UNIT 1.

10 Survival Surgery: Removal of the

Spleen or Thymus
Although for most experiments lymphoid tissue is removed from the freshly sacrificed
animal, there are several circumstances which require the surgical removal, under anes-
thesia, of either the thymus or spleen, and the recovery of the animal for further
investigation. In particular, the removal of the spleen (splenectomy), or a fragment thereof
(hemisplenectomy), can be a valuable source of either T or B lymphocytes, or of a
relatively large amount of tissue for extraction of RNA or DNA. The removal of the
thymus from neonatal or adult animals has profound effects on T cell development and
continues to be an important procedure in studies of T cell ontogeny, tolerance, and
education.
This unit describes survival surgery for removal of the adult spleen as well as the adult
and neonatal thymus of the mouse. Adult mice are anesthetized with pentobarbital or other
injectable anesthesia. Neonatal animals are very difficult to anesthetize and hypothermia
is the method of choice.

BASIC SPLENECTOMY
PROTOCOL
Materials
Mouse (>3 weeks old)
1 mg/ml pentobarbital
70% ethanol
Iris scissors (straight or curved)
Iris forceps (10 cm long with curved ends)
4-0 sutures
Sterile 10 × 10–cm gauze sections
Additional reagents and equipment for anesthesia (UNIT 1.4) and intraperitoneal
injection (UNIT 1.6)
1. Anesthetize the mouse via intraperitoneal injection.
Administer anesthesia 10 to 15 min before surgery. Follow the dosage guidelines listed in
Table 1.4.1. Initial dose should be 40 mg/kg. Observe the animal for response to anesthe-
sia—it should have slow, shallow respirations and be unresponsive to squeezing hind feet.
If these effects are not seen, inject more anesthesia or administer inhalant as in UNIT 1.4.
For some animals, as much as 150 mg/kg may be required.
2. Wet the fur with 70% ethanol, to keep fur from entering the peritoneum.
Water does not wet fur very well because of skin oils.
3. Lay the animal on its right side. With the scissors, make a left-sided skin incision,
∼2.5 cm long, midway between the last rib and the hip joint (Fig.1.10.1A).
4. Loosen any connective tissue under the skin using the blunt end of the forceps.
The spleen is easily seen through the peritoneum attached to the greater curvature of the
stomach by mesentery.
5. Make a 1- to 2-cm incision in the peritoneal wall and gently pull the spleen onto the
exterior surface of the peritoneum.
A small artery, not always visible, is attached to the hilum of the spleen, closest to the
Survival Surgery: stomach.
Removal of the
Spleen or Thymus

1.10.1 Contributed by J.P. Reeves, P.A. Reeves, and L. Thomas Chin

Current Protocols in Immunology (1991) 1.10.1-1.10.11
Copyright © 1991 by John Wiley & Sonc, Inc.
Supplement 2
 6. Tie off the artery with a 4-0 suture by looping the suture through the mesentery. Make
a single knot at the tip of the spleen.
The efferent venule is attached at the other end of the spleen. It may be necessary to tie this
vessel if excessive bleeding occurs when the spleen is removed.
7. Cut away the mesentery and connective tissue, and remove the spleen.
8. Close the peritoneal wall with one or two separate sutures. Close the skin with an
additional two or three sutures.
The ends of the suture should be cut close to the knots of the suture to prevent the mouse
from chewing the ends and loosening the knot. Suture knots should be as tight as possible
to prevent loosening.
9. Remove any blood from the skin by wiping with a sterile gauze section.
10. Place the animal on its side 15 to 22 cm from a desk lamp. Allow the animal to rest
until the anesthesia wears off (30 to 60 min), making sure that the airway is not
obstructed.
11. As the animal comes out of anesthesia, it will begin to shake and then to move around.
When the animal is able to roll over onto its feet on its own, return it to a clean cage.
The animal will begin to groom itself at the incision and may bite the sutures. No more than
five animals should be placed in a large cage for the recovery period.

