Scientific Advances in the Study of Animal Welfare

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Scientific advances in the study of animal welfare

How we can more effectively Why Pain? assess pain…

Matt Leach To recognise it, you need to define it…

‘Pain is an unpleasant sensory & emotional experience associated with actual or potential

tissue damage’ IASP 1979

As it is the emotional component that is critical for our welfare, the same will be true for animals Therefore we need indices that reflect this component! Q. How do we assess experience?

• As it is subjective, direct assessment is difficult.. • Unlike in humans we do not have a gold standard – i.e. Self-report – Animals cannot meaningfully communicate with us… • So we traditionally use proxy indices Derived from inferential reasoning

Infer presence of pain in animals from behavioural, anatomical, physiological & biochemical similarity to humans

In humans, if pain induces a change & that change is prevented by pain relief, then it is used to assess pain

If the same occurs in animals, then we assume that they can be used to assess pain Quantitative sensory testing

• Application of standardised noxious stimuli to induce a reflex response – Mechanical, thermal or electrical… – Used to measure nociceptive (i.e. sensory) thresholds • Wide range of methods used – Choice depends on type of pain (acute / chronic) modeled • Elicits specific behavioural response (e.g. withdrawal) – Latency & frequency of response routinely measured – Intensity of stimulus required to elicit a response • Easy to use, but difficult to master… Value?

• What do these tests tell us:

– Fundamental nociceptive mechanisms & central processing

– It measures evoked pain, not spontaneous pain

• Tests of hypersensitivity not pain per se (Different mechanisms) • What don’t these tests tell us:

– Much about the emotional component of pain

• Measures nociceptive (sensory) thresholds based on autonomic responses (e.g. reflex) directly relating to sensory component

• Not practical for assessing pain outside laboratory… Clinical signs

• Subjective assessments of:

– Appearance: pilo-erection, anorexia etc.

– Posture/Gait: hunched posture, abnormal gait etc.

– Demeanour: aggression, hiding etc.

• Objective assessments of:

– Locomotion / Activity

– Food & water intake / Bodyweight change

– Physiology: Respiration/ Heart rate/ Blood pressure/ Temp Value?

• Little evidence of how these relate to pain experienced:

– Changes could be due to other non-pain related causes

– Changes in measure may not parallel change in pain • Insensitive to individuals’ pain sensitivity or to changes in pain state

• Poor dose-effect relationship with pain relief methods • Retrospective: Poor for immediate assessment

• Problems of reliability between & within observers Specific pain behaviours

• Behaviour-based indices are increasingly being used

– Rodents, rabbits, dogs, cats, lambs and calves etc. • Considered more effective than traditional methods

– Immediate cage/pen side assessment (not retrospective)

– Growing evidence of relationship between pain & behaviour Rat pain behaviours

Stagger Twitch Writhe

Flinch Press Arch 1hr post-laporatomy

Cumulative score of arch, twitch, writhe & stagger/fall 30

25 Roughan & Flecknell 2001, Pain (90), 65-74 1SD - 20

5 Behaviour Frequency +/ Frequency Behaviour

0 Saline 2mg/kg 5mg/kg Saline 2mg/kg 5mg/kg Carprofen Ketoprofen Carprofen Ketoprofen

No Surgery Surgery Potential limitations using behaviour

• Time-consuming to develop & implement:

– Establish which behaviours indicate pain following a procedure

– Often requires >30mins of training1 • Pain behaviours vary markedly between procedures • So behaviour-based schemes only developed for a few species undergoing a limited no. of procedures

– Mostly laboratory rodents & rabbits undergoing castration & lambs undergoing castration & tail docking…

1Roughan & Flecknell (2006) App. Ani. Behav. Sci. 96: 327–342 Potential solution…

• Automated behavioural analysis… • Several commercially available systems

– Development driven by phenotyping of GM mice • Advantages: – Continuous & rapid monitoring of large number of animals Automated systems

• Ethovision (Noldus IT) – Recording of position, velocity & distance travelled • Phenotyper (Noldus IT) – Ethovision in 3D & running wheel • Theme (Noldus IT) – Detection & analysis of patterns in behaviour • Intellicage (New Behavior AG) – Operant testing in social setting • LABORAS (Metris) – Recording 12 behaviours via vibration recognition • HomeCageScan – Recognises 22 categories of behaviour HomeCageScan vs. Manual scoring

C57 C3H 35

1SD 30 - Manual HCS Manual HCS 25

5 Mean Frequency of activity +/ of activity Mean Frequency 0 Saline Mel 5 Mel 10 Mel 20 Saline Mel 5 Mel 10 Mel 20 Treatment

1h post-op Miller et al (2013) Applied Animal Behaviour Science 131: 138-144 Limitations of automated scoring

• So far only scores general behaviour, which may not be pain-specific

– e.g. Activity etc..

