Pain Behaviors

Novel Methods of Evalua0on of Post-­‐Surgical Pain in Female Guinea Pigs Jennie Lofgren D VM, MS, DACLAM Clinical Assistant Professor Refinement and Enrichment Advancements Laboratory (REAL) Unit for Laboratory Animal Medicine (ULAM) University of Michigan Medical School Ann Arbor, MI Outline •  Guinea pigs in research –  Is there a need to iden0fy pain behavior in this species? •  Measuring pain in guinea pigs. •  Poten0al for differences between the sexes. –  Female vs. male guinea pig pain response. Guinea Pig use in Research •  Guinea pigs have been used in research for over 200 years. –  Historically served as models of anaphylaxis, asthma, delayed hypersensi0vity, gene0cs, gnotobio0cs, immunology, infec0ous disease, nutri0on, otology, pharmacology. Changing Use •  Guinea pig use has declined significantly since the 1980s; in part due to the development of the local lymph node assay, replacing the patch test. Guinea Pigs: Pain and Distress •  Despite that decline, guinea pigs are s0ll the most commonly u0lized USDA covered species for research and educa0on. •  Of the 213,029 guinea pigs represented in fiscal year 2010 (USDA annual report): –  Category D: 30% –  Category E: 16% •  Taken together, 46% (97,541 guinea pigs) of the largest represented group of USDA covered species were expected to experience pain or distress. Nocicep0on Research in Guinea Pigs •  Current published methods include: –  modified Randall and Selico –  von Frey –  hot plate Benefits and Limita0ons of Nocicep0ve Tes0ng •  Benefits: –  Gold Standard in analgesia efficacy tes0ng •  Limita0ons: –  Measure reflexive pain •  Hyperalgesia •  Allodynia –  May not reflect spontaneous or post-­‐op pain –  Not prac0cal for clinical evalua0on of pain Modeling Clinical Pain •  Post-­‐surgical pain: OVH, Castra0on, Laparotomy –  Most frequently used for development of Behavioral Ethograms and Grimace Scales (Pain Faces). •  More relevant to detec0on of spontaneous and post-­‐opera0ve pain than injec0on or applica0on of noxious s0mulus. How can we iden0fy clinical pain ? •  Clinical signs: unkempt hair coat, dehydra0on, weight & body condi0on loss –  non-­‐specific and retrospec0ve •  Observed ac0vity: –  Natural tonic immobility (freezing response) to stress •  Need: –  Validated methods to iden0fy guinea pigs with unalleviated pain Possible Clinically Relevant Metrics… •  Observe for new pain behaviors – Subtle, require observa0on of animal – More specific to pain •  Observe for loss of normal behaviors – More obvious than new pain behaviors. – Can be detected using “proxy indicators” – Can be influenced by a mul0tude of factors – pain, illness (fever, nausea), phenotype. Addi0on of new pain behaviors •  Pain Faces •  Body Posture Pain Faces in Humans Pain Faces in Mice •  Langford, et al. 2010. •  5 Facial Ac0on Units •  Each unit scored on a basis of no pain (0), mild to moderate pain (1), severe pain (2). •  Correlated with tradi0onal pain measures used in the laboratory. Mouse Pain Faces are useful for detec0ng Post-­‐Opera0ve Pain •  Leach et al, 2012. Most Painful Analgesia 1 hr post vasectomy Changes in Body Posture in Mice •  In response to abdominal pain, mice demonstrate abdominal contrac0ons, onen accompanied by extension of the hind limbs • 
• 
hcp://www.ahwla.org.uk/site/tutorials/RP/RP09-­‐writhe480/RP09-­‐writhe1.html hcp://www.ahwla.org.uk/site/tutorials/RP/RP09-­‐sian480/RP09-­‐sian1.html
Challenges to observing pain: •  Rodents and rabbits are prey species -­‐ it is central to their survival that they not display painful behaviors. •  -­‐Do NOT exhibit the familiar ‘fight or flight’ response, instead they exhibit ‘Conserva0on withdrawal’ response. –  to many observers a very painful rodent appears to be “QAR, sleeping/res0ng comfortably” What we see… What they perceive… Iden0fying methods to iden0fy pain? •  Observa4on of new pain behaviors – Subtle, require observa0on of animal – More specific to pain •  Observe loss of normal behaviors – More obvious than new pain behaviors. – Can be detected using “proxy indicators” – Can be influenced by a mul0tude of factors – pain, illness (fever, nausea), phenotype. Loss of Normal Behaviors Not interested in play Not taking treats Not interac0ng with cohorts, less ac0ve Can’t get comfortable, not res0ng Loss of Normal Behaviors: think Maslow Organizing cage, nes0ng Taking treats, running on a wheel Sleeping with cohorts Grooming Eat, Drink As PAIN increases, animal will stop performing higher order behaviors such as nes0ng, socializing, grooming, and eventually if severe enough, stop ea0ng and drinking (primal needs). Scoring Nest Complexity Jirkof, et al. 2013 Latency to nest building •  3 days acclima0on to provision of piece of nestlet •  Within 10 minutes 100% of healthy mice across 9 strains would retrieve material and incorporate into their nest. –  1 strain took longer. •  Took 2 days post-­‐surgery to return to baseline TINT scores. Measuring pain in guinea pigs Behavioral Challenges to Overcome •  Guinea pigs have individual personali0es – some very ac0ve, others barely move. Behavior Confounder •  Significant inter-­‐
