8. PYLORUS LIGATION IN RATS
AIM
Pylorus is
ligated over a
certain period of
time
Accumulation
of gastric acid
causes
ulceration
PROCEDURE
Wistar rats weighing 150-200 grams
Fasting 48 hours ; water ad libitum
Under anesthesia, a one inch midline abdominal incision is given.
9. Pylorus is ligated without damaging its blood
supply
Stomach is replaced and abdominal wall closed
with sutures.
Test compounds are given either orally or injected
subcutaneously.
About 17-19 hours after pyloric ligation , rats are
sacrificed and stomach are dissected out.
Stomach is opened along the greater curvature ,
pinned on a cork plate
10. Its inner surface is examined for ulceration with a binocular
microscope
The ulcer index is calculated and the ulcer severity
graded.
EVALUATION
ULCER SEVERITY
0 = No ulcer
1 = Superficial ulcer
2 = Deep ulcer
3 = Perforation
11. Ulcer index (UI)
UI = (UN + US + UP) X 10-1
UN = Average number of ulcer per animal
US = Average of severity scores
Up = Percentage of animals with ulcers
Ulcer index of test drug compared with control group to detect anti-
ulcer effect of test drug.
12. STRESS ULCER MODEL
• Restraint- induced ulcers
AIM
To determine the significant role of stress in the pathogenesis
of gastric ulcers.
For restraint, the rats were placed in a piece of galvanized steel window screen
of appropriate size.
Screen was moulded around the animal and held in place with staples.
Drug is administered orally or subcutaneously.
30 minutes later animals are subjected to restraint.
Albino rats weighing 150-200 grams are taken.
Fasted for 36 hours before experiment.
PROCEDURE
13. Ulcer index and ulcer severity were determined.
Rats were then sacrificed & their stomachs dissected out.
Rats were kept under restraint for 24 hours.
To restrain the rats the limbs were put together in pair and tightened with
adhesive tape.
14. COLD WATER IMMERSION INDUCED ULCER
AIM
Exposure of cold conditions to restrained animals accelerates the
occurrence of gastric ulcers.
Wistar rats
weighing 150-
200 g are used
After fasting the
animals for 16
hours-test drug
given orally
Rats are then
placed
individually in
restraint cages
vertically
Immersed in
water up to the
xiphoid process, at
220 c for 1 hour
PROCEDURE
15. Rats are removed
from the cages, dried.
It is filled with formol
saline & kept
overnight .
On the next day , the
stomach is opened
along the greater
curvature and
examined for
ulcerative lesions.
Evan’s blue (30mg/kg)
injected i.v. via the tail
vein
10 minutes later,
they are sacrificed
The stomach is
removed & ligated
at both ends
16. HISTAMINE- INDUCED GASTRIC ULCER
AIM
Gastric acid secretion is increased when histamine is administered
intraperitoneally.
PROCEDURE
Guinea pig weighing 300-400g are taken
Fasted for 36 hours before experiment; water ad
libitum
1ml of histamine acid phosphate was administered
intraperitonially
Promethazine hydrochloride 5 mg was injected intraperitonially 15
minutes after histamine to protect the animals against histamine toxicity
17. The standard/test drugs were administered
subcutaneously 45 minutes before histamine
injection.
4 hours after histamine injection , guinea pigs were
sacrificed and stomach dissected out.
The gastric contents were subjected to analysis
Stomach was opened along the greater curvature,
ulcers were identified.
18. ULCER SCORING
Type 0 = no visible ulcers
Type 1 = 10 or less small ulcers,1-3 mm in diameter
Type 2 = 11 or more ulcers, 1-3 mm in diameter
Type 3 = 1 or more ulcers, 4-6 mm in diameter
Type 4 = 1 or more ulcers, 7 mm or more in
diameter
Type 5 = Perforation of the gastric wall
19. INDOMETHACIN-INDUCED GASTRIC LESIONS
AIM
NSAID induced gastric damage; by blocking COX enzyme,
endogenous prostaglandin production inhibited.
PROCEDURE
Rats fasted for 36 hours before indomethacin
administration (20 mg/kg ,orally)
30 min prior to the administration of the indomethacin,
standard/test drug is administered.
1 hour after indomethacin administration, rats are
scarified, their stomach dissected out and examined for
the number of lesions under the microscope.
21. CYSTEAMINE-INDUCED DUODENAL ULCER
AIM
Cysteamine can causes the inhibition of alkaline mucus
production, increased gastric acid secretion, delayed gastric
emptying.
PROCEDURE
Female sprague-dawley rats weighing 150-200g are
taken
Test drug and the standard drug are
administered 45 minutes prior to cysteamine
administration
23. • Perforating duodenal ulcers are produced.
• Located 2-4 mm from the pylorus, mainly on the anterior wall of
duodenum
• Necrotic material and acute inflammatory response is present at
ulcer crater.
24. IN VITRO METHODS
125I GASTRIN BINDING ASSAY
• Gastrin bind to CCK2 receptors on parietal cells---release HCl
• Bind 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 agents.
25. PROCEDURE
In presence of test compound (for competition assay)
In presence of unlabeled gastrin (for non specific binding)
In buffer alone (for total binding)
Incubated with 50µl [125I gastrin
Fundic gland suspension (guinea pig stomach)
26. Ice cold buffer, in micro centrifuge tubes, is layered with
incubated mixture.
For 90 minutes at 370 c
EVALUATION
• Total binding ,non specific binding and 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.
27. H+/K+ – ATPase inhibition assay
AIM
H+/K+ – ATPase or proton pump-- final step in the synthesis of
acid by parietal cells. To determine the inhibition of proton pump.
PROCEDURE
Homogenate of 80ng microsomal
gastric H+/K+ – ATPase.(pig gastric
mucosa)
Incubated with 100µl buffer,
ATP and test compound in
microtite plate for 30 minutes at
370c
28. Reaction is stopped by adding
malachite green (colorimetric
agents)
After 10 seconds, 15% sodium
citrate is added for 45minutes
Release of orthophosphate from
ATP quantified by colorimeter at
570 nm.
Percentage inhibition of H+/K+ is
calculated
Lesser the orthophosphate released,
more is the inhibition of H+/K+ –
ATPase by test compound.