When a brain region becomes active, blood flow to the area is increased to ensure cells receive an adequate supply of glucose and oxygen from the surrounding vasculature. This is controlled by complex interactions between the cells of the nervous system and blood vessels, a process termed ‘neurovascular coupling’ (NVC). Impairments in NVC are involved in stroke, hypertension, Alzheimer’s, and vascular dementia. Currently, NVC can only be studied in a living brain with an intact blood supply, so NVC research relies on studies in live animals, commonly mice and rats. Imaging protocols to measure blood flow in the rodent brain require animals to have a section of their skull surgically removed or thinned, and in longitudinal studies, the measurements are made over several weeks.
Why we funded it
This Project Grant aims to replace the use of rodents in the study of neurovascular coupling with a zebrafish larval model. Zebrafish larvae are not classified as protected until five days old when they begin independently feeding.1
A typical NVC study comparing a control and experimental group uses five to eight mice per group, whereas complex experimental designs involving multiple experimental groups examined at multiple timepoints can require up to 160 mice per study. The severity of the model is classified as moderate under the Animals (Scientific Procedures) Act 1986. Approximately 3000 studies were published in 2015 using mammals for neurovascular studies, equating to an estimated 35,500 animals used in these studies annually. Dr. Chico and his colleagues estimate that the zebrafish larvae model could replace up to 10% of animals used in NVC research, removing the requirement of approximately 3500 animals per year globally. This estimation does not include unpublished studies or studies performed in industry.
Compound transgenic zebrafish will be created with genetically encoded fluorescent tags and imaged with light-sheet microscopy enabling simultaneous analysis of cerebral blood flow (CBF), calcium signalling in neurons, and calcium signalling in the endothelium. Neuronal activation in response to a sensory stimulus (light) will be quantified and any effects of this activation on regional CBF and endothelial calcium signalling identified. An increase in regional CBF in response to light provides evidence that NVC also exists in zebrafish. The model will be validated by testing the effect of hypercapnia and two different pharmacological interventions known to affect NVC on evoked blood flow responses and comparing the results with published data from mammals. Unlike mammals, zebrafish can survive in the absence of blood circulation allowing the effect of reversible blood flow reduction on calcium signalling in neurons to be examined by temporarily halting cardiac contraction. Changes in neural activity in response to decreasing CBF gives support to the hemo-neural hypothesis, a novel view of information processing which proposes that hemodynamics serve as more than a metabolic support system for local neural networks. Improved knowledge of how changes in CBF impact neural processing will contribute to current understanding and treatment of diseases such as stroke, epilepsy, and dementia.
1 The Animals (Scientific Procedures) Act 1986 defines protected larval forms of vertebrate animals as independently feeding. Zebrafish larvae begin independently feeding 120 hours (five days) post fertilisation so are not considered protected until this time.
Savage AM et al. (2019). tmem33 is essential for VEGF-mediated endothelial calcium oscillations and angiogenesis. Nature Communications 10:n732. doi: 10.1038/s41467-019-08590-7
Chhabria K et al. (2018). The effect of hyperglycemia on neurovascular coupling and cerebrovascular patterning in zebrafish. Journal of cerebral blood flow and metabolism doi: 10.1177/0271678X18810615
Principal investigatorDr Clare Howarth
InstitutionUniversity of Sheffield
Co-InvestigatorDr Timothy Chico
Dr Vincent Cunliffe
Dr Robert Neil Wilkinson
Professor Oliver Bandmann