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Methods to Measure Gastric Mucosal Lesions in the Rat

Giuseppina Morini1,  Daniela Grandi1

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

Publication Name: 
Current Protocols in Toxicology
Unit Number: 
UNIT 21.2
DOI: 
10.1002/0471140856.tx2102s43
Online Posting Date: 
February, 2010
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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. © 2010 by John Wiley & Sons, Inc.

Keywords: rat; gastric mucosal lesions; macroscopic evaluation; light microscopy; scanning electron microscopy

     
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessing Acute Gastric Lesions Induced by Necrotizing Agents
  • Basic Protocol 2: Macroscopic Evaluation of Gastric Damage
  • Alternate Protocol 1: Evaluation of Gastric Damage by Light Microscopy
  • Alternate Protocol 2: Evaluation of Gastric Damage by Scanning Electron Microscopy
  • Basic Protocol 3: Evaluating Acute Gastric Lesions Induced by Conventional NSAIDs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Topics
    • Toxicology
    • Laboratory Organisms and Animal Models
     
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Materials

Basic Protocol 1: Assessing Acute Gastric Lesions Induced by Necrotizing Agents

 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

Basic Protocol 2: Macroscopic Evaluation of Gastric Damage

 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

Alternate Protocol 1: Evaluation of Gastric Damage by Light Microscopy

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

Alternate Protocol 2: Evaluation of Gastric Damage by Scanning Electron Microscopy

 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

Basic Protocol 3: Evaluating Acute Gastric Lesions Induced by Conventional NSAIDs

 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)
  • Orogastric tube
  • Light microscope
  • Scanning electron microscope
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Figures

  • 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.

    View Image
  • Figure 21.2.2
    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 µm.

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

    View Image
  • 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 µm. (C) Scanning electron micrograph of the fundic mucosa. A crater can be seen, deeply penetrating into the mucosa. Scale bar = 10 µm.

    View Image

Literature Cited

Literature Cited
    Djahanguiri, B. 1969. The production of acute gastric ulceration by indomethacin in the rat. Scand. J. Gastroenterol. 4:265-267.
    Grønbech, J.E. and Lacy, E.R. 1995. Role of gastric blood flow in impaired defense and repair of aged rat stomachs. Am. J. Physiol. 269:G737-G744.
    Jones, M.K., Tomikawa, M., Mohajer, B., and Tarnawski, A.S. 1999. Gastrointestinal mucosal regeneration: Role of growth factors. Front. Biosci. 4:D303-D309.
    Kang, J.Y., Teng, C.H., Wee, A., and Chen, F.C. 1995. Effect of capsaicin and chilli on ethanol induced gastric mucosal injury in the rat. Gut 36:664-669.
    Laine, L., Takeuchi, K., and Tarnawski, A. 2008. Gastric mucosal defense and cytoprotection: Bench to bedside. Gastroenterology 135:41-60.
    Lee, M. and Feldman, M. 1994. Age-related reductions in gastric mucosal prostaglandin levels increase susceptibility to aspirin-induced injury in rats. Gastroenterology 107:1746-1750.
    Majumdar, A.P., Moshier, J.A., Arlow, F.L., and Luk, G.D. 1989. Biochemical changes in the gastric mucosa after injury in young and aged rats. Biochim. Biophys. Acta 992:35-40.
    Morini, G., Grandi, D., Arcari, M.L., and Bertaccini, G. 1995a. Indomethacin-induced morphological changes in the rat gastric mucosa, with or without prior treatment with two proton pump inhibitors. Aliment. Pharmacol. Ther. 9:615-623.
    Morini, G., Grandi, D., and Bertaccini, G. 1995b. (R)-alpha-methylhistamine inhibits ethanol-induced gastric lesions in the rat: Involvement of histamine H3 receptors Digestion 56:145-152.
    Morini, G., Grandi, D., Gentili, S., and Bertaccini, G. 1998. Rapid onset of (R)-alpha-methylhistamine protection in response to ethanol-induced histologic damage in rat gastric mucosa. Life Sci. 62:PL13-PL18.
    Ohning, G.V., Song, M., Wong, H.C., Wu, S.V., and Walsh, J.H. 1998. Immunolocalization of gastrin-dependent histidine decarboxylase activity in rat gastric mucosa during feeding. Am. J. Physiol. Gastrointest. Liver Physiol. 275:G660-G667.
    Paula, A.C., Toma, W., Gracioso, J.S., Hiruma-Lima, C.A., Carneiro, E.M., and Souza Brito, A.R. 2006. The gastroprotective effect of the essential oil of Croton cajucara is different in normal rats than in malnourished rats. Br. J. Nutr. 96:310-315.
    Sander, L.D., Dudrick, S.J., and Johnson, L.R. 1980. Influence of method of feeding on stress ulcer development in the rat. Dig. Dis. Sci. 25:279-283.
    Tarnawski, A. 2005. Cellular and molecular mechanisms of gastrointestinal ulcer healing. Dig. Dis. Sci. 50:S24-S33.
    Tarnawski, A., Pai, R., Deng, X., Ahluwalia, A., Khomenko, T., Tanigawa, T., Akahoshi, T., Sandor, Z., and Szabo, S. 2007. Aging gastropathy-novel mechanisms: Hypoxia, up-regulation of multifunctional phosphatase PTEN, and proapoptotic factors. Gastroenterology 133:1938-1947.
    Wallace, J.L. 2000. How do NSAIDs cause ulcer disease Baillieres Best Pract. Res. Clin. Gastroenterol. 14:147-159.
    Wallace, J.L. 2008. Prostaglandins, NSAIDs, and gastric mucosal protection: Why doesn't the stomach digest itself Physiol. Rev. 88:1547-1565.
    Wallace, J.L. and Granger, D.N. 1996. The cellular and molecular basis of gastric mucosal defense. FASEB J. 10:731-740.
    Zhao, C.M., Chen, D., Yamada, H., Dornonville de la Cour, C., Lindstrom, E., Persson, L., and Hakanson, R. 2003. Rat stomach ECL cells: Mode of activation of histidine decarboxylase. Regul. Pept. 114:21-27.
     
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