PiLAS Issue 1(1) by Anne Jeffery

October 2012

Perspectives in Laboratory Animal Science

PECTIV PERS ES

BORATORY A LA N

SCIENCE AL IM

PiLAS .atl a.or g.

CONTENTS OPINIONS A risk assessment approach to severity classification in animal research Animals are more than tools Animal use in veterinary education — the need for a fourth R: Respect

DISCUSSIONS Automated homecage behavioural analysis and the implementation of the Three Rs in research involving mice Experience of the use of tabletop simulators as alternatives in the primary surgical training of veterinary undergraduate students Toward a humanised alternative to the use of laboratory animals for blood–brain barrier research

POINTS OF VIEW THE WISDOM OF RUSSELL AND BURCH

For professionals in the fields of laboratory animal care and use

The Launch of PiLAS FRAME was founded in 1969, to promote the development and application of sound scientific principles and methodology which could lead to the progressive reduction and replacement of laboratory animal procedures in biomedical research, testing and education. We are not uncritically for or against science, we do not favour humans in competition with animals, and we never put animal welfare above the welfare of humans. Rather, our aim is to avoid the conflicts that can arise between these kinds of competing interests, by encouraging positive scientific developments which are genuinely in the interests of all concerned. While animal procedures continue to be considered necessary in some circumstances, they should be conducted in ways which ensure the highest possible standards of welfare and care for the animals concerned. As members of the Triple Alliance (the BUAV, CRAE and FRAME), which advised the British Government during the passage through Parliament of the Animals (Scientific Procedures) Act 1986 (ASPA), we were totally supportive of the inclusion of requirements for a named day-to-day-care person and a named veterinary surgeon for each animal breeder, supplier or user establishment. Now, 25 years on, we are pleased that similar requirements are spelled out in Article 24 and Article 25 of Directive 2010/63/EU on the protection of animals used for scientific purposes, which applies to all the Member States of the European Union, and which comes into force in January 2013. Like the ASPA and the Directive which preceded it (Directive 86/609/EEC), the new Directive is firmly based on the Three Rs of Russell and Burch, and the principles of Replacement, Reduction and Refinement are clearly spelled out in Article 4. The proper application of the Three Rs involves a wide complexity of ethical, scientific and practical considerations in relation to benefit (to humans) and suffering (of animals). These include: justification of the need for performing the specific procedures; how they should be performed in order to maximise benefit and minimise suffering; the likelihood that worthwhile benefit will be achieved and how that should be weighed against likely animal suffering; the detection, measurement and relief of suffering; the nature and uses of models; the planning of experiments and the analysis of data; the breeding, supply, transport and re-use of animals; species differences among animals and between animals and humans; and conflicts between responsibilities to animals, colleagues, science and medicine, and employers. The aim of PiLAS is to improve the quality of discussion about animal experimentation and alternative approaches, by offering bio-scientists in all relevant fields an opportunity to share their expertise, knowledge and ideas concerning these and other issues raised by laboratory animal use. As well as being circulated along with FRAME’s peer-review, scientific journal, Alternatives to Laboratory Animals (ATLA), articles within PiLAS will be freely available, via open access, on the accompanying website — www.atla.org.uk.

PiLAS has been made possible by a grant from the Phoebe Wortley Talbot Charitable Trust Published by: Fund for the Replacement of Animals in Medical Experiments Russell & Burch House, 96-98 North Sherwood Street, Nottingham NG1 4EE, UK

OPINIONS A Risk Assessment Approach to Severity Classification in Animal Research David B. Morton Assessment of pain, suffering, distress and lasting harm in animal research could be carried out more successfully by adopting the practical risk assessment approach currently in use in farming According to the provisions of EU Directive 2012/63/ EU (Annex VIII), research on animals requires the assessment of pain, suffering, distress and lasting harm. The Directive specifies mild, moderate, and severe levels of these adverse states — but two questions are raised. Firstly, how do we recognise such states, and, secondly, how do we measure them? Recognition is, of course, the key issue, because if researchers and associated care personnel do not recognise these adverse states, then nothing else follows.1 With the exception of lasting harm, pain, distress and suffering have two components — their duration and their intensity or degree. In practice, what is important is the combination of intensity and duration, which is known as the ‘severity’ or ‘magnitude’ of the adverse effect. A third element is the character or quality of the adverse state, as described by humans. Thus, pain can be aching, sharp, shooting, stabbing, throbbing, pins-and-needles, etc. Distress also has different characteristics, and describes mental states such as boredom, frustration, lethargy, malaise, fear, anxiety, and even grief. Suffering can be described in many ways, but perhaps in the context of animals and humans, it is the mental thoughts that may accompany any pain and distress. It is the reflective mental component that will vary according to an animal’s experience, and its cognitive capacity. For example, an animal that has been hurt by someone, is more likely to anticipate pain in future interactions with that specific human, and will show anxiety and fear when they next come into contact. However, I suggest that what is ultimately important is not how we, as humans, define such states for the purposes of research or communication or treatment, but the actual impact that these states have on an animal, i.e. the consequences of animals experiencing feelings of pain, distress and suffering, and lasting harm. It is probably the closest we can get to an understanding of how that animal feels and the

intensity of the adverse state. Duration, by comparison, is simply how long animals are in that state. Furthermore, with knowledge of the biology of that animal species, we can deduce the type of the adverse state and its severity. We can extrapolate from human experiences that might help gain a better insight, so-called “critical anthropomorphism”.2 Animals that don’t eat, or eat less, or that are lame, or that change their behaviour in some way, will show (observable) signs that can be measured. For example, animals that don’t eat lose body weight, so measuring weight loss can be an indicator of pain and distress. If an animal runs away or refuses to cooperate in some way, it is likely to be an indication that it remembers some earlier aversive interaction, i.e. it is feeling fear. (If it has not been exposed to an aversive event, we would call it anxiety or apprehension, as with wild animals.) As well as measuring behaviour and clinical signs, we can take samples of blood, urine or faeces, and measure physiological responses indicative of an adverse state, such as corticosteroid levels (distress) and catecholamine levels (fear, anxiety), and measure ‘end-organ’ responses such as heart-rate or blood pressure. The degree to which a measure has deviated from normality would reflect the intensity of the adverse state. Finally, with what do we compare any observation? The ‘gold standard’ might be the same animal before any experimental interference, or, more likely, control naïve animals that have had nothing done to them. Experimental controls, such as sham-operated or drug vehicle only, are unreliable, as the ‘control procedure’ itself may have had an effect. There will be biological variation among animals, even though scientists try to standardise their protocols. This is even more likely at the laboratory level, in terms of housing, husbandry, care, attitude of carers and so forth. The animal’s psychological state can be as important as it is in humans, so assessment

