Anti-ulcer Activity Using Pylorus Ligation (Shay Rat) and Ethanol Models

1. Introduction

The preclinical evaluation of potential anti-ulcer agents represents a critical phase in the development of new therapeutics for gastroduodenal disorders. Among the various experimental models employed, the pylorus ligation model, commonly known as the Shay rat model, and the ethanol-induced gastric lesion model are considered fundamental and widely utilized. These models serve distinct but complementary purposes in elucidating the pathophysiology of ulcer formation and screening compounds for protective or curative effects. Their continued relevance lies in their ability to simulate specific aspects of human peptic ulcer disease, providing a bridge between basic pharmacological principles and clinical application.

The historical development of these models is noteworthy. The pylorus ligation technique was systematically described by Shay and colleagues in the mid-20th century, establishing a reproducible method for generating gastric hypersecretion and ulceration. Concurrently, the observation that concentrated ethanol reliably produces acute gastric mucosal injury led to the standardization of the ethanol model as a tool for assessing cytoprotective mechanisms. Together, these models have contributed substantially to the understanding of acid-peptic aggression and mucosal defense, underpinning the discovery and validation of major drug classes like proton pump inhibitors and mucosal protectants.

Mastery of these models is essential for students of medical and pharmaceutical sciences, as it provides foundational knowledge in experimental pharmacology, drug screening methodologies, and the integrative physiology of the gastrointestinal tract. The principles demonstrated by these assays extend beyond ulcer research, offering insights into general mechanisms of tissue injury, inflammation, and repair.

Learning Objectives

• Explain the pathophysiological basis and procedural methodology for the pylorus ligation (Shay rat) and ethanol-induced gastric ulcer models.
• Compare and contrast the primary mechanisms of ulcer induction, including hypersecretion, barrier disruption, and oxidative stress, inherent to each model.
• Identify the standard parameters measured to quantify ulcer severity and calculate the protective index of test compounds in both assays.
• Analyze how data from these models can predict potential mechanisms of action for anti-ulcer agents, such as antisecretory, antacid, cytoprotective, or antioxidant effects.
• Evaluate the clinical correlation of findings from these preclinical models with human peptic ulcer disease and current therapeutic strategies.

2. Fundamental Principles

The rational use of any experimental model requires an understanding of its underlying theoretical foundations. Both the pylorus ligation and ethanol models are predicated on disrupting the delicate balance between aggressive factors, which damage the gastroduodenal mucosa, and defensive factors, which maintain its integrity.

Core Concepts and Definitions

Peptic Ulcer: A breach in the mucosal lining of the stomach or duodenum, extending through the muscularis mucosae into the deeper layers. It results from an imbalance between luminal aggressive factors (acid, pepsin, Helicobacter pylori, NSAIDs) and mucosal defensive factors (mucus-bicarbonate barrier, mucosal blood flow, prostaglandins, cellular regeneration).

Pylorus Ligation (Shay Rat Model): An experimental surgical procedure involving the ligation of the pyloric end of the stomach in rats, preventing the emptying of gastric contents. This leads to the accumulation of acid and pepsin, inducing hypersecretory stress, mucosal ischemia, and autodigestion, culminating in glandular ulcer formation.

Ethanol-Induced Gastric Lesion Model: A non-surgical model where the oral administration of a high concentration of ethanol (typically >50% v/v) produces acute hemorrhagic and necrotic lesions in the glandular mucosa. The primary mechanism involves direct cytotoxic damage, disruption of the mucus layer, induction of oxidative stress, and impairment of mucosal microcirculation.

Ulcer Index (UI): A semi-quantitative or quantitative score assigned to grade the severity of gastric lesions. Scoring systems may consider the number, length, and width of lesions, or the percentage of ulcerated area.

Percentage Inhibition (Protective Effect): A key calculated parameter to evaluate drug efficacy. It is derived by comparing the ulcer index in a drug-treated group to that in a control (ulcerated, untreated) group using the formula: Percentage Inhibition = [(UI_control – UI_treated) ÷ UI_control] × 100.

Theoretical Foundations

The theoretical basis for these models rests on distinct pathways to mucosal injury. The Shay rat model primarily amplifies the aggressive factor of acid-pepsin secretion. Ligation creates a closed-loop system where feedback inhibition of gastrin and acid secretion is lost, leading to continuous secretion and luminal distension. Distension, in turn, compromises mucosal blood flow, creating a state of localized ischemia and reduced nutrient delivery, which sensitizes the mucosa to the damaging effects of the accumulated acid and pepsin.

