Pain and distress in laboratory rodents and lagomorphs: Report of the Federation of European...

Pain and distress in laboratory rodents andlagomorphsReport of the Federation of European Laboratory Animal ScienceAssociations (FELASA) Working Group on Pain and Distressaccepted by the FELASABoard of Management November 1992

FELASAWorking Group on Pain and Distress:V. Baumans (NVP) Convenor;P. F. Brain (LASA) Secretary; H. Brugere (SFEA); P. Clausing (GV-SOLAS);T. Jeneskog (Scand-LAS) and G. Perretta (AI SAL)FELASA, BCM Box 2989, London WC1N 3XX, UK

ContentsIntroductionSection ISection ITSection illSection IVSection VSection VISection VITSection VillSection IXConclusionsReferences

Definitions of Pain, Distress and SufferingMechanisms of PainMeasurement of Analgesia and Environmentally-Induced AnalgesiasSensitivity of Tissues and Organs to PainEffects of Pain and DistressLegal ObligationsSources of Pain and DistressSigns of Pain and DistressGrading of Severity of Pain and Distress in Animals

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Introduction

The Working Group considered the natureof pain and distress in laboratory rodentsand lagomorphs because they constitutethe vast majority of subjects used inexperimentation. In order to keep thedocument as practical and as easy to readas possible, the minimum number ofreferences are given in the text. Anextended bibliography of backgroundmaterial consulted by the Working Groupis available from Dr V. Baumans,Rijksuniversiteit Utrecht, BureauProefdierdeskundige, PO Box 80.166, 3508TD Utrecht, Netherlands. An attempt hasbeen made to be consistent when usingdefinitions of the sometimes vague terms

that abound in this subject area. Theinformation is arranged in sections,although these are not always mutuallyexclusive.

There is an inherent humanitarian desireto reduce pain and distress in laboratoryanimals to an absolute minimum, an aimwhich is recognized in European andnational legislation. For example, theofficial guidance (Home Office 1990)attached to the UK Animals (ScientificProcedures) Act 1986 requires licenceholders 'to minimise any pain, suffering ordistress'. One must, however, point outthat pain and distress mechanisms areessentially devices for removing animalsfrom potential sources of tissue damageor mortality: animals lacking such

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mechanisms do not survive long in nature.Further, our desire to eliminate stimuli orsituations producing pain or distress, couldeasily lead to deprivation, self-mutilationand environmental sterility (a lack of'enrichment') if taken to extremes (Brain1992, Dantzer 1991).

I. Definitions of pain, distress andsufferingPain, distress and suffering are termsbasically describing states of the humanmind-human perceptions or experiences.It is difficult to transfer the definitions of'mind' states to comparable states oflaboratory animals. Researchers must,however, be familiar with the difficultconcepts of pain, distress and suffering, andknow how to recognize, assess, controland, preferably, to prevent this experiencein their animals. This topic has also beendiscussed at length e.g. in Morton (1990).There is so far no consensus on definingthese terms but, for the purposes of thisdocument, the following definitions will beused.

PainThe working definition of pain publishedby the International Association for theStudy of Pain (1979) is 'Pain is anunpleasant sensory and emotionalexperience associated with actual orpotential damage or described in terms ofsuch damage.' This and other definitions ofpain emphasize that it is an experience.Physiology and psychology suggest that thisrequires that a perception is evoked, inturn implying that the animal is conscious,with a functioning (alert) cerebral cortex.This seems true at least for mammals. It isalso important that, when judging that ananimal is in pain, the animal shows a painresponse by some changes in behaviour(section Vill).

The definition of pain used here islimited to what is termed physical ornociceptive pain. Thus another importantfeature is that pain is the perception orexperience of nociceptive stimuli, Le.stimuli of a magnitude capable of causing

FELASA Working Group on Pain and Distress

or threatening to cause injury or tissuedamage.

DistressDistress is defined in the Guidelines forthe Recognition and Assessment of Pain inAnimals (UFAW1989J, as a state wherethe animal has to devote substantial effortor resources to the adaptive response tochallenges emanating from theenvironmental situation, a definition verysimilar to that of emotional or mental pain(Spinelli &. Markowitz 1987), perhaps onlyreflecting differences in English andAmerican terminology. Stimuli potentiallyleading to distress are thus more or lessextreme values or levels of the variousfactors constituting the animal'senvironment. This includes also thebehaviour of researchers and technical staffto the animals in their care.

In this document, states that have beentermed anxiety, frustration or depressionare included within the definition ofdistress, as well as discomfort which islooked upon as a mild form of distress.

In attempting to assess the level ofdistress as mild, moderate or severe, it isimportant to realize that certain conditionsmight be more or less distressful to specificanimals depending upon their opportunityand ability to cope with the situation. Thebetter their opportunity and capacity tocope, the less severe the distress.

SufferingSuffering is a specific state of 'mind',which is not identical to, but might be aconsequence of, pain or distress. Physicalpain or distress may result in suffering ifthey are of sufficient intensity or duration,or both. The greater the intensity, the lesstime is needed for pain or distress to leadto suffering. Suffering is reached when painor distress is no longer tolerable to theindividual animal. Physical pain has thenreached a level beyond the pain tolerancethreshold, or distress has passed the levelthat the animal is able to cope with.Detrimental effects including retardedgrowth, impaired breeding and inadequatebody care are obvious at this stage. Clearly,

measures to prevent suffering includekeeping any possible pain or distress tolevels which the animal can tolerate orcope with.

