"Anesthesia". In: Current Protocols in Immunology - IMBA

Anesthesia
Anesthetic agents are used in laboratory animals to prevent pain or distress due to an
experimental procedure or for restraint to facilitate a technically difficult procedure.
Agents may be used alone or in combination to achieve the desired effect. Anesthesia,
analgesia, sedation, and tranquilization are achieved through drug selection and proper
dosing. Investigators using anesthetic agents should familiarize themselves with their
physiologic effects and the risks involved with their use.
This unit provides three basic protocols: injectable anesthesia for mouse, rat, and hamster;
inhalant anesthesia using methoxyflurane for mouse, rat, and hamster; and injectable
anesthesia using ketamine/xylazine for rabbit and an alternate protocol for sedation using
butorphanol/acetylpromazine in the rabbit. The Commentary further describes and compares
these methods of anesthesia for various applications.
INJECTABLE ANESTHESIA FOR MOUSE, RAT, AND HAMSTER
Materials
Anesthetic of choice (Table 1.4.1)
Laboratory balance for weighing the animal
1- or 3-ml syringe with 22-G needle
Additional reagents and equipment for handling and restraint (UNIT 1.3) and
intraperitoneal injection (UNIT 1.6)
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Agent or combination Species Dosage (mg/kg) Duration (min)
Pentobarbitala Mouse 40-70 30-60
Rat 30-40 30-60
Hamster 50-90 30-60
Ketamine + xylazineb Mouse 50-150 15-20
Rat 50-150 15-20
Hamster 50-150 15-20
Tribromoethanolc Mouse 125-160 15-30
Rat 300 15-20
aDilute 1/10 with sterile saline. This is a controlled substance.
bAdd 1.0 ml of xylazine (20 mg/ml) to 10 ml ketamine (100 mg/ml) and dose as if mixture were
100 mg/ml. Dilution 1/10 with sterile saline is recommended for accuracy in dosing mice, hamsters,
and young rats. This mixture is based on the use of commercially available sterile stock solutions
of the two anesthetics (available from veterinary supply companies; see Table 1.4.2), not reagentgrade
compounds. Use of nonsterile dry compounds for making this mixture is contraindicated
because of the risk of introducing bacteria or other contaminants during the preparation process
and because the dry forms of these drugs do not dissolve readily. It is recommended that the
veterinarian member of the institutional Animal Care and Use Committee be contacted to determine
how to obtain these anesthetics legally for use in research. Some institutions maintain institutional
research licenses with the U.S. Drug Enforcement Administration for use of these or other
veterinary drugs; at other institutions, each individual researcher must have a DEA license.
cMake a 100% (w/v) stock solution by dissolving 5 g of 2,2,2-tribromoethanol in 5 ml 2-methyl-
2-butanol (tert-amyl alcohol). Gentle heating may be required. A good working solution is a 1/250
dilution (4 mg/ml) in normal saline; this is stable 4 to 6 months at 4°C in a dark bottle.
Contributed by John Donovan and Patricia Brown
Current Protocols in Immunology (1998) 1.4.1-1.4.5
Copyright © 1998 by John Wiley & Sons, Inc.
UNIT 1.4
BASIC
PROTOCOL 1
Care and
Handling of
Laboratory
Animals
1.4.1
Supplement 27

BASIC
PROTOCOL 2
Anesthesia
1.4.2
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Anesthetic Brand name Supplier a
Sodium pentobarbital,
Schedule II general anesthetic,
65 mg/ml
Ketamine hydrochloride
injection USP, 100 mg/ml
Xylazine hydrochloride,
20 mg/ml
Sodium Pentobarbital Injection
Sodium Pentobarbital Injection
Ketaject
Ketaset
Ketaved
Vetalar
Anased Injection (Dogs)
Gemini SA
Rompun 20 mg/ml Injectable
Solvazine 20 mg/ml
Tranquived Injectable (Dogs)
Xyla-ject Canine/Feline Injectable
Xylazine-20 Injection
Tribromoethanol (mixture) 2,2,2-Tribromoethanol and
tert-amyl alcohol
a See APPENDIX 5 for supplier contact information.
