Chapter 3

Necropsy and Sampling Procedures in Rodents

Laurence Fiette and Mohamed Slaoui

Abstract
Necropsy is a major step of most studies using laboratory animals. During necropsy, tissue and organ
changes noticeable grossly can be recorded, and important tissue samples can be stored for subsequent
evaluation. It is therefore important that the personnel in charge of this key experimentation step be
adequately trained and aware of the study endpoints.

Key words: Necropsy, Histology, Macroscopy, Sampling, Rodents, Mouse, Rat

1. Introduction

Necropsy is a postmortem procedure that consists of observation

of macroscopic changes of tissue and organ in situ with naked
eyes and of collection of key organs and tissues samples for fur-
ther analyses.
Necropsy techniques have been developed to diagnose dis-
eases in animals and are classically used in health status monitor-
ing of domestic and laboratory animals. These postmortem
procedures are also considered as an important step in biomedical
research using laboratory animals and in particular in toxicity
studies where histopathology is a major endpoint. In all cases,
necropsy is the ultimate examination of the animal body. It allows
detecting, describing, and reporting of any gross finding that
could be key to understand changes noted during the in vivo part
of the experiment. It is therefore important that the personnel in
charge of the postmortem procedure have access to the animal
history including clinical examination and behavioral changes
that preceded necropsy as well as to the results of any imaging or
laboratory investigations. On the other hand, the necropsy per-
sonnel should have access to the experiment study plan to harvest
tissue important samples to meet the objectives of the study.

Jean-Charles Gautier (ed.), Drug Safety Evaluation: Methods and Protocols, Methods in Molecular Biology, vol. 691,
DOI 10.1007/978-1-60761-849-2_3, © Springer Science+Business Media, LLC 2011

39
40 Fiette and Slaoui

Since necropsy can be performed only once, it is crucial to

follow a precise procedure that allows identification of gross
abnormalities and adequate sampling of organs.
The necropsy procedure consists of a series of systematic oper-
ations that allow examining all body organs and cavities without
altering the characteristics of any tissue or organ of the animal.
The collection of samples for histology or other complementary
analyses also follow precise rules.
Histopathology is a major endpoint of many experiments. It
is therefore crucial that tissues be sampled and preserved in a
standardized manner especially when the microscopic findings
need to be compared group wise. The amount of tissue that needs
to be sampled should be representative of the whole organ since
the probability of detecting lesions is primarily dependent on the
amount of the tissue examined. For large organs of the body, such
as the lungs or the liver, it is therefore necessary to define the
number of sections and the specific lobe/area to be sampled. The
anatomic characteristics of each organ should be considered. For
example, the kidney is composed of a cortex, a medulla, and a
pelvis. Each of these anatomical regions has distinct histological
characteristics that need to be evaluated, as microscopic changes
may reside only in one or two of these structures. For organs
comprising a lumen or a cavity (such as intestines, urinary blad-
der, uterus, or heart), the amount and type of structures present
on a transversal section may considerably vary and may subse-
quently compromise and sometimes irreversibly hamper adequate
microscopic evaluation. Therefore, the plane of section should be
considered and carefully standardized during sampling. For tis-
sues with multiple or complex anatomical structures (such as the
brain, nasal cavity, or intestines), it is advised to collect multiple
sections to examine all structures of interest.
The probability of detecting lesions in the histological slide is
also influenced by the technical procedures such as method of
preservation and preparation. It is essential that the postmortem
degradation process (otherwise termed as autolysis) be controlled.
This is achieved by thorough immersion of tissue samples in a
fixative like formalin. Other fixation techniques are available and
should be always considered depending on each experiment-
specific endpoints.
The purpose of this chapter is to describe the different steps
of the necropsy, and to give general guidelines for sampling pro-
cedures in Rodents although these can be easily adapted to other
laboratory animal species. As the knowledge of anatomy basics
and species differences is required to perform the necropsy, a
basic anatomical description will also be given. It is extremely use-
ful to be aware of the subsequent histological processing of
organs/tissues for histology to understand the rules of sampling
and trimming. Hence, this chapter is intimately linked to the next
 Necropsy and Sampling Procedures in Rodents 41

one dedicated to Histopathology procedures: from sampling of

tissues to histopathological evaluation.
Well-illustrated descriptions of necropsy protocols are
available (1–5), some of them on the Web (6, 7) that would
complement this chapter. Handbooks on the anatomy of
Rodents are also useful (8, 9). The reader is also strongly
encouraged to refer to the three excellent publications from
the RITA/NACAD group about organ sampling and trim-
ming in rats and mice that give guidelines in a very attractive
manner, with excellent full-color macrophotographs and
microphotographs from the corresponding Hematoxylin and
Eosin (H&E)-stained slides (10–12).

2. Materials

1. Fume hood.
2. Rubber gloves (vinyl gloves in case of allergy to latex), protec-
tive clothing, eyeglasses, and mask.
3. Dissecting board preferentially in plastic, which could be eas-
ily cleaned and autoclaved.
4. Blunt ended forceps. Serrated forceps should be avoided as
they may damage small animal tissues.
5. Small dissecting scissors, surgical scissors, and microsurgical
(ophthalmologic type) scissors (These are very useful espe-
cially during extraction of the central nervous system).
6. Syringes (1 mL, 5 mL, 10 mL) and needles (a 21-gauge
needle is suitable for infusion of the lung with fixative).
7. Scalpels (new blades and handle).
8. Plastic bags and paper towels.
9. Containers for histological specimens, cassettes, and labels.
All containers should be adequately identified before start of
necropsy.
10. Specific containers for other specimens (bacteriology, virol-
ogy, mycology, parasitology, chemistry) should be available.
11. Tubes for liquid samples.
12. Euthanasia solution or suitable source of CO2 and container.
13. Fixative (usually 10% neutral buffered formalin). Unless oth-
erwise specified, the fixative mentioned in the text will be 10%
neutral buffered formalin. Ready-to-use 10% neutral buffered
formalin is commercially available from major suppliers.
However, this fixative can be easily prepared. A detailed
procedure is described in Table 1. Formalin is now included
42 Fiette and Slaoui

in the list of human carcinogens and will be abandoned in the

near future. Different commercial alternatives are proposed
and are currently under testing in many laboratories.
14. Decalcifying solution (in case bones should be examined).
There are several decalcifying solutions. A 26% formic acid
solution (TBD2® Shandon Lipshaw) is routinely used in
histopathology laboratories.
15. Ethanol 95% and 70%.

