All animal procedures were carried out according to Home Office regulations. Animals (Scientific Procedures) Act, 1986. General anaesthesia in rabbits was induced by two different methods depending on the duration of anaesthesia required:
(i) For short-term anaesthesia (1-2 h), rabbits were deeply sedated with an intra-muscular (i.m.) injection of 0.2 ml/kg fentanyl citrate (0.315 mg/ml) + fluanisone (10 mg/ml) (Hypnorm®) followed by slow injection of 1.0 mg/kg diazepam (5 mg/ml) and hypnorm by slow intravenous (i.v.) infusion as required. Animals were supplied with 100% oxygen (2 1/min) via an open face mask throughout the surgical procedure. 1 ml heparin (1000 lU) and 1 ml frusemide (10 mg/ml) were administered i.v. prior to laparotomy.
(ii) For long-term (terminal) anaesthesia (3-6 h), rabbits were deeply sedated with an i.m. injection of 50 mg/kg ketamine hydrochloride (100 mg/ml) and 8 mg/kg xylazine (20 mg/ml), tracheotomized and artificially ventilated (ventilation volume 21 ml) with a 50 : 50 oxygen : nitrous oxide mixture. This method allowed maintainance o f normal blood pressure, and plasma partial pressures o f oxygen and carbon dioxide throughout the surgical procedures. Heparin and frusemide were administered i.v. prior to laparotomy as described above. Surgical anaesthesia was maintained by continuous i.v. infusion of ketamine and xylazine (50mg : 8mg/kg/h).
2.2.2 Physiological Monitoring (During Long-Term Anaesthesia)
The left femoral artery was carefully dissected and cannulated with an 18-gauge plastic cannula, which was inserted for a distance of 5 cm until the tip reached the base o f the aorta. The caimula provided protective housing for a Continucath 1000 oxygen electrode coupled via a 3-way tap to a pressure transducer. Blood pressure (BP) and oxygen partial pressure (PO2) were continuously monitored throughout the study period. To ensure satisfactory renal perfusion, mean arterial systolic/diastolic blood pressure must be maintained above 50 mmHg and preferably above 80 mmHg. The quality of renal cortical perfusion is visible to the eye at low perfusion pressures,
maintained within the range 80-120 mmHg during reperfusion by continuous i.v. infusion o f 30 ml/h Haemaccel (3.5% colloidal infusion), or as required. Intermittent blood samples were taken (from the 3-way tap) for determination of arterial carbon dioxide partial pressure (PCO2), pH, electrolyte levels and conductivity (Hct). Core temperature was monitored using an oesophageal probe and was maintained between 37 and 39®C with a heated pad (37®C). In addition, the electrocardiogram (EGG), percentage inspired oxygen (FiO], maintained at 50%) and percentage expired carbon dioxide (EtCO], maintained at 5%) were continuously monitored.
2.2.3 Nephrectomy and Autografting Procedures
All microsurgical instruments were sterilized by boiling followed by spraying with 0.5% chlorhexidine in ethanol prior to nephrectomy or autografting. The anaesthetized rabbit was shaved abdominally and dressed in previously autoclaved drapes. The right kidney was exposed through a mid-line abdominal incision. The intestines were gently displaced, externalized, swathed in warmed damp sterile swabs and supported on a sandbag raised slightly above the level o f the peritoneum. In the course of long operations, the intestines were additionally wrapped in clingfilm to reduce heat and moisture loss. The connective tissue surrounding the kidney was carefully separated and the renal vessels divided and ligated as close to the aorta as possible using 2/0 silk thread. The renal artery was then cannulated with the rounded glass tip of a pipette inserted into a 2 1-gauge butterfly caimula after removal o f the needle, and flushed with 40 ml ice-cold hypertonic citrate solution (HCA; 0-2°C) from an ice-cooled bag suspended at a height o f 1.5m. The maximum perfusion pressure (measured using a pressure-gauge) was therefore 90 mmHg.
The perfused kidney was plunged into a sterile glass beaker containing HCA (0-2^C) and the beaker was surrounded by ice. The total warm ischaemic time was less than 2 min. Perfused kidneys were then either autografted immediately into the donor rabbit or stored for 24, 48 or 72 h within a closed polystyrene container packed with ice and held in a refrigerator prior to autografting. The storage temperature was thus maintained at a steady 0-2”C. The composition o f HCA solution is shown in Table 2.1.
After the storage period, kidneys were autografted into the donor rabbit. The rabbits were anaesthetized as described above, and the body cavity opened along the same mid-line incision. The left kidney was carefully dissected out from the connective tissue and removed after clamping the renal artery and vein proximal to the kidney using weighted bulldog clamps with
Ch a p t e r 2 Mat e r i a l sa n d Me t h o d s
(stored or unstored) was then autografted into the left renal bursa. The severed ends o f the renal vein were carefully cleaned using isotonic saline to remove blood clots and procaine to prevent nervous or muscular spasm, and placed within the jaws of a double clamp. The ends were clipped with microsurgical scissors to ensure removal o f tissue damaged during handling, and immobilized with stay sutures using 8/0 surgical silk suture under an operating microscope. The vein was then anastomosed using approximately 15 interrupted sutures to ensure strength, and the procedure repeated for the renal artery (with the same number o f sutures). Upon reperfiision o f autografted kidneys (by removal of the clamps), recipient rabbits received a 1 ml i.v. bolus of frusemide and a 60 ml slow i.v. infusion of sodium chloride solution (0.9%). The hypothermic ischaemic time during the autografting operation prior to reperfiision was approximately 2 0 min.
Table 2.1: Composition of Hypertonic Citrate Solution
Solute (mmol/1) Sodium 80 Potassium 80 Magnesium 40 Citrate 55 Sulphate 40 Mannitol 100 Osmolarity (mOsmol/1) 485 pH 7.1
For animal recovery in survival studies, the ureter was also anastomosed. A sterile plastic stent of 1 cm length and 1 mm lumenal diameter was inserted into the two severed ends o f the ureter to prevent blockage o f the lumen, and the ureter wall anastomosed around this using interrupted 8/0 surgical silk sutures. The surrounding fatty and connective tissue were then anastomosed to prevent leakage o f urine from the ureter, and the kidney immobilized by drawing over the surrounding connective tissue and secured with interrupted sutures using 6/0 gut suture. The intestines were carefully replaced within the abdomen, and the peritoneal and muscle wall sutured using interrupted 0/0 gut sutures. Finally, the overlying skin was joined using interrupted 2/0 silk sutures.
P late 2.1 A nastam oses of th e renal a rte ry (right) a n d vein (left).
Ch a p t e r 2 Mat e r i a l sa n d Me t h o d s
2.2.4 Determination o f Survival
Rabbits allowed to recover after autograJfting were treated with: 10 ml i.v. mannitol; 1 ml i.m. frusemide; and 60 ml sub-cutaneous (s.c.) isotonic saline immediately after recovery and on the subsequent four days. Serum samples (2 ml) were taken from the marginal ear vein for analysis of serum urea and creatinine (see Section 2.4.4), on days 1 to 4, 6 and 10 after transplantation. Rabbits developing uraemia were killed and autopsied as soon as clinical symptoms were observed. Renal function was assessed by:
Observation of kidney colour and urine production immediately after revascularization Measurement of serum urea and creatinine
The clinical appearance o f each animal Evidence o f urine production
The macroscopic and microscopic appearance o f each kidney at autopsy.