1. Retrieve the animal from its cage, place in a tared beaker, and determine its weight.
2. Calculate appropriate dose of chosen anesthetic.
Using the guidelines in Table 1.4.1, for a 15-g (0.015 kg) mouse, the amount of pentobarbital
to inject would be (0.015 kg × 40 mg/kg) to (0.015 kg × 70 mg/kg), or 0.6 to 1.0 mg.
Similarly, for the same mouse, using a 100 mg ketamine/20 mg xylazine mixture, (0.015 kg
× 50 mg/kg) to (0.15 kg × 150 mg/kg ) or 0.75 to 2.25 mg, dosed as if the mixture were 100
mg/ml, 0.0075 ml to 0.0225 ml would be the range to use. (Dilution of the mixture 1:10
with sterile saline is recommended to ease administration of such small volumes.)
If tribromoethanol were the anesthetic of choice, (0.015 kg × 125 mg/kg) to (0.015 kg ×
160 mg/kg), or 1.9 to 2.4 mg, would be the appropriate dose.
Sources of anesthetics are listed in Table 1.4.2.
3. Manually restrain the animal. Inject the selected dose intraperitoneally.
4. Return the animal to its cage and monitor for the depth of anesthesia.
INHALANT ANESTHESIA USING METHOXYFLURANE FOR
MOUSE, RAT, AND HAMSTER
Methoxyflurane is a relatively safe anesthetic for rodents, is simple to use, requires no
special equipment, and vaporizes at room temperature to a concentration suitable for
general anesthesia.
Materials
Methoxyflurane
Gauze or cotton sponges
Closed glass container with raised floor
Empty syringe case or 50-ml centrifuge tube (optional)
Butler
J.A. Webster
Phoenix Pharmaceutical
Fort Dodge Labs
Vedco
Fort Dodge Labs
Lloyd Labs
Vetus Animal Health
Bayer
Fort Dodge Labs
Vedco
Phoenix Pharmaceutical
Butler
Commercial chemical
supply houses (e.g.,
Aldrich or Sigma)
CAUTION: All procedures with methoxyflurane should be conducted in a fume hood or
safety cabinet that continuously exhausts anesthetic gases away from personnel.
Supplement 27 Current Protocols in Immunology

1. Pour methoxyflurane on gauze or cotton sponges and place the soaked material on
the bottom of a closed glass container.
2. Place a raised floor over the gauze sponges to permit vaporizing methoxyflurane to
flow through or around it to fill the container.
3. Return the lid to the container and let sit 3 to 5 min to allow methoxyflurane to
vaporize.
4. Place the rodent on the raised floor, close the container, and observe the animal.
5. Allow the animal to become recumbent and reach slow and regular breathing.
6. Remove the animal from the container to perform the appropriate procedure.
If longer periods of anesthetization are needed, fabricate a nose cone from an empty
discarded syringe case or 50-ml disposable centrifuge tube to maintain anesthesia. Place
the methoxyflurane-soaked gauze or cotton sponge in the closed end of the syringe case or
tube. Place the open end over the nose of the animal (Fig. 1.4.1). Remove or replace nose
cone as necessary to maintain adequate anesthesia.
INJECTABLE ANESTHESIA USING KETAMINE/XYLAZINE
FOR THE RABBIT
Materials
20 mg/ml xylazine (see Table 1.4.2 for sources)
100 mg/ml ketamine⋅HCl (see Table 1.4.2 for sources)
Sterile saline or phosphate-buffered saline (PBS; APPENDIX 2)
5-ml syringe with 22- to 25-G needle
Warm water recirculating blanket (optional)
1. Determine the weight of the rabbit in kilograms.
2. Draw up 1 ml xylazine into a 5-ml syringe with a 22- to 25-G needle.
3. Draw up 2 ml ketamine⋅HCl into the same syringe.
4. Draw up 1 ml sterile saline or PBS into the same syringe.
5. Invert syringe several times to mix the solutions.
6. Administer mixture intramuscularly (UNIT 1.6) at a dose of 1 ml/kg (equivalent to 50
mg/kg ketamine and 5 mg/kg xylazine).
