrangement of the dermal collagen fibres. Fissures between collagen fibres are also observed in the dermis and are irregular in size and location. Inflammatory cells can also be observed, distributed across the width of the section to indicate the infiltration of an inflammatory response. These inflammatory cells are smaller in size, allowing them to be distinguished from other dermal cells (Figure 2.3 E-F). Damaged hair follicles contain elongated string-bean nuclei, as well as spacing between cells and in the outer wall. The inner keratin layer is also observed as irregular in shape with clear loss of structure (Figure 2.3 G). Some vascular pathologies identified by H&E staining include blocked BVs observed by aggregation of red blood cells (RBCs) within the lumen of the vessel and free RBCs scattered freely within the dermis (Figure 2.3 H).

H&E is a comprehensive stain and has been proven to visualise collagen denaturation, blood perfusion pathologies and cell death, as well as general

Chapter 2: Selection and Optimisation of Staining Methods 19

Figure 2.3 Reported burn induced damage markers of injury identified with haematoxylin and eosin staining of porcine skin. A) Epidermal loss of adherence from dermis. Clear separation identified between pink dermis and purple epidermal layer (Scale: 50μm); B) Flattening of dermal-epidermal junction with elongated string bean nuclei present in the basal epidermal layer, indicated with arrows (Scale: 200μm); C) Low magnification of collagen denaturation within the dermis (Scale: 50μm); D) High magnification of collagen denaturation, identified by bundles of collagen fibres and irregular fissures between collagen fibres (Scale: 200μm); E) Low magnification of inflammatory band in dermis (Scale: 50μm); F) High magnification of inflammatory band, individual dark inflammatory cells identified by arrows infiltrated within the dermis (Scale: 200μm); G) Hair follicle damage identified by spacing between the border and necrotic cells indicated by the arrow (Scale: 200μm); H) Blocked blood vessels indicated by arrows and free red blood cells found within the dermis, stained a clear bright red. (Scale: 200μm).

Chapter 2: Selection and Optimisation of Staining Methods 20

morphological features of skin. H&E is the most widely reported stain for burn research and it is evidently utilised often as described in this chapter. To observe BWP, it is highly beneficial to include this stain in this study.

2.3 Verhoeff’s Van Gieson

2.3.1 Introduction Verhoeff’s Van Gieson (VVG) is an altered Van Gieson collagen stain, which is an effective connective tissue stain. It combines the Van Gieson stain with a Verhoeff’s haematoxylin, which improves the generic haematoxylin stain with ferric chloride and Lugol’s iodine to stain elastin fibres. Previously, VVG has been used in burns to examine collagen denaturation through a difference in normal collagen being stained red and denatured collagen stained black or purple. However, burn-induced elastin changes have not been investigated to date.

2.3.2 Reagents The reagents used for this stain in optimisation and final stages are as follows: Verhoeff’s Solution A (Haematoxylin) (AC411161000, Thermo Fisher Scientific, Waltham, Massachusetts, USA), Verhoeff’s Solution B (10% Ferric Chloride) (157740, Sigma-Aldrich, St Louis, Missouri, USA), Verhoeff’s Solution C (Lugol’s Iodine) (LU00100500, Chem-Supply, Gillman, South Australia, Australia), graded ethanol (70%, 90%, 100%), xylene, Van Gieson solution (HT254, Sigma-Aldrich, St Louis, Missouri, USA), Depex mounting medium.

2.3.3 Initial Protocol The initial stepwise protocol followed is described below.

1. Rehydrate sections – 2 rinses in xylene (3 minutes each) and 4 rinses in ethanol (1 minute each) in decreasing concentration (100%, 100%, 90%, 70%).

2. Stain sections with Verhoeff’s haematoxylin working solution for 20 minutes using inverted method in Petri dishes:

3. Rinse sections with running water.

4. Differentiate slides using 2% Ferric chloride until elastin fibres appear black on a pale grey background. Start with 15 seconds differentiation and rinse in water before checking under microscope. Repeat until satisfactory differentiation is achieved.

Chapter 2: Selection and Optimisation of Staining Methods 21

5. Quickly rinse sections in 90% ethanol to remove excess iodine staining.

6. Rinse sections with distilled water.

7. Counterstain with Van Gieson solution for 2 minutes.

8. Dehydrate sections – 2 rinses in 100% ethanol (quick as ethanol will wash out Van Gieson) then 2 rinses in xylene (3 minutes each).

9. Coverslip slides using Depex mounting medium. Leave to dry for at least 15 minutes.

Note: The primary point of optimisation was the differentiation of Verhoeff’s Hx in step 4.

2.3.4 Optimisation Compared to other available stains, the VVG is relatively simple, comprising of three major steps in its protocol. For this study, the primary point of optimisation for this stain was its differentiation duration. To optimise this stain, several differentiation durations were tested and evaluated to achieve clear and consistent staining. Sections were initially differentiated for 20 seconds and 30 seconds (Figure 2.4 A-C). These stains were deemed to be under-differentiated as elastin within the dermis is barely visible or difficult to distinguish among the other structures whilst the epidermis is considerably dark, with keratinocytes difficult to identify. Additionally, the internal elastic lamina identified in Figure 2.4 C were unclear and required longer differentiation. The next set of stains consisted of increased differentiation durations of 60 seconds and 90 seconds (Figure 2.4 D-F). A differentiation duration of 90 seconds achieved the desired appearance. This was indicated by a lighter epidermis and clear elastin fibres within the epidermis. An internal elastic lamina is clearly visible within the arteriole wall; however, imaging was difficult to precisely focus the skin.

To resolve the imaging issue and improve staining, the section thickness was decreased from 5 μm to 3 μm. To determine if this change was effective, 3μm sections were stained with an under-differentiated duration of 45 seconds and the optimal duration of 90 seconds (Figure 2.4 G-I). The epidermis appears light grey/purple with visible keratinocytes, and although the 3μm section has a different colour in comparison to Figure 2.4 E (5μm thick), the 90 second differentiation duration

Chapter 2: Selection and Optimisation of Staining Methods 22

Figure 2.4 Verhoeff’s Van Gieson stain optimisation on normal, unburned porcine skin. Staining was optimised primarily through modifying the duration of differentiation and section thickness. Epidermis and elastin appearance indicated optimal differentiation duration, while collagen appearance and clarity of stain was observed to determine optimal section thickness. Optimal staining was observed by a lightly stained epidermis with visible keratinocytes, well-defined collagen fibres and clear individual elastin fibres in dermis and arterioles. The desired stain appearance is presented in H and I. Differentiation durations: A) 20s; B) 30s; C) 30s; D) 60s; E) 90s; F) 90s; G) 45s; H) 90s; I) 90s. Section thickness: A- F) 5μm; G-I) 3μm. C, F, I) Indicate internal elastic lamina of arterioles in the adjacent section. Arrows indicate elastin fibres, stained black. (Scale: 50μm) accurately stains all structures. By decreasing the section thickness, collagen fibres appear better defined and internal elastic lamina is clearly visible.

2.3.5 Evaluation in burned tissue Once the VVG stain was optimised, it was assessed for its ability to detect burn damage markers within the skin sections (see Figure 2.5). The burn markers detected using this stain were collagen denaturation, elastin changes and inflammatory infiltration. Denatured collagen within the dermis is observed by an alteration in the dermal architecture. In Figure 2.5 A, small, inconsistent gaps are observed between the collagen fibres as a result of the thermal injury, this is seen throughout the dermis, and the difference in collagen structure is clear when contrasting with the normal skin in Figure 2.4. In addition to the collagen spaces, individual collagen fibres appear thinner or more condensed. A contrast in collagen staining from the regular fuchsia to yellow is observed in burned tissue (Figure 2.5 C), indicating molecular changes of the collagen fibres, which may be a marker of burn induced injury. Figure 2.5 D shows

Chapter 2: Selection and Optimisation of Staining Methods 23

Figure 2.5 Burn induced injuries visualised with Verhoeff’s Van Gieson staining of burned porcine skin. Collagen changes are identified in A-D. A, B) Altered structure of dermal collagen, with inconsistent spacing between collagen fibres. C, D) Change in collagen colour from fuchsia to yellow located superficial to a band of inflammatory cells within the dermis. E) Low magnification of inflammatory band appears as grey background between collagen fibres; F) High magnification of inflammatory band, individual cell nuclei stained black are infiltrating the dermis. A, C, E) Scale: 50μm; B, D, F) Scale: 200μm. G) Elastin fibres in superficial dermis appear long and thin, similar to elastin fibres in normal skin; H) Elastin fibres in deep dermis are short and thin, appearing fragmented and clustered (Scale: 50μm). Generally, elastin fibres seem unchanged between burned and normal skin, however in some instances, elastin fibres in the deep dermis appear shortened and thicker as shown in H.

Chapter 2: Selection and Optimisation of Staining Methods 24

a high magnification image of the yellow stained collagen, distinct collagen fibres are unidentifiable whilst maintaining irregular spacing within the dermis. A band of infiltrated inflammatory cells were observed beneath this yellow stain colour, as well as in another skin section (Figure 2.5 E).

As described above, the inflammatory cells are observed in a band across the entire width of the section. At low magnification, the band appears as a grey structure dispersed between collagen fibres. Upon closer examination at higher magnifications, individual inflammatory cells can be seen within the dermis by virtue of the haematoxylin staining cell nuclei.

While not entirely specific for elastin fibers, the tendency of Verhoeff’s hematoxylin to be retained more strongly within elastin fibers during treatment with ferric chloride, provided an opportunity to view potential effects of burns on the general structure of elastin fibers. Compared to elastin fibers in normal skin (Figure 2.5 A), the fibers displayed within burned skin (Figure 2.5 B), appeared thicker, more clustered, and relatively shorter and fragmented.

The VVG method therefore displays potential as a method for distinguishing features of burned skin. Nevertheless, prior studies appear to have been focussed on assessment of collagen colour rather than the elastin fibers (20-22). Presumably, the burned collagen fibres have been denatured in a way that promotes retention of the haematoxylin. In the present study, this was observed by the contrast of staining in elastin fibers appearance between unburned skin and burned skin. In conclusion, the altered structure of elastin fibres caused by burns may have an influence on the pathologies in burn wounds.

2.4 Gomori’s Trichrome

2.4.1 Introduction The Gomori’s trichrome is a one-step solution made up of Chromotrope 2R and aniline blue. This trichrome utilises a one-step solution to stain the entire tissue, rather than in separate steps, achieving a faster stain in comparison to other trichrome stains. This stain was chosen to distinguish between collagen states, staining normal collagen blue and denatured collagen red. In addition to denatured collagen, other structures such as muscle, RBCs and fibrin are also stained red.

Chapter 2: Selection and Optimisation of Staining Methods 25

2.4.2 Reagents The reagents used for this stain in optimisation and final stages are as follows: Bouin’s solution (HT10132, Sigma-Aldrich, St Louis, Missouri, USA), Gomori Trichrome (HT10516, Sigma-Aldrich, St Louis, Missouri, USA), Scott’s water (0.35%

NaHCO3, 2% MgSO4) , 0.5% acid alcohol, Weigert’s solution A (Haematoxylin)

(HT107, Sigma-Aldrich, St Louis, Missouri, USA), Weigert’s solution B (FeCl3 + HCl) (HT109, Sigma-Aldrich, St Louis, Missouri, USA), glacial acetic acid (0.5,1%) (Banksia Scientific, Bulimba, Queensland, Australia), graded ethanol (70%, 90%, 100%), xylene, Depex mounting medium.

