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 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 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.

Gastrointestinal Toxicology

21.2.1
Supplement 43

BASIC PROTOCOL 1 ASSESSING ACUTE GASTRIC LESIONS INDUCED BY NECROTIZING AGENTS Necrotizing agents exert a direct damaging effect on the gastric epithelial cells. body weight of 200 to 220 g. House the rats under laboratory conditions for at least 1 week after arrival for adaptation. 25% NaCl) 1 ml/rat via the orogastric tube. They also cause vascular congestion and disruption. gastric damage by these agents can be evidenced only after intragastric administration. House the rats at 22◦ C on a 12-hr light/dark cycle. 3. 0. Return the rat to his cage after the administration of the agent.6 N HCl (APPENDIX 2A) 0. Acute models are largely used for screening potential gastrotoxic or gastroprotective drugs/chemicals and for evaluating the mechanisms responsible for ulcer formation.6 N HCl. open the abdomen through a mid-line laparatomy. Methods to Measure Gastric Mucosal Lesions in the Rat 21.2.2 N NaOH.2 Supplement 43 Current Protocols in Toxicology . to avoid possible diurnal variation. Immediately after the sacrifice. is able to provide an immediate protection against mucosal damage caused by strong irritants administered 1 hr later. Deprive the animals of food but not of water for 24 hr before the experiment. the most commonly used laboratory animal is the rat. 0. 5. under the cutaneous incision. Perform experiments during the same period of the day.ethanol.to 4-cm incision in the skin below the rib cage and then. make another incision of the same length in the abdominal muscle. 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. As a consequence of their topical noxious effect. Make a 3.2 N NaOH (APPENDIX 2A) 25% (w/v) NaCl (APPENDIX 2A) Rat housing Animal balance Orogastric tube Surgical tools 1. Materials Adult rats (male. 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. Weigh each rat on the day of the experiment. Handle the adult male rat gently and administer the necrotizing agent (absolute ethanol. resulting in varying degrees of epithelial rupture and lifting. 9 to 10 weeks old) Necrotizing agent: Absolute ethanol 0. 4. Although experimental models of gastric ulcer were developed in a variety of animal species. 2. 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. Chronic models are mostly used to study drugs/chemicals delaying or accelerating the healing process and the mechanisms underlying ulcer healing and relapse. Experimental models of chronic ulcer have also been developed.

2. Rapidly remove the stomachs of the treated adult male rats.6. Open along the lesser curvature and rinse briefly with 0. Pin the stomach flat onto a board. 3. The transparent grid is self-prepared by making a photocopy on a transparent film of a paper grid. 5. 4. Avoid stretching or distortion of the mucosa. Exposure to ethanol produces the characteristic linear necrotic lesions along the long axis of the glandular stomach.2. Examine the mucosal surface under a stereo microscope. MACROSCOPIC EVALUATION OF GASTRIC DAMAGE Preliminary assessment of gastric damage is done at the macroscopic level.1 Photograph of the stomach of a rat 1 hr after receiving absolute ethanol. Macroscopic gastric damage is defined to be hemorrhagic areas of the mucosa that do not clear on rinsing. Place a transparent plastic grid over the mucosa. light microscopic evaluation (see Alternate Protocol 1). with the mucosal surface uppermost.3 Current Protocols in Toxicology Supplement 43 .9% (w/v) NaCl (APPENDIX 2A) Dissecting board and pins Stereomicroscope Transparent plastic 1-mm grids 1. Proceed to assessment and evaluation of gastric damage—macroscopic evaluation (see Basic Protocol 2). Lesions are absent in the forestomach. Gastrointestinal Toxicology 21. BASIC PROTOCOL 2 Materials Treated adult male rats with open abdomens (see Basic Protocol 1) 0. using a stereomicroscope. Hemorrhagic areas are located mostly in the corpus and to a Figure 21.9% NaCl.2. Score the stomach for macroscopic gastric damage. 1 ml/rat intragastrically. or scanning by electron microscopic evaluation (see Alternate Protocol 2).

