An investigation into the breakdown products of cuprizone and the inflammatory role of RAW 264.7 macrophages in an in vitro culture system

Abstract

The cuprizone mouse model is commonly used to study axonal demyelination/remyelination events that are associated with diseases such as multiple sclerosis (MS). The aetiology of MS remains unclear, but this inflammatory condition involves the crosstalk between neuronal and immune cells. Cuprizone (CPZ) is a reversible demyelinating, toxic, copper-chelating drug that inhibits cell growth and promotes oligodendrocyte death. RAW 264.7 macrophages are innate immune cells that were used in this thesis due to their role in demyelinating diseases. In vivo, following oral ingestion, CPZ is hydrolysed by the acid in the stomach to form the breakdown products, cyclohexanone and oxalyldihydrazide. This thesis tested the hypothesis that one of the breakdown products of CPZ, oxalyldihydrazide, is responsible for the release of pro-inflammatory cytokines, altered morphology and viability of RAW 264.7 macrophages. This hypothesis was tested using enzyme-linked immunosorbent assays (ELISAs) for tumour necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b) and the viability of the cells through the Trypan blue assay. Furthermore, it was hypothesised that protein arginine methylation would change based on the activation status of the RAW 264.7 cells, which was tested using Western blotting. This thesis is important due to the lack of research investigating the roles of the specific breakdown products of CPZ and their effects on cellular viability, morphology, and cytokine production in RAW 264.7 macrophages. This thesis provides methodological considerations that should be acknowledged when optimising protocols in all aspects of cell culture work, when working with immune cells. Pyroptosis was identified as a potential explanation of the collective data obtained in this thesis including the morphological changes and cytokine production in the RAW 264.7 macrophages. This cellular death pathway could be attributed to the apparent toxic nature of CPZ and an overreactive immune system in some disease states. Further cellular work and animal studies are required to discern the effect of the breakdown products of CPZ, which should be compared to human glial cells from MS patients. Such comparative studies will assist in understanding the pathological events associated with demyelination, which could identify more therapeutic targets for numerous demyelinating diseases.

Date of Award	2022
Original language	English