KIDNEY TRANSPLANTATION IN THE RABBIT


David M. A. Francis Department of Surgery, The University of Melbourne, Royal Melbourne Hospital, Vie. 3050, Australia

INTRODUCTION

Appropriate use of an animal model in transplantation research requires that the selected species has anatomical, physiological and immunological characteristics which are similar to those of humans, so that experimental conclusions can form the basis of clinical strategies. Even though the rabbit (Oryctolagus cuniculus) offers some distinct advantages over other species, it has been largely ignored as a model for experimental studies of solid organ trans-plantation. This chapter reviews kidney transplantation in the rabbit as a model for vascular-ised organ transplantation studies. In addition to descriptions of the techniques of the donor and recipient operations, relevant aspects of rabbit biology, pre- and postoperative care, anaes-thesia and management of potential complications are presented, as well as results from a per-sonal series of rabbit renal transplants. KIDNEY TRANSPLANTATION IN THE RABBIT AS AN EXPERIMENTAL MODEL

The rabbit serves as an excellent model for renal transplantation studies. The International Index of Laboratory Animals gives the locations of 116 designated stocks of rabbits, three quarters of which are described as outbred (Adams, 1987). Fully inbred strains are available. Rabbits are of sufficient size to enable the operative procedures to be undertaken readily, and they are easy to house and maintain and to handle in a laboratory setting. The general biology of the rabbit has been well reviewed (Adams, 1987; Harkness and Wagner, 1989). Anatomical Features

The abdominal cavity of the rabbit is large relative to its overall size, a feature which facili-tates intra-abdominal surgery and represents a significant advantage over other small experi-mental animals. The intestine is bulky, being approximately ten times the body length, and must be placed outside the abdominal cavity for access to the kidneys, which are situated on the posterior abdominal wall behind the peritoneum. Each kidney measures approximately 3.5 x 2.5 cm in the adult rabbit. For the purpose of transplantation, the anatomy of the kidney is similar in rabbits and humans. Each kidney is supplied usually by a single artery, arising from the dorsal aorta. The adrenal artery arises from the renal artery and may therefore limit its available length, especially on the right side. The right renal vein is short and directly enters the inferior vena cava (IVC). The left renal vein is longer than the right and receives the left gonadal and adrenolumbar veins before entering the IVC (Kent, 1987). The rabbit has thin walled and fragile veins which tear easily, and so great care must be taken when mobilising and anastomosing these vessels. The vascular supply of the proximal ureter is from branches of the renal vessels and these fine vessels must be preserved during transplantation to avoid ureteric necrosis. Physiological Features

Experimental rabbits exhibit a wide range of body weights, depending on strain, sex and diet. Excessive amounts of intra-abdominal fat in rabbits weighing more than 3.0 kg make surgery difficult. The life span of rabbits is usually quoted as 4-6 years. Blood volume is 57-70 ml/kg body weight. Daily fluid intake is estimated at 100-120 ml/kg depending on ambient temperature, an important point to note when considering intravenous fluid replacement during surgery (see below). Daily urine volume is 50-75 ml/kg. Hematological and biochemical pa-rameters are similar to those of humans, although serum concentrations of creatinine (normal range: 70-170 Цто1/1 10.8-1.9 mg/dl]) and urea (normal range: 7.5-15 mmol/1 [42-80 mg/dll) differ slightly. Survival after bilateral nephrectomy is 4 ± 2 days (Green, 1975). Immunological Features