2. Sum up the lengths of the lesions and obtain an overall total. designated as the lesion index. probably because of the thick layer of cornified epithelium that covers the surface of this region. and 100% (v/v) ethanol Mayer’s 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 Methods to Measure Gastric Mucosal Lesions in the Rat 21. usually parallel to the long axis of the stomach (Fig. epithelial damage may or may not be accompanied by hemorrhage.1). nuclei are stained dark blue whereas the cytoplasm has varying shades of pink. use the transparent grid to measure all individual lesions along their greatest length.to 3-mm wide. Assign a rating of 1 to lesions measuring <1 mm. Typically. By contrast. for each stomach.lesser extent in the antrum. damage to epithelial cells and hemorrhage are not separate independent events. 25% NaCl) or black (0. assign a rating of 2 to lesions measuring 1 to 2 mm. Macroscopically visible hemorrhagic lesions are absent in the forestomach. epithelial damage extending deeply within the mucosa is associated with extensive vascular stasis and hemorrhage.to 10-mm long by 1.4 Supplement 43 Current Protocols in Toxicology . 0. identifying different tissue components. This protocol uses hematoxylin and eosin staining (H&E). When epithelial damage is restricted to the upper mucosa.2 N NaOH). Hematoxylin has a blue color and stains nucleic acids. 6. in H&Estained sections. assign a rating according to their length in millimeters to lesions measuring >2 mm. As a consequence.6 N HCl. Eosin is pink and stains protein nonspecifically. 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%.2. Gastric mucosal damage induced by necrotizing agents is extensive and consists of elongated bands. there is usually no significant hemorrhage from either the superficial or deep vessels. the most common and simple staining method used in light microscopic studies. 21. Only histological examination can more properly establish the extent of damage to epithelial cells. For each stomach. The color varies depending on the necrotizing agent: red (ethanol. 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. 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. However. 96%. 1.

7. infiltrating paraffin will replace the xylene. Adjust the volume of formalin so that the stomach is fully immersed. xylene. Obtain six different tissue samples from each strip (cut six pieces perpendicular to the long axis). Nikon Optiphot) Color image analysis software system (e.. immerse the embedded tissue sample into base molds with lid along with paraffin. 2. Turn on the microtome. Completely separate the mold from the embedding paraffin.5 Current Protocols in Toxicology Supplement 43 . The time needed for tissue processing is usually 12 hr. Section samples 13. Place the cassettes into the automatic tissue processor to remove all water from the tissue and replace it with paraffin wax according to manufacturer’s instructions. Carefully orient the tissue so that histological sections can be cut perpendicular to the epithelial surface. Distend each sample between two biopsy pads and place them in a micromesh biopsy processing/embedding cassette with lid. Mount the sample paraffin block and cut 4-μm thick sections perpendicular to the epithelial surface. Process samples 8. LUCIA G. so that the greater curvature is located in the middle of the strip.45◦ C distilled water bath 37◦ C slide warmer Slide holder Staining dish Coverslips Video camera attached to a light microscope (e. Place the stomach in a 100-ml polypropylene jar filled with 10% formalin for 24 hr at room temperature. 11.2.g. 4. In the processor. Gently inflate the stomach of a treated male rat using a 2-ml syringe with 10% (v/v) neutral buffered formalin. At the end of tissue processing. 5. 15. Nikon Laboratory Imaging) Dissect and fix stomach 1. Finally. Rapidly remove the stomach and open along the lesser curvature. At the end of the embedding process. 9. Place base molds on a cold plate (4◦ C) until the paraffin completely hardens (∼10 min). 5 mm below and parallel to the “limiting ridge” (the border between the forestomach and the glandular stomach). Place cassettes in 10% formalin for an additional 24 hr at room temperature. 3.. 12. place cassettes in the tissue embedding center for tissue embedding. 10. Separate the sections with blunt forceps. Excise a strip (∼5 × 10–mm) from the glandular mucosa. 14. mount disposable stainless steel blades and set the section thickness to 4 μm. 6. Gastrointestinal Toxicology 21.g. the tissue samples are transferred through baths of progressively more concentrated ethanol and subsequently ethanol is removed by the clearing agent.

