Project duration
5 years 0 months
Project purpose
- (a) Basic research
Key words
- Neuroscience
- Cardiovascular
- Stem cells
- Brain
- Spinal cord
Animal types
- Mice
- Rats
Life stages
Mice:
- Neonate
- Juvenile
- Adult
- Embryo
- Pregnant
- Aged
Rats:
- Juvenile
- Adult
- Aged
- Neonate
Retrospective assessment
The Secretary of State has determined that a retrospective assessment of this licence is not required.
Objectives and benefits
Description of the project’s benefits, for example the scientific unknowns or clinical or scientific needs it’s addressing.
What's the aim of this project?
The project aims to increase understanding of how the central nervous system is involved in the control of body functions, such as those controlling heart rate and blood pressure.
In addition, it aims to understand how to induce the adult central nervous system to make new cells that may interact with these circuits.
Potential benefits likely to derive from the project, for example how science might be advanced or how humans, animals or the environment might benefit - these could be short-term benefits within the duration of the project or long-term benefits that accrue after the project has finished.
Why is it important to undertake this work?
Modulating the activity of the nervous system by direct electrical or chemical intervention is increasingly under investigation for treatments of disorders such as hypertension, heart failure and multiple sclerosis.
However, knowledge of the neuronal circuitry underlying such modulation is far from complete and furthering such understanding is likely to contribute to development of more targeted and better treatments. Recent technological advances have improved the ability to gain this understanding and such a project is therefore timely.
What outputs do you think you will see at the end of this project?
The most likely outputs will be new information, which will be made available through publication primarily in scientific journals.
This information may lead to improved or new treatment for diseases that are influenced by circuitry in the spinal cord and brainstem, for example hypertension, heart failure, multiple sclerosis, spinal cord injury.
Who or what will benefit from these outputs, and how?
In the short term the most likely benefits will be to our own lab and to other scientific researchers, who will use the outputs to build upon and further increase knowledge.
In the medium to longer term these advances in fundamental understanding may provide potential translational benefits. For example, understanding of central control of autonomic function gained from previous animal work has encouraged exploration of modulating the activity of the nervous system in humans through non-invasive electrical stimulation with the potential for treatment of diseases such as heart failure, hypertension and arthritis and indeed benefit some signs of ageing.
Our fundamental work will examine the mechanisms and circuits underpinning such functions e.g. by identifying the anatomical and functional properties of constituent cells, how the cells are connected to each other and the functions of the networks. Understanding these will contribute to our ongoing studies in humans.
Potential benefits from work on production of new cells in the nervous system may arise in the treatment of many disorders.
In spinal cord injury, there is a massive upregulation in cell proliferation and if this can be controlled even a little there could be significant improvements to reducing the degree of injury and improving rehabilitation. Pathological increases in neural cell proliferation can also occur and these can underpin brain tumour formation.
Therefore, identifying approaches to increase or decrease cell proliferation and differentiation could have longer term benefits to patients with these conditions.
How will you look to maximise the outputs of this work?
Collaboration is a mainstay in the laboratory - most of our publications have collaborative authors from other labs and indeed, from abroad. This increases the potential for the work to be seen by a larger audience and increase citations, disseminating knowledge effectively. Such collaboration will at least remain at the same level or indeed will likely increase.
We will also aim to present the findings at national and international conferences, where audiences often view outside of their close research area.
When appropriate we also produce press releases, which have led to widespread international media coverage and brought our research to the attention of the public.
We will also continue with our public engagement where presentations based on our work have been made to events such as the national Pint of Science and events held by our University which are run annually, with around 1200 participants per day from the local community.
Species and numbers of animals expected to be used
- Mice: 8600
- Rats: 1500
Predicted harms
Typical procedures done to animals, for example injections or surgical procedures, including duration of the experiment and number of procedures.
Explain why you are using these types of animals and your choice of life stages.
