Characterising the inflammasome response in a zebrafish model of haemorrhagic stroke

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Intracerebral haemorrhage (ICH) is a type of stroke caused by spontaneous bleeding within the brain. The toxic influx of blood into the brain initiates an inflammatory response that induces activation of microglia, infiltration of peripheral monocytes and neutrophils, and the production of inflammatory cytokines. Inflammasomes are cytosolic multimeric protein complexes formed in inflammatory cells in response to pathogenic infection and tissue injury. Recent research highlights an important role of inflammasome activation in progression of acute brain injury after ICH. Indeed, several studies indicate that inhibition of inflammasome activation in experimental models of ICH is beneficial and improves outcomes (Ren et al, 2018) and developing novel inflammasome inhibitors for the treatment of acute brain injury after ICH represents one of the most promising therapeutic avenues. By taking advantage of high-resolution live imaging and utilising a range of transgenic reporter lines that express fluorescent proteins in specific cell/tissue types (e.g. blood, blood vessels, dying cells, inflammatory cells), we have recently characterised the pathological consequences of blood in the brain in zebrafish larval models of ICH (Crilly et al, 2018; Crilly et al, 2019). We discovered that ICH in zebrafish larvae caused a significant increase in brain cell death, increased neuroinflammation, and an impairment in locomotor function, thus mimicking aspects of the injury phenotypes that accompany human ICH. Furthermore, we have shown this model represents a powerful platform for drug screening to identify compounds that reduce brain injury after haemorrhage (Crilly et al, 2022).The next aim of our research is to utilise the zebrafish model of ICH for screening novel inflammasome inhibitors that we are characterising in the lab in parallel studies. Although key components of the inflammasome pathways are functionally conserved (Forn-Cuni et al, 2019), to date, no studies have described the inflammasome response after ICH in zebrafish. Therefore, the aim of this PhD project is to use a combination of genetic (CRISPR-Cas9), pharmacological (novel and existing inflammasome inhibitors) and live imaging (light sheet / confocal microscopy of transgenic zebrafish reporter lines) to characterise the role of inflammasome activation in the progression of brain injury after ICH. The intended project outcome is the development of zebrafish as a new tool to i) interrogate inflammasome-related mechanisms and ii) test new inflammasome inhibitors for the treatment of brain injury after ICH.