CONTENTS

OPINIONSA risk assessment approach toseverity classification in animalresearch

Animals are more than tools

Animal use in veterinaryeducation — the need for afourth R: Respect

DISCUSSIONSAutomated homecagebehavioural analysis and theimplementation of the Three Rsin research involving mice

Experience of the use of table-top simulators as alternativesin the primary surgical trainingof veterinary undergraduatestudents

Toward a humanised alternativeto the use of laboratory animalsfor blood–brain barrier research

POINTS OF VIEW

THE WISDOM OF RUSSELLAND BURCH

Perspectives

in Laboratory

Animal Science For professionals in the fields of laboratory animal care and use

October 2012PERS

PECTIVESIN

LABORATORY ANIM

ALSC

IENCE

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ww.atla.org.uk

PiLAS The Launch of PiLASFRAME was founded in 1969, to promote the development and application ofsound scientific principles and methodology which could lead to the progressivereduction and replacement of laboratory animal procedures in biomedicalresearch, testing and education. We are not uncritically for or against science,we do not favour humans in competition with animals, and we never put animalwelfare above the welfare of humans. Rather, our aim is to avoid the conflictsthat can arise between these kinds of competing interests, by encouraging pos-itive scientific developments which are genuinely in the interests of all con-cerned.

While animal procedures continue to be considered necessary in some circum-stances, they should be conducted in ways which ensure the highest possiblestandards of welfare and care for the animals concerned. As members of theTriple Alliance (the BUAV, CRAE and FRAME), which advised the BritishGovernment during the passage through Parliament of the Animals (ScientificProcedures) Act 1986 (ASPA), we were totally supportive of the inclusion ofrequirements for a named day-to-day-care person and a named veterinary sur-geon for each animal breeder, supplier or user establishment. Now, 25 yearson, we are pleased that similar requirements are spelled out in Article 24 andArticle 25 of Directive 2010/63/EU on the protection of animals used for sci-entific 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 Directivewhich preceded it (Directive 86/609/EEC), the new Directive is firmly basedon the Three Rs of Russell and Burch, and the principles of Replace ment,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) andsuffering (of animals). These include: justification of the need for performingthe specific procedures; how they should be performed in order to maximisebenefit and minimise suffering; the likelihood that worthwhile benefit will beachieved and how that should be weighed against likely animal suffering; thedetection, 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 andbetween animals and humans; and conflicts between responsibilities to ani-mals, colleagues, science and medicine, and employers.

The aim of PiLAS is to improve the quality of discussion about animal experi-mentation and alternative approaches, by offering bio-scientists in all relevantfields an opportunity to share their expertise, knowledge and ideas concerningthese 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 freelyavailable, 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

A Risk Assessment Approach to Severity Classification inAnimal Research

David B. Morton

Assessment of pain, suffering, distress and lasting harm in animalresearch could be carried out more successfully by adopting

the practical risk assessment approach currently in use in farming

OPINIONS

According to the provisions of EU Directive 2012/63/EU (Annex VIII), research on animals requires theassessment of pain, suffering, distress and lastingharm. The Directive specifies mild, moderate, andsevere levels of these adverse states — but two ques-tions are raised. Firstly, how do we recognise suchstates, and, secondly, how do we measure them?Recognition is, of course, the key issue, because ifresearchers and associated care personnel do notrecognise these adverse states, then nothing elsefollows.1

With the exception of lasting harm, pain, distressand suffering have two components — their durationand their intensity or degree. In practice, what isimportant is the combination of intensity and dura-tion, which is known as the ‘severity’ or ‘magnitude’of the adverse effect. A third element is the charac-ter or quality of the adverse state, as described byhumans. Thus, pain can be aching, sharp, shooting,stabbing, throbbing, pins-and-needles, etc. Distressalso has different characteristics, and describesmental states such as boredom, frustration, lethargy,malaise, fear, anxiety, and even grief. Suffering canbe described in many ways, but perhaps in the con-text of animals and humans, it is the mental thoughtsthat may accompany any pain and distress. It is thereflective mental component that will vary accordingto an animal’s experience, and its cognitive capacity.For example, an animal that has been hurt by some-one, is more likely to anticipate pain in future inter-actions with that specific human, and will showanxiety and fear when they next come into contact.

However, I suggest that what is ultimately impor-tant is not how we, as humans, define such states forthe purposes of research or communication or treat-ment, but the actual impact that these states haveon an animal, i.e. the consequences of animals expe-riencing feelings of pain, distress and suffering, andlasting harm. It is probably the closest we can get toan understanding of how that animal feels and the

intensity of the adverse state. Duration, by compar-ison, is simply how long animals are in that state.Furthermore, with knowledge of the biology of thatanimal species, we can deduce the type of theadverse state and its severity. We can extrapolatefrom human experiences that might help gain abetter insight, so-called “critical anthropomor-phism”.2 Animals that don’t eat, or eat less, or thatare lame, or that change their behaviour in someway, will show (observable) signs that can be meas-ured. For example, animals that don’t eat lose bodyweight, so measuring weight loss can be an indicatorof pain and distress. If an animal runs away or refusesto cooperate in some way, it is likely to be an indica-tion that it remembers some earlier aversive inter-action, i.e. it is feeling fear. (If it has not beenexposed to an aversive event, we would call it anxi-ety or apprehension, as with wild animals.)

As well as measuring behaviour and clinical signs,we can take samples of blood, urine or faeces, andmeasure physiological responses indicative of anadverse state, such as corticosteroid levels (distress)and catecholamine levels (fear, anxiety), and meas-ure ‘end-organ’ responses such as heart-rate or bloodpressure. The degree to which a measure has devi-ated from normality would reflect the intensity ofthe adverse state. Finally, with what do we compareany observation? The ‘gold standard’ might be thesame animal before any experimental interference,or, more likely, control naïve animals that have hadnothing done to them. Experimental controls, suchas sham-operated or drug vehicle only, are unreli-able, as the ‘control procedure’ itself may have hadan effect.