Table 1
Protocol for preparation of formalin and modified
Davidson’s fixatives

Protocol for preparation of formalin 10%, buffered

Formaldehyde (37–40%) 100 mL
Distillated water 900 mL
Monosodium phosphate anhydrous 4g
Disodium phosphate anhydrous 6.5 g
Final solution 3.7–4% formaldehyde
To be used preferentially within 6 months after
preparation. Keep at room temperature
Protocol for preparation of paraformaldehyde 4% (PFA 4%)
Paraformaldehyde (powder) 4g
Distillated water 80 mL
Put 6 L of sodium bicarbonate
Put 10 mL of PBS ×10
Filtrate on a paper
Add PBS to obtain 100 mL
Adjust the pH to 7.2–7.4
The solution should be heated to facilitate the dissolution
of the PFA, but below 65°C and in a fume hood
Store at 4°C up to 24 h
Can be also frozen in aliquots
Protocol for preparation of modified Davidson’s fixative
30% of a 37–40% solution of formaldehyde
15% Ethanol
5% Glacial acetic acid
50% Distilled H2O
 Necropsy and Sampling Procedures in Rodents 43

16. Saline solution or Phosphate Buffer Solution (PBS).

17. Weigh scale.
18. Material to take photographs.
19. Metric scale.
20. Recording necropsy cards.
During necropsy, it is recommended to place the instruments
in a stainless steel instrument holder with 70% ethanol. Used nee-
dles and scalpel should be placed in a special container for harmful
material.
There are several hazards related to handling and dissection
of laboratory animals during necropsy that should be considered.
The chemical risk is one of them. For example, formalin causes
eye, skin, nose, and respiratory tract irritation; it is also classified
as a strong skin sensitizer and carcinogen in humans. Therefore,
formalin should not be handled without gloves or outside a fume
hood (13, 14).
Laboratory Rodents can spontaneously carry and transmit
several diseases to man (also called zoonoses) (15). Although
laboratory Rodents are usually tested for these agents, there is
always a risk to contract one of these diseases. In addition, the
allergic risk remains important especially for people with known
animal allergies. Working with laboratory animals can lead to
exposure to allergens via inhalation, direct skin and eye contact
with animal dander, hair, urine, serum, or saliva. It is therefore
essential that all necropsy personnel uses protective equipments
(16, 17) and that cadavers and all waste be eliminated appropriately.

3. Methods

3.1. General It is strongly recommended to carefully read the experimental

Recommendations study plan or protocol. The list of organs that should be exam-
ined and sampled differs from one study to another depending on
3.1.1. Necropsy Protocol
the aim and duration of the study. The following procedure allows
and Specimen Collection
examining and collecting most tissues and organs in Rodents with
the main purpose of histopathology evaluation of tissue samples.
It is very much inspired from necropsy performed in toxicity
studies. The major steps are as follows:
Examination of the live animal
Euthanasia
Exsanguination
Opening of the abdominal cavity
Opening of the thoracic cavity
44 Fiette and Slaoui

Opening of the skull

Examination of muscles and skeleton.
This protocol should be followed step by step. It is the more
convenient way to remove, examine, and sample each organ or
tissue. Alternative methods are available; they will be mentioned
in the notes.
Organs should always be immersed in the fixative immedi-
ately after their removal, macroscopic examination, or weighing.
It is sometimes required to perform necropsy on animals found
dead. Variable degrees of autolysis will inevitably take place,
but it is still useful to perform the autopsy. Carcasses of found
dead animals can be placed in a refrigerator (but NOT frozen).
Necropsies should be performed as soon as practicable (if pos-
sible within the same day).

3.1.2. Weighing of Organs One important part of necropsy is to describe the size of organs.
This requires that the dissector has in reference the normal size of
the organ. Small variation in size can be challenging even for
experienced pathologist especially when organ sizes need to be
compared among several individuals and groups. To accurately
compare organ sizes, it is most helpful to record individual organ
weights. These can be compared either as absolute organ weights
or as ratios of organ weight to total body weight or to brain
weight. Following the recommendations from the Society of
Toxicologic Pathology (STP) (18) liver, kidneys, heart, brain,
adrenal glands, testes, spleen, and thymus should be weighed
routinely in all general toxicology studies with multi-dose admin-
istration in Rodents (Table 2). Thyroid and pituitary gland should
be weighed in rats but not in mice, as handling of these minute
tissues may induce artifacts that can complicate microscopic
assessment. Epididymides and prostate weighing is recommended
in rat studies but only on a case-by-case basis in mouse as well as
other organs including female reproductive organs in Rodents.
Organs weights are not recommended in carcinogenicity studies.
In all cases, organs should be weighed free of surrounding fat
and connective tissues. It is important to remove these tissues in
a standardized manner and without inducing any damage or arti-
fact to the tissue.

3.1.3. Organ Sampling Most of the tissue samples will be immersed in the fixative. Small
for Histology tissues can be kept in histology cassettes at the time of dissection.
The same cassette, adequately identified, can be used for the paraffin
embedding and paraffin block preparation. The number of cassettes
and hence paraffin blocks can be considerably reduced by combin-
ing a few tissues and organs in one cassette. For example, several
small and large intestines can be grouped (19, 20) (Table 3).
 Necropsy and Sampling Procedures in Rodents 45

Table 2
List of organs for weighing

Rat Mice
Liver Liver
Kidney Kidney
Heart Heart
Adrenal glands Adrenal glands
Brain Brain
Testes Testes
Prostate Spleen
Epididymes Thymus
Spleen Thyroid/parathyroid
Thymus Pituitary gland
Thyroid/parathyroid
Pituitary gland
Sellers, R., et al., in Toxicologic Pathology 35, 751–755 (2007)

3.1.4. Examination The description of macroscopic findings should be sufficiently

and Recording detailed to give a mental representation of gross changes.
of Macroscopic Therefore, all criteria should be used to describe the gross changes
Observations of an organ: location, appearance (color, shape, consistency),
demarcation from surrounding tissues, number, distribution and
severity, size, which should always be measured in two or three
dimensions (cm or mm), or in volume (mL).