In contrast, the ethanol model directly assaults defensive factors. Ethanol is a potent necrotizing agent that causes rapid disruption of the surface epithelial cells and the overlying mucus-bicarbonate barrier. It induces vascular injury, leading to increased vascular permeability, stasis, and hemorrhage. A significant component of its action is mediated through the generation of reactive oxygen species (ROS), lipid peroxidation of cell membranes, and depletion of endogenous antioxidants like glutathione and nitric oxide. Thus, while the Shay model is a model of hypersecretion, the ethanol model is a model of direct cytotoxicity and barrier breakdown.

3. Detailed Explanation

A thorough comprehension of the experimental execution, mechanistic pathways, and evaluation criteria is necessary for the critical appraisal of research utilizing these models.

Pylorus Ligation (Shay Rat) Model: Methodology and Mechanisms

The standard protocol employs fasted rats (typically 18-24 hours) to ensure an empty stomach and standardize basal secretion. Under appropriate anesthesia, a midline laparotomy is performed. The pylorus is identified and carefully ligated without occluding blood vessels. The abdominal wall is then sutured closed. Post-operatively, the animals are deprived of water and returned to their cages. After a predetermined period, usually 4 to 19 hours, the animals are euthanized, the stomachs are excised, and the gastric contents are collected for analysis.

Key Measurements:

• Gastric Volume: The total volume of collected gastric contents is measured, indicating secretory activity.
• Total Acidity: Determined by titrating an aliquot of gastric juice with a standard alkali (e.g., 0.01 N NaOH) to a pH endpoint, often using Topfer’s reagent and phenolphthalein. Expressed as mEq/L or total mEq/4h.
• Ulcer Scoring: The excised stomach is opened along the greater curvature, rinsed, and examined for lesions in the glandular portion. A common scoring system is: 0 = no lesions, 1 = superficial mucosal erosion, 2 = deep ulcer or hemorrhagic lesion, 3 = perforated or penetrating ulcer. The sum of scores per animal is the ulcer index.

The pathogenesis of ulcers in this model is multifactorial. The accumulation of hydrochloric acid and pepsin is the primary insult. Luminal distension increases intragastric pressure, which compresses mucosal and submucosal veins, leading to venous congestion, capillary stasis, and tissue hypoxia. This ischemic environment reduces the mucosal capacity to neutralize back-diffusing hydrogen ions (H⁺) and impairs the synthesis of protective prostaglandins and mucus. The combined effect of chemical aggression and compromised defense leads to focal necrosis and ulceration.

Ethanol-Induced Ulcer Model: Methodology and Mechanisms

This model is technically simpler and does not require surgery. Fasted rats are administered a single oral dose of absolute or high-concentration ethanol (e.g., 1 mL/200g body weight of 80-100% v/v ethanol). The test compound is usually administered prophylactically, 30-60 minutes prior to the ethanol challenge. After a short period, typically 1 hour, the animals are euthanized, and the stomachs are removed for examination.

Key Measurements: The primary endpoint is the assessment of gastric lesions. The glandular mucosa typically shows characteristic linear, hemorrhagic streaks. The ulcer index can be calculated based on lesion length (e.g., assigning a score per mm of lesion) or area (using planimetry or digital image analysis). Biochemical analyses of gastric tissue, such as glutathione (GSH) levels, malondialdehyde (MDA, a lipid peroxidation marker), and myeloperoxidase (MPO, a neutrophil infiltration marker), are frequently conducted to elucidate the mechanism of protection.

The mechanism of ethanol injury is rapid and direct. Concentrated ethanol causes immediate dehydration and precipitation of surface epithelial cell proteins, disrupting the cellular membrane. It solubilizes the mucus layer, stripping the mucosa of its first line of defense. This allows back-diffusion of acid and other luminal irritants. Ethanol also induces a robust inflammatory response, with increased production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and recruitment of neutrophils. The recruited neutrophils release ROS and proteolytic enzymes, exacerbating tissue damage. Furthermore, ethanol reduces the synthesis of protective prostaglandins (PGE₂) and nitric oxide (NO), both critical for maintaining mucosal blood flow and integrity.

