Screening Methods for Anti-Ulcer Drugs | Zoology

An ulcer is a local defect, or excavation of the surface of an organ or tissue, which is produced by the sloughing of inflammatory necrotic tissue. The term “peptic ulcer” refers to a group of ulcerative disorders of the upper GIT, which appears to have in common, the participation of acid pepsin in their pathogenesis.

A peptic ulcer probably results due to an imbalance between aggressive (acid, pepsin and H. pylori) and defensive (gastric mucous, and bicarbonate secretion, prostaglandins, innate resistance of the mucosal cells) factors. In gastric ulcers acid secretion is normal or low.

There are various methods for screening anti-ulcer drugs: 1. Pylorus Ligation in Rats 2. Ulcers through Immobilisation Stress 3. Stress Ulcer by Cold Water Immersions 4. Indomethacin Induced Ulcers in Rats 5. Ethanol Induced Mucosal Damage in Rats (Cytoprotective Activity).

Method # 1. Pylorus Ligation in Rats:

The principle is based on ulceration induced by accumulation of acidic gastric juice in the stomach.

The requirements include: stereo microscope, adult albino rats (150-170 g), anaesthetic ether, plastic cylinder, 0.1 N sodium hydroxide.

Adult albino rats weighing 150-170 g are starved for 48 h although they have access to drinking water. Normally ten animals are used per dose and as control. After they are ether anaesthetised, a mid-line abdominal incision is made and the pylorus ligated. The abdominal wall is closed by sutures and test compounds are 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 a wire mesh.

The animals are then sacrificed using carbon dioxide anaesthesia. The abdomen is opened and a ligature is placed around the aesophagus close to the diaphragm. The stomach is removed and the contents drained into a centrifuge tube. Along the greater curvature, the stomach is opened and pinned onto a cork plate. The mucosa is then examined with a stereo microscope.

The evaluation is done by counting the numbers of ulcers and the severity graded according to the following scores:

0 – No ulcers; 1 – Superficial ulcers; 2 – Deep ulcers; 3 – Perforations

The volume of gastric content is measured after centrifugation. Acidity is determined by titration with 0.1 N NaOH.

Ulcer index U₁ is calculated using the following formula:

U₁ = U_n + U_s + U_p x 10^-1

Where,

U_n = the average of the number of ulcers per animal.

U_s = average of severity score, and

U_p = percentage of animals with ulcers.

Method # 2. Ulcers through Immobilisation Stress:

The principle behind this method involves psychogenic factors, such as stress, which play a major role in the pathogenesis of gastric ulcers in man.

The requirements include – adult albino rats, anaesthetic ether, CO₂ anaesthesia, and a stereo microscope.

A group of 10 adult albino rats (150-170 g) per dose of the test drug and for controls are used. Food and water are withdrawn 24 h before the experiment. After oral and subcutaneous administration of the test compound or a placebo solution, the animals are slightly anaesthetised with ether. Both the upper and lower extremities are fixed together and the animals wrapped in wire gauge.

They are horizontally suspended in the dark at 20°C for 24 h and finally sacrificed using carbon dioxide anaesthesia. The stomach is removed, fixed on a cork plate and the number and severity of the ulcers registered with a stereo microscope.

Method # 3. Stress Ulcer by Cold Water Immersions:

The principle behind this assay is that cooling the rats in water when they are restrained according to the previous model accelerates the occurrence of gastric ulcers and shortens the time of necessary immobilisation. In this model, the gastric ulcer formation is mainly due to gastric hypermotility, which could lead to mucosal over-friction.

The requirements include – Wistar rats, cages, Evans blue dye, 2% formol saline, CO₂ anaesthesia, and a magnifier.

Groups of 8-10 Wistar rats weighing 150-200 g are used. After oral administration of the test compounds, the rats are placed vertically in individual restraint cages in water at 22°C for 1 h. They are then removed, dried and injected intravenously through a tail vein with 30 mg/kg Evans blue. 10 min later they are sacrificed using CO₂ anaesthesia and their stomachs removed.

Formol saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greatest curvature, washed in warm water and examined under a three-fold magnifier.

The evaluation is done by measuring the lengths of the longest diameter of the lesions. This is summated to give a total lesion score (in mm) for each animal; the mean count in control rats should be about 25 (range 20-28). Inhibition of the lesion production is expressed as a percentage value.

Method # 4. Indomethacin Induced Ulcers in Rats:

This assay is based on the fact that use of non-steroidal anti-inflammatory agents like indomethacin and acetyl salicylic acid induces gastric lesions in man and in experimental animals by inhibition of gastric cyclo-oxygenase.

The requirements include – Wistar rats, 0.1% tween 80, 2% formol saline and CO₂ anaesthesia.

Groups of 8-10 Wistar rats weighing 150-200 g are used. The test drugs are administered orally in 0.1% tween 80 solutions, 10 min. prior to an oral administration of indomethacin (20 mg/kg). 6 h later, the rats are sacrificed in CO₂ anaesthesia and their stomachs removed. Formol saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomach are opened along the greater curvature, washed with warm water and examined.

The mean score is calculated in control rats. It should be about 25. Inhibition of the lesion production is expressed as a percentage value.

Method # 5. Ethanol Induced Mucosal Damage in Rats (Cytoprotective Activity):

The principle behind this method is that intragastric application of absolute ethanol, which is a reproducible method, produces gastric lesions. The lesions can be inhibited by drugs with cytoprotective activity.

The requirements include – adult albino rats, absolute ethanol, CO₂ anaesthesia, Plexi glass template, densitometer, camera, an Aristo model T-16 cold cathode transilluminator.

Adult albino rats weighing 250-300 g are deprived of food 18 h prior to the test although water is given. The rats are administered the cytoprotective drugs intragastrally 30 min. prior to administration of 1 ml absolute ethanol. Untreated animals are taken as control. 1 h after administration of ethanol, the animals are euthanised with CO₂ and the stomachs excised, cut along the greater curvature and rinsed with tap water.

The stomachs are stretched on a piece of foam core mat. The subjective scores of the treated tissue are recorded; the response is graded from the least (0) to the most (3) damaged. Using a cork borer, a circular area about 13 mm in diameter is cut from each lobe of the fundus just below the ridge dividing the glandular from the non-glandular portion of the stomach.

The excised pairs of tissue from each stomach are placed into the holes of the template. Pairs of tissue from each stomach are examined to minimise sampling errors. The template is positioned on a rectangular central open area of an Aristo model T-16 cold cathode transilluminator (38 × 38 cm) containing a W-45 blue-white lamp.

A camera is mounted on a copy stand directly above the template. Photographs are taken. The film is pressed in a standard manner and the contact sheet made from the negatives. A light transmission densitometer is used to evaluate the negatives and the optical density determined.

The evaluation is done by observing haemorrhagic or damaged areas that appears bright on the negatives, whereas undamaged tissues appear dark showing greater optical density.

The significance of the differences in optical density between ethanol-treated tissue and control is evaluated by non-paired single tail student’s t-test.