Laboratory Animal Services
STANDARD OPERATING PROCEDURES AND POLICIES
ANAESTHESIA and PAIN MANAGEMENT - RODENTS
(Last updated 29 April 2005)
SECTION 1:
GENERAL COMMENTS
The need for pain management
It is a legal requirement that persons using animals for research “must assume that animals experience pain in a manner
similar to humans”. Furthermore, there can be no doubt that the physiological effects of pain are potentially diverse and
can have a major impact on research data. Appropriate attention to pain management is therefore one of the most
important responsibilities of those involved in the use of animals for scientific purposes.
Procedural skills
It is essential that the persons performing any procedure requiring anaesthesia are competent in that procedure. This is
required by legislation and is a condition that must be satisfied when applying to the Animal Ethics Committee for
approval. Apart from the obvious animal welfare implications, poor technique will prolong anaesthetic recovery and
increase the chance of side-effects that can adversely affect the quality of experimental data.
Preparation for anaesthesia





Unlike humans (and certain other monogastrics such as dogs and cats), rodents should not be starved prior to
general anaesthesia.
It is important to have an accurate idea of the animal’s body weight so that the correct dose of drug is administered.
Different strains of rodent vary considerably in the way they handle anaesthesia. Therefore, when using an
anaesthetic drug (or combination of drugs) for the first time, it is always advisable to anaesthetise a single animal first
rather than a group. This enables one to observe the effects of the anaesthetic protocol on that particular strain of
rodent.
When administering anaesthetic drugs, it is best to administer the drug to effect rather than give the full dose all at
once. This helps avoid over-dosing.
The use of pre-anaesthetic medication (‘pre-med’) is not generally practiced with rodents. Frequent handling and
conditioning can be valuable in reducing the amount of restraint and stress for the animal.
What to avoid
1)
Hypothermia
Hypothermia (a potentially fatal reduction in body temperature) develops extremely rapidly in rodents and is
probably the commonest cause of anaesthetic death. In its mildest form, it will lead to a substantial increase in
the time taken to recover from an anaesthetic; this in turn can induce a wide variety of metabolic alterations.
When more severe, animals become more susceptible to anaesthetic overdose and shock.
Hypothermia can be minimised or prevented by the following procedures:
1)
Use of a thermostatically-controlled heating pad.
2)
Use of a heat lamp. This is less controllable than a heat pad and can lead to burns or a dangerous
increase in body temperature (hyperthermia). Careful monitoring of either the animal’s temperature or
the operative field is essential.
3)
Wrapping the animal’s torso in plastic bubble-wrap or cotton wool. This is suitable for short procedures
but should not be relied on too heavily and care should be taken to ensure that breathing movements
can still be observed. The tail should be included in the wrapping since this can be an important route of
heat loss.
Note that procedures such as swabbing with cold (i.e. room temperature) disinfectants, injection of cold solutions,
clipping of hair and haemorrhage will all contribute to the development of hypothermia.
2)
Anaesthetic overdose
2
This most commonly occurs when the animal’s body weight has been overestimated. Another important cause,
however, is metabolic differences between individuals or between strains of animal – such differences can cause
marked variation in susceptibility to anaesthetic drugs.
3)
Eye damage
General anaesthesia can lead to loss of blink reflex or drying of the surface of the eye. It is important to protect
the surface of the eye from drying or injury either through covering the eyes or applying an ointment.
4)
Hepatic enzyme induction
Repeated or prolonged administration of anaesthetic (or other) drugs can lead to alterations in the way the liver
metabolises many substances as well as the drugs responsible. This must be kept in mind when planning
experiments involving liver function or the systemic administration of substances.
Monitoring anaesthesia
Anaesthesia is a dynamic state and the depth of anaesthesia can be altered by many factors. These include intensity of
stimulus, amount of body fat, genetic factors, physiological state, duration of anaesthesia and of course the drugs used. It
is therefore important that anaesthesia be monitored throughout the procedure.
Many devices used for monitoring anaesthesia in larger animals are not sufficiently sensitive for use in rodents. The ability
to make a clinical assessment of anaesthesia by direct observation is therefore very important. Experience and familiarity
with a particular anaesthetic regime are also extremely valuable when ensuring that anaesthesia is maintained at an
appropriate depth.
Specific parameters that can be used to monitor anaesthetic depth in rodents include:

Pinching the skin between the toes or the tail tip: a movement response suggests anaesthesia might be too light;

Changes in the rate or depth of respiration: slow and shallow suggests anaesthesia might be too deep, vice
versa too light;

Colour of tissues, skin or mucous membranes: should be pink, not maroon or bluish.