has to be based on the response of individual animals. A set of animals should be seen as a collection of individuals, just like a herd or a flock, and, in practice, these varying responses should be analysed statistically. In other areas of animal use, such as farming, a risk assessment (RA) approach to the assessment of animal welfare is being taken.3 This involves specific terminology that has been adapted from food and feed safety, and control of infectious diseases.4, 5 At its simplest, the identification of a ‘hazard’ and the likelihood of ‘exposure’ to the hazard(s) are the two basic building blocks of RA. A hazard is an environmental or genetic factor that may cause poor welfare, e.g. pain, distress and suffering. However, with animal welfare, the consequences of exposure are crucial. Will it cause mild, moderate or severe pain? Furthermore, will that occur in all the animals being considered? By asking questions such as the following, a quantitative or a qualitative numerical estimate of the risk of an adverse effect occurring can be made:

breaking up the moderate band will lead to more disagreement). The subsequent actions based on the score should be well-defined and limited, e.g. humane endpoints. The RA approach to measuring the degree of pain, distress, suffering and lasting harm in animals, has yet to be applied to animal research. It is proving to be a transparent and practical system in farmed animals, and would be worth considering in the application of Directive 2010/63/EU in the field of animalbased research.

1. What proportion of animals will be exposed to the ‘hazard’, i.e. the procedure of interest? What is the dose group, control group, sham-treated group?

References

2. How likely is the hazard to produce the anticipated harmful state (in terms of severity), and in what proportion of the animals? 3. What adverse effect is likely to result, how will it be measured, and what impact will it have on the animal? In order to answer these questions, there may be relevant information in the literature (but that is unlikely in animal research, as there is a reluctance to report on the level of suffering involved in any experiment), or expert opinion has to be sought. As indicated above, intensity can be measured by comparing the effects of the procedure on a given parameter/indicator/measure for the experimental group compared with a ‘normal naïve’ control. A system for measuring such effects can then be devised and used for this particular experimental protocol.6 There may well be differences between observers, but these can often be minimised by concentrating on only two categories — those of mild and severe — where normally there is less or little disagreement. Those not falling into either extreme category are scored as moderate (it should be noted that

Professor David Morton c/o FRAME Russell and Burch House 96–98 North Sherwood Street Nottingham NG1 4EE UK E-mail: d.b.morton@bham.ac.uk

Morton, D.B. & Griffiths, P.H.M. (1985). Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record 116, 431–436. 2 Morton, D.B., Burghardt, G. & Smith, J.A. (1990). Animals, science, and ethics — Section III: Critical anthropomorphism, animal suffering, and the ecological context. The Hasting’s Center Report 20 (3), S13– S19. 3 EFSA (2012). Guidance on risk assessment for animal welfare. EFSA Journal 10(1):2513, doi:10.2903/ j.efsa.2012.2513. Parma, Italy: European Food Safety Authority. 4 CAC (1999). Principles and Guidelines for the Conduct of Microbiological Risk Assessment. CAC/GL 30 (1999), 6pp. Rome, Italy: Codex Alimentarius Commission. 5 OIE (2012). Terrestrial Animal Health Code, Chapter 7.1. Paris, France: OIE (Office International des Epizooties) Organisation Mondiale de la Santé Animale. Available at: http://www.oie.int/index. php?id=169&L=0&htmfile=chapitre_1.7.1.htm (Accessed 04.10.12). 6 Hawkins, P. (ed.), Morton, D.B. (Chair), Burman, O., Dennison, N., Honess, P., Jennings, M., Lane, S., Middleton, V., Roughan, J.V., Wells, S. & Westwood, K. (2011). A guide to defining and implementing protocols for the welfare assessment of laboratory animals: Eleventh Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory Animals 45, 1–13.

Animals are More than Tools Coenraad Hendriksen, Vera Baumans and Bas Blaauboer The moral value of animals deserves greater recognition in scientific publications

In the new EU directive on the protection of animals used for scientific purposes (Directive 2010/ 63/EU), it is laid down that â&#x20AC;&#x153;animals have an intrinsic value which must be respectedâ&#x20AC;? (preamble [12]). The adoption of this ethical principle implies the explicit recognition that, beyond their instrumental value, animals also have a moral value. This principle should be reflected in the way we communicate about using animals in biomedical research and testing. In most publications on animal-based research, the information on laboratory animals is summarised under the heading, Material and Methods, which implies that animals have an instrumental value only. We therefore advocate that editors of journals should include a statement in their guidelines for authors, indicating that animals must be described under a separate heading (Animals), thus recognising and emphasising their moral value. Furthermore, and in line with the above, the section on Materials and Methods should specifically contain information on what has been done to implement the Three Rs of Replacement, Reduction and Refinement. Also, we feel that each publication should include a paragraph with an ethical justification of why animals were used. These recommendations are made in the light of the findings of Osborne et al.,1 who evaluated the editorial policies of scientific journals with respect to animal specification criteria, and concluded that

> 50% of journals publishing original research involving animals do not have any editorial policies relating to the use of animals. We believe that confirmation of the moral value of animals reflects a positive attitude toward laboratory animals. Moreover, such information will ultimately result in improvements in animal welfare, as well as in the quality of animal studies in research and testing in terms of reproducibility.

