FP-00013

Section 1 - Basic information about you and your application:

Title of research project
Identifying objective measures of sex-specific pain in humans that can be used diagnostically to target treatment.

Grant Type
The ORUK Inspiration Fund

Research area
Diagnostic and treatment

Duration
9

Start date
February 1, 2024

Have you previously received funding from ORUK?
No.

Profession
Academic scientist

Your current job title/position
Professor

Are you an early-career researcher (ECR)? (definition of ECR)
no

Section 2 - Lay summary

Lay summary:

Osteoarthritis, a painful disease that causes joint destruction, affects 530 million people worldwide. No treatments stop osteoarthritis or manage pain. Pain reduces patient’s quality of life, causing immobility and isolation. Osteoarthritic pain affects nearly twice as many females than males, with pain being particularly common in post-menopause females. Changes in bone structure in osteoarthritis correlate with pain severity, possibly because the bone contains many nerves. Our pilot work shows that joint fluids from 34 patients with knee osteoarthritis contain signals that affect nerve growth and function and that these signals correlate with indicators of bone changes. However, our data only includes 3 females. Fundamental sex differences in pain sensation and bone biology intrinsic to osteoarthritis, are vastly under researched. This project will analyse a further 20 male and 31 female patients’ age matched joint fluids, to assess how factors affecting nerve growth and bone turnover vary together within individuals. These data will indicate which patients are predisposed to nerve growth, bone structural changes and therefore increased pain, and how this varies between males and females, and post-menopause. This could identify patients that would benefit from treatments reducing bone turnover (Risedronate) or preventing nerve growth (Tanezumab). To reveal the biological mechanisms that explain why nerves grow more in some patients to cause more pain, we have developed human cell models that detect interactions between nerves and bone. Mimicking osteoarthritic conditions in our bone model causes it to release factors that influence nerve growth. This project will add joint fluids from 15 male, 10 pre and 10 post-menopause female patients to the nerve model directly, and via the bone model, and measure how this affects nerve growth and function. This will identify mediators that alter nerve activity and determine whether they arise from the bone, or directly affect the nerve. This will provide an objective measure of sex-specific pain that can be used diagnostically to tailor targeted treatments more appropriately to individual patients.

We hold involvement events and consult patient groups via HealthCare Research Wales. Our Patient and Public Involvement event in 2020 involved 32 members of the public with a musculoskeletal condition (55% with osteoarthritis), ranked pain as the most important aspect of their condition to resolve, echoing the 2017 Arthritis Foundation ‘A voice of the patient’, where “reduced pain and regained function, mobility, walking and standing as benefits they would consider to be the most meaningful when considering OA symptoms”. This informs our focus to understand pain mechanisms.

Section 3 - Purpose of research

Purpose of research:

The aim is to elucidate signals that drive pain and bone remodelling in human osteoarthritis and determine how these differ between males and females using patient-based data analysis, and bone/nerve cell model responses.

Objectives

1. To analyse our stored synovial fluids from 41 female and 30 male osteoarthritis patients for neurotrophic, angiogenic and inflammatory signals that alter neural activity or coupling with bone remodelling leading to nerve invasion and pain.
2. To combine this new patient data with our existing synovial fluid analysis of the same markers in 31 males and 3 females and determine sex-specific differences in entire cohort neuronal factors using multivariate analysis.
3. To measure oestrogen levels in all 44 females using ELISA on matched blood samples and determine menopause-specific differences in neuronal factors using multivariate analysis.
4. To determine the responses of our human osteocyte model to 15 male, 10 pre and 10 post-menopausal female signals by direct application of fluids.
5. To determine the responses of our human nerve model to 15 male, 10 pre and 10 post-menopausal female signals in these patients’ synovial fluids.
6. To compare within patient synovial fluid biomarker associations with osteocyte and neuronal responses and identify patient and sex specific mechanisms that link bone remodelling to pain.

Deliverables

1.     Patient synovial fluid neuronal signals in human osteoarthritis
2.     Patient sex and pre/post menopause specific differences in synovial fluid neuronal signals
3.     Patient sex and pre/post menopause specific relationships between neuronal factors and bone remodelling markers
4.     In vitro sex and pre/post menopause specific responses of osteocytes to synovial fluids
5.     In vitro sex and pre/post menopause specific responses of sensory nerves to synovial fluids
6.     Sex and pre/post menopause specific molecular mechanisms associated with osteoarthritic bone remodelling and pain
7.     Sex and pre/post menopause specific diagnostic markers of osteoarthritic pain

Section 4 - Background to investigation

Background to investigation:

Background Chronic pain in osteoarthritis is a severe condition, limiting patient mobility, ability to perform daily activities and live independently. There are currently 530 million osteoarthritis¹, sufferers worldwide. The opioid and Non-Steroidal Anti Inflammatory Drug treatments used to attempt to treat this substantial pain burden carry significant risks², and are ineffective. The incidence and severity of structurally defined osteoarthritis and the associated pain, is increased in females, and post-menopause³. A recent systematic review concluded that females have higher osteoarthritis prevalence, clinical pain and inflammation than males, but molecular mechanisms explaining sex differences were lacking⁴. We propose that the synovial fluids of females and males with osteoarthritis contain different molecular drivers of inflammation and bone remodelling, that influence sensory nerve growth and activation.

Osteoarthritic pain is largely due to intimate associations between nerves and bone. Signalling between skeletal and nervous tissues regulate metabolism, mineral production, and bone remodelling in diseases associated with pain⁵. Skeletal sensory nerve sprouting, invasion and sensitisation are associated with bone pathologies, and intrinsic to pain responses in animal models of osteoarthritis⁶, and in humans⁷. In osteoarthritis, areas of bone remodelling called bone marrow lesions (BML) are characterised by sensory nerve invasion and correlate with pain⁸. Enlargement of BMLs is associated with worsening joint degeneration and increased pain, whereas reduced BML severity relieves pain⁹. Anatomical neuroplasticity of sensory nerves within bone is intrinsic to osteoarthritic pathology and pain¹⁰. The molecular drivers for the changes in neuroanatomy, and sex differences in these responses remain unknown. However, protein expression and sensitivity of nociceptors do display sex-based differences and are regulated by sex-hormone signalling¹¹.

Synovial fluid from human osteoarthritic patients has been shown to activate nociceptors in vitro showing that mediators present in patient synovial fluid are adequate to stimulate nociceptor activity and pain. This has identified mediators present in joints of patients with arthritis that stimulate nociceptors to identify potential drug targets for treating knee pain¹². Our proposal aims to elucidate signals that drive pain and bone remodelling in human osteoarthritis and determine how these differ between males and females by analysing patient joint fluids directly alongside the nociceptor responses they induce.

Track record

Professor Deborah Mason is CoPI and Preclinical Research Director in the Biomechanics and Bioengineering Research Centre Versus Arthritis (12yrs), Translational Theme Lead for Versus Arthritis Musculoskeletal Disease Research Advisory Group, and past President of the British Orthopaedics Research Society. Her research (>30years, EPSRC/MRC/Charity/Industry funded), uses cell and animal models for mechanistic and intervention studies, and human studies in NHS, linking biological, clinical, and biomechanical data. She has identified new osteoarthritis treatments with granted patents. International leadership includes Orthopaedic Research Society Programme Committee and Bone Topic Chair and a Fellow of the International Orthopaedics Research Societies. Provides expertise in osteocyte mechanical responses, glutamatergic signalling, human samples and PPIE.

Ryan Jones (final year PhD student) research (>4 years) generating iPSC-derived sensory neurons to investigate how bone derived axon guidance signalling drives pain in arthritis in comparison to human clinical samples has won several prizes. He has contributed to pilot data and experimental design and will perform experimental work, develop leadership and collaborative opportunities, and lead dissemination to academic and patient audiences.

Preliminary data

Patients We have collected blood and synovial fluid samples, and VAS scores from patients undergoing total knee replacement and high tibial osteotomy (HTO) surgery with informed consent, approved by the Wales Research Ethics Committee 3 (10/MRE09/28) and Cardiff and Vale University Health Board. Inclusion criteria were, age between 18 and 80, undergoing medial opening wedge HTO or total knee arthroplasty, ability to provide informed consent, no neurological/visual conditions affecting movement, no previous injury to the joint under investigation considered unsuitable. ELISA and Mesoscale discovery analysis of 31 male and 3 female patient synovial fluid identified combinations of factors within patient synovial fluid that link bone remodelling, neurotransmitter signalling to markers of OA pain and degeneration (figure 1). We have investigated these signals in our in vitro models of bone and nerve.

