Investigating the effects of Kappa opioid receptor agonists on remyelination in a preclinical model of multiple sclerosis

Abstract

Multiple sclerosis (MS) is a heterogeneous and debilitating neurodegenerative disease of the central nervous system, characterised by chronic inflammation and demyelination. MS is estimated to affect 2.5 million people worldwide and results in motor, sensory, autonomic and cognitive disabilities. There is currently no cure for MS, and the immunomodulatory therapies available are limited in their efficacy to decelerate disability progression. This disease continues to bear a heavy burden on its affected patients. In a recent breakthrough, the kappa opioid receptor (KOR) system has been presented as a potential target for treating MS through promoting remyelination. The KOR agonist, U-50488H, has been shown to enhance remyelination in both immune and non-immune mediated preclinical models of MS by promoting the differentiation of the oligodendrocyte precursor cell (OPC) population into myelinating oligodendrocytes. However, traditional KOR agonists like U-50488H are unable to progress through clinical trials due to centrally mediated side effects such as stress, sedation, dysphoria, aversion, anxiety and depression. Our laboratory has access to highly selective and structurally unique KOR agonists with increased potency and reduced side effect profiles in comparison to U-50488H. This thesis investigates the effects of compound A, B, C and U-50488H on remyelination in a non-immune mediated preclinical model of MS known as the cuprizone (CPZ)-mediated demyelination model. Female C57BL/6 mice were fed a diet containing 0.3% CPZ for 5 to 6 weeks to induce demyelination in the corpus callosum. CPZ was withdrawn, and the mice returned to standard pelleted chow for an additional 4 to 5 weeks. Several techniques were utilised to investigate behaviour, oligodendrocyte numbers, astrogliosis and axonal remyelination. Feeding mice 0.3% CPZ for 5 weeks led to a significant decrease in body weight, extensive astrogliosis and demyelination in the corpus callosum. Withdrawal of CPZ from the diet and treatment with compound A led to a significant recovery in body weight. In the von Frey filament test, CPZ intoxication led to a significant decrease in the threshold to withdrawal following mechanical stimuli. However, CPZ removal from the diet and KOR agonism did not significantly improve threshold. Oligodendrocyte numbers and MBP levels were not significantly different between the CPZ-intoxicated mice and their healthy counterparts following a 4 to 5-week recovery period. This suggests that extensive remyelination had occurred during recovery and the effects of KOR agonism could not be evaluated via immunohistochemistry (IHC) or immunofluorescence (IF). Transmission electron microscopy (TEM) was used to evaluate the G-ratios and the percentage of myelinated and unmyelinated axons in the corpus callosum. The CPZ-intoxicated vehicle mice exhibited significantly higher G-ratios in comparison to the healthy control mice, indicating thinner myelin sheaths. KOR agonism led to a significant decrease in G-ratios and therefore recovery of myelin sheath thickness. The CPZ-intoxicated vehicle mice also exhibited a decrease in the percentage of myelinated axons in comparison to the healthy control mice. KOR treatment with compound A led to a significantly higher percentage of myelinated axons in comparison to the CPZ-intoxicated mice and compound B and U-50488H treatment. In conclusion, we were unable to precisely evaluate the remyelination ability of KOR agonism via IHC or IF. However, TEM allowed us to obtain high-resolution images of axons in the corpus callosum. Compound A showed a significant improvement in myelin sheath thickness and percentage of myelinated axons after toxic demyelination. In conclusion, the use of more objective methods to evaluate demyelination and remyelination, as well as improvements made to the CPZ-mediated demyelination model could prove compound A as a successful remyelination therapy to be used by MS patients in the future.