Scale bar = 250 μm.2. Gland cells are intact. Clear in xylene. Counterstain with eosin 24. gastric pits and glands are normal appearing. Two changes in 100% ethanol. Allow one section to adhere onto the surface of a slide directly out of the bath. Measure damage 29. 21. Keep slides on a slide warmer overnight at 37◦ C. Rehydrate the tissue sections. Add coverslip 27. Counterstain in eosin for 15 min. 17. two changes of 5 min each or alternatively overnight. Grade 0: surface. 3 min each c. Figure 21. 26. 3 min each. 18. There is a marked reduction of the height of the mucosa due to cell sloughing. Three changes in 96% ethanol. Methods to Measure Gastric Mucosal Lesions in the Rat 21.2 (appears on next page) Light micrographs of the fundic mucosa 1 hr after receiving absolute ethanol. Increase or decrease the suggested number of changes and their duration to obtain the removal of excess eosin. Deparaffinize in xylene. One change in 80% ethanol. c. Submucosal edema is prominent. 25. Stain with hematoxylin 19.2. 28. 1 ml/rat intragastrically. ensuring there are no bubbles. 23. by passing the slides through a series of decreasing concentrations of ethanol: a.6 Supplement 43 Current Protocols in Toxicology . 5 min each Two changes in 96% ethanol. Check under a microscope.g. Grade III: note the sloughing of surface cells with necrosis in the midportion of the mucosa corresponding to the parietal cell area (arrows). Three changes in 100% ethanol. Nikon Optiphot) for the morphometric analysis of gastric damage. Grade I: luminal surface mucous cells are damaged and partly exfoliated (arrows). Place a drop of Canada balsam on the slide. Rinse in distilled water. Display the image of each section on a color monitor using a video camera attached to the light microscope (e. The grades of damage used for quantitative analysis are shown. Stain in Mayer’s hematoxylin solution for 10 min. Submucosal edema is prominent. 22. Grade II: luminal surface and pit cells are damaged and exfoliated (arrow). corresponding to the chief cell area (arrows).16. 5 min Rinse in distilled water for 5 min. b. Dehydrate through a series of increasing concentrations of ethanol: a. 1 min b. Wash in running tap water for 10 min. d. 20. two changes of 10 min each. Grade IV: necrosis extends to the base of the mucosa. Allow sections to float in a bath containing distilled water at 45◦ C to allow full distension of the tissue. Insert the slides into a slide holder and then into the staining dish.. 5 min each One change in 80% ethanol. Gently cover all the tissue with a coverslip.

2.7 Current Protocols in Toxicology Supplement 43 .2 (legend appears on previous page) Gastrointestinal Toxicology 21.2.grade 0 250 ␮m grade I grade II grade III grade IV Figure 21.

these cells are also found to be actively migrating. a process known as restitution. The high magnification and the fine-structure resolution of scanning electron microscopy allow study of surface alterations at a cellular level. Materials Treated adult male rat with opened abdomen (see Basic Protocol 1) 10% (v/v) neutral buffered formalin (see recipe) 25%. Moreover. The surface epithelium can be examined by scanning electron microscopy. By migration. 90%. Grade II: extensive luminal surface cell damage plus damage to the cells lining the gastric pits. submucosal edema. Evaluate the severity of mucosal damage on the basis of its depth. LUCIA G. c. Grade III: in addition to surface and pit cell damage. determine the total length of mucosa examined and the length of mucosa with each grade of damage.30. 32. Fig. it represents a barrier separating and protecting epithelial cells from luminal contents. gastric pit cells are undamaged.2. 21. They constantly synthesize and secrete mucus.2. Grade 0: all gastric mucosal cells appear intact.. d. according to the following grading system: a. viable mucous cells can re-establish the epithelial integrity within minutes in areas exfoliated following the exposure to noxious agents.8 Supplement 43 Current Protocols in Toxicology . and 100% acetone Critical point dryer (Leica Microsystems) Aluminum stubs (Electron Microscopy Sciences) Double-sided adhesive tape Sputter coater (Leica Microsystems) Scanning electron microscope Methods to Measure Gastric Mucosal Lesions in the Rat 21. This kind of study has greatly enhanced the knowledge of the mechanisms concerning gastric mucosal damage.g. Grade IV: severe grade III damage extending into the lower portion of the gastric glands (chief cell area. When adherent. The length values of each grade of damage can also be expressed as a percentage of total length of mucosa examined. For each section. e. numerous exfoliated cells and whole layer of necrotic superficial epithelium are also present.2). 31. 50%. b. are central components of local defense mechanisms. For each rat. withstanding damage to the superficial portions of the mucosa. probably occurring frequently during food and drug ingestion. Nikon Laboratory Imaging). Perform quantitations using a color image analysis software system (e. termed surface mucous cells. Surface mucous cells have a short half-life—estimated at 3 to 5 days—and their rapid turnover is considered to be necessary to replace damaged cells lost by exfoliation and to re-establish epithelial continuity. cellular damage is evident in the upper portion of the gastric glands (parietal cell area). gastric gland cells are undamaged. ALTERNATE PROTOCOL 2 EVALUATION OF GASTRIC DAMAGE BY SCANNING ELECTRON MICROSCOPY Epithelial cells facing the gastric lumen. 33. which forms a gel layer adhering to the epithelium and is constantly degraded by gastric enzymes. 75%. Grade I: surface mucous cells on the luminal surface are damaged and partly exfoliated. 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.

2. Scale bar = 10 μm. (C) Grade II: normal epithelial cells cover <50% of the surface. Gastrointestinal Toxicology 21. 6. Lining cells can be seen to partially fill >50% of gastric pits. Epithelial cells can be seen at the mouth of the gastric pits. Rinse in distilled water. Lining cells can be seen to partially fill <50% of gastric pits. 20 min each. d.9 Current Protocols in Toxicology Supplement 43 . 2. Deep craters in completely denuded lamina propria are shown. 1 ml/rat intragastrically. Note that surface mucous cells are fully exfoliated. while the remaining mucosal surface is covered by cells. Excise two to three specimens (∼8 × 8–mm) from the glandular mucosa. A grade 0 B grade I C grade II D grade III Figure 21. Dry by placing the sample in the appropriate holder and then in the critical point drying apparatus following the manufacturer’s instructions. 10 min each two changes in 75% acetone. (B) Grade I: epithelial cells cover >50% of the surface. (D) Grade III: <50% of the surface is covered by normal epithelial cells. yielding a honeycomb appearance of the completely denuded lamina propria. c. Note that a portion of the lamina propria is exposed to the lumen and devoid of epithelial cells.Prepare gastric fundus sample 1. 20 min each two changes in 90% acetone. 5 mm below and parallel to the limiting ridge. 20 min each two changes in 100% acetone. The grades of damage used for quantitative analysis are shown. b. Fix with 10% (v/v) neutral buffered formalin for 2 hr at room temperature. 4. Dehydrate through a series of increasing concentrations of 10 to 15 ml acetone in glass beakers: a. 3. two changes in 25% acetone. 10 min each two changes in 50% acetone. e.3 Scanning electron micrographs of the fundic mucosa 1 hr after receiving absolute ethanol. Dissect the stomach from a treated male rat and open along the lesser curvature.2. 5. (A) Grade 0 = Normal epithelial cells cover >90% of the surface.

b. luminal surface upward. particularly those that are acidic. NSAIDs appear to primarily alter microcirculation.2. 13. Aspirin and indomethacin are the NSAIDS most commonly used to produce gastric damage in experimental conditions.6 N HCl (APPENDIX 2A) Aspirin (Sigma) Adult rats (male. body weight 200 to 220 g. Measure damage 10. while damage induced by indomethacin is largely mediated systematically. Grade III: <50% of the surface is covered by normal epithelial cells. Grade II: normal epithelial cells cover <50% of the surface.2.7. 21. Grade 0: normal epithelial cells cover >90% of the surface. Alternatively. according to the following grading system: a. 9 to 10 weeks old) Indomethacin (Sigma) Methods to Measure Gastric Mucosal Lesions in the Rat Orogastric tube Light microscope Scanning electron microscope 21. (1995). lining cells can be seen to partially fill >50% of gastric pits. based on that previously described by Kang et al. c. can directly damage the epithelial cells. 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. providing evidence that generation of erosions and ulcers can be a systemically mediated effect (Djahanguiri. Assign to each animal a score. such as aspirin. lining cells can be seen to partially fill <50% of gastric pits (Fig. Obtain a photograph and evaluate the severity of mucosal damage. view on the monitor and photograph. following parenteral administration. However. 8. Evaluate damage using a qualitative approach. Insert the sample attached to the stub into the scanning electron microscope. Coat with gold in the sputter coater following the manufacturer’s instructions. 2000. reducing gastric mucosal blood flow and promoting the adhesion of neutrophils to the vascular endothelium. Mount the sample. d. Grade I: normal epithelial cells cover >50% of the surface.3). based on the worst affected area. 9. Materials 1% (w/v) carboxymethylcellulose 0. These agents. 12.10 Supplement 43 Current Protocols in Toxicology . 11. Choose an area with maximum damage from each block of tissue. 2008). on a specimen aluminum stub (specimen holders) using a double-sided adhesive tape. 1969). Topical damage is identified as a prominent feature of aspirin toxicity. despite the evidence that almost all conventional NSAIDs are gastrotoxic in experimental animals. NSAIDs can produce erosions and ulcers in experimental animals. use a modified semiquantitative scoring system. Damage caused by NSAIDs largely differs in its macroscopic and morphological features from that caused by necrotizing agents. These alterations occur as a consequence of inhibition of prostaglandin synthesis by these drugs.

these areas are constituted by amorphous debris. which maintains aspirin in its readily absorbable non-ionized form. 2b.16 N HCl. When administered intragastrically.2. mainly neutrophils. Grade 0: the mucosa is normal appearing. Administer indomethacin at a dose of 20 mg/kg in a volume of 10 ml/kg to the rats via the orogastric tube. clear and cell-free. with pH ranging from 1. 3b. Prepare aspirin solution by suspending the drug in the vehicle to a final concentration of 12 mg/ml. Grade II: vasocongestion and interstitial edema. mainly neutrophils. b. Assign a rating according to their length in millimeters to lesions measuring >2 mm. Suspend indomethacin in 1% (w/v) carboxymethylcellulose to a final concentration of 2 mg/ml. Quantify macroscopically visible damage by NSAIDs using the following grading system: a. 5. Grade III: superficial erosions with the consequent discontinuity of surface epithelial layer. Gastrointestinal Toxicology 21. Sacrifice rat with 70% CO2 or by cervical dislocation 3 to 6 hr after dosing. 1969). and macrophages usually separates the necrotic area from the surrounding mucosal tissue. Measure damage 4. Prepare the vehicle by suspending 1% (w/v) carboxymethylcellulose in 0.3 to 1. 2a. Use the following scale to evaluate damage by light microscopy: a.Treat rats For aspirin treatment 1a. extravasation of red blood cells is rare.11 Current Protocols in Toxicology Supplement 43 . A barrier of leukocytes. Sum the length of the lesions and obtain an overall total. Grade I: vasocongestion not deeper than the pit region—microvessels are dilated and engorged and red blood cells and leukocytes visible inside. both being limited to the subepithelial region. For indomethacin treatment 1b. d. c. 3a. Assign a rating of 2 to lesions measuring 1 to 2 mm. damage is limited to surface and pit cells. Assign a rating of 1 to lesions measuring <1 mm. designated as the lesion index. maximal effect being achieved at 20 mg/kg (Djahanguiri. the interstitial spaces are expanded. by macrophages and by leukocytes. Sacrifice rat with 70% CO2 or by cervical dislocation 3 hr later. indomethacin damages gastric mucosa in a dosedependent way. c. b. Administer aspirin at a dose of 120 mg/kg in a volume of 10 ml/kg to the rats via the orogastric tube.5. d. e. for each stomach. Damage is minimally visible 1 hr after the administration and develops with time. Grade IV: focal necrosis extending into the chief cell area or up to the muscularis mucosae.

Stomachs were removed 6 hr after the administration of indomethacin. A conically shaped necrotic area deeply extending into the chief cell area (grade IV). (A) Macroscopic appearance of the gastric mucosa. 6.4. Determine the total length of the mucosal tissue examined by light microscopy and the length of each grade of damage for each stomach.A B 250 ␮m C Figure 21.2. (B) Light micrograph of the fundic mucosa. is shown.4 The prominent features of gastric damage induced by indomethacin. Edema is present in the submucosa.2.2. Indomethacin causes the formation of macroscopic damage. 7. Scale bar = 10 μm. typically observed at 6 hr after indomethacin administration. (C) Scanning electron micrograph of the fundic mucosa. Scale bar = 250 μm. Methods to Measure Gastric Mucosal Lesions in the Rat The prominent features of gastric damage induced by indomethacin are shown in Figure 21. Lesions are absent in the forestomach. 21. Use a qualitative approach to evaluate damage by scanning electron microscope.12 Supplement 43 Current Protocols in Toxicology . 20 mg/kg intragastrically. A crater can be seen. visible as hemorrhagic points or small lines. deeply penetrating into the mucosa.

. Neutral buffered formalin. 10% (v/v) 4 g sodium phosphate. the plasma concentrations of Current Protocols in Toxicology 21. dibasic 100 ml 37% formaldehyde 900 ml distilled water Mix to dissolve at room temperature. 1998. 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. 1994: Grønbech and Lacy. Gastrointestinal Toxicology Critical Parameters and Troubleshooting Influence of fasting Fasting reduces the density of antral gastrin (G)-cells. see SUPPLIERS APPENDIX. 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. Dissolve hematoxylin into the solution. corpus and antrum. Mayer’s hematoxylin 1 g hematoxylin 50 g aluminum potassium sulphate (alum) 0. 1995. and the activity of the histaminesynthesizing enzyme histamine decarboxylase (HDC). 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. and the glandular stomach. monobasic 6. Store up to 2 months at room temperature. and connective tissue elements. Filter before any use. filter before use using filtration paper and gravity filtration. Fasting conditions apparently activate protective mechanisms by reducing gastric acid output.5 ml glacial acetic acid Mix to dissolve at room temperature. For common stock solutions.5 g sodium phosphate. nerves. see APPENDIX 2A. Influence of aging Aging is associated with increased susceptibility to damage (Majumdar et al. It is critical that the fasting period remains unaltered during the study. gastrin.2 g sodium iodate 50 g chloral hydrate 1 g citric acid 1000 ml distilled water Dissolve alum in distilled water. Store up to 30 days at room temperature. COMMENTARY Background Information The rat stomach may be divided into two main regions: the forestomach. 2003). dissolving each one before adding the next. which consists of two parts. The antrum becomes continuous with the duodenum at the pyloroduodenal junction.13 Supplement 43 .. adjacent to the gastroesophageal junction. for suppliers. Bring the solution to a boil and allow to cool. Eosin 1 g eosin 100 ml distilled water 0. which contains blood and lymph vessels. which could potentially exacerbate gastric mucosal damage. leading to a decrease in histamine content and in gastric acid secretion (Ohning et al. Zhao et al.REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. In its turn. Add remaining chemicals. Store up to 15 days at room temperature. using a filtration paper and gravity filtration. 1989: Lee and Feldman..2. Between the glands there is the lamina propria. a nonglandular region lined by stratified squamous epithelium. The ulcerogenic effect could therefore be modified by alterations related to fasting/ feeding conditions. If necessary. The bottom layer of gastric mucosa consists of the muscularis mucosa.

and Chen. Tomikawa.. The values of total length of damaged mucosa were similar at any time interval. Paraffin blocks may be safely archived up to 1 year.4 ± 8. defensive mechanisms.. a parameter of macroscopically visible lesions. Takeuchi. In aging rats. and Feldman.. 1995. Grønbech.9 and 53.H. and Tarnawski. On the basis of the degree of damage. 92%. Extensive vasocongestion and edema accounted for ∼94% of damaged mucosa at 3 hr.K. Laine. Wee. 20 mg/kg. respec- Literature Cited Djahanguiri.E. The time needed for the semiquantitative measurement of damage require ∼1 to 2 hr per stomach from each rat. 1995. As a consequence. M. which requires 1 to 6 hr.Tarnawski et al.R.. Gastroenterol. When damage was assessed histologically at 5.1 ± 15. Time Considerations Macroscopic evaluation of gastric damage is performed immediately after the sacrifice of the animal. Jones.. A.. and ethanol produce a higher lesion index in old (24-month-old) than in young (3. B. the lesion index. The production of acute gastric ulceration by indomethacin in the rat. 1998). E. while deep hemorrhagic necrosis of mucosal tissue develops. Kang. two to three rats per day are treated. J.. J. 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. and NO synthase activity.. each treatment group is made up of six rats. prostaglandin generation. Biosci. 1980).1 ± 3. 1994. tively. M. Influence of nutrition Susceptibility to damaging agents is influenced by the diet.Y. 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. and in each treatment group. and 60 min after administration of absolute ethanol. embedding. respectively. 15.. 269:G737G744.2.14 Supplement 43 Current Protocols in Toxicology . and Tarnawski. Role of gastric blood flow in impaired defense and repair of aged rat stomachs. Age-related reductions in gastric mucosal prostaglandin levels Methods to Measure Gastric Mucosal Lesions in the Rat 21. 4:D303-D309. Following the intragastric administration of absolute ethanol for 60 min.or 4-month-old) rats. 1995b). such as mucosal blood flow. Correct orientation of histological sections Quality and reproducibility of data by light microscopy are largely affected by the quality of sectioning. 2006). K. sectioning of paraffin blocks. 1999. With time. Physiol. J. 93%. Effect of capsaicin and chilli on ethanol induced gastric mucosal injury in the rat. Anticipated Results The protocols detailed in this unit provide rather simple and highly reproducible methods to measure gastric damage in the rat. 2008. hypertonic saline. the choice of the age of rats is crucial. Teng. 30. 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. A maximum of ten to twelve rats is treated per day. 1969.8 (Morini et al. and staining require ∼2 to 3 days. usually 9 to 12 weeks old. Aspirin. Front. M. C.. Measurement of damage by light microscope takes ∼30 to 60 min per stomach from each rat. B. mucosal restitution. 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. fixation. Evaluation of damage by light microscopy requires ∼4 to 5 days. Gastrointestinal mucosal regeneration: Role of growth factors. vasocongestion and edema are no longer apparent. and 94% of the total mucosal length evaluated (Morini et al. was 87. Usually. F. and Lacy. Am. 4:265-267. Lee.S. A. Gastric mucosal defense and cytoprotection: Bench to bedside.. 1995a) at 3 and 6 hr following indomethacin administration. Scand. Mohajer. After treatment of rats. 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. have been found to be diminished with a parallel increase in hypoxia and expression of preapoptotic proteins in the gastric mucosa. damage respectively involved 96%. Following the intragastric administration of indomethacin. M. the lesion index was 22. L. Gastroenterology 135:41-60. A. 2007). J. Gut 36:664-669.. Most studies are performed in young rats. Due to the age-related responsiveness of the mucosa.3 at 3 and 6 hr.C.. 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. The procedure for obtaining scanning electron micrographs requires ∼1 to 2 days..

