Opioid self-administration in the assessment of post-operative pain in rats

Lead Research Organisation: Newcastle University
Department Name: Neurology Neurobiology and Psychiatry

Abstract

Rats have a vital role in research to investigate new drugs and treatments, but this often requires that they are made unconscious (anaesthetised) before undergoing surgery. A common surgical procedure is laparotomy; an incision whereby the skin and underlying muscles are cut so the organs inside the abdomen are exposed. Scientists have a duty to ensure that after the animals experience little or no pain after they recover from anaesthesia. Because the animals cannot tell us how they feel, however, it is difficult to gauge how painful this is. Scientific research has shown that there are some behaviours that are particularly important for determining how much pain laparotomy surgery causes in rats, because they increase as a result of surgery, but can be reduced if pain relieving drugs (analgesics) are provided (usually injected prior to surgery). This research has shown that different analgesics have different abilities to reduce pain, depending on how much drug the rats are given, and on whether the important (pain-related) behaviours disappear completely or only partially. One major problem, however, is that the researchers are still unsure whether the treatments they use are the most effective, because regardless of the analgesic given to the rats, some subtle behaviour changes usually remain. It is therefore unclear whether the continued presence of these abnormal activities means the rats are still experiencing pain, and so, how effective the analgesic that was given had been. One way to try to fully understand what the rats are experiencing is to give them the opportunity to tell us how they feel, but as they cannot communicate with us, this can only be done indirectly. If the rats are experiencing pain after surgery, then, if given the chance, they should choose to take (self-administer) analgesics to reduce it. The amount of analgesic they choose to self-administer could then be used as an estimate of how much pain was caused by surgery. An essential step in testing this theory, however, is to ensure that the rats know that obtaining the drugs will provide pain relief. Because of this, they will first be trained that poking their nose into a hole in a test chamber will painlessly trigger an injection of a strong pain killer (remifentanil). Once they know this, they should increase the number of times they nose-poke according to how much pain they are experiencing. A very useful aspect of this experiment is that it can also be used to test the pain-relieving properties of other analgesic drugs. The rats will be provided with one of the drugs that, according to the results of behavioural investigations, should be effective for relieving the pain caused by surgery. One such drug is called meloxicam, and another is carprofen. If either of these is given prior to surgery, and the dose given completely eliminates pain, then the rats should not choose to self-administer additional remifentanil when they recover from anaesthesia. Is this occurs, then it will be very good evidence that pain was the reason the rats needed remifentanil. Another way to confirm this is to show that the rats will also consume more remifentanil if the type of surgery causes more pain. The rats will undergo 4 different procedures that will cause different amounts of pain, but none will be highly painful. If the rats continue take more remifentanil even after they have been given meloxicam or carprofen then this will mean that the dose of meloxicam or carprofen was not sufficient, and more was needed. Testing how much remifentanil the rats need over several days following surgery will produce the best possible information on how much pain is caused, how long it lasts and what dose of analgesic is best to prevent it in future. This information would be useful to other researchers who need to conduct laparotomy and other types of surgery as part of their research, and so help to improve the welfare of all rats undergoing surgery in scientific research.

Technical Summary

Our behavioural studies in rats have demonstrated NSAIDs such as meloxicam and carprofen alleviate post-surgical pain; but we cannot confirm that pain was eliminated completely as some subtle effects outlast the main behaviour changes indicating its presence. We will use the rats' requirements for a highly potent and rapid acting opioid analgesic (remifentanil) to investigate this. The rats will 'tell us' if they are experiencing pain by how much remifentanil they choose to self-administer (SA) while having free access to intravenously administered remifentanil. The rats will be trained to SA an analgesic, but minimally rewarding, dose of remifentanil. Remifentanil consumption following painful procedures will then be tested. We already know rats actively seek remifentanil, and we have a 'working' unit dose (one that is analgesic in rats, and that they should self-administer if they are in pain). Rats pre-trained to SA remifentanil will be tested for changes in SA following 4 procedures. We will confirm that pain is the major factor underlying increased SA; 1) by showing remifentanil consumption increases according to how much pain each procedure causes, and 2) by demonstrating that opioid-seeking is reduced by pre-treating with an alternative NSAID analgesic (meloxicam or carprofen) we know relieves post-operative pain in rats. Crucially, we also know remifentanil SA is relatively unaffected by anaesthesia or NSAIDs. Once we know how much remifentanil the rats need to alleviate their pain, we can determine whether standard pre-surgery doses of NSAID provide adequate pain relief. With repeated SA testing, and assessing relative differences in opioid-seeking behaviour between saline and NSAID treated groups we will also be able to determine how long pain lasts, and will be able to establish dose regimens that prevent pain. By refining knowledge on pain and rats requirement for analgesics the project will have a positive impact on the welfare of large numbers of rats.
 