Figure 1.10.1 Surgical removal of the spleen. Through the small incisions in the skin, abdominal
musculature, and peritoneum, the spleen is exteriorized. The splenic artery is ligated and the spleen
removed.
Care and
Handling of
Laboratory
Animals

1.10.2
Current Protocols in Immunology Supplement 2
 ALTERNATE HEMISPLENECTOMY
PROTOCOL This procedure is essentially the same as splenectomy, except that only part of the spleen
is removed.
1. Follow steps 1 to 5 of the basic protocol for splenectomy.
2. Minimize bleeding by placing a small loop of suture across the center of the spleen,
leaving the hilar vessels intact. Gently tighten the suture and tie it.
Tightening too quickly will split the spleen, especially if there is moderate splenomegaly
prior to surgery.
3. Cut the lower part of the spleen with scissors and remove it.
4. Close the animal as in the basic protocol for splenectomy, steps 8 to 11.

BASIC NEONATAL THYMECTOMY

PROTOCOL Neonatal thymectomy is used most often to remove mature T cells without removing T
cell precursors. Mice from birth to day 3 can be thymectomized for this purpose. Removal
of the thymus after day 3 does not alter the influence of the thymus on the development
of later T cell-dependent antibody or cellular immune responses.
Materials
Mouse (0 to 3 days old)
Saline or phosphate-buffered saline (PBS; APPENDIX 2)
Sterile 5 × 5–cm gauze sections
Incandescent desk lamp
Dissecting board (wood or styrofoam)
Dissecting microscope
Iris scissors (angled with extra-fine tips; Roboz #RS-5650, -5658 or equivalent)
Forceps with nonserrated ends (curved with fine tips; Roboz #RS-5045, -5047
or -5057 or equivalent)
Cannula (see support protocol)
Hemostats or forceps
6-0 suture
Cotton-tipped applicators, soaked in saline or PBS
Topical flexible collodion (U.S.P.)
60- or 100-mm Petri plate, sterile
1. Remove newborns from their mother and take them to a separate room for the
operation.
The noises made by the newborns during surgery agitate the mother and may cause the
mother to attack them when they are returned.
2. Place newborns on clean gauze, 10 to 15 cm from the incandescent desk lamp.
3. Anesthetize by hypothermia. Place one newborn at a time in a hole of ice (2.5 cm
diameter, 5 to 8 cm deep) in an ice bucket for 3 to 4 min until general movement of
the limbs ceases.
Tips of the toes and fingers become white. Longer exposure to the ice, causing entire limbs
to become white and frostbitten, adversely affects survival. Placing the mice in the ice a
second time for re-anesthesia substantially reduces survival rates as well.
4. Remove newborns from the ice and place them on the dissecting board between
parallel rubber bands (held in place by two sets of poster pins). Restrain the arms and
Survival Surgery: legs under the rubber bands (Fig. 1.10.2A).
Removal of the
Spleen or Thymus

1.10.3

Supplement 2 Current Protocols in Immunology

 5. Use a dissecting microscope for the surgery. With scissors, make a small incision in
the skin just over the second or third ribs. Elongate the incision toward the clavicle.
6. Puncture the chest cavity as shallowly as possible with the point of the scissors at the
second or third rib on the right side, at the junction of the ribs and sternum.
7. Quickly elongate the incision as shallowly as possible at the junction of the ribs and
the sternum toward the clavicle, cutting through the right edge of the manubrium and
clavicle.
8. Gently separate the ribs with the points of the curved forceps. Partially expose the
top portion of the yellowish bi-lobed thymus just under the ribs, on top of the heart
and toward the head, away from the heart and lungs (blowup, Fig. 1.10.2A).
9. Position the tip of the cannula (rinsed in saline or PBS with suction) near the thymus,
approaching from the head at a very shallow angle (Fig. 1.10.2B).