Cell models Osteocytes¹³ orchestrate bone remodelling in response to mechanical load and inflammation, both mediators of osteoarthritic degeneration.  Mechanical stimulation of our human osteocyte model regulated >200 neuronal genes (figure 2). To determine whether osteocytes drive neuronal invasion, we developed our human iPSC derived nociceptor model (figure 3). Continuous 24 hour confocal microscopy revealed that nociceptors exposed to media from ‘osteoarthritic’ osteocytes displayed more dendritic projections when compared to nerves exposed to media from ‘non-osteoarthritic’ osteocytes (figure 3).

Sex specific pain Although sex differences in pain generation and responses are well established^14,3, the molecular drivers for sex differences remain unknown. However, protein expression and sensitivity of nociceptors do display sex-based differences and are regulated by sex-hormone signalling¹¹. Synovial fluid from osteoarthritic patients can activate nociceptors in vitro to identify potential drug targets for treating knee pain¹². Our proposal aims to elucidate signals that drive pain and bone remodelling in human osteoarthritis and determine how these differ between males and females, both pre and post menopause, by analysing patient joint fluids directly and comparing these to nociceptor responses they induce in vitro.

We hypothesise that the synovial fluids of females and males with osteoarthritis contain different molecular drivers of inflammation and bone remodelling, that influence sensory nerve growth and activation.

References: 1.Long,  et al. Arthritis & Rheumatology 74, 1172–1183 (2022). 2.Davis & Robson, Br J Gen Pract 66, 172–173 (2016).3.Li & Zheng, International Journal of Molecular Sciences 22, 7876 (2021). 4.Tschon, et al. M. J Clin Med 10, 3178 (2021).5.Brazill, et al. J Bone Miner Res 34, 1393–1406 (2019).6.Aso, K. et al. Osteoarthritis Cartilage 28, 1245–1254 (2020).7.Mitchell, et al. Curr Osteoporos Rep 16, 325–332 (2018).8.Kuttapitiya et al. Ann Rheum Dis 76, 1764–1773 (2017).9.Perry et al. Osteoarthritis Cartilage 28, 316–323 (2020).10.Obeidat et al. (2023). doi:10.1101/2023.06.30.547216.11.Tavares-Ferreira et al. Biol Psychiatry 91, 129–140 (2022).12.Chakrabarti et al. Rheumatology 59, 662–667 (2020).13.Delgado-Calle & Bellido, Physiol Rev 102, 379–410 (2022). 14.Bartley & Fillingim British Journal of Anaesthesia 111, 52–58 (2013).

Section 5 - Plan of investigation

Plan of investigation:

This project links five work packages (WP) involving exploratory analysis of human samples to elucidate sex specific signals that drive pain and bone remodelling (WP1&2), and use our in vitro models to characterise sensory nerve responses to osteoarthritic synovial fluid, both directly and via osteocytes (WP3-5). The objectives (Obj) and deliverables (D) are listed in the Gantt chart.

Patient samples have been obtained with informed consent approved by the Wales Research Ethics Committee 3 (10/MRE09/28) and Cardiff and Vale University Health Board. All patients have matched serum (>1 ml Vol) and synovial fluids (>500microl).

 

Patient details	Analysed (fig 1)		For analysis
sex	male	female	male	female
number	31	3	30	41
Age	29-58	44-60	47-80	23-78

WP1 (Obj 1, D1) will analyse female and male osteoarthritis synovial fluids for neurotrophic, angiogenic and inflammatory signals that alter neural activity, or bone remodelling, leading to nerve branching, invasion and pain.   We will analyse synovial fluids that we have already collected, processed and stored from 41 female and 30 male osteoarthritis patients. As with our pilot data (fig 1, n=31 males and 3 females), we will use single/multiplex ELISA/ MesoScale Discovery analysis to quantify 36 proteins that influence angiogenesis and inflammation in osteoarthritis, bone remodelling (OPG, ALP and RANKL) and neurotrophins that alter nerve growth and activity including NGF, Sema3A, BDNF and glutamate.

WP2 (Obj2, D2&3) will combine the new patient data from WP1 with our existing synovial fluid analysis of the same markers in 31 males and 3 females (fig 1).  Sex-specific differences in neuronal factors will be determined using multivariate analysis including principal component analysis (PCA) and multiple linear regression. Models will consider age, sex, weight, BMI, VAS score, KL grade and mediator concentration. For PCA data are Log transformed to ensure equal variance of all continuous variables, and for multiple linear regression, Bonferroni correction is applied for multiple comparisons.

