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Home , Climazolam

Bundesinstitut für Risikobewertung Charite – Universitätsmedizin Berlin Gesellschaft für Versuchstierkunde

38. Seminar über Versuchstiere und Tierversuche Berlin, 27. Mai 2009

Schmerzbehandlung und Anaesthesie bei Labormäusen: Trends und Probleme aus der Praxis

Margarete Arras PD Dr. med. vet. DipECLAM

Institute of Laboratory Animal Science Vetsuisse Faculty University Zurich Goals Anesthesia – species-specific problems in mouse anesthesia – types of anesthesia • injection anesthesia • inhalation anesthesia ¾examples, problems ¾optimized protocols Pain therapy – decide on pain therapy – drugs, side effects – dosages ¾intensive post-operative care in mice Species-specific problems in mouse anesthesia the mouse the drugs – 18 – 50 g body weight – feasibility ® – hypothermia , , di-ethyl-ether, … – availability – access to arteries, veins InnovarVet® > off the market – injection side Hypnorm® > narcotics laws, import from UK i.p., s.c. > off the market? – monitoring Metofane® > import from Australia blood pressure – regulations for protection of the personnel heart rate, ECG secure gas scavenging pulsoximetry – legal restrictions from welfare concerns blood gases and acid base balance Avertin®, di-ethyl-ether, … – intervention cardiac arrest respiratory depression Species-specific problems in mouse anesthesia ® Zeller, W., Meier, G., Bürki, K., Panoussis, B.: Adverse Avertin effects of as used in the production of Tribromoethanol transgenic mice. Laboratory animals, 1998, 32, 407-13 Lieggi, C.C., Fortman, J.D., Kleps, R.A., Sethi, V., Anderson, J.A., Brown, C.E. Artwohl, J.E.: An evaluation of preparation methods and storage conditions of tribromoethanol. Contemporary topics in laboratory animal science, 2005, 44/1, 11-16

Lieggi, C.C., Artwohl, J.E.Leszczynski, J.K., Rodriguez, N.A., Fickbohm, B.L., Fortman, J.D.: Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice. Contemporary topics in laboratory animal science, 2005, 44/1, 17-22

Chu, D.K., Jordan, M.C., Kim, J.K., Couto, M.A., Roos, K.P. Comparing with tribromoethanol anesthesia for echocardiographic phenotyping of transgenic mice. Journal of the American Association for Laboratory Animal Science, 2006 45(4): 8-13

Web page of the german society for laboratory animal science:Stellungnahme des Auschuss für Anesthesie und Analgesie des GV-SOLAS zur Anaesthesie von Labormäusen mit Tribromethanol, www.gv-solas.de Types of anesthesia: route of application Injection Inhalation ease of application application –i.p. – induction chamber –s.c. – face mask special equipment: none special equipment environmental impact: no environmental impact ? cheap ? dangerous for personnel ? not controllable expensive ? controllable

Isoflurane Mono-anesthetics e.g.

