title: Understanding the effects of hypoxia on spinal cord early myelinating oligodendrocytes in vitro
Creators: name: Crawford, Colin Lindsay
Publisher: name: University of Glasgow
Year: 2021

description: In the developing brain, pre-/early myelinating oligodendrocytes (EMOLs) are particularly vulnerable to hypoxia-ischaemia; however, much less is known about how this impacts spinal cord EMOLs. Indeed, in an in vivo mouse model of spinal muscular atrophy, myelinogenesis proceeds normally despite significantly reduced vascularisation of the developing spinal cord. Furthermore, even in the healthy spinal cord, perfusion is up to 60% lower in comparison to the brain. Together, these observations raise the possibility there are anatomically divergent susceptibilities of EMOLs to hypoxia-ischaemia. Thus, this thesis addressed the hypothesis that spinal cord EMOLs are relatively resistant to hypoxia-ischaemia and associated secondary consequences. To test our hypothesis, we used murine spinal cord mixed glial cultures, which we showed contain EMOLs (indicated by BCAS1 expression), astrocytes, and microglia, but lack neuronal cell bodies, making these suitable for modelling spinal cord white matter. The morphology of DAPI-stained nuclei was confirmed to be a useful indicator of cell survival. To examine the response of spinal cord EMOLs to hypoxia, cell cultures were subjected to chemical hypoxia for 5 or 24 hours using azide, or to oxygen deprivation for 24 hours. Five hours chemical hypoxia did not cause death of EMOLs (or astrocytes), whereas they failed to survive 24 hours treatment. Surprisingly, oxygen deprivation had little impact on survival, even when glucose was withdrawn, raising doubts about the efficacy of the method. Together, we conclude that spinal cord EMOLs are tolerant of short-term hypoxia. As neither chemically-induced hypoxia nor oxygen deprivation led to acidosis of the cell culture media, as would occur in the CNS, we next examined the impact of this secondary component of the hypoxic environment. Cells were maintained for 6 days at physiological pH or in increasingly acidic conditions. Reducing pH to 6.8 or lower led to a significant decrease in the densities of EMOLs. Surprisingly, the complete withdrawal of glucose (pyruvate and glutamine) did not compound this and had only a minor effect on cell survival at physiological pH after 6 days. This unexpected observation led us to ask whether our survival assay was sufficiently sensitive. We confirmed this with a lactate dehydrogenase release assay and by further challenging the cells with reintroduction of glucose or complete withdrawal of amino acids and vitamins. The unexpected survival of EMOLs, and also of astrocytes, in the absence of glucose led us to hypothesise that astrocyte energy reserves might sustain both astrocytes and EMOLs under exogenous energy substrate deprivation. To address this, we explored trans-cellular exchange routes and found EMOLs were dye-coupled to astrocytes by nanotube-like structures: a putative conduit for the transfer of energy substrates. Recently, it has been demonstrated that astrocytes can accumulate lipid droplets, the beta-oxidation of which could potentially provide fuel for cell survival in the absence of exogenous energy substrates. Therefore, we next looked for evidence of lipid droplets in these spinal cord-derived cell cultures and found that lipid droplets were abundant in astrocytes. Consistent with this, lipidomic analyses demonstrated a significant reduction in triglycerides (a lipid droplet-associated species) after 6 days of energy substrate deprivation in mixed glial cultures, but not EMOL-enriched cultures, compared to 5 mM glucose controls. In all, these data suggest that spinal cord EMOLs are relatively resilient to hypoxia-ischaemia as demonstrated by their tolerance of short-term hypoxia and exogenous energy substrate deprivation in this in vitro model. The disease-relevant implications remain to be examined and the source of lipid droplet substrates to be determined.