Body temperature. This requires devices such as a rectal probe or infra-red thermometer.
Post-operative care
1)
Anaesthetic recovery
Animals should be placed in a cage lined with a cloth, towel or shredded paper. Dusty materials should not be
used because particles might stick to the eyes. The animal must be kept warm and the environment should be
quiet, free of obtrusive human activity and preferably dimly lit (except when examining the animal).
2)
Respiratory depression
If there appears to be significant respiratory depression (breathing is excessively slow or shallow), recovery from
some anaesthetic drugs can be assisted with the use of specific reversing agents (see table). A possible
alternative is the use of doxapram (a respiratory stimulant) at a dose rate of 5 – 10 mg/kg IV, IM or IP every 15
minutes. Supplying oxygen into the cage at a low slow-rate can also be beneficial.
3)
Fluid replacement
Excessive loss of fluids through bleeding or evaporation (when a body cavity is opened) will rapidly lead to
complications such as circulatory shock. This in turn will affect the function of many organs in a manner that
could have an impact on research data. Note that in a small animal such as a mouse, a few drops of blood
represents a substantial proportion of the animal’s blood volume and can be fatal. Fluid loss should therefore be
minimized and lost fluids replaced. If substantial fluid loss is anticipated, calculation of fluid replacement
requirements can be estimated by weighing the animal before and after the procedure. Lost fluids can be
replaced using solutions such as 0.9% saline or lactated Ringer’s solution preferably warmed to 37C and
administered SC or IP.
Note also that animals recovering from surgery might not be able to eat or drink for some time. In such cases,
they should be provided with moist foods (nutrient agar, jelly or crushed rodent pellets mixed with water)
3
presented in a dish or other manner that does not require the animal to reach up high. Typically, an animal
requires approximately 50 ml of fluid every per kg body weight every 24 hours.
Detection of pain
It should be assumed that animals are affected by pain to a similar extent as humans even though they might perceive it
differently.
Although application of commonsense is one of the most valuable skills in the detection of pain, animals undergoing
procedures should be observed for a range of signs including:

Altered behaviour (more submissive or more aggressive);

Abnormal posture or gait/avoiding the use of a limb;

Decline in body weight;

Dull or roughened coat;

Laboured or rapid respiration;

