5.
2
C H A P T E R
Routes of
Administration
Jiro Hirota, Shinya Shimizu
National Institute of Animal Health, National Agriculture
and Food Research Organization (NARO), Tsukuba, Japan
Introduction
Mice are the most widely used animals for a range
of experiments including medical, chemical,
pharmacological, toxicological, biological and
genetic. The administration of test substances,
such as chemical elements, compounds, drugs,
antibodies, cells or other agents to mice is one of
the major methods for evaluating their biological
activity. A knowledge of available methods and
techniques of administration, as well as knowl-
edge of the deposition and fate of the adminis-
tered substance, will help scientists to select the
most appropriate route for their purpose. The
administration route is largely dependent on the
property of the test substance and the objective
of the experiment. All administration should be
performed with knowledge of the chemical and
physical characteristics of the substance. All
routes have both advantages and disadvantages,
such as the absorption, bioavailability and metab-
olism of the substance. Consideration should be
The Laboratory Mouse
Ó 2012 Elsevier Ltd. All rights reserved.
ISBN 978-0-12-382008-2
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OF
ADMINISTRATION
given to the pH, viscosity, concentration, sterility,
pyrogenicity, irritancy and toxicity, as well as the
existence of hazardous substances. In addition, 709
animal welfare must be taken into consideration
PROCEDURES
in deciding on the administration route, and the
route must be selected before the start of any
experiment.
Proper restraint is the most important tech-
nique when mice are treated as this decreases
stress and increases successful treatment.
Personnel using experimental animals should be
well trained in handling and restraint; they should
be qualified in the responsible use of experi-
mental animals and attain a scientifically high
standard [1, 2]. Further experience will lead to
repeatable and reliable results (see Chapter 5.1).
The foundation of animal welfare is that during
administration mice should be protected from
pain, suffering, distress or lasting harmdor
at least pain and distress must be kept to
a minimum [1]. Some injection sites (such as
footpad injection) are strongly discouraged and
if required must be justified on a case-by-case
DOI: 10.1016/B978-0-12-382008-2.00030-1
 (a) (b)

(c) (d)
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Figure 5.2.1 Manual restraint of a mouse using both hands. (a) The mouse is placed on the cage lid with the
preferred hand. The hand pulls the tail gently back. (b) The mouse is quickly and firmly picked up by the scruff
of the neck behind ears with thumb and index finger of the other hand. (c) The tail is transferred from the
preferred hand to between palm and little or ring finger of the other hand, then held firmly. (d) The mouse is
restrained.
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basis [3]. The regulation of administration sites,
methods, kind of substances and amount to be
administered all have to be reconciled with the
animal welfare board and permission has to be
obtained.
710
Principles of
PROCEDURES
back (Figure 5.2.1a). It is then quickly and firmly
picked up by the scruff of the neck behind the
ears with the thumb and index finger of the
other hand (Figure 5.2.1b). The tail is transferred
from the first hand to between the palm and little
or ring finger of the other hand, then fixed
(Figure 5.2.1c). The mouse is now restrained

(Figure 5.2.1d).

administration Single-handed restraint

Handling and restraint
Good handling and restraint is the most important
technique for correct administration. Proper
restraint leads to successful administration and
varies with the routes of administration. Dispos-
able gloves must be worn, as manual restraint is
frequently used for injections. There are two styles
of manual restraint, one using both hands and the
other single-handed (see Chapter 5.1; [4, 5]).
Two-handed manual restraint
The mouse is lifted by the base of the tail and
placed on the cage lid or other solid surface
with one hand and then its tail is pulled gently
The tail is picked up using thumb and index
finger of the chosen hand (Figure 5.2.2a), then
the mouse is placed on the cage lid or other solid
surface (Figure 5.2.2b). The tail is immediately
grasped by the palm and middle finger, ring
finger and/or little finger, and the thumb and
index finger released (Figure 5.2.2c). The fold
of skin from the scruff of neck down the back
is immediately gripped using the thumb and
index finger (Figure 5.2.2d, e). The mouse is
then restrained (Figure 5.2.2f).
To prevent kicking by the hind legs, the tail is
fixed using the palm and index finger and then
the left hind leg is held firmly between the ring
and little finger (where the mouse is restrained
by the left hand) (Figure 5.2.3).
 (a) (b)

(c) (d)

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(e) (f)

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ADMINISTRATION
Figure 5.2.2 Single-handed restraint of the mouse. (a) The tail is picked up using thumb and index finger of
preferred hand. (b) The mouse is placed on the cage lid or other solid surface pulling gently back by the hand.
(c) The tail is immediately grasped by the palm and middle finger, ring finger and/or little finger and then the 711
tail held between thumb and index finger is released. (d) and (e) The fold of skin from the scuff of the neck
down the back is immediately gripped using the thumb and index finger. (f) The mouse is restrained.

PROCEDURES

Figure 5.2.3 Manual restraint of a mouse to prevent kicks from hind leg. The tail is held using the palm and
index finger and then left hind leg is fixed between the ring and little finger (when the mouse is restrained by
the left hand).
 Site of administration
Among several possibilities for the administra-
tion of substances to mice, the most common
are subcutaneous, intraperitoneal or intrave-
nous injection. Intramuscular administration is
not recommended, as the muscle of the mouse
is too small. Some sites, such as footpad injection
of Freund’s complete adjuvant, intrasplenic
injection and intralymph node injection are
unacceptable nowadays [3], and should be
restricted to cases where they are absolutely
necessary.
Preparation of the site
The area for administration is clipped
ADMINISTRATION
(Figure 5.2.4) or cleaned with warm water if
necessary before cleaning the skin with cotton
wool moistened with alcohol or disinfectant.
Where aseptic skin is necessary the fur must be
clipped, followed by a three-stage surgical prepa-
ration: surgical soap, alcoholic rinse and surgical
preparation solution. The skin is dried immedi-
OF
ately before administration [3]. In some cases
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a local anaesthetic may be applied first to prevent
pain.
Preparation, solubility and
712 safety of solutions
Test substances, solutions and equipment should
PROCEDURES
be prepared aseptically and free from pyrogens,
especially for parenteral injections. Solutions
can be sterilized by filtration (0.22 mm). Living
organisms or cells must be free from contami-
nants when administered. The toxicity of the
substance, the volume and the route of adminis-
tration should be considered to prevent tissue
Figure 5.2.4 Clipping of hair on the back using cordless electric clipper.
damage and to give precise dosage. The
following solvents or vehicles have been found
suitable in most instances and do not greatly
affect drug action because of their own inherent
properties:
• water
• water with 0.85% sodium chloride
• water with up to 50% polyethylene glycol
• water with not over 10% Tween 80
• water with up to 0.25% methylcellulose or
carboxymethylcellulose
• corn oil, vegetable oil or peanut oil (oral and
intramuscular route only).
A small percentage of the lower alcohols,
glycols and acetone can also be used, provided
the volume administered is kept small [6]. Phos-
phate buffered saline (PBS) or various culture
media are also suitable vehicles [2]. Lipid-soluble
substances can only be dissolved in oil but this
delays absorption. Oil-soluble drugs have been
successfully given intravenously in 15% oil–water
emulsions using lecithin as an emulsifier [6].
Until experience indicates otherwise, solutions
or suspensions should be prepared as near to the
time of use as possible because some substances
will deteriorate in solution within a few hours
[6]. When administering drugs, the solvent should
ideally be the same as the one in which the drug is
normally formulated [2]. Although distilled water
can be used under certain conditions, saline is
preferable because water for injections injected
subcutaneously causes pain and intravenous
injection produces haemolysis. Oil and viscous
fluids cannot be injected intravenously [2]. If sus-
pended material is to be used for intravenous
injection, the particles should be removed by
filtration to prevent embolism [6]. The tempera-
ture of fluids must be raised at least to room
 pH of the injected solution
For most administration routes, providing the
solutions are not highly buffered, a pH range
of 4.5–8.0 is satisfactory. For oral administration
a pH as low as 3 can be tolerated, but alkaline
solutions are very poorly tolerated. A rather
wide range of pH is indicated for intravenous
administration, because of the buffering effect
of blood and dilution by blood flow following
use of the intramuscular and subcutaneous
routes. When solutions of low or high pH are
injected intravenously the injection rate is kept
slow and again precautions are taken to avoid
getting solution outside the vein [6].

Figure 5.2.5 Cell strainer (352350, Becton Dickinson).

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Volume and frequency
temperature or better still up to body tempera- of administration

OF
ture before use, because the injection of cold
The injection volume is limited by any toxicity of
fluids is painful [7].

