Methods to Measure Gastric Mucosal

Lesions in the Rat

UNIT 21.2

Giuseppina Morini1 and Daniela Grandi1

1

Department of Human Anatomy, Pharmacology, and Forensic Medicine, University of

Parma, Parma, Italy

ABSTRACT
The maintenance of gastric mucosal integrity is ensured by a dynamic balance between
protective and noxious factors. The gastric mucosa has multiple protective mechanisms
that allow the mucosa to withstand frequent exposure to potentially damaging agents such
as acid and peptic secretions, bacterial products, ingested food, alcoholic beverages, and
certain drugs. The imbalance between defensive and aggressive factors is at the basis
of the formation of erosions/lesions or ulcerations of the gastric mucosa. The difference
between an erosion/lesion and ulceration is that the former is confined to the mucosa,
while an ulceration penetrates to the muscularis mucosae. This unit presents two models
of acute mucosal lesions induced in the rat by gastrotoxic agents acting through different
mechanisms of action. The protocols explain how to measure gastric mucosal lesions by
microscopic examination of the stomach by light microscopy and by scanning electron
C 2010 by John Wiley & Sons,
microscopy. Curr. Protoc. Toxicol. 43:21.2.1-21.2.15. 
Inc.
Keywords: rat r gastric mucosal lesions r macroscopic evaluation r light microscopy r
scanning electron microscopy

INTRODUCTION
The maintenance of gastric mucosal integrity is ensured by a dynamic balance between
protective and noxious factors. The gastric mucosa has multiple protective mechanisms,
including mucus and bicarbonate secreted into the lumen, continuous cell renewal from
mucosal progenitor cells, restitution of the surface epithelium, and mucosal blood flow,
which allow the mucosa to withstand frequent exposure to potentially damaging agents,
such as acid and peptic secretions, bacterial products, ingested food, alcoholic beverages,
and certain drugs (Wallace and Granger, 1996; Jones et al., 1999; Laine et al., 2008).
The imbalance between defensive and aggressive factors is the basis of erosions/lesion
or ulceration formation in the gastric mucosa. The difference between erosions/lesions
and ulcerations is that the former are confined to the mucosa, while ulcerations penetrate
to the muscularis mucosae (Tarnawski, 2005).
Various experimental models of gastric damage have been developed. Almost all models
are directed to the induction of acute damage. Damage to the gastric mucosa can be
acutely produced by a single exposure to a variety of necrotizing agents, such as absolute
ethanol, strong acid or strong base, by a single dose of conventional NSAIDs, by ischemia
of the gastric artery with subsequent reperfusion, or by stressful stimuli. Relatively little
attention has been paid to the progression of gastric damage after repeated exposure to
damaging agents. Unexpectedly, the initial damage exerted by a conventional NSAID,
concentrated ethanol, or hypertonic saline is progressively minimized or absent after
repeated administration over several days of the same or a different damaging agent. The
phenomenon has been described as gastric adaptation and it is characterized by delayed
onset and long persistence. It differs from adaptative cytoprotection, a condition in which
pretreatment of the gastric mucosa with a mild irritant, such as low concentrations of
Current Protocols in Toxicology 21.2.1-21.2.15, February 2010
Published online February 2010 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471140856.tx2102s43
C 2010 John Wiley & Sons, Inc.
Copyright 

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ethanol, is able to provide an immediate protection against mucosal damage caused

by strong irritants administered 1 hr later. Experimental models of chronic ulcer have
also been developed. Two models are well documented in which chronic ulcers are
induced by intraluminal application of acetic acid or application to the gastric wall
of a cryoprobe. Acute models are largely used for screening potential gastrotoxic or
gastroprotective drugs/chemicals and for evaluating the mechanisms responsible for
ulcer formation. Chronic models are mostly used to study drugs/chemicals delaying
or accelerating the healing process and the mechanisms underlying ulcer healing and
relapse.
Although experimental models of gastric ulcer were developed in a variety of animal
species, the most commonly used laboratory animal is the rat.
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved
procedures for the care and use of laboratory animals.
BASIC
PROTOCOL 1

ASSESSING ACUTE GASTRIC LESIONS INDUCED BY NECROTIZING

AGENTS
Necrotizing agents exert a direct damaging effect on the gastric epithelial cells, resulting
in varying degrees of epithelial rupture and lifting. They also cause vascular congestion
and disruption. As a consequence of their topical noxious effect, gastric damage by these
agents can be evidenced only after intragastric administration.