Pasteur pipet used

Iris scissors
to make cannula
cannula
detail of tip
forceps

poster pin

rubber band
A

tips of submaxillary gland

thymus lobes
trachea

cut edge of sternum

Care and
Figure 1.10.2 Neonatal thymectomy. (A) Position of anesthetized newborn mouse secured to the Handling of
operating board with rubber bands. Thymus and surrounding structures following sternal incision Laboratory
(blowup). (B) Position of instruments to remove thymus from neonate is indicated. Animals

1.10.4
Current Protocols in Immunology Supplement 30
 10. Gently suction the thymus to remove it all at once.
CAUTION: If the animal is properly anesthetized, there will be little bleeding when the
thymus is removed. If excessive bleeding occurs, the animal will almost certainly die. Be
careful to apply as little suction as possible to remove the thymus and not lacerate the aorta
or the heart itself. Small amounts of bleeding can be controlled by suction and the use of
cotton-tipped applicators. Check to see if most of the thymus is removed, and resuction any
remaining bits of the thymus. However, do not spend much time exploring, since prompt
closure is critical to survival.
11. Using hemostats or forceps, place a single suture in the skin of the newborn to draw
the sternum and ribs together.
Additional sutures to close the ribs are not necessary.
12. Wipe off any excess blood with a saline- or PBS-soaked cotton-tipped applicator.
13. Cover the incision with a drop of topical collodion to seal the wound and allow it to
dry.
14. Return the mouse to a gauze-lined, sterile Petri plate and place ∼20 cm from an
incandescent desk lamp.
CAUTION: The warming must be gradual since too rapid a rate can induce shock. Do not
use an infrared heat lamp because the heat output is excessive.
15. Return newborns to their mother when their limbs are moving normally, their
breathing is normal, and their color is rosy pink. Place them in a corner of the same
cage from which they were removed in a small depression of the cage bedding.
Lightly cover newborns with cage bedding.
The mother will frequently seek the newborns when the room is quiet and then make a fresh
nest of her own either in the same or another corner. She will then move them by carrying
them in her mouth. (This may appear to be an attack on the newborns.) Do not attempt to
ear- or toe-clip the thymectomized newborns until they are of weanling age because the
mother may attack them.

BASIC ADULT THYMECTOMY

PROTOCOL Removal of the thymus in an adult mouse creates a “status quo” in the immune system:
no new T cells will be generated, allowing the analysis and experimental manipulation of
existing T cells without any dilution by incoming T cells.
Thymectomy of the adult mouse is similar to that of the neonate; however, as the thymus
undergoes considerable enlargement in the first few weeks of life, some adjustments in
technique are required as described below. Protocols for young and adult thymectomy in
rats are described in UNIT 15.12. Two procedures are presented: the “suction” method is
similar to that described in neonatal thymectomy except a larger cannula is used, while
in the “dissection” method, the large adult thymus is manipulated and removed with
forceps.
Materials
Mouse (>3 weeks old)
70% ethanol
Dissecting board (∼7 × 10–cm)
Elastic bands
Iris scissors (10-cm straight)
Iris forceps (2 pair of 10-cm half-curved)
Survival Surgery:
Sterile 5 × 5–cm gauze sections
Removal of the Suction cannula (support protocol)
Spleen or Thymus

1.10.5

Supplement 30 Current Protocols in Immunology

 Rubber tubing, 6-mm diameter
Clay Adams autoclip applier (#7630-000-900) and 9-mm wound clips (#7631)
Incandescent desk lamp
Additional reagents and equipment for anesthesia (UNIT 1.4) and intraperitoneal
injection (UNIT 1.6)

