Anatomy and Neuroscience - Theses
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ItemThe role of neurotrophic factors in osteoarthritis pain( 2023-10)Introduction: Osteoarthritis (OA) is a progressive disease of synovial joints and subchondral bone characterized by swelling, stiffness and pain. Brain-derived neurotrophic factor (BDNF) and artemin (ARTN) are neurotrophic factors that are important regulators of pain, and have recently been implicated in the pathogenesis of OA pain. This study aimed to explore roles for BDNF and ARTN in OA pain, by investigating whether the expression of BDNF and ARTN, and their receptors (TrkB and GFRa3), is altered in different tissues at different stages of OA, and whether blocking their signalling during late-stage OA can alleviate pain. Methods: The monoiodoacetate (MIA)-induced OA of the rat knee joint was used to explore roles for BDNF and ARTN signalling in OA pain. Pain behaviour was assessed using the dynamic weight-bearing apparatus to assay OA-induced changes in hindlimb weight bearing behaviour, at different stages of disease (early vs late). Histopathological alterations in the knee joint and surrounding bones were assayed using Haematoxylin and Eosin staining and scored using a modified OARSI scale. Changes in expression of BDNF/TrkB and ARTN/GFRa3 were explored using Western blot analysis of lysates from different tissues (joint, bone, and DRG), and at different timepoints of the disease (early vs late). The dynamic weight-bearing assay was used to determine if inhibiting BDNF signalling (with a peptide mimetic TrkB inhibitor) or ARTN signalling (with a sequestering antibody) could relieve pain at late-stage disease. Results: The results of this thesis highlight differential histopathological changes occurring in the early and late stages of OA, with joint involvement being prominent in early OA, and bone and cartilage involvement in late OA. BDNF expression was increased in the joint in early OA and in the bone in late OA. ARTN expression was also increased in the joint in both early and late OA and in the bone in late OA. Attempts to alleviate pain in MIA-injected animals by targeting the BDNF/TrkB and ARTN/GFRa3 signalling pathways did not yield pain relief outcomes with the therapeutic approach chosen in this study. Conclusion: Our findings suggest that altered pain behaviour in early MIA-induced OA is associated with changes in the joint not surrounding bones, while altered pain behaviour in late MIA-induced OA are attributable to the surrounding bones. Furthermore, BDNF and ARTN may contribute differentially to pain in early and late stages of MIA-induced OA through actions in joint versus bone. These findings support further investigations into the role of BDNF and ARTN signalling in OA pain and the development of novel targeted therapeutic approaches for managing OA pain.
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ItemInvestigating the role of neuronal TrkB in remyelination( 2023-02)Demyelinating diseases, such as multiple sclerosis (MS), involve damage to the fatty, proteinaceous myelin sheaths surrounding nerve fibres and subsequent development of neurological symptoms related to the damaged area. Repair of the damaged myelin can completely or partially reverse the neurological deficits, however, over time with repetitive demyelinating events and incomplete remyelination this ultimately results in the degeneration of neurons and development of permanent disabilities. Currently, there is a lack of therapies effective in promoting myelin repair. A complete understanding of the factors that promote remyelination is critical for developing therapeutic strategies for myelin repair to prevent permanent neurological disability. Many studies have focused on the potential of oligodendrocytes (the myelin-generating cells in the central nervous system) to enhance the generation of myelin. However, neurons certainly have an intimate relationship with oligodendrocytes and are well-positioned to influence the process of myelination. Previously, neuronal expression of the tyrosine kinase receptor TrkB has been shown to promote myelination in early development and during ageing. However, whether neuronally expressed TrkB receptors also influences the extent of oligodendroglial and myelin damage post a demyelinating injury, and the subsequent process of remyelination has not been elucidated. In this thesis, I generated an inducible, neuron-specific TrkB receptor knockout mouse line to investigate the role of neuronal TrkB receptors in the extent of both demyelination and remyelination following the induction of a demyelinating insult with cuprizone administration. I found that following deletion of TrkB, these mice exhibit a normally myelinated nervous system, but do however develop a motor phenotype consistent with a decrease in precise motor control. Following administration with cuprizone, I revealed that neuronal expression of TrkB is important for the differentiation of OPCs (oligodendrocyte progenitor cells) that are generated in response to the demyelinating insult. Finally, although reduced expression of neuronal TrkB did not influence the overall extent of remyelination, I found that axons with small diameter required expression of neuronal TrkB to be efficiently remyelinated. This work is the first to identify that neuronally expressed TrkB receptors have a role in OPC differentiation following demyelination. This thesis suggests there is therapeutic potential in targeting neuronal TrkB signalling to promote myelin repair, albeit this may only be specific to smaller diameter neurons.