Materials
Adult rats (male, body weight of 200 to 220 g, 9 to 10 weeks old)
Necrotizing agent:
Absolute ethanol
0.6 N HCl (APPENDIX 2A)
0.2 N NaOH (APPENDIX 2A)
25% (w/v) NaCl (APPENDIX 2A)
Rat housing
Animal balance
Orogastric tube
Surgical tools
1. House the rats at 22 C on a 12-hr light/dark cycle. House the rats under laboratory
conditions for at least 1 week after arrival for adaptation. Deprive the animals of
food but not of water for 24 hr before the experiment.
2. Weigh each rat on the day of the experiment.
Perform experiments during the same period of the day, to avoid possible diurnal variation.

3. Handle the adult male rat gently and administer the necrotizing agent (absolute
ethanol, 0.6 N HCl, 0.2 N NaOH, 25% NaCl) 1 ml/rat via the orogastric tube.
4. Return the rat to his cage after the administration of the agent.

Methods to
Measure Gastric
Mucosal Lesions
in the Rat

5. Sacrifice the animal with 70% CO2 or by cervical dislocation at different time
intervals from 5 to 60 min after the instillation of the necrotizing agent. Immediately
after the sacrifice, open the abdomen through a mid-line laparatomy. Make a 3- to
4-cm incision in the skin below the rib cage and then, under the cutaneous incision,
make another incision of the same length in the abdominal muscle.

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6. Proceed to assessment and evaluation of gastric damagemacroscopic evaluation

(see Basic Protocol 2), light microscopic evaluation (see Alternate Protocol 1), or
scanning by electron microscopic evaluation (see Alternate Protocol 2).

MACROSCOPIC EVALUATION OF GASTRIC DAMAGE

Preliminary assessment of gastric damage is done at the macroscopic level, using a
stereomicroscope.

BASIC
PROTOCOL 2

Materials
Treated adult male rats with open abdomens (see Basic Protocol 1)
0.9% (w/v) NaCl (APPENDIX 2A)
Dissecting board and pins
Stereomicroscope
Transparent plastic 1-mm grids
1. Rapidly remove the stomachs of the treated adult male rats. Open along the lesser
curvature and rinse briefly with 0.9% NaCl.
2. Pin the stomach flat onto a board, with the mucosal surface uppermost. Avoid
stretching or distortion of the mucosa.
3. Examine the mucosal surface under a stereo microscope.
4. Place a transparent plastic grid over the mucosa.
The transparent grid is self-prepared by making a photocopy on a transparent film of a
paper grid.

5. Score the stomach for macroscopic gastric damage.

Macroscopic gastric damage is defined to be hemorrhagic areas of the mucosa that
do not clear on rinsing. Hemorrhagic areas are located mostly in the corpus and to a

Figure 21.2.1 Photograph of the stomach of a rat 1 hr after receiving absolute ethanol, 1 ml/rat
intragastrically. Exposure to ethanol produces the characteristic linear necrotic lesions along the
long axis of the glandular stomach. Lesions are absent in the forestomach.

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lesser extent in the antrum. Macroscopically visible hemorrhagic lesions are absent in
the forestomach, probably because of the thick layer of cornified epithelium that covers
the surface of this region.
Gastric mucosal damage induced by necrotizing agents is extensive and consists of
elongated bands, 1- to 10-mm long by 1- to 3-mm wide, usually parallel to the long axis
of the stomach (Fig. 21.2.1). The color varies depending on the necrotizing agent: red
(ethanol, 25% NaCl) or black (0.6 N HCl, 0.2 N NaOH).

6. For each stomach, use the transparent grid to measure all individual lesions along
their greatest length. Assign a rating of 1 to lesions measuring <1 mm; assign a
rating of 2 to lesions measuring 1 to 2 mm; assign a rating according to their length
in millimeters to lesions measuring >2 mm. Sum up the lengths of the lesions and
obtain an overall total, designated as the lesion index, for each stomach.
ALTERNATE
PROTOCOL 1

EVALUATION OF GASTRIC DAMAGE BY LIGHT MICROSCOPY

It is difficult to state the presence or the absence of gastric mucosal damage on purely
macroscopic grounds. Lesion areas become visible because there are focal accumulations
of blood due to hyperemia and hemorrhage and not because of damage to epithelial cells.
Extravasated blood may be in the lamina propria or in the submucosa or both and may
escape into the gastric lumen. However, damage to epithelial cells and hemorrhage are not
separate independent events. When epithelial damage is restricted to the upper mucosa,
there is usually no significant hemorrhage from either the superficial or deep vessels.
By contrast, epithelial damage extending deeply within the mucosa is associated with
extensive vascular stasis and hemorrhage. As a consequence, epithelial damage may
or may not be accompanied by hemorrhage. Only histological examination can more
properly establish the extent of damage to epithelial cells.
This protocol uses hematoxylin and eosin staining (H&E), the most common and simple
staining method used in light microscopic studies. Hematoxylin has a blue color and
stains nucleic acids. Eosin is pink and stains protein nonspecifically. Typically, in H&Estained sections, nuclei are stained dark blue whereas the cytoplasm has varying shades
of pink, identifying different tissue components.

Materials
Treated adult male rats with open abdomens (see Basic Protocol 1)
10% (v/v) neutral buffered formalin (see recipe)
Paraffin wax (melting point 56 to 60 C)
Xylene
80%, 96%, and 100% (v/v) ethanol
Mayers hematoxylin (see recipe)
Eosin (see recipe)
Canada balsam

Methods to
Measure Gastric
Mucosal Lesions
in the Rat

2-ml syringe
100-ml polypropylene jars
Biopsy pads
Micromesh biopsy processing/embedding cassettes
Automatic tissue processor (Sakura Finetechnical)
Tissue embedding center (Sakura Finetechnical)
Base molds
Cold plate
Microtome
Disposable stainless blades
Blunt forceps

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45 C distilled water bath

37 C slide warmer
Slide holder
Staining dish
Coverslips
Video camera attached to a light microscope (e.g., Nikon Optiphot)
Color image analysis software system (e.g., LUCIA G, Nikon Laboratory Imaging)
Dissect and fix stomach
1. Gently inflate the stomach of a treated male rat using a 2-ml syringe with 10% (v/v)
neutral buffered formalin.
2. Rapidly remove the stomach and open along the lesser curvature.
3. Place the stomach in a 100-ml polypropylene jar filled with 10% formalin for 24 hr
at room temperature. Adjust the volume of formalin so that the stomach is fully
immersed.
4. Excise a strip (5 10mm) from the glandular mucosa, 5 mm below and parallel to
the limiting ridge (the border between the forestomach and the glandular stomach),
so that the greater curvature is located in the middle of the strip.
5. Obtain six different tissue samples from each strip (cut six pieces perpendicular to
the long axis).
6. Distend each sample between two biopsy pads and place them in a micromesh biopsy
processing/embedding cassette with lid.
7. Place cassettes in 10% formalin for an additional 24 hr at room temperature.

Process samples
8. Place the cassettes into the automatic tissue processor to remove all water from the
tissue and replace it with paraffin wax according to manufacturers instructions.
In the processor, the tissue samples are transferred through baths of progressively more
concentrated ethanol and subsequently ethanol is removed by the clearing agent, xylene.
Finally, infiltrating paraffin will replace the xylene. The time needed for tissue processing
is usually 12 hr.

9. At the end of tissue processing, place cassettes in the tissue embedding center for
tissue embedding.
10. At the end of the embedding process, immerse the embedded tissue sample into base
molds with lid along with paraffin. Carefully orient the tissue so that histological
sections can be cut perpendicular to the epithelial surface.
11. Place base molds on a cold plate (4 C) until the paraffin completely hardens
(10 min).
12. Completely separate the mold from the embedding paraffin.