Halothane Combination of Combination of dissoziative + alpha-two agonist opioid + alpha-two agonist e.g. ketamin + e.g. fentanyl + medetomidine Di-ethyl-ether Injection anesthesia Combination of substances synergistic action tranquilizer – adverse effects decreased α2-agonist – desired effects increased – analgesia strengthened opioid dissoziative examples – neurolept-analgesia ¾fentanyl + fluanison (Hypnorm®) + medetomidine ¾fentanyl and medetomidine can be antagonized ¾fentanyl + + medetomidine ¾all components can be antagonized [from J. Henke, Munich] – ketamin + xylazine (Rompun®) [+ acepromazine] Table 1. Details of published i.p. injection anesthesia protocols for the mouse. Substances Range of Substance class Avail- Handlingb Lack of toxicityc Efficacyd References dosages abilitya [mg/kg] Mono-anesthetics Pentobarbi- 30 – 90 – n+ – narrow safety margin – analgesia insufficient, long 3, 5, 7, 13, 14, tone sleeping time 15, 16 α- 114 ––difficult to dissolve in saline – matter of controversy – no surgical tolerance 5 hy- 60 - 400 – n – not available in sterile form, – high mortality, intestinal com- – controversial: analgesia 5, 16 drate light sensitive, air sensitive plications, out of use today insufficient 23.7 - 33 Non-barbiturate – c+ – tissue irritant – only hypnosis, no analge- 5, 23, 24 sic properties Combinations of anesthetics Ketamine 50 – 200 + + * analgesia, no relaxation 5, 12, 13, 14, 15, + Xylazine 5 – 20 α2-Agonist * analgesia, sedation 16 Ketamine 75 Dissociative + + * analgesia, no relaxation 14, 15, 18, 19, + Medeto- 1 α2-Agonist + + * analgesia ?, sedation ? 20, 21 midine relaxation ? Ketamine 100 Dissociative + + * analgesia, no relaxation 16, 17 + Azaperone 75 Butyrophenone Tiletamin/ 7.5 – 100 Dissociative/ + + * Telazol alone: only immo- 5, 7 bilization (Telazol™) +Xylazine 7.5 – 45 α2-Agonist + + * analgesia, sedation Fentanyl/ 0.33 – 3.3 Opioid/ – n, c – precipitates out of solution, * neuroleptanalgesia Fluanison [ml/kg] Butyrophenone difficult to mix with other (Hypnorm™) drugs, storage of mixtures not possible 5, 14, 15, 16, 22, + Midazolam 12.5 – 16.6 Benzodiazepine – c * sedation 26 or + Diazepam 5 Benzodiazepine + + * sedation 5, 14 Fentanyl/ 0.0001 – Opioid / – n, + * neuroleptanalgesia 5, 16 droperidol 0.001 Butyrophenone c, o (Innovar- [ml/g] Vet™) + Diazepam 5 Benzodiazepine + + * sedation Fentanyl 0.06 - 0.08 Opioid – n, c + * analgesia 14, 15, 24 + 60 Non-barbiturate – c+ – tissue irritant; hypnosis hypnotic high mortality? Fentanyl 0.08 Opioid – n + * analgesia 15, 23, 24 + Etomidate 18 Non-barbiturate – c+ – tissue irritant hypnosis hypnotic Carfentanyl 0.003 Opioid – n+ – side effects: excitations, analgesia 5, 13, 23, 24 muscle spasms + Etomidate 15 Non-barbiturate – c+ – tissue irritant hypnosis hypnotic a + = commercially available; n = legal restrictions in some countries (narcotics act); c = not available in some countries; o = off the market. b + = available in sterile form, easy to dilute or mix with other drugs, chemically stable, can be stored c Toxicity defined as mortality, tissue irritancy, other side effects. Safety margin = effective dose vs. toxic dose. * = no relevant experimental data in the mouse were found. d Efficacy defined in terms of the 3 components of anesthesia: analgesia, hypnosis, and muscle relaxation from Arras et al. Comp Med 2001 Sensitivity to injectable anesthetics influenced by...

