Trends und Probleme aus der Praxis - GV-Solas

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
‣examples, problems
‣optimized protocols
Pain therapy
– decide on pain therapy
– drugs, side effects
– dosages
‣intensive post-operative care in mice

Species-specific problems in mouse anesthesia
the mouse
– 18 – 50 g body weight
– hypothermia
– access to arteries, veins
– injection side
the drugs
– feasibility
Propofol ® , diazepam, di-ethyl-ether, …
– availability
InnovarVet ® > off the market
Hypnorm ® > narcotics laws, import from UK
–
i.p., s.c.
Halothane > off the market?
monitoring
Metofane ® > import from Australia
blood pressure
heart rate, ECG
– regulations for protection of the personnel
secure gas scavenging
pulsoximetry
blood gases and acid base balance
– legal restrictions from welfare concerns
Avertin ® , di-ethyl-ether, …
– intervention
cardiac arrest
respiratory depression

Species-specific problems in mouse anesthesia

Avertin ®
Tribromoethanol
Zeller, W., Meier, G., Bürki, K., Panoussis, B.: Adverse
effects 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 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
ease of application
– i.p.
– s.c.
special equipment: none
environmental impact: no
cheap ?
not controllable
Inhalation
application
– induction chamber
– face mask
special equipment
environmental impact ?
dangerous for personnel ?
expensive ?
controllable
Mono-anesthetics
e.g. pentobarbital
Isoflurane
Sevoflurane
Desflurane
Combination of
dissoziative + alpha-two agonist
e.g. ketamin + xylazine
Combination of
opioid + alpha-two agonist
e.g. fentanyl + medetomidine
Halothane
Enflurane
Methoxyflurane
Di-ethyl-ether

Injection anesthesia
Combination of substances
synergistic action
– adverse effects decreased
– desired effects increased
– analgesia strengthened
α 2 -agonist
opioid
tranquilizer
dissoziative
examples
– neurolept-analgesia
‣fentanyl + fluanison (Hypnorm ® ) + medetomidine
‣fentanyl and medetomidine can be antagonized
‣fentanyl + midazolam + medetomidine
‣all components can be antagonized
[from J. Henke, Munich]
– ketamin + xylazine (Rompun ® ) [+ acepromazine]

Handling b Lack of toxicity c Efficacy d References
Table 1. Details of published i.p. injection anesthesia protocols for the mouse.
Substances Range of Substance class Availability
dosages
[mg/kg]
a
Mono-anesthetics
Pentobarbitone
30 – 90 Barbiturate – n + – narrow safety margin – analgesia insufficient, long
sleeping time
3, 5, 7, 13, 14,
15, 16
α-Chloralose 114 – – difficult to dissolve in saline – matter of controversy – no surgical tolerance 5
Chloral hydrate
60 - 400 – n – not available in sterile form, – high mortality, intestinal com-
– controversial: analgesia 5, 16
light sensitive, air sensitive plications, out of use today insufficient
Etomidate 23.7 - 33 Non-barbiturate – c + – tissue irritant – only hypnosis, no analgesic
5, 23, 24
hypnotic
properties
Combinations of anesthetics
Ketamine 50 – 200 Dissociative + + * 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,
+ Medetomidine
1 α2-Agonist + + * analgesia ?, sedation ? 20, 21
relaxation ?
Ketamine
+ Azaperone
100
75
Dissociative
Butyrophenone
+ + * analgesia, no relaxation 16, 17
Tiletamin/
Zolazepam
(Telazol)
7.5 – 100 Dissociative/
Benzodiazepine
+ + * Telazol alone: only immobilization
+Xylazine 7.5 – 45 α2-Agonist + + * analgesia, sedation
Fentanyl/
Fluanison
(Hypnorm)
0.33 – 3.3
[ml/kg]
Opioid/
Butyrophenone
– n, c – precipitates out of solution,
difficult to mix with other
drugs, storage of mixtures not
* neuroleptanalgesia
possible
+ Midazolam 12.5 – 16.6 Benzodiazepine – c * sedation 26
or
+ Diazepam 5 Benzodiazepine + + * sedation 5, 14
Fentanyl/
droperidol
(Innovar-
Vet)
0.0001 –
0.001
[ml/g]
Opioid /
Butyrophenone
– n,
c, o
+ * neuroleptanalgesia 5, 16
5, 7
5, 14, 15, 16, 22,
+ Diazepam 5 Benzodiazepine + + * sedation
Fentanyl 0.06 - 0.08 Opioid – n, c + * analgesia 14, 15, 24
+ Metomidate 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
hypnotic
– 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 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
•Rumke, C. L., and J. Noordhoek. 1969. Sex differences in the duration of
hexobarbital narcosis and in serum MUP content in mice. Arch Int
Pharmacodyn 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.
housing conditions
Dairman, W., Balaszs, T. 1970. Comparison of liver microsome enzyme systems and
barbiturate sleeping times in rats caged individually or communally. Biochemical
Pharmacology 19:951-955
Einon, D., Stewart, J., Atkinson, S., Morgan, M. 1976. Effect of isolation on
barbiturate anesthesia in the rat. Psychopharmacology 50:85-88
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
80
n=20
Stock Hsd:NMRI
male
age 3-6 months
[%]
60
40
Ket = ketamin
Xyl = xylazine
Ace = acepromazine
20
Aza = azaperone
0
Ket 100
Ket 100
Xyl 20
Xyl 20
Ket150 Ketamin
Ket 100
Xylazin
Xyl 30 Acepromazin Xyl 20
3mg
Ket 150
Xyl 30
Ace 3
Ketamin
Xylazin
Azaperon Xyl 20
5mg
Ket 100
Aza 5
Telazol
Xylazin
Tel 80
Xyl 20
Ketamin
Azaperon
80mg
Ket 100
Aza 80
Ketamin Ketamin
Ket 100
Medetomidin Medetomidin
1mg 5mg
Med 1
Ket 100
Med 5
Tel = Telazol ®
(tiletamine +
zolazepam)
Med = medetomidine
surgical tolerance
survival rate
dosages in mg/kg bodyweight