Professor Coenraad Hendriksen, Netherlands Vaccine Institute, PO Box 457, 3720 AL Bilthoven, The Netherlands; Professor Vera Baumans, Faculty of Veterinary Medicine, Utrecht University, PO Box 80166, 3508 TD Utrecht, The Netherlands; Professor Bas Blaauboer, Institute for Risk Assessment, Utrecht University, PO Box 80178, 3508 TD Utrecht, The Netherlands; on behalf of the members of the Three Rs Alternatives Initiating Network (TRAIN) Corresponding author: Coenraad.Hendriksen@nvi-vaccin.nl

Reference 1

Osborne, N.J., Payne, D. & Newman, M.L. (2009). Journal editorial policies, animal welfare, and the 3Rs. American Journal of Bioethics 9, 55â&#x20AC;&#x201C;59.

Animal Use in Veterinary Education — The Need for a Fourth R: Respect Catherine Tiplady Animals should not be treated with disrespect, merely because they are surplus to the requirements of others I started my veterinary science training in 2004, and I graduated in 2008. Like many others, my dream since childhood had been to become a veterinarian. However, for me this came at a great cost emotionally, as the constant animal killing for my education took its toll. My graduation day, rather than being a time of jubilation, was a somewhat Pyrrhic victory, which left me wondering if it had all been worth it. Although I believe that some animal use (live animals and cadavers) is essential for educating future veterinarians, I was shocked by the terminal practical classes which were then part of the veterinary curriculum at my university. In these terminal classes, students practised various procedures (such as bladder catheterisation, venepuncture and radiography) on living, anaesthetised animals, before euthanising them. The animals used were usually healthy poundanimals and ex-racing greyhounds. There was no body donation programme for obtaining ethically-sourced cadavers in place at that time, and no ethical objection policy until 2008. Most difficult for me were the terminal surgery practical classes, in which students were required to choose a dog to practise on and later kill. The negligible educational benefits of the terminal practical classes were outweighed by the extreme moral anguish that I felt. What made it even worse was that the terminal surgery classes bore no comparison to the high standards later required for private clients in the university’s veterinary hospital. In terminal surgery classes, cleaning the animal’s skin prior to surgery was not as thorough, heat mats were not provided, and surgical kits were not sterile. The ‘resuscitation’ practical class was horrific. Veterinary students repeatedly overdosed and resuscitated live dogs, before cutting the chest open and squeezing the animal’s heart, in a final, futile attempt to restore a heartbeat in the dying animal. Student attitudes toward the practical classes were diverse. In a previous year, a veterinary student had enjoyed this class so much that she apparently exclaimed afterwards “It was wonderful to hold the dog’s heart in my hand as it died!” Despite the university having a dog resuscitation mannequin, this was not offered to the class.

As each week passed, and the number of dogs that died for the benefit of my education increased, my self-hatred grew. I felt that I would never be able to balance the lives I would later save as a qualified veterinarian with the lives I was now taking as a student. We were told not to discuss the terminal surgery classes ‘socially’, and I believe this stifled the willingness of other students to voice any ethical concerns that they might have had. There is also a risk that educators who mark students for ‘enthusiasm’ during practicals, encourage students to participate in activities that harm animals. Previous studies describe bitterness among students toward those promoting alternatives,1 and a “macho bravado culture” in veterinary school.2 The irony of the terminal surgery practical classes was that, no matter how well or how poorly students performed the surgeries, the outcome for the animals was inevitable — death at the hands of those supposedly being trained to save lives. Such a contradiction was increasingly hard to bear. I requested aged or sick dogs to use in the surgery practical classes, and a staff member supported me by helping to obtain such dogs. Whilst this was slightly easier for me than killing a healthy puppy or a pregnant bitch, as some of my fellow students had to do, it was still taking a life. It seemed that we were encouraged to regard these dogs as somehow deserving to be part of the terminal practicals. One staff member, in defence of the terminal surgery practicals, stated: “the pound dogs have all done something wrong”. I found this statement quite bizarre. If a dog behaves in the ‘wrong’ way, should the animal be sentenced to vivisection and death by lethal injection at the hands of future veterinarians? Does the ‘capital punishment’ of pound dogs serve as a deterrent to other dogs which may dare to perform ‘wrong’ acts? Such comments from staff may indoctrinate students to believe that pound dogs and ex-racing greyhounds are mere tools for our education, and, as stated elsewhere, create a reliance on pounds to supply animals for teaching.3 Being told of the large number of dogs being euthanised at pounds, and that “They’re going to die anyway”, made it no easier for me. I have since worked as a veterinarian

in a pound, but have never felt that pound animals should be used for terminal surgery practice to try and justify their death. I believe that insufficient attention is paid by educators to the encouragement of respect among veterinary students toward animals and people during the veterinary course. I feel that respect is as vital a part of animal use as the other Three Rs. Unfortunately, some veterinary students are disrespectful toward animals and their cadavers. Some students chose to write obscenities on a cow during an anatomy class, and used a stock-marking crayon to draw ‘lipstick’ on her face and ridicule her; others filmed each other fooling around with pieces of a horse cadaver and posted the film on the internet. One student laughed at the female pound dog which was to be euthanised after a terminal surgery practical class — “Ha ha! Your dog’s got tits!” Disturbingly, one student even recommended culling the entire teaching herd of dairy cows as ‘The Final Solution’. These are not incidents isolated to the veterinary school that I attended. Colleagues from other veterinary schools have had similar experiences. One told me how a veterinary student joked about raping the rabbits used in teaching classes, taking photographs of himself squeezing the animal’s genitals to display on the internet. I believe that veterinary educators must constantly work to encourage a climate of respect during veterinary training, as a matter of urgent priority. This respect must extend toward both animals (live and cadavers) and humans (staff and students) in the vet-

erinary course. It is not acceptable that students with compassion are silenced, whether deliberately or not. These are the future veterinarians who can bring about change. Let’s work to nurture their compassion, listen without judgement to their ethical concerns, and continue to develop and embrace humane alternatives in veterinary education.