ADMINISTRATION
the substance and by the size of the mouse. It
Intravenous cell injection has been per-
should be kept as small as possible. Excess volumes
formed in many experiments, such as immuno-
of solution can startle the animal. The frequency
logical or cancer research. In these cases, cells
of administration should be kept to a minimum,
have to be suspended singly in solution because
to avoid unnecessary stress. If solutions are admin-
cell clumps cause embolism, and in some cases
istered intravenously, haemodynamic changes
the mice would die. Consequently, cell suspen-
and pulmonary oedema may occur, while very
sions for injection should be filtered using a cell
rapid injections can produce cardiovascular
strainer just before administration (Figure 5.2.5). 713
failure and be lethal [2]. Maximum volumes are
Meanwhile, preintravenous administration of
shown in Table 5.2.1 [10–12]. For immunization,
heparin before tumour cell injection is reported
the maximum is lower still, because of the mixing

PROCEDURES
to be an effective method to decrease mortality
caused by thromboembolism [8].
TABLE 5.2.1: Guidelines for maximal administra-
tion volumes and needle size
Concentration of substances Maximal
Administration administration
The concentration can vary over a fairly wide route volumes (mL) Needle size
range without greatly influencing the end result
of the experiment. Lower concentrations are Oral 0.2 22G
clearly desirable [9]. Factors limiting the use of
Subcutaneous 2e3 (scruff) 25G
aqueous solutions for parenteral administration 0.2 (inguinal)
are probably related to their osmotic pressure. Intraperitoneal 2e3 23G
Low concentrations can be corrected by the addi- Intravenous 0.2 25G
tion of sodium chloride but ought not to be so
Intradermal 0.05 26G
high as to materially exceed the osmotic pressure
Intramuscular 0.05 25e27G
of 0.15 M sodium chloride [6]. Highly concen-
Intracerebral <0.03 27G
trated solutions can be administered intrave-
nously provided the rate of injection is kept Intranasal <0.02
slow and precautions are taken to avoid getting Source: Flecknell [10], Reeves et al. [11], Wolfensohn and
the solution outside the vein. Lloyd [12].
 with adjuvant. Maximum volumes for injection of size of the substance [2]. Compounds that are
antigen with or without adjuvant per route are highly soluble in the body fluids, will be absorbed
indicated in the section on ‘Immunization of quickly. Substances that are ionized and are not
mice’ in this chapter. lipid soluble can only be absorbed if a specific
carrier exists. In general, the rate of absorption
is in the following order [12]:
Rate of absorption and
iv > ip > im > sc > po:
distribution of administered
substances
The blood flow to the site of administration, the
nature of the substance and its concentration
Needles and
influence the rate of absorption [2, 12]. The
time-course of the effect of the substance is an
syringes
important factor in determining the dosage and
is influenced by the rate of absorption [9]. Nor- Usually, 26–27G, 12.5–15.6 mm (1/2 to 5/8 inch)
mally, injected substances must be absorbed needles are satisfactory for injection. The smallest
ADMINISTRATION

from the site of administration into the blood.
Therefore, the rate of absorption will be deter-
mined by the size of the absorbing surface, the
blood flow and the solubility of the substance in
the tissue fluid. The rate of absorption is also
influenced by lipid solubility, physicochemical
properties, degree of ionization and molecular
OF
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gauge should be selected, as a fine needle prevents
leakage of fluids and will help to minimize
discomfort to the animal [2]. A 1–2 mL syringe is
adequate for most injections. When a small
volume (<1.0 mL) is administered, an insulin
syringe plus needle is convenient (Figures 5.2.6
and 5.2.7; 27–30G, 8.0–12.5 mm (5/16 to 1/2 inch)).

(a)

714

(b)
PROCEDURES

(c)

(d)

(e)

Figure 5.2.6 Insulin syringes. (a) 29G  1/2 in., 0.5 mL, Terumo; (b) 27G  1/2 in., 1.0 mL, Terumo; (c) 29G  1/2 in.,
0.3 mL, Becton Dickinson; (d) 29G  1/2 in., 0.5 mL, Becton Dickinson; (e) 29G  1/2 in., 1.0 mL, Becton Dickinson.