WP3: (Obj3, D2&3) will quantify oestrogen and FSH concentrations in all 44 females (age range 23-78) using ELISA on matched blood samples and determine whether differences in neuronal factors associate with oestrogen and Follicle Stimulating Hormone (FSH) concentrations. Although menopause is not recorded for these patients, females with oestrogen concentrations below 3-pg/ml and FSH concentrations above 20 IU/liter are likely to reflect post-menopausal changes¹. As in WP2, models will consider age, sex, weight, BMI, disease progression, and pain VAS score and data will be tested for compliance with analysis.

WP4 (Obj. 4, D4), Determine the responses of our human osteocyte model to 15 male and 10 pre and 10 post-menopausal female synovial fluids by RTqPCR. Y201 osteocytes (0.05×10⁶ cells/gel) embedded in type I collagen gels will be differentiated in osteogenic medium (Gibco DMEM GlutaMAX supplemented with Dexamethasone, B-Glycerophosphate and Ascorbate) for 7 days according to our methods (Fig 2). 100microL of synovial fluid will be added directly to the osteocyte cell model for 10 mins, removed and cells in gels immediately dissolved in Trizol for RNA extraction. After RNA tape station analysis and conversion of 200μg to cDNA by reverse transcription, RTqPCR arrays reflecting bone remodelling, neuronal factors and inflammation, normalised to the most stable combination of reference genes, will be performed by Platinum RTqPCR following our established protocols.

WP5 (Obj 5, D5) Determine the responses of our human nerve model to 15 male and 10 pre and 10 post-menopausal female synovial fluids. Neural responses to direct application of 100 microL synovial fluid will be analysed by 24-hour confocal microscopy to determine nerve growth and branching (fig 3), and function using calcium-imaging, as well as functional analysis of spontaneous activity using microelectrode array analysis. Single/multiplex ELISA/MesoScale Disovery analysis, and RTqPCR will assess biomarkers expressed/released by the nerve.

WP6 (Obj 6, D6&7) Comparisons between the neuronal, bone remodelling and inflammatory factors identified within the patients synovial fluid in WP1-3, and the molecular responses from osteocytes and sensory nerves to the same patients joint fluid, will identify regulators of bone remodelling and innervation that differ between individuals, the sexes and pre/post menopause. This will allow the elucidation of the factors driving pain processes in male and female OA patients.

 

Opportunities and Risks

Clinical This project will identify important mediators that may drive pain processes in individual patients, and define the variability across patients, between males and females and pre-post menopause. This not only provides potential diagnostic biomarkers, but also new mechanistic understanding, that could be targeted with appropriate drug intervention. This is particularly relevant as there are a range of drugs targeting bone remodelling (e.g. for osteoporosis) and modifying neurotransmitter responses (e.g. for epilepsy, migraine, and central pain) that could be repurposed to prevent neural invasion by intra-articular injection within painful osteoarthritic joints. Testing such drugs in nociceptor growth and function assays, as proposed here may ultimately identify optimal patient-specific treatments.

The project has several risks. It is possible that no associations are found that relate bone remodelling to innervation, although this seems unlikely since our preliminary data indicate within patients associations between IL-6, IL-8, glutamate and ALP and neuronal factors, NGF and Sema3A. We may find no consistent differences between males and females, or pre-post menopause either due to no consistent differences, or insufficient power. However, matching in vitro models responses with biomarker profiles within individual patients, will identify patient specific causative/mechanistic effects.  Variances identified in this exploratory study will define power needed to test specific diagnostic marker combinations in the future. Clinically, it is difficult to sample synovial fluid except at arthroscopy or surgery, although some readouts will be detectable in the blood. Others have shown nerves are viable after application of synovial fluids.

Commercial Exploitation There is an opportunity to develop a commercial diagnostic assay and/or drug selection assay based on the methods we are using and the markers we identify. There would be a large commercial market for such technology and IP would need to be protected with the support of Cardiff Technology Transfer Officers. Mason has successfully protected IP inventing patents granted in Europe, US and China.