Dr Catherine Tiplady Centre for Animal Welfare and Ethics School of Veterinary Science University of Queensland Gatton Queensland 4343 Australia E-mail: catherine.tiplady@uqconnect.edu.au

References 1

Tiplady, C., Lloyd, S. & Morton, J. (2011). Veterinary science students’ preferences for the source of dog cadavers used in anatomy teaching. ATLA 39, 461–469. 2 Woon, S-Y. (2011). A veterinary student’s perspective on educational animal use and the potential for humane alternatives. ALTEX Proceedings, 1/12, Proceedings of WC8, 377–385. 3 Knight, A. (2002). The use of pound dogs in veterinary surgical training. In Learning Without Killing, a Guide to Conscientious Objection (ed. A. Knight), pp. 19–20. Available at: www.LearningWithoutKilling.info (Accessed 03.09.12).

DISCUSSIONS Automated Homecage Behavioural Analysis and the Implementation of the Three Rs in Research Involving Mice Claire A. Richardson Automated homecage behavioural analysis systems have the potential to both refine scientific procedures and reduce the numbers of mice used It is widely accepted that the Three Rs principles of replacement, reduction and refinement should be introduced into experimental procedures involving laboratory animals whenever possible.1 A range of behavioural analysis systems are now available, that can be used to automate the study of behaviour within animalsâ&#x20AC;&#x2122; homecages. Most of these systems are designed for studies on mice, which are the most widely-used laboratory animals,2 and they are frequently marketed as tools that can aid the implementation of the Three Rs. Here, the technology currently available to carry out automated homecage behavioural analysis of mice will be summarised, and the case that these systems have the potential to both refine scientific procedures (minimising pain and distress) and reduce the number of mice used experimentally, will be discussed.

Types of automated homecage behavioural analysis The technology that underlies the automated homecage behavioural analysis of mice is reviewed in detail elsewhere.3,4 Briefly, the methods of automated homecage behavioural assessment include automated video analysis,5,6 radio-frequency identification (RFID) transponders,7,8 infrared beams,9 infrared sensors,10 telemetry,11 and the quantification of homecage vibration.12 Systems vary in the types of behaviours that can be detected, but they all have the capacity to measure some form of spontaneous behaviour. Some of the systems only measure activity (e.g. infrared-based systems), whilst others can detect simple behaviours, such as rearing (e.g. automated video analysis and vibration-based systems). Physiological parameters (e.g. heart-rate and blood pressure) can be measured, in addition to behavioural monitoring, by using telemetry, whilst some RFID transponder systems allow operant behavioural testing to be carried out.

Refinement Automated homecage behavioural analysis is frequently advocated as a refinement to standardised behavioural testing. It is likely that the welfare of the mice can be improved by studying animals in their homecages, as this minimises the necessity for human handling, which may be stressful13 and can act as a potential source of experimental variability. Automated homecage behavioural testing also has many potential applications for mouse welfare assessment. One of the key limitations to implementing refinements to experimental studies involving mice, is the challenge of objectively identifying pain and/or distress.14 It can therefore be difficult to determine when refinements such as changes to husbandry or scientific procedures, should be introduced, and to then evaluate how these refinements affect animal welfare. Examples of how automated homecage technology can be used for welfare assessment include the use of automated video analysis and telemetry to objectively measure post-operative pain and to assess analgesic efficacy,5,11,15 as well as the use of RFID transponder systems to carry out preference testing.16 Automated homecage behavioural analysis systems have also been used successfully to detect subtle behavioural changes that correlate to disease progression in studies involving mouse models of chronic disease,17,18 and this capacity is likely to be useful in refining disease studies. If subtle behavioural changes can be detected, then further intensive monitoring can be carried out on specific individual animals, in study periods where pain and/or distress are likely to occur. The detection of behavioural changes that precede clinical disease also has applications in the implementation of appropriate humane endpoints, increasing the likelihood that mice can be killed prior to the onset of pain or distress, when the scientific objectives of the study have been met. Although there are several ways that automated homecage behavioural analysis can potentially refine

Mice housed in a radiofrequency identiﬁcation (RFID) transponderbased system, the ‘IntelliCage’.

experimental procedures involving mice, there could be some negative aspects of automated homecage behavioural analysis with respect to mouse welfare. The majority of the systems that are currently commercially available require that animals be individually housed, and there may also be limitations in the types of environmental enrichment that can be used. Implants are not required in many of the systems, but RFID-based systems require the implantation of small transponders under a brief general anaesthetic, and telemetry requires the surgical implantation of larger transmitters. As the animals are less frequently handled when automated homecage behavioural analysis systems are used, they are likely to be less acclimatised to humans. As a result, periods when handling is required (e.g. for cage cleaning) may be more stressful to them. Finally, while automation may be a very useful way to complement human observation of animals, it is critically important that it never replaces the direct assessment and care of animals by compassionate and experienced personnel.14

Reduction The main argument that automated homecage behavioural analysis can help in reduction of the number of animals used experimentally, is that the more-effective

use of animals decreases the overall number of animals required. Through the use of more-sensitive assessment techniques, fewer animals may be required to produce statistically-significant results than has been previously obtained by using conventional techniques (e.g. standardised behavioural testing). Scientifically, there is evidence that the standardisation typically carried out for individual behavioural tests may not always be effective in generating reproducible experimental studies between laboratories.19, 20 In contrast, studies carried out by using an automated homecage system show consistency, regardless of the laboratory concerned.21 Many automated techniques also permit investigators to examine several aspects of behaviour simultaneously, which minimises the necessity to carry out several studies examining different behaviours. However, there may be ways in which automated behavioural analysis promotes the use of mice in research, and thus increases the overall number of animals used. Many of these automated techniques are designed to support high-throughput phenotyping, typically involving large numbers of transgenic mice. The generation of genetically modified animals has generally led to increases in the number of animals used, with a 42% increase in animal procedures in the UK since 2001 — when technology to genetically modify animals was introduced.22 It is also possible that more animals may be required to validate

new automated assays, as a result of the necessity of their comparison with standardised behavioural techniques as part of the validation process.

Discussion and conclusion On balance, there is now increasing evidence that automated homecage behavioural analysis can be useful with respect to the implementation of the Three Rs in research involving mice. However, there are some potential areas of concern with respect to the Three Rs that the laboratory animal community should continue to evaluate as this technology develops.

