NIHRDataInsights/HRCSData huggingface dataset:
Title: Assessing cumulative severity in macaques used in neuroscience research.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: It is assumed that there are cumulative effects of repeated invasive procedures and contingent stressors on the welfare of non-human primates used in neuroscience research. The new EU Directive (2010/63/EU) on the use of animals in research emphasises the "lifetime experience" of animals and requires assessment of the "cumulative severity" of experiments. However, there are currently few data on the long-term impacts of repeated procedures to inform severity banding. We aim to develop novel psychobiomarkers of cumulative stress based on our knowledge of major depressive disorder in humans and apply these measures to macaques involved in neuroscience research. We will take two main approaches: (1) measurement via qPCR of changes in leukocyte telomere length, and (2) measurement via MRI of structural and functional changes in the brain. These dependent variables will be taken at 6-monthly intervals from a group of 40+ animals involved in various types of neuroscience research; telomere measurements will also be made in 30+ control animals not subject to invasive procedures. Information on sources of possible stress (our independent variables, e.g. number and type of procedures, days on antibiotics or fluid control) will be collated for all animals in the study. Statistical modeling will be used to explore the relationships between potential stressors and our candidate psychobiomarkers. These analyses will allow us to answer the question of whether the number and/or type of stressors a monkey has experienced predict changes in psychobiomarkers of cumulative stress. If cumulative effects of stressors are found, we will attempt to identify the shape of the relationship between stressors and our psychobiomarkers. Our results will: (1) provide a scientific basis for determining the cumulative severity rating of procedures, (2) reveal procedures that are most in need of refinement, and (3) permit adjudication on the relative costs of continued use of animals.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL
Title: Development of a sensitive in vitro system for anti-alpha-synuclein aggregation drug screening
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Parkinson's disease is a brain disorder characterized by aggregated alpha synuclein (a-syn) inside intraneuronal inclusions known as Lewy bodies. We studied if protein misfolding cyclic amplification (PMCA), used to replicate prion protein aggregates could be used to grow a-syn aggregates. Briefly, proteins are subjected to cycles of sonication and incubation and fibril formation is monitored by increased Thioflavin T fluorescence. Our electron microscopy, circular dichroism and native gels results indicate that stable a-syn fibrils were formed. We also tested the a-syn anti-aggregation activity of several drugs and the results suggest that the a-syn PMCA is robust system for anti-aggregating drug screening. So we propose to use previously approved drugs, with animal testing already done, to repurpose into the treatment for Parkinson disease using the a-syn PMCA. The proposal has two aims: 1. drug screening by high throughput a-syn PMCA: We propose to screen a library from previously FDA approved drugs with high chemical and pharmacological diversity and other with compounds marketed in Europe and/or Asia but not yet in the USA. A cut off of 50% inhibition in the HTS a-syn PMCA will be set up to prioritize the study of the drugs with higher potential. Other prioritization will be done based on the EC50, route of administration and side effects to reduce suffering in mice models. 2. Study of the hits biological relevance in cell and mice models of Parkinson's disease We plan to use PC12 cells overexpressing human alpha synuclein to study the presence or absence of insoluble alpha synuclein in the cells after transducing them with fibrils and in the presence of the hit drugs. We also want to use the A53T mice model that has widespread a-syn aggregation in the brain. The drug effect will be tested, before and simultaneously to the clinical symptoms appearance. With this time points we hope to stop the progression of the disease and reduce animal suffering.


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL
Title: An in vitro model for pain and neurogenic inflammation in the oro-facial region and upper airways
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Advances in our understanding of bi-directional communication between the nervous system and the immune system acknowledges that sensory nerves contribute to the local inflammatory response (neurogenic inflammation) and that inflammatory cytokines contribute to the sensitisation of sensory nerves. As the most densely innervated area of the body, the oro-facial region represents an excellent model for investigating cross-talk between the nervous system and immune system. Research on neurogenic pain and inflammation has conventionally focused on animal models in which nerves are intentionally damaged. However, whether such models reliably predict the human response is questionable. An alternative ex vivo human model using redundant human tissue (dental pulp from extracted human teeth) has the advantage of species specificity but is limited by the fact that only peripheral nerve terminals are present, negating its use in the study of bi-directional communication. We propose to exploit the fact that dental pulp tissue is a rich source of stem cells from which peripheral neurones (with cell bodies), can be differentiated. We propose to functionally characterise peripheral neurones differentiated from dental pulp stem cells as a model system for studying nociception and neurogenic inflammation. This in vitro model will not only allow replacement of animal models, but concomitantly offers important advantages over existing ex vivo models for the study of peripheral nerve injury.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Using non-invasive in vivo imaging to address the 3Rs in high-throughput mouse phenotyping
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: In the wake of the first draft of the full mouse genome sequence, large-scale mutagenesis programmes are underway that will produce mice with gene knockouts/point mutations for each of the approximately 25,000 genes in the mouse genome. Analysis of these mice in coming years will give new insights into the genetic basis of human disease and biology, as novel genes are identified that impact upon mammalian physiology and morphology. This studentship will develop non-invasive imaging of live mice to reduce the numbers of mice generated in high-throughput mutagenesis programs and start to refine experimental strategies for working with mouse models. We will use non-invasive in-vivo magnetic resonance imaging (MRI), to assess if this imaging technology can help us understand the function of genes within the brain, which will enable us remove major “bottlenecks” in the development of new diagnosis tests for genetic disease and human gene therapies.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: NEUROLOGICAL
Title: The development of an in vitro model of spinal cord injury to study aligned neurite outgrowth
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Spinal cord injury (SCI) usually results in devastating paralysis, which in the majority of cases is permanent due to the limited capacity of central nervous system (CNS) axons to regenerate. Laboratory models of SCI generally involve the use of animals and are technically demanding, time consuming and are followed by substantial disability in the animals with long-term post-operative care. Furthermore, biological variations in the level of injury necessitate the use of large groups of up to 10 animals in each cohort in order to impart statistical power into the data. It is now apparent that therapies for SCI will require combined strategies which require even larger cohorts of animals. We have developed a cell based assay of SCI which reduces and refines the number of animals necessary to test potential therapeutic agents for SCI as well as removing any suffering to the animal. These cultures comprise of a monolayer of astrocytes on top of which are plated dissociated rat mixed spinal cord cells. The cultures produce a carpet of neurites which become ensheathed with myelin internodes by oligodendrocytes interspaced with nodes of Ranvier. A lesion is created across the culture with a scapel blade, which is initially cell free and persistently lacks neurite outgrowth although many reactive astrocytes enter expressing markers associated with astrogliosis and inhibition of axonal outgrowth. These findings are in agreement with that seen for in vivo models of SCI. The cultures were validated as a SCI model by treating with agents known to promote repair in vivo. These include a cell-permeable form of the C. botulinum-derived Rho antagonist, C3 ADP-ribosyltransferase (C3), which promoted neurite outgrowth in these cultures with a concomitant reduction in CSPG levels. However, myelination was only enhanced when C3 was introduced together with the ROCK inhibitor Y-27632 or the PDE4 inhibitor rolipram. We now wish to develop these cultures using topographical cues to focus specifically on neurite outgrowth and regeneration after cutting. We will use novel constructs made from polydimethylsiloxane (PDMS) with topography to promote aligned neurite outgrowth and restraining pillars at on end of the device to isolate the neuronal cell bodies from their processes. The cultures will be standardised and validated with a range of agents known to promote axonal outgrowth. We aim to identify a combination of agents which could potentially be used in the clinic to promote SCI repair.