3.1.5. Orientation It is critical for the necropsy personnel to correctly use the terms
that define the orientation of an organ or a section (Fig. 1):
1. Dorsal refers to the back of the animal whereas ventral refers
to the abdomen.
2. Cranial refers to the skull, whereas caudal refers to the tail.
3. Rostral refers to the organs or structures situated toward the
front of the head.
4. Lateral pertains to a side, medial is related to, situated in, or
extending toward the middle, closer to the body’s midline.
5. Proximal is located nearer to a point of reference; distal is
located far from a point of reference (origin, point of attach-
ment, or midline of the body).
Table 3
Example of blocking scheme

Cassette Tissues
1 Heart
Aorta
Vena cava
2 Muscle skeletal (diaphragm, tongue and soleus)
3 Lung: entire (all lobes)
4 Thyroid with trachea (specimen immediately caudal to larynx, cross section)
5 Kidneys (cross-sections of left and right kidneys)
Urinary bladder
6 Ureters
7 Adrenal glands
8 Stomach: glandular and nonglandular portions
Duodenum
Jejunum
9 Ileum (with Peyer’s patch)
Cecum
Colon
Rectum
10 Salivary glands (Mandibular, sublingual and parotid)
Cervical lymph nodes
11 Pancreas
Mesenteric lymph node
Thymus
Spleen
12 Liver: left lateral (largest) lobe (one section cut from hilus to periphery), median lobe (one
section to include gall bladder)
13 Ovaries
Oviducts
Uterus (two cross sections through horns and one longitudinal section through body and
cervix)
Vagina (cross section)
14 Testes (cross sections, left and right)
Epididymides
Seminal vesicles with coagulating glands (cross sections or longitudinal sections depending
on size)
Prostate (cross section)
15 Skin ventral inguinal specimen (one section parallel to hair growth, to include mammary glands)
16 Clitoral or preputial glands
17 Femur with knee and tibia with bone marrow
18 Sternum with bone marrow
19 Brain (2 transversal sections through cerebrum including olfactory lobes and
hippocampus, and one through cerebellum and pons)
20 Spinal cord
21 Pituitary
22 Eyes with optic nerves and lacrimal glands
 Necropsy and Sampling Procedures in Rodents 47

Lateral

Caudal Cranial

Proximal

Distal

1: Median plane
2: Sagittal plane
3: Transversal plane

Fig. 1. Anatomical orientation descriptors usually used at necropsy.

The major planes of sections are defined as follows:

1. Median plane, passing longitudinally through the middle of the

body from front to back, dividing it into left and halves right.
2. Frontal (coronal) planes, passing transversally through the
body from side to side, perpendicular to the median plane,
dividing the body into front and back parts.
3. Sagittal planes are vertical planes passing through the body
parallel to the median plane dividing the body into left and
right portions.
4. Transverse plane (dorsoventral), passing vertically through
the body, perpendicular to the sagittal planes, and dividing
the body into front and back portions.
5. Horizontal plane, passing through the body, perpendicular to
both the frontal and median planes, dividing the body into
upper and lower parts.
6. Vertical plane, perpendicular to a horizontal plane, dividing
the body into left and right or front and back portions.

3.2. Examination 1. Observe the behavior of the animal and check its response to
of the Live Animal external stimuli.
2. Observe the fur and the skin to detect any changes.

3.3. External 1. Weigh the animal. As indicated in Subheading 3.1.2, this will
Examination be useful to calculate organ weight to body weight ratios.
2. Examine the animal before proceeding to the necropsy.
48 Fiette and Slaoui

3. Assess the general condition of the animal (emaciated, thin,

adequate/good condition, obese).
4. Check and record any external lesions (skin lesions, fur loss or
discoloration, and subcutaneous masses). Record sizes and
surfaces whenever possible and record their gross appearance
and precise location.
5. Check and record the color changes of the skin and mucosae
(gingival, genital mucosae, conjunctiva).
6. Examine the eyes, mouth, teeth, and nasal openings and
record any abnormality.
7. Examine the ano-genital region to look for signs of diarrhea,
rectal, or vaginal prolapse and record any abnormality.
8. Gently palpate the abdomen to reveal abdominal masses or
presence of fluid.
9. If the abdomen is distended by fluid, take a sample with a
sterile needle and syringe for further evaluation.
10. Palpate any mass and record its consistency (soft, fluctuant,
firm, or hard).

3.4. Euthanasia Various methods of euthanasia are available for Rodents. The
selected method should induce death quickly with minimal ani-
mal pain or distress and should not interfere with the gross obser-
vation and microscopic evaluation of the tissues. Euthanasia
protocols should be approved by the local ethical committee.
We describe in this chapter the euthanasia method which is
commonly used in toxicological studies: exposure to carbon diox-
ide (CO2). This method requires only a source of CO2 and a poly-
carbonate box of a size that is well-matched with the size of rodents.
The euthanasia procedure is as follows:
1. Place a wet sponge in one of the corners of the box.
2. Place the lid with the CO2 tube attachment on the box.
3. Charge the chamber with CO2 gas for 1–2 min.
4. Place animals in the box.
5. Turn gas on low so as not to frighten the animals.
6. Administer CO2 until deep sedation is observed.
7. Death is induced by maximal exsanguination from the abdominal
aorta (this can be the occasion to collect blood for hematology,
coagulation, and clinical chemistry parameters).
8. Check heart beat and respiration to verify death.

3.5. Incision of the Skin, subcutaneous tissue, mammary glands, salivary glands,
Skin and Examination superficial cervical lymph nodes, extraorbital lacrimal gland, clito-
of the Subcutaneous ral glands, preputial glands, penis, and prepuce.
Tissues/Organs
 Necropsy and Sampling Procedures in Rodents 49

3.5.1. Incision of the Skin 1. Pin the animal on the dissection board, ventral side up and
and Examination of the head in front of you. Remember that from this point, the left
Subcutaneous Tissue side of the animal is on your right side and vice versa.
2. Moisten the skin and hairs with 70% alcohol.
3. Incise the skin with a scalpel on the midline, from the mandibles
to the pubis. In males, incision should end on both sides of the
penis.
4. Reflect the skin on both sides of the incision.
5. Examine the skin and the subcutaneous tissue. Record any
lesion; confirm skin changes observed in the previous step.

3.5.2. Examination 1. Examine the mammary glands: the mouse has five pairs of
and Sampling of the mammary glands (three thoracic and two abdominal), while the
Mammary Glands rats have six pairs (three thoracic and three abdominal). There
are six or seven pairs in hamsters (21, 22). In females, the mam-
mary tissue extends from the salivary gland region to the base
of the tail. When lactating, the mammary gland occupies almost
all the abdominal and thoracic ventral subcutaneous area in the
mouse and in the rat.
2. Harvest the mammary gland from the inguinal region where
the mammary tissue is abundant in both rats and mice. Take
a transverse section (1 cm × 3 cm) including the associated
nipple and skin (see Note 1). This applies to males as well as
to females.

3.5.3. Examination Examine the superficial lymph nodes. Under normal conditions,
and Sampling of the lymph nodes are grayish organs, bilateral, and have the size and
Superficial Lymph shape of a small pea. Major superficial lymph nodes, located in the
Nodes subcutaneous area are the cervical superficial, axillary in the axil-
lary fossa, brachial, inguinal, and popliteal. The peripheral lymph
nodes that are most often examined are the mandibular, axillary,
and/or popliteal lymph nodes (see Note 2). The location of these
lymph nodes is detailed in Table 4 (23).