Dehydration (skin is not easily pliable, eyes appear sunken).
Surgical wounds should also be inspected regularly for signs of inflammation (swelling, discharge etc).
Record keeping
It is a legal requirement that animals used in research are appropriately monitored and that this is properly recorded. This
is particularly important in cases where animals are undergoing invasive procedures such as surgery. Failure to meet this
obligation may result in withdrawal of animal ethics approval.
In cases where animals are undergoing potentially painful procedures, the Animal Ethics Committee will usually require
that an appropriate and clear record be kept for each animal. These records must be readily available in the animal room
for inspection by members of the animal house staff and the Animal Ethics Committee. Records for animals no longer in
the animal house should be filed in the researcher’s office. A template record and other information is available from the
Laboratory Animal Services Director or the Ethics Office.
Security and occupational safety
All anaesthetic drugs hold considerable potential for serious occupational injury and abuse. It is essential that they are
only supplied to and handled by persons named on the Animal Ethics approval. It is a condition of supply that they be kept
in a secure location that is not accessible to persons .
Obtaining anaesthetic drugs
Researchers may be supplied with prescription-controlled drugs for anaesthesia of animals for approved research
purposes. These can usually be ordered through LAS upon application to the Director. You may be required to provide a
copy of the AEC letter approving your project as well as a full copy of the AEC protocol in which the proposed use of the
drug is described. You must also provide an account code to which the cost of the drug(s) can be charged.
SECTION 2:
DRUGS AND DOSE RATES
Choice of drugs
There are many drugs available for anaesthesia of rodents. Those listed in the table below are suggested as a starting
point only. They have been chosen because they are relatively free of undesirable side effects, are commonly used and
are simple to administer. Alternative drugs might be more suitable for certain applications. Note that the membership of
the University of Sydney’s Animal Ethics Committee includes a veterinary anaesthetist with extensive experience in the
anaesthesia of laboratory animal species.
Long-term (greater than 1 hour) anaesthesia.
The drugs listed in the table below are best used for procedures of short to medium duration. Longer term procedures will
usually require a different approach.
4
For many longer term procedures, an inhalation anaesthetic delivered via a well made face mask connected to an
anaesthetic machine with suitable vapouriser is the most reliable. Note that any method of delivering anaesthetic gases
must ensure adequate removal (‘scavenging’) of waste gases to avoid possible short-term and long-term health effects on
the operator.
Long-term anaesthesia can also be provided using injectable agents administered IM or IP on a top-up basis although this
results in variable anaesthetic depth. A more precise alternative among the injectable procedures is continuous
intravenous administration. This usually requires an infusion pump. Drugs suited to intravenous infusion in rodents include
alphaxalone and propofol.
MICE
Type of
anaesthesia
Light sedation
Light sedation
Light to deep
sedation
Sedation
Sedation
Deep sedation or
restraint for noninvasive
procedures
Restraint for
minimally invasive
procedures
Duration
Drug
Dose (mg/kg)
Comment
20 – 30 min
20 – 30 min
20 – 30 min
Acetylpromazine
Xylazine
Meditomidine
2 – 5 mg/kg IP or SC
5 mg/kg IP
30 – 100 g/kg SC
20 – 30 min
45 min
Diazepam
Alphaxalone
Ketamine
5 mg/kg IM or IP
100 mg/kg IP
150 mg/kg IP
20 – 40 min
(sleep time >
2 hr)
Pentobarbitone
IP
Light surgical
anaesthesia
20 – 30 min.
Sleep time 1
– 2 hrs.
Ketamine and
acetylpromazine
Ket 100 mg/kg
Ace 5 mg/kg IP
Surgical
anaesthesia
20 – 30 min.
Sleep time 1
– 2 hrs.
Ketamine
(100mg/ml) and
xylazine
(20mg/ml)
Ket 75 mg/kg
Xyl 10 mg/kg IP
Does not provide analgesia
Provides mild analgesia
Provides mild analgesia. Reversed with
atipamezole (1 mg/kg IP)
Does not provide analgesia
Poor analgesia; can be inconsistent.
Produces increase in muscle tone and slight
increase in blood pressure. Must not be used for
invasive procedures unless combined with
analgesic drugs (see below).
Provides poor analgesia and can cause
significant cardiovascular and respiratory
depression. Commercial production of
anaesthetic grade pentobarbitone is now very
limited. Other, shorter-acting barbiturates are
available but references usually cite these for IV
use.
Note that ketamine is now a restricted drug and
requires Department of Health approval. Contact
Laboratory Animal Services for further
information.
Xylazine can be partially reversed with
atipamezole.
Note that ketamine is now a restricted drug and
requires Department of Health approval. Contact
Laboratory Animal Services for further
information.
0.3 ml undiluted
Dilute ketamine
(100mg/ml) 1 in 5 to