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ItemHomeostatic and activity-dependent oligodendrogenesis in the adult central nervous system( 2021)Aging induces a decline in cognitive function and is the primary risk factor for neurodegenerative disease, and both are associated with impairments in myelination. Oligodendrocytes and myelin in the central nervous system (CNS) are now recognised as important for plasticity in mediating learning, memory and cognitive function. It appears that with age, myelin plasticity declines in a region-specific manner and myelin loss increases. This suggests a slow decline in oligodendrogenesis occurs during aging, but the underlying cellular and molecular properties dictating the onset, progression and regional specificity of decline remain unknown. A complete understanding of oligodendrogenesis throughout the CNS over the course of healthy aging is critical in understanding the lifelong capacity for myelin plasticity, and may identify important therapeutic avenues for preventing age-related cognitive decline or onset of neurodegeneration. In this thesis, I used C57BL/6 mice to assess the homeostatic, activity-dependent and molecular basis of oligodendrogenesis throughout the CNS during adulthood and healthy aging. I used immunohistochemistry and confocal microscopy to identify oligodendroglial lineage cells, and transmission electron microscopy to investigate myelinated axon structure and myelin sheath thickness. First, I identified newly-formed oligodendrocytes with a cumulative labelling strategy, by administering thymidine analogue EdU for 6-weeks beginning at each representative timepoint of; 2-months (young-adulthood), 12-months (early aging) and 18-months (aging). I investigated homeostatic oligodendrogenesis in three distinctly myelinated regions of the CNS; the optic nerve (an almost fully myelinated white matter tract), the corpus callosum (a partially myelinated white matter tract) and the somatosensory cortex (a sparsely myelinated grey matter area). Second, I investigated activity-dependent changes in oligodendroglial behaviour in young-adult mice by inducing CNS plasticity using a physiologically relevant, environmental enrichment (EE) paradigm at 2-months of age. Finally, I generated and characterised an inducible, neuronal-specific TrkB knock-out mouse to investigate neuronal TrkB as a molecular mediator of oligodendrogenesis across 5-months of aging, after inducing deletion in the aged-adult, at 12-months. First, I observed a spatiotemporal decline in oligodendrogenesis during healthy aging, to a very low, but uniform plateau of <2% adult-born post-mitotic oligodendrocytes, which was maintained until 18-months. This occurred in the optic nerve by 2-months, and in the corpus callosum and somatosensory cortex by 12-months, as at 2-months I identified 11% of adult-born post-mitotic oligodendrocytes in the corpus callosum and 6% in the somatosensory cortex. Accordingly, I observed a decrease in the population of EdU+ dividing OPCs with aging, falling from ~80% to stabilise at 60% at 12- and 18-months in the corpus callosum and somatosensory cortex, and from 2-months in the optic nerve, suggesting age-related OPC quiescence. Importantly, this decline in oligodendrogenesis aligns with the known spatiotemporal development of myelin, suggesting it is tailored regionally to the requirements of lifelong myelination. Interestingly, I observed that inherent overproduction in oligodendroglia continues throughout adulthood, as there was incomplete integration of the pre-myelinating adult-born oligodendroglia at 12-months observed 5-months later, suggesting an inherent cellular reservoir for activity-dependent myelination during adulthood. Second, I observed that the differentiation of these pre-myelinating oligodendroglia was increased in adulthood after 6-weeks of physiological, EE-induced myelin plasticity, which disrupted the maintenance of OPC density homeostasis. This contrasts to mechanisms of myelin plasticity in the juvenile CNS that first involve large amounts of OPC proliferation. Interestingly, these data also provided some of the first experimental evidence for an additional form of myelin plasticity, in the coincident remodelling of the myelinated axon diameter and pre-existing myelin sheath. Third, I was one of the first to identify a neuronally-expressed molecule, neuronal TrkB, as an indirect mediator of adult-born oligodendrocyte differentiation and/or survival during aging, and specifically in the somatosensory cortex. This provides evidence for spatiotemporal molecular regulation of myelination and suggests a bias for activity-dependent myelination in the somatosensory cortex during aging. Together, these data highlight the inherent spatiotemporal regulation of oligodendrogenesis throughout the lifespan and implicate neuron-oligodendroglial communication in orchestrating lifelong myelination, with neuronal TrkB as an important molecular mediator. Importantly, these data define anatomical limits that constrain the capacity for physiological myelin plasticity and emphasise the subtlety of a remodelling response during adulthood. These data comprise an important resource for age- and region-related considerations that should inform future experimental design in adult myelin research and propose further investigation of the therapeutic utility of BDNF-TrkB signalling, via mediating myelination, during aging to prevent onset of cognitive decline or neurodegenerative disease.