Herrgen, Leah

Becker, Catherina

Herzog, Chiara Maria Stella

2019-07-30T09:52:45Z

2019-07-30T09:52:45Z

2019-06-29

Traumatic brain injury (TBI) is a major cause of death and long-term disability worldwide. It is elicited by an external force injuring the brain, and leads to two phases of neuronal cell death: primary cell death, resulting from direct mechanical disruption of the tissue, and secondary cell death, caused by delayed cytotoxic cascades. While primary cell death can only be prevented by avoiding physical injury, secondary cell death occurs within hours to weeks following the initial injury and therefore lies within a therapeutic window during which pharmacological agents targeting the pathomechanisms of TBI could be applied. Pre-clinical models of brain injury in rodents have identified several neuroprotective compounds, but not a single drug has shown improved patient outcome over placebo in multi-centre phase III clinical trials. Intriguingly, the largest randomised controlled trial for TBI to date reported a higher mortality in patients treated with an immunosuppressive drug compared to patients treated with placebo, suggesting that the immune system may play a neuroprotective role following brain injuries. To investigate the role of the immune system in more detail, I developed a model of brain injury in larval zebrafish. Larval zebrafish are amenable to genetic and pharmacological manipulation, and their optical transparency in combination with the availability of a multitude of transgenic reporter lines allows for efficient in vivo (timelapse) microscopy. This allowed me to visualise the cellular and molecular reactions to brain injury in real-time in a living vertebrate organism, which provided an advantage compared to the majority of studies so far, which had been done either in rodents with static end points, or in vitro. Characterisation of cell death dynamics revealed that larval zebrafish reproducibly exhibit primary and secondary cell death following brain injury. In line with mammalian TBI models, I demonstrated that excitotoxicity contributes to secondary cell death. Furthermore, I described early calcium waves in reponse to injury that may instruct repair mechanisms; using pharmacological agents, I identified the release of glutamate, acting on neurons surrounding the injury site, and ATP, acting predominantly on glial cells, as the upstream mechanisms of calcium waves. In contrast to observations in rodent models and human TBI patients, I observed little infiltration of peripheral macrophages or neutrophils. Microglia, the resident immune cells of the brain, were rapidly recruited to the injury site and significantly increased their phagocytic activity upon injury. Inhibition of microglial phagocytosis by targeting phosphatidyl serine receptors either pharmacologically or genetically via CRISPR/Cas9-mediated gene editing resulted in a significant increase in the rate of secondary cell death. This result demonstrated a role for rapid phagocytosis of debris in limiting the extent of secondary cell death, and suggested an overall neuroprotective role for microglial phagocytosis. Probing into the transcriptome of microglia/macrophages following injury using RNA sequencing of sorted cells revealed profound transcriptomic changes within two hours of injury, and will aid the future investigation of the role of microglia in neuroprotection. To summarise, this body of work provides evidence for a neuroprotective role of microglia in a new larval zebrafish model of brain injury, and the transcriptomic analysis will form the basis of a more in-depth study of microglia-derived neuroprotective molecules. Using CRISPR/Cas9-guided mutation of genes of interest in larval zebrafish, we will be able to investigate the role of these genes following injury in microglia-mediated neuroprotection. Following further validation, these findings could potentially be exploited for prevention of secondary cell death after human TBI.

http://hdl.handle.net/1842/35921

en

The University of Edinburgh

traumatic brain injury

zebrafish

microglia

excitotoxicity

secondary cell death

calcium waves

Molecular and cellular mechanisms of microglia-mediated neuroprotection

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy