Methods to Measure Gastric Mucosal Lesions in the Rat

Giuseppina Morini1 and Daniela Grandi1

Department of Human Anatomy, Pharmacology, and Forensic Medicine, University of Parma, Parma, Italy
1

UNIT 21.2

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 conned 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 ow, 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 conned 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 Copyright C 2010 John Wiley & Sons, Inc.

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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 rst be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow ofcially 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. 5. Sacrice 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 sacrice, 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 briey with 0.9% NaCl. 2. Pin the stomach at 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 lm of a paper grid.

5. Score the stomach for macroscopic gastric damage.

Macroscopic gastric damage is dened 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 cornied 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 difcult 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 signicant hemorrhage from either the supercial 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 nonspecically. 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) Parafn wax (melting point 56 to 60 C) 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 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 x stomach 1. Gently inate 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 lled 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 parafn 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, inltrating parafn 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 parafn. 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 parafn completely hardens (10 min). 12. Completely separate the mold from the embedding parafn.

Section samples 13. Turn on the microtome, mount disposable stainless steel blades and set the section thickness to 4 m.
14. Mount the sample parafn 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 oat 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. Deparafnize 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.
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 supercial 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 supercial 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 magnication and the ne-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. 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.

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 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 ll >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 ll <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 modied 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 ll >50% of gastric pits. d. Grade III: <50% of the surface is covered by normal epithelial cells, lining cells can be seen to partially ll <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 signicant 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 ow 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 identied 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 nal 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. Sacrice rat with 70% CO2 or by cervical dislocation 3 hr later.

For indomethacin treatment 1b. Suspend indomethacin in 1% (w/v) carboxymethylcellulose to a nal 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. Sacrice 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: supercial 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-puried 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 ltration paper and gravity ltration.

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, lter before use using ltration paper and gravity ltration.

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 stratied 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. 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 modied 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. Inuence of aging Aging is associated with increased susceptibility to damage (Majumdar et al., 1989: Lee and Feldman, 1994: Grnbech and Lacy, 1995;
Gastrointestinal Toxicology

Critical Parameters and Troubleshooting

Inuence of fasting Fasting reduces the density of antral gastrin (G)-cells, the plasma concentrations of
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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 ow, 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. Inuence of nutrition Susceptibility to damaging agents is inuenced 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.

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 sacrice 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, xation, embedding, sectioning of parafn blocks, and staining require 2 to 3 days. Parafn 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.

Anticipated Results
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-

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Methods to Measure Gastric Mucosal Lesions in the Rat

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Gastrointestinal Toxicology

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