make 20 mg/ml;
administer 0.1 ml per
mouse.
OR
2.5mg/33g
mouse=0.025ml ket
0.33mg/33g
mouse=0.017ml xyl
Surgcial
20 – 30 min.
Ketamine and
Ket 75 mg/kg
For multiple mice: using
a 1 ml syringe,
administer
0.04ml/mouse of a
solution of ket:xyl in
ratio of
0.5mlket+0.34mlxyl
Dilute ketamine
Medetomidine can be reversed with atipamezole.
6
anaesthesia
Sleep time 1
– 2 hrs.
medetomidine
Med 1 mg/kg IP
Surgical
anaesthesia
60 – 70 min if
not reversed.
Sleep time 4
– 6 hrs if not
reversed.
Fentanyl and
medetomidine
Not for use in mice?
Surgical
anaesthesia
15 -45 min
(sleep time 1
-2 hr)
30 – 60 sec
Tribromoethanol
(‘Avertin’)
250 mg/kg IP
Isoflurane
Surgical
anaesthesia
Determined
by duration of
administration
Isoflurane
Surgical
anaesthesia
Determined
by duration of
administration
Halothane OR
enflurane
Best done by running
anaesthetic gas into a
transparent container
using an anaesthetic
machine. Simple but
crude alternative is to
place cotton wool with
a few drops of
isoflurane in large jar
or transparent
chamber then place
mesh over cotton
wool. Animal is
placed on mesh and
removed as soon as
consciousness is lost.
Delivered by
anaesthetic machine
with oxygen. Must
use vapouriser
calibrated to
isoflurane. Usually
administered via face
mask which must be
well-fitting.
Delivered by
anaesthetic machine
with oxygen. Must
Very brief
anaesthesia or
induction
(100mg/ml) 1 in 5 to
make 20 mg/ml; dilute
medetomidine (1mg/ml)
1 in 3 to make 0.3
mg/ml. Mix with equal
part of diluted ketamine.
Administer 0.2 ml of
mixture per 30 g mouse.
Note that ketamine is now a restricted drug and
requires Department of Health approval. Contact
Laboratory Animal Services for further
information.
Medetomidine is reversed with atipamezole (1
mg/kg IP). Fentanyl can be reversed by opioid
antagonists.
Note that fentanyl is a restricted drug and
requires Department of Health approval. Contact
Laboratory Animal Services for further
information.
Contentious because can cause irritation of the
peritoneum. Must be prepared fresh and stored
correctly.
Only suitable for very brief, minimally invasive
procedures or for initial induction of anaesthesia
with subsequent maintenance by other means. If
not using anaesthetic machine, must avoid direct
contact between isoflurane liquid and animal
because drug is very irritant to mucous
membranes. Need to take care to avoid
occupational exposure.
Causes negligible induction of hepatic enzymes
therefore is sometimes recommended over other
inhalation agents. Rapid induction and recovery.
Widely used in the past; now being superceded
by isoflurane. Note halothane is associated with
possible long term occupational health risks.
7
Surgical
anaesthesia
Determined
by duration of
administration
Methoxyflurane
Post-operative
pain relief
24 hr
Carprofen,
ketoprofen or
meloxicam
Post-operative
pain relief
12 hr
Buprenorphine
(‘Temgesic’)
use vapouriser
calibrated to
halothane or
enflurane. Usually
administered via face
mask which must be
well-fitting.
Delivered by
anaesthetic machine
with oxygen. Must
use vapouriser
calibrated to
methoxyflurane.
Usually administered
via face mask which
must be well-fitting.
3 – 5 mg/kg SC, IM or
PO
0.05 mg/kg SC or IM.
Can also be offered in
jelly form for postoperative
administration.
Induction of anaesthesia is slow and therefore
safer than other inhalation agents. Good for
anaesthesia of neonates.
All these are non-steroidal anti-inflammatory
drugs (NSAIDs) that act on peripheral pain
mechanisms; also have anti-inflammatory and
anti-pyretic (fever-reducing) effects. May be used
in association with opioids or as an alternative if
opioids would interfere with procedure. Oral
administration can be achieved by supplying
drug in flavoured jelly (make up jelly double
strength and add drug so that cubes of jelly
contain one 24 hour dose); must be only one
animal per cage and animals should be
accustomed to jelly be feeding drug-free jelly 2 to
3 days beforehand.
Opioid agonist i.e. acts on central pain receptors.
Note that the undesirable side-effects of opioids
observed in humans tend to be less marked in
animals. Buprenorphine may be used with or as
an alternative to NSAIDs. Can be delivered in
jelly (see above). Exceeding the recommended
dose can lead to a reduction in analgesic effect.
Note that buprenorphine is a restricted drug and
requires Department of Health approval. Contact
Laboratory Animal Services for further
information.
RATS
Type of anaesthesia
Duration
Drug
Dose (mg/kg)
Comment
8
Light sedation
Light to heavy
sedation
Light to heavy
sedation
Light sedation
Deep sedation or
restraint for noninvasive procedures
20 – 30 min
20 – 30 min
Acetylpromazine
Xylazine
2 – 5 mg/kg IM, IP or SC
1 - 5 mg/kg IM or IP
Does not provide analgesia
Provides mild analgesia
20 – 30 min
Meditomidine
30 – 100 ug/kg IM, IP or SC
20 – 30 min
20 – 30 min
Diazepam
Ketamine
2.5 – 5.0 mg/kg IM or IP
75 mg/kg IM or IP
Restraint for
minimally invasive
procedures
20 – 40 min
(sleep time >
2 hr)
Pentobarbitone
IP
Light surgical
anaesthesia
20 – 30 min.
2 – 4 hrs
sleep time.
Ketamine and
acetylpromazine
Ket 75 mg/kg
Ace 2.5 mg/kg IP