Dr Claire A. Richardson Comparative Biology Centre Medical School Framlington Place Newcastle University Newcastle upon Tyne NE2 4HH UK E-mail: claire.richardson@ncl.ac.uk

References 1

Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen & Co. Ltd. Home Office (2012). Statistics of Scientific Procedures on Living Animals Great Britain 2011, 56pp. London, UK: The Stationery Office. Spruijt, B.M. & DeVisser, L. (2006). Advanced behavioural screening: Automated home cage ethology. Drug Discovery Today: Technologies 3, 231–237. Schaefer, A.T. & Claridge-Chang, A. (2012). The surveillance state of behavioral automation. Current Opinion in Neurobiology 22, 170–176. Miller, A.L., Flecknell, P.A., Leach, M.C. & Roughan, J.V. (2011). A comparison of a manual and an automated behavioural analysis method for assessing post-operative pain in mice. Applied Animal Behaviour Science 131, 138–144. Maroteaux, G., Loos, M., van der Sluis, S., Koopmans, B., Aarts, E., van Gassen, K., Geurts, A., The NeuroBSIK Mouse Phenomics Consortium, Largaespada, D.A., Spruijt, B.M., Stiedl, O., Smit, A.B. & Verhage, M. (2012). High-throughput phenotyping of avoidance learning in mice discriminates different genotypes and identifies a novel gene. Genes, Brain & Behavior [In press.] doi: 10.1111/j.1601-183X.2012.00820.x. Branchi, I., D’Andrea, I., Cirulli, F., Lipp, H.P. & Alleva, E. (2009). Shaping brain development: Mouse communal nesting blunts adult neuroendocrine and behavioural responses to social stress and modifies chronic antidepressant treatment outcome. Psychoneuroendocrinology 35, 743–751. Winter, Y. & Schaefers, A.T.U. (2011). A sorting system with automated gates permits individual operant experiments with mice from a social home cage. Journal of Neuroscience Methods 196, 276–280.

Moretti, P., Bouwknecht, J.A., Teague, R., Paylor, R. & Zoghbi, H.Y. (2005). Abnormalities of social interactions and home-cage behaviour in a mouse model of Rett Syndrome. Human Molecular Genetics 14, 205–220. Dell’Omo, G., Vannoni, E., Vyssotski, A.L., Di Bari, M.A., Nonno, R., Agrimi, U. & Lipp, H.P. (2002). Early behavioural changes in mice infected with BSE and scrapie: Automated home cage monitoring reveals prion strain differences. European Journal of Neuroscience 16, 735–742. Goecke, J.C., Awad, H., Lawson, J., Caldwell, J. & Boivin, G.P. (2005). Evaluating postoperative analgesics in mice using telemetry. Comparative Medicine 55, 37– 44. Razzoli, M., Carboni, L., Andreoli, M., Ballottari, A. & Arban, R. (2011). Different susceptibility to social defeat stress of BalbC and C57BL6/J mice. Behavioural Brain Research 216, 100–108. Hurst, J.L. & West, R.S. (2010). Taming anxiety in laboratory mice. Nature Methods 7, 825–826. Hawkins, P., Morton, D.B., Burman, O., Dennison, N., Honess, P., Jennings, M., Lane, S., Middleton, V., Roughan, J.V., Wells, S. & Westwood, K. (2011). A guide to defining and implementing protocols for the welfare assessment of laboratory animals: Eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory Animals 45, 1–13. Dickinson, A.L., Leach, M.C. & Flecknell, P.A. (2009). The analgesic effects of oral paracetamol in two strains of mice undergoing vasectomy. Laboratory Animals 43, 357–361. Tsai, P.P., Nagelschmidt, N., Kirchner, J., Stelzer, H.D. & Hackbarth, H. (2012). Validation of an automatic system (DoubleCage) for detecting the location of animals during preference tests. Laboratory Animals 46, 81–84. Rudenko, O., Tkach, V., Berezin, V. & Bock, E. (2009). Detection of early behavioural markers of Huntington’s disease in R6/2 mice employing an automated social home cage. Behavioural Brain Research 203, 188–199. Steele, A.D., Jackson, W.S., King, O.D. & Lindquist, S. (2007). The power of automated high-resolution behaviour analysis revealed by its application to mouse models of Huntington’s and prion diseases. Proceedings of the National Academy of Sciences of the USA 104, 1983–1988. Cryan, J.F. & Holmes, A. (2005). The ascent of mouse: Advances in modelling human depression and anxiety. Nature Reviews Drug Discovery 4, 775–790. Richter, S.A., Garner, J.P. & Würbel, H. (2009). Environmental standardization: Cure or cause of poor reproducibility in animal experiments? Nature Methods 6, 257–261. Krackow, S., Vannoni, E., Codita, A., Mohammed, A., Circulli, F., Branchi, I., Alleva, E., Reichelt, A., Willuweit, A., Voikar, V., Colacicco, G., Wolfer, D.P., Buschmann, J-U.F., Safi, K. & Lipp, H-P. (2010). Consistent behavioural phenotyping differences between inbred strains in the IntelliCage. Genes, Brain & Behavior 9, 722–731. NC3Rs (2012). Evaluating progress in the 3Rs: The NC3Rs framework. London, UK: National Centre for the Replacement, Refinement and Reduction of Animals in Research. Available at: http://www.nc3rs.org.uk/ document.asp?id=1810 (Accessed 06.09.12).