There will be biological variation among animals,even though scientists try to standardise their proto-cols. This is even more likely at the laboratory level,in terms of housing, husbandry, care, attitude ofcarers and so forth. The animal’s psychological statecan be as important as it is in humans, so assessment

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has to be based on the response of individual animals.A set of animals should be seen as a collection of indi-viduals, just like a herd or a flock, and, in practice,these varying responses should be analysed statisti-cally.

In other areas of animal use, such as farming, a riskassessment (RA) approach to the assessment ofanimal welfare is being taken.3 This involves specificterminology that has been adapted from food andfeed safety, and control of infectious diseases.4, 5 Atits simplest, the identification of a ‘hazard’ and thelikelihood of ‘exposure’ to the hazard(s) are the twobasic building blocks of RA. A hazard is an environ-mental or genetic factor that may cause poor wel-fare, e.g. pain, distress and suffering. However, withanimal welfare, the consequences of exposure arecrucial. Will it cause mild, moderate or severe pain?Furthermore, will that occur in all the animals beingconsidered?

By asking questions such as the following, a quan-titative or a qualitative numerical estimate of therisk of an adverse effect occurring can be made:

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

2. How likely is the hazard to produce the antici-pated harmful state (in terms of severity), and inwhat proportion of the animals?

3. What adverse effect is likely to result, how will itbe measured, and what impact will it have on theanimal?

In order to answer these questions, there may be rel-evant information in the literature (but that isunlikely in animal research, as there is a reluctanceto report on the level of suffering involved in anyexperiment), or expert opinion has to be sought.

As indicated above, intensity can be measured bycomparing the effects of the procedure on a givenparameter/indicator/measure for the experimentalgroup compared with a ‘normal naïve’ control. Asystem for measuring such effects can then bedevised and used for this particular experimentalprotocol.6 There may well be differences betweenobservers, but these can often be minimised by con-centrating on only two categories — those of mild andsevere — where normally there is less or little dis-agreement. Those not falling into either extreme cat-egory are scored as moderate (it should be noted that

breaking up the moderate band will lead to more dis-agreement). The subsequent actions based on thescore 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, hasyet to be applied to animal research. It is proving tobe a transparent and practical system in farmed ani-mals, and would be worth considering in the applica-tion of Directive 2010/63/EU in the field of animal-based research.

Professor David Mortonc/o FRAME

Russell and Burch House96–98 North Sherwood Street

Nottingham NG1 4EEUK

E-mail: d.b.morton@bham.ac.uk

References1 Morton, D.B. & Griffiths, P.H.M. (1985). Guidelines onthe recognition of pain, distress and discomfort inexperimental animals and an hypothesis for assess-ment. Veterinary Record 116, 431–436.

2 Morton, D.B., Burghardt, G. & Smith, J.A. (1990).Animals, science, and ethics — Section III: Criticalanthropomorphism, animal suffering, and the ecologi-cal context. The Hasting’s Center Report 20 (3), S13–S19.

3 EFSA (2012). Guidance on risk assessment for ani malwelfare. EFSA Journal 10(1):2513, doi:10.2903/j.efsa.2012.2513. Parma, Italy: European Food SafetyAuthority.

4 CAC (1999). Principles and Guidelines for the Con ductof 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. Availableat: http://www.oie.int/index. php?id=169&L=0&htm-file=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 protocolsfor the welfare assessment of laboratory animals:Eleventh Report of the BVAAWF/FRAME/RSPCA/UFAWJoint Working Group on Refinement. LaboratoryAnimals 45, 1–13.

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In the new EU directive on the protection of ani-mals used for scientific purposes (Directive 2010/63/EU), it is laid down that “animals have an intrin-sic value which must be respected” (preamble[12]). The adoption of this ethical principle impliesthe explicit recognition that, beyond their instru-mental value, animals also have a moral value. Thisprinciple should be reflected in the way we com-municate about using animals in biomedicalresearch and testing.

In most publications on animal-based research, theinformation on laboratory animals is summarisedunder the heading, Material and Methods, whichimplies that animals have an instrumental value only.We therefore advocate that editors of journals shouldinclude a statement in their guidelines for authors,indicating that animals must be described under aseparate heading (Animals), thus recognising andemphasising their moral value.

Furthermore, and in line with the above, the sec-tion on Materials and Methods should specificallycontain information on what has been done to imple-ment the Three Rs of Replacement, Reduction andRefinement. Also, we feel that each publicationshould include a paragraph with an ethical justifica-tion of why animals were used.

These recommendations are made in the light ofthe findings of Osborne et al.,1 who evaluated theeditorial policies of scientific journals with respectto animal specification criteria, and concluded that

Animals are More than Tools

Coenraad Hendriksen, Vera Baumans and Bas Blaauboer

The moral value of animals deserves greater recognition

in scientific publications

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> 50% of journals publishing original research involv-ing animals do not have any editorial policies relatingto the use of animals.

We believe that confirmation of the moral value ofanimals reflects a positive attitude toward laboratoryanimals. Moreover, such information will ultimatelyresult in improvements in animal welfare, as well asin the quality of animal studies in research and test-ing in terms of reproducibility.

Professor Coenraad Hendriksen, NetherlandsVaccine Institute, PO Box 457, 3720 AL Bilthoven,The Netherlands; Professor Vera Baumans, Facultyof Veterinary Medicine, Utrecht University, PO Box80166, 3508 TD Utrecht, The Netherlands;Professor Bas Blaauboer, Institute for RiskAssessment, Utrecht University, PO Box 80178, 3508TD Utrecht, The Netherlands; on behalf of themembers of the Three Rs Alternatives InitiatingNetwork (TRAIN)

Corresponding author:Coenraad.Hendriksen@nvi-vaccin.nl

Reference1 Osborne, N.J., Payne, D. & Newman, M.L. (2009).Journal editorial policies, animal welfare, and the 3Rs.American Journal of Bioethics 9, 55–59.