II. Mechanisms of pain

PeripheryNociceptive (pain-related) signals aregenerated in sense organs callednociceptors, in the skin connected to thin,myelinated Ac5-fibres(conduction velocity:4-30 m/ s) or to unmyelinated C-fibres(conduction velocity: 0.4-2m/s). Theexistence of two sorts of afferent fibres(carrying information towards the centralnervous system) may explain why briefcutaneous nociceptive stimuli are able toproduce a double pain sensation. The firstor fast pain is a well-localized, distinctpricking sensation evoked by activation ofnociceptors connected to Ac5-fibres.Thesecond or slow pain is a burning, morediffuse sensation ('ache') evoked by activityin C-fibres. C-fibres constitute the majorityof cutaneous nociceptive and almost allvisceral nociceptive afferents (Besson &Chaouch 1987, Campbell et al. 1989,Kitchell & Guinan 1990, Jeneskog 1991).

Several endogenous substances appearinvolved in pain sensations associated withtissue damage (traumatic or inflammatory),including potassium (K+ I and hydrogen(H+) ions and bradykinin released byinjured cells, histamine and serotoninreleased from degranulated mast cells, andserotonin also from aggregation ofthrombocytes. Some prostaglandins andleukotrienes are produced duringinflammation and sensitize the nociceptorsleading to lower threshold for theiractivation. The Ac5-and C-fibres alsocontain neuropeptides, such as substanceP, Calcitonin Gene-Related Peptide andneurokinin A, which are released attheir central terminals within thedorsal horn of the spinal cord as wellas at their peripheral terminals. Thesesubstances are implicated in 'neurogenicinflammation' through so-called axonreflexes.

Dorsal horn

The vast majority of fine afferent nervefibres enter the spinal cord through thedorsal roots and split into ascending anddescending branches which may run forsome segments in a tract beforeterminating in the grey substance of thespinal cord's dorsal horn. Two main typesof substances released from the nerveendings probably act as neurotransmittersand neuromodulatorsi these are excitatoryamino-acids (e.g. glutamate) and peptides(substance P and several others). Two mainclasses of dorsal horn relay cells aredirectly or indirectly activated bynociceptive inputs, 'nociceptive specific'(NS) neurones, activated only bynociceptive stimuli, and 'wide dynamicrange' (WDR)or 'convergent' neurones,activated, to some extent also by non-nociceptive stimuli.

As soon as it enters the central nervoussystem, the nociceptive message is subjectto a variety of control mechanisms. Theseinclude segmental modulation (suppression)via activity in thick, myelinated skinfibres, and descending modulation throughcontrol systems of supraspinal origin,notably from the brain stem'speriaqueductal grey substance. Another,seemingly global, system where onenociceptive input may inhibit another(even a remote nociceptive input) viasegmental as well as supraspinalcomponents is termed Diffuse NoxiousInhibitory Controls rONIC).

The dorsal horn is rich in opioidreceptors and their endogenous ligands,notably those derived from pro-enkephalinA and pro-dynorphin. The analgesic (pain-relieving) effects elicited by segmental aswell as descending modulatory systems andalso by administration of opioids at leastpartly depend on activation of such spinalreceptors.

The axons [long nerve fibres) of the NSand WOR cells ascend, mainly contra-laterally, through the spinal cord. Althoughmany interspecies differences exist, datarelated to the rat, the monkey and manappear to be to some extent homogeneous.

The main pathways are the spino-thalamicand the spino-reticular tracts. Otherpathways, studied in animals, include thespino-(ponto)-mesencephalic, spino-solitarius, spino-cervical (Morin's) tractsand post-synaptic fibres in the dorsalcolumns. The nociceptive messages thusreach the brain through a multiplicity ofpathways. The neo-spino-thalamicpathways, which terminate in the lateralthalamus, preserve a reasonable amount ofsomatotopy (localization). Signals aretransmitted from the thalamus to thesomatosensory cortex in the parietal lobes,where the sensory-discriminativedimension of pain is probably elaborated orat least initiated. Both the palaeo-spino-thalamic and the spino-reticulo-thalamicpathways terminate in the medialthalamusj somatotopy is no longerpreserved in these systems.

The message eventually reaches wideareas of the frontal cerebral cortex andcortical and subcortical parts of the limbicsystem. The limbic system and the frontalcortical areas are implicated in themotivational-affective (emotional)dimension of all kinds of sensoryinformation, including nociceptiveinformation (Willis 1989, Willis &Coggeshall 1991).

Reticular formationThe medial ascending system of pathwayslargely connects the dorsal horn tomedullary centres in the reticularformation regulating circulation andrespiration. Consequently, autonomous[sympathetic) reflexes are elicited bynociceptive stimuli, such as increased heartrate and blood pressure, altered respiration,pupil dilation, and inhibition ofgastrointestinal motility. Theseconnections, furthermore, possibly activatethe previously-mentioned descendingmodulatory systems.

Parts of the reticular formation arecomponents of the so-called Iascendingreticular activating system' which istonically active, being excited by differentforms of ascending sensory (includingnociceptive) information, and is

indispensible for keeping the cerebralcortex alert. Nociceptive information seemsparticularly effective in arousing [alerting)the cerebral cortex. An aroused, alertcerebral cortex is the basis forconsciousness and thus for perceptions-among them pain - to be produced.