Section samples
13. Turn on the microtome, mount disposable stainless steel blades and set the section
thickness to 4 m.
14. Mount the sample paraffin block and cut 4-m thick sections perpendicular to the
epithelial surface.
15. Separate the sections with blunt forceps.
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16. Allow sections to float in a bath containing distilled water at 45 C to allow full
distension of the tissue.
17. Allow one section to adhere onto the surface of a slide directly out of the bath.
18. Keep slides on a slide warmer overnight at 37 C.

Stain with hematoxylin

19. Insert the slides into a slide holder and then into the staining dish.
20. Deparaffinize in xylene, two changes of 10 min each.
21. Rehydrate the tissue sections, by passing the slides through a series of decreasing
concentrations of ethanol:
a.
b.
c.
d.

Two changes in 100% ethanol, 5 min each

Two changes in 96% ethanol, 5 min each
One change in 80% ethanol, 5 min
Rinse in distilled water for 5 min.

22. Stain in Mayers hematoxylin solution for 10 min.

23. Wash in running tap water for 10 min. Rinse in distilled water.

Counterstain with eosin

24. Counterstain in eosin for 15 min.
25. Dehydrate through a series of increasing concentrations of ethanol:
a. One change in 80% ethanol, 1 min
b. Three changes in 96% ethanol, 3 min each
c. Three changes in 100% ethanol, 3 min each.
Increase or decrease the suggested number of changes and their duration to obtain the
removal of excess eosin. Check under a microscope.

26. Clear in xylene, two changes of 5 min each or alternatively overnight.

Add coverslip
27. Place a drop of Canada balsam on the slide, ensuring there are no bubbles.
28. Gently cover all the tissue with a coverslip.

Measure damage
29. Display the image of each section on a color monitor using a video camera attached
to the light microscope (e.g., Nikon Optiphot) for the morphometric analysis of
gastric damage.

Methods to
Measure Gastric
Mucosal Lesions
in the Rat

Figure 21.2.2 (appears on next page) Light micrographs of the fundic mucosa 1 hr after receiving
absolute ethanol, 1 ml/rat intragastrically. The grades of damage used for quantitative analysis
are shown. Grade 0: surface, gastric pits and glands are normal appearing. Grade I: luminal
surface mucous cells are damaged and partly exfoliated (arrows). Grade II: luminal surface and
pit cells are damaged and exfoliated (arrow). Gland cells are intact. Grade III: note the sloughing
of surface cells with necrosis in the midportion of the mucosa corresponding to the parietal cell
area (arrows). Submucosal edema is prominent. Grade IV: necrosis extends to the base of the
mucosa, corresponding to the chief cell area (arrows). There is a marked reduction of the height
of the mucosa due to cell sloughing. Submucosal edema is prominent. Scale bar = 250 m.

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grade 0

250 m

grade I

grade II

grade III

grade IV

Figure 21.2.2

(legend appears on previous page)

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30. Evaluate the severity of mucosal damage on the basis of its depth, according to the
following grading system:
a. Grade 0: all gastric mucosal cells appear intact.
b. Grade I: surface mucous cells on the luminal surface are damaged and partly
exfoliated, gastric pit cells are undamaged.
c. Grade II: extensive luminal surface cell damage plus damage to the cells lining
the gastric pits, gastric gland cells are undamaged.
d. Grade III: in addition to surface and pit cell damage, cellular damage is evident
in the upper portion of the gastric glands (parietal cell area), numerous exfoliated
cells and whole layer of necrotic superficial epithelium are also present.
e. Grade IV: severe grade III damage extending into the lower portion of the gastric
glands (chief cell area; Fig. 21.2.2), submucosal edema.
31. Perform quantitations using a color image analysis software system (e.g., LUCIA G,
Nikon Laboratory Imaging).
32. For each section, determine the total length of mucosa examined and the length of
mucosa with each grade of damage.
The length values of each grade of damage can also be expressed as a percentage of total
length of mucosa examined.

