MSACL 2024 Abstract

INTRODUCTION: Tissue injury, including burns, are a major trauma affecting millions worldwide every year. These injuries cause death and morbidity, resulting in huge healthcare costs and impacting both patients and their families. After injury, intracellular molecules are released, triggering inflammatory reactions to restore tissue function and resulting in persistent pain. This pain can persist for months or years after the injury itself, affecting quality of life. However, these pain-inducing molecules are largely unidentified, hampering understanding of tissue injuries and the ability to treat them effectively.

OBJECTIVES: Using a mass spectrometry-based workflow, we aim to improve understanding of molecular changes during tissue injury, to advance treatment of tissue injuries, reduce pain and improve patient outcomes.

METHODS: We have developed a scalding burn injury model and metabolomics workflow for analysing dermal microdialysate and skin from burn-injured and control subjects. The burn injury model facilitates collection of samples immediately after injury (minutes) and enables molecular changes to be followed over time, while clinical samples provide insight into longer term metabolic changes (days). We combined ultra-performance liquid chromatography (UPLC) and desorption electrospray ionisation (DESI) imaging mass spectrometry to elucidate metabolite and lipid changes after burn injury. For metabolite and lipid profiling, microdialysate and skin samples from burned and non-burned subjects were extracted and subjected to reversed-phase chromatography employing in-house methods, using a UPLC Acquity system coupled to a Synapt G2-S mass spectrometer. DESI-MS imaging was performed on sections of burned and non-burned skin from patients (n=72) in positive and negative ion mode using a Waters Xevo G2 QTOF instrument. Data were pre-processed as required and further analysed using univariate and multivariate approaches to identify significant molecular alterations due to burn injury.

RESULTS: Metabolic changes were observed in response to burn injury, including elevated uric acid and niacinamide, known to be involved in tissue repair and wound healing. A novel and significant finding was that lysophosphatidylcholine (LPC) species were significantly altered in both microdialysate and skin. Of specific interest were 14:0, 16:0 and 18:0-LPCs, all elevated following burn injury, due to oxidative stress. These pro-inflammatory lipids can be hydrolysed to lysophosphatidic acid, which causes pain through demyelination and activation of pain-related molecules on primary sensory neurons. DESI-MS showed significant alterations in skin lipid content 5-9 days after scalding burn injury, also revealing the increase in 18:0-LPC to be in the upper and middle third of the dermis. RNAseq data indicated several LPC metabolic enzymes to be differentially expressed after burn injury. Subsequent in vitro and in vivo studies revealed that administration of 18:0-LPC induced immediate pain and development of hypersensitivities to mechanical and heat stimuli, through the modification of specific ion channels.

CONCLUSION: Burn injury causes significant and consistent alterations in small molecules and lipids, in both microdialysate and skin samples. Importantly, 18:0-LPC was observed to contribute to the development and persistence of pain. These lipids have also been found to increase in other tissue injuries (e.g. ischemia, surgical injuries), and contribute to the development of inflammation, insulin resistance, and atheroschlerotic plaques. Therefore, increased 18:0 LPC likely to be an important mechanism for the development of hypersensitivities in tissue injuries. These novel findings could potentially improve patient care and outcomes by reducing pain and inflammation and providing better treatments.