3. INTRODUCTION
• Peptic ulcer is one of the most prevalent chronic gastrointestinal
disorder.
• It is a sore that develops on the lining of the stomach or small
intestine.
• More common in middle- age to older age
• Site : duodenum/ stomach, in a ratio of about 4:1
• Male/ Female ratio is 3:1
9. PYLORUS LIGATION IN RATS
• PRINCIPLE:
Pyloric ligation in rats leads to accumulation of gastric acid in the
stomach leading to acute gastric ulceration.
• REQUIREMENTS:
Albino Wistar Rats (150-200g)
Drugs: Ether (Anesthetic)
Saline (control)
Omeprazole (standard) / Test drug
11. PROCEDURE
Albino Wistar Rat (150-200gm) is taken
Rats are divided into two groups
Control Group Test Group
Both Groups being fasted for 24h before ligation
Vehicle is administered Test drug is administered
Once for 2days and 30minutes before ligation
12. Both group rats are anesthetized with ether
Pyloric ligation procedure done
19 hrs after pyloric ligation,
animals sacrificed by decapitation
Abdomen opened and stomach dissected out
Contents of the stomach collected
Stomach opened along greater curvature
Ulcers observed under 10x magnification
13. Cont…
• Ulcer severity is graded as:
0- normal stomach 1- superficial ulcder 2- deep ulcer 3- penetrated
or perforated ulcer
• The Ulcer Index (U1 )is calculated by the following equation:
U1 = UN +US +UP X 10-1
Where UN = Average no. of Ulcers/animal
US = Average of Severity scores
UP = Percentage of animals with ulcers
14.
15. Cont…
Contents of the stomach analyzed for :-
• Volume
• pH : pH of it is noted with pH meter
• Free acidity & total acidity
• Mucin & prostaglandin levels can be estimated to detect
cytoprotective effects.
INFERENCE:
• Ulcer index of test drug compared with control group to detect
anti- ulcer effect of test drug.
16. Cont.…
• Other parameters help to infer the mechanism of ulcer protection.
e.g. Decrease in vol of gastric acid: antisecretory action
Rise in pH : acid neutralising action
Increase in mucin, PGs : cytoprotective effect
17. NSAIDs INDUCED GASTRIC LESIONS
• Gastric ulceration is produced in rats by certain drugs i.e NSAIDs like
Aspirin, Indomethacin, Ibuprofen.
• The ability of the test drug to protect against the ulceration is observed.
• PRINCIPLE:-
Inhibition of endogenous prostaglandin production and consequent loss of
gastric mucosal defence.
• Important model for identifying drugs that could be effective in NSAID
induced gastropathy
18. Procedure
Wistar Rats (150-200g)
Rats are divided into two groups
Control group Test group
Both Groups being fasted for 24-36 h before ligation
Vehicle administered Test agent (1% carboxymethyl cellulose)
via gastric intubation.
19. Cont…
Administration of Ulcerogenic i.e. NSAIDs
intraperitoneally.
Placing the rats in cages and immersed in water upto the level
of xiphoid process at 23 C for 7h.
Animals are sacrificed
Stomach removed and evaluated for Ulcer Index
20. Stress Ulcer Models
• Stress plays a significant role in the pathogenesis of gastric ulcers in human
being.
• The involvement of psychogenic factors and the ease of production of
gastric ulcers using these models is a real advantage over the pyloric
ligation method of gastric ulcer production.
• 2 type of Stress Ulcer Model
Restraint Induced Ulcers
Cold Water Immersion-Induced Ulcers
21. Procedure Of Restraint induced ulcers
Albino Rat (either sex of 150-200g)
Animals are fasted for 36 h and divided into 2 groups:
Test Group Control Group
Test agent administered saline administered
Both limbs of the rats are tied in pair so that they cannot move
22. Cont…
Animals are subjected to restraint by molding
galvanized steel window around rats
Animal kept under restraint for 24h
Animals are sacrificed and stomach are
dissected out.
Stomach opened along greater curvature
Ulcer index, Ulcer severity & gastric content of the test group is determined
and compared with control group.
23. Cold Water Immersion Induced Ulcers
Wistar rats (150-200g)
Fasting the animal for 16h
Test compound is administered orally to test group
Saline is administered to Control group
Animals are placed individually in restraint
cages vertically and then immersed in water
at 22 C for 1h
Rats are removed from restraint
Evan’s blue injected IV via tail vein
v
24. Cont…
10min Later, Animals are sacrificed
Stomach is removed and filled with formol saline & kept overnight
Next day, stomach is opened along greater curvature, washed in warm water
examined for ulcerative lesions and compared with control group
26. [125I] Gastrin Binding Assay
• PRINCIPLE:
Gastrin (G cells of gastric antrum)
1. Binds to (Cholecystokinin) CCK2 receptors on parietal cells, release HCl
2. Binds to CCK2 receptors on ECL cells Histamine act on H2
receptors of parietal cells release HCl
• Compounds with gastrin receptor antagonistic activity can be potential
antiulcer agent
27. Procedure
• Fundic gland suspension is obtained from Guinea Pig stomach.
The gland suspension is incubated with
50µ𝑙 of [125I] Gastrin Unlabeled Gastrin In the presence of
in the presence of buffer for non specific test compound
(for total binding) binding
for 90 minutes at 37 C
• In microcentrifuge tube, ice cold buffer+ incubated mixture is taken and
centrifuged for 5min at 10,000g
• Radioactivity is quantified in pellet after discarding the supernatant.
28. Evaluation
• Total binding, non- specific binding & specific binding are determined
• Percentage of specifically bound [125I]Gastrin displaced by a given
concentration of the test compound calculated.
• The higher the displaced [125I] Gastrin, more is the gastrin antagonistic
activity of test compound.
29. H⁺ / K⁺- ATPase Inhibition Assay
• H⁺/K⁺ - ATPase or proton pump is the final step in the synthesis of acid by
parietal cells.
• Procedure:
Microsomal gastric H⁺/K⁺ - ATPase obtained from pig gastric mucosa
Incubated with buffer, ATP and Test compound
After 30min.of incubation, Add malachite green to stop the reaction
30. Cont…
After 10 seconds, Add 15% sodium citrate for 45min
Release of orthophosphate from ATP is quantified by calorimeter
EVALUATION:-
• Percentage inhibition of H⁺/K⁺ - ATPase is calculated
• Lesser the orthophosphate release, more is the inhibition of H⁺/K⁺ - ATPase
by test compound
31. REFERENCES
• K.D Tripathi, Drugs for Peptic Ulcer: Essentials of Medical
Pharmacology.2014; Eight edition: 647-649.
• SK Gupta et al, Screening Of Antiulcer Agents: Drug screening
methods.2016;3rd edition: 527-539.
• https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.resear
chgate.net%2Ffigure%2FThe-cold-restraint-stress-induced-acute-
gastric-injury-model-in-mice-A-A-
simple_fig9_300001236&psig=AOvVaw0qbWy3GY3fn69_5DS-
ddsH&ust=1703859165928000&source=images&cd=vfe&opi=899784
49&ved=0CBQQjhxqFwoTCJD0rPbMsoMDFQAAAAAdAAAAABAD