Toma.15 Current Protocols in Toxicology Supplement 43 . Cellular and molecular mechanisms of gastrointestinal ulcer healing. L. J. Gastroenterology 133:1938-1947.L. and proapoptotic factors.. Pharmacol.A.. 50:S24-S33..D. Sci.. Akahoshi.R. Arlow. A. Grandi.. Chen. C.. J. 2005. and Johnson. A.V. Wallace. Gentili...H. Moshier. G. Zhao.. H. A. Life Sci. Indomethacin-induced morphological changes in the rat gastric mucosa. R. Ohning.C.J.N. L. Tanigawa. Gastroenterology 107:1746-1750.S. G. E. 25:279-283. M. and Bertaccini.. Paula. J.. A... Morini. Sandor.. J.. D. L..C. G. Res.. Carneiro. G. J. Biochim. G. 1980. NSAIDs.L. Gastroenterol. Sci. 2006. J. D.. Br. Arcari.L. R. Rev. The gastroprotective effect of the essential oil of Croton cajucara is different in normal rats than in malnourished rats. Wallace. 2007. Sander. T. T. Biophys. Dudrick. Z. E. Grandi. (R)-alpha-methylhistamine inhibits ethanolinduced gastric lesions in the rat: Involvement of histamine H3 receptors? Digestion 56:145152.. 1995b.. S. Deng. 1989.P. 9:615-623. FASEB J. C. Gracioso. Song. with or without prior treatment with two proton pump inhibitors.. Pai. Regul. Yamada. Wong. and Granger. upregulation of multifunctional phosphatase PTEN. Wu.L. S. 96:310-315. X... 10:731-740. and Bertaccini. G. H. Liver Physiol. 2008. Dornonville de la Cour. Persson. and Szabo. Rapid onset of (R)alpha-methylhistamine protection in response to ethanol-induced histologic damage in rat gastric mucosa. Gastrointest. T. D. 2003. How do NSAIDs cause ulcer disease? Baillieres Best Pract.. The cellular and molecular basis of gastric mucosal defense.. 1998. 1995a. Dis. and Hakanson. F. 1998. M. D. Clin.M. and Bertaccini. Aging gastropathy-novel mechanisms: Hypoxia. Ther. 2000. 114:21-27.. Tarnawski. and gastric mucosal protection: Why doesn’t the stomach digest itself? Physiol. Grandi. Morini.. J. 275:G660G667. Majumdar. C.M. A.L. 1996.. Gastrointestinal Toxicology 21.. Immunolocalization of gastrin-dependent histidine decarboxylase activity in rat gastric mucosa during feeding. Dis. Aliment. G. Nutr.. W. Dig. S.. 62:PL13-PL18. Pept. 14:147-159. J.. Influence of method of feeding on stress ulcer development in the rat.. Biochemical changes in the gastric mucosa after injury in young and aged rats. Wallace. D..A. Lindstrom. Acta 992:35-40.. Tarnawski. Khomenko. A.D. Rat stomach ECL cells: Mode of activation of histidine decarboxylase. Prostaglandins. Morini. HirumaLima. G.R. Ahluwalia. 88:15471565. and Luk.V. and Walsh. Am.2..increase susceptibility to aspirin-induced injury in rats. Physiol. Dig. and Souza Brito. S.

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