Understanding brain regeneration in a zebrafish larval model of intracerebral haemorrhage

Brain haemorrhages are the most severe type of stroke, and patients are often left with disabilities due to brain damage. We currently do not know much about how the brain tissue adapts after the bleed. Presently, the research into brain regeneration following haemorrhage mostly uses invasive, surgically-induced rodent models that do not accurately recreate the spontaneous nature of the human condition. Experimentally, stem cells and synthetic scaffolds are injected into the brain to encourage regrowth, however once delivered into the body, the response is very difficult to measure accurately. Zebrafish can regenerate from injuries, including damage to the central nervous system. We have previously shown that zebrafish larvae, a small, transparent, immature organism can exhibit spontaneous brain bleeds like humans and then quickly recover from injury, growing into healthy adults. I think that we can learn more from the zebrafish brain recovery after a haemorrhage, and utilise their unique transparency, to non-invasively visualise the live recovery response, something that is impossible in rodent models. This work would lead to a reduction in the number of protected animals required for such experiments, as a single pair of breeding zebrafish can produce ~200 embryos.
The first step of my project will be to image the cells inside the brain responding to the bleed, to answer the following questions: How quickly do immune cells clear the blood? What fills the space left by the haematoma? Do new brain cells form in the space, or do existing ones spread across the gap? The second part of my project will require extracting these brain stem cells from the zebrafish and investigating the genetic and proteomic factors that influence their behaviour. In the final stage of my project I will apply what I have learnt to human brain stem cell cultures in a dish and determine if turning on the same genes, or exposing them to the same proteins, encourages the same regenerative behaviours we observe in the zebrafish. The ultimate aim of this work is to enable scientists to develop better models of brain regeneration in which to investigate regenerative therapies for patients that could potentially prevent long-term disability after a brain haemorrhage, and reduce the need for animal models in the long term.

Intracerebral haemorrhage (ICH) is the most devastating subtype of stroke in terms of mortality and morbidity with limited treatment options. Very little is understood about the rehabilitation in patients from neurophysiological, anatomical and behavioural perspectives. Currently, pre-clinical regenerative medicine methods require the use of invasive collagenase-induced/autologous blood injection models of ICH in rodents. Injection of stem cells, hydrogel scaffolds and functional biomaterials show promising results in vivo promoting the repair of damaged neurons and reducing neuroinflammatory responses. However, elucidation of stem cell fate requires ex vivo methods and longitudinal studies of recovery, and mechanisms of repair are still unknown. I propose that we can utilise a spontaneous, non-invasive model of ICH in zebrafish larvae to investigate the inherent regenerative properties and better understand these mechanisms. Preliminary evidence shows that brain damage and functional deficits observed 24 hours after ICH (72 hours post fertilisation) recover in the following days before 5 days post fertilisation, at which zebrafish larvae acquire protected status. Utilising this model would reduce the number of protected species required for experimentation as breeding adult zebrafish have high fecundity. The objectives of this project are threefold. Firstly, to image the immune and neuronal cellular response to the brain bleed in a live organism, secondly to identify the genetic and proteomic changes in neural progenitor cells over the period of recovery, and thirdly to explore these mechanisms in in vitro cultures of human neural progenitor cells. Collectively, these experiments will contribute to a better understanding of the regenerative processes after ICH, validate the zebrafish model further reducing the need for protected species, and potentially advance the regenerative medicine strategies for ICH patient recovery.