We will use predominantly juvenile and adult mice and on some occasions, rats. Many of the fundamental processes that are controlled by neuronal circuits that we aim to understand are comparable to those in humans e.g. the baroreceptor reflex that controls blood pressure.
In addition, increasing availability of transgenic mouse lines which enable specific cells to be identified and/or manipulated increase precision and specificity of studies in a manner that is not available in other species. To allow for development of circuitry we will mostly use adult animals, but in some cases they will require intervention at a younger age (from neonate) to promote expression of genes in specific cells.
Typically, what will be done to an animal used in your project?
In a typical experiment an animal will be terminally anaesthetised and either perfused with a fixative for anatomical studies, or will have tissue removed and sliced or en bloc for recording activity of cells ex vivo.
In some cases there will be a prior surgical intervention under anaesthesia, to inject a vector that will allow expression of genes in specific cells into the brain or spinal cord.
Another intervention will be injection of a substance to influence production of new cells, usually intraperitoneally via a short procedure that does not require anaesthesia.
Animals will recover for up to 6 months, but more usually less than 4 weeks. Following this animals will be terminally anaesthetised and prepared as above.
What are the expected impacts and/or adverse effects for the animals during your project?
Animals used only for anatomical studies or tissue slices do not recover from anaesthesia.
Post-surgical animals routinely receive suitable analgesia. Weight loss which is typically less than 10% is often seen on the first day following surgery and weight gain is within two days post surgery.
Expected severity categories and the proportion of animals in each category, per species.
What are the expected severities and the proportion of animals in each category (per animal type)?
Preparing animals for anatomical studies or tissue slices is non-recovery. This is likely to account for 40% of the animals used in this project.
Animals which have had injections into the CNS may exhibit moderate severity (hunched posture, piloerection, transient anorexia, up to 10% weight loss) outcomes for a day following procedures. This is likely to account for 30% of the animals used.
Animals which have had interventions to modulate production of new cells typically exhibit mild severity (transient underactivity, piloerection), likely to account for approximately 30% of the project.
What will happen to animals at the end of this project?
- Killed
Replacement
State what non-animal alternatives are available in this field, which alternatives you have considered and why they cannot be used for this purpose.
Why do you need to use animals to achieve the aim of your project?
The work will study how the CNS controls body functions, and at times how manipulating outflows of these circuits can influence progression of disease.
It will also investigate how the production of new cells in the central nervous can be manipulated in vivo.
These will necessarily involve placing tracers onto peripheral nerves to identify the cells or their origin, determine the effects of stimulating particular nerves or manipulate the signalling in the CNS to influence stem cells.
Therefore, to provide physiological relevance whole animals are required. However, where in vitro work can be undertaken we will do so e.g. we use acutely prepared spinal cord slices for electrophysiology.
In addition, we have cultured spinal cord slices to facilitate some experiments in vitro, particularly for studying neurogenesis. This allows for greater numbers of tests to be performed in vitro, reducing the number required in vivo.
Which non-animal alternatives did you consider for use in this project?
A possible longer term alternative is to use organoids derived from induced pluripotent stem cells or via cell reprogramming.
Why were they not suitable?
It is not yet possible to derive organoids that recapitulate the circuitry as they do not have the appropriate physiological inputs, such as fluctuations in blood pressure, changes in respiration etc.
In addition, there is not enough information on circuitry to know if such organoids are faithful to the intact brainstem or spinal cord. Cell lines and cell cultures also do not have the necessary connections and appropriate circuitry.
Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce animal numbers, and principles used to design studies.
Describe practices that are used throughout the project to minimise numbers consistent with scientific objectives, if any. These may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
How have you estimated the numbers of animals you will use?
The numbers have been estimated with our experience of previous projects and accounting for breeding of transgenic animal lines. They allow for appropriate blinding of researchers as well as the use of either sex.
We will conduct our experiments to be able to publish to the Arrive guidelines: https://www.nc3rs.org.uk/arrive- guidelines.
What steps did you take during the experimental design phase to reduce the number of animals being used in this project?