Time of surgical tolerance and immobilization
420
360
300
surgical tolerance
immobilization
n=20
Stock Hsd:NMRI
male
age 3-6 month
time
[min]
240
180
Ket = ketamin
Xyl = xylazine
Ace = acepromazine
120
Aza = azaperone
60
Tel = Telazol ®
0
Ket 100
Xyl 20
Ket 150
Xyl 30
Ket 100
Xyl 20
Ace 3
Ket 100
Xyl 20
Aza 5
Tel 80
Xyl 20
Ket 100
Aza 80
Ket 100
Med 1
Ket 100
Med 5
(tiletamine +
zolazepam)
Med = medetomidine
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:
Restraint:
50 min
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
i.m.
Meerschweinchen
i.m.
Chinc hilla
i.m.
Ratte
i.m.
Hamster
i.p.
Gerbil
s.c.
Maus
i.p.
Fentanyl
0,02
0,025
0,02
0,005
0,033
0,03
0,05
Anästhesie
Midazolam
1,0
1,0
1,0
2,0
3,3
7,5
5,0
Medetomidin
0,2
0,2
0,05
0,15
0,33
0,15
0,5
Antagonisierung
Naloxon
0,03
0,03
0,05
0,12
0,8
0,5
1,2
Flumazenil
0,1
0,1
0,1
0,2
0,33
0,4
0,5
Atipamezol
1,0
1,0
0,5
0,75
1,7
0,375
2,5
Antagonisierung routinemäßig s.c., in Notfällen i.v. mit halber Dosis
Statt Midazolam auch Climazolam, nicht Diazepam, statt Flumazenil auch Sarmazenil
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:
Restraint:
15 min
30 min
Propofol
Medetomidine
Fentanyl
75 mg/kg bodyweight
1 mg/kg bodyweight
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
‣ pregnant women should stay away from rooms in
which inhalation anaesthesia is performed
‣ 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 longterm
interventions
Sevoflurane, 4% - 8%
via face mask
death rate:

pressure
reducing
valve
flow meter 1L
inhalation anesthesia machine
O 2
vaporizer
for
isoflurane,
sevoflurane
nose
mask
filter
O 2
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
– application route
– frequency of
application
select an
analgesic
drug
interference with the
experiment

Types of analgesic drugs
Opioids
(acting mainly on the central
nervous system)
severe pain
availability?
side effects
– respiratory depression
– obstipation
– rat: pica behavior if
overdosed, = rat eats
bedding, papers, towels
etc.!
– Buprenorphin:
behavioural abberations
NSAID
(peripheral action)
anti-inflammatory
anti-pyrogenic
side effects
– inhibition of platelet
aggregation!
– long-term application: kidney
function decreased
– stomach ulceration
– bleeding in the gastrointestinal
tract

Trends in rodent
pain therapy
Table from
Claire A. Richardson and
Paul A. Flecknell:
Anaesthesia and postoperative
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
s.c.
5 mg/kg
s.c.
www.ahwla.org.uk/site/t
utorials/RP/RP11-
Where.html
2005
10 mg/kg
s.c.
? daily
5 mg/kg
s.c.
? daily
Claire A. Richardson
and Paul A. Flecknell
ATLA 33, p. 119-127,
2005
2000
5 mg/kg
s.c.or
by mouth
daily
?
?
2.5 mg/kg
s.c.;
? 12-24
hourly
Pain Management in
AnimalsbyP. Flecknell
and A. Waterman-
Pearson, Harcourt
Intern., London, 2000
1996
-
2.5 mg/kg
s.c., i.m.;
? 12 hourly
Laboratory animal
Anaesthesia, by Paul A.
Flecknell, Harcourt
International, London,
2nd ed. 1996

Intensive care in mice after major surgery
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. 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