Description Human cancer patients will consume (self-administer) more of a painkilling drug (such as morphine) to prevent pain. How much is consumed gives an indication of how severely the patient's pain is. However, as they can't speak, little is known about how severely rats experience pain following common laboratory procedure such as surgery. We planned to use drug self-administration to measure how severe their pain might be. This would be measured by determining how much drug they need following surgery or application of another unpleasant (noxious or painful) stimulus. They were initially trained to obtain the drug by pressing a lever that would activate a pump and deliver the drug directly into their bloodstream. This training in normal rats required several weeks longer than anticipated. During this they injected themselves with large quantities of the drug, however, once exposed to surgery, unlike in humans, their drug intake dropped rather than increased. As we were able to partially prevent this drop in intake using another painkiller we showed that the rats probably do experience pain, and may even be one similar to how humans perceive it. However, it was important to determine why their intake initially reduced rather than increased when they may have been experiencing pain. Eventually we found that this was because the numerous injections they received during training probably caused them to enter a state where they become more sensitised to pain; called hyperalgesia. That meant that following surgery, exposure to the morphine-like drug probably intensified their pain rather than prevented it. The consequence of this was that we chose an alternative method of training using a drug that would not sensitise the rats to pain. These results show that it is probably not advisable for scientists to expose animals to morphine-like substances for long periods, in other words, we helped refine the methods used for research of a similar type in future. We then used a new method of testing called conditional drug discrimination. The basis of this was that rats would be required to press a lever if they were in pain, or another if they were not, in order to receive a food reward. We tried to control the state they were in (i.e. painful or not) by injecting them with morphine or only a placebo (saline). This allowed us to establish more about whether rats can experience pain. It was more successful than the self-administration method because the time needed for training was much shorter. We were able to determine that the rats were aware of pain, but we could not tell how severe it may have been. This knowledge was nevertheless important, and led the research into a new direction that we are currently exploring.
Exploitation Route The findings made on the project showed that we needed to find training methods that do not involve any drug exposures prior to pain testing. The findings are therefore just as important for other researchers that might attempt a similar approach to pain assessment. One possibility is to train the animals to lever press to obtain pleasurable brain stimulation, which should also change in response to pain. We hope to begin preliminary work on the new approach very soon. If this were successful it should be possible to learn a lot more about how severely laboratory rodents experience pain and what scientists need to do (i.e. what drugs to give) to prevent it.
Sectors Education,Pharmaceuticals and Medical Biotechnology,Other
 
Description We now have a greater understanding of many pitfalls arising as a consequence of attempting to train rats to perform an operant task using an opioid as the reinforcer. Provision of the opioid reminfentanil was supposed to enable behavioural responding such that rats would self-medicate if experiencing pain. The major problem was that they became less, and not as originally hypothesised, more willing to respond by increasing their rate of drug consumption once in a painful state. The long exposure to opioids for training purposes also had other detrimental consequences. One of these was that the rats probably became more susceptible to pain during testing due to the development of a drug-induced hyperalgesic state. Due to this we devised an alternative method or training using amphetamine as the reinforcer. This, however, also had confounding effects by causing the animals to become hyperactive. These findings are directing us towards trying to develop a training method that completely avoids the need to use drugs during training by using intra-cranial self-stimulation (ICSS). The scientific benefits of this could be considerable, but discovering that the welfare of the animals is adversely affected by using opioids is equally if not more important. Avoiding use of these drugs is advisable. These findings have been widely disseminated via invited lectures to colleagues engaged in similar studies, but taken alone, are not well suited to the usual route of publication by peer review. We nevertheless are still attempting to do so.
First Year Of Impact 2011
Sector Education,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Policy & public services
 
Description Newcastle Pain Workshop - Refining Best Practices in animal pain management
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
Impact The annual Newcastle workshop that the PI on the project helped to establish is updated as 3Rs advances emerge and these are communicated to Students, Veterinarians, Researchers and others involved in biomedical science applications involving animals. They are given information on how to recognise the occurrence of pain or poor welfare in laboratory animals and how to prevent this happening using analgesics or other no-pharmacological solutions; such as housing refinements. A great deal of this information has arisen from the research. This has influenced the attendees directly and by reporting back to their own research facilities as to to how to implement welfare refinements. The success of this program is recognised in the University's 2014 Research Excellence Framework submission when it was part of a chosen impact case. It has also been commended by the Home Office Animals in Science Regulation Unit.
URL http://www.ahwla.org.uk/site/workshops.html
 