Anesthetize and dissect the mouse

1. Place scissors and forceps in a small beaker with 70% ethanol.
2. Anesthetize mouse by intraperitoneal injection.
Pentobarbital is recommended for this procedure (see Table 1.4.1). The anesthesia must be
deep enough to avoid any spontaneous movement by the animal. As with all surgical
procedures, anesthesia is crucial for the success of an operation (see UNIT 1.4). Mice should
have slow, shallow breathing and should not respond to squeezing the hind feet.
3. Place the mouse on the dissecting board in the dorsal position with its head facing
the operator. As illustrated in Figure 1.10.3, place a rolled-up gauze pad under the
mouse’s shoulders to aid in pushing the heart and thymus forward for easier access.
Restrain the arms and legs by placing them under parallel elastic bands. Extend the
mouse’s neck by securing the head with a third rubber band placed in its mouth.
4. Swab neck and upper chest area with 70% ethanol. With scissors, begin a midline
longitudinal skin incision over the suprasternal notch. Extend the incision 2 to 3 cm
down the chest.
5. Loosen the skin from the underlying muscle using the blunt end of the forceps. Reflect
the skin to expose the thoracic cage.
6. Stabilizing the thoracic cage with your free hand, insert scissors under the sternum
and cut to the second or third rib.
In some mice the sternum and thus the midline are difficult to identify; in this case, it is
prudent first to identify the trachea by freeing and retracting the submaxillary glands. The
trachea will mark the midline as well as the point of insertion of the scissors into the chest.
The second ribs are easily identified as they form the first prominent “v” from the
suprasternal notch where they join the sternum.

2nd rib
3rd rib sternal notch
trachea

rolled up gauze pad incision

length

Care and
Figure 1.10.3 Adult thymectomy. (A) Position of the adult mouse secured to the operating board Handling of
with rubber bands. (B) Location of the sternal notch. The incision is made in the midline from the Laboratory
Animals
sternal notch to the second or third rib (blowup).
1.10.6
Current Protocols in Immunology Supplement 2
 7. Turn the mouse board 90° so that the mouse’s side now faces the operator; hold
forceps in each hand, points down.
Hereafter, the forceps held in the hand closest to the mouse’s head will be referred to as
the cranial forceps; the forceps held in the hand closest to the mouse’s tail will be referred
to as the caudal forceps.
8. Insert the tips of the closed caudal forceps into the incision. Expose the chest by
allowing the forceps to open. The elastic recoil of the forceps will hold the chest open.
Generally the thymus is not visible at this stage as the strap muscles from the neck overlie
the thymus.
9. Using the cranial forceps, retract the strap muscles by gently inserting the closed
forceps through the muscle layer and then allowing forceps to open. Insert the caudal
forceps and open so that the strap muscles as well as the chest is held open.
The lobes of the thymus should now be visualized as two thin white discs overlying the
heart. Retraction of the strap muscles is not always necessary: the strap muscles attach at
a point near the second rib, so retraction of a chest incision made beyond this point often
moves the strap muscles aside.

Remove the thymus

Either of two methods can be used to remove the thymus. The “dissection” method is
described in steps 10a to 12a and the “suction” method is described in steps 10b to 12b.
Whichever technique is chosen, practice should reduce the total time required to <1 min,
to preclude collapse of the lungs (see commentary).
Dissection method
10a. Gently grasp the thymic lobes at the lower pole with the cranial forceps and lift up
and toward the head.
The thymus overlies the great vessels and atria cranially, and the ventricles caudally.
11a. Gently pull the thymus out of the chest as follows: switch grasp of the thymus to
the caudal forceps which had been holding open the chest wall—the chest wall will
partially close on the thymus. Remove the cranial forceps and flip so that the points
now face up. Grasp the thymus at a point below the caudal forceps, remove the caudal
forceps, and lift the thymus further. Flip the caudal forceps so that the points also
face up. Again grasp the thymus at a point below the other forceps. By alternately
grasping and lifting the thymus at successively lower positions, remove the thymus
from the chest cavity. Rotating the forceps slightly to the left and the right will also
aid in bringing the thymic lobes out of the chest.
The thymus has been successfully mobilized when both lobes are evident outside of the
chest, and only thin translucent connective tissue remains attaching the underside of the
thymus to the mouse.
12a. One pair of forceps will now be under the thymus and the thin connective tissue
should be visible. Anchor the connective tissue by grasping with the other pair of
forceps and pull the thymus free. Hold the skin closed and proceed to step 13.
Step 12 is essential as vital structures such as major blood vessels are attached to this
connective tissue and may be damaged by simply pulling the thymus free.
If the mouse has been positioned correctly, it is generally possible to remove both lobes
simultaneously. In some cases, especially when the chest is rotated or if the chest incision
was made off of the midline, it is not possible to grasp both lobes simultaneously. In this
case, remove each lobe separately.
Survival Surgery:
Removal of the Often, thymic remnants fall between the heart and the lungs because of elastic recoil
Spleen or Thymus

1.10.7

Supplement 2 Current Protocols in Immunology

 of the pleura and movement of the heart. Do not attempt to retrieve thymic remnants blindly
(i.e., without clear visualization), as vital structures are often damaged in this manner.
Suction method
10b. Prepare cannula and tubing as described in the support protocol. Insert the suction
cannula and place the tip over the lower pole of one of the thymic lobes in a manner
such that the thymic lobe totally occludes the cannula opening.
11b. Apply suction by mouth or vacuum pump as described in the support protocol,
controlling the amount of suction by varying the degree of occlusion of the side
hole. Simultaneously lift the lobe up and toward the head so the thymic lobe lifts
off the heart and with gentle manipulation is aspirated into the cannula.
12b. Repeat for the other lobe.
13. Inspect the thymus. Both lobes should appear intact. Inspect the chest cavity and
check for thymic remnants, especially if the thymus appears to be missing portions.
Often, thymic remnants fall between the heart and the lungs because of elastic recoil of the
pleura and movement of the heart. Do not attempt to retrieve thymic remnants blindly (i.e.,
without clear visualization), as vital structures are often damaged in this manner.
14. Remove forceps and hold the skin closed to seal the chest.
15. Release the arms from the rubber bands and secure the skin with one or two 9-mm
wound clips.
It is not necessary to suture the chest closed.
16. Wipe excess blood from the incision site and place mouse on its side in a clean cage.
Warm with lamp.
Postsurgery breathing should be regular yet rapid. The skin and tongue should regain a
normal pink color. These parameters along with the absence of bleeding from the incision
generally indicate a favorable postoperative course. Depending on the anesthetic used, full
recovery from surgery and anesthesia takes ∼60 min.
If spontaneous respirations do not return after closing the thoracic cavity, breathing
through a tube placed against its nose or inserted in its mouth may revive the mouse.

CONSTRUCTION OF CANNULA FOR SUCTION THYMECTOMY SUPPORT

PROTOCOL
The following describes cannula construction for suctioning the thymus of neonatal or
adult mice. Most commonly, the suction cannula is constructed from either a Pasteur
(glass) pipet or a disposable plastic pipet. Either may be used successfully for thymec-
tomy, although neonatal thymectomies are best performed with a glass cannula because
a narrower tip can be made and is more easily inserted into the neonate’s chest. The critical
parameter is the size of the opening used for suction. The opening should be small enough
so that the thymic lobe occludes the lumen of the cannula, thereby creating sufficient
suction for removal of the thymus. However, if the opening is too small the thymus will
merely plug the cannula and will not be aspirated.
1. Cut off the last ∼6 cm of the tip-end from a 51⁄4-in. (∼13-cm) Pasteur pipet for neonatal
surgery, or from a 10-ml pipet for adult surgery, just above the taper. In either case,
discard the wide-bore half and fire polish the cut end.
Care and
Handling of
Laboratory
Animals

1.10.8
Current Protocols in Immunology Supplement 2
 2a. For neonatal surgery, after the cut end has cooled, gently heat the other pointed end
until the inner diameter is 1 mm. Proceed to step 3.
2b. For adult surgery, after the cut end has cooled, cut ∼5 mm off of the end of the tip.
The opening should be ∼4 mm in diameter although larger diameters may be needed
for especially large thymi. Smooth the tip with a file or fine sandpaper.
3. Drill a hole ∼6 mm in the side of the cannula to allow for better control of the amount
of suction.
4. Attach the wide end to rubber tubing, a vacuum flask, and house or pump vacuum.
Alternatively, suction by mouth can be applied for better control; however, a relatively
long piece of tubing (>0.6 m or 2 ft) is recommended in this case to prevent accidental
aspiration of the thymus.
Mouth suction is not recommended for neonatal thymectomies.
A second precaution when employing mouth suction is to prepare a small trap consisting
of a 15-ml plastic centrifuge tube, a two-holed rubber stopper, and two pieces of glass or
plastic tubing.