• Not able to be ‘trained’ to score more pain specific behaviours…

• But these automated systems may offer a ‘triage’ method at least for new procedures

– Indicate when more specific analysis is needed… Behaviour limitations: species differences

Species differences are important!

Rodents primary response: Rabbits primary response: • Explore & move around their cage • Become inactive • Seems to occur when observer • Rabbits freeze & are motionless present when observer present Behaviour limitations: strain differences

1hr post abdominal vasectomy

30 1SD - 25 C57 C3H 20

15 C57 C3H 10

5 Behaviour Frequency +/ Frequency Behaviour

0 Saline Melox 5mg/kg Melox 10mg/kg Melox 20mg/kg Tr eatmen t Wright-Williams et al (2007) Pain, 130, 108-118 Indices of pain ‘experience’?

• So far the indices covered at best can be used as an indirect measure of the emotional component

– Could just measure responses to sensory component

– Some authors (e.g. Rose et al. 2012) argue that these indices simply represent ‘complex’ reflex responses…

– No higher processing, so no emotional reaction…

• We would argue this is an extreme view…

– Principle of triangulation (Bateson 1991)

Rose et al. (2012) Fish & Fisheries, doi: 10.1111/faf.12010, Bateson (1991) Ani Behav 42: 827-839. So how can we assess ‘experience’?

Use approaches ulitlised with non verbal humans can provide a framework for animal pain assessment

Grunau & Craig (1987) Pain 28 (3):395-410. Complex behaviour

• In humans, pain effects complex behavioural patterns – Locomotion, getting dressed, washing, keeping house clean • As behavioural complexity of responses increase – Responses go beyond a “stimulus-response” reflex… – Likelihood of responses requiring higher processing increases.. – So more likely to be direct indices of pain experience Complex behaviour

• Pain also influences complex behaviour in animals: – Locomotion, explorative behaviour & rearing (most species) – Nest building (Mice) – Grooming & Burrowing (rodents) • Represent highly motivated behavioural needs – Alterations likely indicate important impacts on welfare Rearing

• Rats placed into novel environment explore & rear • Pain induced by inflammation of the knee joints (CFA) – + /- pain relief (i.e. NSAIDs) • Horizontal locomotion & vertical rearing measured

Matson et al. (2017) Journal of Pharmacology and Experimental Therapeutics 363 Naturalistic behaviours

• Burrowing & Nest Building • Highly motivated behavioural needs – Alterations likely indicate important impacts on welfare • Wild mice build nests for warmth – Lab mice prefer near 30oC (1) , not 20-24oC – Lab usually 20-24oC – Nests create microclimates within the cage • Burrowing expression = global wellbeing1

1Deacon (2006) Nature Protocols 1(3) 1117-1119, 2Gaskill et al (2012) PLOS one 7(3) e32799 Nest building

Vol 47, No 6 Journal of the American Association for Laboratory Animal Science November 2008

Figure 1. The naturalistic nest score system. Scores are based on the shape of the nest as well as on how much the walls are built up around the nest cavity in order to form a dome. Both a top view and a side view are shown.

Hess et al. (2008) JAALAS 47(6) 25-31, Deacon (2006) Nature Protocols 1(3) 1117-1119

Figure 2. Sample nests and their corresponding scores.

28 Well-made nests

Poorly-made nests No. of cages showing poor nest building (/8)

Arras et al. 2007 BMC Veterinary Research 3: 16 Burrowing Burrowing

Jirkof et al. (2010) Frontiers in Behavioural Neuroscience, doi: 10.3389 Self-grooming (?)

• Complex & evolutionary conserved behaviour in rodents – 15-50% of awake time spent grooming • Stereotyped sequence: – Nose ➤ Eyes ➤ Ears ➤ Body ➤ Tail/genitals Self-grooming (?)

• Complex & evolutionary conserved behaviour in rodents – 15-50% of awake time spent grooming • Stereotyped sequence: – Nose ➤ Eyes ➤ Ears ➤ Body ➤ Tail/genitals

Kalueff et al. (2007) Nature Protocols Volume 2 Issue 10: 2538-2544 Self-grooming (?)