animal varia0on in behavior while in observa0on cage. •  Animals serve as their own controls in a longitudinal study. Solu0on Behavioral Challenges to Overcome • Guinea pigs have a significant freeze response to anything new. Behavior Confounder •  Will remain mo0onless in a new seung or with new people. •  Daily acclima0on •  2 weeks prior •  Every day during Solu0on Behavioral Challenges to Overcome •  Guinea pigs have individual responsiveness to touch. Behavior Confounder •  Varia0on in response to von Frey probe. •  Compare a painful (peri-­‐incisional) and non-­‐painful area of the body. Solu0on Behavioral Challenges to Overcome Confounder • Guinea pigs are easily stressed. Behavior • Anesthesia could significantly alter behavior • Anesthesia only condi0on. Solu0on Behavioral Challenges to Overcome Confounder • Wild guinea pigs are crepuscular Behavior • Behavior could alter depending on 0me of day • Include a crepuscular 0me point Solu0on Male guinea pig study 0me line Acclima0on (2 weeks) Anesthesia Condi0on Washout (2 weeks) N = 10 pigmented males At 2, 8, 24 hours aner anesthesia/surgery (1pm, 7pm, 11am) Evaluated by eVF, remote video, proxy indicators Post-­‐
Castra0on Condi0on Methods: Guinea Pig Behavioral Ethogram Category Inclusive Behaviors Movement Forward or backward mo0on, turning the body or head, head or neck extensions, ambula0on, and if present, interac0on with enrichment Chew May or may not be observed with coprophagy behavior Bite or Lick Bars Bite or lick mesh floor of observa0onal cage Pain Behaviors Abdominal tuck, twitching, and weight shining Methods: Electronic von Frey (eVF) •  Measures peak force in grams •  2 areas: peri-­‐incisional caudal abdomen (Ca) and cranial abdomen (CrAb) •  Threshold differen0al= CrAb-­‐Ca •  Repeated 3x with washout period Methods: Proxy Indicators Time to Hut (TTH) Time to Consump0on of a preferred treat (parsley) Results: Subtle body movement behaviors increased aner surgery Results: Mechanical threshold decreased aner surgery Proxy indicators failed to delineate between anesthesia and surgery condi0ons. Time to Consump0on (of preferred treat) Time to Hut entry Male Guinea Pig Findings •  ↓eVF threshold and ↑pain behaviors aner surgery validates novel behavioral ethogram –  Playorm for analgesia efficacy studies and clinical use •  Proxy indicators (TTC and TTH) failed to iden0fy pain. –  Highlights challenge of accurately iden0fying pain behaviors cage-­‐side in this species BUT… Pain in Female Guinea Pigs aner Surgery •  10 intact adult albino female guinea pigs •  Op0mize behavioral tes0ng units to increase quality of image. Observa0on cages: external view HD Cameras Black mace paper to reduce reflec0on Angled mirrors for visualiza0on during von Frey Observa0on cages: inside Intra-­‐cage ligh0ng increases visibility of fine details. Mace black plas0c to reduce glare and increase contrast with albino guinea pigs. What the camera sees Pigmented vs. Albino Condi0ons & Methods •  Condi0ons: –  Baseline –  Anesthesia (isoflurane) plus asep0c prep •  Shave and prep skin with alterna0ng iodine scrub and alcohol –  Anesthesia & hysterectomy surgery Acclima0on (2 weeks) Baseline Anesthesia Condi0on Washout (2 weeks) At 2, 8, 24, 32, 48 hours aner anesthesia/surgery (1pm, 7pm, 11am, 7pm, 11am) Evaluated by eVF, remote video, proxy indicator Post-­‐
Hysterectomy Condi0on Hysterectomy Methods •  Metrics: –  Behavioral Ethogram –  Grimace Score –  Proxy Indicator – TTC (with increased challenge) –  Electronic von Frey (eVF) Behavioral ethogram Ac0ve Behaviors Mouth Behaviors Passive Behaviors Scoring Behavior Proxy indicators Electric von Frey Probe gently applied perpendicular to the incision •  Increasing pressure slowly applied un0l the guinea pig responds (jump, vocalize, or moves away) •  3 measurements taken, averaged •  To avoid sensi0za0on, a 5-­‐minute rest period elapsed between probe applica0ons eVF Results: Increased sensi0vity aner surgery Post-­‐Surgery: decreased ac0ve behaviors, increased passive behaviors Possible pain behaviors iden0fied: Female Guinea Pig Results •  Significantly increased sensi0vity for at least 48 hours aner surgery. •  However, by TTC and post-­‐opera0ve veterinary exam animals appeared normal by 48 hours. –  Underscores difficulty in accurately iden0fying presence of pain on cage-­‐side exam •  Videos currently being scored by ethogram –  Early results indicate increased pain behaviors •  Grimace scale –  Colleagues in Newcastle currently reviewing video Female vs. Male differences? Strain differences! Use male albino guinea pigs for comparison. Male vs. Female EVF Behavioral differences? •  TBD… –  Full male and female ethogram and grimace scale pending; to be completed by end of Summer 2015. •  Need to assess differences in responses to analgesia –  GLAS funded analgesia efficacy study found buprenorphine and buprenorphine plus carprofen was best analgesia regimen for post-­‐castra0on pain in males. –  Females may need 48 hours or more of analgesia post laparotomy. Thank You! •  The ACLAM Founda0on •  Refinement and Enrichment Advancements Lab –  Dr. Jean Nemzek –  Chris Fry •  Unit for Laboratory Animal Medicine, University of Michigan •  ULAM Residents: –  Jennifer Beninson, Jane Na, and Misha Dunbar •  ASLAP Veterinary Students: –  Stephanie Spignesi and Marian Aline •  University of Michigan Pre-­‐veterinary Students: –  Emily David, Jonathan Zhou, Maddie Hileman, Alec Wishie, Courtney Wright, Olivia Gignilliat