I started my veterinary science training in 2004, andI graduated in 2008. Like many others, my dreamsince childhood had been to become a veterinarian.However, for me this came at a great cost emotion-ally, as the constant animal killing for my educationtook its toll. My graduation day, rather than beinga time of jubilation, was a somewhat Pyrrhic vic-tory, which left me wondering if it had all beenworth it.

Although I believe that some animal use (live ani-mals and cadavers) is essential for educating futureveterinarians, I was shocked by the terminal practicalclasses which were then part of the veterinary cur-riculum at my university. In these terminal classes,students practised various procedures (such as blad-der catheterisation, venepuncture and radiography)on living, anaesthetised animals, before euthanisingthem. The animals used were usually healthy pound-animals and ex-racing greyhounds. There was no bodydonation programme for obtaining ethically-sourcedcadavers in place at that time, and no ethical objec-tion policy until 2008. Most difficult for me were theterminal surgery practical classes, in which studentswere required to choose a dog to practise on andlater kill.

The negligible educational benefits of the terminalpractical classes were outweighed by the extrememoral anguish that I felt. What made it even worsewas that the terminal surgery classes bore no com-parison to the high standards later required for pri-vate clients in the university’s veterinary hospital. Interminal surgery classes, cleaning the animal’s skinprior to surgery was not as thorough, heat mats werenot provided, and surgical kits were not sterile.

The ‘resuscitation’ practical class was horrific.Veterinary students repeatedly overdosed and resus-citated live dogs, before cutting the chest open andsqueezing the animal’s heart, in a final, futileattempt to restore a heartbeat in the dying animal.Student attitudes toward the practical classes werediverse. In a previous year, a veterinary student hadenjoyed this class so much that she apparentlyexclaimed afterwards “It was wonderful to hold thedog’s heart in my hand as it died!” Despite the uni-versity having a dog resuscitation mannequin, thiswas not offered to the class.

Animal Use in Veterinary Education — The Need for a FourthR: Respect

Catherine Tiplady

Animals should not be treated with disrespect, merely because they are

surplus to the requirements of others

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As each week passed, and the number of dogs thatdied for the benefit of my education increased, myself-hatred grew. I felt that I would never be able tobalance the lives I would later save as a qualified vet-erinarian with the lives I was now taking as a student.We were told not to discuss the terminal surgeryclasses ‘socially’, and I believe this stifled the will-ingness of other students to voice any ethical con-cerns that they might have had. There is also a riskthat educators who mark students for ‘enthusiasm’during practicals, encourage students to participatein activities that harm animals. Previous studiesdescribe bitterness among students toward those pro-moting alternatives,1 and a “macho bravado culture”in veterinary school.2

The irony of the terminal surgery practical classeswas that, no matter how well or how poorly studentsperformed the surgeries, the outcome for the animalswas inevitable — death at the hands of those suppos-edly being trained to save lives. Such a contradictionwas increasingly hard to bear. I requested aged orsick dogs to use in the surgery practical classes, anda staff member supported me by helping to obtainsuch dogs. Whilst this was slightly easier for me thankilling a healthy puppy or a pregnant bitch, as someof my fellow students had to do, it was still taking alife.

It seemed that we were encouraged to regardthese dogs as somehow deserving to be part of theterminal practicals. One staff member, in defenceof the terminal surgery practicals, stated: “thepound dogs have all done something wrong”. I foundthis statement quite bizarre. If a dog behaves in the‘wrong’ way, should the animal be sentenced to vivi-section and death by lethal injection at the handsof future veterinarians? Does the ‘capital punish-ment’ of pound dogs serve as a deterrent to otherdogs which may dare to perform ‘wrong’ acts? Suchcomments from staff may indoctrinate students tobelieve that pound dogs and ex-racing greyhoundsare mere tools for our education, and, as statedelsewhere, create a reliance on pounds to supplyanimals for teaching.3 Being told of the largenumber of dogs being euthanised at pounds, andthat “They’re going to die anyway”, made it noeasier for me. I have since worked as a veterinarian

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in a pound, but have never felt that pound animalsshould be used for terminal surgery practice to tryand justify their death.

I believe that insufficient attention is paid by edu-cators to the encouragement of respect among veteri-nary students toward animals and people during theveterinary course. I feel that respect is as vital a partof animal use as the other Three Rs. Unfortunately,some veterinary students are disrespectful toward ani-mals and their cadavers. Some students chose to writeobscenities on a cow during an anatomy class, and useda stock-marking crayon to draw ‘lipstick’ on her faceand ridicule her; others filmed each other foolingaround with pieces of a horse cadaver and posted thefilm on the internet. One student laughed at thefemale pound dog which was to be euthanised after aterminal surgery practical class — “Ha ha! Your dog’sgot tits!” Disturbingly, one student even recommendedculling the entire teaching herd of dairy cows as ‘TheFinal Solution’.

These are not incidents isolated to the veterinaryschool that I attended. Colleagues from other veteri-nary schools have had similar experiences. One told mehow a veterinary student joked about raping the rab-bits used in teaching classes, taking photographs ofhimself squeezing the animal’s genitals to display onthe internet.

I believe that veterinary educators must constantlywork to encourage a climate of respect during vet-erinary training, as a matter of urgent priority. Thisrespect must extend toward both animals (live andcadavers) and humans (staff and students) in the vet-

erinary course. It is not acceptable that students withcompassion are silenced, whether deliberately ornot. These are the future veterinarians who can bringabout change. Let’s work to nurture their compas-sion, listen without judgement to their ethical con-cerns, and continue to develop and embrace humanealternatives in veterinary education.

Dr Catherine TipladyCentre for Animal Welfare and EthicsSchool of Veterinary ScienceUniversity of QueenslandGattonQueensland 4343AustraliaE-mail: catherine.tiplady@uqconnect.edu.au

References1 Tiplady, C., Lloyd, S. & Morton, J. (2011). Veterinaryscience students’ preferences for the source of dogcadavers used in anatomy teaching. ATLA 39, 461–469.

