Eight- to 10-week-old male and female Wistar rats, bred within the Biomedical Services Unit at the Queens Medical Centre, Nottingham, were used. Rats were immunized by subcutaneous injection with rat Gastrimmune (rat Gastrimmune differs from human Gastrimmune by a single amino acid substitution) at 3-week intervals. Gastric fistulas were inserted between the second and third immunizations, at approximately week 5. After four immunizations, acid secretion studies were performed. Further immunizations were performed at 3-weekly intervals. Four groups of six Wistar rats were used. A control group was immunized with diphtheria toxoid. There were three rat Gastrimmune dose groups: 10 μg, 100 μg and 200 μg.
Gastric fistula insertion
The technique for insertion of the gastric fistula was based on a method reported by Dimaline et al.14 The day before fistula insertion, food was withdrawn, but the animals were allowed free access to water.
The rat’s abdomens were shaved and Hypnorm/Hypnoval (1:1:2) dilution in water (Janssen/Roche, 3 mL/kg) was administered intraperitoneally. An adequate depth of anaesthesia was assessed by the loss of the foot pinch reflex. Animals received supplemental oxygen throughout the procedure.
A right paramedian incision was made through which the stomach was delivered. A stainless steel Gregory cannula (Medical School Workshop, University of Nottingham) was then inserted into the body of the stomach along the greater curve using vicryl purse string sutures. Three interrupted sutures were placed in the ‘neck’ of the cannula to prevent rotation. The cannula was exteriorized via a stab incision made in the midline. The abdominal cavity was closed in layers with interrupted chromic catgut. Local anaesthetic (1% lignocaine) was infiltrated over the incision. The cannula was closed with a threaded brass cap, lubricated with silicone grease.
In the immediate post-operative period, animals were given prophylactic antibiotics (Penbritin, 80 mg/kg intramuscularly) and 10 mL of phosphate-buffered saline subcutaneously at two sites. The animals were placed in a warming cabinet and observed at regular intervals. Insertion of the cannula appeared not to cause the animals distress and no further analgesia was required.
In the post-operative period, food was withdrawn over the first 24 h; however, water was freely available. The subcutaneously administered phosphate-buffered saline, together with antibiotics, was continued over the following 7 days. Rats were weighed daily to ensure that no greater than 20% of their body weight was lost. Weight gain recommenced approximately 5–7 days following surgery.
Following insertion of the cannula, rats were housed separately in 9.5 in × 18 in high plastic cages with stainless steel tops. They were fed standard pelleted diet and given normal tap water to drink. Room temperature was maintained at 19–23 °C, with the animals exposed to 12 h light and 12 h dark cycles.
Pharmacological stimulation of acid secretion in terminally anaesthetized rats
Twenty-four hours prior to the procedure, the stomach of each rat was washed out with warm saline via the gastric fistula. The rats were then transferred to a cage with a wire bottom and fasted, although water was freely available. The animals were weighed on the day of the study.
Intraperitoneal anaesthetic was administered (Hypnorm/Hypnoval (1:1:2) dilution in water, 3 mL/kg, Janssen/Roche) and the rats were transferred to a warming cabinet in order to dilate the tail vein. If necessary, additional intraperitoneal anaesthetic was given during the experiment until the rat no longer exhibited a foot pinch reflex.
The tail vein was cannulated using a catheter (made from silastic tubing, 0.02 in × 0.37 in, and 23 gauge needle with hub removed), which was placed in the tail vein and flushed with phosphate-buffered saline. This catheter was used for the injection of all test reagents. A 5 cm midline incision was made through the skin and peritoneal wall of the rat, extending from just below the xiphoid process and around the indwelling midline fistula.
The stomach was mobilized, as some rats had developed marked adhesions around the fistula, often involving the liver. A transverse incision was made 0.5 cm above the oesophagogastric junction. A 0.05 in × 0.09 in silastic tube was passed down the oesophagus, until it could be felt in the stomach, and was held in place by two 4/0 vicryl ties; this also prevented back flushing. A vicryl tie was also placed distal to the pyloric junction to block flow into the duodenum. The abdominal wall was loosely closed with a single suture and a piece of phosphate-buffered saline soaked gauze was placed over the incision.
The cap from the previously inserted gastric fistula was removed and connected to tubing to allow drainage and collection of gastric contents. The stomach was irrigated to remove food particles by flushing with 0.9% saline at 35 °C. The oesophageal tubing was connected to a pump and 0.9% saline, maintained at 35 °C in a constant water bath, was infused at a constant rate (0.5 mL/min). The body temperature of the rat was monitored by a thermometer attached to the chest wall. Normal body temperature was maintained by placing the rat on a heating pad with insulation.
Approximately 30 min prior to the administration of any test solution, 10.8 μg of atropine sulphate was injected to stabilize the acid secretion baseline. Once regular baseline readings had been established, infusion of acid stimulants was performed. Rat amidated gastrin-17 was dissolved in phosphate-buffered saline to a concentration of 120 ng/mL. In ascending dose order, gastrin was administered at 30, 60 and 120 ng over a 5-min period.
Pentagastrin was used as a positive control as it is composed of the 5-carboxy-terminal amino acids of gastrin-17 and is therefore unaffected by Gastrimmune, which is directed against the amino-terminal of gastrin-17. Pentagastrin was dissolved in phosphate-buffered saline at a concentration of 600 ng/mL; 1 mL was infused slowly over a period of 5 min.
Acid was collected at 5-min intervals and the volume of each sample was measured gravimetrically. A 1-mL aliquot of each perfusate sample was titrated with 0.01 M NaOH to determine the micromoles of acid per aliquot. The total number of micromoles of acid per sample was then calculated from the total volume of the perfusate sample (micromoles per 1-ml aliquot × number of millilitres in sample=total micromoles).