Factors Affecting Model Outcomes

The reproducibility and interpretation of data from these models are influenced by numerous variables.

4. Clinical Significance

The translational value of preclinical models lies in their ability to predict therapeutic efficacy and mechanism in human disease. Both the Shay and ethanol models have played instrumental roles in the development and understanding of major anti-ulcer drug classes.

Relevance to Drug Therapy and Mechanism Elucidation

The pylorus ligation model is highly predictive for agents that work primarily by reducing gastric acid secretion. A compound that significantly decreases gastric volume and total acidity in this model, while reducing ulcer index, likely possesses antisecretory activity. This model was crucial in characterizing histamine H₂-receptor antagonists (e.g., cimetidine, ranitidine) and proton pump inhibitors (e.g., omeprazole). These drugs show a dose-dependent reduction in both secretory parameters and ulcer formation in the Shay rat. Furthermore, the model can identify antacid agents, which would neutralize existing acid but may not affect volume, and anticholinergic agents, which inhibit neural stimulation of secretion.

The ethanol-induced ulcer model, conversely, is the benchmark for identifying so-called “cytoprotective” agents—those that protect the mucosa from injury without necessarily reducing acid secretion. The prototypical cytoprotective agent is prostaglandin analogs like misoprostol. These agents enhance mucosal defense by stimulating mucus and bicarbonate secretion, increasing mucosal blood flow, and promoting epithelial cell renewal. A drug that potently inhibits ethanol-induced lesions but shows weak effects in the Shay model likely acts via cytoprotective, antioxidant, or anti-inflammatory pathways. This model is also sensitive to antioxidants (e.g., flavonoids, sulforaphane) and agents that enhance endogenous prostaglandin synthesis.

Practical Applications in Drug Discovery

In a standard screening cascade for a new anti-ulcer compound, these models are employed sequentially to build a pharmacological profile. Initial screening might use the rapid and inexpensive ethanol model to identify any protective activity. Active compounds are then progressed to the pylorus ligation model to determine if the protection is mediated through antisecretory mechanisms. This two-model approach allows for the preliminary classification of a compound’s primary mode of action. Additional models, such as indomethacin-induced or stress-induced ulcers, are then used to further refine the mechanistic understanding and predict efficacy against NSAID-associated or stress-related ulcers, respectively.

The biochemical parameters measured alongside ulcer indices, particularly in the ethanol model, provide direct evidence for the mechanism. A test compound that normalizes depleted glutathione levels and reduces elevated malondialdehyde strongly suggests an antioxidant mechanism. A reduction in myeloperoxidase activity indicates an anti-inflammatory effect via inhibition of neutrophil infiltration.

5. Clinical Applications and Examples

The following scenarios illustrate how data from these models inform the understanding and application of anti-ulcer therapies.

Case Scenario 1: Evaluation of a Novel Synthetic Compound

A pharmaceutical research team synthesizes a new chemical entity (NCE) believed to have anti-ulcer potential based on its structural similarity to known H₂-antagonists. The team first evaluates the NCE in the ethanol-induced ulcer model at three doses (25, 50, 100 mg/kg). The results show a modest, dose-dependent reduction in ulcer index (15%, 35%, and 50% inhibition, respectively). Biochemical analysis reveals a slight increase in gastric glutathione levels.

The compound is then tested in the pylorus ligation model at the same doses. Here, it produces a potent, dose-dependent inhibition of both gastric volume (30%, 55%, 75% reduction) and total acidity (25%, 60%, 80% reduction), with a corresponding high degree of ulcer protection (70%, 85%, 95% inhibition).

Interpretation: The strong activity in the Shay model, coupled with significant reductions in secretory parameters, confirms the hypothesized antisecretory action, likely via H₂-receptor blockade or a similar mechanism. The weaker but present activity in the ethanol model suggests the compound may also possess secondary antioxidant or mild cytoprotective properties, possibly unrelated to its primary antisecretory effect. This profile would justify further investigation as a potential acid-suppressive drug.

Case Scenario 2: Investigation of a Herbal Extract

A research group investigates an aqueous extract of a traditionally used medicinal plant for gastroprotective effects. In the pylorus ligation model, the extract (500 mg/kg) shows no significant effect on gastric volume or acidity but still produces a 40% reduction in ulcer index. In the ethanol model, the same dose demonstrates a very strong protective effect (85% inhibition). Biochemical analysis shows the extract markedly elevates gastric PGE₂ levels, normalizes glutathione, and reduces lipid peroxidation and MPO activity.

