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I Int J Med Lab Res 2017, 2(3):44-54
INTRODUCTION:
Gastro-intestinal disorders 1 are one of the severe acid and pepsin. There are several causes
classes of human ailments causing maximum smoking, H.pylori infection, ingestion of drugs
discomfort, morbidity and mobility. Peptic ulcer and chemicals. Especially consumption of
2-3
is one such GIT disorder. Peptic ulcer is alcohol for a prolonged period, smoking of
considered as a major health problem, both in Cigarette, or chronic consumption of NSAIDs
terms of morbidity and mortality and it is very are causing peptic ulcers. The peptic ulcer is
common in the present day life of industrialized mainly caused because of irregulated acid
and civilized countries. Available statistical secretion in the body. The symptoms of peptic
reports indicate that10% or more of adult ulcer are: severe pain and irritation in the upper
population are affected within their life time and abdomen. If it is not treated properly, it may
peptic ulcer affects individuals invariably from result in perforations in the wall of the
20 to 60 years of age with males being gastrointestinal tract. Because of the severity of
predominantly affected. Peptic ulcer is benign the problem, now world is awaiting for the
lesion of gastric or duodenal mucosa occurring at treatment of it. Before going to do the treatment
a site where the mucosal epithelium is exposed on the human, we need to check the effects of the
Corresponding Author:
Sai Datri Arige
Department of Pharmaceutical Analysis, V.V. Institute of Pharmaceutical
Sciences, Gudlavalleru, A.P., India.
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Compare the test and standard with that of the after the infusion and measure the inhibition of
control to access the anti-ulcer activity of the acid secretion and compare the results with that
drug. of standard.
GOSH AND SHILD PERFUSED RAT PYLORUS LIGATION INDUCED ULCERS
STOMACH PREPARATION IN RATS
This method is first demonstrated by Shay
PRINCIPLE: and co-workers in 1945. Pyloric ligation in rats
The anti-ulcer agent screening is done by leads to accumulation of gastric acid in the
continuous reading of acid secretion in the rat, stomach leading to acute gastric ulceration.
which is stimulated by histamine, acetylcholine
REQUIREMENTS:
and gastrine and that acid secretions can be
Albino Wistar Rats (150-200 g), Ether
blocked by anti-ulcer drugs.
(Anesthetic), Saline (control), Omeprazole
PROCEDURE: (standard), Test drug (3 different concentrations
Male Sprague Dawley or Wistar rats of weighing x, 2x, 4x), NaOH (0.01N), Topfer’s reagent
180 g are taken for the procedure. Prior to the (dimethyl amino azo benzene) and
experiment, starved the animal overnight by Phenolphthalein.
withdrawing the food. Group the rats randomly
PROCEDURE:
as four rats in a group. Anaesthetize the animals
Wistar albino rats of either sex weighing 150 –
with 25% urethane solution (0.6 ml/100 g, i.m.).
200 g are taken for the procedure. Rats fasted for
The body temperature is artificially stabilized by
24 hrs prior to pyloric ligation.
means of a heating coil using a rectal
Randomly divided into 5 groups of 10 animals
thermometer and also trachea is exposed and
each
cannulated for
Group I: Control vehicle
artificial respiration. The external jugular vein is
Group II: Standard drug (Omeprazole)
exposed and cannulated with polythene tubes
Group III: concentration of test drug
leveled at the tip. The abdomen is opened
Group IV: concentration of test drug
through midline incision, and pyloric end of the
Group V: concentration of test drug
stomach is cannulated. A polythene tube is
Drugs administered once for 2 days and 30 mins
passed down the oesphagus and tied in the
prior to ligation. Rats anesthetized with ether.
cervical region. The stomach is washed out
Pyloric ligation procedure:
thoroughly by passing distilled water to the tube.
o The abdomen is opened by a small midline
Perfuse the stomach with N/4000 sodium
incision.