3.5.4. Examination, 1. Examine the three pairs of salivary glands located on both
Removal, and Sampling sides in the cranioventral region of the neck: mandibulary
of Salivary Glands, lacrimal glands, sublingual glands, and parotid glands (24, 25). The
glands, clitoral or preputial mandibulary gland is the largest one, located in the ventral
glands region of the neck. Sublingual glands are situated on the top
of mandibulary glands. Parotid glands are the most lateral
ones; they extend to the base of the ear.
2. Collect salivary glands by gently grasping the tip of the clos-
est salivary gland to the thorax with forceps. Then slowly pull
toward cutting the surrounding tissues with scissors and
immerse in fixative.
50 Fiette and Slaoui

Table 4
Nomenclature and location of major lymph nodes

Name Location

Superficial Mandibular Rostromedial to the sublingual and mandibu-

lar salivary glands
Axillary Axillary fossa
Brachial Proximity to the angle of the capsula, upon
the biceps, underneath the pectorals
Inguinal Adherent to the skin of the groin
Popliteal Between the adductor muscle and the
semimembranous muscle behind the knee
Deep Deep cervical Behind the salivary glands hidden in the
connective tissue encircling the trachea
Lumbar, caudal Anterior to the bifurcation of the abdominal
aorta
Mediastinal Posterior face of the thymus
Mesenteric Lengthened shape, with the mesentery, close
to the ascending portion of the colon
Pyloric (pancreatic) Anterior end of the pancreas, near the
pylorus
Renal Between the aorta and hilum of the kidneys
Sacral Posterior to the bifurcation of the abdominal
aorta
Sciatic Below the sciatic nerve on the back

3. Examine extraorbital lacrimal glands. These glands are located

on the ventro-lateral aspect of the head. They appear as flat,
brown-gray glands next to the parotids.
4. Remove extraorbital glands on both sides by gently grasping
the tip of the gland with forceps, isolate the gland from its
attachment in the eye socket using scissors, and immerse the
two glands in fixative (see Note 3).
5. Examine clitoral glands in females or preputial glands in
males. Clitoral/preputial glands are modified sebaceous
glands that are included in the subcutaneous adipose tissue.
They can be found cranial to the vulva in females and lateral to
the penis in males. Preputial glands are leaf-shaped and dark-
gray color with a soft consistency.
6. Remove whole glands on both sides by gently grasping the tip
with forceps to isolate them from the surrounding tissue.
Immerse the clitoral/preputial glands in fixative.

3.5.5. Examination Examine the penis and the prepuce in males. These organs will be
of the Penis harvested with the remaining genital organs.
 Necropsy and Sampling Procedures in Rodents 51

3.6. Opening Abdomen is opened and abdominal organs such as peritoneum,

of the Abdomen spleen, mesenteric lymph nodes, pancreas, digestive tract, liver,
and Examination adrenal glands, kidneys, and genital organs are examined.
of Abdominal Organs

3.6.1. Opening of the 1. Grasp the abdominal wall with forceps near the sternal
Abdominal Cavity xyphoid appendix and lift firmly.
2. Make a small incision to let air into the abdomen. This will allow
abdominal viscera to be separated from the abdominal wall.
3. Cut the abdominal wall on the midline with scissors from the
pelvis to the xyphoid appendix. Make sure not to cut any of
the abdominal organs that lie underneath.
4. Reflect the abdominal wall on the sides.
5. Examine the abdominal serous membrane (peritoneum) and
look for the presence of abnormal contents such as serous
fluid, blood, or fibrin as well as to any adhesion between
abdominal wall and abdominal organs.
6. Check the position of the different organs in situ.
7. Check fat deposits and score the nutritional conditions (1.
Obese, 2. Good nutritional condition, 3. Poor nutritional
condition, 4. Bad nutritional condition, 5. Emaciation, absence
of fat in the body deposits). The amount of fat is dependent
on the age and strain of the animal: adults and aged animals
tend to have more fat deposits in the abdominal cavity.

3.6.2. Removal, 1. The spleen is situated on the left superior abdominal quad-
Examination, and Sampling rant. To remove it, grasp the connective tissues and fat sur-
of the Spleen rounding the spleen with the forceps. Cut them along the
hilus of the spleen and cut the gastrosplenic ligament as well.
2. Examine the spleen. It is a lengthened, oval, slightly curved
shape organ, has a dark-red color, and is soft in consistency
with a thin transparent capsule (26). It is attached to the
stomach by the gastro-splenic ligament.
3. Should spleen be weighted, it should be carefully freed from
all remnants of connective and adipose tissues, in particular
along the hilus.
4. The entire spleen can be sampled and immersed in fixative
although a 2-mm thick transverse mid-section can be
sufficient.

3.6.3. Removal of the Abdominal part of the esophagus, stomach, small intestine (duo-
Digestive Tract and denum, jejunum, ileum), large intestine (cecum, colon, rectum),
Mesenteric Lymph Nodes and mesenteric lymph nodes.
1. Dissect the anus free from the surrounding skin.
52 Fiette and Slaoui

2. Insert the tip of heavy duty scissors between the colon and
the pelvis to cut the pelvic arch on both sides. Take off the
resulting bone chip to facilitate the subsequent removal of
the genital tract and rectum.
3. Hold the rectum with forceps and lift it upwards.
4. Isolate the rectum from the vagina in females.
5. Gradually extract the intestines in a caudal-cranial direction,
while cutting the insertion of the mesentery as close to the
intestines as possible.
6. Cut the esophagus just below the diaphragm in the abdomen
leaving a small portion of esophagus connected to the
stomach.
7. Isolate the whole digestive tract together with the pancreas.
8. Examine the deep abdominal lymph nodes (23, 27) for
nomenclature and location before unzipping the intestines.
The mesenteric lymph nodes are usually the largest lymph
nodes of the abdominal cavity. They drain the duodenum,
ileum, cecum, and colon.
9. Starting at the rectum, progressively unzip the coiled intes-
tines by pulling gently with scissors.
10. Samples of intestine segments as well as mesenteric lymph
nodes should be immersed in the fixative as soon as possible.

3.6.4. Examination and 1. In Rodents, the pancreas is very diffuse and completely
Sampling of the Pancreas enclosed in the mesentery. It is tan in color and therefore easily
identifiable from the surrounding fat. It has one left lobe
located close to the spleen in the great omentum, and one
right lobe adjacent to the duodenum.
2. Lift up the pancreas attached to the spleen and separate them
from the intestines by cutting its insertion with scissors.
3. Isolate the spleen from the pancreas with the scissors.
4. Take a sample from the left pancreatic lobe to have the largest
surface. The right pancreatic lobe will be sampled with the
duodenum.
3.6.5. Examination and 5. In small Rodents (mice, rats, hamsters, and gerbils), the stomach
Sampling of the Stomach has two distinct regions: the proventricular or anterior region
that has a white appearance, and the glandular region which
has a reddened and thicker wall (28, 29). Both are clearly
demarcated by a limiting ridge in these species but not in the
guinea pigs.
6. Isolate the stomach from the esophagus and the intestines.
7. In mice, open the stomach along the greater curvature, mount
on a plastic card, fix it with pins, and immerse it in the fixative.
In rats, open the stomach of the rat along or para-median
 Necropsy and Sampling Procedures in Rodents 53

to the greater curvature and placed on a corkboard. Remove

the gastric content and, if necessary, clean carefully the mucosa
with saline solution or fixative. Spread out the stomach and
pin it on the cardboard. This procedure avoids folds in
the mucosa and therefore is essential for the macroscopic
orientation and allows reproducible microscopic evaluation of
the thickness of gastric mucosa.