2.4.3 Initial Protocol Two different Gomori Trichrome (GT) protocols were used to optimise this stain. The first protocol, the “Gibson Protocol”, was provided by Dr Angela Gibson (Burn, Trauma, Emergency General Surgery and Surgical Critical Care, University of Wisconsin – Madison) who has conducted porcine burn wound analysis using this stain, therefore this was used as the primary protocol. The second protocol comes from Sigma-Aldrich using its standard procedure, the “Sigma Protocol”. The GT stain consists primarily of a post-fixation, haematoxylin, trichrome solution, and acetic acid rinse. These stages were modified to optimise the stain and achieve the desired appearance. The details of the initial protocols provided are described in Table 2.2.

Table 2.2 Gomori’s trichrome initial protocols Protocol Steps Gibson Protocol Sigma Protocol 1. Rehydrate slides Xylene to graded ethanol Xylene to graded ethanol

2. Post-fixation in Bouin’s 60 minutes at 60°C 15 minutes at 56°C Solution 3. Weigert’s Haematoxylin 10 minutes 5 minutes

4. Differentiation No No

5. Gomori one-step solution 20 minutes 5 minutes

6. Glacial Acetic Acid 1%, 2x rinses for 1 minute 0.5%, dipped for 1 minute

7. Dehydrate and Mount Graded ethanol to xylene Graded ethanol to xylene

2.4.4 Optimisation To optimise the GT staining, each individual protocol was optimised separately before comparing the two staining protocols to each other.

Chapter 2: Selection and Optimisation of Staining Methods 26

To optimise Angela Gibson’s protocol (Figure 2.6 A-C), the effectiveness of post-fixation was assessed, and a differentiation step was introduced. To observe the influence of post-fixation, the protocol was followed with one stain omitting post- fixation and one stain completing the entire 60 minutes. The section stained with no post-fixation, shows an intense blue collagen stain within the dermis and a purple/red epidermis (Figure 2.6 A). Although structures are stained vibrantly, the intensity of the colours mask the details of the structures, as individual collagen fibres are not stained clearly. As the epidermis was expected to be stained red, the purple appearance indicates a possible over staining in haematoxylin or a mixture of red and blue dyes within the trichrome solution.

With the addition of a 60 minute post-fixation step, the sections were unable to maintain adherence to the slide, resulting in poor structural appearance and in some cases folding and overlapping as shown in Figure 2.6 B. This occurred due to the sections being mounted on uncharged slides, where prolonged heat contact caused the sections to lose adherence. The sections further lost structural integrity with subsequent washes during the protocol. Nevertheless, sections stained with 60 minutes post-fixation show better distinction between structures as shown by a higher intensity of red in the epidermis and hair follicle.

To remedy the loss of adherence to the slide, sections were then mounted onto charged slides to withstand the post-fixation. Charged slide sections were stained with the identical protocol, however the more durable sections could withstand thorough rinsing in acetic acid following Gomori’s Trichrome staining. A significant difference in staining outcome was observed primarily in the intensity of collagen appearance. Individual collagen fibres are clearly distinguishable, stained lightly blue with red staining of dermal structures, in and around the collagen (Figure 2.6 C).

The standard Sigma-Aldrich protocol is generalised and non-specific to porcine skin, however the protocol was tested to determine its effectiveness in comparison to the Gibson protocol. Following the protocol outlined in Table 2.2, sections appeared similar to initial stains in the Gibson protocol. Collagen is stained an intense blue, with dark purple epidermis and slight fissures between collagen (Figure 2.6 D). Fissures within the dermis were likely a result of post-fixation of sections on uncharged slides causing loss of adherence.

Chapter 2: Selection and Optimisation of Staining Methods 27

Figure 2.6 Gomori Trichrome Optimisation using either the Gibson protocol (A, B, C, F) or Sigma Aldrich protocol (D, E) on normal, unburned porcine skin. To optimise the Gibson protocol, the post- fixing step was omitted or included for 60 minutes and charged slides were used. A) No post-fixing, uncharged slide, acetic acid dip; B) 60 minutes post-fixing, uncharged slide, acetic acid dip; C) 60 minutes post-fixing, charged slide, acetic acid rinse. Optimal staining as indicated in (C), with collagen fibres stained light blue, and blood vessels, muscle and other dermal structures stained red. This was achieved by using charged slides which enabled thorough acetic acid rinsing of Gomori trichrome solution (Scale: 200μm). To optimise the Sigma Aldrich protocol, the haematoxylin was differentiated, and the section was carefully handled during the acetic acid stage. D) Protocol was followed directly; E) Protocol was altered with the addition of haematoxylin differentiation and an additional acetic acid rinse. The inclusion of a haematoxylin differentiation stage allows cell nuclei to be more clearly distinguished from the surrounding structures. The addition of an acetic acid rinse allowed for a less intense collagen stain to better visualise other structures, optimal staining is not achieved due to variance in epidermal staining and fissure formation within the dermal tissue (Scale: 200μm). F) High magnification of collagen fibres with optimal staining using Gibson protocol, individual fibres clearly stained blue with fragments of red staining (Scale 50μm).

To optimise this protocol, a differentiation step was added following haematoxylin staining to better define nuclei within the epidermis. More care was taken with the slides after post-fixing with subsequent washing and staining to reduce section loss of adherence. An additional acetic rinse was implemented to further rinse off the trichrome solution prior to dehydrating. However, observed in Figure 2.6 E, fissures within the dermis continued to develop. By comparing the initial and optimised protocol, several observations can be made. Both the red staining in the epidermis and surrounding structures, and the blue collagen staining are stained with less intensity in the optimised protocol. This is likely due to the additional acetic acid rinse restricting overstaining of structures. The light epidermis may also be due to differentiating haematoxylin, providing a clear image of cell nuclei within the epidermis. Although the structure of the skin should be improved with using charged slides, the staining intensity and accuracy is adequate for the structures in porcine skin.

To determine which optimised protocol was superior, the staining intensity, specificity and clarify was considered. An optimal stain shows primarily a light blue

Chapter 2: Selection and Optimisation of Staining Methods 28

collagen, with visible distinct fibres and a light pink epidermis with clear keratinocytes. These colours should be sufficiently distinct with clear contrast and minimal cross-staining which is indicated by purple staining. When observing the epidermis, the Gibson stain shows a consistent colour throughout, with clear keratinocytes. In comparison, the appearance of the epidermis with the Sigma-Aldrich protocol has a gradient in staining, with more lightly stained keratinocytes. Epidermis staining in the Gibson stain was assisted by a longer duration of haematoxylin staining combined with selective differentiation, which resulted in an overall superior epidermal stain.

In the dermis, the collagen staining between the two protocol is consistent in visibility of individual fibres and intensity, however the Gibson stain appears to have additional red staining of collagen in some areas. We believe this red collagen staining may be a result of the one-step solution overstaining, as it eliminates the opportunity to differentiate between stains. The Gomori’s one-step solution was applied for 20 minutes which is likely to contribute to overstaining. Unlike the Sigma-Aldrich protocol, the trichrome solution was applied for 5 minutes, producing a slightly lighter blue stain, resulting in low contrast between the epidermis and dermis. The light intensity of purple staining observed in the epidermis and hair follicle of the Sigma- Aldrich stain may be an effect of minimal post-fixation. An extended post-fixation period of 60 minutes allows the red and blue within the trichrome solution to be distinct and separate as seen in Figure 2.6 C.

The use of a post-fixation step combined with longer staining times in haematoxylin and Gomori’s solution most likely accounts for the improved staining outcomes achieved using this method.

2.4.5 Evaluation in burned tissue The optimised stain was applied to burned sections to evaluate its ability to detect burn pathologies in skin (see Figure 2.7). Collagen denaturation is identified in the dermis by presence of light and dark blue staining of collagen. The variance in intensity indicates collagen bundling due to heat-induced denaturation (Figure 2.7 A-B). Among the structural changes, Figure 2.7 C shows a tear in the epidermis of the skin. Epidermal loss of adherence is a common feature seen burns which may appear as in Figure 2.3 A, however, may also appear as tears in the tissue structure, as clearly demonstrated with the GT staining. Similar to the results published by Watts et al.

Chapter 2: Selection and Optimisation of Staining Methods 29

Figure 2.7 Burn induced injuries visualised with Gomori’s Trichrome staining using optimised Gibson protocol on burned porcine skin. A) Low magnification of collagen within the dermis (Scale 50μm); B) High magnification of collagen, with damage indicated by darker blue stained bundled fibres (Scale: 200μm); C) Epidermal loss of adherence from the basal layer observed within red stained epidermis (Scale: 200μm) ; D) Clustering of red blood cells within a blood vessel, appearing as a blocked vessel indicated by the arrow (Scale: 200μm); E) Low magnification of inflammatory band located within the dermis (Scale: 50μm); F) High magnification of inflammatory band, showing individual inflammatory cell infiltration into the dermis surrounded by red background staining (Scale: 200μm); G) Change of collagen colour from blue to red, located superficial to an inflammatory band, due to altered, burned collagen structure (Scale: 50μm); H) Web-like structures located in the deep dermis stained red, their appearance is consistent with neutrophil extracellular traps, previously identified in literature (Scale: 200μm).

Chapter 2: Selection and Optimisation of Staining Methods 30

(59), GT staining was effective in detecting blocked vessels, visualised by a cluster of RBCs located within a BV in Figure 2.7 D. Red staining in the vessel walls may be indicative of fibrin deposits in damaged vessels.

A band of inflammatory cells infiltrating the dermis was also observed in some burns 72 hours post injury (Figure 2.7 E-F). A purple band is seen in low magnification, however under higher magnification, the band is comprised of many individual cells surrounded by red staining within the collagen. Another section shows bright red staining of collagen densely packed towards the surface of the skin (Figure 2.7 G). Literature states that normal collagen and denatured collagen is stained blue and red, respectively. Although these results showcase a combination of blue and red staining, the presence of red staining is not consistent with denatured collagen. As seen in Figure 2.7 E-G, blue staining is observed superficial to red staining, suggesting that collagen which has closest proximity to the burn source has not denatured.

Figure 2.7 H shows web-like structures located in the deep dermis, bordering the adipose layer. The appearance of these structures is consistent with Neutrophil Extracellular Traps (NETs) observed histologically by Shiogama et al. in human lung tissue, described as clusters of fibrillar meshwork (69). The presence of NETs has been detected in trauma and burns patients’ blood (70, 71), however, only one study by Korkmaz et al. investigates NETs histologically in burned skin. NETs were observed within intravascular thrombi and believed to play a role in inducing secondary thrombosis BWP (72). NETs were found to mimic fibrin structure, with no morphologic differences, unable to be discerned by electron microscopy (73). The relationship between NETs and fibrin was observed with electron microscopy discovering that NETs and fibrin fibrils were co-deposited to form web-like structures (74). Overall, GT is a quick stain, made simple with a one-step solution and effectively shows collagen denaturation, blocked vessels, inflammatory infiltration and may even indicate fibrin deposits within the dermis. However, as observed in Figure 2.7, many structures seemed to be unnecessarily stained red, which introduces some doubt regarding the specificity of this stain.

GT staining has been beneficial in skin research for illustrating depth of injury as changes in collagen colour staining. Its use in burn research is limited, however it has similar results to MT staining. Through optimisation and evaluation of this stain in burned porcine skin, it is evident that burn markers of injury are clearly identified.

Chapter 2: Selection and Optimisation of Staining Methods 31

The simplicity of this stain’s method to identify multiple markers of injury suggest this stain would be effective in further assessing burns.

2.5 MARTIUS SCARLET BLUE

2.5.1 Introduction The Martius Scarlet Blue (MSB) stain is a reliable fibrin stain, utilising trichrome properties to additionally stain collagen and RBCs. This stain was chosen for this study due to its popularity in fibrin detection, so we could determine fibrin’s relevance in BWP. This stain consists of three major stains, Martius yellow (to stain RBCs), brilliant crystal scarlet (to stain fibrin) and methyl blue (to stain collagen). A reference section of porcine placental tissue, gifted by the histology teaching laboratory at QUT, was used as a positive control for fibrin.