• Complex & evolutionary conserved behaviour in rodents – 15-50% of awake time spent grooming • Stereotyped sequence: – Nose ➤ Eyes ➤ Ears ➤ Body ➤ Tail/genitals • Sensitive to various experimental manipulations – Complex neurobehavioral disorders, e.g. basal ganglia disorders, autism, OCD & attention-deficit/hyperactivity disorder – Stress

• Could it be used to assess pain? Using complex behavioural tests

• Pain experience assessed using operant testing • Animals use their pain to inform their decisions – Requires animals to use their underlying ‘experience’ – This goes beyond even complex reflex response & requires cerebral processing Self-administration

• People in pain can self-medicate to reduce their pain – Animals have similar receptors for pain relief – Pain relief alters behaviour of animals in pain • Will animals in pain give themselves pain relief? Suprofen intake in rats with arthritis

15.0 Colpaert et al (1980) Life Sciences 27, 921-928

12.0

Control 9.0 Arthritic

6.0 Intake (ml)

3.0

0.0 2 3 4 5 6 7 8 9 10 11 Weeks after challenge Conditioned discriminations • Methods borrowed from drug abuse literature – Used to establish affective value of drugs • Conditioned place aversion: – Animals choose to spend less time in a location previously associated with a negative experience (e.g. pain) • e.g. Being in pain is a negative experience, so pain should induce a place aversion • Conditioned place preference: – Animals choose to spend more time in a location previously associated with a rewarding experience (e.g. analgesia) • e.g. Relief from pain is a rewarding experience, so pain relief should induce a place preference Conditioned discriminations • Conditioned drug discrimination: – Animals use the feeling evoked by a drug to solve a conditional discrimination – If pain is a feeling that animals can access, they should be able to use the presence/absence of the feeling to solve a discrimination

• Animals in pain discriminate the difference in the way they feel when given pain relief?

Lal, H. (1977) Discriminative Stimulus Properties of Drugs. Plenum Press, New York. Conditioned place aversion • Animals confined in a chamber & given a noxious stimulus (CPA), or no stimulus • When animals given a free choice of chamber, they should choose the one where they did not receive noxious stimulus • May be combined by making the “no pain chamber” environment one that is mildly aversive (eg light rather than dark) Conditioned place aversion

Training (?) Tests

Formalin (S+) Measures: Odd days - Time preference - Choice preference

S+ ? ? S-

Vehicle (S-) Even days Compare: - Un-lesioned rats - Lesioned rats Conditioned place aversion

3 Time after injection (min) 300 250 2 200 150 100 1 50 0 0 -50 5-10 15-20 25-30 35-40 45-50 -100 Sham Lesion

• Spontaneous nociceptive behaviours (licking foot) unchanged in rats with anterior cingulate cortex lesions • Conditioned place aversion blocked by lesioning

Johanssen et al, 2001 PNAS 98, 8077-8082 Conditioned place preference • Animals given analgesic & placed in one chamber, or saline & placed in other chamber • When animals in pain given free choice of chamber, they should choose the one where they received analgesic • Animals must be able to use their “feeling” of pain, and its change in intensity, in order to make this choice Conditioned place preference

Rats in pain show a preference for a context previously associated with analgesic administration

Inflamed Non-inflamed 0.75

S+ (analgesia) 0.5 S- (saline)

0.25

0.0mg/kg 3.0mg/kg 10.0mg/kg Morphine dose

Sufka, K. J. 1994. Pain 58, 355-366. Conditioned drug discrimination • When given analgesic receive food reward when the press left lever (only) • When given vehicle receive food reward when the press right lever (only) • When animals in pain given free choice of levers, they should choose the one associated with analgesic • Animals must be able to use their “feeling” of pain, and its change in intensity to make the correct choice

Are arthritic (painful) rats better than normal/pain free rats at discriminating between analgesic and placebo? Conditioned drug discrimination

Train (14 days) Test

Aspirin Right lever Food Measures: injection FR10 reward - Lever presses (56 mg/kg i.p) Group: Left lever Nothing - Control: untreated - Pain: Arthritis-induced Right lever Nothing Saline Compare: Discrimination injection Left lever Food performance in two groups (same volume i.p.) FR10 reward

Weissman (1976) Pharmacol Biochem Behav 5, 583-586. Conditioned drug discrimination

80 70 60 choices 50 40 correct 30 Arthritic rats Non-arthritic rats 20 Mean % 10 0 1 2 3 4 5 6 7 8 Block of 7 sessions Arthritic rats better at learning the discrimination: Used “how they felt” to make the correct choice

Weissman (1976) Pharmacol Biochem Behav 5, 583-586. Cognitive bias • New method to identifying affective states in animals – In humans, emotions are associated with adaptive biases in information processing • Emotional state influences decision making – Negative affective states associated with expectations of poor outcomes & makes individuals more risk averse

Someone will I’m doomed! come! Put another way…

• Emotional state can influence decision making • Optimists (or a positive emotional state) sees the glass as half-full • Pessimists see the glass as half-empty • Can we assess whether rats are optimists or pessimists? Rat enrichment & cognitive bais

Train Test (5x)

Choose Chocolate ++ Reward black

button 60 Grit Ambiguous probes 60 Grit (cinnamon) 80 Grit Choose orange Nothing (coriander) 120 Grit

150 Grit

Choose + Reward Nothing 1200 Grit black 1200 Grit (cinnamon) Record which bowl Choose orange (coriander) Cheerio is chosen?