3.6.6. Examination and 1. The small intestine has three different regions: duodenum,
Sampling of the Intestines which is very short (1 cm) in Rodents, jejunum and ileum
that are not recognizable grossly. The large intestine is com-
posed of the cecum, colon, and rectum (short segment in
Rodents, included in the pelvis) (30). Lymphoid tissue asso-
ciated with the small intestine (jejunum and ileum) is also
called gut-associated lymphoid tissue (GALT) or Peyer’s
patches. These appear as slightly elevated lighter fields in the
intestine’s wall and can be discernible as prominent areas
when activated.
2. Separate carefully the intestines from the mesentery during
necropsy (or after fixation).
3. Examination of Intestines should comprise at least the con-
tent, wall thickness, color, aspect of mucosa, or any notice-
able lesion in each intestinal segment.
4. If the microscopic evaluation of the whole intestine and
GALT on a single section is preferred, the so-called “Swiss
roll method” can be performed (31, 32) (see Note 4).
5. Routine intestine samples for histopathology evaluation usually
consists of one transverse section (2–3-mm thick) from each
part of the bowel without opening it: duodenum (1 cm distal
to the pyloric sphincter), jejunum (central section), ileum
(1 cm proximal to cecum), cecum, colon (central section), rec-
tum (2 cm proximal to the anus). For small intestines, it is also
essential to sample the GALT. Standardized sampling proce-
dure of the bowel segments is necessary to guarantee examina-
tion of each required segment.
6. After sample collection for histopathology, the remaining
intestine should be opened longitudinally and examined for
abnormalities. To better examine the mucosa, a gentle rinse
of the ingesta with saline solution may be necessary. This lat-
ter procedure is time consuming and should be considered
on a case-by-case basis only.

3.6.7. Removal, 1. The liver has four lobes: medial lobe, right medial lobe, right
Examination, and Sampling lateral lobe, caudate lobe, plus a papillary process (see Note 5).
of the Liver There is no gallbladder in the rat. The liver is normally dark-
reddish and has a hard, but friable consistency. A thin trans-
parent capsule covers the liver.
54 Fiette and Slaoui

2. The liver is a fragile organ that should be handled with

caution.
3. Remove the liver gently out the way with the forceps.
4. Grasp the xyphoid process firmly with the forceps.
5. Puncture the diaphragm with scissors and trim it completely
away from the ribs.
6. Pull upward to create a negative pressure in the thorax.
7. Using the diaphragm as a handle, pull the liver out of the
abdominal cavity. Separate the liver from the diaphragm by
cutting the falciform and coronary ligaments that attach the
liver to the diaphragm.
8. Before weighing the liver, remove all remnants of the dia-
phragm and ensure that the small lobes are present to weigh
the entire organ.
9. Sampling of the liver (see Note 6). In the rat, take a piece
from the left lateral lobe (transverse) and the right medial
lobe (transverse); in the mouse from the left lateral lobe
(transverse), from the left and right medial lobe including
gall bladder (longitudinal-vertical preferentially to keep the
gallbladder with the liver). In both species, a transverse sec-
tion from the caudate lobe is optional. Size of samples should
be as large as possible, but all pieces should fit into one
cassette.
10. Immerse the two or three liver samples in the fixative. It is
advised to keep the remaining liver tissue in fixative.

3.6.8. Removal, 1. Remove the kidney and adrenals, grasp the caudal part of the
Examination, and Sampling ureter with forceps near its opening, and keep the adrenals
of the Kidneys and attached to the kidneys. Adrenal glands and kidneys are
Adrenals Glands located deep in the retroperitoneal space; they should be
recognized as early as possible during the process.
2. Separate the adrenals from kidneys.
3. Examine each adrenal gland. These glands are small white
structures within the perirenal fat. In male, adrenal glands
tend to be large, often rose-colored and translucent while in
females, they are smaller and, due to high lipid content, have
an opaque pale color (24, 33, 34). Adrenals have an external
cortex and central medulla.
4. Check their shape, volume, as well as the presence of nodular
formations.
5. Before weighing adrenal glands, remove carefully all rem-
nants of fat and connective tissues. As for all paired organs,
unless otherwise specified in the study plan, the weight of the
pair is recorded.
 Necropsy and Sampling Procedures in Rodents 55

6. Immerse the adrenal glands in the fixative. Due to their

relative small size in Rodents, they will be embedded in toto.
7. Examine the kidneys. Kidneys are located on the dorsal wall
of the abdominal cavity. They are bean-shaped pair organs,
with a hilus in the concave margin from which main vessels,
nerves, and ureters exit. Their color is brownish red and their
consistency is firm. The right kidney is the more cranially
located, usually larger and heavier than the left one (35, 36).
The kidneys are surrounded by a capsule and have three dif-
ferent regions: cortex, medulla, and papilla.
8. Before weighing, kidneys must be freed of all remnants of
connective and adipose tissues.
9. Kidneys can be immersion fixed in toto or after trimming
(e.g., take a longitudinal section from the left kidney and a
transverse section from the right kidney).

3.6.9. Removal, 1. Examine the urinary bladder (12, 37).

Examination, and Sampling 2. The urinary bladder can be freed of urine after incision of the
of the Urinary Bladder wall and then sampled in toto. Alternatively, the bladder can
be sampled after instillation of the fixative. In the mouse,
instill the fixative (0.05 mL) through the bladder wall after
ligation of the urethra with a ventral knot. In the rat, it is
more convenient to instill the fixative (0.2 mL) with a needle
inserted via the urethra. In both rats and mice, fixative instil-
lation should not be performed when the bladder is distended
with urine.
3. Then continue the fixation by immersion in a container of
fixative.
4. It may be helpful also to mark the ventral side of the bladder
with a stick of silver nitrate.