2 Woon, S-Y. (2011). A veterinary student’s perspectiveon educational animal use and the potential forhumane alternatives. ALTEX Proceedings, 1/12,Proceedings of WC8, 377–385.

3 Knight, A. (2002). The use of pound dogs in veterinarysurgical training. In Learning Without Killing, a Guideto Conscientious Objection (ed. A. Knight), pp. 19–20.Available at: www.LearningWithoutKilling.info(Accessed 03.09.12).

DISCUSSIONS

It is widely accepted that the Three Rs principles ofreplacement, reduction and refinement should beintroduced into experimental procedures involvinglaboratory animals whenever possible.1 A range ofbehavioural analysis systems are now available, thatcan be used to automate the study of behaviourwithin animals’ homecages. Most of these systemsare designed for studies on mice, which are the mostwidely-used laboratory animals,2 and they are fre-quently marketed as tools that can aid the implemen-tation of the Three Rs. Here, the technologycurrently available to carry out automated homecagebehavioural analysis of mice will be summarised, andthe case that these systems have the potential toboth refine scientific procedures (minimising painand distress) and reduce the number of mice usedexperimentally, will be discussed.

Types of automated homecagebehavioural analysisThe technology that underlies the automatedhomecage behavioural analysis of mice is reviewedin detail elsewhere.3,4 Briefly, the methods of auto-mated homecage behavioural assessment includeautomated video analysis,5,6 radio-frequency identi-fication (RFID) transponders,7,8 infrared beams,9

infrared sensors,10 telemetry,11 and the quantificationof homecage vibration.12 Systems vary in the types ofbehaviours that can be detected, but they all havethe capacity to measure some form of spontaneousbehaviour. Some of the systems only measure activity(e.g. infrared-based systems), whilst others candetect simple behaviours, such as rearing (e.g. auto-mated video analysis and vibration-based systems).Physiological parameters (e.g. heart-rate and bloodpressure) can be measured, in addition to behav-ioural monitoring, by using telemetry, whilst someRFID transponder systems allow operant behaviouraltesting to be carried out.

RefinementAutomated homecage behavioural analysis is fre-quently advocated as a refinement to standardisedbehavioural testing. It is likely that the welfare of themice can be improved by studying animals in theirhomecages, as this minimises the necessity for humanhandling, which may be stressful13 and can act as apotential source of experimental variability. Auto -mated homecage behavioural testing also has manypotential applications for mouse welfare assessment.One of the key limitations to implementing refine-ments to experimental studies involving mice, is thechallenge of objectively identifying pain and/or dis-tress.14 It can therefore be difficult to determine whenrefinements such as changes to husbandry or scientificprocedures, should be introduced, and to then evalu-ate how these refinements affect animal welfare.Examples of how automated homecage technology canbe used for welfare assessment include the use ofautomated video analysis and telemetry to objectivelymeasure post-operative pain and to assess analgesicefficacy,5,11,15 as well as the use of RFID transpondersystems to carry out preference testing.16 Automatedhomecage behavioural analysis systems have also beenused successfully to detect subtle behavioural changesthat correlate to disease progression in studies involv-ing mouse models of chronic disease,17,18 and thiscapacity is likely to be useful in refining disease stud-ies. If subtle behavioural changes can be detected,then further intensive monitoring can be carried outon specific individual animals, in study periods wherepain and/or distress are likely to occur. The detectionof behavioural changes that precede clinical diseasealso has applications in the implementation of appro-priate humane endpoints, increasing the likelihoodthat mice can be killed prior to the onset of pain ordistress, when the scientific objectives of the studyhave been met.

Although there are several ways that automatedhomecage behavioural analysis can potentially refine

Automated Homecage Behavioural Analysis and theImplementation of the Three Rs in Research Involving Mice

Claire A. Richardson

Automated homecage behavioural analysis systemshave the potential to both refine scientific procedures

and reduce the numbers of mice used

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experimental procedures involving mice, there couldbe some negative aspects of automated homecagebehavioural analysis with respect to mouse welfare.The majority of the systems that are currently com-mercially available require that animals be individu-ally housed, and there may also be limitations in thetypes of environmental enrichment that can be used.Implants are not required in many of the systems, butRFID-based systems require the implantation of smalltransponders under a brief general anaesthetic, andtelemetry requires the surgical implantation of largertransmitters. As the animals are less frequently han-dled when automated homecage behavioural analysissystems are used, they are likely to be less acclima-tised to humans. As a result, periods when handlingis required (e.g. for cage cleaning) may be morestressful to them. Finally, while automation may bea very useful way to complement human observationof animals, it is critically important that it neverreplaces the direct assessment and care of animalsby compassionate and experienced personnel.14

ReductionThe main argument that automated homecage behav-ioural analysis can help in reduction of the number ofanimals used experimentally, is that the more-effective

use of animals decreases the overall number of animalsrequired. Through the use of more-sensitive assess-ment techniques, fewer animals may be required toproduce statistically-significant results than has beenpreviously obtained by using conventional techniques(e.g. standardised behavioural testing). Scientifically,there is evidence that the standardisation typically car-ried out for individual behavioural tests may not alwaysbe effective in generating reproducible experimentalstudies between laboratories.19, 20 In contrast, studiescarried out by using an automated homecage systemshow consistency, regardless of the laboratory con-cerned.21 Many automated techniques also permitinvestigators to examine several aspects of behavioursimultaneously, which minimises the necessity to carryout several studies examining different behaviours.

However, there may be ways in which automatedbehavioural analysis promotes the use of mice inresearch, and thus increases the overall number ofanimals used. Many of these automated techniquesare designed to support high-throughput phenotyp-ing, typically involving large numbers of transgenicmice. The generation of genetically modified animalshas generally led to increases in the number of ani-mals used, with a 42% increase in animal proceduresin the UK since 2001 — when technology to geneti-cally modify animals was introduced.22 It is also pos-sible that more animals may be required to validate

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

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new automated assays, as a result of the necessity oftheir comparison with standardised behavioural tech-niques as part of the validation process.

