Schmerzbehandlung und Anaesthesie bei Labormäusen:

Trends und Probleme aus der Praxis

Margarete ArrasPD Dr. med. vet. DipECLAM

Institute of Laboratory Animal Science Vetsuisse Faculty University Zurich

Bundesinstitut für RisikobewertungCharite – Universitätsmedizin BerlinGesellschaft für Versuchstierkunde

38. Seminar über Versuchstiere und TierversucheBerlin, 27. Mai 2009

GoalsAnesthesia

– species-specific problems in mouse anesthesia– types of anesthesia

• injection anesthesia• inhalation anesthesia

examples, problemsoptimized protocols

Pain therapy– decide on pain therapy– drugs, side effects– dosages

intensive post-operative care in mice

the drugs– feasibility

Propofol®, diazepam, di-ethyl-ether, …– availability

InnovarVet® > off the marketHypnorm® > narcotics laws, import from UK Halothane > off the market?Metofane® > import from Australia

– regulations for protection of the personnelsecure gas scavenging

– legal restrictions from welfare concernsAvertin®, di-ethyl-ether, …

the mouse– 18 – 50 g body weight– hypothermia– access to arteries, veins– injection side

i.p., s.c.– monitoring

blood pressureheart rate, ECGpulsoximetryblood gases and acid base balance

– interventioncardiac arrestrespiratory depression

Species-specific problems in mouse anesthesia

Species-specific problems in mouse anesthesia

Zeller, W., Meier, G., Bürki, K., Panoussis, B.: Adverseeffects of tribromoethanol as used in the production of 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 isoflurane 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 animalscience:Stellungnahme des Auschuss für Anesthesieund Analgesie des GV-SOLAS zur Anaesthesie von Labormäusen mit Tribromethanol, www.gv-solas.de

Avertin®

Tribromoethanol

Types of anesthesia: route of applicationInjectionease of application

– i.p.– s.c.

special equipment: noneenvironmental impact: nocheap ?not controllable

Inhalationapplication

– induction chamber– face mask

special equipmentenvironmental impact ?dangerous for personnel ?expensive ?controllable

Mono-anestheticse.g. pentobarbital

Combination ofdissoziative + alpha-two agonist

e.g. ketamin + xylazine

Combination of opioid + alpha-two agonist

e.g. fentanyl + medetomidine

IsofluraneSevofluraneDesflurane

HalothaneEnflurane

MethoxyfluraneDi-ethyl-ether

Injection anesthesiaCombination of substances

synergistic action– adverse effects decreased– desired effects increased– analgesia strengthened

examples– neurolept-analgesia

fentanyl + fluanison (Hypnorm®) + medetomidinefentanyl and medetomidine can be antagonized

fentanyl + midazolam + medetomidineall components can be antagonized[from J. Henke, Munich]

– ketamin + xylazine (Rompun®) [+ acepromazine]

tranquilizer

dissoziative

α2-agonist

opioid

Table 1. Details of published i.p. injection anesthesia protocols for the mouse.Substances Range of

dosages[mg/kg]

Substance class Avail-abilitya

Handlingb Lack of toxicityc Efficacyd References

Mono-anestheticsPentobarbi-tone

30 – 90 Barbiturate – n + – narrow safety margin – analgesia insufficient, longsleeping time

3, 5, 7, 13, 14,15, 16

α-Chloralose 114 – – difficult to dissolve in saline – matter of controversy – no surgical tolerance 5Chloral hy-drate

60 - 400 – n – not available in sterile form,light sensitive, air sensitive

– high mortality, intestinal com-plications, out of use today

– controversial: analgesiainsufficient

5, 16

Etomidate 23.7 - 33 Non-barbituratehypnotic

– c + – tissue irritant – only hypnosis, no analge-sic properties

5, 23, 24

Combinations of anestheticsKetamine 50 – 200 Dissociative + + * analgesia, no relaxation 5, 12, 13, 14, 15,+ Xylazine 5 – 20 α2-Agonist * analgesia, sedation 16Ketamine 75 Dissociative + + * analgesia, no relaxation 14, 15, 18, 19,+ Medeto-midine

1 α2-Agonist + + * analgesia ?, sedation ?relaxation ?

20, 21

Ketamine+ Azaperone

10075

DissociativeButyrophenone

+ + * analgesia, no relaxation 16, 17

Tiletamin/Zolazepam(Telazol™)

7.5 – 100 Dissociative/Benzodiazepine

+ + * Telazol alone: only immo-bilization

5, 7

+Xylazine 7.5 – 45 α2-Agonist + + * analgesia, sedationFentanyl/Fluanison(Hypnorm™)

0.33 – 3.3[ml/kg]

Opioid/Butyrophenone

– n, c – precipitates out of solution,difficult to mix with otherdrugs, storage of mixtures notpossible

* neuroleptanalgesia

5, 14, 15, 16, 22,+ Midazolam 12.5 – 16.6 Benzodiazepine – c * sedation 26or+ Diazepam 5 Benzodiazepine + + * sedation 5, 14Fentanyl/droperidol(Innovar-Vet™)