Interpretation: The lack of antisecretory activity in the Shay model rules out a primary acid-reducing mechanism. The potent activity in the ethanol model, supported by biochemical data, indicates a multifaceted cytoprotective mechanism. The proposed mechanisms include: 1) Stimulation of endogenous prostaglandins (increased PGE₂), enhancing mucus and bicarbonate secretion and blood flow. 2) Antioxidant activity (normalized GSH, reduced MDA), scavenging free radicals. 3) Anti-inflammatory activity (reduced MPO), limiting neutrophil-mediated damage. This profile is characteristic of a mucosal defense enhancer, similar to prostaglandin analogs or certain flavonoids, and would be particularly relevant for ulcers where acid suppression is not the primary goal, such as NSAID-induced gastropathy.

Application to Specific Drug Classes

6. Summary and Key Points

The pylorus ligation and ethanol-induced gastric ulcer models are cornerstone techniques in experimental gastroenterology and anti-ulcer drug discovery. Their complementary nature allows for a comprehensive initial pharmacological profiling of test substances.

Summary of Main Concepts

• The pylorus ligation (Shay rat) model induces ulcers primarily through hypersecretion and accumulation of acid-pepsin, coupled with mucosal ischemia from luminal distension. It is the model of choice for identifying antisecretory agents like PPIs and H₂-antagonists.
• The ethanol-induced ulcer model causes damage via direct cytotoxicity, barrier disruption, oxidative stress, and inflammation. It is the premier model for detecting cytoprotective, antioxidant, and anti-inflammatory agents.
• Standard evaluation parameters include the Ulcer Index (UI) for both models, with additional measurement of gastric volume and total acidity in the Shay model, and often biochemical markers (GSH, MDA, MPO) in the ethanol model.
• Drug efficacy is quantitatively expressed as Percentage Inhibition, calculated from the ulcer indices of control and treated groups.
• Interpretation of results requires careful consideration of confounding factors such as animal strain, fasting state, dose, and timing of drug administration.

Clinical and Experimental Pearls

• A compound showing strong activity in both models may possess a dual mechanism (e.g., antisecretory plus antioxidant), which could be advantageous for broad-spectrum ulcer therapy.
• The ethanol model’s sensitivity to prostaglandin-based cytoprotection makes it an essential tool for screening agents intended to prevent NSAID-induced gastropathy, a condition where acid suppression alone is often insufficient.
• While these acute models are invaluable for screening, they do not replicate chronic ulcer conditions or the role of Helicobacter pylori. A complete preclinical package requires additional chronic and infection-based models.
• Understanding the mechanistic basis of these models allows for the rational design of combination therapies that simultaneously target aggressive factors and bolster mucosal defense, mirroring the multifaceted approach often required in clinical management.

In conclusion, mastery of the principles, execution, and interpretation of the pylorus ligation and ethanol-induced ulcer models provides a fundamental framework for understanding the pathophysiology of gastric mucosal injury and the preclinical assessment of therapeutic interventions. This knowledge forms an essential component of the education of future pharmacologists, pharmaceutical scientists, and clinicians engaged in gastroenterological therapeutics.

References

1. Rang HP, Ritter JM, Flower RJ, Henderson G. Rang & Dale's Pharmacology. 9th ed. Edinburgh: Elsevier; 2020.
2. Whalen K, Finkel R, Panavelil TA. Lippincott Illustrated Reviews: Pharmacology. 7th ed. Philadelphia: Wolters Kluwer; 2019.
3. Katzung BG, Vanderah TW. Basic & Clinical Pharmacology. 15th ed. New York: McGraw-Hill Education; 2021.
4. Trevor AJ, Katzung BG, Kruidering-Hall M. Katzung & Trevor's Pharmacology: Examination & Board Review. 13th ed. New York: McGraw-Hill Education; 2022.
5. Golan DE, Armstrong EJ, Armstrong AW. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. 4th ed. Philadelphia: Wolters Kluwer; 2017.
6. Brunton LL, Hilal-Dandan R, Knollmann BC. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 14th ed. New York: McGraw-Hill Education; 2023.