strain, sex, age circadian rhythm, health, •Rumke, C. L., and J. Noordhoek. 1969. Sex differences in the duration of narcosis and in serum MUP content in mice. Arch Int pregnancy, socio-physiological Pharmacodyn Ther 182(2):399-400. •Green, C. J. 1979. Chapter 1: General principles, p. 9-16. In C. J. Green conditions, adaptation (ed.), Animal Anaesthesia, Laboratory animal handbooks 8. Laboratory •Davis, W. M. 1962. Day-night periodicity in pentobarbital response of mice and the Animals Ltd., London. influence of socio-physiological conditions. Experientia 18:235-237. •Lovell, D. P. 1986. Variation in pentobarbitone sleeping time in mice. 1. •Green, C. J. 1979. Chapter 1: General principles, p. 9-16. In C. J. Green (ed.), Animal Strain and sex differences. Lab Anim 20(2):85-90. Anaesthesia, Laboratory animal handbooks 8. Laboratory Animals Ltd., London. •Lovell, D. P. 1986. Variation in pentobarbitone sleeping time in mice. 2. • Furukawa, S., M. J. MacLennan, and B. B. Keller. 1998. Hemodynamic response to Variables affecting test results. Lab Anim 20(2):91-96. anesthesia in pregnant and nonpregnant ICR mice. Lab Anim Sci 48(4):357-363. •Cruz, J. I., J. M. Loste, and O. H. Burzaco. 1998. Observations on the use of medetomidine/ketamine and its reversal with atipamezole for chemical restraint in the mouse. Lab Anim 32(1):18-22. •Homanics, G. E., J. J. Quinlan, and L. L. Firestone. 1999. Pharmacologic and behavioral responses of inbred C57BL/6J and strain 129/SvJ mouse housing conditions lines. Pharmacol Biochem Behav 63(1):21-26. Dairman, W., Balaszs, T. 1970. Comparison of liver microsome enzyme systems and •Zambricki, E. A., D’Alecy, L. G. 2004. Rat sex differences in anesthesia. barbiturate sleeping times in rats caged individually or communally. Biochemical Comp Med 54 (1): 49-53 Pharmacology 19:951-955 • Avsaroglu, H, van der Sar, A.S., van Lith, H.A., van Zutphen, L.M.F., Einon, D., Stewart, J., Atkinson, S., Morgan, M. 1976. Effect of isolation on Hellebrekers, L.J. 2007. Differences in the response to anaesthetics and barbiturate anesthesia in the rat. Psychopharmacology 50:85-88 analgesics between inbred rat strains. Lab Anim 41:337-344 Watanabe, H., Ohdo, S., Ishikawa, M., Ogawa, N. 1992. Effects of social isolation on pentobarbital activity in mice: relationship to racemate levels and enantiomer levels in brain. Journal of Pharmacology and Experimental Therapeutics 263:1036-1045

genetic modification •Quinlan, J. J., G. E. Homanics, and L. L. Firestone. 1998. Anesthesia sensitivity in mice that lack the beta3 subunit of the gamma- aminobutyric acid type A receptor. Anesthesiology 88(3):775-780. •Xie, W., J. L. Barwick, M. Downes, B. Blumberg, C. M. Simon, M. C. Nelson, B. A. Neuschwander-Tetri, E. M. Brunt, P. S. Guzelian, and R. M. Evans. 2000. Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435-439. •Jurd, R., Arras, M., Lambert, S., Drexler, B., Siegwart, R., Crestani, F., Zaugg, M., Vogt, K.E., Ledermann, B., Antkowiak., B., Rudolph, U. 2003. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. Faseb J 17(2): 250-2 •Takei, T., Saegusa, H., Zong, S., Murakoshi, T., Makita, K., Tanabe, T. 2003. Increased sensitivity to halothane but decreased sensitivity to propofol in mice lacking the N-type Ca channel. Neuroscience letters 350: 41-45 Percentage of mice reaching surgical tolerance and survival rate

100 n=20 Stock Hsd:NMRI male 80 age 3-6 months

60 Ket = ketamin [%] Xyl = xylazine 40 Ace = acepromazine Aza = azaperone 20 Tel = Telazol® (tiletamine + 0 KetKet 100 100 KetKet150 150 KetKetamin 100 KetKetamin 100 TelazolTel 80 KetKetamin 100 KetaminKet 100 KetaminKet 100 zolazepam) Xylazin Xylazin Xylazin Azaperon Medetomidin Medetomidin Xyl 20Xyl 20 XylXyl 30 30 AcepromazinXyl 20 AzaperonXyl 20 Xyl 20 Aza80mg 80 Med1mg 1 5mgMed 5 Med = medetomidine Ace3mg 3 Aza5mg 5

surgical tolerance survival rate dosages in mg/kg bodyweight Time of surgical tolerance and immobilization

