Developing a zebrafish model of neurovascular coupling (NVC)
Lead Research Organisation:
University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease
Abstract
The blood supply to the brain is tightly controlled by a process called "neurovascular coupling". This increases blood supply to areas of the brain which are active, although how this is achieved is not completely understood. Neurovascular coupling is defective in conditions such as stroke and dementia, and understanding this process might ultimately lead to strategies to improve these diseases.
Because neurovascular coupling can currently only be studied in a living brain with an intact blood supply, its study requires the use of animals, commonly mice and rats. These experiments require surgery to expose the brain, and imaging of the brain in animals that are either awake and restrained, or anaesthetised, which inevitably induces distress and suffering. We therefore wish to develop a model that can provide scientific insight into neurovascular coupling but reduces the number of invasive animal experiments.
The zebrafish has two key advantages for the study of neurovascular coupling. Firstly, because it is transparent and we can generate "transgenic" animals in which certain cells fluoresce different colours, we can image the brain without any instrumentation. Secondly, we can stop the heart without the animal dying, as it obtains sufficient oxygen from the water surrounding it. The tiny zebrafish embryo is not considered to be a free living animal until it is older than 5d, and using these is not considered to be an animal experiment by government legislation (called non-protected).
We will exploit these advantages to develop a model of neurovascular coupling in non-protected zebrafish embryos. We have generated genetically modified zebrafish in which either the blood vessels or the nerves fluoresce brightly when levels of calcium in these cells increase. Because calcium levels increase in cells that are "activated", these transgenic lines allow us to image which parts of the brain are activated in response to a stimulus such as light or sound. We can then measure blood flow in different parts of the brain in response to this stimulus, and perform experiments to test whether this response is affected by certain drugs or alterations in conditions such as increased levels of carbon dioxide or alteration in blood flow.
If we find that neurovascular coupling exists and is controlled in the same ways in zebrafish as in mammals, our new model would be able to replace the use of protected animals and to provide new scientific insights into neurovascular coupling.
Because neurovascular coupling can currently only be studied in a living brain with an intact blood supply, its study requires the use of animals, commonly mice and rats. These experiments require surgery to expose the brain, and imaging of the brain in animals that are either awake and restrained, or anaesthetised, which inevitably induces distress and suffering. We therefore wish to develop a model that can provide scientific insight into neurovascular coupling but reduces the number of invasive animal experiments.
The zebrafish has two key advantages for the study of neurovascular coupling. Firstly, because it is transparent and we can generate "transgenic" animals in which certain cells fluoresce different colours, we can image the brain without any instrumentation. Secondly, we can stop the heart without the animal dying, as it obtains sufficient oxygen from the water surrounding it. The tiny zebrafish embryo is not considered to be a free living animal until it is older than 5d, and using these is not considered to be an animal experiment by government legislation (called non-protected).
We will exploit these advantages to develop a model of neurovascular coupling in non-protected zebrafish embryos. We have generated genetically modified zebrafish in which either the blood vessels or the nerves fluoresce brightly when levels of calcium in these cells increase. Because calcium levels increase in cells that are "activated", these transgenic lines allow us to image which parts of the brain are activated in response to a stimulus such as light or sound. We can then measure blood flow in different parts of the brain in response to this stimulus, and perform experiments to test whether this response is affected by certain drugs or alterations in conditions such as increased levels of carbon dioxide or alteration in blood flow.
If we find that neurovascular coupling exists and is controlled in the same ways in zebrafish as in mammals, our new model would be able to replace the use of protected animals and to provide new scientific insights into neurovascular coupling.
Technical Summary
Increased regional activity in the brain is accompanied by an increase in regional blood flow, controlled by "neurovascular coupling" (NVC). This is impaired in a range of human diseases including stroke and dementia.
Current animal models of neurovascular coupling generally use mice or rats, in which the brain is surgically exposed prior to sensory stimulation and measurement of cerebral blood flow. These are usually of moderate severity under ASPA and incur significant distress and discomfort.
Zebrafish embryos allow unparalleled in vivo imaging due to the ability to generate transgenic lines labelling specific cell types. It is also not protected by ASPA until 5.2d old. We therefore wish to develop a zebrafish model of NVC of minimal severity that could replace some rodent studies and provide novel scientific insights.
We have generated novel transgenic lines in which genetically encoded calcium indicators are expressed specifically in neurons or endothelial cells. Crossing these with other existing transgenics stably labelling erythrocytes and endothelium provides the ability to simultaneously image cerebral blood flow, neuronal and endothelial calcium signalling in the living embryo without instrumentation or distress to the animal.
We will determine whether and at what stage zebrafish exhibit NVC. We will administer a sensory stimulus to embryos of different developmental stages, and quantify the effect on cerebral blood flow and regional endothelial calcium signalling.
We will consider NVC to be detected if in response to a sensory stimulus that increases regional neuronal calcium signalling, we detect regional increases in blood flow and/or increased endothelial calcium signalling. To validate the model we will test the effect of nitric oxide synthase and cyclo-oxygenase inhibition and hypercapnia on NVC and compare our results with published data from mammals. Lastly, we will test the effect of reducing blood flow on neuronal activation.
Current animal models of neurovascular coupling generally use mice or rats, in which the brain is surgically exposed prior to sensory stimulation and measurement of cerebral blood flow. These are usually of moderate severity under ASPA and incur significant distress and discomfort.