3.6.10. Removal, 1. Remove the testes. They are oval-shaped paired organs, a few
Examination, and Sampling millimeters in diameter that lay inside the scrotum. They are
of the Male Genital Organs covered by a smooth and transparent membrane (tunica
albuginea). They are grayish-white with a soft elastic consis-
tency. When the abdominal cavity is opened, testes are often
found outside the scrotum, in an intra-abdominal position.
Grasp them delicately by the inguinal fat pad and cut them
away from the viscera. If the testes are still in the scrotum,
open the scrotum and extract the testes with the epididymides
by cutting the fibrous ligaments anchoring the tail of the
epididymis to the scrotum. Cut the vas deferens.
2. Examine the testes (38–41). Check the shape, volume,
weight, consistency, and presence of masses.
3. Weigh the testes individually or as a pair. Weigh the epididymis
(in rats only) separately if needed. Before weighing the testes
56 Fiette and Slaoui

and epididymes, all remnants of connective and adipose tis-

sues should be removed.
4. Place the testes with the epididymis on a piece of cardstock.
Orient testis and epididymis in the same plane so that they can
be trimmed simultaneously later (see Note 7). Fix the testes
and epididymis as a whole, without cutting the testes before
fixation to prevent them from rupturing (see Note 8).
5. It is suggested to fix testes and epididymis in Davidson
solution instead of formalin (Table 1).
6. Remove the male accessory glands: seminal vesicles, coagulat-
ing glands (dorsocranial lobe of the prostate), prostate (two
ventral lobes and two dorsolateral lobes). The dorsolateral
and ventral lobes lie in a vertical axis above each other with
urinary bladder and seminal vesicles in between.
7. Remove the group of adjacent organs consisting of prostate,
urinary bladder, seminal vesicles, and coagulating glands.
8. Examine the male accessory glands (38, 39, 42, 43). Check
any size or color changes or presence of nodular masses.
9. Before weighing the prostate (rats only), all remnants of con-
nective and adipose tissues should be carefully removed.
10. Immerse these organs into the fixative in toto if weights are
not required to prevent leakage of the glandular secretions.
11. Remove the penis, prepuce, and urethra.

3.6.11. Removal, 1. Dissect the vulva and vagina free from the skin and cut the
Examination, and Sampling supporting ligaments of the vagina, uterus, and oviducts.
of the Genital Organs in 2. Cut the ligaments and isolate the ovaries with the oviduct and
Female the whole genital tract.
3. Examine the ovaries and oviducts. Ovaries are small oval red-
dish organs found within the fat tissue caudally to the kidneys
and attached to the inferior poles of the kidneys, and to the
posterior wall of the abdomen by ligaments. Check the shape,
volume, consistency and presence of any gross lesions (24,
38, 44, 45).
4. If ovaries are to be weighed, isolate them from the oviducts
before weighing.
5. Examine the uterus and the vagina. Record any enlargement,
fluid, or mass (24, 38, 44, 45).
6. If uterus has to be weighed, uterine horns and the cervix
should be weighed together but separated from the vagina.
7. The uterine body (fused part of the uterus) and the vagina
should be placed with their dorsal aspect on cardboard before
fixation.
 Necropsy and Sampling Procedures in Rodents 57

3.7. Opening of the The thorax is opened and the thoracic organs such as tongue,
Thorax and larynx, trachea, thymus, mediastinal lymph nodes, lungs, heart,
Examination of and thyroid gland (with parathyroids) are examined.
Thoracic Organs
3.7.1. Opening of the 1. Open the thorax by first lifting the sternal xyphoid process
Thorax with forceps. Then cut the ribs starting from the xiphoid
process and up to the first rib to remove the sternum and rib
cage to reveal the thoracic organs.
2. The sternum is a convenient organ for bone marrow exami-
nation after decalcification. Take a piece containing two to
three sternebrae and immerse this sample into fixative.
3. Examine the thoracic serous membrane (pleura) and pres-
ence of abnormal contents such as serous fluid, blood, fibrin,
or adhesions between organs.
4. Check the position of the different organs in situ.

3.7.2. Removal of the 1. Cut the muscles of the lower jaw with a scalpel.
Tongue, Trachea, 2. Cut the soft palate and pharynx.
Esophagus, and Thoracic
3. Grasp the tip of the tongue with forceps and retract gently to
Organs
remove the tongue, larynx, trachea, and esophagus from the
head and neck.
4. Continue retracting to remove the heart and the lungs from
the thorax. Use scissors to perform a blunt dissection to free
these tissues.
5. Cut the thoracic aorta and posterior vena cava at the level of
the diaphragm

3.7.3. Isolation, 1. Isolate the heart from the lungs by delicately cutting the main
Examination, and Sampling vessels with scissors.
of the Heart and Aorta 2. Examine the heart without opening the inner cavities (atria
and ventricles) (24, 46, 47). Before weighing the heart, all
blood should be removed. In Rodents, this can be easily
achieved by placing the base of the heart over a piece of clean-
ing paper.
3. Immerse the heart in toto in the fixative.
4. Take a section from the thoracic aorta (in the middle of the
last 1 cm caudal segment). This region is closely attached to
dorsal vertebrae and can easily be removed.

3.7.4. Isolation, 1. Isolate the thymus by gently grasping one of the two lobes in
Examination, and Sampling its inferior part and cut the ligament connecting it to the
of the Thymus pericardium.
58 Fiette and Slaoui

2. Examine the thymus (48). In rodents, the thymus is an oval-

shaped lobulated organ with a whitish-translucent color. With
age, the thymus shrinks but remains grossly visible.
3. Before weighing the thymus, all remnants of connective and
adipose tissues should be carefully removed.

1. Examine the tongue (28).

3.7.5. Examination and
2. Make a transversal incision to sample half the tongue and
Sampling of the Tongue
immerse it in the fixative (see Note 9).

3.7.6. Examination and 1. Examine the thyroid gland. Thyroid gland has two symmetric
Sampling of the Thyroids oval lobes, adherent to the lateral and dorsal surfaces of the
(Parathyroids), Trachea, trachea and has a translucent, tan yellowish color. The para-
and Esophagus thyroids are located around or within the thyroids but are not
visible grossly (49, 50), (see Note 10).
2. Take the larynx including epiglottis, ventral pouch, and cricoid
cartilage (rats only) and immerse it in the fixative.
3. If the thyroid is weighed (rat only), carefully remove all rem-
nants of connective and adipose tissues. Immerse the thyroids
in the fixative. It is advised to weigh the thyroid gland after 1
or 2 min of fixation.
4. Then take a transverse sample section of trachea and esopha-
gus. If the thyroids are not weighed, leave them on the tra-
chea. Take a transverse section of the esophagus, trachea,
including the thyroids (and parathyroids) (oral studies).
Tracheal content should be noted and reported.