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Experience of the Use of Table-top Simulators as Alternatives in the Primary Surgical Training of Veterinary Undergraduate Students Juan Jose Perez-Rivero and Emilio Rendon-Franco Table-top simulators are valuable alternatives to animal models in health sciences training, by virtue of their low cost, ease of construction and ability to successfully fulfil teaching objectives

Currently, simulators are being used more and more in the training of health sciences students, including veterinarians. These simulators range from the simplest and cheapest ones (so called ‘table-top simulators’) to the most sophisticated, such as computerised models, to which access is limited, due to their cost.1, 2 A table-top simulator is usually constructed by using readily-available materials in common use, is generally used on the table (hence its name), and is useful for the training of isolated procedures that require, as part of their learning process, the coordination of movements, such as the formation of sutures and surgical knots, the creation and maintenance of intravenous cannulae, and the use of surgical instruments.3 Due to their origin, table-top simulators have the advantage of being economical and portable. This means that each student can have his or her own simulator, can transport it easily, practise with it, and replace it, whenever necessary.4 The use of a table-top simulator enhances the acquisition of skills, because the students can perform the necessary number of repetitions during their training, to become familiar with the correct use of materials and to improve the procedure they are practising.5 At the Universidad Autónoma Metropolitana Unidad Xochimilco (UAM-X), third-year veterinary students who attend classes within the monogastrics module, are taught basic surgical skills. As part of this process, it is important to train their hands and coordinate their movements. To facilitate this, the students are trained by using simulators, the table-top simulator being the most accessible version. The use of these simulators contributes to the reinforcement of the Three Rs principles with regard to the reduction and replacement of the use of animals for training. The importance of using simulators lies in the fact that students acquire skills, prior to their contact with real animals.4, 6 Training takes place in groups of no more than 20 students. Before starting the training, an introductory session must be performed, supported by a multimedia presentation, to explain in detail the surgical procedure which is going to be learned. When using the simulator, the training is usually supervised by an instructor, who will help make the necessary correc-

tions until the student performs the procedure without any flaws. This provides the students with better skills than if they were only to perform the training without assessment.7 The average training time in the surgery classroom is three hours for each type of simulator (suture or venipuncture). As the final part of the training, it is necessary to evaluate whether the students have acquired the expected skills, and the only way of doing this with this type of simulator is by direct observation of the student while actually working with the simulator. The members of the Surgical Teaching Unit of the UAM-X have created and used a range of different tabletop simulators, among which the venipuncture simulator,4 basic suture simulator, and the advanced simulators for suturing tubular and parenchymal organs, stand out (Figure 1). In our experience, the use of these simulators helps the undergraduate veterinary students to improve their skills in managing the materials used in venipuncture, as well as improving their knowledge and handling of suture instruments. Moreover, the skills of the students performing venipunctures, and the placement of peripheral venous catheters and their correct fixation, are improved. Suture simulators also enhance the correct use of different suturing patterns and surgical knots, as are required in the anaesthesia room or in the operating theatre. For surgical training, it is necessary to move forward in the development of simulators that can satisfy different scenarios, e.g. from the viewpoints of physiology, anaesthesia, bleeding control, and the handling of delicate tissues, which are currently only learned in real surgery.8 If this were to be achieved, then it would become possible to replace the use of animal models in this phase of the training of veterinarians. Dr Juan Jose Perez-Rivero Universidad Autónoma Metropolitana Unidad Xochimilco Calzada del Hueso 1100 Colonia Villa Quietud Delegación Coyoacán Ciudad de México C.P. 04960, D.F. México México E-mail: jjperez1_1999@yahoo.com

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Figure 1: A range of table-top simulators a)

a) A tabletop simulator for venipuncture — note the catheters on the sinuous venous path; b) a foam sheet used for practising basic sutures; c) and d) a latexglove finger filled with gelatine to practice suturing on parenchymatous organs; e) a latex tube used to practise suturing in tubular organs.

References 1

McGaghie, W.C., Siddall, V.J., Mazmanian, P.E. & Myers, J. (2009). Lessons for continuing medical education from simulation research in undergraduate and graduate medical education: Effectiveness of continuing medical education: American College of Chest Physicians Evidence-Based Educational Guidelines. Chest Journal 135, 62S–68S. 2 Parkes, R., Forrest, N. & Baillie, S. (2009). A mixed reality simulator for feline abdominal palpation training in veterinary medicine. Studies in Health Technology & Informatics 142, 244–246. 3 Hammoud, M.M., Nuthalapaty, F.S., Goepfert, A.R., Casey, P.M., Emmons, S., Espey, E.L., Kaczmarczyk, J.M., Katz, N.T., Neutens, J.J. & Peskin, E.G.; Association of Professors of Gynecology and Obstetrics Undergraduate Medical Education Committee (2008). To the point: Medical education review of the role of simulators in surgical training. American Journal of Obstetrics & Gynecology 199, 338–343.

Perez-Rivero, J.J. & Rendon-Franco, E. (2011). Validation of the education potential of a simulator to develop abilities and skills for the creation and maintenance of an intravenous cannula. ATLA 39, 257–260. 5 Scalese, R.J., Obeso, V.T. & Issenberg, S.B. (2008). Simulation technology for skills training and competency assessment in medical education. Journal of General Internal Medicine 23, 46–49. 6 Engum, S.A., Jeffries, P. & Fisher, L. (2003). Intravenous catheter training system: Computer-based education versus traditional learning methods. American Journal of Surgery 186, 67–74. 7 Govaere, J.L.J., de Kruif, A. & Valcke, M. (2012). Differential impact of unguided versus guided use a multimedia introduction to equine obstetrics in veterinary education. Computers & Education 58, 1076– 1084. 8 Calasans-Maia, M.D., Monteiro, M.L., Áscoli, F.O. & Granjeiro, J.M. (2009). The rabbit as an animal model for experimental surgery. Acta Cirúrgica Brasileira 24, 325–328.