COMMENTARY
Background Information
The spleen is often used as a source of
active lymphoid cells because it is the only
easily available non-vital organ aside from
peripheral blood or the tail. One spleen can
yield 5 × 107 to 5 × 108 white blood cells, as
compared to 5 × 106 cells per ml of peripheral
blood. More DNA can be extracted from the
spleen than from a piece of tail (0.5 to 1 mg
DNA per spleen versus 10 to 100 µg per cm of
tail).
Although the usual means for obtaining
spleen cells is by first sacrificing the animal
and then removing the spleen as described in
UNIT 1.9, special circumstances demand surgi-
cal removal in order to preserve an irreplace-
able animal. Hence, the splenectomy and
hemisplenectomy procedures described here
have become valuable tools to the immunolo-
gist. Although these procedures are designed
for adult mice, they should also be effective on
newborn (i.e., 3 to 5 day old) mice. The protocol
for neonatal splenectomies would be the same
as presented here except for the type of anes-
thesia used. Because neonatal animals are
difficult to anesthetize, hypothermia is usually
chosen. However, if pentobarbital anesthesia is
desired, the proper dosage would have to be
determined empirically using the indicated
amounts for adults as guidelines (see also UNIT
1.4).
Because development of most T cells is
dependent on the thymus, neonatal thymec-
Survival Surgery: tomy has been used extensively to create mice
Removal of the that are completely devoid of T cells and there-
Spleen or Thymus
fore can be used to study the role of these cells
in immune responses in vivo. Neonatal thymec-
tomy can also be used to obtain donors of
tissues that are depleted of T cells for in vitro
experimentation, and was the procedure that
established the role of the thymus in the devel-
opment of T cells (Miller, 1964, 1965).
In order to successfully create a T cell–de-
pleted mouse, the procedure needs to be per-
formed at a very early age—from birth to 3
days. Some strains of mice often present more
problems than others when performing
thymectomies. The birth weight of the mice is
the factor that correlates most closely with
success. Larger mice are usually more tolerant
of the surgery than smaller ones. BALB/c,
C57Bl/6, and C57Bl/10 mice tend to have
lower birth weights and thus tend to be poorer
candidates for survival of the operation than
other strains. Larger-birth-weight mice such as
the autoimmune MRL, NZB, and (NZB ×
NZW)F1 strains are often excellent survivors
of the operation. However, actual practice and
experience with a single strain can overcome
the difficulties described, and survival rates of
>95% can be achieved. For many purposes, a
survival rate of 75% is more than adequate.
Mothers frequently do not provide optimal
care and nursing to their newborns following
neonatal surgery. Residual blood left on the
newborns can be smelled or tasted and the
mother may attack such offspring. Human
scent from the operator’s hands can also be
detected. This scent can be obscured by roll-
ing the newborns in the dirty cage bedding