3.7.7. Examination and 1. Examine the lungs (51, 52). In Rodents, lungs have three
Sampling of the Lungs right lobes (right cranial lobe, right middle lobe, right caudal
lobe), an accessory lobe, and one left lobe (see Note 11).
2. The lung parenchyma has normally a smooth surface, a nice
light pink color (but that depends on air and blood present if
not inflated), and a spongy consistency. Examine all lung
lobes and note any changes.
3. For optimal microscopic evaluation, instillation of the lung by
the fixative is strongly recommended. This can be performed
easily through the trachea. A 21-gauge needle is placed on a
3-mL syringe filled with the fixative. The needle is introduced
in the trachea at its open end. Clamp gently around the nee-
dle with forceps and inflate the lung by depressing the plunger
of the syringe very slowly until excess fixative refluxes up the
trachea. The lung with the trachea should be then immersed
in the fixative. If the procedure is correctly executed, there is
no need to place a ligature on the trachea (see Note 12).
 Necropsy and Sampling Procedures in Rodents 59

3.8. Head and Central Brain, cerebellum, spinal cord, pituitary gland, eyes, Harderian
Nervous System glands, nasal cavities, and Zymbal‘s gland.
The most optimal way the fix to central nervous system is
intracardiac perfusion of fixative (see Note 13). However, fixation
of the nervous system by immersion allows acceptable preserva-
tion of tissues and is routinely used in toxicity studies.

3.8.1. Removal of the Brain 1. Cut the skin over the head with a median-longitudinal incision
and Spinal Cord from the nape to the snout.
2. Reflect the two edges of the skin and pull them to better
observe the entire skull.
3. Remove any excess tissue or muscle from the cranium and
neck.
4. Keeping the head firmly with large forceps, insert the tip of heavy
duty scissors in the left eye socket (avoid any damage to the eye-
ball), and cut the nasal bone transversely at the level of the nasal
septum between the two orbital cavities. The used scissors for
this operation should be exclusively dedicated to this step.
5. Then, with the ophthalmologic scissors, cut progressively the
parietal, interparietal, and occipital bones in a craniocaudal
direction on both sides to isolate a bone cap and reveal the
brain. Be very careful to avoid damaging the brain beneath
the skull bones during this operation.
6. Pull the skullcap caudally with forceps and take it away.
7. Use the small ophthalmologic scissors to cut the vertebrae.
Start from the occipital bone level and then, with the tip of
the scissors, alternate right and left side cuts of the vertebral
bodies to progressively remove the vertebral arches. In this
way, the spinal cord will be uncovered in the vertebral canal.
8. Raise the brain by gently introducing forceps under the fron-
tal lobe of the encephalon, then cut intracranial vessels and
nerves at the brain base.
9. Gently handle the brain between the thumb and forefinger
and cut successively the spinal nerves coming from the ventral
aspect of the spinal cord on each side of the brain, and hold
the detached segment of the spinal cord. In the area of the
cauda equina (where nerves are numerous), cut the spinal
cord transversely (see Note 14).
10. Isolate the brain and the spinal cord by a scalpel frank trans-
versal section at the junction between the medulla oblongata
and the brain.

3.8.2. Examination of the 1. Examine the brain (cerebrum and cerebellum) (53, 54). The
Brain and Spinal Cord brain is covered by the meninges: the dura mater is fibrous
and in direct contact with the skull, the pia mater is highly
60 Fiette and Slaoui

vascularized and adheres intimately to the surface of the brain,

the arachnoid is a transparent membrane located between the
dura mater and the pia mater, but too thin to be visualized
grossly.
2. The cerebrum has two cerebral hemispheres, separated by a
longitudinal fissure. There are no cerebral circumvolutions
in Rodents that have a lissencephalic brain. Rodent brains
have two large olfactory bulbs located in the front. The cer-
ebellum shows thin convolutions and lays in the caudal part
of the brain.
3. On the ventral part of the brain, the optic chiasma, the median
eminence (representing a part of the hypothalamus), the pons,
and the medulla oblongata can be observed.
4. The brain is a very fragile tissue, especially if not fixed. It is
therefore important not to handle this organ with the forceps
but rather lift it carefully using the scalpel blade.
5. Before weighing the brain, carefully remove all remnants of
connective tissue (see Note 15). To achieve accurate brain
weights, the spinal cord should be cut off at a consistent level.
6. It is preferable that the brain (undetached from the cerebel-
lum) be immersion fixed in toto. The brain is an anatomically
complex tissue. Therefore, microscopic examination of the
brain should be performed at standardized section levels:
transverse section of the cerebrum at the optic chiasma, cerebrum
at the base of the posterior hypothalamus, midcerebellum, and
medulla oblongata (12). Therefore, the brain slices are routinely
prepared after fixation.
7. Examine the vertebral canal on the cadaver.
8. Examine the spinal cord.
9. Take three transverse sections of the spinal cord for microscopic
examination at the upper cervical, mid-thoracic, and lumbar
levels. Put all three segments in a cassette and immerse in the
fixative (see Note 16). As for the brain, spinal cord is a very
fragile tissue and should not be handled with the forceps.

3.8.3. Examination, 1. Examine the pituitary gland (24, 55). The pituitary gland can
Removal, and Sampling of be easily seen on the ventral aspect of the cranial cavity after
the Pituitary Gland removal of the brain. It appears as a small spherical gland,
covered by a thin layer of dura matter and located behind the
optic chiasma in the sella tursica, a small depression of the
sphenoid bone.
2. The fixation in situ of the gland is recommended before
removal or weighing. Weighing after fixation provides accurate
weight measurements and improves morphology. Fixation can
be performed by a few drops of fixative on the ventral aspect of
 Necropsy and Sampling Procedures in Rodents 61

the cranial cavity or by immersion of the remaining skull in the

fixative; the gland can be removed later (11) (see Note 17).
3. Weighing of the pituitary gland is not required in mice and
usually performed after fixation.

3.8.4. Removal, 1. Remove the eyes by sinking a pair of curved forceps behind
Examination, and Sampling the orbit. Gently grasp the optic nerve, isolate the eye by pull-
of the Eyes, Optic Nerve, ing it outward and cutting its attachments to the socket (see
Harderian Gland, and Note 18).
Lacrymal Glands (Internal/ 2. Examine the eyes (56).
External)
3. Immerse the eyes with the optic nerve in the fixative (see
Note 19).
4. Examine the Harderian glands (or retroorbital gland). The
Harderian gland lies intraorbitally behind the eyes and
embraces the back of the ocular globe. It is a gray-colored
gland under normal conditions, cone-shaped in the rat, and
horseshoe-shaped in the mouse.
5. Remove the Harderian glands and immerse them in the
fixative.

3.8.5. Sampling of the 1. Remove the skin and muscles and immerse the head in the
Nasal Cavity and Zymbal’s fixative if further histological examination of the nasal cavity
Glands and paranasal sinuses, nasal cavity, nasopharynx, and paranasal
sinuses is needed (57, 58).
2. Zymbal’s glands are modified sebaceous glands located at
the base of the external ear in anterio-ventral position (59).
A section through the base of the skull after gentle decalcifi-
cation will allow histological examination of these glands.

