Harnessing fluorescence microscopy to elucidate the mechanisms of metabolic dysfunction-associated steatotic liver disease

Thesis

Metabolic dysfunction-associated steatotic liver disease is defined by hepatic lipid overload resulting in a metabolic shift and subsequent mitochondrial impairment. To facilitate the investigation of disease mechanisms and the identification of novel, translational therapeutic targets, it is essential to have an arsenal of effective models that recapitulate human disease. Here, a broad range of models is utilised to study steatotic liver disease, with a key focus on metabolic dysfunction. The models span from a hepatocyte cell line to mouse models to the establishment of a human tissue slice model. To quantify mitochondrial dysfunction across these systems, a range of microscopy techniques are employed. Fluorescence microscopy reveals lipid-induced mitochondrial fragmentation and membrane depolarisation in cellular and tissue contexts. Substantiating these findings, fluorescence lifetime imaging microscopy of autofluorescent metabolic cofactors detects shifts in hepatocyte metabolism without the need for exogenous dyes. We show that measuring fluorescence lifetime can capture features of metabolic change that standard histological methods used in diagnosis do not. A robust lifetime-based pipeline is developed and applied across in vitro, in vivo, and ex vivo models, culminating in the generation of a quantitative index of mitochondrial dysfunction capable of identifying disease-associated changes. Integrated into these experiments is the investigation of High Mobility Group Box 1 protein and its potential reparative effects in models of steatotic liver disease. Fully reduced forms of this protein show functional benefits in acute or lipid-damaged models, but limited effects in chronic disease suggest a need to refine dosing regimens or better account for cell-type-specific responses. Together, this work establishes a versatile platform for investigating mitochondrial dysfunction in steatotic liver disease and highlights the potential of fluorescence lifetime metabolic imaging to enhance current methods of histopathologic assessment in diagnosis.

Authors: Purdie, K

• DOI: 10.5287/ora-yrdmep94a
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: liver steatosis; fluorescence lifetime imaging microscopy; mitochondrial dysfunction; precision-cut liver slices
• Subjects: Liver; Mitochondria; Microscopy