Experiment no 1

To study anti-ulcer activity of a drug by using–
Pylorus ligand (SHAY) rat model
NSAIDS induced ulcer model.
Experiment No. 1
Pharmacology-III Practical
B.Pharm Semester-VI
Dr. Sameer H. Sawant,
Assistant Professor Pharmacology,
Dr. Sameer. H. Sawant, SIOP

References:
1.S.K. Kulkarni, Handbook of experimental pharmacology,
2009, Vallabh Prakashan, Delhi, 3rd Edition, Pg. no. 148-150.
2. W.H. Vogel, Drug Discovery and Evaluation, 2002,
Springer, Berlin, 2nd Edition, Pg. no. 867 – 870.

Requirements:
1. Animals – Female Wistar rats weighing 150-170 g
2. Drugs – Anesthetic ether, ranitidine, normal saline.
3. Reagents – 0.1 N NaOH, phenolphthalein, Topfer’s reagent
4. Instruments – Stereomicroscope, burette, pH meter, dissecting instruments

Theory:
• Peptic ulcer : Most prevalent gastrointestinal disorders
• Peptic ulcers are open sores that develop on the inside lining of your
stomach and the upper portion of your small intestine.
• The most common symptom of a peptic ulcer is stomach pain.
• Peptic ulcers include:
 Gastric ulcers that occur on the inside of the stomach
 Duodenal ulcers that occur on the inside of the upper portion of
your small intestine (duodenum)

Causes of Peptic Ulcer
• The most common causes of peptic ulcers are infection with the
bacterium Helicobacter pylori (H. pylori)
• Long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs)
such as ibuprofen and naproxen sodium
• Stress and spicy foods do not cause peptic ulcers
• They can make your symptoms worse

Pylorus ligand (SHAY) rat model
• A simple and reliable method for production of gastric ulceration in
the rat
• based on ligature of the pylorus has been published by Shay et al.
(1945).
• The ulceration is caused by accumulation of acidic gastric juice in the
stomach.

Procedure
• Female Wistar rats weighing 150–170 g are starved for
48 h having access to drinking water ad libitum
• During this time they are housed single in cages with
raised bottoms of wide wire mesh in order to avoid
cannibalism and coprophagy.
• Ten animals are used per dose and as controls
• Under ether anesthesia a midline abdominal incision
is made

Cont.
• The pylorus is ligated
• Care being exercised that neither damage to the blood
supply nor traction on the pylorus occurs
• Grasping the stomach with instruments is to be
meticulously avoided
• Ulceration will invariably develop at such points
• The abdominal wall is closed by sutures

• Standard drug ranitidine (20mg/kg, p.o.), test compounds & saline to
control group is given either orally by gavage or injected
subcutaneously.
• The animals are placed for 19 h in plastic cylinders with an inner
diameter of 45 mm being closed on both ends by wire mesh.
• Afterwards, the animals are sacrificed in CO2 anesthesia.
• The abdomen is opened and a ligature is placed around the esophagus
close to the diaphragm.
• The stomach is removed, and the contents are drained in a centrifuge
tube.
• Along the greater curvature the stomach is opened and pinned on a
cork plate.

Cont.
• The mucosa is examined with a stereomicroscope
• In the rat, the upper two fifths of the stomach form the rumen with
squamous epithelium and possess little protective mechanisms against
the corrosive action of gastric juice.
• Below a limiting ridge, in the glandular portion of the stomach, the
protective mechanisms are better in the mucosa of the medium two
fifths of the stomach than in the lowest part, forming the antrum.
• Therefore, lesions occur mainly in the rumen and in the antrum.
• The number of ulcers is noted and the severity recorded with the
following scores:-

 0 = no ulcer
 1 = superficial ulcers
 2 = deep ulcers
 3 = perforation.
• The volume of the gastric content is measured.
• After centrifugation, acidity is determined by titration with 0.1 n NaOH.
• Ulcer index and acidity of the gastric content of treated animals are compared
with controls.
• Using various doses, dose-response curves can be established for ulcer formation
and gastric acid secretion.

B. Indomethacin (NSAIDS) induced ulcer model.
• Principle:
• Nonsteroidal anti-inflammatory drugs (NSAIDS),
• like indomethacin and acetyl-salicylic acid, induce gastric lesions in
man and in experimental animals by inhibition of gastric cyclo-
oxygenase resulting in less formation of prostacyclin
• The predominant prostanoid produced in the gastric mucosa

PROCEDURE
1. Groups of 8–10 Wistar rats weighing 150–200 g are used.
2. The test drugs are administered orally in 0.1% Tween 80 solution 10 min
prior to oral indomethacin in a dose of 20 mg/kg (4 mg/ml dissolved in
0.1% Tween 80 solution).
3. Six hours later, the rats are sacrificed in CO2 anesthesia and their
stomachs removed.
4. Formol-saline (2% v/v) is then injected into the totally ligated stomachs
for storage overnight.

5. The next day, the stomachs are opened along the greater curvature,
then washed in warm water, and examined under a 3-fold magnifier.
6. The lengths of the longest diameters of the lesions are measured and
summated to give a total lesion score (in mm) for each animal, the
mean count for each group being calculated.
7. The mean score in control rats is about 25 (range 20–28). Inhibition
of the lesion production is expressed as percentage value

Observation table
Model A - Pylorus ligand (SHAY) rat model
Group Volume of gastric
contents (ml)
pH Ulcer Index
Control (Saline)
Standard (Ranitidine,
20 mg/kg p.o.)
Test

Calculations: ulcer index UI :
An ulcer index UI is calculated:
UI = UN + US+ UP x 10-1
UN= average of number of ulcers per animal
US = average of severity score
UP = percentage of animals with ulcers
Acidity = vol.of NaOH x normality x 100 / 0.1 mEq/l/100g

Model B - Indomethacin (NSAIDS) induced ulcer model
Group Volume of
gastric
contents (ml)
pH Ulcer Score
(mm)
Control (Saline)
Ulcer Control (Indomethacin,
20 mg/kg p.o.)
Test (Indomethacin + test
drug)

Conclusion:
1.Comparison of ulcer index between study groups estimates
the potency of antiulcer activity of test drug.
2.Decrease in volume of gastric contents, free and total
acidity determines anti-secretory activity of test drug.
3.Rise in pH evaluates acid neutralizing action of the drug.

Experiment no 1

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