Cerebral cortexAs mentioned earlier, the cerebral cortex isindispensible for all kinds of perceptions,including pain (Zieglgansberger 1986).However, as a result of clinicalobservations in man, the role of the cortexin pain has been the subject of debate.First, during neurosurgical operations onpatients who had not previously presentedwith deafferentation pain, pain was veryrarely evoked by cortical stimulation}although it is very easy to evoke othersomaesthetic sensations in this way.Second, most attempts (many years ago) toalleviate chronic pain by cortical ablationhave failed. Interestingly, the use ofmagnetic resonance imaging and positronemission tomography have recentlydemonstrated that painful stimuli activatethe contralateral sensory and, above all,anterior cingulate cortices (Jones et al.1991, Talbot et al. 1991, Kenshalo & Willis1991).

Several different methods have been usedto help people with chronic painconditions, where conventional treatmentshave failed. These methods includeprefrontal lobotomy and, more recently,cingulotomy. An interesting feature ofusing these kinds of lesions to treat achronic pain condition is that patientsreport that they still feel pain but that itdoes not bother them, i.e. they no longersuffer from the pain. This indicates thatsuch surgical interventions may separatethe two dimensions of pain and that themotivational-affective component isdependent upon intact connectionsbetween the frontal cortex and underlyingparts of the forebrain. It is, however, veryuncertain if and how we can transfer thisknowledge from human physiology toconditions in animals. The frontal lobes ofthe cerebral cortex are the parts which

have most conspicuously increased in sizeand development during vertebrateevolution. If the capacity for amotivational-affective interpretation of anociceptive message increases with the sizeof these cortical areas, this ability wouldbe highly developed in humans (and apes).However, all mammals may be assumed toperceive and experience pain, and further-more remember situations associated withthis sensation. This should be our basis forappropriate action, even if in mammalssuch as rodents and lagomorphs thecapacity for making advanced interpreta-tions of a pain situation are likely to beinferior to our own.

III. Measurement of analgesia andenvironmentally-induced analgesias

Many assays have been used in rodents totest the efficacy of putative analgesiccompounds. These include: hot-water tailimmersion (Janssen 1963); acetic acidwrithing (Koster et al. 1959)j exposure tounavoidable electroshock using ascendingand descending current values; pressuremethods (use of forceps or artery clamps);intra-plantar injection of yeast orcarageenin (Taber 1974)j radiant-heat tailflick (D'Amour & Smith 1941); and thehot-plate test (Woolfe &. MacDonald 1944).Some of these methods are severe, difficultto control and of very limited utility whenassessing the impact of subtle changes inhousing or scientific procedures involvinganimals. The last two are most likely to beuseful in welfare research as they areparticularly sensitive to opiates, easy toperform, require simple apparatus and havea well-defined end-point.

In the tail-flick assay, pain sensitivity isdetermined by focusing radiant-heat froma strong light bulb on to the rodent's tailtip until there is a reflexive flick awayfrom the heat source or the appearance ofsmall white blisters. Semi-automateddevices are available. In the hot-plate test,individual animals are tested for nociceptiveresponses on a hot-plate maintained at55°C, a temperature which althoughuncomfortable will not cause serious

damage. The end-points described areforepaw licking (animal sitting on hind legslicking forepaws in a washing action), hindpaw lick (head orientated to hind paw withventral surface angled upwards) and escapejumping (animal jumping upwards withboth hind paws away from the hot-platesurface) .

Using such tests, it has been possible todemonstrate that a wide range ofenvironmental factors (some associatedwith animal husbandry or laboratoryprocedures) can influence pain sensitivity.This is of crucial importance in evaluatingthe impact of procedures on pain anddistress. Amongst the diverse items shownto produce clear analgesias in rodentspecies (see Rodgers & Randall 19871 areacupuncture, anxiety, brain stimulation,body pinch, centrifugal rotation, classicalconditioning, copulation (males), electricfootshock, exercise, electroconvulsiveshock, food deprivation, forced swim, heatexposure, hypertonic saline stimulation,insulin, irradiation, novelty, opiates,pregnancy Iparturition, presence of apredator, restraint, social conflict, socialisolation, stress odours, tail pinch, tailshock, territorial scent marking, trans-cutaneous nerve stimulation and vaginalstimulation. Some analgesias involve endo-genous opioids while others are not opioiddependent. Some are controlled by neuralfactors and others by hormones, so thatthere are many different forms of analgesia.In spite of the considerable technicaldifficulties mentioned earlier, their analysisseems essential in order to be able tospecify and control pain levels associatedwith husbandry or with experimentalprocedures. The basic point which must benoted is that a wide range of experienceshave dramatic influences on analgesio-metric tests, and the possibility exists thatthey change an animal's perception ofpain.

IV. Sensitivity of tissues and organsto pain

Sensitivity of particular tissues and organsdepends on the innervation of tissues

[types of receptors, sensitivity to stimuli,density, size ,of the receptive field); thecharacteristics of the stimulus and possiblesensitization at receptor level resultingfrom pathological tissue reactions (such asinflammation and ischaemia). Somevariation of sensitivity can also occurbecause of processes affecting conductivityin nerves, such as nervous systemmaturation in the young animal, oralterations of nerves and of their myelinsheaths in metabolic diseases.

Information about tissue sensitivity canbe obtained from clinical observations andfrom surgical experience in man andanimals. More accurate knowledge can beexpected from histological and electro-physiological studies of nociceptors, butthere is still a gap between theexperimental approach and the clinicaldata.