 

1. Randolph et al. J Clin Endocrinol Metab. 2004 Apr;89(4):1555-61.

Section 6 - Research environment and resources

Research environment and resources:

Biological Sciences at Cardiff University was ranked in the top 25 worldwide and the top 5 in the UK In the 2019 Academic Ranking of World Universities (research strength and performance). The School of Biosciences hosts the UK Centre of Excellence in Biomechanics and Bioengineering funded by Versus Arthritis (BBRCVA) with DM as Co-PI and Preclinical Research Director. BBRCVA provides a mechanical loading laboratory for cell models and the School provides service hubs for the real time confocal imaging used to quantify neuronal responses, RNA quality assessments and multiplex protein analysis using the mesoscale discovery platform. Laboratory space, and all necessary equipment for RNA/protein extraction and RTqPCR are available in DMs laboratory. Patient samples are held by DM and have been obtained with fully informed consent approved by the Wales Research Ethics Committee 3 (10/MRE09/28) and Cardiff and Vale University Health Board. All patients have matched serum (>1 ml Vol) and synovial fluids (>500microl).

 

BBRCVA provides long-standing, academic links with Cardiff and Vale University Health Board (C&VUHB), Cardiff and Vale Orthopaedic Centre (CAVOC), Physiotherapy, and the Rehabilitation Engineering Unit, through the BBRCVA Umbrella Ethics (since 2010), allowing multiple protocols (motion analysis, imaging, samples, patient reported questionnaires) for the same patients. NHS support – Chris Wilson, Rhys Williams, Rhydian Morgan- Jones, Consultant Orthopaedic Surgeons (Knee), provide clinical expertise, and have provided clinical data biological samples from their knee surgery patients. Some HTO patients in this proposal have additional data and samples that could be used to relate findings to longitudinal motion analysis, blood samples and bone biopsies taking before and after joint realignment surgery in future work.

Additional in kind funding is provided by bit.bio who have agreed to supply 1 vial free of charge of io EA1024 iPSC-Derived nociceptor sensory neurons (ioSensory Neurons) to support the project. Additional vials of these cells will be provided at a discounted rate of 50% off the current list price. Based on the number of vials needed to complete the work, we calculate in kind support of £2,500. The company will not hold any IP in return for this support.

 

 

Section 7: Research impact

Who will benefit from this research?

Patients and Clinicians We will identify new mechanisms that explain how bone remodelling and pain are linked within individual patients, and whether this differs between sexes and with menopause. The benefit for people with osteoarthritic pain, and their clinicians, will be selective diagnostic and prognostic indicators of pain, linked to new drug targets.

 

Researchers The new models and protocols will improve understanding of differences between male and female bone derived osteoarthritic pain.

 

Applicants Mason benefits from Jones’ cutting-edge sensory nerve model. Jones benefits from access to patient samples, bone model, and Mason’s lab facilities, and her scientific/industry collaborations. As a grant holder, Jones will develop leadership and collaborative opportunities.

 

Animals Our bone/nerve models could reduce early-stage preclinical research animal usage and replace animal models used to test new treatments for osteoarthritic pain.

 

Industry Bit.bio will benefit from our validation of their sensory nerves as reporters for human pain.

How can your research be translated in real-life?

Females, especially post-menopausal females, are particularly susceptible to both osteoarthritis and associated pain. Sex/menopause specific relationships between indicators of bone remodelling and neuronal invasion provide objective measures of pain that can be used diagnostically to tailor targeted treatments to individual patients. Patients predisposed to nerve growth, or bone structural changes and therefore increased pain, may benefit from treatments reducing bone turnover (e.g. Risedronate) or preventing nerve growth (e.g. Tanezumab).  Approved drugs that target bone remodelling (e.g. osteoporosis, bone metastasis) or modify neurotransmitter responses (e.g. epilepsy, migraine) could be repurposed to prevent neural invasion by intra-articular injection within painful osteoarthritic joints.

A commercial diagnostic assay and/or drug selection assay based on the methods we are using and the markers we identify could be developed. There would be a large commercial market for such technology. IP would be protected with Cardiff Technology Transfer Officers.

How will your research be beneficial for ORUK and its purpose?

Our project directly addresses ORUK Strategy 2020-23 to ‘pioneer new forms of orthopaedic diagnosis and treatments’. This project addresses a challenging unmet need;  that of osteoarthritic pain, especially the susceptibility of females and the influence of menopause. Our project uses patient samples and cell models to reveal molecular mechanisms that relate bone remodelling to pain within individual osteoarthritic patient joint fluids. Our human bone/nerve cell models represent innovative, state of the art technologies to reveal such mechanisms.

