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UNIT 21.2Methods to Measure Gastric MucosalLesions in the Rat

Giuseppina Morini1 and Daniela Grandi1

1Department 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

microscopy. Curr. Protoc. Toxicol. 43:21.2.1-21.2.15. C 2010 by John Wiley & Sons,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 littleattention 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

Copyright C 2010 John Wiley & Sons, Inc.

GastrointestinalToxicology

21.2.1

Supplement 43

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Methods toMeasure GastricMucosal Lesions

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21.2.2

Supplement 43 Current Protocols in Toxicology

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 Insti-

tutional 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 NECROTIZINGAGENTS

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 balanceOrogastric tube

Surgical tools

1. House the rats at 22C 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 varia-

tion.

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.

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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Current Protocols in Toxicology Supplement 43

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).

BASIC

PROTOCOL 2

MACROSCOPIC EVALUATION OF GASTRIC DAMAGE

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

stereomicroscope.

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/ratintragastrically. Exposure to ethanol produces the characteristic linear necrotic lesions along thelong 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 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&E-

stained sections, nuclei are stained dark blue whereas the cytoplasm has varying shades

of pink, identifying different tissue components.

MaterialsTreated adult male rats with open abdomens (see Basic Protocol 1)

10% (v/v) neutral buffered formalin (see recipe)

Paraffin wax (melting point 56 to 60C)

Xylene

80%, 96%, and 100% (v/v) ethanol

Mayers hematoxylin (see recipe)

Eosin (see recipe)

Canada balsam

2-ml syringe

100-ml polypropylene jars

Biopsy padsMicromesh 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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45C distilled water bath

37C 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 (4C) 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 45C 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 37C.

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. Two changes in 100% ethanol, 5 min each

b. Two changes in 96% ethanol, 5 min each

c. One change in 80% ethanol, 5 min

d. 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.

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

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21.2.7


grade 0

grade I

grade II

grade III

grade IV

250 m

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 ELECTRONMICROSCOPY

Epithelial cells facing the gastric lumen, termed surface mucous cells, are central compo-

nents 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 sepa-rating 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 conti-

nuity. 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

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. two changes in 25% acetone, 10 min each

b. two changes in 50% acetone, 10 min each

c. two changes in 75% acetone, 20 min each

d. two changes in 90% acetone, 20 min each

e. 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.

A

grade 0

B

grade I

C

grade II

D

grade III

Figure 21.2.3 Scanning electron micrographs of the fundic mucosa 1 hr after receiving absoluteethanol, 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 devoidof epithelial cells, while the remaining mucosal surface is covered by cells. ( C) Grade II: normalepithelial cells cover 50% of gastricpits. Note that surface mucous cells are fully exfoliated, yielding a honeycomb appearance of thecompletely denuded lamina propria. Epithelial cells can be seen at the mouth of the gastric pits.(D) Grade III:

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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 previ-

ously described by Kang et al. (1995).

12. Choose an area with maximum damage from each block of tissue. Obtain a pho-

tograph 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 cover50% of gastric pits.

d. Grade III:

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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 theorogastric 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 dose-

dependent 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 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 subep-

ithelial region, the interstitial spaces are expanded, clear and cell-free.

d. Grade III: superficial erosions with the consequent discontinuity of surface ep-

ithelial layer, damage is limited to surface and pit cells.

e. Grade IV: focal necrosis extending into the chief cell area or up to the muscularismucosae, 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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A

B

C

250 m

Figure 21.2.4 The prominent features of gastric damage induced by indomethacin, 20 mg/kgintragastrically. Stomachs were removed 6 hr after the administration of indomethacin. (A) Macro-scopic appearance of the gastric mucosa. Indomethacin causes the formation of macroscopicdamage, 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 thechief 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.

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


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, filterbefore 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


Mix to dissolve at room temperature. Store up to 2 months at room temperature.

COMMENTARY

Background InformationThe rat stomach may be divided into

two main regions: the forestomach, a non-

glandular 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 duode-

num 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 ves-

sels, nerves, and connective tissue elements.

The bottom layer of gastric mucosa consists

of the muscularis mucosa.

Critical Parameters andTroubleshooting

Influence of fastingFasting reduces the density of antral gas-

trin (G)-cells, the plasma concentrations of

gastrin, and the activity of the histamine-

synthesizing enzyme histamine decarboxylase

(HDC), leading to a decrease in histamine con-tent 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 po-

tentially 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 evaluat-ing chemicals potentially damaging to the gas-

tric 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 agingAging is associated with increased suscep-

tibility to damage (Majumdar et al., 1989: Lee

andFeldman, 1994: Grnbech andLacy, 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, defen-

sive 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 hy-

poxia 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 per-

formed in young rats, usually 9 to 12 weeks

old.

Influence of nutritionSusceptibility to damaging agents is in-

fluenced 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 increasedin rats on parenteral

nutrition in comparison with animals fed the

identical diet orally (Sander et al., 1980).

Correct orientation of histological sectionsQuality 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 theregions in which full-length glandsare

oriented perpendicular to the luminal surface

should be considered for quantitative analysis.

Anticipated ResultsThe 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 ab-

solute ethanol, damage respectively involved

96%, 93%, 92%, and 94% of the total mucosal

length evaluated (Morini et al., 1998). The val-

ues of total length of damaged mucosa weresimilar 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 dam-

age 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 vaso-

congestion and edema accounted for94%

of damaged mucosa at 3 hr. With time, vaso-

congestion and edema are no longer apparent,

while deep hemorrhagic necrosis of mucosal

tissue develops.

Time ConsiderationsMacroscopic evaluation of gastric damage

is performed immediately after the sacrifice

of the animal. As a consequence, treatment of

rats and evaluation of lesions by stereomicro-

scope are usually performed within 2 to 3 hr

for necrotizing agents and within 4 to 8 hr for

longer-acting agents like indomethacin. Usu-

ally, 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 totwelve rats is treated per day.

Evaluation of damage by light microscopy

requires4 to 5 days. After treatment of rats,

which requires 1 to 6 hr, fixation, embedding,

sectioning of paraffin blocks, and staining re-

quire 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 pro-

cedure for obtaining scanning electron micro-

graphs requires1 to 2 days. The time needed

for the semiquantitative measurement of dam-

age require 1 to 2 hr per stomach from eachrat.

Literature CitedDjahanguiri, B. 1969. The production of acute gas-

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Kang, J.Y., Teng, C.H., Wee, A., and Chen, F.C.1995. Effect of capsaicin and chilli on ethanolinduced gastric mucosal injury in the rat. Gut36:664-669.

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Morini, G., Grandi, D., Arcari, M.L., andBertaccini, G. 1995a. Indomethacin-inducedmorphologicalchangesin therat gastric mucosa,with or without prior treatment with two pro-ton pump inhibitors. Aliment. Pharmacol. Ther.

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normal rats than in malnourished rats.Br. J. Nutr.96:310-315.

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Tarnawski, A., Pai, R., Deng, X., Ahluwalia,

A., Khomenko, T., Tanigawa, T., Akahoshi,T., Sandor, Z., and Szabo, S. 2007. Aginggastropathy-novel mechanisms: Hypoxia, up-regulation of multifunctional phosphatasePTEN, and proapoptotic factors. Gastroenterol-ogy 133:1938-1947.

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Zhao, C.M., Chen, D., Yamada, H., Dornonvillede la Cour, C., Lindstrom, E., Persson, L., andHakanson, R. 2003. Rat stomach ECL cells:Mode of activation of histidine decarboxylase.

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