Finding effective analgesics in zebrafish by analysing effects on the nervous system (CNS)

The zebrafish is becoming increasingly popular in biomedical research (more than 400.000 individuals were used in 2011 according to the Home Office), as it is relatively small and still shares many features with other back-boned animals (vertebrates). This makes scientific findings from the zebrafish translatable for medical research. Adult and larval fish are being used in diverse fields, including research in cancer and neurodegenerative diseases and undergo potentially painful procedures, yet most procedures are done without proper analgesia (pain relief). This is mainly because not much is known about how fish process potentially harmful input. Efforts are underway to look, for example, at changes in behaviour after potentially painful experiences and how specific drugs (analgesics) mitigate these, in order to develop protocols for effective analgesia (pain relief) in fish. However, the central nervous system, which receives these stimuli, processes them and directs the behavioural changes, is not being analysed. We believe that analysing changes in the central nervous system is indispensable to directly demonstrate that harmful stimuli affect the brain, and that analgesic drugs are effective at the level of brain. In this pilot project, we aim to support efforts analysing behaviour and other parameters by finding changes in the activity of different genes in areas of the spinal cord and brain after potentially painful experiences. This will give direct information on the processing of such input. Moreover, by trying a number of potential analgesics we will determine which of these work most effectively by directly measuring gene activity in the brain. Our preliminary analyses show that application of mustard oil to muscle tissue leads to increased activity of genes in the spinal cord and brainstem that are often activated in the context of harmful stimuli. Ideally, we will find analgesics that block this reaction already at the level of the spinal cord, where stimuli are first processed. In subsequent research, our findings from the brain will be compared to data from behaviour and other non-invasive measures to determine optimal protocols for analgesia in fishes and derive non-invasive indicators of successful analgesia in future zebrafish research. Our data will thus help to refine experiments in fishes in the sense of Refinement as defined in the 3Rs, by devising protocols for analgesia that are supported by neurobiological observations and thus inform scientists and policy makers on best practice in fish experiments.

The zebrafish is increasingly being used in biomedical research, because it is a cost-effective vertebrate model organism. However, most of the more than 400.000 procedures per year are performed without analgesia, because there is a lack of knowledge regarding nociception and effective analgesics in this important laboratory species. Efforts are underway to establish non-invasive measures of nociception, such as altered behaviour and cortisol measurements. To firmly link such measures to nociception, changes in genes expression elicited by noxious stimuli in the CNS need to be established. In this pilot project, we propose to analyse gene expression changes of immediate early genes and endogenous opiates in the dorsal horn of the spinal cord, where primary nociception processing takes place, and in higher brain centres. Our preliminary findings indicate that c-fos expression is selectively increased in the dorsal horn and pro-enkephalin expression is increased in the brainstem of adult fish that received an intramuscular injection of mustard oil, compared to those that received a vehicle injection. This suggests that gene expression changes specifically related to nociception and not just to handling stress can be measured. We will conduct similar experiments in larval zebrafish and, importantly, will determine how potential analgesics reduce nociception-related gene expression. Our experiments will elucidate central nociception in zebrafish and will inform other studies using non-invasive measurements regarding the relevance of these measures to nociception. Our ultimate goal is to contribute to the refinement of procedures conducted on zebrafish by devising effective ways of analgesia and validating non-invasive measurements of these.

The present project aims to identify changes in CNS gene expression representing nociception, with the ultimate aim of finding drugs that can attenuate these CNS changes and act as analgesics. We expect a major benefit in terms of Refinement in the sense of the 3Rs.

Over 400'000 fish are used in procedures in the UK every year. More than 75% of these fish are used in anatomical and physiological research which is likely to involve noxious procedures. An unspecified percentage of these procedures are classified as "substantial" and without appropriate/proven analgesia, cannot easily be refined. Therefore, any scientist performing invasive procedures on zebrafish would benefit from our findings. We could reduce the severity of 500 procedures per year in our own laboratory from substantial to moderate. Our Edinburgh colleagues Dr Lyons (performing cell ablation in larval zebrafish CNS) and Dr Denvir (performing heart lesions in adult zebrafish), would also profit from our findings. We are liaising with Ms Gidona Goodman, who is currently conducting NC3Rs-funded research into the effect of fin clips, used in husbandry, on zebrafish behaviour. This will provide a solid basis for the combination of our findings on gene expression changes in the brain during nociception with non-invasive measures of nociception in the zebrafish. We further have a collaboration with Prof. J. Douglas Armstrong, who has developed software for zebrafish tracking.

Our results are relevant both for researchers in the field and policy makers (e.g. the Home Office). Successfully defining effective analgesia after invasive procedures in zebrafish would inform best practise both in the UK (legislated/monitored through the Home Office), in Europe (e.g. recommended by FELASA) and elsewhere, where zebrafish are used in research.

Furthermore, the public has a great interest in research and best practise in research animal welfare. The University's press officer, Miss Tara Womersley has written several press releases about our work. I have spoken about our research findings on two different radio shows (including the BBC). Our research has been featured by newspapers (Telegraph, Daily Mail, Herald) and online media (BBC). Furthermore, a non-technical summary of our work is published on the web sites of the Centre for Neuroregeneration and School of Biomedical Sciences. We will further ensure dissemination by publications in the EuFishBioMed Society newsletter read by scientists and the University's "Friends" magazine, read by Alumni and donors.

This research combines our knowledge in neuroanatomy with the search for effective analgesics in the zebrafish, employing in situ hybridisation, immunohistochemistry, and tract tracing. A postgraduate fellow will be employed and we will train at least one MSc student in these techniques.

Commercial impact of the present study is unlikely. However, we have quarterly meetings with our ERI/BioQuarter officer Dr. Amy Lam to identify potential commercial leads of any of our research. She is keeping a research summary of our work, which we regularly update, and which is sent out to companies. I have met with several pharmaceutical companies (3 in 2012; 2 in 2013) to discuss our work. A patent covering a drug screening procedure we developed using transgenic zebrafish embryos has been applied for with the University of Edinburgh in 2009.

The most important impact measure will be how many groups adopt analgesia practise that we find effective. We will obtain information about analgesia in Edinburgh from our colleagues and through the University's Ethical Review Committee of which Dr Thomas Becker is a member. National numbers will be reported to the Home Office. A drop in the number of procedures/entire licenses classified as "substantial" would indicate the impact of verified analgesia methods. Impact of publications will be assessed by the citations and/or coverage in other publications and the press.