0.0001 –0.001[ml/g]

Opioid /Butyrophenone

– n,c, o

+ * neuroleptanalgesia 5, 16

+ Diazepam 5 Benzodiazepine + + * sedationFentanyl 0.06 - 0.08 Opioid – n, c + * analgesia 14, 15, 24+ Metomidate 60 Non-barbiturate

hypnotic– c + – tissue irritant;

high mortality?hypnosis

Fentanyl 0.08 Opioid – n + * analgesia 15, 23, 24+ Etomidate 18 Non-barbiturate

hypnotic– c + – tissue irritant hypnosis

Carfentanyl 0.003 Opioid – n + – side effects: excitations,muscle spasms

analgesia 5, 13, 23, 24

+ Etomidate 15 Non-barbituratehypnotic

– c + – tissue irritant hypnosis

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 storedc 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•Rumke, C. L., and J. Noordhoek. 1969. Sex differences in the duration of hexobarbital narcosis and in serum MUP content in mice. Arch IntPharmacodyn Ther 182(2):399-400.•Green, C. J. 1979. Chapter 1: General principles, p. 9-16. In C. J. Green (ed.), Animal Anaesthesia, Laboratory animal handbooks 8. Laboratory Animals Ltd., London.•Lovell, D. P. 1986. Variation in pentobarbitone sleeping time in mice. 1. Strain and sex differences. Lab Anim 20(2):85-90.•Lovell, D. P. 1986. Variation in pentobarbitone sleeping time in mice. 2. Variables affecting test results. Lab Anim 20(2):91-96.•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 lines. Pharmacol Biochem Behav 63(1):21-26.•Zambricki, E. A., D’Alecy, L. G. 2004. Rat sex differences in anesthesia. Comp Med 54 (1): 49-53• Avsaroglu, H, van der Sar, A.S., van Lith, H.A., van Zutphen, L.M.F., Hellebrekers, L.J. 2007. Differences in the response to anaesthetics and analgesics between inbred rat strains. Lab Anim 41:337-344

circadian rhythm, health, pregnancy, socio-physiological conditions, adaptation •Davis, W. M. 1962. Day-night periodicity in pentobarbital response of mice and the influence of socio-physiological conditions. Experientia 18:235-237.•Green, C. J. 1979. Chapter 1: General principles, p. 9-16. In C. J. Green (ed.), Animal Anaesthesia, Laboratory animal handbooks 8. Laboratory Animals Ltd., London.• Furukawa, S., M. J. MacLennan, and B. B. Keller. 1998. Hemodynamic response to anesthesia in pregnant and nonpregnant ICR mice. Lab Anim Sci 48(4):357-363.

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

housing conditionsDairman, W., Balaszs, T. 1970. Comparison of liver microsome enzyme systems and barbiturate sleeping times in rats caged individually or communally. BiochemicalPharmacology 19:951-955Einon, D., Stewart, J., Atkinson, S., Morgan, M. 1976. Effect of isolation on barbiturate anesthesia in the rat. Psychopharmacology 50:85-88Watanabe, 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

Percentage of mice reaching surgicaltolerance and survival rate

[%]

dosages in mg/kg bodyweight

0

20

40

60

80

100

Ket 100

Xyl 20

Ket150

Xyl 30

KetaminXylazin

Acepromazin3mg

KetaminXylazin

Azaperon5mg

TelazolXylazin

KetaminAzaperon

80mg

KetaminMedetomidin

1mg

KetaminMedetomidin

5mg

surgical tolerance survival rate

Ket 100Xyl 20

Ket 150Xyl 30

Ket 100Xyl 20Ace 3

Ket 100Xyl 20Aza 5

Ket 100Aza 80

Tel 80Xyl 20

Ket 100Med 1

Ket 100Med 5

Ket = ketamin

Xyl = xylazine

Ace = acepromazine

Aza = azaperone

Med = medetomidine

Tel = Telazol®

(tiletamine +zolazepam)

n=20Stock Hsd:NMRImale age 3-6 months

Ket = ketamin

Xyl = xylazine

Ace = acepromazine

Aza = azaperone

Med = medetomidine

Tel = Telazol®

(tiletamine +zolazepam)

Time of surgical tolerance and immobilization

dosages in mg/kg bodyweight

time[min]

0

60

120

180

240

300

360

420

Ket 100Xyl 20

Ket 150 Xyl 30

Ket 100Xyl 20Ace 3

Ket 100Xyl 20Aza 5

Tel 80Xyl 20

Ket 100Aza 80

Ket 100Med 1

Ket 100Med 5

immobilization

surgical tolerance

n=20Stock Hsd:NMRImale age 3-6 month

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

Improved ketamin xylazine anesthesia

recommended dosageKetamin 65 mg/kg bodyweightXylazine 13 mg/kg bodyweightAcepromazine 2 mg/kg/bodyweight

for results and details, see Arras et al. Comp Med, 2001

example

Surgical tolerance: 50 minRestraint: 120 min

2,50,3751,70,750,51,01,0Atipamezol

0,50,40,330,20,10,10,1Flumazenil

1,20,50,80,120,050,030,03Naloxon

0,50,150,330,150,050,20,2Medetomidin

5,07,53,32,01,01,01,0Midazolam

0,050,030,0330,0050,020,0250,02Fentanyl

Mausi.p.