Most of our experiments do not have prior knowledge of effect size. When this can be estimated from our pilot studies we have used online resources such as the NC3Rs Experimental Design Assistant to help calculate the number of animals.
What measures, apart from good experimental design, will you use to optimise the number of animals you plan to use in your project?
We aim to breed as efficiently as possible. For example, cross breeding from homozygotes is preferred as this ensures that offspring carry the desired genes. Our institution has an online database that enable us to follow such breeding carefully.
If possible we conduct pilot studies on tissue slices, since many slices can be obtained from one animal and this enables pilot testing where several animals may otherwise be required.
The number of animals used will be optimised in several ways:
- Tissue from the same animal will be used for different objectives where possible. This reduces the total number of animals that would otherwise be required if a single animal was used for each objective.
- We have also developed a method of storing tissue for long term which also reduces animal use - this tissue is also available to other labs.
- In addition, each experiment is designed to maximise the amount of information gleaned since they often combine different approaches to verify this information.
Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals.
Describe the mechanisms in place to take up emerging refinement techniques during the lifetime of the project.
Which animal models and methods will you use during this project? Explain why these models and methods cause the least pain, suffering, distress, or lasting harm to the animals.
Rats and mice will be used since they contain cell types and assemblies of cells that are similar to those known in humans as far as can be told so far. Transgenic animals will be used when they make identifying and/or manipulating specific cell types possible with fluorescent markers, helping to minimise numbers used.
Suffering of animals will be minimised as the majority of the procedures performed will have a minor severity where the animal is killed by anaesthesia prior to organ removal. In some cases experiments will require recovery from anaesthesia following surgical procedures. Post-operative analgesia will be applied in these cases to limit suffering of the animals.
Why can’t you use animals that are less sentient?
We cannot use more immature animals as we are studying the control of the autonomic nervous system and this is not properly developed until the adult stages. Similarly, the study of adult neurogenesis (the production of new neural cells) requires adult animals since such cells can be more readily produced in the neonate.
Less sentient species (such as flies and worms) do not have the same autonomic nervous system control.
We will use terminally anaesthetised animals in many of our experiments, but some will require surgical intervention with recovery to allow expression of markers or functional controllers in sets of cells beforehand or to mark the terminations of specific nerves. There is no current approach to do this without surgery.
How will you refine the procedures you're using to minimise the welfare costs (harms) for the animals?
Suffering of animals will be minimised as the majority of the procedures performed will have a minor severity where the animal is killed by anaesthesia prior to organ removal. In some cases experiments will require recovery from anaesthesia following surgical procedures. Post-operative analgesia will be applied in these cases to limit suffering of the animals.
Examples of refinements that we have made include:
- peri--operative administration of warm saline to ensure that the animals remain hydrated
- adding food mashed with water to the cages after surgery so that there is easy access for eating and maintaining hydration post-surgery
- housing with other mice wherever possible (which is usually the case).
What published best practice guidance will you follow to ensure experiments are conducted in the most refined way?
The NC3Rs have published best practice guidance: (Prescott MJ, Lidster K. Improving quality of science through better animal welfare: the NC3Rs strategy. Lab Anim (NY). 2017 Mar 22;46(4):152-156. doi: 10.1038/laban.1217. PMID: 28328893) and this and subequent publications will be referred to.
We attend webinars from Nc3R like "the best practice in experimental design":
In addition, the ARRIVE 2.0 guidelines will be referred to assist with good design as well as reporting.
How will you stay informed about advances in the 3Rs, and implement these advances effectively, during the project?
I, and my lab members, take part in CPD regularly. This includes courses run internally as well as reading appropriate literature including N3CR resources such as websites and the newsletters we receive. In some conferences, there are posters and sessions dedicated to the 3Rs.
We discuss our experiments with other researchers, NACWO and the vet, implementing appropriate changes as we go through projects.
Download central nervous system control of physiological functions non-technical summary (PDF).