Zebrafish embryos allow unparalleled in vivo imaging due to the ability to generate transgenic lines labelling specific cell types. It is also not protected by ASPA until 5.2d old. We therefore wish to develop a zebrafish model of NVC of minimal severity that could replace some rodent studies and provide novel scientific insights.
We have generated novel transgenic lines in which genetically encoded calcium indicators are expressed specifically in neurons or endothelial cells. Crossing these with other existing transgenics stably labelling erythrocytes and endothelium provides the ability to simultaneously image cerebral blood flow, neuronal and endothelial calcium signalling in the living embryo without instrumentation or distress to the animal.
We will determine whether and at what stage zebrafish exhibit NVC. We will administer a sensory stimulus to embryos of different developmental stages, and quantify the effect on cerebral blood flow and regional endothelial calcium signalling.
We will consider NVC to be detected if in response to a sensory stimulus that increases regional neuronal calcium signalling, we detect regional increases in blood flow and/or increased endothelial calcium signalling. To validate the model we will test the effect of nitric oxide synthase and cyclo-oxygenase inhibition and hypercapnia on NVC and compare our results with published data from mammals. Lastly, we will test the effect of reducing blood flow on neuronal activation.
Planned Impact
The number of animal studies performed worldwide in areas relevant to our application is very large. Table 1 (see case for support) shows the results of a PubMed search combining an animal species with a single search term restricted to publications in 2015. Although these have not been further filtered, it is clear that annually there are hundreds of animal studies (including non-human primates). It is noteworthy that there were fewer studies using zebrafish than macaques. Stroke studies make up the majority, with dementia second, though as discussed in the Case for Support neurovascular coupling is directly relevant to these diseases.
A zebrafish model of neurovascular coupling would fulfil all the 3Rs, in descending order of impact;
1) Replacement: by using non-protected embryos before free-living status, we will entirely replace the use of protected animals for experiments.
2) Refinement: A major refinement of our model is the lack of surgery or instrumentation, either to alter blood flow, stimulate neural activity, or access the brain for imaging. These are important considerations in view of the need to perform invasive surgery and image brain physiology in awake or anaethetised mammals via a cranial window as used in current mammalian studies. All our work would be mild in severity (simply maintaining and breeding adult transgenic zebrafish) compared with the moderate ASPA severity in place for existing work. The use of an organism of lower neurophysiological sensitivity is an additional refinement.
3) Reduction: The use of compound transgenic zebrafish expressing multiple cell-specific trangenes in a range of fluophores will increase the amount of data obtained per animal and so reduce overall animal numbers.
If one assumes 50% of the 3,224 mammal and primate studies published in 2015 in table 1 are primary research, and that each study uses 12-32 animals, an estimated 35,464 animals are currently used per year, or 354,640 in a ten-year period. We anticipate a validated and well-disseminated zebrafish model might replace somewhere between 1-10% of such work, or between 3,546-35,646 animals over 10 years; this figure does not include unpublished studies or studies in industry, so it is likely to be an underestimate. Since both Dr Howarth and other colleagues in the NICAD network currently use rodent models of neurovascular coupling we are in an excellent position to immediately apply the zebrafish model to current research projects, initially in parallel, and subsequently in place of, mouse studies. Dr Howarth and colleagues are internationally prominent researchers using mammalian models of neurovascular coupling, the development of an alternative model by a group already engaged in mammalian work is likely to facilitate uptake. Although we will publish and disseminate our model once validated, its uptake may be promoted best by proving it can replace the use of mice and rats within our own institution. As well as publishing our model we will therefore disseminate our experience via media such as Lab Animal, & the NC3Rs newsletter.
A zebrafish model of neurovascular coupling would fulfil all the 3Rs, in descending order of impact;
1) Replacement: by using non-protected embryos before free-living status, we will entirely replace the use of protected animals for experiments.
2) Refinement: A major refinement of our model is the lack of surgery or instrumentation, either to alter blood flow, stimulate neural activity, or access the brain for imaging. These are important considerations in view of the need to perform invasive surgery and image brain physiology in awake or anaethetised mammals via a cranial window as used in current mammalian studies. All our work would be mild in severity (simply maintaining and breeding adult transgenic zebrafish) compared with the moderate ASPA severity in place for existing work. The use of an organism of lower neurophysiological sensitivity is an additional refinement.
3) Reduction: The use of compound transgenic zebrafish expressing multiple cell-specific trangenes in a range of fluophores will increase the amount of data obtained per animal and so reduce overall animal numbers.
If one assumes 50% of the 3,224 mammal and primate studies published in 2015 in table 1 are primary research, and that each study uses 12-32 animals, an estimated 35,464 animals are currently used per year, or 354,640 in a ten-year period. We anticipate a validated and well-disseminated zebrafish model might replace somewhere between 1-10% of such work, or between 3,546-35,646 animals over 10 years; this figure does not include unpublished studies or studies in industry, so it is likely to be an underestimate. Since both Dr Howarth and other colleagues in the NICAD network currently use rodent models of neurovascular coupling we are in an excellent position to immediately apply the zebrafish model to current research projects, initially in parallel, and subsequently in place of, mouse studies. Dr Howarth and colleagues are internationally prominent researchers using mammalian models of neurovascular coupling, the development of an alternative model by a group already engaged in mammalian work is likely to facilitate uptake. Although we will publish and disseminate our model once validated, its uptake may be promoted best by proving it can replace the use of mice and rats within our own institution. As well as publishing our model we will therefore disseminate our experience via media such as Lab Animal, & the NC3Rs newsletter.