1.10.9

Supplement 2 Current Protocols in Immunology

prior to surgery or by rubbing gloved hands in
the cage bedding. Loud noises inside the animal
area can be stressful to the mother and lead to
attacks on the newborns. It is well known that
some strains function better as mothers than
others. Lactating outbred Swiss mice can be
used as foster mothers.
In contrast to neonatal thymectomy, adult
thymectomy does not create a fully T cell–de-
pleted mouse: T cells generated until the time
of thymectomy will persist for a long time
(some estimates put the average lifespan of
recirculating T cells at 4 to 6 months). Adult
thymectomy therefore merely serves to prevent
generation of any new T cells, “freezing” the T
cell population at a certain point in time. This
procedure has been particularly useful in study-
ing the effects of immunological manipulations
in the absence of newly developed T cells that
have not been subject to such manipulations.
As such, adult thymectomy has been especially
useful in the study of tolerance induction (Qin
et al., 1989; Webb et al., 1990).
In addition to adult thymectomy, other mod-
els have been used to study the role of the
thymus in immune responsiveness. One of
these models is the nude mouse, that has a
genetic defect in the formation of a normal
thymus (Pantelouris, 1968). However, some
objections to this system have been raised be-
cause these mice appear to have a defect in
epithelial development. This epithelial defect
may have effects independent of the lack of
thymus tissue. Thymectomy in conjunction
with anti–T cell monoclonal antibody treat-
ment (see UNIT 4.1) would, in essence, create a
nude mouse lacking the epithelial defect and
thus presents a viable alternative. The most
stringently T cell–depleted mouse is created
through the construction of radiation bone mar-
row chimeras in thymectomized mice: these
mice are thymectomized at 4 to 6 weeks of age,
lethally irradiated to remove remaining T cells,
and reconstituted with T cell–depleted bone
marrow (Sprent, 1977; UNIT 4.3). Transplantation
of allogeneic or syngeneic thymi into such
thymectomized and irradiated bone marrow
chimeras has been used—e.g., to delineate the
contribution of various extrathymic elements
to T cell development (Bandeira et al., 1991).
Choice of method for adult thymectomy is
ultimately determined by personal preference.
However, each method is better suited for par-
ticular tasks. If an intact thymus is required,
then dissection is the method of choice. While
it is possible to recover an intact lobe with the
suction cannula, frequently the thymus disinte-
Current Protocols in Immunology
grates as it is aspirated. The dissection method
also allows complete visualization of the thy-
mus as it is being removed, ensuring complete
removal. However, in younger mice, ages 1 to
3 weeks, the thymus is friable and often breaks
apart when grasped. Thus, for the younger mice
the suction method is preferred.
Critical Parameters
As with all survival surgery, the most critical
parameter is anesthesia. For splenectomies and
hemisplenectomies, anesthetic doses should be
determined carefully (UNIT 1.4) so that additional
anesthesia is unnecessary. In neonatal thymec-
tomies, excessive exposure to ice reduces the
chances for survival. Preanesthesia of neonates
is discouraged. Placing the animals on ice
ahead of time—while attention is directed to
finishing the current mouse—risks frostbite
and hence, reduced survival. The neonates are
ready for surgery when absence of limb move-
ment is observed, but before the limbs turn
white. The amount of time between these two
events is very short, and if attention is on
another mouse, the risk of frostbite is greatly
increased.
The equipment used in neonatal thymec-
tomy is critical. Several pairs of instruments
should be kept on hand for the surgery. The tips
are very delicate and should be handled accord-
ingly. General laboratory use of these instru-
ments should be discouraged, and they should
be reserved for surgical procedures.
There is no substitute for a high-quality
dissecting microscope with 200× to 400× mag-
nification in neonate surgery. Microscopes with
no stage and a 60-W lamp (Nicholas illumina-
tor) are the best. These offer the ability to use
a large, flat, well-lit surface for the dissection.
It is desirable to be able to move the body of
the microscope both horizontally and verti-
cally.
To minimize damaging vital structures
while preparing mice for adult thymectomy,
incisions should be made with a single cut and