2.5.2 Reagents The reagents used for this stain in optimisation and final stages are as follows: Picric acid (Sigma-Aldrich, St Louis, Missouri, USA), Weigert’s solution A

(Haematoxylin) , Weigert’s solution B (FeCl3 + HCl), Martius yellow (377767, Sigma- Aldrich, St Louis, Missouri, USA), brilliant crystal scarlet (C0644, Sigma-Aldrich, St Louis, Missouri, USA), methyl blue (A18174, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1% phosphotungstic acid (PTA) (Sigma-Aldrich, St Louis, Missouri, USA), 1% acetic acid, 0.5% acid alcohol (Sigma-Aldrich, St Louis, Missouri, USA), 0.1% ammonia (Sigma-Aldrich, St Louis, Missouri, USA), graded ethanol (70%, 90%, 100%), xylene, Depex mounting medium.

2.5.3 Protocol The initial stepwise protocol followed is described below.

1. Rehydrate sections – 2 rinses in xylene (3 minutes each) and 4 rinses in ethanol (1 minute each) in decreasing concentration (100%, 100%, 90%, 70%).

2. Post-fix sections in Picric Acid for 60 minutes at 60°C.

3. Rinse sections in tap water until sections appear clear.

4. Stain with Weigert’s Iron working solution for 10 minutes using inverted method in petri dishes.

5. Rinse sections in tap water.

Chapter 2: Selection and Optimisation of Staining Methods 32

6. Differentiate in 0.5% acid alcohol (leave slightly overstained)

7. Wash well in tap water and then blue with 0.1% ammonia.

8. Stain with Martius yellow solution for 10 minutes using inverted method.

9. Rinse sections in de-ionised water.

10. Stain with brilliant crystal scarlet solution for 5-7 minutes.

11. Rinse sections quickly in de-ionised water.

12. Differentiate with 1% PTA until RBC are yellow and fibrin is a clear red.

13. Stain in methyl blue solution for 5-8 minutes.

14. Rinse quickly in 1% acetic acid.

15. Dehydrate sections – 2 quick rinses in 100% ethanol then 2 rinses in xylene (3 minutes each).

16. Coverslip slides using Depex mounting medium. Leave to dry for at least 15 minutes.

Note: This protocol was optimised by using porcine placental tissue sections as a positive control during staining procedures.

A standard MSB protocol was followed, with the specific protocol retrieved from the QUT Manual of Histological Techniques (Appendix A). Initial Weigert’s iron haematoxylin was applied to stain cell nuclei. Sections were then stained with Martius yellow followed by brilliant crystal scarlet which were differentiated using 1% PTA. Methyl blue staining was then conducted before rinsing in 1% acetic acid.

2.5.4 Optimisation In optimising this stain, both unburned skin and burned skin was used. Unburned skin generally should not contain fibrin and the presence of fibrin in burned skin is unconfirmed. Therefore, to produce an accurate staining protocol, porcine placenta was used as a positive control to determine staining and differentiation times. Placenta makes an excellent positive control for MSB staining as it is known to include all relevant structures MSB intends to stain RBCs, fibrin, and collagen. The current protocol was employed on the placental tissue section, showing clearly defined staining of yellow RBCs, red fibrin, and blue collagen on a clear background (Figure 2.8 A). From the appearance of the placenta, differentiation steps were omitted, and

Chapter 2: Selection and Optimisation of Staining Methods 33

Figure 2.8 Optimising Martius Scarlet Blue stain in porcine skin, compared to porcine placenta. A) Porcine placenta positive control (Scale: 200μm) Optimal staining is shown with yellow red blood cells, red fibrin and blue collagen.; B) Burned skin exhibiting red fibrin deposits, indicated by the arrow (Scale: 50μm); C) Unburned skin, superficial dermis; D) Unburned skin, deep dermis (Scale: 200μm). Optimal staining within the skin can be observed as individual red blood cells within the vessels, clear collagen fibres and fibrin deposits adjacent to blood vessels. staining times were modified: scarlet red was stained for six minutes and methyl blue was reduced to three minutes of staining time. After staining in Weigert’s Hx, sections were blued and observed, staining was light and thus acid alcohol differentiation was unnecessary. Fibrin was stained with brilliant crystal scarlet for 6 minutes achieving a clear, effective stain of appropriate intensity, requiring no differentiation step. Methyl blue staining of collagen was reduced to 3 minutes to match the intensity of fibrin, resulting in optimal staining of all structures. This same protocol was employed on the porcine skin in which the MSB was effective in staining collagen, RBCs, and fibrin as well as other structures. Red staining surrounding BVs is indicative of fibrin deposits present in burned tissue (Figure 2.8 B).

Normal, unburned skin was stained to visualise the appearance of a healthy epidermis and dermis. Low magnification images of the sections are observed in Figure 2.8 C-D, the dermis is stained primarily blue, with no yellow or red staining (red staining of stratum corneum is not indicative of fibrin). This is indicative of healthy, uninjured skin as there is no evidence of injury. The correct protocol required

Chapter 2: Selection and Optimisation of Staining Methods 34

for optimal MSB staining of porcine placenta has shown to accurately stain porcine skin and identify fibrin deposits.

2.5.5 Evaluation in burned tissue The MSB stain is a valuable stain in skin analysis, however it appears to be of little value for observing burn injuries. The presence of collagen denaturation is identified in Figure 2.9 A and B, by bundling of collagen fibres and inconsistent spacing within the dermis. Free RBCs, indicated by arrows, are also observed in the dermis, located within and outside dermal BVs. Figure 2.9 C displays red staining of collagen fibres present superficial to a band of inflammatory infiltration. However, red staining of this structure is not consistent with the appearance of fibrin within the skin. This suggests that the scarlet red stains the denatured collagen due to increased aggregation of collagen, most likely resulting from heat displacement of water. A band of inflammatory cells can also be identified in another section, shown in Figure 2.9 E- F. A high magnification image shows inflammatory cells as small dark cells occupying the dermis, in and around collagen fibres.

Evidence of fibrin staining can be identified by red staining of fibrous like structures commonly adjacent to a BV stained red-orange. As observed in Figure 2.9 D, red staining is present surrounding a BV previously observed by immunostaining for fibrin in contact porcine burns (53). Within the inflammatory band, red staining can be observed in collagen fibres (Figure 2.9 F). Due to the structure, appearance and location, this red staining indicates fibrin presence, but this is not confirmed. Figure 2.9 G-H presents images taken of deep dermal structures, located bordering the adipose layer. Scarlet red staining is observed as web-like structures present within collagen fibres. These structures are similar in appearance to GT staining (Figure 2.7H), and consistent with NETs. Due to the indication of fibrin, these structures identified in MSB staining further support the presence of NETs in burns.

Chapter 2: Selection and Optimisation of Staining Methods 35

Figure 2.9 Burn induced injuries visualised with Martius Scarlet Blue staining. A, B) Change in collagen structure indicated by irregular spacing between collagen fibres. Arrows indicate free red blood cells within the dermis, stained yellow (Scale: 50μm); C) Collagen colour changed from blue to bright red (arrows) superficial to inflammatory cells; D) Completely blocked blood vessel indicated by an arrow, with red fibrin deposit staining adjacent to the vessel (Scale: 200μm); E) Low magnification image of inflammatory band (Scale: 50μm); F) High magnification of inflammatory band, showing individual inflammatory cell nuclei stained black and arrows indicating red fibrin deposits in the dermis (Scale: 200μm); G,H) Web-like structures located in the deep dermis and adipose tissue, stained red. Webs are consistent in appearance to neutrophil extracellular traps reported in literature (69, 74) (Scale: 200μm).

Chapter 2: Selection and Optimisation of Staining Methods 36

MSB staining has demonstrated its effectiveness in identifying markers of damage in burned porcine skin. Its specificity for fibrin allows for in depth analysis of vascular pathologies leading to BWP. However, red staining is observed in areas not consistent with fibrin deposits. The effectiveness of MSB staining is dependent on the relative molecular densities of RBCs, fibrin, and collagen. Although certain structures stained red are consistent in structure and location to fibrin, the presence of fibrin is not conclusive. To verify the presence of fibrin, an antibody specifically for fibrin would confirm the identity of these structures. In the next section, a polyclonal anti- fibrinogen antibody was selected due to its common use in IHC and immunofluorescence (IF) staining of FFPE sections.

2.6 IMMUNOHISTOCHEMISTRY

2.6.1 Introduction The presence of fibrinogen and fibrin deposition play a role in wound healing and haemostasis. Detecting fibrinogen and fibrin may indicate specific mechanisms in wound healing which may or may not contribute to BWP within the burn. The observations of burned tissue with GT and MSB staining suggest the presence of fibrin deposits within the tissue at 72 hours post-burn. Trichrome stains are dependent on discrepancies in density to selectively distinguish different structures. Although the deposition of fibrin is probable in burns, these stains are not conclusive in their identification of fibrin. A polyclonal anti-fibrinogen antibody was utilised to accurately stain early stages of thrombosis and fibrin formation (75). Porcine placental tissue sections were used as controls and burn samples were 72 hours post-burn.

2.6.2 Reagents The reagents used for this stain in optimisation and final stages are as follows: xylene, graded ethanol (70%, 90%, 100%), Tris Buffered Saline (TBS) (Invitrogen, Carlsbad, California, USA), Triton X-100 (Sigma-Aldrich, St Louis, Missouri, USA), sodium citrate (Chem-Supply, Gillman, South Australia, Australia), Tween 20 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 2.5% trypsin (Sigma- Aldrich, St Louis, Missouri, USA), calcium chloride (Thermo Fisher Scientific, Waltham, Massachusetts, USA), bovine serum albumin (BSA) (10099141, Thermo Fisher Scientific, Waltham, Massachusetts, USA), goat normal serum (X0907, Agilent Technologies, Santa Clara, California, USA), rabbit polyclonal anti-fibrinogen

Chapter 2: Selection and Optimisation of Staining Methods 37

antibody (ab34269, Abcam, Cambridge, United Kingdom), goat anti-rabbit Alexa Fluor 488 (A11008, Invitrogen, Carlsbad, California, USA).

2.6.3 Protocol The initial protocol followed is as described below.

1. Rehydrate sections – 2 rinses in xylene (3 minutes each) and 4 rinses in ethanol (1 minute each) in decreasing concentration (100%, 100%, 90%, 70%), then into water.

2. Rinse slides in TBT-T (TBS + 0.025% Triton X-100) for 3 x 5 min washes.

3. Follow antigen retrieval methods (heat or enzymatic)

4. Wash in TBS-T in 3x 5 min washes.

5. Using a Kimwipe, wipe excess TBS off the slide and around the section leaving a small amount of liquid remaining over the entire section. Place slides in humidifying chamber.

6. Apply 100μl per slide of 10% goat normal serum in 1% BSA in TBS for 1 hour at room temperature.

7. Remove by tapping the slide and using a Kimwipe to wipe excess solution as in Step 5.

8. Apply 100μl per slide of primary antibody diluted in 1% BSA in TBS. Incubate overnight at 4°C.

9. Tap slides to remove excess solution before washing in TBS-T in 3x 5 min washes.

10. Repeat step 5.

11. Apply 100μl per slide of fluorophore-conjugated secondary antibody to the slide diluted in 1% BSA in TBS. Incubate for 1 hour at room temperature (should be done in the dark to prevent photobleaching).

12. Tap slides to remove excess solution before washing in TBS-T in 3x 5 min washes.

13. Mount with fluorescent mounting medium and coverslip.

Antigen retrieval protocols

Chapter 2: Selection and Optimisation of Staining Methods 38

Heat antigen retrieval – microwave method

1. Add sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0) to microwaveable vessel.

2. Place slides in the microwaveable vessel and vessel into microwave. Allow buffer to boil for 20 minutes. Check periodically to ensure buffer does not completely evaporate. Refill buffer if necessary, to avoid slides drying out.

