Identifying more clinically effective analgesic regimens and potential strain differences in pain perception in mice using a novel Operant Pain Assay

This project aims to improve the pain relief administered to laboratory mice that undergo potentially painful procedures in scientific research. We estimate that annually ~13% (~400k UK, >4 million worldwide) of mice used in research, will undergo potentially painful procedures where effective pain relief could reduce or prevent the pain experienced. Unfortunately, many of the current pain relieving drugs administered to mice appear to be partially or completely ineffective. This represents a major challenge for both animal welfare and the value of the scientific data collected from these mice.

To achieve improved pain relief for mice we need:

- A better means of testing pain-relieving drugs for their effectiveness in treating procedure-related pain.
- To determine whether new pain-relieving drugs are better than those currently being used.
- To determine whether or not the differences in the effectiveness of pain-relieving drugs observed between mouse strains relates to differences in the underlying experience of pain.

To answer these fundamental questions, we have two interlinked objectives. Objective 1 will establish the effectiveness of routinely used and new pain-relieving drugs in three common strains of laboratory mice. This will be accomplished in 3 stages, which will ensure that the minimum number of mice are used, and that those mice are exposed the mildest pain possible to achieve of the objectives. Stage 1 will rapidly assess the effectiveness of currently used and new painkillers in the most common strains of mice using a new automated method (Orofacial Operant Pain Assay). This is considered better than currently used methods as it only produces a mild and short-lived painful stimulus that the mouse is able switch off and is a better model of the pain experienced after an actual procedure. Stage 2 will test the effectiveness of the three most effective drugs identified in stage 1 in two other common strains of mice. Stage 3 will test the effectiveness of the three most effective forms of pain-relief (from stages 1 & 2) in treating post-surgical pain in the same common strains of mice. These mice will be undergoing routine vasectomy as part of a breeding programme and the effectiveness of pain-relieving drugs will be tested using a range of validated pain measures for mice, including: spontaneous behaviour (e.g. walking and rearing), presence of pain-related behaviours (e.g. twitching and writhing), pain-related facial expressions, and the willingness and ability to nest build and burrow (innate mouse behaviours). The effectiveness of the drugs will be determined by identifying which drug(s) restore and maintain normal patterns of behaviour and reduce the expression of pain-related behaviours.

Objective 2 will establish whether the pain experienced potentially differs between the common mouse strains being used in this project. Using the Orofacial Operant Pain Assay data from stages 1 and 2 allows us to investigate pain experience in these mice because it asks each mouse to balance their motivation to seek palatable reward against the mild pain they perceive. Such a complex task cannot be accomplished with a simple reflex response since these are not dependent on the animal consciously experiencing the pain. Using the post-surgical data from stage 3 enables us to determine whether strain differences in the expression of pain related behaviours following surgery correlate with strain differences in reward seeking observed at stages 1 and 2. This enables us to determine if the differences are potentially related to differences in pain experience between the strains.

This project will determine which pain relieving drugs are the most effective, for a specific type (strain) of mouse following a routinely used surgical procedure allowing recommendations to be made and implemented to improve the welfare of mice used in scientific research and the quality of the science carried out on these animals.

This project has two inter-linked objectives: [1] to establish more effective analgesic regimens for laboratory mice and [2] to establish whether pain perception differs between mouse strains. We estimate that annually ~13% (~400k in the UK, >4 million worldwide) of mice used in regulated research undergo potentially painful procedures. Critically, we cannot currently manage post-procedural pain effectively in mice, making this a significant focus for 3Rs research. Unalleviated and uncontrolled pain is a considerable welfare and scientific concern affecting a range of disciplines. Effective pain management is therefore integral to the 3Rs that underpin in vivo research. We will first rapidly screen routinely used (meloxicam, morphine), novel (Tapentadol) and multi-modal (combinations of the above) analgesic regimens to assess their efficacy in three common strains of laboratory mice (C57Bl/6, CD1 and C3He) using the Orofacial Operant Pain Assay. This novel automated nociceptive test is considered more clinically relevant for developing analgesia. We will then establish which of these regimens demonstrate efficacy following routine surgery (vasectomy) in these strains using established and validated behavioural indices (behaviour, facial expressions, burrowing, nest building).

As the Orofacial Operant Pain Assay uses an operant-conflict paradigm requiring higher cognitive processing, it can be used to determine whether pain perception differs between these mouse strains when exposed to a standard nociceptive stimulus. This data will be compared to that of the post-surgical study to determine whether any strain differences observed in the behavioural indices relate to differences in pain perception. Establishing whether common strains differ in their analgesic requirements and potentially their pain perception is critical for effective pain management and to comply with the 3Rs, improve welfare and enhance scientific outcomes.

A wide range of stakeholders will directly benefit from the outputs of this project. Laboratory animal veterinarians and animal technicians who conduct potentially painful procedures and care for laboratory mice that have undergone painful procedures will directly benefit. The outputs will provide essential guidance as to which analgesics and what dose will be effective for preventing or alleviating post-procedural pain in the most commonly used strains of mice.

This project will benefit scientists undertaking in-vivo research that involves potentially painful procedures. Uncontrolled pain has a detrimental effect on animal welfare, particularly where pain is avoidable and could be effectively controlled through the administration of appropriate analgesics. The project outputs will not only enable research to more closely comply with the principles of the 3Rs (and regulatory requirements) but will also ensure that those who use animals in research do so with the minimum possible compromise to animal welfare. The latter is of importance to many of those who use animals. Uncontrolled pain not only poses a significant threat to animal welfare but also to the validity of the data collected from those animals. This can lead to poor quality data and may increase the number of animals required to achieve specific scientific outcomes. The financial costs and time saving associated with studying fewer mice, along with the welfare improvements to those that are used, will be widespread across a range of disciplines where mice are currently used in the UK and worldwide.

Policy makers, regulatory bodies and advisory boards will also benefit from the outputs of this project as it will provide clear scientific data on which to evaluate and refine guidelines on appropriate analgesic regimens for commonly used strains of laboratory mice. This is particularly important following revision of the European Directive (2010/63/EU) that encourages an evidence-based approach to the development and implementation of an EU-wide codes of practice (and following modification to A(SP)A, this will continue to apply in a post-Brexit UK).

We have a strong track record in disseminating our research findings to ensure that a wide range of stakeholders gain access to the results that will be produced. These will include publication in peer reviewed journals and presentations at national and international scientific conferences. We will also incorporate the outputs into the range of training materials that we produce including the 'Pain and distress' workshops we run annually that are attended by a wide range of stakeholders from around the world (over 400 delegates to date), and our 4 web resources: www.digires.co.uk, www.ahwla.org.uk, www.procedureswithcare.org.uk, and www.flairelearning.com that provide online training and teaching resources to underpin implementation of refinement of in-vivo studies (viewed by over 500,000 people to date).

Finally, the outputs will also be of significant public interest. The use of animals in scientific research is a continuing area of public concern, and it is important that the public is informed about how the scientific community is working to reduce the negative effects of the research on the animals used. If it can be scientifically demonstrated that pain is being prevented or alleviated and that the absolute minimum number of mice are being used to obtain reliable scientific outcomes, public perception of research will improve. Our project will demonstrate how scientific research can improve the welfare of mice, the species that comprises the greatest proportion of animals used worldwide. This will be achieved through both publication in general science magazines, social media (e.g. Twitter), presentations at science events attended by the general public (e.g. British Science Festival) and presentations to school children, lay-members of ethics committees and university students.