Acetic acid – induced ulcer in rats
Induction of ulcer is performed according to the method described by Millar et al., 1996. Each rat is sedated by brief inhalation of 20-30% CO2 followed by intra peritoneal injection of phenobarbitone (35 mg / kg). Two ml acetic acid (3% v/v in 0.9% saline) is infused for 30 seconds through the rectum into the colon using a polyethylene tube 2 mm in diameter inserted to a distance of 8 cm. The rats are then returned to their cages. They are killed 24 h using ether anaesthesia. Macroscopic, and microscopic evaluations are assessed by appropriate methods described below.
Assessment of Colitis
At postmortem laparotomy, 6 cm of colon extending proximally from 2 cm above the anal margin is removed, splited longitudinally, pinned out on card, and the macroscopic appearance of the colonic mucosa scored by an independent observer on a scale ranging from 0 to 4 as follows: 0 = no macroscopic change, 1 = mucosal erythema alone, 2 = mild mucosal oedema, slight bleeding or small erosions, 3 = moderate edema, slight bleeding ulcers or erosions, 4 = severe ulceration, erosions, edema and tissue necrosis (Millar et al., 1996).
Following macroscopic scoring, tissue samples or mucosal scrapping are stored immediately at – 200C for biochemical and microscopic reading.
Full thickness biopsy specimens from animals are fixed in 10% formol saline prior to wax embedding, sectioning and staining with haematoxylin and eosin. Microscopic assessments are carried out by a histopathologist who is unaware of the treatment protocol.
Determination of drug concentrations in plasma by HPLC
The plasma concentrations of a drug is determined by using a modification of the high-performance liquid chromatographic method described by Asberg and Haffner (21). Briefly, blood samples are centrifuged at 2100 g for 10 min on a Gallenhamp Angle Head Centrifuge. An aliquot (200 µl) of the plasma is added to a 200 µl of a chromatography grade acetonitrile solution. The suspension is thoroughly shaken and then centrifuged at 5400g for 5 min on a Select-a-fuge 24 Biodynamics. Twenty microlitres of the acetonitrile supernatant are injected into an HPLC system consisting of a Waters (Milford, Massachusetts M-510) pump and a Waters reverse phase NOVA-PAK C-18 (3.9 mm x 1.5 cm) column. The column is eluted at the rate of 1.5 ml/min with a prefiltered and degassed mobile phase consisting of 45 per cent methanol and 0.01 M potassium dihydrogen phosphate.
The drug is detected by a Waters M-441 variable wave length UV absorption detector set at 214 nm with a sensitivity of 0.01 absorbance units of full scale (AUFS) and a chart speed of 2 mm/min. To determine the plasma concentrations of a drug, several standard solutions of the drug are freshly prepared in a concentration range of 50-1000 µg/ml. Twenty microlitres of these drug concentrations are injected into the column using a 50 µl Hamilton syringe. A linear relationship is obtained when these concentrations of the drug are plotted against their corresponding peak heights. The concentration of the drug in plasma is calculated from a previously constructed standard curve.
Determination of drug concentration in urine using HPLC
Urine samples are collected at different timing following the administration of the drug. Briefly, 0.5 ml of urine samples (in duplicates) are buffered with 1ml of 3M Tris - HCL buffer, PH 7.5, and extracted by mixing for 15min with 5ml of diethyl ether (HPLC grade).The suspension is then centrifuged at 5400g for 5 min on a select - a - fuge 24 Biodynamics. After centrifugation, a 4ml aliquot of the organic layer is evaporated and then reconstituted with the mobile phase.Twenty microlitres are injected into the HPLC system and the concentration of drug in urine is determined as described above.