3.9. Muscles Bone (femur, sternum), skeletal muscle (biceps femoris), periph-
and Skeleton eral nerve (sciatic nerve), bone marrow (femur, sternum).
1. Remove the skin from the hind leg.
2. Transversally cut the biceps femoris with a scalpel (12, 27,
60). Gently grasp one edge with the forceps and immerse it
into the fixative.
3. Take a sample of the sciatic nerve (1 cm long) (12) (see Note
20). Gently grasp one edge with forceps and fix it on a card
board.
4. Remove the distal portion of one femur, the knee joint with
the proximal portion of the adjacent tibia. This will allow
microscopic examination of the bone, joint tissues, and bone
marrow (12, 61, 62). For routine bone and bone marrow
microscopic examination, a sternum section should be sam-
pled (see Note 21).
5. Immerse the samples in the fixative.
62 Fiette and Slaoui

4. Notes

1. A longitudinal section, vertical to the direction of the hair

flow can be taken to examine the skin and mammary glands;
in this case, the nipple is not included if the lymph node is
enclosed.
2. In case of parenteral application, one lymph node draining the
application site and another distant one could be collected.
3. The extraorbital glands can be removed and embedded
together with the salivary glands.
4. ‘‘Swiss roll’’ technique: this technique allows examining the
whole intestine and the GALT on a single section. However,
these transverse sections when they are made properly will
often provide a better morphology. Strip the intestines off the
mesentery, open it with a pair of scissors, and gently rinse the
intestine to remove the content. Then recoil the intestine
except cecum on cotton swabs and put in the fixative. After
fixation, detach the spooled intestine and proceed to embed-
ding. This procedure is not required in routine microscopic
examination of the intestines. Bear in mind that with this
technique, the intestinal mucosa and the lymph follicles will
often be cut tangentially.
5. Nomenclature of hepatic lobes can differ. We have elected to
use the anatomical terms we recommend: medial lobe, right
medial lobe, right lateral lobe, caudate lobe, and papillary
process.
6. If major bile duct is required, take a section through the left
lateral lobe of the liver.
7. Optionally epididymides can be isolated from the testes, fixed,
and trimmed separately
8. In short-term studies, fixation of the testes with modified
Davidson’s or Bouin’s solutions is highly recommended to
detect less extensive early and subtle changes (63).
9. The longitudinal vertical section of the tongue covers a large
part of the dorsum including the dorsal prominence. The sec-
tion also includes the lingual lesser salivary glands and should
be slightly lateral to the median sulcus. A transverse section of
the tongue is recommended if blood sampling from the
tongue is performed.
10. There are variations in position and number of parathyroids
in Rodents. In the rat, there is one pair of parathyroids, with
a variable position but usually on the anterior lateral aspect of
the thyroid lobes. In the mouse, there are usually two para-
thyroids (sometimes more than two), situated bilaterally just
under the capsule near the dorsolateral border of each thyroid
 Necropsy and Sampling Procedures in Rodents 63

lobe. They are rarely at the same level and may be deeply
embedded in the thyroid tissue.
11. Nomenclature of lung lobes can differ. We have elected to use
the anatomical terms that we recommend: right cranial, right
middle, right caudal lobe, accessory lobe, and left lobe.
12. Alternative procedure for the lung in oral studies in the rat:
right lobes embedded, ventral surface down and in the mouse:
whole lung embedded, ventral surface down.
13. The optimal way to fix the central nervous system is to per-
fuse it in situ while the animal is deeply anesthetized. The
procedure is as follows: anesthetize the animal (for example)
with intraperitoneal injection of Nembutal working solution
(1.6 mL stock solution with 8.4 mL PBS), 0.1 mL/10 g body
weight. Then, pin the mouse on a dissection board ventral
side up. Trim back the skin from the thorax to the mandible.
Open the thorax following the necropsy protocol (herein
described). If blood is needed, collect it by cardiac puncture
at this time. With small scissors, cut the right auricle (64).
Blood will start to flow from the heart. Insert a 23-gauge
needle within the left ventricle, with the syringe containing
PBS. Perfuse the PBS with steady pressure, strong enough to
wash the blood out from the body, but not so much that it
causes damages. The liver (and other organs) would normally
go pale very quickly. The lungs should not bulge; if they do,
your syringe has perfored the interventricular septum. Slowly
remove the needle and repeat the perfusion. After injecting
15 mL of PBS, change the syringe and perfuse 4% paraform-
aldehyde in the same way. If fixation is successful, the body
will stiffen from the tip of the tail to the tip of the nose.
Proceed as usual for tissue collection.
14. Alternatively remove the vertebral bodies with the spinal cord.
15. Changes in brain weights are rarely associated with neurotox-
icity. The utility of brain weight rests in the ability to calculate
organ to brain weight ratios which is helpful when terminal
body weights are affected or to normalize organ weight data.
16. To avoid artifactual vacuolation in the white matter, do not
store specimens from nervous tissue in alcohol.
17. The pituitary gland can be trimmed in situ as transverse sec-
tion of skull. This procedure is not recommended.
18. Each eye can be removed from the socket together with the
optic nerve and the Harderian gland after fixation of the head.
19. For long-term studies, formalin fixation of the eyes is gener-
ally sufficient. For other study types, fixation in Davidson’s
fixative is recommended to avoid detachment of the retina.
20. Alternatively, the skeletal muscle and sciatic nerve can be sampled
together. In this case, the gracilis, adductor, semimembranous,
64 Fiette and Slaoui

and semitendinous muscles are removed from the medial aspect

of the thigh to get access to the sciatic nerve running along the
medial surface of the biceps femoris muscle. After fixation,
transverse and longitudinal sections are prepared.
21. The bone marrow is generally examined concurrently with the
bone tissue after decalcification. On this specimen, it is possi-
ble to evaluate the cellularity, the number of megakaryocytes,
and the stromal compartment. If evaluation of the iron con-
tent and more precise cytology are needed, examination of a
bone marrow smear of a core sample from the femur may be
useful. This method requires training to obtain satisfactory
results. Prepare bone marrow smears as fresh as possible to
avoid blood clotting. Cut off the proximal and distal epiphyses
with scissors. Then blew air from one end into the marrow
cavity and collect the marrow cast onto a glass slide. The smear
is prepared conventionally with a cover glass. A smear of ade-
quate quality contains grossly visible particles. Alternatively,
aspiration with a pipette containing anticoagulated serum or a
small paint brush or a cotton bud from the longitudinally
opened femur can also be performed (this procedure implies
that the contro-lateral femur, knee joint, and proximal tibia
should be sampled).

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