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Toward a Humanised Alternative to the Use of Laboratory Animals for Blood–Brain Barrier Research Adjanie Patabendige The development of a novel flow-based, three-dimensional human blood–brain barrier model for studying brain infections is now under way Central nervous system (CNS) diseases have a devastating impact on the quality of life of those affected, with large health economic and social costs globally. There are no effective treatments for many neurological diseases, including almost all viral brain infections. A major hurdle in treating CNS disease is transporting therapeutics across the blood–brain barrier (BBB). The BBB is formed by the endothelial cells that line cerebral capillaries, supported by cells of the neurovascular unit (NVU) that induce and maintain the properties of the BBB. The main function of the BBB is to restrict the entry of molecules and pathogens into the CNS and maintain brain homeostasis, which is essential for normal neuronal function.1 The BBB is a major challenge in drug discovery programmes, as many potential CNS drugs cannot cross the BBB due to its strict regulation of paracellular and transcellular entry of molecules. To develop better treatment strategies, we need to increase our understanding of BBB function during health and disease.2 Evidence of the role of the BBB during viral pathogenesis has traditionally come from studies on animals, because of the difficulties associated with conducting clinical studies in humans and obtaining human brain tissue for in vitro investigations. There are several good in vitro BBB models derived from animal tissue, but as I have outlined in a Comment to be published in ATLA, there is an urgent need for the development of realistic human BBB models that can mimic the in vivo characteristics of the BBB.3 BBB research is a comparatively new field, as the existence of a BBB was only discovered just over 100 years ago. Publications on the BBB have steadily increased — from just one paper in 1947 to over 1500 papers so far this year — as we begin to understand the importance of the BBB in neurological disease (Figure 1). My interest in BBB research, and particularly in the development of in vitro BBB models, stemmed from discovering at the very beginning of my research career that only a handful of good in vitro BBB models were available. A major limitation was that most of these models were not robust and simple enough to use in the pharmaceutical industry for drug permeability studies. Furthermore, many pharmaceutical companies used in vitro BBB models derived from epithelial tissue (e.g. MDCK cells, Caco-2 cells), rather than from brain endothelial origin for their early-phase CNS drug discovery studies. To address these issues, I established a simple to use and robust in vitro BBB model, derived from porcine

brain endothelial cells that expressed major BBB features.4 Porcine brain material was used, because it was a by-product of the meat industry, so the availability of this type of brain tissue was not an issue. Furthermore, the anatomy, physiology and genome of the pig, all reflect those of the human more closely than most laboratory animal models.5 Therefore, a porcine BBB model seemed to be the best alternative to a human model for drug discovery studies. Having established a porcine BBB model, I moved onto developing an in vitro human model, because of my interest in studying the role of the BBB during CNS infection. Mechanisms of pathogen entry to the brain are poorly understood for many brain infections (e.g. viral encephalitis, bacterial meningitis, parasitic brain infections). I used this static human model to investigate how viruses that cause encephalitis cross the BBB. The static human model has given some interesting insights into the complex interactions between the virus and the BBB, and the role of inflammatory cytokines in viral pathogenesis (manuscript in preparation). However, replicating all the main features of the human BBB in vitro is not an easy task. The main challenges are the availability of fresh human brain tissue and maintaining BBB morphology and function in culture, following isolation of the cells. Immortalised human brain endothelial cells are an alternative, but these may lack certain important BBB features because of the immortalisation process. To overcome these challenges, I am now attempting to establish a flow-based three-dimensional (3-D) BBB model for studying the role of the BBB in brain infections (funded by an NC3Rs David Sainsbury fellowship).6 A flow-based model is preferred to a static model, as the shear stress caused by blood flow is important in maintaining the BBB features of brain endothelial cells, and will also provide the right environment for leukocyte transmigration across the endothelium, which is an important physiological process during infection. A realistic human BBB model will help increase our understanding of the importance of the BBB in protecting the brain during infection, as well as the damage caused to the BBB during the course of the infection. Furthermore, this flow-based 3-D human BBB model will potentially lead to the generation of physiologically-relevant human data and the identification of targets for developing novel therapeutics. If successful, it will be a useful alternative to laboratory animals for studying the BBB in CNS disease.

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Figure 1:

Blood–brain barrier research papers published, by year

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A PubMed search was performed on 19.10.12. Search term = blood–brain barrier; Field = Title/Abstract.

Dr Adjanie Patabendige University of Liverpool Brain Infections Group Institute of Infection & Global Health The Apex Building 8 West Derby Street Liverpool L69 7BE, UK E-mail: adjanie@liverpool.ac.uk

References 1

Abbott, N.J., Patabendige, A.A.K., Dolman, D.E.M., Yusof, S.R. & Begley, D.J. (2010). Structure and function of the blood–brain barrier. Neurobiology of Disease 37, 13–25.

Hawkins, B.T. & Davis, T.P. (2005). The blood–brain barrier/neurovascular unit in health and disease. Pharmacological Reviews 57, 173–185. 3 Patabendige, A. (2012). The value of in vitro models of the blood–brain barrier and their uses. ATLA [In press.] 4 Patabendige, A., Skinner, R.A. & Abbott, N.J. (2012). Establishment of a simplified in vitro porcine blood–brain barrier model with high transendothelial electrical resistance. Brain Research [In press.] http://dx. doi.org/10.1016/j.brainres.2012.06.057. 5 Walters, E.M., Agca, Y., Ganjam, V. & Evans, T. (2011). Animal models got you puzzled?: Think pig. Annals of the New York Academy of Sciences 1245, 63–64. 6 Janigro, D., Leaman, S.M. & Stanness, K.A. (1999). Dynamic in vitro modeling of the blood–brain barrier: A novel tool for studies of drug delivery to the brain. Pharmaceutical Science & Technology Today 2, 7–12.

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POINTS OF VIEW The following words of support for PiLAS have been received: Bill Russell was extremely gratified when Michael Balls, among others, rediscovered The Principles of Humane Experimental Technique, decades after it was published, not for reasons of personal vanity, but because it gave him hope that the rigorous science that it contained might yet be fully integrated into laboratory science, resulting in more-humane, and better, research. PiLAS is a further, and much needed step along that road. Bill would be pleased.