Discussion and conclusionOn balance, there is now increasing evidence thatautomated homecage behavioural analysis can beuseful with respect to the implementation of theThree Rs in research involving mice. However, thereare some potential areas of concern with respect tothe Three Rs that the laboratory animal communityshould continue to evaluate as this technology devel-ops.

Dr Claire A. RichardsonComparative Biology CentreMedical SchoolFramlington PlaceNewcastle UniversityNewcastle upon Tyne NE2 4HHUKE-mail: claire.richardson@ncl.ac.uk

References1 Russell, W.M.S. & Burch, R.L. (1959). The Prin ciples of

Humane Experimental Technique, 238pp. London, UK:Methuen & Co. Ltd.

2 Home Office (2012). Statistics of Scientific Procedureson Living Animals Great Britain 2011, 56pp. London,UK: The Stationery Office.

3 Spruijt, B.M. & DeVisser, L. (2006). Advanced behav-ioural screening: Automated home cage ethology. DrugDiscovery Today: Technologies 3, 231–237.

4 Schaefer, A.T. & Claridge-Chang, A. (2012). The surveil-lance state of behavioral automation. Current Opinionin Neurobiology 22, 170–176.

5 Miller, A.L., Flecknell, P.A., Leach, M.C. & Roughan, J.V.(2011). A comparison of a manual and an automatedbehavioural analysis method for assessing post-opera-tive pain in mice. Applied Animal Behaviour Science131, 138–144.

6 Maroteaux, G., Loos, M., van der Sluis, S., Koop mans,B., Aarts, E., van Gassen, K., Geurts, A., The NeuroBSIKMouse Phenomics Consortium, Largae spada, D.A.,Spruijt, B.M., Stiedl, O., Smit, A.B. & Verhage, M.(2012). High-throughput phenotyping of avoidancelearning in mice discriminates different genotypes andidentifies a novel gene. Genes, Brain & Behavior [Inpress.] doi: 10.1111/j.1601-183X.2012.00820.x.

7 Branchi, I., D’Andrea, I., Cirulli, F., Lipp, H.P. & Alleva,E. (2009). Shaping brain development: Mouse commu-nal nesting blunts adult neuroendocrine and behav-ioural responses to social stress and modifies chronicantidepressant treatment outcome. Psychoneuro -endocrinology 35, 743–751.

8 Winter, Y. & Schaefers, A.T.U. (2011). A sorting systemwith automated gates permits individual operantexperiments with mice from a social home cage.Journal of Neuroscience Methods 196, 276–280.

9 Moretti, P., Bouwknecht, J.A., Teague, R., Paylor, R. &Zoghbi, H.Y. (2005). Abnormalities of social interactionsand home-cage behaviour in a mouse model of RettSyndrome. Human Molecular Genetics 14, 205–220.

10 Dell’Omo, G., Vannoni, E., Vyssotski, A.L., Di Bari,M.A., Nonno, R., Agrimi, U. & Lipp, H.P. (2002). Earlybehavioural changes in mice infected with BSE andscrapie: Automated home cage monitoring revealsprion strain differences. European Journal of Neuro -science 16, 735–742.

11 Goecke, J.C., Awad, H., Lawson, J., Caldwell, J. &Boivin, G.P. (2005). Evaluating postoperative analgesicsin mice using telemetry. Comparative Medicine 55, 37–44.

12 Razzoli, M., Carboni, L., Andreoli, M., Ballottari, A. &Arban, R. (2011). Different susceptibility to socialdefeat stress of BalbC and C57BL6/J mice. BehaviouralBrain Research 216, 100–108.

13 Hurst, J.L. & West, R.S. (2010). Taming anxiety in lab-oratory mice. Nature Methods 7, 825–826.

14 Hawkins, P., Morton, D.B., Burman, O., Denni son, N.,Hon ess, P., Jennings, M., Lane, S., Middleton, V.,Roughan, J.V., Wells, S. & West wood, K. (2011). A guideto defining and implementing protocols for the welfareassessment of laboratory animals: Eleventh report ofthe BVAAWF/FRAME/RSPCA/UFAW Joint Working Groupon Refinement. Laboratory Animals 45, 1–13.

15 Dickinson, A.L., Leach, M.C. & Flecknell, P.A. (2009).The analgesic effects of oral paracetamol in two strainsof mice undergoing vasectomy. Laboratory Animals 43,357–361.

16 Tsai, P.P., Nagelschmidt, N., Kirchner, J., Stelzer, H.D.& Hackbarth, H. (2012). Validation of an automaticsystem (DoubleCage) for detecting the location of ani-mals during preference tests. Laboratory Animals 46,81–84.

17 Rudenko, O., Tkach, V., Berezin, V. & Bock, E. (2009).Detection of early behavioural markers of Huntington’sdisease in R6/2 mice employing an automated socialhome cage. Behavioural Brain Research 203, 188–199.

18 Steele, A.D., Jackson, W.S., King, O.D. & Lindquist, S.(2007). The power of automated high-resolution behav-iour analysis revealed by its application to mousemodels of Huntington’s and prion diseases. Proceedingsof the National Academy of Sciences of the USA 104,1983–1988.

19 Cryan, J.F. & Holmes, A. (2005). The ascent of mouse:Advances in modelling human depression and anxiety.Nature Reviews Drug Discovery 4, 775–790.

20 Richter, S.A., Garner, J.P. & Würbel, H. (2009). Environ -mental standardization: Cure or cause of poor repro-ducibility in animal experiments? Nature Methods 6,257–261.

21 Krackow, S., Vannoni, E., Codita, A., Mohammed, A.,Circ ulli, F., Branchi, I., Alleva, E., Reichelt, A., Will -uweit, A., Voikar, V., Colacicco, G., Wolfer, D.P.,Buschmann, J-U.F., Safi, K. & Lipp, H-P. (2010). Con -sistent behavioural phenotyping differences betweeninbred strains in the IntelliCage. Genes, Brain &Behavior 9, 722–731.