3. Once completed, remove vessel from microwave and run cold tap water for 10 minutes to cool down slides.

Enzymatic antigen retrieval – pipetting method

1. Carefully blot excess water from slide and around the tissue.

2. Pipette trypsin working solution (2.5% trypsin diluted to 0.5% in calcium chloride stock solution, pH 7.8) to cover the entire section.

3. Place slides in a humidified container and then into preheated 37°C incubator for 20 minutes.

4. Rinse by running tap water for 3 minutes.

2.6.4 Optimisation A rabbit polyclonal anti-fibrinogen antibody was selected due to its ability to detect fibrinogen from multiple species and its successful application in paraffin embedded IHC and IF. Fibrinogen and fibrin detection using IF staining has not been utilised previously to examine burns pathology. Therefore, the use of the antibody is experimental and optimisation processes are based on the standard protocol provided by the supplier with some slight modifications. In all optimisation stages, a porcine placental tissue section control and 72 hour post-burn samples were stained. Antigen retrieval techniques were tested to determine which method is most appropriate for this protocol. No antigen retrieval, trypsin antigen retrieval and heat antigen retrieval (Figure 2.9 C) were assessed. Primary antibody (ab34269, Abcam, Cambridge, United Kingdom) at 1:100 was incubated overnight at 4°C and secondary antibody (A11008, Invitrogen, Carlsbad, California, USA) at 1:500 was incubated for one hour at room temperature. No antigen retrieval (Figure 2.9 A) and trypsin mediated antigen retrieval (Figure 2.9 B) showed dull green, auto fluorescent background staining of skin

Chapter 2: Selection and Optimisation of Staining Methods 39

Figure 2.10 Fibrinogen immunofluorescence staining optimisation. Porcine skin and porcine placenta controls were stained with a rabbit anti-human fibrinogen primary antibody (ab34269, Abcam, Cambridge, United Kingdom) and a goat anti-rabbit secondary antibody tagged with Alexa Fluor 488 (A11008, Invitrogen, Carlsbad, California, USA) to identify fibrin and fibrinogen deposits. A) No antigen retrieval, primary antibody (1:100) overnight, secondary antibody (1:500) for 1 hour; B) Antigen retrieval using 0.05% trypsin, primary (1:100) overnight, secondary (1:500) 1 hour; C) Heat antigen retrieval by microwaving for 20 minutes, primary (1:100) overnight, secondary (1:500) 1 hour; D) Heat antigen retrieval, primary (1:100) 1 hour, secondary (1:500) 1 hour; E) Heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour; F) Heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour; G, H) Porcine placenta positive controls, heat antigen retrieval, primary (1:200) overnight, secondary (1:500) 1 hour. Arrows indicate positive fibrinogen/fibrin staining in the sections (Scale: 50μm).

Chapter 2: Selection and Optimisation of Staining Methods 40

structures, with no distinct positive staining. Microwave heat antigen retrieval was conducted using one-minute intervals for the first five minutes, followed by five- minute intervals to reach a total of 20 minutes. Figure 2.10 C shows comparatively better contrast between brighter components and background staining, however further optimisation of the heat antigen retrieval method was required to reduce background staining. Microwaving time intervals were set to five-minute intervals to make up 20 minutes total, to allow for refilling of the sodium citrate buffer to prevent the slides drying out. By continually refreshing the buffer, the boiling was able to be continued for 20 minutes to maximise antigen retrieval. To further reduce background staining, the primary antibody incubation time was decreased to one hour or the primary antibody concentration was reduced to 1:200 and left overnight. Reducing the incubation time to one hour instead of overnight, reduced background staining, however it also reduced positive staining of fibrin as seen in Figure 2.10 D. Using a dilution of 1:200 primary antibody resulted in brighter, positive staining of fibrin in deep dermis (Figure 2.10 E-F). Figure 2.10 F shows distinct fluorescent staining of fibrin indicated by arrows, with dark background within burned skin sections.

Optimal staining of fibrinogen in porcine skin was obtained with microwave heat antigen retrieval, primary antibody at 1:200 overnight at 4°C, and secondary antibody at 1:500 for one hour at room temperature. Concurrent optimal fluorescent staining of porcine placenta using the same technique verifies the fibrinogen staining identified in burned porcine skin. Figure 2.10 G-H show distinct bright fluorescent staining of fibrinogen against dull background staining in the placenta sections.

2.6.5 Evaluation in burned tissue The fibrin observed in burned porcine skin using the MSB staining was red and resembled web-like structures (Figure 2.8 F-H). Positive IF staining of fibrinogen in is similar in appearance to this, as seen in Figure 2.10 E-F. Fibrin deposition identified in injured skin 72 hours post-burn by MSB staining was confirmed by IHC staining of fibrinogen using a polyclonal anti-fibrinogen antibody. The use of this antibody in porcine burn models is novel and should be further optimised in future studies. Additionally, the use of 4′,6-diamidino-2-phenylindole (DAPI) to counterstain nuclei would assist with defining cell boundaries.

Chapter 2: Selection and Optimisation of Staining Methods 41

2.7 STAIN ASSESSMENT

Five stains were selected and optimised for porcine skin analyses – H&E, VVG, GT, MSB and anti-fibrinogen antibody. These stains were selected based on their previous use in literature and their ability to identify the damage associated with, collagen denaturation, cell death and vascular pathologies. The H&E stain was evaluated as an effective stain to identify all these markers of damage in a single stain. VVG, GT and MSB were chosen for their ability to visualise collagen denaturation. However, for the Chapter 3 analyses, which are more detailed and time-intensive, the number of stains will be decreased.

When comparing the collagen stains, they all share the ability to identify collagen denaturation, but can also visualise other features. Therefore, in the selection of staining for Chapter 3, the stains were assessed on the value of the other features stained. The specificity of elastin staining in VVG stains was of interest, as elastin’s biomechanical properties preventing wound contraction and scar formation (76). However, the current optimal staining of porcine skin showed low elastin content, and the elastin was located primarily in the superficial dermis. The VVG staining of elastin in burned tissue indicated elastin fibres were inconsistent in length and appearance (Figure 2.5 B, G, H). Therefore, the VVG stain was not continued for BWP analysis. GT staining of burned sections highlighted fibrous structures adjacent to BVs deep in the dermis (Figure 2.7 D). These structures were consistent in location and structure of fibrin deposition in burn injuries. This suggested fibrin staining might be relevant for observing pathological mechanisms in burns and consequently led to the inclusion of MSB, as this stain is known for accurately detecting fibrin. After observations of damage markers in the skin, it is evident that GT is less specific compared to MSB. Background red staining of inflammatory cells in GT can be observed in Figure 2.7 F, in comparison to the MSB staining in Figure 2.8 F, where individual cells are easily identified. The NETs observed in the deep dermis in both GT and MSB stains also demonstrate the overintensity in GT. The clarity of the NET structures with the GT stain in Figure 2.7 H is noteworthy, however the more specific nature of MSB only subtly stains these structures, visualised in Figure 2.8 G and H. The MSB staining suggests that these structures may not be as large as initially observed with the GT stain. The GT staining protocol involves a one-step solution, and there are no differentiation steps to wash away excess staining and eliminate background staining.

Chapter 2: Selection and Optimisation of Staining Methods 42

MSB reduces this through its protocol by individually applying each stain to achieve a more accurate result. The use of specific anti-fibrinogen antibody staining effectively verified the presence of fibrin observed by MSB in these burn models.

H&E, MSB and fibrinogen IHC staining were the optimal stains for this study, as they accurately stained the burn damage markers identified in literature: collagen denaturation, cell death and vascular pathologies; with a specificity for fibrin identification.

2.8 CHAPTER 2 DISCUSSION

In previously published burns research, an assortment of histological techniques has been used to measure burn depth and analyse the pathological environment of burn wounds. After critically reviewing the literature, a combination of histochemical and immunohistochemical stains were chosen for this project. These stains included: H&E, VVG, and GT. MSB and IHC staining for fibrinogen were added to this project to further examine the involvement of fibrin in burn wounds. The chosen stains were optimised to achieve the appropriate protocol for burn wound analysis in porcine skin. Once optimised, each stain was evaluated for its capacity to assess burn wounds and their intended burn markers. HMGB1 and cleaved Caspase 3a IHC stains were also initially chosen from the literature as potentially useful techniques to identify burn wound damage. However, they were excluded from this study as the optimisation process would have been extensive and time-consuming.

H&E was evaluated as a comprehensive stain and was able to visualise general structures in skin and identify multiple burn induced pathologies such as collagen denaturation, cell death and vascular pathologies. VVG and GT staining was previously identified in literature to differentiate between healthy and denatured collagen. However, unlike reports in the literature, the appearance of denatured collagen in the burn wounds in this study was not visualised by a consistent difference in staining colour, but by identifying the change in structure of the collagen fibres stained (e.g. collagen bundles and fissures between fibres). This was identified as collagen denaturation in burns of all depths, from superficial to full thickness burns. Our results are similar to those reported by Sheu et al. who conducted MT staining of contact burns, in which superficial and superficial-partial thickness burns exhibited collagen fibrils that were not clearly discerned, with some collagen fibres appearing to

Chapter 2: Selection and Optimisation of Staining Methods 43

fuse together. Changes in collagen staining colour, from blue to red, has previously only been identified in contact DDPT burns (15). This may indicate that the visualisation of collagen denaturation in DDPT to FT burns may differ between scald and contact burns. GT staining aimed to surpass specificity of staining collagen denaturation in comparison to MT staining. The results of this stain show that collagen denaturation does not simply cause a change in staining colour. As GT staining produces similar results in collagen assessment to MT staining, it cannot be concluded that GT staining is better. VVG staining is advantageous for assessing elastin contact within the wound. No research has been conducted in determining the role of elastin in BWP, however its role in preventing and reducing wound contraction and scar development is recognised (76-78). The effects of wound contraction may be associated with BWP, and the presence or change in elastin content may be relevant. However, the VVG staining indicated that elastin fibres were inconsistent in length and appearance. The GT staining highlighted the potential importance of fibrin deposits, which lead to the adoption of an MSB stain.

The MSB stain primarily distinguishes three difference structures: RBCs, fibrin, and collagen. In burn wounds, the MSB stain is a valuable stain for its ability to identify vascular pathologies, collagen denaturation and fibrin deposition. The role of fibrin and fibrinogen formation is linked to BV occlusion and microthrombi formation through various pathways (53). IHC staining for fibrinogen was utilised to further verify the presence of fibrin in porcine skin.

The fibrillar structure observed by GT (Figure 2.7 H) and MSB (Figure 2.8 G, H) staining is consistent with NETs observed in literature. NETs have been found to have antimicrobial properties to trap and kill bacteria, fungi, and parasites (79-81). The presence of NETs and fibrinogen formation have been linked with promoting prothrombotic molecules and platelet aggregation, triggering coagulation cascades (82). NETs have also been associated with neutrophil death, described as NETosis, which differs from necrotic or apoptotic cell death as nuclear and granular membranes disintegrate but plasma integrity is maintained (83, 84). The detection of NETs or NET like structures in burn wounds which resemble NETs observed with electron microscopy has not been previously reported. More research into the role of these structures may indicate mechanisms responsible for BWP.

Chapter 2: Selection and Optimisation of Staining Methods 44

For the purpose of this study, due to time restraints, only H&E staining will be used for Chapter 3 in assessing burn depth and intensity. These burn wound assessments require examination of all dermal and epidermal structures, and H&E staining effectively enables identification of these structures.