SkinCutaneous nociceptors include receptorsactivated by mechanical (e.g. pressureLthermal, and some chemical influences(Besson et 01. 1986, Raja et 01. 1988). Thesemechano-heat nociceptors, constitute twodifferent groups, associated respectivelywith Ab and C fibres [p 5). The C fibremechano-heat nociceptors are alsostimulated by some chemicals and canshow a sensitization phenomenon (theirthreshold is decreased when they areexposed to mediators of inflammation orother substances). Another type ofnociceptor, the 'high thresholdmechanoreceptor' is activated only byintense mechanical stimuli. Painsensations arising from skin (superficialpain), which can be of high intensity, arediverse and provide accurate localization ofthe stimulation.

MusclesMuscles are rarely very sensitive. Themajority of receptors are mechanoreceptorsand 75% originate from blood vessels,tendons and connective tissue (Raja et 01.1988). More than half of muscle receptorsare stimulated by intravascular injection ofpain-producing substances. Sensitivity of

muscle receptors is increased by local ,inflammatory processes, and especially byischaemia induced by sustained toniccontractions ('cramps').

Joints and bonesJoints and bones are normally relativelyinsensitive. Their receptors are activatedonly in inflammatory or degenerativepathological processes. Bones are sensitiveto injuries particularly of their coating (theperiosteum) which accounts for the sharppains occurring after fractures or aftersurgical section. If, however, receptors ofthe periosteum are destroyed by thepathological process, pain sensitivity maybe negligible (Crane 1987).

Teeth and corneaTeeth and the cornea of the eye areamongst the most sensitive tissues. Thedensity of nerve endings in dental pulp is20 to 40 times greater than in skin whilethe cornea contains a still higher density ofreceptors (300-600 times that of skin) (Rajaet a1. 1988). The majority of corneal nerveendings [70%) are only sensitive tomechanical stimuli, others (17%) areactivated only by cold and the remainderare activated by both.

VisceraViscera are less sensitive to pain than isthe skin, explaining the clear-cutdistinction between 'superficial' and'visceral' pain. Parenchymatous organs [e.g.liver and kidney) do not produce painexcept in the case of pathological injuriesor inflammation. Hollow viscera (e.g. thedigestive and urinary tractsl give rise topainful sensations only if a mechanicalstimulus produced by distension or spasmsoccurs simultaneously with ischaemia. Theexistence of specific visceral nociceptors isnot fully established: pain in the visceracan also result from paroxysmalstimulation of receptors and nervesinvolved in other functions, for instanceregulating motility or producing vascularadjustments. Pain from viscera poorlydiscriminates type of stimulus and lacksprecise localization. Referred pain is

FElASA Working Group on Pain and Distress

frequently encountered with visceralinjury. True nociceptors have beendiscovered only in two viscera: testis andgall bladder (Besson et al. 1986) areas,known to produce very sharp pain in man.In the bodily cavities (e.g. abdomen andchest), serous membranes (peritoneum andpleura) are considered sites of great sensi-tivity to pain, a feature which becomesobvious with injury (wounding or surgery)or inflammation.

Nervous tissueNervous tissue varies in its sensitivity topain. The stimulation of peripheralreceptors and of nerves (including A-a andC afferents) induces a sharp pain sensationvia the spinal cord. Stimulation of spinalcord dorsal columns elicits painful feelingsas if to an electric discharge. In contrast,stimulation of encephalic (brain) tissuedoes not produce any pain, and one canperform stereotaxic surgery on conscioushuman patients.

General commentsA classification of tissues and organs interms of a decreasing sensitivity can begenerated: cornea, dental pulp, testis,nerves, spinal cord, skin, serous mem-branes, periosteum and blood vessels,viscera, joints, bones, and encephalictissue. Although such a classification mayseem useful, it is unrealistic becausesensitivities can be greatly modified bypathological processes or experimentalprocedures. For these reasons, it is necessaryto consider all data provided by practicalexperience. For instance, it is known thatthoracotomy (thoracic surgery) is more pain-ful in quadrupeds if performed by cuttingthe sternum than if an incision is made inmuscles between two ribs (Haskins 1987,Johnson 1991, Sackman 1991). It is moreimportant to evaluate overall severity ofindividual experimental procedures than toclassify tissue sensitivity.

V. Effects of pain and distress

The affective-motivational (mood-related)and cognitive-evaluative (thought-related)

dimensions of pain are of particular interestin assessing its effects on body function.Affective-motivational processes are relatedto release of neurotransmitters andultimately adrenocortical and adreno-medullary hormones as well as endogenousopiates. The result may be stress oranalgesia. Cognitive-evaluation processesmay modulate these neuroendocrineresponses, thereby altering pain detectionthresholds. Little has been published onthe effects of pain on physiologicalfunctions, but there is a substantialliterature on the endocrine correlates ofstress. Thus, pain is generally inferred fromthe activation of autonomic (= stresslresponses (Manser 1992).