33. For each rat, calculate the mean length of gastric mucosa examined from the different
sections of each stomach and the mean length (or percentage) of mucosa with each
grade of damage.
ALTERNATE
PROTOCOL 2

EVALUATION OF GASTRIC DAMAGE BY SCANNING ELECTRON

MICROSCOPY
Epithelial cells facing the gastric lumen, termed surface mucous cells, are central components of local defense mechanisms, withstanding damage to the superficial portions of the
mucosa, probably occurring frequently during food and drug ingestion. They constantly
synthesize and secrete mucus, which forms a gel layer adhering to the epithelium and
is constantly degraded by gastric enzymes. When adherent, it represents a barrier separating and protecting epithelial cells from luminal contents. Surface mucous cells have
a short half-lifeestimated at 3 to 5 daysand their rapid turnover is considered to be
necessary to replace damaged cells lost by exfoliation and to re-establish epithelial continuity. Moreover, these cells are also found to be actively migrating. By migration, viable
mucous cells can re-establish the epithelial integrity within minutes in areas exfoliated
following the exposure to noxious agents, a process known as restitution. The surface
epithelium can be examined by scanning electron microscopy. The high magnification
and the fine-structure resolution of scanning electron microscopy allow study of surface
alterations at a cellular level. This kind of study has greatly enhanced the knowledge of
the mechanisms concerning gastric mucosal damage.

Materials
Treated adult male rat with opened abdomen (see Basic Protocol 1)
10% (v/v) neutral buffered formalin (see recipe)
25%, 50%, 75%, 90%, and 100% acetone

Methods to
Measure Gastric
Mucosal Lesions
in the Rat

Critical point dryer (Leica Microsystems)

Aluminum stubs (Electron Microscopy Sciences)
Double-sided adhesive tape
Sputter coater (Leica Microsystems)
Scanning electron microscope

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Prepare gastric fundus sample

1. Dissect the stomach from a treated male rat and open along the lesser curvature.
2. Excise two to three specimens (8 8mm) from the glandular mucosa, 5 mm
below and parallel to the limiting ridge.
3. Fix with 10% (v/v) neutral buffered formalin for 2 hr at room temperature.
4. Rinse in distilled water.
5. Dehydrate through a series of increasing concentrations of 10 to 15 ml acetone in
glass beakers:
a.
b.
c.
d.
e.

two changes in 25% acetone, 10 min each

two changes in 50% acetone, 10 min each
two changes in 75% acetone, 20 min each
two changes in 90% acetone, 20 min each
two changes in 100% acetone, 20 min each.

6. Dry by placing the sample in the appropriate holder and then in the critical point
drying apparatus following the manufacturers instructions.

B
grade 0

grade I

D
grade II

grade III

Figure 21.2.3 Scanning electron micrographs of the fundic mucosa 1 hr after receiving absolute
ethanol, 1 ml/rat intragastrically. The grades of damage used for quantitative analysis are shown.
(A) Grade 0 = Normal epithelial cells cover >90% of the surface. (B) Grade I: epithelial cells cover
>50% of the surface. Note that a portion of the lamina propria is exposed to the lumen and devoid
of epithelial cells, while the remaining mucosal surface is covered by cells. (C) Grade II: normal
epithelial cells cover <50% of the surface. Lining cells can be seen to partially fill >50% of gastric
pits. Note that surface mucous cells are fully exfoliated, yielding a honeycomb appearance of the
completely denuded lamina propria. Epithelial cells can be seen at the mouth of the gastric pits.
(D) Grade III: <50% of the surface is covered by normal epithelial cells. Lining cells can be seen to
partially fill <50% of gastric pits. Deep craters in completely denuded lamina propria are shown.
Scale bar = 10 m.

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7. Mount the sample, luminal surface upward, on a specimen aluminum stub (specimen
holders) using a double-sided adhesive tape.
8. Coat with gold in the sputter coater following the manufacturers instructions.
9. Insert the sample attached to the stub into the scanning electron microscope, view
on the monitor and photograph.