22 NC3Rs (2012). Evaluating progress in the 3Rs: TheNC3Rs framework. London, UK: National Centre for theReplacement, Refinement and Reduction of Animals inResearch. 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 Alternativesin the Primary Surgical Training of Veterinary UndergraduateStudents

Juan Jose Perez-Rivero and Emilio Rendon-Franco

Table-top simulators are valuable alternatives to animal models inhealth 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 inthe training of health sciences students, including vet-erinarians. These simulators range from the simplestand cheapest ones (so called ‘table-top simulators’) tothe 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 usingreadily-available materials in common use, is generallyused on the table (hence its name), and is useful forthe training of isolated procedures that require, as partof their learning process, the coordination of move-ments, such as the formation of sutures and surgicalknots, the creation and maintenance of intravenouscannulae, and the use of surgical instruments.3

Due to their origin, table-top simulators have theadvantage of being economical and portable. Thismeans that each student can have his or her own sim-ulator, can transport it easily, practise with it, andreplace it, whenever necessary.4 The use of a table-topsimulator enhances the acquisition of skills, becausethe students can perform the necessary number of rep-etitions during their training, to become familiar withthe correct use of materials and to improve the proce-dure they are practising.5

At the Universidad Autónoma Metropolitana UnidadXochimilco (UAM-X), third-year veterinary studentswho 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 theirmovements. To facilitate this, the students are trainedby using simulators, the table-top simulator being themost accessible version. The use of these simulatorscontributes to the reinforcement of the Three Rs prin-ciples with regard to the reduction and replacement ofthe use of animals for training. The importance of usingsimulators 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 stu-dents. Before starting the training, an introductory ses-sion must be performed, supported by a multimediapresentation, to explain in detail the surgical proce-dure which is going to be learned. When using the sim-ulator, the training is usually supervised by aninstructor, who will help make the necessary correc-

tions until the student performs the procedure withoutany flaws. This provides the students with better skillsthan if they were only to perform the training withoutassessment.7

The average training time in the surgery classroom isthree hours for each type of simulator (suture orvenipuncture). As the final part of the training, it is nec-essary to evaluate whether the students have acquiredthe expected skills, and the only way of doing this withthis type of simulator is by direct observation of the stu-dent while actually working with the simulator.

The members of the Surgical Teaching Unit of theUAM-X have created and used a range of different table-top simulators, among which the venipuncture simula-tor,4 basic suture simulator, and the advancedsimulators for suturing tubular and parenchymal organs,stand out (Figure 1). In our experience, the use of thesesimulators helps the undergraduate veterinary studentsto improve their skills in managing the materials usedin venipuncture, as well as improving their knowledgeand handling of suture instruments. Moreover, the skillsof the students performing venipunctures, and theplacement of peripheral venous catheters and their cor-rect fixation, are improved. Suture simulators alsoenhance the correct use of different suturing patternsand surgical knots, as are required in the anaesthesiaroom or in the operating theatre.

For surgical training, it is necessary to move forwardin the development of simulators that can satisfy dif-ferent scenarios, e.g. from the viewpoints of physiol-ogy, anaesthesia, bleeding control, and the handling ofdelicate tissues, which are currently only learned inreal surgery.8 If this were to be achieved, then it wouldbecome possible to replace the use of animal modelsin this phase of the training of veterinarians.

Dr Juan Jose Perez-RiveroUniversidad Autónoma Metropolitana UnidadXochimilcoCalzada del Hueso 1100Colonia Villa QuietudDelegación Coyoacán Ciudad de MéxicoC.P. 04960, D.F. MéxicoMéxicoE-mail: jjperez1_1999@yahoo.com

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References1 McGaghie, W.C., Siddall, V.J., Mazmanian, P.E. & Myers,J. (2009). Lessons for continuing medical educationfrom simulation research in undergraduate and gradu-ate medical education: Effectiveness of continuingmedical education: American College of ChestPhysicians Evidence-Based Educational Guidelines.Chest Journal 135, 62S–68S.

2 Parkes, R., Forrest, N. & Baillie, S. (2009). A mixedreality simulator for feline abdominal palpation train-ing in veterinary medicine. Studies in HealthTechnology & 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.; Assoc -iation of Professors of Gynecology and ObstetricsUndergraduate Medical Education Committee (2008).To the point: Medical education review of the role ofsimulators in surgical training. American Journal ofObstetrics & Gynecology 199, 338–343.

4 Perez-Rivero, J.J. & Rendon-Franco, E. (2011). Val -idation of the education potential of a simulator todevelop abilities and skills for the creation and main-tenance 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 compe-tency assessment in medical education. Journal ofGeneral Internal Medicine 23, 46–49.

6 Engum, S.A., Jeffries, P. & Fisher, L. (2003). Intra ven -ous catheter training system: Computer-based educa-tion versus traditional learning methods. AmericanJournal of Surgery 186, 67–74.

7 Govaere, J.L.J., de Kruif, A. & Valcke, M. (2012).Differential impact of unguided versus guided use amultimedia introduction to equine obstetrics in veteri-nary 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 modelfor experimental surgery. Acta Cirúrgica Brasileira 24,325–328.