The length of anesthesia will vary from 20 to 75 min. The ketamine/xylazine combination
provides analgesia with muscle relaxation. Hypothermia can be a problem and use of a
warm water recirculating blanket to maintain body temperature is recommended.
metophane-soaked gauze
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BASIC
PROTOCOL 3
Care and
Handling of
Laboratory
Animals
1.4.3
Current Protocols in Immunology Supplement 27

ALTERNATE
PROTOCOL
Anesthesia
1.4.4
INJECTABLE SEDATION USING BUTORPHANOL/
ACETYLPROMAZINE FOR THE RABBIT
This mixture provides good vasodilation and sedation with analgesia to allow for blood
collection from the ear blood vessels without additional physical restraint of the animal.
It should not be used for more invasive procedures such as cardiac puncture.
Materials
10 mg/ml butorphanol
10 mg/ml acetylpromazine
1- or 3-ml syringe with 22- to 25-G needle
1. Determine the weight of the rabbit in kilograms.
2. Draw up 0.5 ml butorphanol into a 1- or 3-ml syringe with 22- to 25-G needle.
3. Draw up 0.5 ml acetylpromazine into the same syringe.
4. Invert syringe several times to mix the solutions.
5. Administer mixture subcutaneously or intramuscularly (UNIT 1.6) at a dose of 0.2 ml/kg
(equivalent to 1 mg/kg each butorphanol and acetylpromazine).
COMMENTARY
Background Information
The intensity and duration of an animal’s
response to anesthetic agents is affected by
species, age, genetic background, health or nutritional
status, environmental conditions, and
route of administration.
The route of administration is largely determined
by the formulation of the agent and the
recommendations found on the package insert.
If the formulation allows for multiple routes of
administration, the choice is generally based on
the species and desired duration of effect.
The most commonly used route in rodent
species is intraperitoneal injection because it is
technically simple and results in generally safe
and uniform absorption with minimal and predictable
periods of induction and recovery. In
rodents, the intramuscular route is difficult to
achieve due to the small muscle mass available,
and frequently results in unpredictable induction
times, duration periods, and intensity of
effect. The intravenous route requires technical
expertise and skill, but has very rapid induction
and recovery phases. However, dosages must
be carefully calculated to ensure adequate anesthesia
without overdose.
Subcutaneous injection of anesthesia, while
very safe, results in nonuniform or poor absorption
and quite unpredictable levels of anesthesia.
Because of the small size of rodent species,
most injectable agents should be diluted 5- to
10-fold to enable accurate dosing.
Various inhalant anesthetics are available for
animals but most are unsuitable for rodents. The
most commonly used agents include methoxyflurane,
ether, and isoflurane. As noted in Basic
Protocol 2, methoxyflurane is generally the
safest and most convenient for use with rodents.
Ether and isoflurane are not recommended for
general use in rodents. Ether is both flammable
and explosive. Safe handling and storage are a
constant concern. Additionally, ether is highly
irritating to the respiratory tract and excess
respiratory tract secretions can be troublesome
for the animal during its use. Physicochemical
characteristics of isoflurane make it difficult to
administer. Because isoflurane easily reaches
lethal concentrations at room temperature, a
sophisticated and calibrated vaporizer is
needed for its use.
Because of their larger size, rabbits can be
effectively anesthetized with intramuscular administration
of a ketamine/xylazine mixture
(see Basic Protocol 3). The method described
in the Alternate Protocol is recommended for
sedation of rabbits to facilitate blood withdrawal
from ear vessels.
Critical Parameters
Risk is inherent with all methods of anesthesia
in all species, but is particularly significant
when anesthetizing small laboratory species.
Methods used in larger animals to reduce
the risk of anesthetization—e.g., endotracheal
intubation, sophisticated cardiopulmonary
Supplement 27 Current Protocols in Immunology

monitoring, and systemic support through indwelling
catheters and respiratory support—
are often impractical, inefficient, or nearly impossible
in rodents.