(a)
(b)
(c)
(d)
Figure 5.2.7 Needles for Insulin syringes. (a) 30G  3/8
in., 0.3 mL, Becton Dickinson; (b) 29G  1/2 in., 0.5 mL,
Becton Dickinson; (c) 29G  1/2 in., 0.5 mL, Terumo;
(d) 27G  1/2 in., 1.0 mL, Terumo.
These syringes can be obtained from various
companies (e.g. Terumo, Japan; Becton Dickinson,
USA). Intradermal needles are practical for intra-
cerebral injections (Figure 5.2.8; Top, Tokyo,
Japan). Plastic syringes cannot be used with
solvents such as acetone.
The withdrawal of hazardous substances
from bottles requires great care. An alcohol-
moistened cotton pledget can be kept at the point
where the needle enters the stopper in order to
minimize the inadvertent formation of aerosols
(a)
(b)
Figure 5.2.8 Intradermal needle. (a) 26G  3/8 in.
needle (Terumo); (b) 1/2 in. intradermal needle.
[13]. Because of the risk of embolism, air bubbles
in fluid, syringe and needle must be purged.
Gently tapping the side of the syringe and slowly
expelling the air into absorbent tissue to prevent
any dispersal of the contents until fluid appears
at the tip of the needle can purge air bubbles.
The needle size will vary with the viscosity of
the substance being used; the greater the
viscosity, the larger the needle required [11]. If
blood or body fluid flows back into the needle,
it must be discarded and a fresh attempt made.
Enteral
administration
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Enteral administration has the advantages that it
is possible to give quite large amounts of non-
OF
sterile substances or solution and that a pH as
low as 3 can be administered by this route. On
ADMINISTRATION
the other hand, alkaline solutions are very poorly
tolerated by this route [6]. When using the oral
route it should be understood that substances
can be destroyed by the gastric juices and that
the food content of the stomach influences both
rate and order of the gastric emptying. The rate
of absorption is markedly influenced by its time
of residence in the stomach and is also directly 715
related to the rate at which substances are passed
from the stomach into the intestine [14]. Enzymes
PROCEDURES
of the host and microflora of the digestive tract
can also metabolize the substance. On the other
hand, some insoluble substances may become
solubilized as the result of enzymatic activity
during their passage through the stomach and
intestine, making absorption possible [2]. The
two major methods for enteral administration
are mixing the substance with food or water or
direct administration using gavage. Rectal admin-
istration is also possible [6].
Oral administration
The simplest method for administration is giving
the substance with food or drinking water.
However, this is not practicable with substances
that are unpalatable, insoluble or chemically
unstable in drinking water or when they irritate
the mucosa of the gastrointestinal tract [2]. The
 daily food and water intake of the mice should be
known before the experiment, to calculate the
quantity of substance to be added. Because
wastage of food and water happens all the time,
it is difficult to determine the precise amount
of food and water intake and therefore the
precise intake of the substance. The only way
this can be done is by keeping mice in metabolic
cages and recording the wastage.
Intragastric administration
Direct administration by oral gavage is preferred
to mixing substances with food or drinking water
because the intake of the substances is precisely
measured. A ball-tip needle is used to prevent
ADMINISTRATION
damaging the oesophagus or passing through the
glottal opening into the trachea (Figure 5.2.9). A
22G ball-tip needle is suitable for administration
to adult mice and can be obtained from suppliers
such as Cadence Inc., or Fine Science Tools Inc.
The conscious mouse is manually restrained by
firmly gripping a fold of skin from the scruff of
OF
the neck down the back (Figure 5.2.10a); immobili-
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zation of the head is essential for this procedure
(Figure 5.2.10b). When the neck is extended, the
position is vertical and there is a straight line
from the mouth to the cardiac sphincter through
the oesophageal orifice (Figure 5.2.10b). The needle
716
is passed gently through the mouth and pharynx
into the oesophagus (Figure 5.2.10c). The mouse
PROCEDURES
usually swallows as the feeding needle approaches
the pharynx, and these swallowing movements can
help the probe to slip through the oesophageal
opening. The substance is then administered
slowly. If any obstruction is felt, if the mouse
(a)
(b)
(c)
Figure 5.2.9 Syringe with a gavage needle. (a) 1.0 mL
syringe with 22G  1.0 in. feeding needle; (b) 1.0 mL
syringe with 22G  11/2 in. feeding needle; (c) 1.0 mL
syringe with 20G  11/2 in. disposable feeding needle.
coughs, chokes or begins to struggle vigorously
after the gavage begins, or if fluid is seen coming
out of the nose, it may indicate that the needle
has entered the lungs. Any of these signs necessi-
tates immediate withdrawal of the needle,
and the mouse must be observed very carefully.
If there is any sign that fluid has got into the
lungs, the mouse should be euthanized. As soon
as administration is finished, the needle must be
withdrawn [5, 15]. A volume of less than 0.2 mL is
recommended.
Parenteral
administration
Administration of substances to the body other
than via the alimentary canal includes injection,
infusion, topical application, inhalation and
implantation of an osmotic pump or
a controlled-release drug delivery pellet. Small
amounts of solution are injected, and large
volumes are infused. In both cases a needle
must penetrate the skin. Subcutaneous, intraperi-
toneal and intravenous administration are the
most common and important routes to inject
substances in solution or suspension into the
mouse. The rate of absorption is dependent on
the administration route. Following intravenous
injection the substance will disperse immediately;
this route therefore achieves the most rapid
absorption. The large surface area of the abdom-
inal cavity and its abundant blood supply also
facilitate rapid absorption; absorption from this
route is usually 25–50% as rapid as that from
the intravenous route [6].
Subcutaneous
administration
Subcutaneous administration is easy. As it is rarely
painful [12], a conscious mouse can usually be used.
The rate of absorption is lower than with intraper-
itoneal or intramuscular injections [16]. Subcuta-
neous injections are made into the loose skin over
the interscapular (Figure 5.2.11a) or inguinal area
(Figure 5.2.11b). Subcutaneous administration
 (a) (b) (c)