Figure 1: A range of table-top simulators

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

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a) b) c) e)

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Central nervous system (CNS) diseases have a devastat-ing impact on the quality of life of those affected, withlarge health economic and social costs globally. Thereare no effective treatments for many neurological dis-eases, including almost all viral brain infections. Amajor hurdle in treating CNS disease is transportingtherapeutics across the blood–brain barrier (BBB). TheBBB is formed by the endothelial cells that line cerebralcapillaries, supported by cells of the neurovascular unit(NVU) that induce and maintain the properties of theBBB. The main function of the BBB is to restrict theentry of molecules and pathogens into the CNS andmaintain brain homeostasis, which is essential fornormal neuronal function.1 The BBB is a major challengein drug discovery programmes, as many potential CNSdrugs cannot cross the BBB due to its strict regulationof paracellular and transcellular entry of molecules. Todevelop better treatment strategies, we need toincrease our understanding of BBB function duringhealth and disease.2 Evidence of the role of the BBBduring viral pathogenesis has traditionally come fromstudies on animals, because of the difficulties associ-ated with conducting clinical studies in humans andobtaining human brain tissue for in vitro investigations.There are several good in vitro BBB models derivedfrom animal tissue, but as I have outlined in a Commentto be published in ATLA, there is an urgent need for thedevelopment of realistic human BBB models that canmimic the in vivo characteristics of the BBB.3

BBB research is a comparatively new field, as theexistence of a BBB was only discovered just over 100years ago. Publications on the BBB have steadilyincreased — from just one paper in 1947 to over 1500papers so far this year — as we begin to understand theimportance of the BBB in neurological disease (Figure1). My interest in BBB research, and particularly in thedevelopment of in vitro BBB models, stemmed from dis-covering at the very beginning of my research careerthat only a handful of good in vitro BBB models wereavailable. A major limitation was that most of thesemodels were not robust and simple enough to use in thepharmaceutical industry for drug permeability studies.Furthermore, many pharmaceutical companies used invitro BBB models derived from epithelial tissue (e.g.MDCK cells, Caco-2 cells), rather than from brainendothelial origin for their early-phase CNS drug discov-ery studies.

To address these issues, I established a simple to useand robust in vitro BBB model, derived from porcine

brain endothelial cells that expressed major BBB fea-tures.4 Porcine brain material was used, because it wasa by-product of the meat industry, so the availability ofthis type of brain tissue was not an issue. Furthermore,the anatomy, physiology and genome of the pig, allreflect those of the human more closely than most lab-oratory animal models.5 Therefore, a porcine BBBmodel seemed to be the best alternative to a humanmodel for drug discovery studies.

Having established a porcine BBB model, I movedonto developing an in vitro human model, because ofmy interest in studying the role of the BBB during CNSinfection. Mechanisms of pathogen entry to the brainare poorly understood for many brain infections (e.g.viral encephalitis, bacterial meningitis, parasitic braininfections). I used this static human model to investi-gate how viruses that cause encephalitis cross the BBB.The static human model has given some interestinginsights into the complex interactions between thevirus and the BBB, and the role of inflammatorycytokines in viral pathogenesis (manuscript in prepa-ration). However, replicating all the main features ofthe human BBB in vitro is not an easy task. The mainchallenges are the availability of fresh human braintissue and maintaining BBB morphology and functionin culture, following isolation of the cells.Immortalised human brain endothelial cells are analternative, but these may lack certain important BBBfeatures because of the immortalisation process. Toovercome these challenges, I am now attempting toestablish a flow-based three-dimensional (3-D) BBBmodel for studying the role of the BBB in brain infec-tions (funded by an NC3Rs David Sainsburyfellowship).6 A flow-based model is preferred to astatic model, as the shear stress caused by blood flowis important in maintaining the BBB features of brainendothelial cells, and will also provide the right envi-ronment for leukocyte transmigration across theendothelium, which is an important physiologicalprocess during infection. A realistic human BBB modelwill help increase our understanding of the importanceof the BBB in protecting the brain during infection, aswell as the damage caused to the BBB during thecourse of the infection. Furthermore, this flow-based3-D human BBB model will potentially lead to the gen-eration of physiologically-relevant human data and theidentification of targets for developing novel thera-peutics. If successful, it will be a useful alternative tolaboratory animals for studying the BBB in CNS disease.

Toward a Humanised Alternative to the Use of LaboratoryAnimals 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

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Dr Adjanie PatabendigeUniversity of LiverpoolBrain Infections GroupInstitute of Infection & Global HealthThe Apex Building8 West Derby StreetLiverpool L69 7BE, UKE-mail: adjanie@liverpool.ac.uk

References1 Abbott, N.J., Patabendige, A.A.K., Dolman, D.E.M.,Yusof, S.R. & Begley, D.J. (2010). Structure and functionof the blood–brain barrier. Neurobiology of Disease 37,13–25.

2 Hawkins, B.T. & Davis, T.P. (2005). The blood–brain bar-rier/neurovascular unit in health and disease.Pharmacological Reviews 57, 173–185.

3 Patabendige, A. (2012). The value of in vitro models ofthe 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–brainbarrier model with high transendothelial electrical resist-ance. 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 theNew 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: Anovel tool for studies of drug delivery to the brain.Pharmaceutical Science & Technology Today 2, 7–12.

Figure 1: Blood–brain barrier research papers published, by year1947

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Bill Russell was extremely gratified when Michael Balls, among others, rediscoveredThe Principles of Humane Experimental Technique, decades after it was published,not for reasons of personal vanity, but because it gave him hope that the rigorousscience that it contained might yet be fully integrated into laboratory science,resulting in more-humane, and better, research. PiLAS is a further, and muchneeded step along that road. Bill would be pleased.

Cleo Paskal Literary Executor of Bill and Claire Russell

The issues raised by animal experimentation are complex and emotive, yet it iscrucial to consider and debate these, if we are to find alternative methods andreduce the need for laboratory animal procedures. PiLAS will provide anexcellent 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 lookforward to reading about the important topics it deals with.

Professor David GreenawayVice Chancellor, University of Nottingham

As Chair of the All-Party Group on Replacement of Animals in Experiment ation,I am delighted to welcome the launch of PiLAS. This will create a significantnew forum for professionals from all disciplines to share their expertise andideas about how to further improve the quality of experiments to the benefitof both animals and humans. I anticipate that the quality of discussion itgenerates will be very high indeed. It is likely to make an exciting contributionto a subject of great interest to the general public, as well as to the scientificcommunity.

