Unhealthy ageing and memory loss associated with vascular insufficiency

Perivascular astrocytes are abundant in the brain and form a physical bridge between neurons and blood vessels. In addition, astrocytes control neurovascular coupling that serves to match local cerebral blood flow to regional neuronal energy use and ensures normal functioning of the brain. Vascular insufficiency takes place over several years without any obvious clinical symptoms and could be exacerbated by environmental stress or lifestyle choices (Ritchie et al. 2001). Astrocytes in particular respond to endothelial cell activation that arises from vascular insufficiency, and collectively these processes have a huge impact on brain function as we age. Recently, we have shown that when astrocytes react to pathological changes, they have dysregulated oxidative and nitric oxide pathways and this prevents astrocytes to regulate neurovascular coupling accordingly (Sarmiento et al. 2020). Therefore, we seek to further investigate the link between compromised energy resources and perivascular brain cells to better understand how neurovascular coupling may be affected and predispose the brain to damage and reduced function during unhealthy ageing.
1) You will be trained to optimise the culture of brain cells and/or brain slices from postnatal and adult mice. This is called primary culture, and it is an essential laboratory technique that requires a high level of skill. Subsequently, you will be trained to investigate the effects of oxygen-glucose deprivation (OGD). This is an experimental model of environmental stress that mimics what happens in the brain when there are insufficient nutrients, which may occur as a result of vascular insufficiency associated with age and may be compounded by lifestyle. You will be trained to assess the reactivity and health of astrocytes and endothelial cells (i.e., blood vessels) using specific cell markers (e.g., GFAP, STAT3, angiopoietin-2, CD-31), as well as signalling pathways associated with neurovascular coupling in the same cells (vasoactive molecules such as COX-1, iNOS, 20-HETE). In addition, electrophysiological measurements of endothelial and astrocytic functions in conjunction with measurements of intracellular calcium (Fura 2) and nitric oxide (DAF-FM) will be correlated to changes in cell markers and signalling pathways in the same environment stress.
2) Following characterisation of astrocytic responses to ODG, you will use the optimised in vitro protocols to culture perivascular cells and/or brain slices from a mouse model that recapitulates features of vascular insufficiency such as endothelial activation, neuroinflammation and memory deficits (Boehm-Sturm et al. 2017, Stroke). This is a surgical model, and the possibility exists to be trained in animal handling and surgical procedures if you desire. Brain regions will be isolated for the culture of primary cells and/or slices to examine changes in the molecular pathways involved in response to vascular insufficiency as the next step in the translational pipeline. We hypothesise that the astrocytes will be primed towards a reactive phenotype as a result of the surgical procedure, and this will allow us to investigate molecular mechanisms that underpin this and may be amenable to treatment. Our groups have been working with several compounds that influence oxidative stress pathways in the brain, and these will be examined in the cultured cells, potentially in conjunction with further threshold manipulations of OGD with neuroprotective drugs.
3) Ultimately, compounds that provide benefit for the tissue culture could be tested in the same surgical mouse model. Functional outcomes will be assessed at the whole organism level using either magnetic resonance imaging (MRI) (which could include training in computational image analysis) or behaviour testing (depending on funding, and preferences of the student). However, mechanistic insight into the compound's efficacy will be confirmed using histology, and molecular biology techniques.