This project addresses patients’ unmet need, informed by patient events identifying the importance of pain. The projects will identify new diagnostic and treatment approaches to reduce MSK pain and improve quality of life. This research has the potential to reduce the burden of osteoarthritic pain on individuals and be translated into benefits for people. We can contribute to ORUK ‘influencer network’ through our scientific society, charity and industry connections, seminars and PPIE activities.

Section 8: Outreach and engagement

We will proactively disseminate our research to researchers, public, academic, clinical, patients and industry using our established communication routes within Cardiff University, the Biomechanics and Bioengineering Research Centre Versus Arthritis, and through our Society memberships and industry connections (Mason’s current Industry partners, Orphelion, Novartis and Hospital Innovations) as well as through direct communication with ORUK.
Local dissemination will be achieved through planned public engagement events hosted by the Biomechanics and Bioengineering Research Centre Versus Arthritis in Cardiff for 2024, 2025 and 2026. These will be utilised to display the potential of our findings to highlight new mechanisms underlying bone derived pain and how this differs in males and females. Webinars and workshops based on our cell models of pain will be made available internationally via https://www.orthomedia.org.uk/ and NC3Rs website. Direct engagement with ORUK will allow us to provide information for researchers and public through submission of our findings to the ESCAPE-Pain Newsletter and contributions to the BOMSA | BOTA | ORUK National Research Webinar Series to access orthopaedic trainees (Mason last gave a Research Webinar in May 2023). Internationally, we intend to run a discussion forum in the Orthopaedic Research Society (ORS) Preclinical Research Section (Mason is a member) 2024 to compare in vitro and in vivo models of pain and the importance of sex-specific differences. We intend to apply to run a workshop to OARSI (2026), via the Osteoarthritis Pain Discussion Group (Jones is a member), involving industry and regulators to fully realise the potential of in vitro models in testing new treatments for osteoarthritic pain in context of FDA ‘New Alternative Methods Program’. Mason has previously organised 5 international workshops involving researchers, orthopaedic surgeons, rheumatologists, physiotherapists, industry, and patients. In addition, we will communicate comparisons of the bone-nerve model to in vivo data to our current industry partners to demonstrate efficacy as a drug screening tool. Both applicants have access to Healthcare research Wales training in public involvement.
We have held involvement events and consulted with patient groups via HealthCare Research Wales. Our Patient and Public Involvement event in 2020 involved 32 members of the public with a musculoskeletal condition (55% with osteoarthritis), ranked pain as the most important aspect of their condition to resolve, echoing the 2017 Arthritis Foundation ‘A voice of the patient’, where “reduced pain and regained function, mobility, walking and standing as benefits they would consider to be the most meaningful when considering OA symptoms”. This informs our focus to understand pain mechanisms.

Section 9: Research budget

Requested funding from ORUK

University fees (if any)
£0

Salary
£39834

Consumables
£10000

Publications
£0

Conference attendance
£150

Other items
£

Total 'requested fund'
£49984



Other items




Other secured funds

Internal funding
£0

Partner (University)
£0

Partner (Commercial)
£2500

Partner (Charity)
£0

Other sources
£0

Total 'other funds)
£2500

Section 10: Intellectual property and testing on animal

Is there an IP linked to this research?
No

Who owns and maintains this patent?


Does your research include procedures to be carried out on animals in the UK under the Animals (Scientific Procedures) Act?
No

If yes, have the following necessary approvals been given by:

The Home office(in relation to personal, project and establishment licences)?


Animal Welfare and Ethical Review Body?


Does your research involve the use of animals or animal tissue outside the UK?
No

Does the proposed research involve a protected species? (If yes, state which)



Does the proposed research involve genetically modified animals?


Include details of sample size calculations and statistical advice sought. Please use the ARRIVE guidelines when designing and describing your experiments.


There should be sufficient information to allow for a robust review of any applications involving animals. Further guidance is available from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), including an online experimental design assistant to guide researchers through the design of animal experiments.

Please provide details of any moderate or severe procedures


Why is animal use necessary, are there any other possible approaches?


Why is the species/model to be used the most appropriate?