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Figure 5.2.10 Procedure for intragastric administration using ball-tip needle. (a) Before extending mouse’s
neck; (b) A straight line is formed between mouth and stomach; (c) Intragastric injection is made using 1.0 mL

OF
syringe with 22G  1.0 in. feeding needle.

ADMINISTRATION
over the interscapular area is performed as follows. leakage, the needle should be advanced several
The mouse is manually restrained and then placed millimetres through the subcutaneous tissue [5, 15].
on a clean towel or solid surface. The needle is
inserted under the skin of the interscapular area
tented by the thumb and index finger and the
substance is then injected. A volume of less than
Intraperitoneal
3 mL is recommended. Subcutaneous administra-
tion over the inguinal area is done as follows. The
administration 717
mouse is restrained manually and the head tilted

downwards. Holding the hind leg firmly helps
this procedure (Figure 5.2.3). The needle is inserted
into the lower left or right quadrant of the
abdomen, avoiding the abdominal midline, and
the substance is injected. A volume of less than
0.2 mL per site is recommended. To minimize
PROCEDURES
This is the most common route, being technically
simple and easy. It allows quite long periods of
absorption from the repository site. The rate of
absorption by this route is usually 25–50% as
rapid as by the intravenous route [6]. Its limita-
tions are the sensitivity of the tissue to irritating

(a) (b)

Figure 5.2.11 Subcutaneous injection. (a) Subcutaneous injection at the base of a fold of loose skin (area at the
neck) using an insulin syringe: 27G  1/2 in., 1.0 mL; (b) Subcutaneous injection at the lower left quadrant using
an insulin syringe: 27G  1/2 in., 1.0 mL.

Figure 5.2.12 Intraperitoneal injection to lower left
quadrant using insulin syringe: 27G  1/2 in., 1.0 mL.
substances and less tolerance to solutions of non-
ADMINISTRATION
physiological pH. Solutions should be isotonic
and quite large volumes can be administered
via this route.
The conscious mouse is manually restrained
[16] and held in a supine position with its posterior
end slightly elevated, or the head can be tilted
lower than the body (Figure 5.2.12). The needle
OF
and syringe should be kept almost parallel to
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the mouse’s vertebral column in order to avoid
accidental penetration of the viscera [17]. The
needle is pushed in at an approximately 10 angle
between the needle and the abdominal surface in
the lower left quadrant of the abdomen [16]. To
718
avoid leakage from the puncture point, the nee-
dle is run through subcutaneous tissue in a cranial
PROCEDURES
direction for 2–3 mm and then inserted through
the abdominal wall [15]. The recommended
volume is less than 2.0 mL for a 40 g mouse.
Intravenous
administration
Intravenous injection has advantages over other
routes. There is one primary route, the tail vein;
another possibility is the ophthalmic plexus route.
Solutions that are highly concentrated, high or
low pH, or irritating, can be administered intrave-
nously provided that the rate of injection is kept
slow and precautions are taken to avoid getting
the solution outside the vein. Compounds that are
poorly absorbed by the digestive tract may be given
intravenously but intravenous administrations
require technical expertise and skill. The syringe
plus needle or the catheter must first be filled
with the solution to remove air bubbles. Adminis-
tration is usually into the lateral tail vein, not the
dorsal tail vein (Figure 5.2.13a), as it is not straight.
The lateral veins are readily visualized, but are
quite small in diameter. If anaesthesia is not used,
a restraining device is usually necessary (see
Chapter 5.1; [5, 11, 18]).
The mouse is either placed in the restrainer or
anaesthetized and the tail is then warmed with
a lamp or warm towel, or immersed in warm water
(40–45  C) in order to dilate the vessels [10]. The tail
is swabbed with 70% alcohol on a gauze sponge or
swab. The needle is inserted parallel to the tail vein,
penetrating 2–4 mm into the lumen, while keeping
the bevel of the needle face upwards
(Figure 5.2.13b). The solution is then injected slowly
and no resistance should be felt if the solution is
properly administered (Figure 5.2.13c). The
injected solution temporarily replaces the blood,
but should then be washed away by the blood
stream. If this does not happen, the position of
the needle is certainly not in the vein but in the
surrounding tissue, so it must be either moved in
the surrounding tissue in such a way that it then
enters the vein, or a new attempt must be made.
When the intravenous administration is finished
or the cannula is pulled out, the injection site
must be pressed firmly with a swab or fingers to
prevent backflow of the administered solution
and/or blood [2, 5]. If the same vein must be used
several times the first administration should be
made as distal as possible in relation to the heart
and subsequent administrations should be placed
progressively more proximally. Because vene-
puncture and the administration of substances
can damage and/or block the vein, the distal part
of the vein may no longer be used [2]. The recom-
mended volume is less than 0.2 mL.
The ophthalmic plexus route is also used for
fluid administration [19, 20], but this method of
application is controversial and is under discussion
or even forbidden in various countries for reasons
of animal welfare. This route may be suitable in
case of emergency for rescuing a mouse that shows
signs of anaphylactic shock. Details of the tech-
nique are described in the section on ‘Rescue
from anaphylactics’ later in this chapter.
Other routes for intravenous administration
via the external jugular vein [21], the dorsal
 (a) (b)

