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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