If experimenters cannot avoid usingpotentially painful or stressful procedures,they should be aware of the effects of stresson cardiovascular, respiratory, gastro-intestinal and other functions. There isextensive evidence that different qualities(e.g. physical versus psychosocial) anddurations (acute versus chronic) of stressmay have variable influences (Adams et al.1987, Cabib et ai. 1988, Melia & Duman1991). Moreover, the impact of thesevariables on the stress response may bemodified by genetic background (Marek etal. 1991) or physiological state e.g.pregnancy [Pascoe et al. 1991). Such factorsalso influence pain sensitivity (Zamir et al.1980, Gintzler & Bohan 1990) and, it mustbe assumed, alter effects of pain on bodyfunctions. Virtually contradictory outcomesof procedures in terms of pain and distressare therefore unsurprising. For instance,increased rates of major infections inindividuals have been noted in patientssubjected to pain (Benedetti 1990), perhapsdue to the long-known immunosuppressiveeffects of corticosteroids (Berczi 1986). Thiscontrasts with observations that pain(Fujiwara & Orita 1987) and stress [Jessopet al. 1987) may result in immuno-enhancement. All these processes are time-dependent, a feature exemplified bycontrasting effects of acute versus chronicstress [Cabib et al. 1988), as well as bystudies showing that stress responses aredynamic events (Reznick 1989). Moreover,

104 FELASA Working Group on Pain and Distress

Table 1 References to terms included in national legislations on the use of experimental animals by Europeancountries*

Pain Suffering Distress Grading of severity Cost-benefit analysis

Austria Yes Yes Yes No YesBelgium Yes No No No NoDenmark Yes Yes Yes No NoFinland Yes Yes Yes Yes NoFrance Yes Yes No No NoGermany Yes Yes Yes No YesIreland** Yes No No No NoItaly Yes Yes No No YesLuxembourg*** Yes Yes No No NoNetherlands Yes Yes Yes Yes YesNorway**** Yes Yes Yes No NoSpain Yes Yes Yes No YesSweden No Yes Yes Yes YesSwitzerland Yes Yes Yes No YesUnited Kingdom Yes Yes Yes Yes Yes

*Greece & Portugal have legislation in preparation**Uses 1876 UK legislation***Uses the general law on protecting animals****The 1974 law is under revision

functional responses to pain and distresscan be conditioned (Siegfriedet al. 1984). Ifpain or stress is repeatedly matched withenvironmental cues, the functionalresponse [analgesia or corticosteronereleasel may finally become elicited bypresenting the environmental (orexperimental) feature alone. In addition,putative antinociceptive neurotransmittersaffect other systems e.g. endorphinsinfluence immunological factors (Johnson& Torres 1988) and substance Palterscardiovascular function (Urbanski et al.1989).

Effects of impairment of muscularactivity in the pain-affected area, must alsobe taken into consideration. Otherfunctions may suffer secondaryimpairment, depending on the muscularregion affected [e.g. motor behaviour orrespiration). Finally, recent investigationson post-operative pain in man show thatmorbidity and mortality are drasticallyreduced when proper postoperative anal-gesia is provided (Benedetti 1990).

In summary, it is difficult to predict thedetailed effects of pain and distress inindividuals or in specific experimentalsituations. One must consider individual

cases. Pain and distress generally increasevariability in experimental results, becauseof the various neurotransmitter andhormonal responses they elicit. Conse-quently, an animal in pain or distress is apoor research subject, except when painitself is investigated. This practical featuremust reinforce the ethical reasons forminimizing such conditions inexperimentation.

VI. legal obligations

A survey of national legislations ofEuropean countries on the protectionof animals used for experimental purposesreveals that all texts include at least one ofthe terms 'pain', 'distress' or 'suffering'ITable 1). All European countries are,however, signatories to EuropeanCommunities Directive 86/609 IEEC or theCouncil of Europe European Convention forthe protection of vertebrate animals usedfor experimental and other purposes (1986).Both the Directive and the Convention useall three terms. Projects are generallyrequired to avoid or minimize suchexperiences, consistent with the researchaim. Alternatives to procedures that might

produce pain, suffering or distress must beconsidered and adopted whenever appro-priate to the purposes of the investigation.Experiments should also be performed inspecies with the lowest degree of neuro-logical development consistent with theprocedure's aim.

Few of the legal texts surveyed requiredassessment and grading of severity ofexperimental procedures. Where it isrequired, assessment has to reflect thepotential adverse effects the animals mightexperience. Examples of severity ofdifferent procedures are given in theguidance (Home Office 1990) to the UKAnimals (Scientific Procedures) Act 1986.Collecting small blood samples, skin testswith substances expected to be only mildlyirritant, conventional minor surgicalprocedures under anaesthesia such aslaparoscopy, small superficial tissuebiopsies or cannulation of principal bloodvessels are considered mild unless they arerepeated or combined in the same animal.Procedures described as moderate include:screening and developing of potentialpharmaceutical agents, toxicity testsavoiding lethal endpoints, and mostsurgical procedures followed by post-operative analgesia and treatment.Substantial severity is assumed to beinvolved in procedures such as acutetoxicity tests with significant morbidity ordeath as an endpoint, some efficacy tests ofantimicrobial agents and vaccines, somemodels of disease and major surgeryresulting in severe post-operative suffering.

Another requirement included in mostEuropean and national laws is thatappropriate analgesia, sedation or anaes-thesia have to be used in procedurescausing actual or potential suffering toanimals. In addition, some laws require acost-benefit analysis in which painful anddistressful procedures have to be balancedagainst expected positive results of thestudy.

VII. Sources of pain and distress

Various features of the operations of animalfacilities can give rise to pain and distress.

Some are obvious; others are less evidentto all workers.

TransportUsing measures of mortality, condition,behaviour and endocrine changes it is clearthat transport to animal houses fromsuppliers can be a significant source ofstressful experiences. Obviously, factorssuch as cage and vehicle design, provisionof food and water, time involved, care withhandling and exposure to fumes, differenttemperatures or noises determine thedegree to which transport constitutes aproblem. Transport within a facility canalso stress animals.