(c)

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OF
ADMINISTRATION
Figure 5.2.13 Intravenous administration. (a) Transverse section view of the mouse tail; (b) Sagittal view of the
mouse tail (the tail is turned 90 ); (c) Intravenous injection to lateral tail vein of an anaesthetized mouse using
an insulin syringe: 27G  1/2 in., 1.0 mL.

metatarsal vein [22] and the sublingual vein [23] injections may be given into the thigh muscle
have been reported. with injection volumes of less than 0.05 mL. The
tip of the needle should be directed away from
the femur and sciatic nerve (Figure 5.2.14). The 719
Intramuscular mouse is anaesthetized or is manually restrained

administration
The intramuscular route should usually be
avoided, as mouse muscles are small. If necessary,
PROCEDURES
by another person. The needle tip is inserted
through the skin and into the muscle and aspi-
rated briefly with the syringe before injection.
If there is backflow of blood or body fluid, the
procedure should be stopped; the needle must
be moved or a fresh attempt must be made.
Good technique and restraint are necessary and
this method should only be performed by well-
trained personnel [2, 4, 6, 15].

Intradermal
administration
This route is not recommended in general and
should be restricted to cases of absolute necessity
Figure 5.2.14 Intramuscular injection into the leg [3, 24]. It is a very difficult route in the mouse
muscle. because of the animal’s very thin skin. A fine

Figure 5.2.15 Intradermal injection into the skin of
the back.
needle (29G or less) is recommended. The mouse is
anaesthetized, the fur clipped or hair removed
ADMINISTRATION
The site of injection is approximately half way
between the eye and ear and just off the midline
(Figure 5.2.16a). The recommended maximum
volume per suckling mouse is 0.01 mL and that
for weanling or older mice is up to 0.03 mL. The
needle directly pierces the cranium
(Figure 5.2.16b). An intradermal needle
(Figure 5.2.8) is convenient in order to prevent the
needle from extending too deeply into the brain.
To inject into a specific region of brain, mice
have to be restrained using a small animal stereo-
taxic instrument (Figure 5.2.17), as supplied for
example by David Kopf Instruments, NARISH-
IGE or Stoelting. The injection region is decided
with reference to a brain atlas [27]. This atlas

from an area on the back, ventral abdomen, or

hind footpad, which is wiped with 70% ethanol
on a gauze sponge or swab. The skin is held taut
with thumb and index finger and the needle
inserted, bevel up and at a shallow angle, just under
the superficial layer of epidermis. The volume
should be less than 0.05 mL per site. Resistance
OF

should be felt both as the needle is advanced and

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as the compound is injected. A hard bleb will be

seen upon successful intradermal injection of
even a small quantity of fluid (Figure 5.2.15) [5]. If
multiple sites are injected, adequate separation is
720 necessary to prevent coalescing of lesions.
PROCEDURES

Intracerebral
administration
Figure 5.2.17 Stereotaxic alignment system (Model
For this procedure the mouse is anaesthetized and 940, David Kopf Instruments).
then restrained manually on a solid surface [25, 26].