Brydes et al. (2010) Animal Behaviour 81: 169-175 Predictions…

‘Optimistic’ 0.5

‘Pessimistic’ Probability of choosing ++ bowl choosing of Probability

1200 150 120 80 60

Ambiguous probes Floor texture (Grit) Findings

Pre-treatment Post-treatment 5 P=0.02 P=0.02 4 1SE ± 3

1 Optimistic choices choices Optimistic 0 Enriched Unenriched

Has not been used to assess pain yet, but we are working on it… Cognitive bias & pain? • Well recognised in humans… • Pre-operative affective state determines; – Perceived pain severity, surgical outcome

• Anxious, depressed or painful humans (–ve emotions) – Report more severe pain & require more analgesia • Is this also true in animals? • In theory yes… – We are working on this… New (ish) operant test • Orofacial Operant Pain Assay (OOPA) • Uses a reward-conflict paradigm – Ask animal to titrate pain (heat) against its willingness to access a palatable reward (e.g. condensed milk) • Requires higher-level cognitive processing – The animal decides, according to its perceived pain, whether it will complete the task of maintaining contact with the thermode while obtaining a palatable reward • Minimally invasive using transient pain that the animals terminate themselves

Neubert et al. (2006) Behav Brain Res,170: 308–315 New (ish) operant test • Mice/rats trained to seek a fluid reward – To access sweetened condensed milk they have to keep cheeks in contact with heated bars (e.g. 37oC) • As temperature increases (41oC) licking decreases • Animals show less licking when orofacial pain was induced with capsaicin

Photos and data from Neubert et al. (2008) Molecular Pain, 4: 1-14 New (ish) operant test

Effect of buprenorphine dose on the lick:face ratio in male mice & rats. Rodents were more willing to complete the task (higher lick/face ratio) with pain relief New NC3Rs grant to further validate this technique & to identify more effective analgesia Ramirez et al. (2015) JALALAS 54: 426-432 Limitations of complex behaviour

• Impractical at the cage-side, i.e. a ‘clinical’ scenario! • May not be pain-specific, rather negative state specific – i.e. change in response to pain, distress, anxiety or combinations of these states! – But does this matter! • But could be used as adjuvants to other cage side methods for the purposes of validation! Assessing pain in non-verbal species?

Q. How do we assess pain in non-verbal humans clinically? A. We use facial expressions Q. Why use facial expressions?

• Considered as ‘Gold Standard’ assessment:

– Effective using a limited number of indicators

– Rapid & easy to carry out after minimal training

– Represents ‘generic’ response to pain or distress

– Good accuracy (>80%)

– Uses the human tendency to fixate on faces

• e.g. the human face on the moon! Facial expressions have evolved…

• Share chages in many components of expressions (species generic) – Eyes, cheeks, ears, whiskers, mouth, jaw etc. Current grimace scales

Mouse (MGS) Langford et al. (2010) Nature Methods 7: 447 ? Rat (RGS) Sotocinal et al. (2011) Molecular Pain 7: 55 ? Rabbit (RbtGS) Keating et al. (2012) PLoS ONE 7: e44437 ? Horse (HGS) Dalla Costa et al. (2013) PLoS ONE 9: e92281 ? Gleerup et al. (2014) Veterinary Anaesthesia and Horse (EPF) Analgesia 42, 103–114 ? Holden et al. (2014) Journal of Small Animal Practice 55: Cat 615-621 ? McLennan et al. (2016) Applied Animal Behaviour Sheep (SPFS) Science 176: 19–26 Gleerup et al. (2014) Applied Animal Behaviour Science Cow (CPS)* 171: 25–32 ? Methodology

• Development: – Comparison of images (same individual) before vs. after painful event (blinded observer) to identify what changes… • Validated method used by human facial coding experts • Current scoring method: – Intensity (presence) of AUs measured on 3-point scale: • 0= not present, 1= moderately present, 2= obviously present – Overall ‘Grimace score’ is either: • An average of all AUs in the scale • The total (composite) of all AUs in the scale Mouse grimace scale (MGS)

Orbital tightening: • Closing of the eyelid (narrowing of orbital area) • A wrinkle may be visible around the eye