Physical factors associated with themacroenvironmentMajor variations of ambient temperature,lighting [especially for nocturnal animals),relative humidity and noise can beimportant sources of distress in laboratoryanimals and it is conceivable that someprocedures cause actual pain. Someapparently innocuous activities such ashaving mice in close proximity to rats [anatural predator) and use of unscreened(electronically) visual display units,operating vacuum cleaners or running taps(all sources of ultrasound) can causedistress and associated abnormal behaviourin laboratory rodents. The commonpractice of playing transistor radios inanimal rooms is also a distinctlycontentious activity.

Physical factors associated with themicroenvironmen tCage design and construction can have amajor impact on an animal's well-being.Factors such as opportunities for exerciseor retreat out of direct contact withcagemates can be extremely important.Although cages have to be cleaned toprevent exposure to high levels ofammonia, the act of cleaning can causegreat disturbance to some animals,especially those who leave odour patterns.Rehousing rodents from established groupsin new associations is an intensely stressfulprocedure as measured by physiological and

behavioural changes. Cleaning is oftenfollowed by short bouts of fighting ingroup-housed male rodents. Certain timesof the light/dark cycle minimize thisresponse. Strangely, animals often appeareven more distressed if existing substratesare disrupted but left in place than if thecage is cleaned. Cage-mates [especiallymales and lactating females) are potentialsources of pain and distress for rodents andlagomorphs [Brain 1990). Again, one mustunderstand requirements of particularspecies and use great care when housinganimals together. Although fighting isrelatively common in some species, mostanimals tend to use relatively non-injuriousmodes of attack on conspecifics, and haveantinociceptive mechanisms whichameliorate the stress caused. Even 'goodfaith' procedures such as grouping animalsfor purposes of mating can generatefighting and chasing. As much of animalbehaviour (whether one is looking at sexualor agonistic activity) involves initial ambi-valence before the 'desired' activity, it ishardly remarkable that procedures can anddo occasionally generate distress. Thedesignating of individually-housed animalsas abnormal is also dubious. The responseto individual housing depends on thespecies and stage of development-amajority of group-housed male mice maybe at a disadvantage vis-a-vis 'isolated'counterparts in that they show higheradrenocortical and lower gonadal activities(Brain 1975). Many workers fail toappreciate that rodents and lagomorphsoften communicate between neighbouringcages using a variety of auditory (includingultrasound) and olfactory cues. It has, forexample, been demonstrated that rats andmice can communicate pain and distress(presumably producing anxiety in neighbour-ing conspecifics) via a variety of olfactorymessages. Some visual (probably a veryminor sensory modality in rodentsl separa-tion is hardly 'isolation' in such species.

Factors associated with experimentalproceduresTechniques used in husbandry and inlaboratory practice may be sources of pain

or distress. It is difficult to produce adefinitive list of these very variedprocedures but they. obviously differ inseverity. We might judge that efficientgeneral and specialist husbandry,behavioural studies, and food and waterdeprivation for up to 24 h are relativelypainless and distress free. Tattooing andother methods of marking animals,administration of substances (see Table Alin LASA 1990), anaesthesia and recoveryfrom anaesthesia, and surgical techniques[see Table A3 in LASA1990)-are likely,however, to be painful and stressful.Collection of tissues and body fluids [seeTable A2 in LASA1990), post-surgical care,restraint (see Table A4 in LASA1990),physiological, pharmacological and toxico-logical studies, and humane killing ofanimals - are difficult to evaluate. Betterinformation is required on the severitiesassociated with all scientific procedures.

VIII. Signs of pain and distress

A relatively simple means of accuratelygrading levels of pain and distress is neededthat can be applied to the wide range ofcircumstances and procedures used inanimal laboratories throughout Europe byanimal care specialists, technicians andscientists. As rodent and lagomorph specieslack the ability to communicate verballytheir state of 'mind' or experience, wegenerally rely on modifications ofbehaviour, notably changes which arerecognized as active, aversive (nocifensive)reactions to infer a state of pain in them.Although this might be regarded as crudevis-a-vis humans, the most effectivemedical screening systems for human painare also visual rather than verbal.

Protective motor actions include genuinewithdrawal reflexes as well as less specificfight or flight reactions. Such reactions arelargely species-specific. The tendency toreact to pain by inhibition of motoractivity leading to a passive, immobileappearance rather than by withdrawal,must not be forgotten. This behaviour ismore prevalent in rabbits and poultry, butdoes occur in rodents.

FELASAWorking Group on Pain and Distress

Visceral reactions, mainly caused byincreased activity in parts of the sympa-thetic nervous system, may result inchanges in heart rate, blood pressure,respiratory pattern, pupil size, sweating orgastrointestinal motility. These visceralreactions are not, however, conclusiveevidence for a perception of pain, as theymay be evoked by nociceptive stimuli inanimals under an anaesthesia deep enoughto suppress the cerebral cortex.