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL
Title: Refinement of the use of fluid control as a motivational tool for non-human primates in neuroscience research
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Behavioural neuroscience is important for understanding how the brain works, and aids the discovery of appropriate treatments for human brain diseases and conditions1. Non-human primates have similar neural structures to humans2, and are therefore used as a model species in this type of research. They are often motivated to perform in behavioural neuroscience experiments through the use of fluid rewards during the tasks, and an associated control of fluid intake outside of the experimenteg.3. Fluid control protocols are contentious, and yet there are very few scientific data to inform how these protocols could be refined or replaced4. This project is the first to systematically measure how fluid protocols impact on the physiology and behaviour of individual animals, and tests whether more palatable fluids or social rewards might increase motivation, and reduce or eliminate the need for fluid control. In addition, by refining current motivational methods, it may also be possible to reduce the number of non-human primates used in behavioural neuroscientifc studies through improved data collection.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: NEUROLOGICAL
Title: drosophila as a model to understand the role of glial cells in neurodegeneration
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Glial cells have evolved in close association with neurons in most sophisticated nervous systems and are essential for the development, proper functionality and maintenance of neuronal networks and of the whole nervous system. One of their key functions is to provide trophic support and buffering ability over time to neurons, which makes them a critical factor in homeostasis of the nervous system in ageing and for the onset and progression of neurodegenerative diseases. Importantly for the 3Rs, because glial cell function is inevitably linked to that of its impact on neuronal cells and circuits, traditional cell culture approaches as a way or replacing animal experiments are less straightforward. As a consequence, virtually all information about glial cells in neurodegeneration has come from mouse studies. We plan instead to to exploit our drosophila model for dentatorubral-pallidoluysian atrophy (drPLA) and a collection of microRNAs transgenic strains as a tool for discovery, to shed light on glial function and glia-neuron interactions in neurodegeneration, and as a solution for promoting replacement, and encouraging reduction, of animal experiments. Our specific aims are: 1.To identify miRs and targeted genes of relevance in glia for polyglutamine neurodegeneration. 2.To identify miRs and targeted genes of relevance in neurons for polyglutamine neurodegeneration driven in glial cells. 3.To study the mechanism of action of the top miR(s)>genes network in glia-neuron interactions. Although the paradigm we use is a rare human disease, our system is capable of identifying also general mechanisms that are relevant to any disease that involves glial malfunctioning and degeneration. In conclusion, in this project we will develop a research strategy of paramount importance for the 3R agenda, with a great potential for major findings in a key area of bio-medical research, of proven interest to biotechnological and pharmacological industry.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Development of an in vitroscreening system to minimise animal use in the search for factors that modulate (re)myelination
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: The aim is to develop a high throughput screen, that uses a minimal number of animals, to identify therapeutic agents that will enhance remyelination in the context of a complex cellular environment mimicing that of the CNS in vivo The objectives are: (i) to adapt an established myelinating cell culture system, from embryonic murine spinal cord, to a multi-well (96 or 384) format, that will minimise animal usage by a factor of at least 10. (ii) automate handling and analysis for high throughput (iii) automate data acquisition and analysis for high throughput and to eliminate user bias (iv) validate the methodology, using factors known to modulate myelination in vitro, to confirm it provides the sensitivity and specificity required for high throughput screen (v) to achieve i-iv using minimum numbers of experimental animals Methodology (i) adapt existing myelinating cell culture techniques into a multi-well format to minimise animal usage (ii) establish and validate the use of a robotic integrated liquid handling platform to automate all dispensing, feeding and processing steps (iii) accelerate objective data acquisition using an IN CELL Analyzer 2000 high content screening microscope (GE Healthcare) (iv) develop template analysis protocols using currint IN CELL level 3 software to quantify effects on myelination Scientific and medical opportunities The proposed project will provide ultimately a high throughput screening protocol that will have diverse scientific/medical applications. In the first instance it will provide, for the first time, the opportunity to screen molecular libraries for compounds that stimulate myelination by endogenous progenitor cells; an approach predicted to provide substantial clinical benefits in multiple sclerosis and other de- or dysmyelinating disorders of the central nervous system. It will also facilitate studies of autoantibody mediated effects in neurological diseases.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL
Title: Applying the 3Rs to elucidate the mechanisms of tau pathology using DRG neurons in culture