Chapter 2: Selection and Optimisation of Staining Methods 45

Chapter 3: Burn Wound Progression Analysis

3.1 INTRODUCTION

In this chapter, haematoxylin and eosin stained burn tissue was analysed to identify the mechanisms responsible for BWP. Burn wound samples from burn wounds that progressed and became deeper over a period of three days, were compared to a control group with no progression. The severity of these burns was assessed using depth and intensity measurement techniques to identify markers of burn damage. Burn depth is the most common metric for burn severity, however measuring intensity (the amount of total damage to the tissue), provides a greater insight into the wound pathology. By identifying burn damage markers consistently present within burns that progress or deepen in depth, the mechanisms responsible for BWP can be determined.

This chapter is divided into two main parts. The first part describes the process of developing the novel burn rubric and its application for assessing burn damage in skin. The second part utilises the burn rubric to evaluate the extent of burn damage within each skin section and compare progression burns to non-progression burns.

3.2 BURN MODEL SELECTION

To effectively investigate BWP, biopsies from porcine burn wounds were used. Porcine scald burn models were developed previously by Dr. Christine Andrews (39). Using this scald burn model, burns were applied with different temperatures and durations to gain a range of burn conditions, including low temperature – long duration, or high temperature – short duration burns. Biopsies were collected post-burn at four-time intervals: 1 hour, 24 hours, 72 hours, and 7 days. These biopsies were fixed in formalin, sectioned by QUT histology services at 3 μm thickness and then were stained using the optimised protocol developed in Chapter 2.

The set of slides chosen for this chapter were selected because they showed evidence of BWP, where the depth of the burn deepened over time. For this study, progression burns were classified as burns which at one-hour post-burn were less than 50% burn depth and progressed to more than 75% burn depth at 72 hours post-burn.

Chapter 3: Burn Wound Progression Analysis 47

Figure 3.1 Burn depth information data for the sections chosen for this study, based on data previously calculated by Christine Andrews (68). Each line represents a specific group chosen for analysis, including progression (pink) and non-progression (green) burns, and burns which are of low temperature – long duration (circles or triangles) and burns which are high temperature – short duration (squares or diamonds), dotted lines represent criteria thresholds for each group, progression (<75% at 1 hour and ൒75% at 72 hours and non-progression (between 25% and 75% over 72 hours). A) Combined burn depth, points are represented as the mean ± standard deviation, with n=3 replicates for each condition; B-C) Individual burn data for progression burns; D-E) Individual burn data for non-progression burns.

Non-progression burns were classified as burns which maintained a burn depth of between 25% and 75% over the 72 hours post-burn. The 75% threshold was based on results from a previous study by Dr. Christine Andrews which examined the association between histological burn depth assessment and time to re-epithelialisation (34). Depth measurements provided by Christine’s data of the selected sections are plotted in Figure 3.1, detailing the depth of burns over 72 hours post-burn.

Chapter 3: Burn Wound Progression Analysis 48

The study reported that wounds which had burn depths of ൒75% assessed histologically took longer than 21 days to re-epithelialise, indicating they are clinically more serious, would definitively scar and would require more aggressive treatment such as surgery in order to heal (34). Day 7 sections were omitted from this study as burn wounds were found to progress to the full extent of damage by 72 hours post- burn (39), and BWP occurred mostly between 1 hour and 72 hours post-burn. Therefore, only biopsies collected at 1 hour, 24 hour and 72 hours post-burn were selected for this study. A total of 12 burns were examined, split equally into two groups: progression and non-progression with six burns in each. Within each group, burn conditions were chosen so that there were three replicates each of low temperature – long duration and high temperature – short duration burns. With each burn consisting of three sections at each time point, the total working set for this chapter is made up of 36 sections. The distribution of sections per group are provided in Figure 3.2. In total, the selected burns were conducted on 8 pigs. Sections from four normal, unburned skin biopsies were selected as a control from the same animals. Specific burn conditions of burn temperature and duration are described in Table 3.1.

Progression (6 burns, at 3 timepoints, n=18)

Low temperature – Long High temperature – Short duration (3 burns, n=9) duration (3 burns, n=9)

1 hour 24 hours 72 hours 1 hour 24 hours 72 hours (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3)

Non-Progression (6 burns, at 3 timepoints, n=18)

Low temperature – Long High temperature – Short duration (3 burns, n=9) duration (3 burns, n=9)

1 hour 24 hours 72 hours 1 hour 24 hours 72 hours (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3) (3 burns, n=3)

Figure 3.2 Burned sections data representing burn treatment group, burn conditions and timepoints. The number of burns represented in each group are shown, n equals number of sections for each group.

Chapter 3: Burn Wound Progression Analysis 49

Table 3.1 Burns selection information detailing animal number, burn conditions (temperature and duration) and progression criteria. LT+LD: low temperature/long duration, HT+SD: high temperature/short duration. Animal Burn Progression Temperature Duration and Conditions (൒75% at 72 (°C) (seconds) Wound hours) #4-16 ri A 50 600 LT+LD Yes #3-16 ri A 60 60 LT+LD Yes #3-16 ri C 60 30 LT+LD Yes #3-16 ri D 90 5 HT+SD Yes #3-16 le A 90 5 HT+SD Yes #7-14 ri C 100 5 HT+SD Yes #7-15 ri A 55 120 LT+LD No #8-15 ri A 60 30 LT+LD No #12-15 ri B 60 30 LT+LD No #6-15 le D 85 5 HT+SD No #6-15 ri A 85 5 HT+SD No #11-15 le D 85 5 HT+SD No

3.3 BURN ASSESSMENT METHODOLOGY

The burn conditions (temperature, duration), timepoints and treatment group of each section were blinded from the scorer to prevent any biases from prior knowledge of the sections, as some sections scored in Chapter 3 were previously utilised to optimise stains in Chapter 2. I was the sole scorer in assessing burn depth and burn intensity during this study.

3.3.1 Burn Depth The severity of burns is routinely categorised and distinguished by burn depth, in which deeper burns are more severe. There are several methods of assessing burn depth, as described in the literature. Burn depth can be measured by gross examination, observing the border contraction, scarring, colouration, and reepithelialisation. This method is most often used clinically, as it is non-invasive. However, this assessment is limited as it cannot assess what is happening in the skin under the skin surface. Punch biopsies and histological staining have been described as the gold standard for burn depth assessment as it provides a comprehensive snapshot on the wound, for the entire depth of the skin (11, 12).

Burn depth in tissue biopsies was measured histologically in this project by using CaseViewer (v2.4, 3DHISTECH, Budapest, Hungary) software. Injury area was defined by observing the deepest points of structural injury across the section and

Chapter 3: Burn Wound Progression Analysis 50

drawing a green line which represented the border between damaged and undamaged tissue (Figure 3.3). The surface area above the line of injury was defined as the area of injury and was calculated as a percentage of the entire section area (blue line). Points of injury observed as damage to dermal structures, including BVs, hair follicles and glandular structures were used to create the line of injury. Damage of these structures were observed as cellular necrosis, structural damage and coagulated or partially coagulated BVs. As different tissue sections were of different sizes, the section area was determined prior to measurements. The total section area was drawn from the top of the epidermis (top of dermis in case of absent epidermis) to the bottom of the dermis. Left and right borders were drawn vertically to resemble a rectangular shape.

Area measurements of the area of injury and total area were exported from CaseViewer in square microns. The area of injury was divided by the total area to calculate the burn depth as a percentage of total tissue area.

Alternate methods of digitally calculating burn depth were tested to determine the most accurate measurement. Aperio ImageScope (v12.4.3, Leica Microsystems, Wetzlar, Germany) was also tested to calculate the injury depth. This software utilises independent distances between lines and measured the injury distance as a percentage of the total distance of the section (i.e. distance, rather than area). However, Aperio ImageScope is limited to only including 25 distance calculations between injury and tissue lines. This causes less units of measurement between lines, limiting the accuracy of the average measurement as it may not consider unusual fluctuations in distance.

Figure 3.3 Screen captures of the burn depth area measurements, calculated in CaseViewer. Area of structural tissue injury is defined by the green line; the total area of the section is defined by the blue line. Burn depth was calculated as injury area/total area. Scale: 200μm.

Chapter 3: Burn Wound Progression Analysis 51

Therefore, this software was not considered an appropriate tool to measure depth due to its limitations in accuracy in comparison to CaseViewer.

3.3.2 Development of Burn Damage Rubric The current methods of measuring burn injury and severity revolve around burn depth, and the deepest points of damage across the section. During burn depth measurements, it was observed that structures located superficial to the deepest point of damage were healthy and uninjured, despite the injury progressing deeper. To consider the presence of healthy structures within the injury area, a measure of burn intensity was developed. Burn intensity measures the severity of the burn, irrespective of its depth, allowing burns to be analysed with more detailed information than just depth. We hypothesised that this measure of burn injury could identify differences in wounds with identical depth measurements.

A burn injury rubric was developed to identify markers of burn induced damage, through initial observations of burned skin in Chapter 2. Features were defined as major structures or components within the skin. Damage to the features were identified by specific injury markers, describing the type of injury observed in the feature. Table 3.2 lists the features and burn markers utilised in the burn injury rubric for assessing burn intensity. Large blood vessels were defined as vessels with diameter >100 microns and small blood vessels were defined by lumen diameter >10 microns but <100 microns. Glandular structures included apocrine and merocrine glands within the dermis. Collagen indicated the appearance of collagen denaturation. Red blood cells describe the presence of extravasated red blood cells in the dermis. Inflammatory response describes the infiltration of inflammatory cells and their expression within the dermis.

To measure the burn intensity using this rubric, the section was divided into four sections, from superficial to deep (Table 3.3). These quadrants were developed to assist with scoring, and to also be representative of the different burn classifications.

Quadrants were divided using CaseViewer, in two techniques. Figure 3.4 A utilises straight horizontal lines at depths of 25, 50 and 75%, measured from the left and right borders, to develop four quadrants across the section. This technique was used in sections which best resembled a rectangle however, due to the morphology of skin biopsies, sections would often present as an arc (Figure 3.4 B). In these tissue

Chapter 3: Burn Wound Progression Analysis 52

ures and burn markers in burn intensity measurement. Table 3.2 Burn injury rubric utilised to identify feat

Chapter 3: Burn Wound Progression Analysis 53

sections, quadrant lines were made up of two straight lines, connecting at the median point of the section. Depths of 25, 50 and 75% were measured at the left border, right border, and centre line.

Table 3.3 Burn intensity scoring quadrants. Depth measured from superficial to deep epidermis, with quadrants indicating damage associated to burn classifications. Quadrant Depth Burn Indication 1 0 – 25% Superficial 2 25 – 50% Superficial partial thickness 3 50 – 75% Superficial partial thickness 4 75 – 100% Deep dermal partial thickness – full thickness

A

B

Figure 3.4 Screen captures of two techniques utilised to draw quadrants in burn intensity assessments. Lines were measured and drawn at depths 25, 50, and 75%, forming four quadrants across the section. A) Quadrant lines are straight horizontal lines, depths measured at left and right borders; B) Quadrant lines are made up of two lines joined at the median, depths measured at left border, right border, and centre line.

Chapter 3: Burn Wound Progression Analysis 54

Burn intensity assessment was conducted by individually observing each feature present in the section and examining what burn markers were present as damage within that feature. Injury markers were scored on a binary scale as present (1) or absent (0), providing an injury score for each burn marker. This score was entered into an Excel Worksheet (v2008, Microsoft Corporation, Albuquerque, New Mexico, USA) into each cell corresponding to the specific marker (e.g. partially blocked vessels) for that feature (e.g. small BVs), for that quadrant, in that tissue section. The burn rubric scoring template is provided in Appendix B. Scores were standardised to account for the number of features present in each tissue section, and the number of features across all sections. For epidermis, BVs, hair follicles and glandular structures, this was calculated by dividing the sum of the marker score by the number of features, producing an injury score from zero to one. For RBCs, collagen and inflammatory response, damage markers were unable to be assessed individually. Therefore, the presence of the injury (0-1) in each quadrant, was then divided by number of quadrants (4), also producing an injury score from zero to one. By standardising scores, sections can be appropriately compared against other sections.