Measure damage
10. Evaluate damage using a qualitative approach.
11. Alternatively, use a modified semiquantitative scoring system, based on that previously described by Kang et al. (1995).
12. Choose an area with maximum damage from each block of tissue. Obtain a photograph and evaluate the severity of mucosal damage, according to the following
grading system:
a. Grade 0: normal epithelial cells cover >90% of the surface.
b. Grade I: normal epithelial cells cover >50% of the surface.
c. Grade II: normal epithelial cells cover <50% of the surface, lining cells can be
seen to partially fill >50% of gastric pits.
d. Grade III: <50% of the surface is covered by normal epithelial cells, lining cells
can be seen to partially fill <50% of gastric pits (Fig. 21.2.3).
13. Assign to each animal a score, based on the worst affected area.
BASIC
PROTOCOL 3

EVALUATING ACUTE GASTRIC LESIONS INDUCED BY CONVENTIONAL

NSAIDS
Conventional NSAIDs are largely recognized to cause significant damage to the gastric
mucosa (Wallace, 2000, 2008). These agents, particularly those that are acidic, such as
aspirin, can directly damage the epithelial cells. However, NSAIDs can produce erosions and ulcers in experimental animals, following parenteral administration, providing
evidence that generation of erosions and ulcers can be a systemically mediated effect
(Djahanguiri, 1969). NSAIDs appear to primarily alter microcirculation, reducing gastric
mucosal blood flow and promoting the adhesion of neutrophils to the vascular endothelium. These alterations occur as a consequence of inhibition of prostaglandin synthesis by
these drugs. Aspirin and indomethacin are the NSAIDS most commonly used to produce
gastric damage in experimental conditions, despite the evidence that almost all conventional NSAIDs are gastrotoxic in experimental animals. Topical damage is identified as
a prominent feature of aspirin toxicity, while damage induced by indomethacin is largely
mediated systematically. Damage caused by NSAIDs largely differs in its macroscopic
and morphological features from that caused by necrotizing agents.

Materials
1% (w/v) carboxymethylcellulose
0.6 N HCl (APPENDIX 2A)
Aspirin (Sigma)
Adult rats (male, body weight 200 to 220 g, 9 to 10 weeks old)
Indomethacin (Sigma)
Methods to
Measure Gastric
Mucosal Lesions
in the Rat

Orogastric tube
Light microscope
Scanning electron microscope

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Treat rats
For aspirin treatment
1a. Prepare the vehicle by suspending 1% (w/v) carboxymethylcellulose in 0.16 N HCl,
with pH ranging from 1.3 to 1.5, which maintains aspirin in its readily absorbable
non-ionized form.
2a. Prepare aspirin solution by suspending the drug in the vehicle to a final concentration
of 12 mg/ml.
3a. Administer aspirin at a dose of 120 mg/kg in a volume of 10 ml/kg to the rats via the
orogastric tube. Sacrifice rat with 70% CO2 or by cervical dislocation 3 hr later.

For indomethacin treatment

1b. Suspend indomethacin in 1% (w/v) carboxymethylcellulose to a final concentration
of 2 mg/ml.
2b. Administer indomethacin at a dose of 20 mg/kg in a volume of 10 ml/kg to the rats
via the orogastric tube.
When administered intragastrically, indomethacin damages gastric mucosa in a dosedependent way, maximal effect being achieved at 20 mg/kg (Djahanguiri, 1969).

3b. Sacrifice rat with 70% CO2 or by cervical dislocation 3 to 6 hr after dosing.
Damage is minimally visible 1 hr after the administration and develops with time.

Measure damage
4. Quantify macroscopically visible damage by NSAIDs using the following grading
system:
a. Assign a rating of 1 to lesions measuring <1 mm.
b. Assign a rating of 2 to lesions measuring 1 to 2 mm.
c. Assign a rating according to their length in millimeters to lesions measuring
>2 mm.
d. Sum the length of the lesions and obtain an overall total, designated as the lesion
index, for each stomach.
5. Use the following scale to evaluate damage by light microscopy:
a. Grade 0: the mucosa is normal appearing.
b. Grade I: vasocongestion not deeper than the pit regionmicrovessels are dilated
and engorged and red blood cells and leukocytes visible inside, extravasation of
red blood cells is rare.
c. Grade II: vasocongestion and interstitial edema, both being limited to the subepithelial region, the interstitial spaces are expanded, clear and cell-free.
d. Grade III: superficial erosions with the consequent discontinuity of surface epithelial layer, damage is limited to surface and pit cells.
e. Grade IV: focal necrosis extending into the chief cell area or up to the muscularis
mucosae, these areas are constituted by amorphous debris, by macrophages and
by leukocytes, mainly neutrophils.
A barrier of leukocytes, mainly neutrophils, and macrophages usually separates the
necrotic area from the surrounding mucosal tissue.