Title Intracranial self-stimulation (ICSS) for welfare and affective state assessment in rodents 
Description Developing capability to assess correlates of emotional/affective state towards identifying refinements to the analgesic treatment of post-procedural or post-surgical pain in rodents. The equipment housing the modules needed for establishing our ability to record intra-cranial electrical self-stimulation were purchased using this BBSRC award. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2016 
Provided To Others? Yes  
Impact The ICSS methodology was used by undergraduate students, informing them on the welfare needs of rodent and how such innovative methods can be applied in the assessment thereof. 
 
Description Intra-cranial self-stimulation (ICSS) as a measure of altered affective state caused by pain 
Organisation Virginia Commonwealth University
Country United States 
Sector Academic/University 
PI Contribution Subjective (emotional or 'affective') aspects of pain are the most difficulty to investigate. This was initially attempted using the self-administration methods made available by BBSRC, and subsequently the CPP paradigm used for the NC3Rs work. However, the drawback of both is the need to train animals using drugs such as opioids that also alter affective state, and may also impact negatively on welfare. This ground work led to the involvement of colleagues both at Newcastle and at Virginia Commonwealth University USA who use ICSS.
Collaborator Contribution The VCU partners provided access to mice already trained for ICSS behaviour, provided some necessary hardware and demonstrated the techniques for electrode implantation and use of the software needed for data acquisition.
Impact The work has begun and is progressing well. The rats are now undergoing ICSS training and we anticipate findings withing 2 months.
Start Year 2014
 
Description Using operant learning paradigms to invistigate the affective properties of pain in rodents 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Our Pain and Animal Welfare Science (PAWS) group has extensive experience of developing methods fo assessing the welfare of laboratory rodents. with particualar emphais on whether the procedures they are commonly exposed to causue pain. This information is used to establish whether there are any needs to refine these procedures such as by provising the animals with analgesics. We have provided animals to a colleague within the Institute of Neuroscience to develop operant approaches to investigate the affective properties, or the 'feeling' of pain in rats and mice. This is a long standing close partnership that should continue for the forseeable future.
Collaborator Contribution The partner brings essential technical expertise in operant methods of investigating the neuropharmacological properties of drugs, especially those that could be effective for refnining the provision of pain relief to laboratory rodents. Their input provided invaluable help developing the Conditional Drug Discrimination studeis that were undertaken during the latter part of the project and contributed directly to the student obtaining their MPhil degree.
Impact Successful completion of the BBSRC project on assessing self-administration behaviour in rats. Their input was also important to help a student complete an MPhil based on research conducted on the project. This led to essential knowledge about methods of pain testing that, although relatively innovative, are unlikely to be successful in the future.
Start Year 2006
 
Description 6th UK-China Seminar for Lab Animal Welfare and Ethical Use 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I gave 2 presentations at the 6th UK-China Seminar for Lab Animal Welfare and Ethical Use; Nanjing. The first was on 'Experimental pain assessment and practical management', and this was followed by another relating to my most recent NC3Rs award exploring the welfare advantage to tunnel handling in mice: 'Benefits of Tunnel handling.'
Year(s) Of Engagement Activity 2019
 
Description Animal welfare and the 3Rs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Describing how to implement refinements to improve welfare and to increase staff thinking about possible options to be applied to their own animal models. Increasing awareness of methods of assessing the affective properties of pain in rats and mice was a major objective of this. I used results obtained via use of CPP testing in cancer bearing mice and self-administration and conditional drug discrimination in rats to achieve this.

Summary of talk was included in the Abbott Newsletter. This was distributed across all Abbott Research and Development sites.
Year(s) Of Engagement Activity 2010
 
Description Detecting pain in laboratory animals 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Informing veterinarians about best practise in pain assessment in laboratory and other animal species via implementation of methods developed following NC3Rs and BBSRC funding

As a group mainly comprised of veterinarians, the AVA benefited by being informed about the most refined methods of caring for and utilising animals whilst maximising the gain of scientific knowledge. Members lacked knowledge on up-to-date methods of assessing subjective states linked to pain. This presentation focussed on examples of methods drawn from both the NC3Rs funded cancer studies in mice (Conditioned Place Preference testing and automated behaviour analysis) and the self-administration and Conditional Drug Discrimination testing conducted as part of the BBSRC project.
Year(s) Of Engagement Activity 2011
URL http://www.liverpoolavacongress2011.appspot.com/AVA_Liv_Programme.pdf
 
Description Effective and emerging methods for assessing and refining the welfare of laboratory animals 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact At dinner following the talk I realised that the welfare monitoring procedures described in the talk nere relatively new to German technicians and staff present reported they were keen to apply them.