An overall score of 1 for a particular feature would indicate that all burn markers in that feature would also have a maximum score, and all damage markers were observed throughout the section. There were 12 replicate slides scored for each timepoint, and 36 slides in total. For each marker and feature, the average and standard deviation were calculated for each timepoint and progression or non-progression burns, and these were plotted on graphs using GraphPad Prism (v8.2.1, GraphPad Software, San Diego, California, USA).

Statistical analyses were carried out using GraphPad Prism. Burn depth and intensity correlation analyses were conducted with Spearman correlation of burn depth and intensity correlations were conducted using non-parametric Spearman correlation with a confidence interval of 95%. Comparisons between timepoints and progression or non-progression burns were conducted using nonparametric, unpaired Mann- Whitney tests with 95% confidence level. P-values less than 0.05 were considered statistically significant.

Chapter 3: Burn Wound Progression Analysis 55

3.4 RESULTS

A total of 40 sections were analysed in depth and intensity using methods described in sections 3.3.1 and 3.3.2, including 36 burned sections and 4 normal control sections. Burn depth results were recorded in progression and non-progression burns to determine any correlation between depth and intensity. Intensity scores are shown by feature and divided by each individual damage marker and overall feature damage.

3.4.1 Burn Depth In section 3.2, burn model selection was determined by previous burn depth measurements conducted by Dr. Christine Andrews (39) of the selected sections. Burn depth was required to be measured and recorded for these sections to account for sectioning variability (different parts of wound being sectioned) and individual variability in burn depth assessment (between scorers). As shown in Figure 3.5, progression and non-progression burns maintain initial burn depth assessments. Progression burns are initially below 50% and convert to over 75% in depth, whilst non-progression burns sustain a burn depth between 25% and 75% over 72 hours.

Time Post-burn 1 hour 24 hours 72 hours Progression 0.42±0.18 0.83±0.18 0.92±0.13 Non-Progression 0.39±0.17 0.57±0.16 0.44±0.11 Figure 3.5 Burn depth results for burned sections analysed in Chapter 3. Non-progression burns (green) and progression burns (pink) plotted on as mean ± standard deviation across 1 hour, 24 hours and 72 hours post-burn with 6 replicates for each point. Dotted lines represent criteria thresholds for each group, progression (<75% at 1 hour and ൒75% at 72 hours and non-progression (between 25% and 75% over 72 hours).

Chapter 3: Burn Wound Progression Analysis 56

3.4.2 Burn Intensity Epidermis Epidermal markers of burn damage include elongated string bean nuclei, shrunken nuclei, loss of adherence and no epidermis, with no epidermis indicating the most intense injury score. Sections with an absent epidermis were scored 1 for all epidermal damage markers. No statistically significant differences were found between different timepoints or between progression and non-progression burns. Large variances are also observed in overall epidermal damage and all burn markers except shrunken nuclei at some time points, as observed by standard deviation bars in Figure 3.6. The large variances in data is a result of only one epidermis per section. Therefore, in evaluating the markers of damage in each section, intensity scores for each section were either 0 or 1 as represented by data points in Figure 3.6 B-D.

The overall epidermal damage graph shows mean intensity scores of burns at all timepoints in both progression and non-progression groups are greater than 0.5, indicating large epidermal damage across all burns. Epidermal markers provide no substantial data between treatment groups or timepoints to be appropriately used for assessing BWP.

Blood Vessels In observing BVs, burn damage markers from the rubric included in burn intensity assessment were partially blocked BV, blocked BV, shrunken nuclei and structural damage. Markers of damaged endothelial cells and absence of endothelial cells were omitted due to limited observations of these markers in burned samples. In observing shrunken nuclei and structural damage of BVs, no statistically significant changes were found between time points or treatment groups. However, large variances are seen in progression burns at 72 hours, indicating that the presence of these markers in these burns are highly variable at this timepoint. Partially blocked vessels were seen to increase statistically significantly from one hour in progression burns. Progression burns showed significant increases in intensity of partially blocked vessels from one hour post-burn (0.41±0.23) to 24 hours (0.79±0.13, p=0.011) and 72 hours (0.84±0.17, p=0.009) post-burn. No significant changes of partially blocked vessels were observed in non-progression burns. Significantly higher intensity scores were found in progression burns (0.84±0.17) in comparison to non-progression burns (0.58±0.09) at 72 hours, indicating that increased partially blocked vessels are

Chapter 3: Burn Wound Progression Analysis 57

Figure 3.6 Intensity scores for epidermal burn damage markers, of non-progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total epidermal damage, calculated as the average of all the individual marker intensity scores (string bean nuclei, shrunken nuclei and loss of adherence) at each timepoint; B) Intensity score for elongated string bean nuclei in the epidermis; C) Intensity score for shrunken nuclei; D) Intensity score for loss of adherence of the epidermis from the dermis. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each timepoint. Statistical analysis demonstrated no statistically significant differences in epidermal burn damage markers. indicative of burns that progressed. Blocked vessels were also observed to significantly increase in intensity scoring in progression burns from one hour post-burn (0.13±0.16) to 24 hours (0.72±0.13, p=0.002) and 72 hours (0.70±0.39, p=0.041) post-burn. Non- progression burns found no significant changes in blocked BV intensity. Progression burns had significantly lower intensity scores of blocked BVs at one hour post-burn in comparison to non-progression burns (p=0.004).

In overall BV damage, significant differences were only observed at 72 hours between the two groups. Progression burns had increased overall intensity scores at 72 hours (p=0.002) in comparison to non-progression burns. Large standard deviation is observed in this group (0.38±0.14) indicating high variability of BV damage at 72 hours in progression burns. Across the remainder of data for overall BV damage, no

Chapter 3: Burn Wound Progression Analysis 58

Figure 3.7 Intensity scores for blood vessel burn damage markers of non-progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total blood vessel damage, calculated as the average of all the individual marker intensity scores (partially blocked vessels, blocked vessels, shrunken nuclei and structural damage at each time point; B) Intensity score for partially blocked vessels; C) Intensity score for blocked vessels; D) Intensity score for shrunken nuclei; D) Intensity score for structural damage. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant differences assessed using Mann- Whitney test between timepoints and treatment group indicated as * = p<0.05, ** = p<0.01.

Chapter 3: Burn Wound Progression Analysis 59

statistically significant differences were observed. However, due to low variances in overall BV damage, these markers are consistent assessing burn wound intensity.

Hair Follicles Hair follicle damage was observed as elongated string bean nuclei, structural damage, and shrunken nuclei. Burned samples had 3.39±2.05 hair follicles per section across progression and non-progression burns. Large variances are observed in the data in Figure 3.8, this is representative of the variance of hair follicles in each section. Statistically significantly higher intensity scores were observed in progression burns compared to non-progression burns at 72 hours, in structural damage and shrunken nuclei. No other statistically significant differences were recorded.

Figure 3.8 Intensity scores for hair follicle burn damage markers, of non-progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total hair follicle damage, calculated as the average of all the individual marker intensity scores (elongated string bean nuclei, structural damage and shrunken nuclei) at each timepoint; B) Intensity score for elongated string bean nuclei; C) Intensity score for structural damage; D) Intensity score for shrunken nuclei. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections (for all except 1-hour NP, 1-hour P and 72-hour P, where n=5) at each time point. Significant differences assessed using Mann-Whitney test between treatment groups indicated as * = p<0.05.

Chapter 3: Burn Wound Progression Analysis 60

Glandular Structures Glandular damage was represented by burn markers of elongated string bean nuclei, structural damage, and shrunken nuclei. Intensity scores recorded for elongated string bean nuclei were low with only four sections scoring more than zero. String bean nuclei in glandular structures may not be an appropriate marker of burn damage. Similar results to hair follicle damage were observed in glandular structures, where intensity scores in progression burns were statistically significantly higher at 72 hours for structural damage (p=0.028) and shrunken nuclei (p=0.028) in comparison to non- progression burns. However, these groups have high variances in structural damage (0.52±0.43) and shrunken nuclei (0.47±0.37).

Figure 3.9 Intensity scores for glandular burn damage markers, of non-progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Total glandular damage, calculated as the average of all the individual marker intensity scores (elongated string bean nuclei, structural damage and shrunken nuclei) at each timepoint; B) Intensity score for elongated string bean nuclei; C) Intensity score for structural damage; D) Intensity score for shrunken nuclei. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant differences assessed using Mann-Whitney test between treatment groups indicated as * = p<0.05, ** = p<0.01.

Chapter 3: Burn Wound Progression Analysis 61

Red Blood Cells Red blood cells as a feature were any extravasated RBCs identified in the dermis. The burn damage markers are differentiated by the location of RBCs with respect to BVs. Nearby BVs describe red blood cells located directly adjacent to any BVs in the dermis, whereas free red blood cells describe RBCs in the dermis not located near distinct BVs.

Figure 3.10 Intensity scores of red blood cell presence in dermis as burn damage markers, non- progression (NP) and progression (P) burns, at 1 hour (black), 24 hours (pink) and 72 hours (green) post-burn. A) Overall red blood cells in dermis, calculated as the average of all the forms of extravasated red blood cell intensity scores (nearby blood vessels and free red blood cells) at each timepoint; B) Red blood cells nearby blood vessels; C) Intensity score for free red blood cells in dermis. Intensity scores are represented as the mean ± standard deviation for 6 replicate sections at each time point. Significant differences assessed using Mann-Whitney test between timepoints indicated as ** = p<0.01.

Chapter 3: Burn Wound Progression Analysis 62

RBC presence nearby BVs significantly increased from non-progression burns at one hour (0.04±0.10) to 24 hours (0.46±0.19, p=0.006) and 72 hours (0.42±0.13, p=0.006). However, no other statistically significant changes between timepoints and burn groups were observed. High variances are observed in progression burns represented in overall RBC presence in dermis by standard deviation bars in Figure 3.9A. These are also reflected in each RBC marker, describing the variability of extravasated RBC in the dermis of progression burns.

3.4.3 Burn Severity Correlation Burn severity can be measured in two metrics: burn depth and burn intensity, as shown in sections 3.4.1 and 3.4.2. Burn severity correlation analyses were carried out between depth scores (Figure 3.11) and burn marker intensity scores. A two-tailed nonparametric Spearman correlation test showed that total BV damage was observed to best correlate with burn depth in progression burns at 24 hours as observed in Figure 3.10 with r2=0.71 (p=0.044).

Figure 3.11 Correlation data of progression burns at 24 hours between depth score and total blood vessel damage intensity scores. Two-tailed nonparametric Spearman correlation was conducted with 95% confidence interval. * = p<0.05.

3.5 CHAPTER 3 DISCUSSION

Burn wound progression was investigated by comparing burn damage markers between progression burns and non-progression burns determined by whether these burns progressed to over 75% burn depth within 72 hours post-burn, as described in section 3.2. The severity of these burns was measured as burn depth and burn intensity utilising a novel rubric for burn intensity scoring.

Chapter 3: Burn Wound Progression Analysis 63

Blinded burn depth scoring of these sections in this project was consistent with initial scoring of the same sections conducted by Dr Christine Andrews (68), demonstrating that the burn depth assessment technique used here is accurate, and enabling accurate detection of different structural features and markers associated with burn damaged tissue. Across replicates at each timepoint for progression (n=6) and non-progression (n=6) burns, burn depth measurements (means) of these sections maintained the same trend of progression to >75% area damage or remained <75% area damaged for non-progression burns.