(a) Ear line Eye line (b)

Figure 5.2.16 Intracerebral injection. (a) Injection site of the head for intracerebral injection; (b) Intracerebral
injection into an anaesthetized mouse using an intradermal needle.
relates to the adult male C56BL/6J mouse, there-
fore some experiment is needed if other mouse
strains are to be used.
Intrathoracic
administration
Intrathoracic injection is restricted to special
experiments. It can be made in mice with a slightly
bent or curved needle, which should be inserted
between the ribs at approximately the midpoint
of the rib cage. Caution must be taken to insert
the needle at an angle, thus preventing injection
directly into lung tissue. The speed of absorption
is similar to the intraperitoneal route [16].
Intranasal
administration
This is usually done with the mouse lightly anaes-
thetized. The animal is manually restrained and
the tail anchored between the small finger and
the palm [16]. The mouse is held in a supine posi-
tion with the head elevated. The end of the
micropipette is placed at or in the external nares,
and then the solution is poured in slowly
(Figure 5.2.18) [25, 28, 29]. The volume should be
less than 0.02 mL. Excess volume or rapid injec-
tion will induce suffocation and death.
Figure 5.2.18 Intranasal instillation into an anaes-
thetized mouse using a pipette (Gilson P-20).
Topical application
It is not often realized that the skin is the largest
organ of the body and survival depends on its
patency perhaps more than for most other organs.
An animal (or human) can survive with only
about one-seventh of its liver or one-fourth of
its kidney functioning, but destruction of more
than 50% of the skin usually results in death [6].
The skin is also a convenient site for the adminis-
tration of drugs. Numerous factors, such as the
physicochemical properties of the substance, the
attributes of the vehicle and the permeability of
the skin, can affect the degree of percutaneous
absorption [30, 31]. The ability of a substance to
ROUTES
be absorbed through the skin and enter the
systemic circulation is determined by its ability
to partition into both lipid and aqueous phases [2].
The usual site is the skin of the back or the
OF
abdomen. After clipping the hair for topical
ADMINISTRATION
administration (Figure 5.2.4), the hairless area
should be cleaned of any fat and grease and
other debris. The substance to be administered
should be dissolved in a volatile solvent or mixed
in a suitable cream before application and then
applied with a dropper or smeared onto the
skin with a swab [2]. Some precautions are usually
necessary to prevent the animal from licking or
scratching the application sites [6].
721
PROCEDURES
Inhalation
This route is used for experiments on asthma, air
pollution or respiration [32, 33]. The inhalation
route is, incidentally, the most akin to an intrave-
nous injection because of the relatively large
area presented for absorption by a membrane
that is separated from the blood by only one or
two cell layers. Consequently, absorption of gases
and aerosols that reach the alveoli is virtually
complete. The greatest problems surrounding
the use of the inhalation route are the generation
of a suitable aerosol of the test substance, if it is not
sufficiently volatile, a constant and suitable air
level of the material under study and the determi-
nation of the dosage given. Particle sizes that are
too small or too large are not suitable; it is generally
believed that a particle size of 0.5–2.0 mm diameter
 is optimum [6]. Equipment is available to purchase
e.g. from Omuron or Buxco Electronics.
Other routes
Other routes of administration have been
reported such as intra-arterial administration
using the femoral artery [16] or the carotid artery
[34], intrathymic injection [4], intraspinal injec-
tion [35], intrathecal injection [36] or intracardiac
injection [8].
The dosing and treatment of newborn mice
provides special problems not only because of
their size or but also because the dam is apt to
reject or cannibalize neonates that have been
ADMINISTRATION
handled. Subcutaneous injections can be made
over the neck and shoulders using a less than
30G  5/16 inch needle. Up to 0.1 mL (depending
on the age of the infant mice) may be adminis-
tered orally using a piece of plastic tubing inserted
over a needle [15, 37]. Direct injection into the
stomach of infant mice can be made through
OF
the abdominal wall [38]. Intravenous injection
ROUTES
into infant mice has also been reported [15, 39, 40].
Implantable pumps,
722
controlled-release
drug delivery pellets
PROCEDURES
and cannulas
The delivery of substances at a slow, steady rate
over a period of days, weeks or months without
the need for external connection or frequent
animal handling can be accomplished by using
an osmotic pump or controlled-release drug
delivery pellets.
Osmotic pumps (ALZET pumps) can be used
for systemic administration when implanted
subcutaneously or intraperitoneally, or can be
attached to a catheter for intravenous, intracere-
bral or intra-arterial infusion. The pumps have
been used to target delivery to a wide variety of
sites including the spinal cord, spleen, liver, organ
or tissue transplants and wound healing sites.
ALZET pumps are supplied by DURECT.
Controlled-release drug delivery pellets
effectively and continuously release the active
product into the animal. The pellets are intended
for, although not limited to, simple subcutaneous