Surgical anaesthesia
20 – 30 min.
2 – 4 hrs
sleep time.
Ketamine and xylazine
Ket 75 mg/kg
Xyl 10 mg/kg IP
Surgical anaesthesia
20 – 30 min.
2 – 4 hrs
sleep time if
not reversed.
Ketamine and
medetomidine
Ket 75 mg/kg
Med 0.5 mg/kg IP
Does not provide analgesia. Reversed
with atipamezole (1 mg/kg IP)
Does not provide analgesia
Produces increase in muscle tone and
slight increase in blood pressure. Must
not be used for invasive procedures
unless combined with analgesic drugs
(see below).
Note that ketamine is now a restricted
drug and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
Provides poor analgesia and can cause
significant cardiovascular and respiratory
depression. Commercial production of
anaesthetic grade pentobarbitone is now
very limited. Other, shorter-acting
barbiturates are available but references
usually cite these for IV use.
Note that ketamine is now a restricted
drug and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
Xylazine can be partially reversed with
atipamezole.
Note that ketamine is now a restricted
drug and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
Medetomidine can be reversed with
atipamezole.
Note that ketamine is now a restricted
drug and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
Surgical anaesthesia
60 – 70 min if
not reversed.
4 – 6 hrs if
not reversed.
Fentanyl and
medetomidine
Fen 0.3 mg/kg
Med 0.3 mg/kg IP
Administer 0.25
ml ketamine
(100mg/ml)
mixed with 0.15
ml medetomidine
(1mg/ml) in
same syringe.
Medetomidine is reversed with
atipamezole (1 mg/kg IP). Fentanyl can
be reversed by opioid antagonists.
Note that fentanyl is a restricted drug
and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
9
Surgical anaesthesia
15 -45 min
(sleep time 1
-2 hr)
Tribromoethanol
(‘Avertin’)
240 mg/kg IP
Contentious because can cause irritation
to peritoneum. Must be prepared fresh
and stored correctly.
Very brief
anaesthesia or
induction
30 – 60 sec
Isoflurane
Only suitable for very brief, minimally
invasive procedures or for initial
induction of anaesthesia with
subsequent maintenance by other
means. If not using anaesthetic machine,
must avoid direct contact between
isoflurane liquid and animal because
drug is very irritant to mucous
membranes. Need to take care to avoid
occupational exposure.
Surgical anaesthesia
Determined
by duration of
administration
Isoflurane
Surgical anaesthesia
Determined
by duration of
administration
Halothane OR enflurane
Surgical anaesthesia
Determined
by duration of
administration
Methoxyflurane
Post-operative pain
relief
24 hr
Carprofen, ketoprofen or
meloxicam
Best done by running
anaesthetic gas into a
transparent container using
an anaesthetic machine.
Simple but crude alternative
is to place cotton wool with
a few drops of isoflurane in
large jar or transparent
chamber then place mesh
over cotton wool. Animal is
placed on grid and removed
as soon as consciousness
is lost.
Delivered by anaesthetic
machine with oxygen; must
have vapouriser calibrated
to isoflurane. Usually
administered via face mask
which must be well-fitting.
Delivered by anaesthetic
machine with oxygen. Must
use vapouriser calibrated to
halothane or enflurane.
Usually administered via
face mask which must be
well-fitting.
Delivered by anaesthetic
machine with oxygen. Must
use vapouriser calibrated to
methoxyflurane. Usually
administered via face mask
which must be well-fitting.
3 – 5 mg/kg SC, IM or PO
Causes negligible induction of hepatic
enzymes therefore is sometimes
recommended over other inhalation
agents. Rapid induction and recovery.
Widely used in the past; now being
superceded by isoflurane. Note
halothane is associated with possible
long term occupational health risks.
Induction of anaesthesia is slow and
therefore safer than other inhalation
agents. Very good for anaesthesia of
neonates.
All these are non-steroidal antiinflammatory drugs (NSAIDs) that act on
peripheral pain mechanisms; also have
anti-inflammatory and anti-pyretic (feverreducing) effects. May be used in
association with opioids or as an
alternative if opioids would interfere with
procedure. Oral administration can be
achieved by supplying drug in flavoured
jelly (make up jelly double strength and
add drug so that cubes of jelly contain
10
Post-operative pain
relief
12 hr
Buprenorphine
Long term, nonrecovery
Several hours
Urethane
0.05 – 0.05 mg/kg SC or
IM. Can also be offered in
jelly form for post-operative
administration.
one 24 hour dose); must be only one
animal per cage and animals should be
accustomed to jelly be feeding drug-free
jelly 2 to 3 days beforehand.
Opioid agonist i.e. acts on central pain
receptors. Note that the undesirable
side-effects of opioids observed in
humans tend to be less marked in
animals. Buprenorphine may be used
with or as an alternative to NSAIDs. Can
be delivered in jelly (see above).
Exceeding the maximum dose can lead
to a reduction in the analgesic effect.
Note that buprenorphine is a restricted
drug and requires Department of Health
approval. Contact Laboratory Animal
Services for further information.
Note occupational health
(carcinogenesis) risk.