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250 m

Figure 21.2.4 The prominent features of gastric damage induced by indomethacin, 20 mg/kg
intragastrically. Stomachs were removed 6 hr after the administration of indomethacin. (A) Macroscopic appearance of the gastric mucosa. Indomethacin causes the formation of macroscopic
damage, visible as hemorrhagic points or small lines. Lesions are absent in the forestomach. (B)
Light micrograph of the fundic mucosa. A conically shaped necrotic area deeply extending into the
chief cell area (grade IV), typically observed at 6 hr after indomethacin administration, is shown.
Edema is present in the submucosa. Scale bar = 250 m. (C) Scanning electron micrograph of
the fundic mucosa. A crater can be seen, deeply penetrating into the mucosa. Scale bar = 10 m.

6. Determine the total length of the mucosal tissue examined by light microscopy and
the length of each grade of damage for each stomach.
7. Use a qualitative approach to evaluate damage by scanning electron microscope.
Methods to
Measure Gastric
Mucosal Lesions
in the Rat

The prominent features of gastric damage induced by indomethacin are shown in

Figure 21.2.4.

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REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock
solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Eosin
1 g eosin
100 ml distilled water
0.5 ml glacial acetic acid
Mix to dissolve at room temperature. Store up to 15 days at room temperature.
Filter before any use, using a filtration paper and gravity filtration.

Mayers hematoxylin
1 g hematoxylin
50 g aluminum potassium sulphate (alum)
0.2 g sodium iodate
50 g chloral hydrate
1 g citric acid
1000 ml distilled water
Dissolve alum in distilled water. Dissolve hematoxylin into the solution. Bring the
solution to a boil and allow to cool. Add remaining chemicals, dissolving each one
before adding the next. Store up to 30 days at room temperature. If necessary, filter
before use using filtration paper and gravity filtration.

Neutral buffered formalin, 10% (v/v)

4 g sodium phosphate, monobasic
6.5 g sodium phosphate, dibasic
100 ml 37% formaldehyde
900 ml distilled water
Mix to dissolve at room temperature. Store up to 2 months at room temperature.
COMMENTARY
Background Information
The rat stomach may be divided into
two main regions: the forestomach, a nonglandular region lined by stratified squamous
epithelium, adjacent to the gastroesophageal
junction, and the glandular stomach, which
consists of two parts, corpus and antrum. The
antrum becomes continuous with the duodenum at the pyloroduodenal junction. A simple
layer of columnar specialized epithelial cells
lines the luminal surface of both regions and
invaginates to form the oxyntic pit-gland unit
in the corpus and the shorter antral pit-gland
unit. Between the glands there is the lamina
propria, which contains blood and lymph vessels, nerves, and connective tissue elements.
The bottom layer of gastric mucosa consists
of the muscularis mucosa.

Critical Parameters and

Troubleshooting
Influence of fasting
Fasting reduces the density of antral gastrin (G)-cells, the plasma concentrations of
Current Protocols in Toxicology

gastrin, and the activity of the histaminesynthesizing enzyme histamine decarboxylase

(HDC), leading to a decrease in histamine content and in gastric acid secretion (Ohning et al.,
1998; Zhao et al., 2003). Fasting conditions
apparently activate protective mechanisms by
reducing gastric acid output, which could potentially exacerbate gastric mucosal damage.
In its turn, the presence of food in the lumen
exerts a buffering effect on the acid secreted
by parietal cells and can reduce the contact
between the potentially damaging compounds
present in the lumen and the surface mucous
cells. The ulcerogenic effect could therefore
be modified by alterations related to fasting/
feeding conditions. Studies aimed at evaluating chemicals potentially damaging to the gastric mucosa are usually performed on fasted
animals and the fasting period can vary from
24 hr to 48 hr. It is critical that the fasting
period remains unaltered during the study.
Influence of aging
Aging is associated with increased susceptibility to damage (Majumdar et al., 1989: Lee
and Feldman, 1994: Grnbech and Lacy, 1995;