Nose bulge: • Bulging of the bridge of the nose • Vertical wrinkles extend down the side of the nose

Cheek bulge: • Bulging of the cheeks

Ear position: • Ears move back from facing forward to lay on body • Ears rotate outwards & space between the ears increases

Whisker change: • Whiskers either being pulled back against the cheek or; • Pulled forward to “stand on end” & clump together • Whiskers lose their natural ‘downward’ curve

0 1 2 Langford et al. (2010) Nature Methods 7: 447-449 Mouse Grimace Scale (MGS)

★ 4 2ml/kg Saline (sc) ★ ★: P< 0.001 3.5 20mg/kg Meloxicam (sc) 5mg/kg Bupivacaine (lc) 3

2SE 2.5 ± 2 Non-significant 1.5 Mean MGS 1

0.5

0 Pre-op Post-op Session Leach et al. (2012) PLoS ONE 7(4): e35656. doi:10.1371 Are they reliable & accurate?

• Inter-rater reliability: – Interclass correlation: 0.90 to 0.91 (High) • Accuracy: 72% to 97% – Depends on image quality & species – Higher after 5mins of training with grimace scale manual…

*Langford et al. (2010), Sotocinal et al. (2011), Keating et al. (2012) Could assess pain experience! • Ibotenic lesioning of the rostral anterior insula – Activation associated emotional component of pain (humans) MGS: Writhing test:

Attenuation ofLangford pain etface al. (2010) Nature NoMethods effect 7: 447 on-449 writhing Could assess pain experience! • Ibotenic lesioning of the rostral anterior insula – Activation associated emotional component of pain (humans)

MGS: Writhing test:

• Lesion could have reduced emotional but not sensory component of pain in mice… – Facial expressions are a direct measure of experience… • But this is one study in only 6 mice… 682 E.B. Defensor et al. / Physiology & Behavior 107 (2012) 680–685

