2. Peptic ulcer is a break in the inner lining of the stomach, first part of the small
intestine or sometimes the lower esophagus. An ulcer in the stomach is known as
a gastric ulcer while that in the first part of the intestines is known as a duodenal ulcer.
Causes:
Acid, pepsin, bile
Helicobacter pylori
NSAIDs & other drugs
Smoking, Alcohol, stress
Free radicals
Oily, spicy, irregular dietary habit
Hereditary factors
2Peptic ulcer
4. Gastrin Binding Assay
Principle:
Major stimuli for gastric acid secretion.
Gastrin ( G cells of gastric antrum)
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
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5. Procedure:
Fundic gland suspension (Guinea pig stomach)
Incubated with 50μl [125I] Gastrin -->
1) In buffer alone (for total binding)
2)In presence of unlabeled gastrin (for non-specific binding)
3) In presence of test compound (for competition assay)
For 90 minutes at 37°C
Ice cold buffer, in Microcentrifuge tubes, is layered with incubated mixture.
Centrifuged for 5 minutes at 10,000 g
Radioactivity is quantified in pellet after discarding the supernatant.
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6. Evaluation:
Total binding, non-specific binding and specific binding are determined.
Percentage of specifically bound Gastrin displaced by a given concentration of
the test compound calculated.
The higher the displaced Gastrin , more is the gastrin antagonistic activity of test
compound.
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7. H+ / K+-ATPase Inhibition Assay
H+/K+ - ATPase or proton pump --> final step in the synthesis of acid by parietal
cells
Procedure:
Homogenate of 80 ng Microsomal gastric H+/K+ - ATPase (pig gastric
mucosa) incubated with 100μl buffer, 1mM ATP and Test compound in
microtiter plate for 30 mins at 37°
Reaction is stopped by adding Malachite green (colorimetric agent)
After 10 seconds, 15% sodium citrate is added for 45 minutes
Release of orthophosphate from ATP quantified by colorimeter at 570 nm
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8. Evaluation:
Percentage inhibition of H+/K+ - ATPase is calculated.
Lesser the orthophosphate released, more is the
inhibition of H+/K+ - ATPase by test compound.
%Inhibition =
8
[activity (control)- activity(test)]
Activity (control)
* 100
10. Pylorus Ligation in Rats
This procedure was described by Shay et al.
Principle:
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.
Housed singly in cages with raised bottoms of wide wire mesh to avoid
coprophagy.
Under anaesthesia, a one-inch midline abdominal incision is given below
the xiphoid process.
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11. Under anaesthesia, a one-inch midline abdominal incision is given
below the xiphoid process.
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 s.c.
About 17-19 hours after pyloric ligation, rats are
sacrificed and stomachs are dissected out.
Contents of the stomach are drained into a graduated centrifuge tube
and acidity determined by titration with 0.1 N NaOH.
Stomach is opened along the greater curvature, pinned on a cork
plate.
Its inner surface is examined for ulceration with a binocular
microscope.
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13. The Ulcer Index is calculated and the Ulcer Severity graded.
Ulcer severity is graded as:
0 - No ulcer
1 - Superficial ulcer
2 - Deep ulcer
3 - Perforation
Ulcer index:
UI = UN+ US + UP * 10-1
Where, UN = Avg. of number of animals
US = Avg. of severity scores
UP = Percentage of animals with ulcer
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14. Stress Ulcer Model
Selye (1936) , for the first time , described the use of
restraints for production of Gastric ulcers
Uses:
Technically simple
Do not require anesthesia or surgery
Lesions located in glandular region of stomach
As psychogenic factors are involved in the
pathogenesis of gastric ulcers, psychotropic drugs
could be evaluated.
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15. Restraint- induced ulcers
Principle:
Stress plays a significant role in the pathogenesis
of gastric ulcers.
Procedure:
Albino rats weighing 150-200 g are used
Fasted for 36 hours before experiment
Drug was administered orally or subcutaneously
30 min later animals were subjected to restraint
A special galvanized steel window screen molded
around the animal
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16. The limbs of the animal are tied ,so that it cannot move
Rats were kept under restraint for 24 hours
Animals are sacrificed and their stomach is dissected& opened along greater
curvature. UI & ulcer severity are determined
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17. Cold water immersion induced ulcers
Principle:
Exposure of cold conditions to restrained animals accelerates the occurrence of
gastric ulcers.
Procedure:
Wistar rats weighing 150-200 grams are used.
After fasting the animals for 16 hours, the test compound is administered orally
Rats are then placed individually in restraint cages vertically, and then
immersed in water upto abdominal, at 22°C for 1 hour.
Then rats are removed from the cages, dried
Evan’s blue (30mg/kg) injected i.v. via the tail vein
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18. 10 min later, they are sacrificed
The stomach is removed & ligated at both
ends
It is filled with Formol saline & kept
overnight
On the next day, the stomach is opened
along the greater curvature, washed in
warm water and examined for ulcerative
lesions
Evan’s blue helps in evaluation of lesion
score.