n=20 Stock Hsd:NMRI 420 surgical tolerance male 360 age 3-6 month immobilization 300 Ket = ketamin 240 time Xyl = xylazine [min] 180 Ace = acepromazine 120 Aza = azaperone 60 Tel = Telazol® (tiletamine + 0 Ket 100 Ket 150 Ket 100 Ket 100 Tel 80 Ket 100 Ket 100 Ket 100 zolazepam) Xyl 20 Xyl 30 Xyl 20 Xyl 20 Xyl 20 Aza 80 Med 1 Med 5 Med = medetomidine Ace 3 Aza 5

dosages in mg/kg bodyweight Summary

ketamin + xylazine – surgical tolerance: 25 min – immobilization: approx. 2 hours

ketamin + xylazine + acepromazine – surgical tolerance: 50 min – immobilization approx. 2 hours – low death rate – acceptable safety margin

for results and details, see Arras et al. Comp Med, 2001 example Improved ketamin xylazine anesthesia

Surgical tolerance: 50 min Restraint: 120 min

recommended dosage Ketamin 65 mg/kg bodyweight Xylazine 13 mg/kg bodyweight Acepromazine 2 mg/kg/bodyweight for results and details, see Arras et al. Comp Med, 2001 Example for neuroleptanalgesia with antagonization

Dosierung VAA Kleinsäuger (in mg/kg) Julia Henke, Wolf Erhardt

Kaninchen Meerschweinchen Chinc hilla Ratte Hamster Gerbil Maus i.m. i.m. i.m. i.m. i.p. s.c. i.p.

e Fentanyl 0,02 0,025 0,02 0,005 0,033 0,03 0,05

esi

h t

äs Midazolam 1,0 1,0 1,0 2,0 3,3 7,5 5,0

n A

Medetomidin 0,2 0,2 0,05 0,15 0,33 0,15 0,5

g n

u Naloxon

r 0,03 0,03 0,05 0,12 0,8 0,5 1,2

e

i

s i

n 0,1 0,1 0,1 0,2 0,33 0,4 0,5

o

g

a t

n Atipamezol 1,0 1,0 0,5 0,75 1,7 0,375 2,5 A

Antagonisierung routinemäßig s.c., in Notfällen i.v. mit halber Dosis Statt Midazolam auch Climazolam, nicht Diazepam, statt Flumazenil auch Bei jungen und leichten Meerschw. evtl. auch Flumazenil weglassen Bei Be darf 1/3 de r Ausgangsdosis nachdosieren Zwergkaninchen nur 2/3 der Dosis aus: Erhardt, Henke, Lendl: Narkosenotfälle, ENKE 2002 example Short intraperitoneal injection anesthesia

Surgical tolerance: 15 min Restraint: 30 min

Propofol 75 mg/kg bodyweight Medetomidine 1 mg/kg bodyweight Fentanyl 0.2 mg/kg bodyweight

from Alves, HC, Valentim, AM, Olsson, IAS, Antunes, LM Laboratory Animals, 2009, 43: 27-33 General drawbacks

Injection anesthesia in mice – narrow safety margin – special care: hypothermia

Volatile anesthetics: halothane, isoflurane – affect fertility? – mutagenic?, teratogenic? – hepatotoxicity? Isoflurane: long-term exposition of staff, health risks from daily work with Isoflurane

¾ pregnant women should stay away from rooms in which inhalation anaesthesia is performed ¾ for pregnant women it is prohibited to work in rooms, in which halothane is used Consider specific regulations if working with volatile anesthetics!!!