I am very pleased that PiLAS is being launched by FRAME, because, unfortunately, many of the issues raised by those concerned about the use of animals in laboratory experimentation have not been adequately addressed. Although I am not qualified to speak in depth about the need for the use of animals as models for human beings, I have discussed this with many people who are, and I have read extensively about the subject. And it seems conclusive that animals can never be really good models for human beings, since they are anatomically, physiologically and behaviourally adapted to ways of life very different from our own. In short, experiments on animals lead to too much pain for too little gain. As technical advances provide for new ways of gaining knowledge, the use of animals is becoming increasingly unnecessary. And as we learn more about the capacity of animals to suffer â&#x20AC;&#x201D; physically and psychologically â&#x20AC;&#x201D; increasingly unethical. Almost never can the basic needs of laboratory animals be adequately addressed. In the 21st century, it is imperative that we move away from the use and abuse of animals for this purpose. Jane Goodall PhD DBE Founder of the Jane Goodall Institute; UN Messenger of Peace As Chair of the All-Party Group on Replacement of Animals in Experimentation, I am delighted to welcome the launch of PiLAS. This will create a significant new forum for professionals from all disciplines to share their expertise and ideas about how to further improve the quality of experiments to the benefit of both animals and humans. I anticipate that the quality of discussion it generates will be very high indeed. It is likely to make an exciting contribution to a subject of great interest to the general public, as well as to the scientific community. Nic Dakin MP House of Commons The issues raised by animal experimentation are complex and emotive, yet it is crucial to consider and debate these, if we are to find alternative methods and reduce the need for laboratory animal procedures. PiLAS will provide an excellent platform to facilitate high quality unbiased and informed discussion, and build on the outstanding work of the Alternatives to Laboratory Animals (ATLA) journal over the past 30 years. I welcome the new supplement and look forward to reading about the important topics it deals with. Professor David Greenaway Vice Chancellor, University of Nottingham

Photo Stuart Clarke

Cleo Paskal Literary Executor of Bill and Claire Russell

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THE WISDOM OF RUSSELL AND BURCH 1. The Concept, Sources and Incidence of Inhumanity and its Diminution or Removal Through Implementation of the Three Rs

R.L. Burch and W.M.S. Russell

The concepts expounded by W.M.S. Russell and R.L. Burch in the 1950s in their outstanding book, The Principles of Humane Experimental Technique,1 are now the basis of many national and international laws and regulations on the proper use of laboratory animals. They were the outcome of a project proposed by Charles Hume, the founder of the Universities Federation for Animal Welfare.2 Their underlying philosophy concerned the concept of inhumanity, which they saw as “an objective assessment of the effects of any procedures on the animal subject”, without implying “any criticism or even psychological description of persons practising any given procedure”. They judged the central problem to be “that of determining what is and what is not humane, and how humanity can be promoted without prejudice to scientific and medical aims”. Chapter 2 of The Principles comprises a brilliant discussion on inhumanity, which considers pain and fear, and the “rather more general notion of distress”,

taking into account the different levels of consciousness and intelligence in animals in relation to our concern for their welfare. It introduces the provocative thought that “inhumane procedures are those which drive the animal’s mood down. Removing inhumanity must ultimately mean driving the animal as near the other end of the scale as we can. More humane means less inhumane.” However, it is Chapter 4, on The Sources, Incidence and Removal of Inhumanity, which is of vital significance. It begins with a discussion of the important distinction between direct inhumanity, “the infliction of distress as an unavoidable consequence of the procedure employed”, and contingent inhumanity, “the infliction of distress as an incidental and inadvertent by-product of the use of the procedure, which is not necessary for its success”. Russell and Burch emphasised that contingent inhumanity is almost always detrimental to the achievement of the objective of an experiment, but that much could be done to avoid it. Direct inhumanity, being unavoidable, is a totally different matter, and they discussed it in terms of incidence (e.g. in control and experimental groups), severity (e.g. the severity of a procedure in those animals that are affected), and special character (e.g. post-operative pain and distress, effects of particular pathogens, or death due to various types of toxic chemical). They saw the avoidance of contingent inhumanity as mainly a matter of good husbandry, diligent care and common sense, but their priceless gift, of equal value to biomedical science and animal welfare, was offered when they said: “We turn now to consideration of the ways in which [direct] inhumanity can be and is being diminished or removed. These ways can be discussed under the three broad headings of Replacement, Reduction, and Refinement, [which] have conveniently been referred to as the Three Rs of humane technique.”3 Russell and Burch’s definitions and discussions on the Three Rs will be considered in future issues of PiLAS, but let us close this short introduction with the words of Article 4: Principles of replacement, reduction and refinement, in Directive 2010/63/EU on the protection of animals used for scientific purposes,4 the provisions of which will come into force in the Member States of the European Union in January 2013:

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“1. Member States shall ensure that, wherever possible, a scientifically satisfactory method or testing strategy, not entailing the use of live animals, shall be used instead of a procedure. “2. Member States shall ensure that the number of animals used in projects is reduced to a minimum without compromising the objectives of the project. “3. Member States shall ensure refinement of breeding, accommodation and care, and of methods used in procedures, eliminating or reducing to the minimum any possible pain, suffering, distress or lasting harm to the animals.” All those who are in any way responsible for activities related to this Directive or the national laws and regulations of the Member States which are in accordance with it, have a legal duty and a moral obligation to act according to these principles.

References and Notes 1

Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique. xiv + 238 pp. London, UK: Methuen. 2 Balls, M. (2009). The origins and early development of the Three Rs concept. ATLA 37, 255–265. 3 Russell, W.M.S. (1957). The increase of humanity in experimentation: Replacement, Reduction and Refinement. Collected Papers of the Laboratory Animals Bureau 6, 23–25. 4 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79. The Principles of Humane Experimental Technique is now out of print, but the full text can be found at http://altweb.jhsph.edu/pubs/books/humane_exp/ het-toc. An abridged version, The Three Rs and the Humanity Criterion, by Michael Balls (2009) can be obtained from FRAME.

Submissions for consideration for publication in PiLAS are welcome. Please send articles to susan@frame.org.uk, or by post to Susan Trigwell, FRAME, Russell and Burch House, 96–98 North Sherwood Street, Nottingham NG1 4EE, UK. Instructions to Authors are available from the above, or from the PiLAS website, www.atla.org.uk. All articles considered for publication will be peer-reviewed.

Some possible topics for consideration in future issues are: v

the value of models and their uses

the planning of experiments

the analysis of scientific data

the use of non-human primates and, in particular, great apes as laboratory animals

the breeding, supply and transport of laboratory animals

the re-use of animals

re-homing

the humane killing of animals

the rodent bioassay for carcinogenicity

reproductive toxicity tests

animal experimentation for the benefit of animals

the importance of species differences

whether the use of humane endpoints is always humane

who actually are the vets’ clients?

is more suffering for the few better than less suffering for the many?

do some animals matter more than others?

should there be limits on genetic modification?

is it acceptable to humanise animals?