Understanding the cellular neurobiology of paediatric stroke and moyamoya disease

Stroke is often thought of as a disease affecting older adults, but it is a significant condition in children as well - with several hundred affected in the UK annually. Paediatric (children) and neonatal (newborn baby) stroke can lead to significant disabilities including cerebral palsy - a lifelong condition that costs the NHS millions of pounds per year. Although in adults stroke is most often due to atherosclerosis (narrowing of the arteries due to high cholesterol, smoking and other risk factors), in children an underlying condition such as moyamoya disease is often found. In moyamoya, the arteries of the brain narrow, causing a chronic reduction of blood flow to the brain. The presentation, course of, and recovery from stroke are different in children and adults, for reasons we do not yet fully understand.

I am a paediatric neurosurgeon who looks after children with moyamoya and stroke. I am part of a multidisciplinary neurovascular team that has the largest caseload of moyamoya and paediatric stroke in the UK. Early in my training, I undertook a PhD project where I used basic neuroscience techniques to examine the response to injury of both neurons (nerve cells) and glial cells (specialized supporting cells) in the brain. I have always been interested in understanding the mechanisms in the brain that are involved in the response to injury and stroke, and hope to use this understanding to improve our treatments for children with these conditions. In this project I wish to link my clinical paediatric neurovascular practice with my basic neuroscience training to elucidate these mechanisms in the world-renowned laboratory of my research partner. The Attwell lab has great expertise in studying neurons, glial cells, and brain blood flow and its disorders, using state of the art techniques including patch-clamping, 2-photon microscopy and a range of transgenic animals allowing cell identification.

Capillaries are tiny blood vessels which deliver oxygen and nutrients to brain tissue. My research partner has discovered that these are regulated by surrounding cells called "pericytes" which, in part via glial cells, respond dynamically to changes in blood flow and oxygen levels. I will study the responses of pericytes to ischaemia (when the brain does not get enough blood, e.g. during stroke or in moyamoya) in juvenile rodents of different ages, using 2-photon and confocal microscopy.

Axons are the tiny "wires" that send signals between brain cells. During childhood the initially bare axons are progressively wrapped in an insulation called myelin, which is necessary for development of brain function. Employing rodent models of stroke and moyamoya, I will use immunohistochemical labelling, calcium imaging, mathematical conduction modelling and electrophysiology to examine the effect of ischaemia on axons, myelination and the nodes of Ranvier (gaps between myelin) in the developing brain.

I will also characterise the stroke-relevant properties of microglia, which are the immune cells of the brain, carrying out surveillance of the microenvironment and responding to injury and cell death, as well as pruning unnecessary synapses. Using brain slices from transgenic rodents I will examine with 2 photon microscopy the responses of microglia to ischaemia in the developing brain and compare them to those of adults.

As a neurosurgeon, I have access to brain tissue from operations: small pieces removed to access vascular malformations and tumours, which would otherwise be discarded. This gives me a unique opportunity to replicate rodent experiments on human brain slices and test whether the mechanisms are the same.

These experiments will delineate the cellular mechanisms underlying the response of the child's brain to ischaemia, and may lead to the identification of targets for novel drug and surgical therapies to reduce the brain damage caused by, and improve recovery from, paediatric stroke and moyamoya.

This research project will examine the physiology of the response of young brains to ischaemia - seen in acute arterial stroke and moyamoya disease. Both rodent tissue and human tissue (discarded from neurosurgery) from individuals of different ages will be studied (with rodent age chosen to mimic human ages). Rodent models of ischaemia, in brain slices and in vivo in anaesthetised animals, will be used. My research partner's laboratory has transgenic mice labelling pericytes (NG2-dsRed) and sensing pericyte [Ca2+] (NG2-GCaMP6), and labelling microglia (Iba-1-GFP), and paranodes (Caspr-GFP, to define the node of Ranvier) to facilitate the work.

The experiments will examine 4 themes:

(1) Responses of brain capillaries to ischaemia in the juvenile brain. Two-photon microscopy will be used to assess pericyte-mediated capillary constriction in normal tissue from juveniles of different ages and tissue which has been subjected to ischaemia.

(2) Responses of axons (myelinated and unmyelinated) to ischaemia during development. Immunohistochemistry and electrophysiology will be used to examine axonal function and myelination following ischaemia.

(3) Responses of the node of Ranvier to ischaemia during development. Immunohistochemisty and mathematical modelling of conduction will be used to examine the effect of ischaemia on the structure and function of the node.

(4) Microglial function in the juvenile ischaemic brain. Two-photon microscopy and assays of microglial surveillance activity will be used to examine the response to ischaemia.

The mechanistic information from these experiments will identify potential therapeutic targets for intervention, and will synergise the basic science expertise of the host lab with my clinical experience and membership of the UK's largest paediatric neurovascular service.

The NIHR GOSH BRC is the only BRC in the UK solely devoted to children's disease. This project offers a potential new collaborative workstream focussing on neural injury and repair in children. Most importantly, understanding and elucidating the basic cellular mechanisms underlying the response of the brain to ischaemia and its revascularisation will lead to benefit to children and families affected by stroke and moyamoya disease. This will be through increased understanding of the pathophysiology, potentially leading to new and refined pharmacological, surgical and rehabilitative interventions.

Paediatric and neonatal stroke and its aftermath, such as cerebral palsy, developmental delay and motor disorders, inflict a massive financial burden on the NHS and social care. Because these diseases affect children,they limit the children's capacity to enter education and the workforce, adding a further economic penalty. Any reduction in the damage, or improvement of the recovery, from paediatric stroke made possible by the results of this study would have significant and long-term economic repercussions.

The cellular mechanisms we are planning to study are likely to also apply to some other causes of brain injury, implying that the results will be of benefit to both the basic and clinical neuroscience research communities.

For the NHS Consultant, the training and mentorship received from the academic partner will be invaluable in establishing an independent research group. The UK neurosurgery community has identified a need for surgeon-scientists who can "bridge the gap" between basic science and clinical neurosurgical practice. Surgeon-scientists can bring a unique clinical perspective to laboratory science. The long-term aim of the NHS Consultant is to have an active translational research programme to investigate clinically-relevant basic science aspects of all paediatric neurovascular disease, and this project is a critical step in achieving that goal.

For the academic partner's laboratory, having an experienced clinician as part of the team can bring benefits in several ways. Materially, it facilitates access to human tissue which, as well as being used in the primary project, may be very useful for other laboratory members working on related projects. In addition, clinical experience and feedback from children and families can be helpful in guiding research questions and potential therapeutic applications of both the primary project and allied work in the lab.

The Department of Neurosurgery at Great Ormond Street Hospital is the largest in the UK (and one of the largest in the world) in terms of clinical workload. The Department has made increasing its academic profile a priority. By funding dedicated research time, and by building collaboration with the well-respected research programme of the academic partner and within the UCL Neuroscience Domain, the NHS Consultant will be able, via publication and presentation of results, to establish an international profile in translational neuroscience research. This will benefit colleagues, trainees and students in the Department of Neurosurgery.

The project will generate opportunities for BSc, MSc and PhD students to contribute to research in the laboratory, to the benefit of their education. Due to the clinical background of the NHS Consultant we expect that clinical students undertaking a period of research, such as UCL intercalating medical students and GOS-ICH and Institute of Neurology MSc and PhD students, would be interested in undertaking research for their degree as part of this study. In the longer term, the NHS Consultant would apply for his own grants to fund PhD students. Expertise in supervising PhD and other students will be provided by the research partner who has supervised many such students, and has received the FENS-Kavli award for mentoring neuroscientists.