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Good cellular models of tauopathy would be hugely beneficial as a platform for understanding the causes of tau-related dysfunction and neuron degeneration, and for testing potential drugs that ameliorate tauopathies. Several cellular models of tauopathy have been described but none appears to sustain spontaneous tau aggregation and filament formation, and few, if any, of these die as a consequence of tau expression. In preliminary studies, we have found that we can culture DRG neurons from adult transgenic P301S human mutant tau for several weeks during which they develop pathological forms of tau and they go on to die. Given that we can obtain several replicate cultures form a single mouse prior to the stage when the mice develop severe pathology, this means that we can meet the aims of the 3R programme as well as test fundamental questions concerning the mechanisms of tau-induced neurodegeneration. In this proposal we aim to further characterise these cultures and uncover how the neurons die, which intracellular processes are disturbed such that they contribute to the ultimate pathology, whether inflammatory cytokines or microglia/macrophages exacerbate the pathology, and finally test drugs we have been given to see if the platform can be used to uncover potential anti-tau therapies. Our research will significantly reduce and replace the number of animals needed for tau-related research, and refine husbandry because DRG can be cultured from the mice prior to overt pathology. At the same time, we will contribute to the basic understanding of mechanisms of tau pathology and hopefully to the identification of therapies for these devastating diseases.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Motor neuron regeneration in larval zebrafish
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Zebrafish, in contrast to mammals, regenerate spinal motor neurons. Previous work on spinal-lesioned adult zebrafish has led to important insights into the signals that allow spinal progenitor cells to produce new motor neurons after injury and in human embryonic stem cell culture. In order to replace animal experiments with a substantial severity banding and to significantly speed up experiments, we suggest to develop protocols for spinal lesions in larval zebrafish, most of which are completed before larvae become protected under the Animals (Scientific Procedures) Act 1986. We hypothesise that larval spinal lesion paradigms reproduce the salient features of adult lesions and lead to the discovery of differentiation pathways that are relevant for human stem cell differentiation. Therefore, the PhD student will 1) demonstrate that pathways known from adult regeneration have similar effects in larval regeneration; 2) establish a genetic lesion paradigm for motor neurons; 3) test pathways that are effective in zebrafish motor neuron regeneration and in motor neuron differentiation from human embryonic stem cells. This project will lead to the replacement of up to 400 fish currently undergoing procedures of substantial severity in our laboratory alone, and has the potential to reduce the number of CNS procedures (420'000 per year) overall. Data from this project will improve our understanding of vertebrate motor neuron (re-) generation, and may ultimately contribute to therapeutic approaches to motor neuron disease and spinal cord injury.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: NEUROLOGICAL
Title: Developing microfluidic systems for high-throughput studies of functional neuronal networks
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: In vitro studies are invaluable as a primary tool for investigating the cellular and sub-cellular changes implicated in CNS disorders. Whilst many biological experiments use primary cultures, it is difficult to gain control over the parameters that influence the cellular microenvironment and interaction. As a consequence, it is often impossible to decouple and simplify the mechanisms underlying cellular activity, obtaining only averaged responses observed on global cell populations allied to low-throughput and time consuming studies. A viable solution to these problems lies in the use of in vitro miniaturised procedures which enable the formation of simplified structured neuronal networks thus allowing the controlled, localised application of compounds or the localised transfection of cell populations to identify the consequence on cell acivity and communication. The aim of this project is to develop novel and high-throughput microfluidic systems for advanced patterning of neuronal cultures that will allow simultaneous optical and electrical recording of cellular activity, as well as enabling CNS drug discovery studies to be conducted using small amounts of novel compounds and the investigation of the consequence on cellular activity of altered protein expression (achieved by knock-down/overexpression) proposed to underlie certain CNS disorders. Finally,combining this system with novel patterning of neuronal cultures will enable the examination of localised neurotoxicity and how it spreads to affect neighbouring microfluidically isolated cultures. Taken together, this project will develop a novel microfluidic platform ideal for in vitro neurobiological studies.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL
Title: Find the target of valproic acid; pioneering the use of a non-animal model for basic biomedical (epilepsy) research
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: We have recently shown that valproic acid functions in the rodent hippocampus to block a seizure-induced reduction in PIP3. This research arose from the identification of an inhibitory effect of valproic acid on phosphoinositide turnover in Dictyostelium. The mechanism of this effect remains unknown. Thus the overall goal of this studentship is to identify this mechanism in Dictyostelium to reduce the use of animals in this field. In pursuit of this, the first step will be to develop a sensitive phosphoinositide turnover screen, where multiple species of phosphoinositides including the low concentration PIP3 can be quantified, and we have proof-of-principle data for this approach. The project will continue by analysing Dictyostelium knockout mutants, where encoded proteins have been implicated as a target for valproic acid in both our studies and from other groups, through simple cell movement and development assays, where we have well-established expertise. The project will continue by analysing phosphoinositide turnover in potentially resistant mutants, replacing the use of moderate to severe animal epilepsy experiments. Finally, this project will develop Dictyostelium as an expanding new approach for biomedical research into human protein function by heterologous expression of human phosphoinositide turnover proteins in this model.


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: NEUROLOGICAL