hydroxide at a uniform rate. The concentration of
o Pyloric portion of stomach is slightly lifted
sodium hydroxide is adjusted so that the
out & ligated avoiding traction to pylorus or
perfusate under basal conditions has a pH. The
damage to its blood supply.
perfusate emerges out bathes the micro
o The stomach is replaced carefully and
electrodes which are directly connected to the pH
abdominal wall is closed by interrupted
meter. Changes in acid secretion are accessed by
sutures.
pH changes. The gastric secretion is stimulated
The drugs are administered subcutaneously
by continuous i.v. infusion of 100
immediately after pyrolus. Rats placed in
microgram/kg/hr of pentagastrin or 3 mg/kg/h of
separate cages and allowed to recover. After 19
histamine hydrochloride or 30 microgram/kg/h
hrs to pyloric ligation, animals sacrificed by
of carbachol. The test is injected either prior or
decapitation. Abdomen opened and stomach
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experiment to the fed rats. For the treated 2 – Deep ulcer usually with transmural
animals: Administer the drugs before 30 min of 3 – Perforated or penetrated ulcer
first dose and again after 24 hrs on the second
day. The rats are sacrificed after 48hrs of the first APPLICATIONS:
dose of cysteamine Hcl. The duodenal ulcers are The lesions developed are analogus to the
developed 2 -4 mm away from the pylorus on the clinical disease with respect to location and
anterior wall of the duodenum and frequently histology.
perforate the liver. A small ulcer present on the The factors responsible for producing the
posterior wall (kissing ulcer) of the duodenum pathological changes are similar in man and
and it is invariably penetrates the pancreas. animal used.
The intensity of the duodenal ulcers is evaluated The drugs effective against experimental
as 0 – No ulcer ulcer could be clinically useful.
1 – Superficial mucosal erosion The method is dependable, reproducible and
2 – Deep ulcer usually with transmural easy to perform and results are obtained with
3 – Perforated or penetrated ulcer in 24hrs.
INDOMETHACIN AND HISTAMINE
APPLICATIONS: INDUCED DUODENAL ULCER IN RATS
Suitable method for studying the
pathogenesis of duodenal ulcer PRINCIPLE:
An effective method for anti-ulcerogenic A single administration of indomethacin and
regimes detection. subsequent dosing with histamine consistently
Since some of such chemicals might have a produce lesions at the opposite site of the
role in the etiology of human duodenal ulcers mesenteric attached in the proximal duodenum of
as well, the structure activity conclusions rats. The development of duodenum lesions by
may helps in the identification of ulcerogenic indomethacin + histamine induction in rats is
chemicals in our diet and environment. due to both an increase in gastric secretion and
an impairment of acid induced duodenal HCO3
DUCLEROZINE INDUCED DUODENAL secretion.
ULCER IN RATS PROCEDURE:
Wistar or Sprague Dawley rats weighing 150-
PROCEDURE: 200 g are taken for the procedure. Grouped rats
Male wistar or Sprague dawley rats weighing as such 6-8 rats must in a group. Fast the rats for
around 200 g are taken for the procedure. 24 hrs before the experiment with water ad
Duclerozine 300 mg/kg, suspended in 5% gum libitum. Administered 5 mg/kg Indomethacin s.c
acacia solution, is administered orally as a single and subsequently administered 40 mg/kg
dose. The drugs (test/standard) are given 30 min histamine dihydrochloride 3 times in a day at 2.5
prior to the administration of Duclerozine. The hours interval, beginning after 30 min of the
animals are sacrificed after 18 hr of indomethacin injection. Sacrifice the animal after
administration. 24 hrs of the procedure and observe it for ulcers.
Due to this combined treatment, two lesions are
SCORING: formed at the proximal duodenum and also few
0 – No ulcer lesions at corpus and antrum of the stomach.