Recommended specific literature, job security: Umgang mit Anästhesiegasen; Gefährdung, Schutzmassnahmen Schweizerische Unfallversicherungsanstalt, Abteilung Arbeitsmedizin, Postfach, 6002 Luzern Tel.: 041 419 51 11, Fax 041 419 58 28 Contemporary anesthesia protocol for short- and long- term interventions

Sevoflurane, 4% - 8% via face mask death rate: <1% costs: < 10 CHF/ h anesthesia inhalation anesthesia machine pressure reducing flow meter 1L valve

nose mask vaporizer filter for isoflurane,

O2 sevoflurane

O2

oxygen pump

power supply Optimization of inhalation anesthesia in laboratory mice (balanced anesthesia)

Injection of fentanyl 0.4 mg/kg + midazolan 4 mg/kg, s.c., at 10 to 15 minutes prior to induction with volatile anesthetics, e.g. sevoflurane (3.5%),or isoflurane

Injection of ketamin 30 mg/kg body weight subcutanously, at 10 to 15 minutes prior to induction with volatile anesthetics, e.g. sevoflurane (4.9%), or isoflurane How to decide on pain therapy experimental design – degree of pain – duration of pain animal species select an – application route – frequency of analgesic application drug interference with the experiment Types of analgesic drugs Opioids NSAID (acting mainly on the central (peripheral action) nervous system) anti-inflammatory severe pain anti-pyrogenic availability? side effects side effects – respiratory depression – inhibition of platelet – obstipation aggregation! – rat: pica behavior if – long-term application: kidney overdosed, = rat eats function decreased bedding, papers, towels – stomach ulceration etc.! – bleeding in the gastro- – Buprenorphin: intestinal tract behavioural abberations Trends in rodent pain therapy

Table from Claire A. Richardson and Paul A. Flecknell: Anaesthesia and post- operative Analgesia following experimental surgery in laboratory rodents: Are we making progress? ATLA 33, p. 119-127, 2005 Dosages of NSAID for mice, taken from literature

Carprofen Meloxicam Meloxicam Flunixin Reference s.c. Per os

2007 10 mg/kg 5 mg/kg www.ahwla.org.uk/site/t s.c. s.c. utorials/RP/RP11- Where.html 2005 10 mg/kg 5 mg/kg Claire A. Richardson s.c. s.c. and Paul A. Flecknell ? daily ? daily ATLA 33, p. 119-127, 2005 2000 5 mg/kg ? ? 2.5 mg/kg Pain Management in s.c.or s.c.; AnimalsbyP. Flecknell by mouth ? 12-24 and A. Waterman- hourly Pearson, Harcourt daily Intern., London, 2000 1996 - 2.5 mg/kg Laboratory animal s.c., i.m.; Anaesthesia, by Paul A. ? 12 hourly Flecknell, Harcourt International, London, 2nd ed. 1996 Intensive care in mice after major surgery prior to surgery –providehigh energy food (e.g. solid drink®-Energy) and glucose 15% in the water bottle during anesthesia/surgery – after induction of anesthesia: injection of 1 mL NaCl 0.9% i.p. – induction of post-operative analgesia at 20-30 minutes before anesthesia is finished, e.g. flunixin 5-7 mg/kg body weight s.c. or buprenorphine 0.1 mg/kg body weight s.c. – prevent hypothermia during anesthesia and in the post-operative phase – put mice back in their home cage, not in a new territory Intensive care in mice after major surgery after surgery (for up to 7 days) – heating pad underneath the cage – oxygen supply in the cage – in 12-hours intervals: flunixin 5 mg/kg BW s.c. (or buprenorphin 0.1 mg/kg BW s.c.) 0.3 mL NaCl 0.9% s.c. and 0.3 mL glucose 5% s.c. – provide high energy food, and food pellets; glucose 15%, in the drinking bottle and in dishes on the cage ground – 1-2 times per day: check body weight, food and water consumption, and the animals outer appearance and moving behavior

Schuler, B. et al., submitted Fluid therapy

Ringer-Lactat or NaCl 0.9% (37°C)

in general 10 ml/kg/h i.v. rat 5-10 ml i.p. during laparatomy mouse 0.5-1.0 ml i.p. during laparatomy 0.5-0.7 ml s.c. after surgery in 12 hour-intervals Thank you for your attention