Calculation: Adding the lengths of the
longest diameter of the lesions
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19. Swimming stress ulcer
Procedure :
Albino rats ; either sex
Fasted for 24 hrs; free access to water
Rats forced to swim in deep concrete
tube filled with water at 230C for 5 hrs
Animals removed and Sacrificed &
stomach removed ; opened along
greater curvature
Severity grading done ; Ulcer Index
calculated
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20. Histamine induced gastric stress
Principle:
Gastric acid secretion is increased when histamine is administered
intraperitoneally
Procedure:
Guinea pig weighing 300-400 grams are taken
Fasted for 36 hours before experiment; water ad libitum
Histamine acid sulphate (50 mg ) was administered i.p.
Promethazine hydrochloride 5 mg is injected i.p. 15 min before and 15 min
after histamine to protect the animals against histamine toxicity.
The test drugs is administered before 30-45 mins before the histamine
injection.
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21. Four hours after histamine injection, guinea pigs are sacrificed and
stomach dissected out.
The animals are sacrificed and their stomachs dissected out.
The degree of ulceration grading is done as follows
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 bigger ulcers, 7 mm or more in diameter
Type 5 : Gastric or esophageal wall perforation
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22. Ethanol-Induced Mucosal Damage
Principle:
Using transmission densitometer it is possible to quantify the extent of gastric lesion
induced by ethanol, by measuring the optical density of photographic negatives of
gastric mucosa.
Procedure:
Wistar rats weighing 150-200 grams are taken
Fasted for 18 hours before experiment; water ad libitum.
Rats are given test drugs or standard drug orally.
30 mins later 1 ml/200gm of 99.80% alcohol is administered orally.
After 1 hour, Rats are sacrificed and stomachs dissected out.
Severity score and ulcer index are calculated
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23. Using a transmission densitometer,the photo graph of the tissue were taken.
Damaged areas have lower optical density values.
EVALUATION:
The significance of differences in optical density between control and
ethanol-treated tissue is evaluated.
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24. 2424Acetic acid induced gastric ulcers
Principle:
Acetic acid enhances the ulceration in stomach by increasing the acidity of
stomach contents.
This model produces chronic ulcers that resembles human ulcers and it is used to
screen drugs for gastroprotective effect.
Procedure:
Albino rats used
0.05 ml of Acetic acid (1-30%) injected in the submucosal layer of the stomach
Penetrating peptic ulcers : adhered to Liver
Chronic ulcers with repeated healing and re-aggravation.
Effect of test drug given twice daily for 10-15 days is noted.
25. Indomethacin -induced chronic ulcers
Principle:
NSAID induced gastric damage ; by blocking COX enzyme, cytoprotective
prostaglandin production inhibited.
Procedure:
Oral administration of Indomethacin (25 mg/kg) after 24h fasting male albino rats.
Indomethacin is dissolved in sodium bicarbonate to form a clear solution.
Gastric ulcer can be examined after 4h and opening the stomach along the greater
curvature & washed in normal saline.
Wounds are located in the glandular region under a simple microscope.
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27. MODEL ANIMAL ROUTE DOSE DURATION
Cysteamine
10% in
Normal saline
Sprague-
Dawley
rats
Orally 28mg/100g 3 times at
intervals of
3.5 hours -->
animals
sacrificed after
28
hours of 1st
dose
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Procedure:
28. Perforating duodenal ulcer are produced that are located 2-4mm from the
pylorus, mainly on the anterior wall of the duodenum.
The ulcer and its features in test group are compared to those in control group.
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29. Dimaprit-Induced Duodenal Ulcer
Principle:
H2 receptor agonist
Induced gastric erosion in rats after single i.v. dose.
Duodenal ulcer in guinea pigs after repeated s.c. dose.
Procedure:
Female Sprague rats (150-200 g)or female guinea pigs (250-300 g) are taken
Fasted for 24 hours before experiment; free access to water
In rats ,Dimaprit is given in a dose of 100mg/kg intravenously, single dose
The animal is sacrificed one hour later & stomach dissected out for evaluation
of gastric erosions
The test drug or vehicle is given orally 60 min before injecting dimaprit
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30. Mepirizole -Induced Duodenal Ulcers
Okabe et al(1982)- described the production of Duodenal Ulcers in rats by
mepirizole , a NSAIDS
This is used in screening of antacids and H2 receptors antagonist
Procedure:
Male Sprague – Dawley rats (200-220g)
Mepirizole(200mg/kg) in 1% carboxymethyl cellulose solution via gastric
intubation
Subsequently, rats kept in cages with raised mesh bottom anddeprived of food and
water for 24 hrs
Leads to, ulceration in proximal duodenum anderosions in antrum.
Anti ulcer therapy started 24 hrs afterMepirizole administration
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31. 11th day, Animal sacrificed
Duodenum and Stomach evaluated for ulcer area under microscope
Ulcer or erosion indices are calculated from the sum of area of ulcers &
erosions respectively
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32. Reserpine-Induced Chronic Ulcers
The mechanism of ulcer formation is by histamine liberation from the mast cells
in stomach wall which increase in gastric acid secretion
Procedure:
Female Sprague – Dawley rats; 130-180g
Fasted for 48 hrs, Free access to 0.8% sucrose in 0.2% NaCl w/v
Liquid diet withdrawn 1hr before starting
Animals injected with Test drug i.p. ½ hr later, Reserpine(5mg/kg) or Vehicle
injected i.p.
4 hrs later, Animal sacrificed, Stomach removed, Examined for mucosal lesions
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