Changes in general or social behaviour orin learned avoidance reactions have beenconsidered expressions of both physical andemotional pain. Great familiarity withnormal behaviour and its variation in theparticular species is needed in order to beable to recognize and assess suchalterations. The Laboratory Animal ScienceAssociation (LASA1990) has proved avaluable starting point in this considerationand their recommendation that evaluationof well-being necessitates workersbecoming 'familiar with the normalbehaviour, appearance, physiological andanatomical characteristics of each species'is extremely pertinent. One should addthat individual strains lespecially of mice)show enormous variability in behaviourand physiology, suggesting that one needsfamiliarity at the strain level (Jones &.Brain 1985). It is important to understanddifferences between nocturnal and diurnalspecies and to recognize the simpleimportance of subjects being warm to thetouch, showing good muscle tone, a well-groomed coat and maintenance of bodyweight. LASA (1990) record that mostlaboratory rodents will make positiveattempts to avoid capture and handlingwhen they are healthy. Subjects shouldmake movements without reluctance,without favouring a particular limb and notshow staggering or circling. Dantzer (1991)and Mason (1991) have recently challengedthe view that stereotypies (repetitivemovements) provide indices of distress inlaboratory and farm species.

LASA(19901suggest that many animalsrespond to acute pain by simple reflexactions including 'withdrawal from thesource, accompanied by vocalization and

followed by licking, shaking or scratchingthe affected part'. Although this impliesthat animals are consistent responders tospecific stimuli, Rodgers (1989) hasreviewed the wide range of analgesicmechanisms found in rodents, as well asthe extremely diverse environmentalfactors associated with pain inhibition (seesection IIII. The same stimulus may thuscause different perceptions of pain indifferent individuals. LASA(1990) suggestthat a second stage of response involves'some changes in behaviour, reluctance tomove, combined with vocalization, irrita-bility, short-term anorexia and abnormalposture'. Unusual noises such as'chattering, bubbling or wheezing' mayalso be diagnostic. Ultrasound productionmight also be of utility here (Sales &. Pye1974) and pharmacological evidence hasbeen produced that the ultrasoundsproduced by infant mice are reliable indicesof 'anxiety' [Nastiti et ai. 1991).

LASA(1990) suggested that chronic painor distress is usually more insidious in itsearly stages than acute pain and requirescareful observation to detect changes in ananimal's appearance and behaviour whichindicate deterioration. Behaviour andappearance are actually promising candi-dates for routine assessments of pain anddistress.

What other features might be used toassess pain and distress? It has beensuggested that hormones (especially gluco-corticoids) signify 'stress' (reviewed byBrain, 19901but many hormonal factorschange in a complex way and samplingitself can be stressful. Broom (1991) hassuggested combining behavioural indiceswith physiological (hormones, heart rateland immunological (stressed animals oftenshow impaired antibody production)measurements, as well as injury, disease,mortality risk, growth and reproductionmeasures to obtain complete indications ofwelfare. One might also add that detailedpathological investigation can also proveuseful in (retrospectively) establishingeffects of housing conditions andprocedures on distress. Several of thesemight be used to confirm the severity of

pain and distress. There is, however, adanger of intervening too much with theanimal and one can make a convincingcase for intelligently used behaviouralindices, simple appearance and rate ofbody-weight change, combined withpathology, being the best indicators ofhealth and welfare. Good stockmanshipmay, in the end, prove the best we canlegitimately expect on a routine basis.There is an urgent need for systematicevaluation of the usefulness of the postu-lated indicators, combined with animperative to increase the sophistication ofbehavioural assessment in animal labora-tories. Remote controlled videotaperecording of activity can be very revealingin this respect.

IX. Grading of severity of pain anddistress in animals

A variety of schemes for scoring pain anddistress in laboratory animals have beenpresented. Such assessments may beimportant in three stages of experimentalprocedures namely: in gaining approvalfrom ethical committeesj during theexperiment per sej and in postmortemexaminations. The response to paindepends on age, sex, health status, speciesand strain of the animal. Criteria used inpain evaluation are applied differently indifferent schemes. All current methods areunsatisfactory in that they are relativelysubjective but most workers feel that theyare considerably better than nothing.

In general, discomfort can be assessed ina qualitative and a-more or less-quantitative way. In both the qualitativeand the quantitative way, assessment ofdiscomfort contains two steps: collection ofdata, which can be regarded as an objectiveprocess, followed by 'translation' into adegree of discomfort, which is a subjectiveprocess (Beynen et al. 1989a,b,c).

Morton & Griffiths (1985), Beynen et ai.(1988a,b,c,d, 1989a,b,c), LASA(1990) andthe Disturbance Index used by Barclay et al.11988)have tried to score signs of pain anddistress. LASAidentified components ofseverity and gave a numerical rating

reflecting its potential range. Morton &Griffiths (1985) tried to correlate clinicalsigns and severity of pain and distress,while the Disturbance Index used changesin number of movements made by anormal laboratory rat or mouse introducedinto an unfamiliar cage as a method forassessing severity of procedures. Otherauthorities (e.g. Wright & Woodson 1990)use qualitative criteria from clinicalexamination or physiological signsincluding pupillary dilation, degree ofopening of the eyelids, transient increasesor decreases in blood pressure, heart rate,increases and alterations in respiration(including panting and gasping), whiskermovements, piloerection, increases in bodytemperature, increased muscle tone,sweating, changes in skin temperature,evacuation of the rectum, ungroomedappearance or excessive licking, decrease inappetite, and abnormal stances. All theseschemes suggest an objectivity which isapparent rather than real as, in most cases,diverse items are assumed to be related topain and/or distress without statisticalweightings being applied. Flecknell (1986j,Sanford et al. (1986) and Zimmermann(1986) have all provided additional materialon behavioural indicators of pain inanimals. Biochemical signs may also beused, including increases in plasma ACTH,corticosteroids and catecholamines as wellas decreases in plasma sex steroids, buttheir correlation with pain is imprecise atbest. Presumed 'mental' status may also beassessed with animals classified asdepressed, unaware, unresponsive, anxious,alert, excitable or aggressive. These ratingshave to be compared with previousbehaviour and normal behaviour of thespecies, breed, or strain. The state ofconsciousness may be assessed in testsusing e.g. visual or auditory threats andreflexes (palpebral or flexor withdrawal).Abnormal activity [inactivity to hyper-activity) may give clues about pain ordistress as may posture, facial expression(where appropriate), gait (e.g. lamenessj,reluctance to accept handling (showing e.g.vocalization or attack) or vocalization(noises may characterize particular