High variances were observed in epidermal burn markers, elongated string bean nuclei, shrunken nuclei and loss of adherence. Loss of adherence as a damage marker differs from absence of epidermis, as individual burn damage markers could not be identified in sections with no epidermis. To remedy this, sections with no epidermis were scored with maximum injury intensity across all epidermal burn markers to indicate maximal burn intensity. In this rubric, those sections with a score of 1 due to an absent epidermis do not accurately reflect the total damage to the epidermis. This demonstrates the limitation of this rubric for effectively identifying burn damage markers in the epidermis. As most burns analysed in this study were superficial partial thickness burns at one hour and either progressed or maintained depth at later time points, changes in the epidermis were not often apparent between sections. Assessing the epidermis for burn intensity may not be as relevant for BWP but more applicable for superficial thickness burns.

Correlation analysis showed statistically significant high correlation between overall BV damage and burn depth in progression burns at 24 hours post-burn. Therefore, BV damage intensity can be representative of burn depth in progression burns, however, this was not observed in non-progression burns. Significantly higher intensity scores of blocked BVs observed at one hour post-burn were indicative of no BWP, suggesting that decreased blood flow prevented any progression at later time points. Prolonged inflammation is known to have a role in BWP, with infiltration of pro-inflammatory mediators and production of oxygen-derived free radicals further injuring dermal tissue (6, 7). Progression burns were found to have initially low intensity of blocked vessels at one hour post-burn but then had significantly more increased blocked vessels at later time points. This is supported by findings from Clark et al. where burns at earlier timepoints contained RBC aggregates in the superficial

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dermis, with deep dermal BV occluding at 24 hours post-burn. However, this RBC aggregation was observed to lead to BV occlusion and subsequent hypoxia and ischemic necrosis (52). Infiltration of inflammatory cytokines in burns were found in higher concentrations at day one in comparison to later times (85). The increased proportion of blocked BV at earlier time points suggests that blocked BVs in non- progression burns may prevent the infiltration of harmful inflammatory mediators via BVs. This may indicate that BV occlusion or microthrombi formation does not completely occur within the first hour post-burn, but instead occur at later timepoints to cause ischemic necrosis. Partially blocked vessels, although not completely patent, were indicative of progression at 72 hours. Aggregation of RBC in partially blocked BVs reduces blood flow to viable tissue, causing hypoxia. This would induce fibrinogen and fibrin deposition for microthrombi formation and lead to ischemic necrosis at later time points (52). Prolonged inflammatory responses that were not prevented by early onset of blocked BVs, may be introduced by partially blocked BVs to cause BWP due to the incomplete obstruction of the BV. The inflammatory response after burn injury includes infiltration of interleukin, tumour necrosis factor, intercellular adhesion molecule-1 and reactive oxygen species (7). BWP was characterised by a statistically significant increase in complete and partial occlusion of BVs in the first 24 hours. This is believed to occur due the action of the coagulation cascade, initiating formation of cross-linked fibrin, leading to ischemic necrosis (53).

The burn intensity scores represented by hair follicles and glandular structures were limited to structural damage and necrotic (represented as shrunken) nuclei. Significant differences between groups were only observed at 72 hours, with progression burns containing higher intensity scores. In observing these differences, the dermal location of these epithelial structures needs to be considered. Glandular structures are primarily found in the deep dermis, often bordering the adipose layer. Hair follicles, although varied in location, were primarily located in the mid to deep dermis. Therefore, the significant increase in damage intensity observed is likely to be a result of only deep-dermal to full thickness burns exhibiting damage, at 72 hours post- burn. Hair follicle frequency was highly variable, ranging from 0 to 7 individual hairs across 40 sections, resulting in high variances in damage intensity data. Damage of glandular structures was only evident at later time points (24 hours and 72 hours) in progression burns and had large variance. The burn damage markers associated with

Chapter 3: Burn Wound Progression Analysis 65

these features are highly indicative of deep-dermal to full thickness burns and can be useful in assessing burn depth (12, 23, 34, 38, 39). However, due to the varied frequency and dermal location, hair follicles and glandular structures may not be ideal for assessing total burn intensity or BWP analysis.

Extravasated red blood cells within the dermis showed an increase in intensity from one hour post-burn to 24 hours post-burn in both non-progression and progression burns. However, statistically significant increases were only detected in non-progression burns. Extravasated red blood cells have not been reported previously as a marker of burn damage. Salibian et al. posits BWP is linked to oedema-mediated hypoperfusion. The release of prostaglandins, histamine, and bradykinin increase vascular permeability, thus releasing fluid into interstitial spaces (6). This may be a result of increased partially blocked vessels previously reported permitting blood flow into leaky vessels. Although fluid shifts do not necessarily cause RBC extravasation into the tissue, this may indicate leaky vessels, caused by direct damage from the burn (7), release RBCs into the dermis. RBCs nearby BVs had high variances in intensity score in progression burns, indicating that the presence of extravasated RBCs in burned tissue is highly variant. The underlying impact of RBCs located within the dermis should be further investigated to determine its role in BWP.

The rubric developed in this project for assessing burn intensity is novel, with no previously published study reporting the assessment of the entire section and all its features. The only analysis similar to this was conducted by Asif et al. which assessed all partially and completely occluded BVs in peri-burn tissue (53). This was conducted on a porcine comb model, designed to examine areas between burns as the “zone of stasis”. The emphasis on BV assessment affirms the findings evaluated in Chapter 3, where vascular features (i.e. BV damage, extravasated RBCs) were most significant in observing differences between non-progression and progression burns. This was also supported by significant correlation between burn depth and total BV damage at 24 hours in progression burns. The analyses regarding other features including epidermis, collagen denaturation, and inflammatory response, found little to no significance between timepoints and treatment groups. This indicates either limitations in the ability of the rubric to assess this damage, or that these features serve no impact in assessing burn damage and predicting BWP. Markers of damage previously utilised in burn depth rubrics may be inclusive of all types of damage, however, are only applicable

Chapter 3: Burn Wound Progression Analysis 66

when located at the deepest point of injury. This was observed in glandular structures in which damage was only observed in deep-dermal thickness burns due to their dermal location. In this study, although the damage markers were counted within each quadrant, the data from each quadrant was combined to obtain a total score for each section. The comparison of damage features between the dermal quadrants (from superficial to deep) would be beneficial in future research to determine the location of burn damage markers and their role in BWP. The assessment of burn damage in the entire section allows for distinct differences to be observed between burns that progressed and burns that did not, in different quadrants/regions and in different features within the section. To further investigate these mechanisms, other stains such as MSB should be used with this rubric to observe vascular damage markers, and another damage marker for fibrin deposition should be added to the rubric.

The statistical analyses conducted in this study were limited by the number of replicates of each burn (n=6). This was further limited by the number of animal replicates, with some burns occurring on the same animal, meaning that inter-animal effects would need to be considered. Therefore, statistical analyses were limited to Mann-Whitney tests and Spearman correlations between singular groups, rather than across several timepoints. In future research, additional replicates would allow for more extensive analyses (i.e. repeated measures) to observe intensity scores across the three timepoints as well as interquadrant data. The damage was assessed in this project by only one assessor. Repeat scoring by the same assessor would be valuable to determine intra-rater reliability, while additional scorers would also be valuable to determine inter-rater reliability for the rubric and results seen here.

Chapter 3: Burn Wound Progression Analysis 67

Chapter 4: Discussion and Conclusion

4.1 DISCUSSION

Throughout burns research in both experimental models and clinical assessment, BWP has been observed to occur following the initial injury from 24 hours to up to seven days post-burn. There are various mechanisms at play within the wound to cause this progression. This study aimed to utilise various histological stains to observe burn damage markers, with the development of a novel burn damage rubric using H&E to further assess burn wounds. Several different burn markers used previously include collagen denaturation (Table 1.1), cell death (Table 1.3, Table 1.4), collagen denaturation, and vascular pathologies (Table 1.4), however their respective roles in BWP is unclear.

In Chapter 2, various stains were optimised and compared to determine which were appropriate in observing burn damage markers. Collagen denaturation was not observed as clearly as described in literature, where denatured collagen was identified by distinct change in staining colour (12, 16, 17, 21). In this project, staining for collagen denaturation was observed using VVG, GT and MSB stains, and only one section contained changes in staining colour (Figure 2.5 C-D, Figure 2.7 G, Figure 2.8 C). Other markers for burn induced collagen denaturation were collagen bundles and irregular fissures observed within the dermis. The disparity observed between burns analysed in this study and burns reported in literature, is likely a result of different burn injury inducement techniques. The studies which reported collagen denaturation as a change in staining colour, was conducted using contact burns. The histopathological features in contact burns show clear demarcation between necrotic and viable tissue, possibly due to considerably higher burning temperatures, however scald burns present differently (67). Hirth et al. reports that staining colour change of collagen was only evident in burns made at 165-205°C (12). Alternative collagen stains should be investigated in future research to better identify discrete changes in collagen structure not only between healthy and denatured collagen, but possibly identify intensity differences between different burn conditions. Other trichrome stains may be useful, as well as more specific techniques such as second-harmonic generation (SHG) and picrosirius red to observe collagen type composition.

Chapter 4: Discussion and Conclusion 69

GT and MSB staining of burn induced, vascular pathologies was evident in the Chapter 2 evaluation in burned tissue stages as these stains could identify vascular markers of occluded BVs, free RBCs, fibrin deposition, and NET formation. Although MSB staining is recognised to detect fibrin, its specificity in porcine skin has not been verified. Therefore, immunostaining for fibrinogen was necessary to further confirm the presence of fibrin in burns. In confirming fibrin presence, this opens up further avenues of investigation into the role of fibrin in burn wounds. This aligns with other findings observed in Chapter 3, indicating the importance of investigating vascular pathologies. Intensity scoring of sections determined that burns which progressed in the first 24 hours, reported significant differences in BV damage. In investigating BWP, H&E staining deemed vascular pathologies to be of importance in observing burn wounds. Thus, utilising these techniques with MSB staining to detect fibrin deposition may further investigate these mechanisms.

The significant difference in BV occlusion observed between non-progression and progression burns suggests that certain mechanisms may be responsible for BWP, including RBC aggregation, platelet activation, microthrombus formation and fibrinogen deposition (6, 52, 53, 86). The detection and presence of NETs in burn wounds has been linked to fibrin deposition and associated with pro-coagulant factors (72). The formation of NETs, fibrin and RBC aggregation may all be involved in the development of BV occlusion and BWP. Fibrin and fibrinogen are important factors that initiate haemostasis and clot, to aid in wound healing and prevent infection (55). Fibrin deposition creates a temporary scaffold, providing an environment for endothelial cell stimulation and vascular growth. Research has shown that in fibrin rich matrices at early timepoints (3 days post-damage), microvascular endothelial cells invaded the fibrin clot to initiate angiogenesis (87). As the fibrin matrix degrades, the formation of tubular capillary-like endothelial structures has been observed (88). Thus, fibrin deposition is associated with angiogenesis. BV occlusion observed in early timepoints of non-progression burns could be indicative of fibrin deposition in these vessels resulting in angiogenesis and aiding in the recovery of these vessels to prevent progression of burn injury.

