Optimized surgical techniques and postoperative care
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1 Optimized surgical techniques and postoperative care Margarete Arras PD Dr. med. vet. DipECLAM University Hospital Zurich Center for Clinical Reserach Division of Surgical Research
2 Optimized surgical techniques and postoperative care Surgery - Optimized anaesthesia - Pre-emptive analgesia - Anti-infective prophylaxis - Experienced personnel - Surgical skills per se Postoperative care Pain alleviation in laboratory mice - Pain killers Supportive measures - warmth, fluid, housing conditions, Humane endpoints Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
3 The laboratory mouse represents probably 70% of laboratory animals used in biomedical research and testing thousands of genetically modified lines/models available Examples for the need of surgery in research Organ transplantation liver lung stem cells Open chest surgery myocardial infarction Stroke Physiology Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
4 Agenda: Pain alleviation in laboratory mice Problems detection of pain in mice treating pain in mice Example: Use of a minor surgery model for assessing (1) postsurgical pain and (2) the efficacy of the pain relief regimen Read-out: Clinicial investigation: cage-side observations, body weight, food consumption Telemetry: Heart rate, heart rate variability Natural behaviors: Nest building, burrowing Where are we now? Next steps Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
5 Detection of postoperative pain in mice Examples (video recordings): No effect of pain killers visible by cage-side observations of typical clinical signs Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
6 Pain treatment Claire A. Richardson and Paul A. Flecknell; ATLA 33, p , 2005 Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
7 Detecting ongoing pain by its effects on body weight? food, water? pain spontaneous behavior? Clinical symptoms; appearance? experimental or GM induced changes? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
8 Clinical signs Appearance fur - rough, soiled, hair loss, piloerection body - weight loss - decreased food intake - sunken abdomen - hunched posture - sunken eyes - dehydration Behavior aggressive - biting response; automutilation apathy - unresponsive; isolation from the cage mates increased activity - restlessness; sudden running movements prolonged times for sleeping or resting Constraint = fear, distress, suffering, pain, disease, Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
9 Food intake after surgery Decrease of food consumption after surgery Alteration of body weight, water intake, wheel runing activity Adamson 2010, Bourque 2010, Arras 2007, Bomzon 2006 Side-effects Example (picture): Pica behaviour in the rat from Buprenorphine (Opioid) high dosage? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
10 Detecting ongoing pain by its effects on body weight? food, water? pain pain killers behavior? appearance? experimental or GM induced changes? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
11 Obstacles in pain relief regimens Table from Claire A. Richardson & Paul A. Flecknell, ATLA 33, p , 2005 Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
12 Pain alleviation in mice Opioid e.g. Burpenorphine NSAID e.g. Carprofen, Meloxicam, Metamizol, 0.1 mg/kg BW, s.c., 2/die 5 mg/kg BW, s.c., 1-2/die? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
13 Problems of pain alleviation Difficulties in detecting pain in the mouse Side-effects of analgesics sometimes masking symptoms No control of success efficacy of pain treatment remains questionable Regimens (agents, dosages, application intervals) mostly rely not on systematic studies or scientific investigations Knowledge about efficient pain treatment in mice: Evidence-based regimens = agents, dosages and intervals Animal-friendly application routes Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
14 Agenda: Pain alleviation in laboratory mice Problems detection of pain in mice treating pain in mice Example: Use of a minor surgery model for assessing (1) postsurgical pain and (2) the efficacy of the pain relief regimen Read-out: Clinicial investigation: cage-side observations, body weight, food consumption Telemetry: Heart rate, heart rate variability Natural behaviors: Nest building, burrowing Where are we now? Next steps Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
15 Recovery of mice after minor surgery with and without pain killers (NSAID: Carprofen, Flunixin) Telemetric measurements preliminary implantation of transmitters Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
16 Change of heart rate Δ heart rate [bpm] * * * * without pain therapy (n=8) with Carprofen (n=8) with Flunixin (n=8) 0 1 [day] 2 3 * p s.c. injection (vehicle/analgesic) Vasectomy * p=0.003 Control groups (anesthesia + pain therapy only) not shown Arras, M. et al. BMC Vet Res; 3:16 (2007) Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
17 Time domain analysis of heart rate variability: IBI 10 5 ΔInterbeat interval [ms] 0 without pain therapy (n=8) * * with Carprofen (n=8) with Flunixin (n=8) s.c. Injection (vehicle/analgesic) Vasectomy 1 [Tag] 2 3 Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec * p Control groups (anesthesia + pain therapy only) not shown Arras, M. et al. BMC Vet Res; 3:16 (2007)
18 Time domain analysis of heart rate variability: SDANN ΔSDANN [ms] without pain therapy (n=8) with Carprofen (n=8) * * with Flunixin (n=8) [Tag] 2 3 * p s.c. Injection (vehicle/analgesic) Vasectomy Control groups (anesthesia + pain therapy only) not shown Arras, M. et al. BMC Vet Res; 3:16 (2007) Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
19 Body weight and food consumption [%] [%] * * * * * Body weight Food intake [hours] without pain therapy with Carprofen 5 mg/kg, 2/die, 24 h with Flunixin 5 mg/kg, 2/die, 24 h Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec (n=8) * p Control groups (anesthesia + pain therapy only) not shown Arras, M. et al. BMC Vet Res; 3:16 (2007)
20 Recovery of mice after minor surgery with and without pain killers (NSAID: Carprofen, Flunixin) Attenuation of aberrations in heart rate and heart rate variability with analgesia Inhibition of the reduction in body weight (~2%) and food consumption (~20%) with analgesia Clinical investigation: no difference in outer appearance, posture, spontaneous behaviors Arras et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