Anesthetic risk can be reduced by proper
selection of an anesthetic agent appropriate for
the species, careful titration of dosage and
monitoring of the desired effect, and use of an
appropriate route of administration. Dosage
titration should begin at the low end of the range
suggested. Anesthetic risk is reduced and recovery
is quicker and safer when systemic support
such as warmth and systemic fluid therapy
is provided. Small rodents have a high ratio of
body surface area to weight, which favors heat
loss. Cold counter tops or stainless steel tables
also speed heat loss. Anesthetized or recovering
animals should be kept warm (but not hot) with
heat lamps, commercially available water circulating
heating pads, or other devices. In addition,
attention should be given to keeping the
animal’s airway clear of debris. Gentle extension
of the chin and neck will help overcome
respiratory difficulties.
Surgical procedures result in fluid loss due
to hemorrhage or evaporation from open body
cavities. Replacement therapy may aid in the
successful recovery from anesthetization and
surgery. Normal saline or balanced electrolyte
solution can be given subcutaneously (or in
rabbits, intravenously) to facilitate recovery.
Investigators should consult with a veterinarian
regarding dosage and frequency.
Recovering animals should also be turned
from side to side every 5 to 10 min to assist
circulation and speed recovery. Finally, animals
should be observed until recovery is nearly
complete and animals are able to maintain sternal
recumbency or move about without falling.
Because the permutations of species, age,
strain, and route of administration make it difficult
to give precise recommended dosages for
injectable anesthetic agents, it may be necessary
to vary combinations in the context of the
experiment, procedure, and desired effect. Table
1.4.1 provides guidelines for dosages and
routes of administration of commonly used
injectable anesthetics in mice, rats, and hamsters.
When titrating an anesthetic regime through
trial and error, monitoring the animal’s level of
anesthesia is necessary to assess its effect. Two
methods enable the investigator to estimate the
suitability of a specific dosage and to monitor
the animal throughout a period of anesthesia.
The rate and depth of respiration are used to
crudely assess cardiorespiratory function. Induction,
recovery, and inadequate anesthesia
are generally characterized by rapid or irregular
breathing patterns, while an adequate level of
anesthesia is indicated by slower and more
regular respirations. Stimuli such as a toe pinch
will elicit a reflex extension of the hind limb
and will also result in a noticeable quickening
of respirations or movement, until adequate
anesthesia is achieved.
It is often necessary to supplement incomplete
anesthesia obtained with an injectable
such as tribromoethanol with a small dose of
the inhalable methoxyflurane. This is more
advisable than a second smaller dose of the
injectable, which can be lethal. Nevertheless,
with experience, some investigators prefer to
give a second injectable dose. For some mouse
strains, the margin of safety between effective
and lethal dose of an anesthetic is particularly
narrow.
Key References
Green, C.J. 1987. Anesthesia and analgesia. In
Laboratory Animals: An Introduction for New
Experimenters (A.A. Tuffery, ed.) pp. 261-301.
John Wiley & Sons, Chichester, England.
Manning, P.J., Ringler, D.H., and Newcomer, C.E.
(eds.) 1994. The Biology of the Laboratory Rabbit,
2nd ed., ACLAM Series, pp. 104-105. Academic
Press, San Diego.
White, W.J. and Field, K.J. 1987. Anesthesia and
surgery of laboratory animals. In The Veterinary
Clinics of North America, Small Animal Practice,
Exotic Pet Medicine, 17:5 (J.E. Harkness,
ed.) pp. 989-1017. W.B. Saunders, Philadelphia.
Wixson, S.K. and Smiler, K.L. 1997. Anesthesia and
analgesia in rodents. In Anesthesia and Analgesia
in Laboratory Animals (D.F. Kohn, S.K. Wixson,
W.J. White, and G.J. Benson, eds.) pp.165-
200. Academic Press, San Diego.
Contributed by John Donovan
Rhone-Poulenc Rorer
Collegeville, Pennsylvania
Patricia Brown
National Cancer Institute
Bethesda, Maryland
Care and
Handling of
Laboratory
Animals
1.4.5
Current Protocols in Immunology Supplement 27