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Tarnawski et al., 2007). Aspirin, hypertonic

saline, and ethanol produce a higher lesion
index in old (24-month-old) than in young
(3- or 4-month-old) rats. In aging rats, defensive mechanisms, such as mucosal blood flow,
mucosal restitution, prostaglandin generation,
and NO synthase activity, have been found to
be diminished with a parallel increase in hypoxia and expression of preapoptotic proteins
in the gastric mucosa. Due to the age-related
responsiveness of the mucosa, the choice of
the age of rats is crucial. Most studies are performed in young rats, usually 9 to 12 weeks
old.
Influence of nutrition
Susceptibility to damaging agents is influenced by the diet. Gastric lesions induced
by indomethacin are lower in number in rats
maintained on a low-protein diet than in rats
maintained on a normal protein diet (Paula
et al., 2006). Gastric mucosal lesions have
been found to be increased in rats on parenteral
nutrition in comparison with animals fed the
identical diet orally (Sander et al., 1980).
Correct orientation of histological sections
Quality and reproducibility of data by light
microscopy are largely affected by the quality
of sectioning. It is mandatory that sectioning
is perpendicular to the mucosal surface and
only the regions in which full-length glands are
oriented perpendicular to the luminal surface
should be considered for quantitative analysis.

Anticipated Results

Methods to
Measure Gastric
Mucosal Lesions
in the Rat

The protocols detailed in this unit provide

rather simple and highly reproducible methods
to measure gastric damage in the rat.
Following the intragastric administration of
absolute ethanol for 60 min, the lesion index, a
parameter of macroscopically visible lesions,
was 87.1 15.8 (Morini et al., 1995b). When
damage was assessed histologically at 5, 15,
30, and 60 min after administration of absolute ethanol, damage respectively involved
96%, 93%, 92%, and 94% of the total mucosal
length evaluated (Morini et al., 1998). The values of total length of damaged mucosa were
similar at any time interval. On the basis of the
degree of damage, no major differences were
observed by comparing lesions of the same
grade at the different time intervals or lesions
of different grade in the group examined at the
same time.
Following the intragastric administration of
indomethacin, 20 mg/kg, the lesion index was
22.1 3.9 and 53.4 8.3 at 3 and 6 hr, respec-

tively. Quantitative analysis of mucosal damage evaluated by light microscopy revealed

that damage involved 99% and 23% of the
total mucosal length evaluated (Morini et al.,
1995a) at 3 and 6 hr following indomethacin
administration, respectively. Extensive vasocongestion and edema accounted for 94%
of damaged mucosa at 3 hr. With time, vasocongestion and edema are no longer apparent,
while deep hemorrhagic necrosis of mucosal
tissue develops.

Time Considerations
Macroscopic evaluation of gastric damage
is performed immediately after the sacrifice
of the animal. As a consequence, treatment of
rats and evaluation of lesions by stereomicroscope are usually performed within 2 to 3 hr
for necrotizing agents and within 4 to 8 hr for
longer-acting agents like indomethacin. Usually, each treatment group is made up of six
rats, and in each treatment group, two to three
rats per day are treated. A maximum of ten to
twelve rats is treated per day.
Evaluation of damage by light microscopy
requires 4 to 5 days. After treatment of rats,
which requires 1 to 6 hr, fixation, embedding,
sectioning of paraffin blocks, and staining require 2 to 3 days. Paraffin blocks may be
safely archived up to 1 year. Measurement
of damage by light microscope takes 30 to
60 min per stomach from each rat. The procedure for obtaining scanning electron micrographs requires 1 to 2 days. The time needed
for the semiquantitative measurement of damage require 1 to 2 hr per stomach from each
rat.

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Gastrointestinal
Toxicology

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