Nic Dakin MPHouse of Commons

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 inlaboratory experimentation have not been adequately addressed. Although I amnot qualified to speak in depth about the need for the use of animals as modelsfor human beings, I have discussed this with many people who are, and I haveread extensively about the subject. And it seems conclusive that animals cannever be really good models for human beings, since they are anatomically,physiologically and behaviourally adapted to ways of life very different from ourown. 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 ofanimals is becoming increasingly unnecessary. And as we learn more about thecapacity of animals to suffer — physically and psychologically — increasinglyunethical. Almost never can the basic needs of laboratory animals be adequatelyaddressed. In the 21st century, it is imperative that we move away from the useand abuse of animals for this purpose.

Jane Goodall PhD DBE Founder of the Jane Goodall Institute; UN Messenger of Peace

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The following words of support for PiLAS have been received:

THE WISDOM OF RUSSELL AND BURCH

The concepts expounded by W.M.S. Russell and R.L.Burch in the 1950s in their outstanding book, ThePrinciples of Humane Experimental Technique,1 arenow the basis of many national and internationallaws and regulations on the proper use of labora-tory animals. They were the outcome of a projectproposed by Charles Hume, the founder of theUniversities Federation for Animal Welfare.2

Their underlying philosophy concerned the con-cept of inhumanity, which they saw as “an objec-tive assessment of the effects of any procedures onthe animal subject”, without implying “any criti-cism or even psychological description of personspractising any given procedure”. They judged thecentral problem to be “that of determining what isand what is not humane, and how humanity can bepromoted without prejudice to scientific and med-ical aims”.

Chapter 2 of The Principles comprises a brilliant dis-cussion on inhumanity, which considers pain and fear,and the “rather more general notion of distress”,

taking into account the different levels of conscious-ness and intelligence in animals in relation to our con-cern for their welfare. It introduces the provocativethought that “inhumane procedures are those whichdrive the animal’s mood down. Removing inhumanitymust ultimately mean driving the animal as near theother end of the scale as we can. More humane meansless inhumane.”

However, it is Chapter 4, on The Sources, Incidenceand Removal of Inhumanity, which is of vital signifi-cance. It begins with a discussion of the importantdistinction between direct inhumanity, “the inflic-tion of distress as an unavoidable consequence of theprocedure employed”, and contingent inhumanity,“the infliction of distress as an incidental and inad-vertent by-product of the use of the procedure,which is not necessary for its success”.

Russell and Burch emphasised that contingent inhu-manity is almost always detrimental to the achieve-ment of the objective of an experiment, but thatmuch could be done to avoid it. Direct inhumanity,being unavoidable, is a totally different matter, andthey discussed it in terms of incidence (e.g. in controland experimental groups), severity (e.g. the severityof a procedure in those animals that are affected),and special character (e.g. post-operative pain anddistress, effects of particular pathogens, or deathdue to various types of toxic chemical).

They saw the avoidance of contingent inhumanityas mainly a matter of good husbandry, diligent careand common sense, but their priceless gift, of equalvalue to biomedical science and animal welfare, wasoffered when they said: “We turn now to considera-tion of the ways in which [direct] inhumanity can beand is being diminished or removed. These ways canbe discussed under the three broad headings ofReplacement, Reduction, and Refinement, [which]have conveniently been referred to as the Three Rsof humane technique.”3

Russell and Burch’s definitions and discussions onthe Three Rs will be considered in future issues ofPiLAS, but let us close this short introduction withthe words of Article 4: Principles of replacement,reduction and refinement, in Directive 2010/63/EUon the protection of animals used for scientific pur-poses,4 the provisions of which will come into forcein the Member States of the European Union inJanuary 2013:

1. The Concept, Sources and Incidence of Inhumanity andits Diminution or Removal Through Implementation ofthe Three Rs

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

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“1. Member States shall ensure that, wherever pos-sible, a scientifically satisfactory method or test-ing strategy, not entailing the use of live animals,shall be used instead of a procedure.

“2. Member States shall ensure that the number ofanimals used in projects is reduced to a mini-mum without compromising the objectives of theproject.

“3. Member States shall ensure refinement of breed-ing, accommodation and care, and of methodsused in procedures, eliminating or reducing tothe minimum any possible pain, suffering, dis-tress or lasting harm to the animals.”

All those who are in any way responsible for activ-ities related to this Directive or the national lawsand regulations of the Member States which are inaccordance with it, have a legal duty and a moralobligation to act according to these principles.

References and Notes1 Russell, W.M.S. & Burch, R.L. (1959). The Principles ofHumane Experimental Technique. xiv + 238 pp.London, UK: Methuen.

2 Balls, M. (2009). The origins and early development ofthe Three Rs concept. ATLA 37, 255–265.

3 Russell, W.M.S. (1957). The increase of humanity inexperimentation: Replacement, Reduction andRefinement. Collected Papers of the LaboratoryAnimals Bureau 6, 23–25.

4 Anon. (2010). Directive 2010/63/EU of the EuropeanParliament and of the Council of 22 September 2010on the protection of animals used for scientific pur-poses. Official Journal of the European Union L276,20.10.2010, 33–79.

The Principles of Humane Experimental Technique isnow out of print, but the full text can be found athttp://altweb.jhsph.edu/pubs/books/humane_exp/het-toc. An abridged version, The Three Rs and theHumanity Criterion, by Michael Balls (2009) can beobtained from FRAME.

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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 BurchHouse, 96–98 North Sherwood Street, Nottingham NG1 4EE, UK. Instructions to Authors are availablefrom the above, or from the PiLAS website, www.atla.org.uk. All articles considered for publicationwill be peer-reviewed.

Some possible topics for consideration in future issues are:v the value of models and their uses

v the planning of experiments

v the analysis of scientific data

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

v the breeding, supply and transport of laboratory animals

v the re-use of animals

v re-homing

v the humane killing of animals

v the rodent bioassay for carcinogenicity

v reproductive toxicity tests

v animal experimentation for the benefit of animals

v the importance of species differences

v whether the use of humane endpoints is always humane

v who actually are the vets’ clients?

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

v do some animals matter more than others?

v should there be limits on genetic modification?

v is it acceptable to humanise animals?

ISSN 0261 1929 ©2012 FRAME FRAME Charity Registration Number 259464