implantation in laboratory animals. Pellets are
available from Innovative Research of America.
The dosage can be selected (from 1 mg to 200 mg
per pellet), and the release period is also select-
able (21, 60 or 90 days).
Implantable cannulas permit continuous
access to the venous or arterial system for either
intravenous substance administration or blood
withdrawal. Using strict aseptic techniques, the
cannula is inserted into a vein or artery (the
femoral vessels, jugular vein and carotid artery
are common sites) and secured in place. The other
end of the cannula is attached to a small port that
is secured in a subcutaneous location, most often
over the shoulders [5]. See Desjardins [41] for
more information on implantable cannulas.
Immunization
Mice are not used for the production of polyclonal
antibodies because of the small amounts
produced. On the other hand, they are a good
source of antibody-producing lymphoid cells or
hybridomas [42]. In general, immunization consists
of two stages; primary and booster. The primary
antigen is usually injected with adjuvant. Boosters
are injected once or more with or without adjuvant
depending on the immunogen. Footpad, intra-
splenic [43] or intralymph node [44] injection is
not recommended in general. If required, the
investigators should provide scientific justification
to ethics committees for such protocols (such as the
need to use valuable, unique and irreplaceable
antigens, or extremely small quantities of antigen).
The injection of immunogens at the base of the tail
or in the popliteal area substitutes for footpad
injections with much less distress to the animal,
because immunogens injected into the footpad
are processed by the popliteal lymph node [3, 45].
The intraperitoneal route for injection of Freund’s
complete adjuvant (FCA) is permitted in small
rodents only. FCA should be administered only
once, and be limited to minimal volumes of up to
0.1 mL. In the mouse, up to 0.1 mL with adjuvant
may be administered subcutaneously in the neck
 TABLE 5.2.2: Maximum volumes of antigen, with or without adjuvant, per route
Administration route
Subcutaneous
Intramuscular
Intraperitoneal
Intravenous
Intradermal
The intradermal route should be restricted to cases where it is absolutely necessary [3].
Source: Warsson et al. [48]; Van Zutphen et al. [49].
region. Oil-based or viscous gel adjuvants should
not be injected by the intramuscular route [46].
The intravenous route should also not be used
for oil-based adjuvants, viscous gel adjuvants or
large-particle antigens, due to the risk of pulmo-
nary embolism [47]. Though FCA is the strongest
adjuvant, use of other adjuvants can be recom-
mended. Mice must be closely monitored immedi-
ately after injection for any anaphylactic
reactions, both after the primary and any booster
injections [3]. The recommended route and
volumes are shown in Table 5.2.2.
Rescue from
anaphylaxis
In some cases, anaphylactic shock happens in
hyperimmunized mice. Intravenous fluid replace-
ment of isotonic solution is effective to rescue
mice, but the tail vein of mice in anaphylactic shock
is often collapsed by the low blood pressure. In this
case, peripheral intravenous access via the sublin-
gual vein is the preferred route [23]. Alternatively,
fluid replacement may also be achieved using the
retro-orbital sinus route [19, 20]. In a normal-sized
mouse, the injection of 0.5–1.0 mL of warmed
isotonic solution is enough for rescue. In detail,
the technique resembles blood collection by
retro-orbital sinus puncture (see Chapter 5.3). The
mouse is manually restrained on a solid surface,
being held gently but firmly by the nape of the
neck. By pressing down with the thumb and fore-
finger in the occipital area and pulling back the
skin, the point of the needle can be directed toward
the back of the orbit at a 20–40 angle. The needle is
Maximum volume (mL)
With adjuvant Without adjuvant
0.1 0.5
Not recommended 0.05
0.2 1
Not recommended 0.2
Not recommended 0.05
inserted medially through the conjunctiva on the
inner side of the ocular cavity. If entry is blocked
by bone, the needle is withdrawn slightly. Fluid is
ROUTES
injected slowly, loosening the neck skin slightly.
Mice in anaphylactic shock are unable to maintain
their body temperature, therefore warming them
OF
is an effective means of recovery.
ADMINISTRATION
Acknowledgement
We are grateful to Mr T. Fujisawa for his skil-
ful photography.
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PROCEDURES
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ADMINISTRATION
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Suppliers’ websites
Becton Dickinson, Franklin Lakes, USA: http:// 725
www.bd.com
Buxco Electronics, Inc., Sharon, USA: http://
PROCEDURES
www.buxco.com/
Cadence, Inc., Staunton, USA: http://www.
cadenceinc.com/
David Kopf Instruments, Tujunga, USA:
http://www.kopfinstruments.com/
DURECT Corporation, Cupertino, USA: http://
www.alzet.com/index.html
Fine Science Tools Inc., Canada: http://
www.finescience.ca
Innovative Research of America, Sarasota, USA:
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NARISHIGE, Tokyo, Japan: http://narishige-
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Omuron, Kyoto, Japan: http://www.healthcare.
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Stoelting Co., Wood Dale, USA: http://www.
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