FElASA Working Group on Pain and Distress

physiological functions). Responses toanalgesics - to reverse the signs of pain-may also be revealing. Some peopledifferentiate between signs of acute painsuch as guarding, crying, mutilation(licking, bitingL restlessness, sweating,recumbency, ambulation (reluctance tomove) and abnormal positions (head downetc.) and chronic pain expressed by featuressuch as limping, licking area, reluctance to

move, loss of appetite, changes in boweland urinary activity, accumulation of bodysecretions (decrease in grooming) andchanges of behaviour towards attendance.

Buckwell (1992) listed physical signs forrodents that can be related to mild,moderate and substantial severity,categories used in UK legislation. Theapproach is included in a slightly modifiedform below.

Mild Moderate Substantial

Reduced weight gainFood and water consumption

40-75% of normal for 72 h

Partial piloerection

Subdued but responsive, animalshows normal provoked patternsof behaviour

Interacts with peersHunched transiently especially afterdosing

Transient vocalization

Oculo-nasal discharge transient(typically signs of chromorhino-dacryorrhoea in rodents)

Normal respiration

Transient tremorsNo convulsionsNo prostration

No self-mutilation

Weight loss of up to 20%Food and water consumption lessthan 40% of normal for 72 h

Staring coat- marked piloerection

Subdued animal shows subduedbehaviour patterns even whenprovoked.

little peer interactionHunched intermittently

Intermittent-vocalization whenprovoked

Oculo-nasal discharge persistent

Intermittent abnormal breathingpattern

Intermittent tremorsIntermittent convulsionsTransient prostration (less than

1 h)No self-mutilation

Weight loss greater than 25%Food and water consumption lessthan 40% for 7 days, or anorexia(total inappetence) for 72 h

Staring coat-marked piloerection-with other signs of dehydrationsuch as skin tenting

Unresponsive to extraneous activityand provocation

Hunched persistently ('frozen')

'Distressed' -vocalizationunprovoked

Oculo-nasal discharge - persistentand copious

laboured respiration

Persistent tremorsPersistent convulsionsProlonged prostration (more than

1 h)Self-mutilation

A degree of retrospective assessment ofdistress is possible by using datafrom postmortem examination (Walvoort1991). The following criteria can beused: anabolic/catabolic balance e.g.body weight, muscle volume, fluid balance,fatty depots, fur condition and gastro-intestinal contentsj neuroendocrine balancee.g. gonad and adrenocorticalsizes; immunological balance e.g.intactness of gastric mucosal barrier,lymphoid organ size and presence ofinfections as well as behaviour balance

e. g. wear of toenails and chromorhino-dacryorrhoea.

Morton (1990) has also highlighted areasin which refinement, with the specific aimof reducing laboratory animal pain, distressand anxiety, can be achieved. He alsosuggested that good husbandry and housingare of paramount importance. It isbelieved that choice tests may have a rolenot only in determining preferred environ-ments for laboratory animals but also inassessing the aversiveness of proceduresIBaumans et 01. 1987, 1990).

Conclusions

Pain, distress and suffering are all difficultconcepts to apply to laboratory animals.However, as European workers have bothan ethical and a legal requirement to limitthe impact of such factors it seems timelyto consider how much can be achieved.This report attempts to set out the natureof the problems and suggests ways inwhich progress can be made.

The essential problem is to find a rela-tively simple means of accurately gradingthe levels of pain and distress, a meansthat can be applied to the wide range ofcircumstances and procedures used inanimal laboratories throughout Europe byanimal care specialists, technicians andscientists. This is essential if one hopes tofulfil the natural humanitarian desire tominimize the pain and distress in labora-tory animals that is enshrined in recentlegislation and to perform the cost [interms of pain and distress)/benefit(in terms of hoped for gains in knowledge)analyses of research that are advocated bymany national authorities. Certainly,scientists require more quantifiabledata on the welfare repercussions of manyof the currently used procedures inhusbandry and experimentation (Brain1992).

In spite of improved laboratorytechniques, it seems that good stockman-ship and laboratory animal technology(generally based on appropriatelyinterpreted behaviour, general conditionand body weight changes) combined withdetailed pathology provides the best meansof assessing attempts to improve theconditions of laboratory animals. Thisis not only ethically desirable butwould tend to improve the quality ofresearch.

It is essential to recognize the variationsbetween species (and even strains of thesame species) and for workers to avoidanthropocentrism where possible.Legislators and scientists also have a dutyto warn the general public about thedangers of too readily applying humanvalues to other species. Action based on

human values is not always helpful toanimals and can actually prove deleterious.

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Note: Reprints of this Report are available free of charge from the Secretary, FELASA,Rijksuniversiteit Utrecht, Bureau Proefdierdeskundige, Postbus 80.166,3508 TO Utrecht, TheNetherlands.

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