1 – Superficial mucosal erosion Statistically compare the data of test and
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standard then conclude the activity of the test. expressed as unit (U) of SOD activity/mg
The standards for treating duodenum and antrum protein.
ulcers are oral cimetidine and 16, 16 – Estimation of mucosal glycoprotein’s:
dimethylprostaglanin E2 in a dose dependent Sample of gastric mucosal scraping is
manner and also for corpus ulcer is cimetidine. homogenised in distilled water and treated with
HISTAMINE INDUCED DUODENAL 90% ethanol and are subjected for the estimation
ULCERS IN GUINEA PIGS of carbohydrates and proteins using the methods
described above for gastric juice contents.
PROCEDURE: Statistical analysis was done by student’s t-test.
Guinea pigs fasted for 48 hrs are taken for Estimation of free acidity and total acidity:
the procedure. Drugs (control, standard and test) One ml of gastric juice is pipette into 100 ml
given once daily for 2 days & AMP; before 30 conical flask, added 2 to 3 drops of topfer’s
mins to drug, administrate histamine. Ulceration reagent and treated with 0.01 N sodium
induced by injecting 1 ml of histamine solution hydroxide until all traces of red colour
intraperitoneally. Promethazine 5 mg injected disappears and the colour of the solution turns to
intraperitoneally 15 mins before and after yellowish orange. The volume of the alkali added
histamine. Animals sacrificed after 4 hrs. Degree was noted. This volume corresponds to free
of ulceration & AMP; gastric contents assessed. acidity. Then 2 to 3 drops of phenolphthalein
solution is added and titration is continued until a
ESTIMATION OF PARAMETERS: definite red tinge reappears. Again the total
Estimation of free radical generation: The volume of alkali added is noted. Acidity is
fundic part of the stomach is homogenised (5%) calculated by: Acidity = Volume of NaOH ×
in ice cold 0.9% saline with a Potter-Elvehzem Normality of NaOH × 100 / 0.1 × meq/L/100
glass homogeniser for 30 second. The gm.
homogenate is then centrifuged at 800× g for 10 Estimation of DNA in gastric mucosa: DNA
min followed by centrifugation of the and protein are estimated in the gastric fundal
supernatant at 12000× g for 15 min and the mucosal scrap homogenised in 2.5 ml of ice
obtained mitochondrial fraction is used for the cooled 0.6N perchloric acid (PCA). The
following estimation. Statistical analysis was concentration of DNA is expressed as µg
done by student’s t-test. DNA/mg protein.
Lipid peroxides (LPO): LPO product Estimation of glandular weights of
malondialdehyde (MDA) is estimated using 1, 1, stomach: The weight of the glandular portion of
3, 3-tetraethoxypropane as the standard and is stomach is calculated by subtracting the weight
expressed as n mol/mg protein. of the whole stomach minus rumen and is
Super oxide dismutase (SOD) activity: expressed as mg/100 g body weight of the
Inhibition of reduction of nitro blue tetrazolium animals. Statistical analysis was done by
(NBT) to blue coloured Formosan in presence of student’s t-test.
phenazine methasulphate (PMS) and NADH is Estimation of Ulcer index (Ui):
measured at 560 nm using n-butanol as Ui = mean degree of ulceration × % group of
blank. One unit of enzyme activity is defined as ulceration /100
the amount of enzyme that inhibits rate of %inhibition = (ulcer index in control - ulcer
reaction by 50% in one min under the define index in test) ulcer index in control x 100
assay condition and the results have been
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Estimation of gastrin: In order to determine the suggested that the above mentioned all types of
gastrin levels in plasma, blood is collected by induced models procedure meets the established
cardiac puncture, centrifuged, and the plasma is criteria for a useful experimental ulcer model and
analysed for gastrin levels with double-antibody thus represents a viable research tool [22]. The
liquid phase radioimmunoassay. natural and synthetic products can be
Histological studies: scientifically evaluated to establish therapeutic
Gastric tissue samples from each group were efficacy by the above mentioned techniques.
fixed in 10% formalin for 24 h. The formalin
fixed specimens are embedded in paraffin and REFERENCE:
section (3-5µm) and stained with haematoxylin
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