Giessen University (Germany) requested a presentation on pain and welfare assessment in laboratory animals as a means of compliance with Directive EU/2010/63. This talk provided German animal technicians and veterinarians with knowledge about relatively simple welfare measures that could be applied widely (e.g. nest-building, burrowing, MGS scoring). Methods developed and applied as a consequenc of all recent funding was presented including CPP testing, drug self-adminiatration and Conditional discrimination.
Year(s) Of Engagement Activity 2013
 
Description FGB Laboratory animal welfare and pain assessment. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Raised several specific question about how the latest methods in pain detection and analgesic efficacy testing could be applied in other laboraties. The methods proposed were those developed following BBSRC funding (Self-administration and Conditional Drug Discrimination) and automated behaviour analysis and Conditioned Place Preference testing methods developed for NC3Rs.

Following the presentation I was approached by a staff member of Cancer Research UK for more information about pain testing and whether I would be willing to visit the UK facility to discuss this further. In 2013 two members of our group visited CRUK to discuss possible contributions to developing refined methods for welfare monitoring in a spontaneous human pancreatic cancer model. Further details are in the Collaboration/Partnerships section.
Year(s) Of Engagement Activity 2011
URL http://www.fondazioneguidobernardini.org/en/foundation/video.aspx
 
Description FOR 2591: Severity assessment in animal based research 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This was a meeting held in Rostock (Germany) bringing together a highly interdisciplinary consortium with the aim of identifying objective, classifiable, and standardized severity assessment parameters in animal research models. The aim of the consortium is to put in place a means of validating currently used severity assessment methods and develop new minimally- and non-invasive surveillance techniques. A scientific network meant to determine animal welfare-based recommendations for severity assessment, and determine if these can enable improvement in the quality of animal-based research data and whilst minimizing the severity of any suffering experienced by laboratory animals.
Year(s) Of Engagement Activity 2018
URL https://severity-assessment.de/
 
Description Innovative Strategies for Severity Assessment in Laboratory Animals 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Round table discussion resulted to try to achieve some degree of Europe wide methods for severity assessment. I presented findings on methods of assessment developed in all previous projects including affective state assessment, imaging of cancer, CPP testing, self-administration and Conditional drug discrimination.

Increased agreement about the need to development more innovative methods of welfare assessement to achieve retrospective severity reporting
Year(s) Of Engagement Activity 2014
URL http://www.mh-hannover.de/symposiumztl.html
 
Description Practical approaches to pain assessment and management in laboratory animals and compliance with the 3Rs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This annual event allows engagement with future stakeholders; students thinking of undertaking careers in Animal Welfare Science. The student catchment is mainly from across Europe and the USA. It is given to final year MSc students on the course in Animal Welfare at Edinburgh University. It stimulates discussion about possible refinements for in-vivo research and focusses on the problem of assessing pain in research animals.

Having been given information on the currently most relevant methods of assessing pain/welfare three students have visited Newcastle after graduating from their courses to help on welfare research projects.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2013,2014
URL http://www.ed.ac.uk/studying/postgraduate/degrees?id=238&cw_xml=details.php
 
Description Refining understanding of rodent pain and welfare assessment 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Increasing the likelihood that participants think more pro-actively about the need for refinement in their work with laboratory animals, and methods available for assessing the subjective properties of pain and its impact on laboratory rodents (Conditioned Place Preference testing (NC3Rs) and analgesic self-administration (BBSRC).

Disseminating knowledge on welfare concerns in rodent pain models: The specific aim was to communicate contemporary possibilities for refinement strategies and methods of reducing animal numbers.
Year(s) Of Engagement Activity 2010
 
Description Sainsbury Wellcome Centre; 27th Feb 2019: Lecture on welfare assessment in terms of subjective state evaluation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact I gave a presentation at the Sainsbury Wellcome Centre on 'Welfare assessment in terms of subjective state evaluation' This covered much of the research findings obtained across multiple previous awards The intention was to educate those currently responsible for the care and welfare of laboratory rodents about the assessment and refinement techniques that are currently available.
Year(s) Of Engagement Activity 2019