Gerbils.c.

Hamsteri.p.

Rattei.m.

Chinchillai.m.

Meerschweincheni.m.

Kanincheni.m.

Antagonisierung routinemäßig s.c., in Notfällen i.v. mit halber DosisStatt Midazolam auch Climazolam, nicht Diazepam, statt Flumazenil auch SarmazenilBei jungen und le ichten Meerschw. evtl. auch Flumazenil weglassenBei Bedarf 1/3 der Ausgangsdosis nachdosierenZwergkaninchen nur 2/3 der Dosis

Anä

sthe

sie

Ant

agon

isie

rung

aus: Erhardt, Henke, Lendl: Narkosenotfälle, ENKE 2002

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

Example for neuroleptanalgesia with antagonization

Short intraperitoneal injection anesthesia

Propofol 75 mg/kg bodyweightMedetomidine 1 mg/kg bodyweightFentanyl 0.2 mg/kg bodyweight

fromAlves, HC, Valentim, AM, Olsson, IAS, Antunes, LMLaboratory Animals, 2009, 43: 27-33

Surgical tolerance: 15 minRestraint: 30 min

example

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

pregnant women should stay away from rooms in which inhalation anaesthesia is performedfor pregnant women it is prohibited to work in rooms, in which halothane is used

Consider specific regulations if working withvolatile anesthetics!!!

Recommended specific literature, job security:Umgang mit Anästhesiegasen; Gefährdung, Schutzmassnahmen Schweizerische Unfallversicherungsanstalt, Abteilung Arbeitsmedizin, Postfach, 6002 LuzernTel.: 041 419 51 11, Fax 041 419 58 28

Sevoflurane, 4% - 8% via face mask

death rate: <1%costs: < 10 CHF/ h anesthesia

Contemporaryanesthesia protocol for short- and long-term interventions

inhalation anesthesia machine

O2

O2

oxygen

flow meter 1L

vaporizerfor

isoflurane,sevoflurane

filter

pump

power supply

pressurereducingvalve

nosemask

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 withvolatile anesthetics, e.g. sevoflurane (3.5%),or isoflurane

Injection of ketamin 30 mg/kg body weightsubcutanously, at 10 to 15 minutes prior to induction with volatile anesthetics, e.g. sevoflurane (4.9%), or isoflurane

experimental design– degree of pain– duration of pain

animal species– application route– frequency of

application

interference with theexperiment

How to decide on pain therapy

select an analgesicdrug

Types of analgesic drugsOpioids(acting mainly on the central nervous system)severe painavailability?

side effects– respiratory depression– obstipation– rat: pica behavior if

overdosed, = rat eatsbedding, papers, towelsetc.!

– Buprenorphin: behavioural abberations

NSAID(peripheral action)anti-inflammatoryanti-pyrogenic

side effects– inhibition of platelet

aggregation!– long-term application: kidney

function decreased– stomach ulceration– bleeding in the gastro-

intestinal tract

Trends in rodentpain therapy

Table fromClaire A. Richardson and Paul A. Flecknell: Anaesthesia and post-operative Analgesiafollowing experimental surgery in laboratoryrodents: Are we makingprogress?ATLA 33, p. 119-127, 2005

Laboratory animalAnaesthesia, by Paul A. Flecknell, Harcourt International, London, 2nd ed. 1996

2.5 mg/kg s.c., i.m.; ? 12 hourly

-1996

Pain Management in Animals by P. Flecknelland A. Waterman-Pearson, Harcourt Intern., London, 2000

2.5 mg/kg s.c.; ? 12-24 hourly

??5 mg/kg s.c.orby mouthdaily

2000

Claire A. Richardsonand Paul A. FlecknellATLA 33, p. 119-127, 2005

5 mg/kg s.c.? daily

10 mg/kg s.c.? daily

2005

www.ahwla.org.uk/site/tutorials/RP/RP11-Where.html

5 mg/kg s.c.

10 mg/kg s.c.

2007

ReferenceFlunixinMeloxicamPer os

Meloxicams.c.

Carprofen

Dosages of NSAID for mice, taken from literature

prior to surgery– provide high 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. orbuprenorphine 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 thedrinking bottle and in dishes on the cage ground

– 1-2 times per day: check body weight, food and waterconsumption, and the animals outer appearance and movingbehavior

Intensive care in mice after major surgery

Schuler, B. et al., submitted

Fluid therapy

Ringer-Lactat or NaCl 0.9% (37°C)

in general10 ml/kg/h i.v.

rat5-10 ml i.p. during laparatomy

mouse0.5-1.0 ml i.p. during laparatomy0.5-0.7 ml s.c. after surgery in 12 hour-intervals

Thank you for your attention