NIAA Final Report
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1 NIAA Final Report Project title: Metabolic characterisation of human models of burn pain Author: Dr Carsten Bantel, MD, PhD, FRCA, FFPMRCA Short Summary Background Burn injury associated pain is complex and often difficult to treat. To be able to personalise therapies, more knowledge is required about injury induced mechanisms in the skin. Methods Dermal microdialysis was performed in healthy human volunteers exposed to either thermal, capsaicin or UVB induced burn. Dialysates were analysed for cytokine and metabolite (metabonomic) profiles. Conclusion This study provides evidence that different models of mild burn injury induce unique patterns cytokine and metabolite compositions in the interstitial fluid of damaged skin. 1
2 Background Burn injury induced pain is complex and often severe. It likely arises from an incompletely defined milieu of inflammatory mediators sensitising peripheral sensory nerve fibres. This pain represents a major clinical challenge. Understanding its underlying inflammatory responses may help guide clinicians with care, leading to true personalised analgesic management. Pilot work highlighted the inflammatory response 2 hours after experimental burn injury is different between the type of burn imparted. This study aimed to investigate the inflammatory response in three human burn models at three time points following burn injury and to determine if these changes were time as well as model dependent. Methods A total of 45 healthy volunteers (15 per model) received either a thermal, topical capsaicin or ultraviolet light B (UVB) induced skin injury on their non-dominant forearm. All volunteers underwent sensory testing and dermal microdialysis in both the burn and a control area in the forearm. Diasylate samples taken at 60, 120 and 180 minutes following burn injury were analysed using a 41 cytokine / chemokine multiplex microbead array assay and ultraperformance liquid chromatography / mass-spectrometry (UPLS-MS) for metabonomic characterization. Changes in the metabonomic profile between burn injuries were analysed using principal component analysis (PCA). To be able to characterise the cytokine profiles for each model first ratios of the individual cytokine and its respective control were calculated. This allowed assessment of the magnitude of change. Then the direction of change was analysed by subtracting unit (1) from the calculated ratios (ratio-1). 2
3 Area [mm 2 ] Results Somatosensory changes All models demonstrated erythematous flare and sensory changes similar to clinical burn injuries (primary and secondary hyperalgesia and allodynia, figure 1) Thermal vs Capsaicin vs UVB Somatosensory areas volunteers 6-15 for each model Thermal 1500 Capsaicin UVB statistical significance Flare Primary Hyperalgesia Secondary hyperalgesia Primary Allodynia Secondary Allodynia Figure 1: Erythematous (flare) and somatosensory changes (hyperalgesia, allodynia) of burn models. p>0.05, Kruskal-Wallis test 3
4 Cytokines Of the 41 cytokines tested only 3 were common to all burn models (table 1). Three different cytokines were exclusive to both thermal and capsaicin induced injury whereas only 1 cytokine (IL1-RA) was solely released after UVB exposure. When considering model and sampling time, each injury displayed a unique profile at each time (table 2). Adding the direction of change these profiles became even more distinct (table 3). Cytokines that were seen in all models (burn and control) and at all time points were EGF, FGF2, PDGFAA, PDGFAA, IL6, IL8, MCP1, RANTES. The only molecule not present in any model was GMCSF. Common to all Common to Thermal Common to Capsaicin Common to UVB EGF FGF-2 FGF-2 G-CSF IL-10 IL-10 MCP-3 MCP-3 MDC IL-12P70 PDGF-AA PDGF-AA scd40l scd400l IL-1RA IL-3 IL-3 IL6 IL6 IL6 IL6 IL8 IL8 IP10 IP10 MCP1 MCP1 MCP1 MCP1 MIP-1alpha RANTES RANTES RANTES RANTES TNFalpha Table 1: Cytokines common to each model of burn (data pooled over the entire observation period) 4
5 Table 2: Cytokine profiles for each model at each time point without considering direction of change. Purple: capsaicin (C); red: thermal (T); blue: UVB (U) 5
6 Table 3: Cytokine release pattern over time for each burn model including direction of change. Red: increased cytokine release; blue: decreased cytokine release; capsaicin: C; thermal: T; UVB: U 6
7 Metabonomics Similar to what was shown for the cytokine release patterns principal component analysis also suggests unique metabolic profiles for each individual burn model in general (pooled data, figure 2A-C) but also for the different time points (data not shown). Figure 2A: Differences in metabolic profiles of capsaicin and UVB models. Orange (1): capsaicin; blue (3): UVB Figure 2B: Differences in metabolic profiles of thermal and UVB models. Orange (1): thermal; blue (3): UVB 7
8 Figure 2C: Differences in metabolic profiles of thermal and UVB models. Orange (1): capsaicin; blue (2): thermal Conclusion This study provides evidence that different mild burn injury models induce unique patterns of both cytokine and metabolite compositions in the interstitial fluid of damaged skin. In the future this might help to rationalise treatments of burn victims according to the stimulus induced specific pathomechanisms. 8
MCP uL 25uL 25uL 25uL Typical Dilution 1:2 None None None Manufacturer Defined Minimum Detectible Concentration
Units: pg/ml pg/ml pg/ml pg/ml Assay Name RnD Systems Millipore Magnetic Hormone ELISA Adipokine Panel 2 Panel Catalog Number DCP HADK2MAG-61K HCYTOMAG-6K HMHMAG-34K EGF, FGF-2, Eotaxin, TGF-a, G-CSF,
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