2.4. Statistical analysis 3.2. Cat odor exposure test

Data for the vibrissae contact test, the social proximity test, the cat When exposed to a cat odor cloth (Fig. 2c), mice displayed nose odor exposure test and the rat exposure test were analyzed using a swells [t(17)=2.42, pb0.05] and cheek swells [t(17)=8.40, paired t-test, comparing test trials with pretest trials. The resident– pb0.0001], compared to the pretest trial. No significant changes to intruder test was analyzed using a two-way repeated-measures anal- the eyes [t(17)=1.33, p>0.05] or ears [t(17)=1.58, p>0.05] were ysis of variance (ANOVA) with status (resident or intruder) as the be- observed. tween subjects factor and trial (pretest or test) as the within subjects factor. A probability of pb0.05 was adopted as the level of statistical 3.3. Rat exposure test significance. Subsequent analysis on a significant ANOVA was con- ducted using a Newman–Keuls post-hoc test. When exposed to a rat (Fig. 2d), mice displayed tightened eyes [t(17)=2.47, pb0.05], flattened ears [t(17)=3.63, pb0.01] and nose swells [t(17)=3.46, pb0.01], compared to the pretest trial. No 3. Results significant changes to the cheek [t(17)=1.90, p>0.05] were observed. When a medium bristle brush made contact with the mystacial682 vi- E.B. Defensor et al. / Physiology & Behavior 107 (2012) 680–685 brissae (Fig. 2a), mice displayed changes to all facial components2.4. Statistical analysis 3.2. Cat odor exposure test measured. Mice showed tightened eyes [t(17)=9.16, pb0.0001], flat- 3.4. Resident–intruder test Data for the vibrissae contact test, the social proximity test, the cat When exposed to a cat odor cloth (Fig. 2c), mice displayed nose tened ears [t(17)=3.04, pb0.0001], nose swells [t(17)=3.78,odor exposure test and the rat exposure test were analyzed using a swells [t(17)=2.42, pb0.05] and cheek swells [t(17)=8.40, pb0.0001] and cheek swells [t(17)=2.43, pb0.0001] inpaired thet-test, test comparingBoth test trials resident with pretest and trials.intruder The resident mice– showedpb0.0001], changes compared in facial to the expres- pretest trial. No significant changes to trial when compared to the pretest trial. intruder test was analyzedsion during using a two-way the test repeated-measures trial as compared anal- tothe the eyes pretest [t(17)=1.33, trialp (>0.05]Figs. 3 or and ears [t(17)=1.58, p>0.05] were ysis of variance (ANOVA) with status (resident or intruder) as the be- observed. tween subjects factor4). and trial (pretest or test) as the within subjects factor. A probability of pb0.05 was adopted as the level of statistical fl Only residents showed tightened eyes3.3. and Rat exposureattened test ears (Fig. 3). 3.1. Social proximity test significance. SubsequentA main analysis effect onfor a signi trialficant was ANOVA signi wasficant, con- showing that eye and ear scores ducted using a Newman–Keuls post-hoc test. were higher in the test trial when comparedWhen to exposedthe prestest to a rat trial (Fig. 2 [eyed), mice displayed tightened eyes 682 E.B. Defensor et al. / Physiology & Behavior 107 (2012) 680–685 [t(17)=2.47,fl pb0.05], flattened ears [t(17)=3.63, pb0.01] and When two mice were placed together in social proximity (Fig. 2b), tightening, F(1,16)=117.60, pb0.0001;nose ear swells [attening,t(17)=3.46, Fp(1,16)=b0.01], compared to the pretest trial. No 3. Results 2.4. Statistical analysis mice displayed changes to all facial3.2. Cat components odor exposure test measured. Mice 33.21, pb0.0001]. A main effect for statussignifi wascant also changes signi tofi thecant cheek with [t(17)=1.90, p>0.05] were showed tightened eyes [t(17)=10.83, pb0.0001], flattened ears residents showing higher eye and earobserved. scores than intruders [eyes, Data for the vibrissae contact test, the social proximity test, the cat When exposed to a cat odor clothWhen (Fig. a 2 mediumc), mice bristle displayed brush nose made contact with the mystacial vi- odor exposure test and the rat[t(17)=4.10, exposure test werepb analyzed0.001], using nose a swellsswells [tt(17)=2.42,(17)=3.28,pb0.05]pb0.01]brissae and cheek and(Fig. 2 swellsa), miceF(1,16)=79.45, [t displayed(17)=8.40, changesp tob0.0001; all facial components ears, F(1,16)=33.21, pb0.0001]. Post- measured. Mice showed tightened eyes [t(17)=9.16, pb0.0001], flat- 3.4. Resident–intruder test paired t-test, comparing test trials with pretestt trials. The residentpb– pb0.0001], compared to the pretest trial. No signifihoccant changes to fi cheek swells [ (17)=2.31, 0.05] in the test trial when comparedtened ears [t(17)=3.04,testspb0.0001], following nose a swells signi [tcant(17)=3.78, interaction between status and trial intruder test was analyzed using a two-way repeated-measures anal- the eyes [t(17)=1.33, p>0.05] or ears [t(17)=1.58, p>0.05] were to the pretest trial. No aggressive behaviors were observed.pb0.0001] and cheekindicated swells [t(17)=2.43, that residents,pb0.0001] but in not the test intruders,Both had resident higher and intruder eye and mice ear showed changes in facial expres- ysis of variance (ANOVA) with status (resident or intruder) as the be- observed. trial when compared to the pretest trial. sion during the test trial as compared to the pretest trial (Figs. 3 and tween subjects factor and trial (pretest or test) as the within subjects 4). factor. A probability of pb0.05 was adopted as the level of statistical 3.3. Rat exposure test Only residents showed tightened eyes and flattened ears (Fig. 3). significance. Subsequent analysis on a significant ANOVA was con- 3.1. Social proximity test A main effect for trial was significant, showing that eye and ear scores ducted using a Newman–Keuls post-hoc test. When exposed to a rat (Fig. 2d), mice displayed tightened eyes were higher in the test trial when compared to the prestest trial [eye [t(17)=2.47, pb0.05], flattened earsWhen [t(17)=3.63, two mice werepb placed0.01] together and in social proximity (Fig. 2b), tightening, F(1,16)=117.60, pb0.0001; ear flattening, F(1,16)= nose swells [t(17)=3.46, pb0.01],mice compared displayed to the changes pretest to trial. all facial No components measured. Mice 33.21, pb0.0001]. A main effect for status was also significant with 3. Results significant changes to the cheekshowed [t(17)=1.90, tightened eyesp>0.05] [t(17)=10.83, were pb0.0001], flattened ears residents showing higher eye and ear scores than intruders [eyes, observed. [t(17)=4.10, pb0.001], nose swells [t(17)=3.28, pb0.01] and F(1,16)=79.45, pb0.0001; ears, F(1,16)=33.21, pb0.0001]. Post- When a medium bristle brush made contact with the mystacial vi- cheek swells [t(17)=2.31, pb0.05] in the test trial when compared hoc tests following a significant interaction between status and trial brissae (Fig. 2a), mice displayed changes to all facial componentsThings weto the pretest don’t trial. No aggressive behaviors know were observed. indicated… that residents, but not intruders, had higher eye and ear measured. Mice showed tightened eyes [t(17)=9.16, pb0.0001], flat- 3.4. Resident–intruder test tened ears [t(17)=3.04, pb0.0001], nose swells [t(17)=3.78, pb0.0001] and cheek swells [t(17)=2.43, pb0.0001] in the test Both resident and intruder mice showed changes in facial expres- trial when compared to the pretest trial. sion during the test trial as compared to the pretest trial (Figs. 3 and 4). Only residents showed tightened eyes and flattened ears (Fig. 3). 1 3.1. Social proximity• test MGS changesA main effect in for trialresponse was significant, showing that eye andto ear scores other emotional states were higher in the test trial when compared to the prestest trial [eye When two mice were placed together in social proximity (Fig. 2b), tightening, F(1,16)=117.60, pb0.0001; ear flattening, F(1,16)= mice displayed changes to all facial components measured. Mice 33.21, pb0.0001]. A main effect for status was also significant with showed tightened eyes [t(17)=10.83,– Currentpb0.0001], flattened scales ears residents showing only higher eyeseem and ear scores thansensitive intruders [eyes, to negative states [t(17)=4.10, pb0.001], nose swells [t(17)=3.28, pb0.01] and F(1,16)=79.45, pb0.0001; ears, F(1,16)=33.21, pb0.0001]. Post- cheek swells [t(17)=2.31, pb0.05] in the test trial when compared hoc tests following a significant interaction between status and trial to the pretest trial. No aggressive behaviors• wereNo observed. changeindicated seen that residents, in butRGS not intruders, with had higher positive eye and ear emotions2