21 Recovery of mice after minor surgery with and without pain killers (NSAID: Carprofen, Flunixin) with pain treatment without pain treatment Arras et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
22 Nature Protocols 1, (2006) Burrowing in rodents: a sensitive method for detecting behavioral dysfunction Robert M J Deacon mice, rats, hamsters, gerbils home cage based read out: aberration of natural, highly motivated behavior Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
23 Burrowing Species-specific, spontaneously occurring behaviour High motivation to dig burrows persists during domestication Deacon et al > neurodegenerative diseases Useful as indicator for impaired wellbeing??? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
24 Hypothesis: Abberration of a natural behavior is an indicator of pain, constraint, or impaired well-being Aim: Changes in burrowing behavior as indicator of pain after minor surgery Pre-study burrowing test Time of testing (circadian rhythm), Setup (apparatus), Sex bias, Strain bias C57BL/6J, DBA/2J, 129Sv/Ev-IFNabRtmAgt Study design 32 male & 32 female adult C57BL/6J Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
25 Burrowing test: study design acclimatisation individual housing with burrowing apparatus baseline measurement experimental measurement n = 16 n = 16 surgery + anaesthesia surgery + anaesthesia + analgesia n = 16 anaesthesia n = 16 anaesthesia + analgesia day 0 day 1 day 2 day 3 day 4 day 5 day 6 video recording video recording Jirkof et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
26 Burrowing test: study design Surgical intervention Injection of analgesia (+/- carprofen, 5 mg/kg BW, s.c.) transport (5 min) inhalation anaesthesia (sevoflurane, 15 min) +/- one side minor laparotomy recovery on warming mat (at least 15 min) Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
27 Burrowing test: set up Experimental Setup. Burrowing test apparatus (A), shelter (B) and nesting material (C) in home cage. Jirkof et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
28 Burrowing test: analysis Before & after treatment Video recording Analyses: software based, ObserverXT (Noldus) Burrowing parameter: latency to burrow duration of burrowing weights of removed pellets after 2h Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
29 Results: burrowing performance Almost all (98 %) healthy mice burrowed during baseline measurement Example: infrared video recording of burrowing. All parameters (latency to burrow, duration of burrowing, weight of removed pellets) responded to the different experimental treatments. Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
30 Results: burrowing performance fraction of animals starting burrowing [%] baseline surgery + anaesthesia surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia time [h] Jirkof et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
31 Results: baseline vs experimental Increase in latency & duration of burrowing after treatment latency to burrow duration of burrowing (h) surgery + anaesthesia surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia (h) surgery + anaesthesia surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
32 Results: baseline vs experimental weight of removed pellets after 2h (gram) surgery + anaesthesia surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
33 Results: group comparisons 1.0 Time to effect: Kaplan-Meier analysis probability of non-burrowing Procedural control treatments did not differ in latency time [h] surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia Jirkof et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
34 Results: group comparisons A B C probability of non-burrowing p = χ 2 = p = χ 2 = probability of non-burrowing probability of non-burrowing p = χ 2 = probability of non-burrowing probability of non-burrowing p = χ 2 = time [h] [h] time [h] [h] time [h] time [h] surgery + anaesthesia surgery + anaesthesia surgery + anaesthesia surgery + anaesthesia + analgesia anaesthesia anaesthesia + analgesia Surgery without pain treatment causes significant longer latencies Jirkof et al Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
35 Burrowing test: summary & conclusion Easy to perform in the animals home cage under routine laboratory conditions Burrowing parameters react sensitive on treatments Results allow to discriminate between groups of mice that are treated with a pain killer or not before surgery Results imply a balance between the motivation to burrow and an individuals level of constraint experienced In conclusion, postsurgical impairment, mainly attributable to pain, can be assessed on the basis of the burrowing test Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
36 Where are we now? Currently: new approaches and methodologies for the assessment of pain and constraint in mice Conditioned place preference Frank Porreca [King et al. 2009] Facial expression (mouse grimace scale) Jeff Mogil [Langford et al. 2010] General changes in behaviors (rearing, climbing, etc.) van Loo et al. 2007, Roughan et al Abberrant behaviors (e.g. twitching, flinching, ) Paul Flecknell [Roughan et al. 2009] Drug self administration Vera Baumans [Pham et al. 2010] Alterations of natural behaviors Jirkof et al Sympathetic reaction (telemetry) Arras et al Ultrasound vocalisations? Feasibility, validity, benefits, limitations in routine laboratory conditions Reliability of specific tests for estimating the efficacy of pain relief regimens Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
37 Long-term goals Utilization of new tests for verifying pain relief regimens which are in use to-date Establish efficient, evidence-based modern pain relief regimens Sustained-release drugs - Foley et al Administration of analgesia via drinking water, food, or mini-pumps Multimodal analgesia protocols Is there a market for analgesics or other drugs for use in mice??? Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
38 Thank you for your attention Paulin Jirkof Andreas Rettich Nikola Cesarovic Thea Fleischmann Beat Schuler Paolo Cinelli Robin Schneider Kurt Bürki Gregor Zünd Our work was supported by Federal Veterinary Office Switzerland; UBS Foundation; ESLAV and ECLAM foundation, 3R research foundation Switzerland and Central Biological Laboratory and Division of Surgical Research at University Hospital Zurich; Institute of Laboratory Animal Science, University of Zurich. Welfare of Laboratory Animals Role of Refinement? Berlin 13 Dec
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