kept as close to the midline as possible, ideally
bisecting the sternum. It also is important to
keep the tips of the scissors pointed up, as this
will lessen the chance of damage to the heart
and great vessels. In fact, it is best to lift the
thoracic cage up with the scissors prior to cut-
ting. Mice generally fare better with small in-
cisions. During initial training for this proce-
dure, larger cuts may be required for better
visualization. The chest should not be left open Care and
Handling of
>1 to 2 min as the lungs will eventually col- Laboratory
lapse. Although speed is required, the operation Animals
1.10.10
Supplement 2
 must be performed carefully and deliberately
to avoid disrupting vital structures. However, if
the mouse begins gasping for air or stops
breathing altogether and more time is needed
to continue the operation, the skin should be
grasped at the edges of the incision and the
incision sealed. Negative intrathoracic pressure
is reestablished as the mouse gasps for air. After
a few minutes the mouse generally recovers,
and the operation may continue.
Prior to using the thymectomized mice for
experiments, it is important to confirm the ab-
sence of thymic remnants by gross and/or mi-
croscopic examination, as remnants often get
pulled between the heart and the lung and might
not be obvious immediately after surgery. It is
not uncommon for thymic remnants to give rise
to a regenerated thymus. These should not be
confused with cervical lymph nodes, which
may approach a size as large as the thymus. To
verify the thymectomy and to distinguish thy-
mus and lymph nodes in this region, gross and
microscopic examination of the animals fol-
lowing their sacrifice is helpful. An alternative
is to perform flow cytometry of splenic or
peripheral blood cells for Thy-1+ cells (UNITS 5.3
& 5.4), or anti-Thy-1 antibody-dependent cyto-
toxicity assays of spleen cells of the putatively
thymectomized animals (UNIT 3.4). This latter
technique will only be informative for neonatal
thymectomized mice, because adult thymec-
tomy will leave T cells for 4 to 6 months.
Anticipated Results
After sufficient practice, a survival rate of
greater than 95% can be achieved following
adult splenectomy or thymectomy. For neona-
tal thymectomy, a survival rate of 95% can be
obtained, but for many purposes, a 75% sur-
vival rate is more than adequate.
Time Considerations
Clearly, the time required for neonatal or
adult thymectomy and splenectomy depends on
the skill of the surgeon. With practice, dissec-
tion and removal of the organ will take 1 to 2
min. Induction of anesthesia and postoperative
observation bring the total time for the opera-
Survival Surgery:
Removal of the
Spleen or Thymus
1.10.11
Supplement 2
tion to ∼10 min. By having one person operate
and another administer anesthesia and observe
postoperatively, twenty mice can be easily
thymectomized or splenectomized in 1 hr. For
neonatal thymectomy, 6 to 10 mice can be done
in 1 hr. The lower rate reflects the time needed
to individually anesthetize the neonates (see
critical parameters).
Literature Cited
Bandeira, A., Itohara, S., Bonneville, M., Burlen-
Defranoux, O., Mota-Santos, T., Coutinho, A.,
Tonegawa, S. 1991. Extrathymic origin of intes-
tinal epithelial lymphocytes bearing T cell anti-
gen receptor γδ. Proc. Natl. Acad. Sci. U.S.A.
88:43-47.
Miller, J.F.A.P. 1964. The thymus and the develop-
ment of immunologic responsiveness. Science
144:1544-1547.
Miller, J.F.A.P. 1965. Effects of thymectomy in adult
mice on immunological responsiveness. Nature
208:1337-1339.
Pantelouris, E.M., 1968. Absence of thymus in a
mouse mutant. Nature 217:370-371.
Qin, S., Cobbold, S. Benjamin, R., Waldmann, H.
1989. Induction of classical transplantation tol-
erance in the adult. J. Exp. Med. 169:779.
Sprent, J. 1977. Migration and lifespan of lympho-
cytes. In B and T Cells in Immune Recognition,
pp. 59-82. John Wiley & Sons, London.
Webb, S.R., Morris, C., Sprent, J.S. 1990. Ex-
trathymic tolerance of mature T cells: Clonal
elimination as a consequence of immunity. Cell
63:1249-1256.
Key Reference
Sjokin, K., Dalmasso, A.P., Smith, J.M., and
Martinez, C. 1963. Thymectomy in newborn and
adult mice. Transplantation 1:521-525.
Classic description of method of neonatal and adult
thymectomy.
Contributed by J.P. Reeves
and P.A. Reeves
Food and Drug Administration
Bethesda, Maryland
L. Thomas Chin (adult thymectomy)
University of Massachusetts
Worcester, Massachusetts
Current Protocols in Immunology