Fig. 2. Facial scores in the (A) vibrissae contact, (B) social proximity, (C) cat odor exposure and (D) rat exposure tests presented as mean±SEM. Mice showed changes in the eyes, Fig. 2. Facial scores in the (A) vibrissae contact, (B) social proximity, (C) catears, odor nose, exposure and cheek when and contacted (D) rat with exposure a brush and tests when presented placed in social as mean±SEM. proximity. Only nose Mice and showed cheek changes changes were observed in the when eyes, exposed to a cloth containing cat odor. Changes to the eyes, ears, and nose, but not cheek, were displayed by mice when exposed to a live rat. *pb0.05. ears, nose, and cheek when contacted with a brush and when placed in social proximity. Only nose and cheek changes were observed when exposed to a cloth containing cat odor. Changes to the eyes, ears, and nose, but not cheek, were displayed by mice when exposed to a live rat. *pb0.05. • Current scales more pain related rather specific! – At least true for MGS, but for the others we don’t know! Fig. 2. Facial scores in the (A) vibrissae contact, (B) social proximity, (C) cat odor exposure and (D) rat exposure tests presented as mean±SEM. Mice showed changes in the eyes, ears, nose, and cheek when contacted with a brush and when placed in social proximity. Only nose and cheek changes were observed when exposed to a cloth containing cat odor. Changes to the eyes, ears, and nose, but not cheek, were1Defensor displayed by miceet al. when (2012) exposed toPhysiology a live rat. *pb0.05. & Behavior 107 (2012) 680–685, 2Finlayson et al. (2017) PLoS ONE 11(11): e0166446, 3 Things we don’t know…

• Currently it seems practical in experimental context

www.nature.com/scientificreports/ – i.e. where we can take images/video & are not time Observation type Bias Upper limit Lower limit

RT-interval10 − 0.09 0.46 − 0.63 − − restricted RT-interval5 0.11 0.44 0.65 RT-interval2 − 0.14 0.43 − 0.71

RT-point10 − 0.07 0.49 − 0.63

RT-point5 − 0.08 0.47 − 0.63

RT-point2 − 0.09 0.50 − 0.68

• Clinical scoring seems toTable be 1. Bland andvariable Altman method comparing… each real-time (RT) observation method with image (IMG) scores. Bias is the mean diference between RT and IMG Rat Grimace Scale scores. Upper and lower limits of agreement are mean diference ± 2 SD.