Immunofluorescence staining of fibrinogen captured by Rybarczyk et al. (55) has many similarities in appearance to NET (69, 74) detection in literature and from MSB staining seen in this project (Figure 2.8 G-H). Although similar in appearance,

Chapter 4: Discussion and Conclusion 70

NETs are comprised of chromatin released by neutrophils (89). NETs have the capacity to impede blood flow in capillaries without thrombosis formation, as in vitro NETs have enabled plasma to flow through microfluidic networks and block RBCs (90). Tritiated-Citrulline was previously used by Korkmaz et al. to identify NET formation in burned skin amongst intravascular thrombi, implicating neutrophil activity in wound progression (72). However, the histological staining in the detection of NETs by Korkmaz et al. does not resemble NET-like structures previously reported with electron microscopy and other IHC techniques (69, 73, 74, 81). The formation of NETs has been associated with neutrophil death through stimulation of phorbol 12- myristate 13-acetate (PMA) or interleukin (IL), described as “NETosis” (89). NETosis has also been observed to occur without neutrophil death, allowing normal function of neutrophils to continue (91). Inflammatory response was assessed using our burn injury rubric through identifying inflammatory cells presence with H&E staining. This burn marker did not produce any significant result however, examining other inflammatory markers may find different results. The occurrence of NETosis in the formation of NETs and BV occlusion, may have an impact on the inflammatory response involved in burns. BV occlusion intensity was significantly different between progression and non-progression burns, indicating the role of increased BV occlusion in preventing BWP. Fibrin and thrombosis formation to create clots may prevent prolonged inflammatory response, whilst creating a scaffold for the rebuilding of vascular structures. The function of NETs may be advantageous to investigate, however, the presence of NETs needs to be verified using IHC staining.

The importance of perfusion and patent BVs has shown to be influential on whether burns progress or maintain depth. Many different treatments have been investigated to prevent or minimise BWP. Optimal first aid for burns (i.e. cool running water for 20 minutes) has been shown to reduce burn depth and hasten time to re- epithelialisation (40, 92, 93). The mechanisms of cool running water in burn wounds to reduce burn severity is not well understood, however there are several theories. Optimal first aid provides analgesia and cooling to the wound, which may reduce burn wound damage by decreasing inflammatory response and cellular energy requirements in the zone of stasis (40). Cool running water has been shown to resolve vascular pathologies by reducing stasis, oedema and haemorrhage in the microvascular of bat- wing tissue (94). This effect may work to prevent RBC aggregation and leukocyte

Chapter 4: Discussion and Conclusion 71

adhesion to increase dermal perfusion, whilst minimising vascular resistance and reducing capillary damage and extravasation (94). The application of optimal first aid has been shown to reduce BWP, suggesting that these mechanisms are of importance to investigate. Treatments that improve perfusion levels and anti-coagulation agents have been investigated to prevent BWP. The effect of erythropoietin (EPO) was considered as it induces vasodilation and angiogenesis. Low doses (500 IU/kg) were observed to increase perfusion within the wound and reduce BWP, however, also resulted in an increased haematocrit, which was suspected to impair blood flow and may increase the risk of thrombosis formation (95). Nevertheless, higher EPO doses were found to restrict blood flow into the zone of stasis and the angiogenic effects were not observed until day 4 post-burn (5). Schmauss et al. reviewed treatments targeting anticoagulation agents including recombinant tissue–type plasminogen activator (r- tPA), activated protein C (APC), beraprost-sodium and poloxamer-188 (P-188) (5). The burn wound assessments in these studies were conducted at 5 hours (APC) or 7 days (r-TPA and beraprost-sodium). These timepoints fail to identify the effects of BWP as it usually occurs within 24-72 hours post-burn. Administration of r-TPA and beraprost-sodium increased perfusion levels within the zone of stasis and minimised progression, while administration of APC caused decreased levels of perfusion and deepened burns (96-98). However, these treatments should be carefully assessed to consider the effects of excess bleeding within the tissue. As observed in this study, the incidence of BV occlusion can be a fine balance between preventing or inducing BWP.

When designing the rubric to assess total section burn damage, the tissue section is divided into depth quadrants (Table 3.2) and damage markers were recorded accordingly. Further analysis of the presence of burn damage with respect to each quadrant may give more detail into the activity of the wound. Jackson (3) refers to the zone of stasis as the area surrounding the immediate zone of necrosis in which tissue may be salvageable before undergoing further necrosis (8). In observing burn wounds histologically, different burn markers appear to determine burn depth differently. Identifying the burn marker that best represents burn depth is contentious, as research into measuring and observing burn depth is inconsistent. Burn depth has previously been measured by collagen denaturation (13), blood vessel occlusion (59), cellular necrosis (12), or a combination of all dermal markers (23, 39). This raises the questions: What burn marker is most representative of damaged skin? Can the zone of

Chapter 4: Discussion and Conclusion 72

necrosis or zone of stasis be identified, histologically? The rubric could be used to determine burn intensity in each quadrant and identify the zone of stasis histologically. Hirth et al.’s research suggested that endothelial necrosis was indicative of deeper burn depths, compared to collagen denaturation (12), however no comparisons have been conducted between endothelial necrosis and blood vessel occlusion. The burn rubric utilised in Chapter 3 was limited to H&E staining resulting in BV damage primarily recorded as occlusion (partially or completely blocked vessels) with minimal data regarding endothelial cell death. Cellular necrosis arguably defines irreversible damage, whereas BV occlusion may be indicative of the zone of stasis. BV occlusion may act as a protective barrier to minimise prolonged inflammatory responses leading to mechanisms causing BWP. However, BV occlusion has also shown to prevent blood flow and cause ischemic necrosis. The porcine comb model utilised by Asif et al. (53) aims to identify the zone of stasis by creating space between burns, forming what is theorised as the immediate area surrounding the zone of necrosis. Although horizontal progression is a valid concept, the assessment of this region may include pathologies associated with the area surrounding the burn instead of specifically the zone of stasis. It is believed that the distinction of the zone of necrosis could be indicated by evidence of cellular necrosis, whereas zone of stasis may contain vascular pathologies, except for endothelial cell death. Identifying intensity of cellular necrosis in comparison to intensity of BV occlusion in individual quadrants may identify the area in which tissue is salvageable to determine the mechanisms responsible for BWP. Cellular necrosis and apoptosis were identified in literature as markers of burn damage, and necrosis can be detected with H&E stains. However, immunostaining techniques were the primary method for detecting these mechanisms in the other studies. Necrotic cells were observed in H&E staining and assessed in the burn rubric as shrunken nuclei or pyknotic in appearance with these burn markers only significantly exhibited in epithelial structures like hair follicles and glandular structures. Research suggests that endothelial necrosis observed at one hour post-burn predicts BWP (37), however this rubric failed to identify this mechanism. Further research in utilising IHC to detect cell death with respect to burn intensity and dermal location (per quadrant) may be valuable in assessing BWP.

The rubric applied in this study was developed using staining of burn markers identified in scald burns. The assessment of the entire section was designed to consider

Chapter 4: Discussion and Conclusion 73

differences in burn marker appearance previously described in scald burns. Brans et al. (67) describes contact burns as having clear demarcation between healthy and damaged tissue, whilst scald burns are not homogenous in exhibiting damage. Recent studies from Singer et al. (99) compared contact and scald burns in a porcine model, with identical temperatures and durations. The findings contradicted conclusions made by Brans et al. and concluded that scald and contact burns were in fact similar in pathological appearance with no differences in structure. However, contact burn temperatures between the two studies were vastly different, and may explain the difference in histological appearance. The contrasting results between these two papers demonstrate that the complex pathology of burns can be influenced by many factors including burn mechanism, duration, temperature and can vary greatly in intensity. Regardless of the burn mechanism, it is important to assess the entire tissue section to determine what mechanisms occur, not only at the deepest point of injury but at all depths of the wound, to cause BWP. As contact burns have also shown to be variable in appearance, this rubric would be effective in assessing contact burns as well as scalds.

In this study, burns were categorised as low temperature, short duration or high temperature, long duration to mimic different types of scald burns. The differences in damage markers between these two burn conditions were not investigated due to time restraints, and small sample sizes (n=3 per burn condition, per treatment group) however, may be valuable in the assessment of burn damage. Within each treatment group (progression or non-progression), burns followed the same trend with similar intensities over 72 hours. However, it would be interesting to investigate any differences in the pathologies observed between the groups. A systematic review conducted by Andrews and Cuttle (66), found that of the limited number of porcine scald burn models published in the literature, burn conditions and histological depth data were not reported in detail. From the porcine scald burn models in this review, the progression of burn depth was only mentioned by Henze et al. (100) and Brans et al. (67), but no comments are made on the mechanisms responsible. The study from which these porcine samples come from, observed that these burns progressed until day 3 post-burn (34). Further research into the differences in burn pathologies between different burn conditions should be investigated to determine what changes with respect to scald temperature and duration.

Chapter 4: Discussion and Conclusion 74

This rubric was an effective tool for assessing burn wounds by using a novel approach. Histological staining of burns has the capacity to detect several mechanisms within the wound. This rubric advances the standard burn assessment protocol by analysing the entire tissue section and all individual features, instead of only burn markers at the deepest points of injury. This allows for different burns with similar depths to be differentiated by the intensity of overall amount of pathology of the burn. The practice of dividing the tissue section into quadrants, from superficial to deep, has recently been used by Gibson et al. to measure burn damage (101). To verify deep partial thickness porcine burn models, assessors were asked to identify markers of damage and report the location with respect to quadrants within the section (101). This provided an intensity score and more accurate reporting of burn depth scores across the assessors. Although Gibson et al.’s paper was focussed on the accuracy of the scoring rubric, this scoring method verifies the effectiveness in using intensity to score burn wounds and BWP. The rubric developed and utilised in this study can be validated to investigate burn intensity to predict time to heal. This can be further researched investigating burn wounds at later time points post-burn.

4.2 CONCLUSION

The complexity of burn wounds can be assessed effectively using the novel rubric developed in this study, as it allows for full assessment of the entire wound. This rubric utilises H&E staining to identify burn markers, however further work involving more specific stains such MSB stain could identify more vascular pathologies. Presence of BV occlusion in progression burns suggests the involvement of fibrin and NETs, which could be detected with the MSB stain. There is clear evidence for the involvement of BV occlusion in progression burns however, burn intensity assessment may specify the zone of stasis in which tissue can be recovered. Using histological techniques and novel assessment methods, the mechanisms responsible for BWP can be more accurately determined. This research adds value to literature by evaluating several histological stains to observe burns in porcine scald models and the development of a novel rubric for burn wound assessment.

Chapter 4: Discussion and Conclusion 75

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Appendices

Appendix A

Martius Scarlet Blue (MSB) Staining Protocol from QUT Manual of Histological Techniques.

METHOD:

1. Bring sections to water (Post-fixing in Picric Acid (p10) can be done at this point. Drain slide over coplin jar and carry on a tissue back to your station. Wash well (5 – 10min) in running water before Step 2)

2. Stain with Weigert’s Iron Haematoxylin for 10 min 3. Rinse in tap water 4. Differentiate in Acid Alcohol (leave slightly overstained) 5. Wash well in tap water and then blue with ammonia 6. Stain with Martius Yellow solution for 10 min 7. Rinse in de-ionised water and stain in Brilliant Crystal Scarlet solution for 5 - 7 min 8. Rinse QUICKLY in de-ionised water and differentiate with 1% Phosphotungstic Acid until RBC are yellow and fibrin is a clear red (quick microscope check as you may not need to diff). If the fibrin has some Orange/Yellow colouring then you have either under stained in Brilliant Crystal Scarlet OR you have over differentiate with 1% Phosphotungstic Acid, go back and try re-staining in Brilliant Crystal Scarlet for 1-3mins. 9. Stain in Methyl Blue solution (5min- 8min pending your intensity preference). 10. Rinse quickly in 1% acetic acid 11. Dehydrate in ABSOLUTE Alcohol, clear and mount

RESULTS:

Nuclei Blue - Black Fibrin Red Connective tissue and very old fibrin Blue Red blood cells Yellow

Appendices 85

Appendix B

Burn Rubric Template. Score of zero (0) or one (1) were recorded for each damage marker present. New rows were added in Column A for each individual feature observed in each section.

Appendices 86