P<0.05 P<0.05 P<0.05 P<0.05

Figure 4. Bland and Altman plots comparing image and real-time scores. and RT-interval5 or RT-point5. Te Bland-Altman analysis indicates that the limits of agreement between (A) Real-time interval observation over 5 minutes (RT-interval5) with a bias (underestimation) by real-time scores of −0.11 and limits of agreement ranging from −0.65 to 0.44. (B) Real-time point observation over 5 minutes (RT-point5) with a bias − − Figure. Mean Mouse Grimace Scale scores in 4 non-painful mouse strains. MGS scores are (underestimation) by real-time scores of 0.08 and limits of agreement ranging from 0.63 to 0.50. presented on the y-axis (±2 SE) for C57, C3H, CD1 & BALB/c on the x-axis.

variability around these diferences. Tere was a small systematic underestimation by all the real-time methods, showing that on average, real-time scores are very close to image-generated scores. Te similarity between 5 and 10-minute real-time observation periods indicates that 10-minute observation periods are unnecessary if the 1 2 3 Miller et al. (2015) PLoS ONE 10(9): e0136000. Leung et al. (2016) ScientificRGS is Reportsbeing applied 6:31667, as a tool to guideFaller pain et management al. (2015) (rather Experimental than as a research Physiology tool). Furthermore, 100: 163 the- sim172- ilarity between RT-interval5 and RT-point5 observations ofers alternative means of scoring depending on user preference. Te acceptability of a new (real-time) technique over a criterion standard (image-based) depends on a subjective assessment of the limits of agreement. For RT-interval5 and RT-point5 observations, the limits of agreement span a 0.5 score range either side of the bias. Terefore, there is the possibility of a single observation either over or underestimating the true score. Furthermore, the Bland and Altman plots show that data variability increases at RGS scores > 0.5. Interpreting these observations together, a practical approach could be a planned reassessment of any animal with an initial RGS score > 0.5 within a relatively short period (e.g. 1 hour), taking in to account the potential for sufering if providing analgesia is delayed against any side-efects associated with analgesic use. As RGS scores exceed a previously identifed threshold for intervention (RGS score > 0.67)16, the likelihood of an animal experiencing pain increases, in which case the reassessment interval should be kept short or analgesia provided immediately and the animal reassessed for an improvement in RGS score. Te agreement between RT scores and IMG scores was not refected in their ability to discriminate treatment efects statistically as observations decreased to 2 minutes. Both interval and point observation methods (RT-interval10 and RT-point10) were able to discriminate between the saline and analgesic treatments at the 6 and 9 hour time points, when peak RGS scores are expected13,22 and did not difer signifcantly from the standard RGS scoring method. Furthermore, the mean scores at these times exceeded a proposed analgesic intervention threshold16, providing evidence for the relevance of this decision-making tool. However, when the observation period was decreased to 5- or 2-minutes (RT-interval5,2 and RT-point5,2) only the interval scoring methods were able to reliably discriminate between saline and analgesia treatment groups, though the pattern of RGS scores did exhibit the expected time courses of the diferent treatment groups. Tis inability to discriminate was likely due to insufcient power when scoring with RT-point5,2 as the Bland and Altman results showed similar agreement to the equivalent interval scoring methods. Our fndings agree with those of Ballantyne et al.23, where a multidimensional 7 item pain scale, of which 3 items were facial action units, was evaluated in neonatal infants during painful and non-painful procedures23. Te authors showed that real-time (bedside) observations (over a 45 s period) did not difer signifcantly from the standard video-based assessments and were able to discriminate between predicted painful and non-painful states. Tis assessment method is similar to the successful interval method we employed. Faller et al.21 successfully used the mode of observed scores (scored from 10 photographs taken over a 15–20 minute observation period) to identify a reduction in the MGS score following buprenorphine

Scientific RepoRts | 6:31667 | DOI: 10.1038/srep31667 6 Things we don’t know…

• Grimace scales may be influenced by other factors? – Breed/strain & gender of animals (MGS)1,2

– Anaesthesia & analgesia (MGS)1,2

– Presence & gender of observers (MGS, RGS)3

◼

, ◼

Figure. Mean Mouse Grimace Scale scores in CBA & DBA/2 mouse strains. MGS scores for mice receiving 0.05mg/kg buprenorphine with the post-vasectomy recording sessions on the x-axis (P<0.001,P<0.01, ◼P<0.05).

1Miller et al. (2015) PLoS ONE 10(9): e013600, 2Miller at al. (2015) Applied Animal Behaviour Science 181: 160-165, 3Sorge et al. (2014) Nature Methods 11 (6): 629-632 Limitations

• Should only be used in awake animals… – False positives: falling asleep, asleep or waking up… – So sedation & anaesthesia have a confounding effect… • Most scales developed to assess acute pain (10mins to 24h)1-4 – SPFS developed for chronic pain (lameness & mastitis) • Currently we still need baseline observations… Maximising pain assessment • Tailor assessment to the context/study etc. • Use a combination of indices

– As all the methods have their limitations

– But one index can often compensate for limitation of another! • Be familiar with:

– Different methods of assessing pain

– Species & strain being assessing & how this affects pain assessment Thank you listening…. If you any questions or wish to discuss anything, then either chat to me or email me at: [email protected]