The rabbit white blood cell count is 5-10 x 10 leucocytes per litre, of which 80% are lym-phocytes (Hinton et al., 1982). Т cells comprise approximately 63% of unseparated lympho-cytes and, in contrast to В cells, do not express la-like alloantigens in the non-activated state or surface immunoglobulin molecules (Knight, 1980). Ponsard et aL (1986) reported that most types of» helper cells for mitogen-induced proliferation of rabbit spleen Т cells were la-positive, although there was evidence that some regulator cells were la-negative. ‘Null’ lym-phocytes have been characterised in rabbits (Jackson et aL, 1983). Natural killer cell activity and antibody-dependent cell-mediated cytotoxicity have not been detected in rabbits (Lay-boum et aL, 1990). The rabbit major histocompatibility complex (MHC) consists of three polymorphic loci, RLA-A, RLA-B and RLA-D. The RLA-A and В loci code for serologically defined alloanti-gens and are analogous to human class I genes, and D т д ,п regulates mixed lymphocyte reac-tivity and is analogous to class II. Genetic control of immune responsiveness (Ir genes) has not been accurately assigned within the rabbit MHC (Lancki et aL, 1979). As in humans, RLA-D gene products are encoded by genes in three subregions of the rabbit MHC, namely DP, DQ and DR. Fourteen class II genes have been defined in the rabbit MHC: R-DQ—one alpha and one beta gene; R-DR—one alpha and eight beta genes; and R-DP—two alpha and one beta gene (Spieker-Polet et aL, 1990). Alpha and beta genes from each subregion have been shown to hybridise with the corresponding human alpha and beta chain cDNA probes from the same subregion (Sittisombut and Knight, 1986). On the basis of the multiplicity of rabbit alpha chain loci (Sittisombut and Knight, 1986) and analysis of nucleotide sequences of class I genes (Tykocinski et al., 1984), it has been suggested that the rabbit MHC has greater homol-ogy with the human than the mouse MHC. Also, it is believed that the mouse does not possess homologues of R-DP genes (Spieker-Polet et aL, 1990). Allograft rejection has been well documented in rabbits. As in humans, rabbits exhibit hy-peracute rejection ofallografts if active or passive immunisation occurs prior to transplantation (Klassen and Milgrom, 1969; Ueno et at., 1972; McDowall et aL, 1973). In contrast, hy-peracute rejection can be induced in presensitised rodents only after provision of exogenous complement, because of the ineffective rodent complement system (Koene et aL, 1973). Thus, the rabbit is a useful model for studies of pre-transplant sensitisation (Francis et al., 1987). Acute allograft rejection has been characterised in rabbits (Hobbs and Cliff, 1973), and histo logical evidence of acute rejection is observed uniformly six days after renal transplantation between unmodified outbred rabbits (Dunn, 1976). Renal Disease

Hinton (1981) found only one case of unilateral aplasia in a series of post-mortem examina-tions of 312 rabbits, 96% of which were laboratory animals. In 75 apparently healthy adult laboratory rabbits which had been disposed of as surplus to requirements, histological exami-nation of the kidneys revealed no abnormality in 56 rabbits (75%), cortical fibrosis with or without calcification in 8 (10%), colloid material in renal tubules in 5 (7%), evidence ofpye-litis in 3 (4%), focal interstitial nephritis in 2 (3%), and cortical cysts in 1 (1%). Hinton (1981) concluded that the rabbit appeared to suffer from a similar range of naturally occurring kidney disorders as other mammalian species. The possibility of a preexisting abnormality should be borne in mind when interpreting histological features of rabbit kidneys following transplanta-tion. PREOPERATIVE PREPARATION OF RABBITS

Housing, caging and dietary requirements of laboratory rabbits have been well reviewed (Adams, 1987). Rabbits are fasted overnight prior to operation so that the stomach is empty and does not reduce access to the abdominal cavity, but water is allowed freely. Rabbits should be handled as little as possible and care should be taken in restraining rabbits preopera-tively to reduce the risk of cardiac arrhythmia and injury to the vertebral column. Rabbits with respiratory problems or gastrointestinal infections should not be used. Premedication is usu-ally not required but can be achieved with acepromazine or ketamine. MATERIALS Surgical Instruments

1 electric animal hair clippers

2 Dust Buster’ manual vacuum cleaner

3 electric heating mat

4 Zeiss diploscope 12.5 x operating microscope

5 Fibre optic light source

6 Castroviejo microvascular needle holder

7 4 pairs of pointed microsurgical forceps—JF-5

8 1 pair ophthalmic scissors

9 1 pair small straight scissors

10 1 pair large (Mayo) scissors

11 1 pair large toothed (Bonney) tissue forceps

12 4 pairs (Mosquito) artery forceps

13 4 straight microvascular 1.5 x 8.0 mm occlusion clamps

14 2 medium size (Langenbeck) retractors

15 medium size self-retaining retractor

16 ophthalmic cautery

Materials

17 sutures: 3/0 black silk ligatures, 3/0 monofilament prolene on a straight needle 9/0 monofilament nylon on a 6 mm taper needle 8/0 virgin black silk on a 6 mm taper needle

18 gauze swabs

19 large and small cotton bud swab sticks

20 polyethylene tubing, 0.96 mm ED, 0.58 mm ID

21 absorbable haemostatic gelatin sponge (Gelfoam)

22 scalp vein (‘butterfly’) 25 gauge needles

23 hypodermic 19 and 25 gauge needles

24 1 ml and 20 ml syringes

25 Trucut’ biopsy needle (Travenol)

26 thin coloured plastic squares, 10 x 20 mm

Drugs and Reagents

27 normal saline for intravenous use

28 Hartmann’s compound sodium lactate solution for intravenous use

29 70% alcohol solution

30 Ross’s tissue perrusion fluid

31 Saffan (9 mg/ml Alphaxalone, 3 mg/ml Alphadolone

32 Papaverine hydrochloride (12 mg/ml)

33 Lethabarb (pentobarbitone sodium—325 mg/ml)

34 Penicillin (40 000 units/kg)

35 Temgesic (buprenorphine hydrochloride—0.3 mg/ml)

ANAESTHESIA

Safe anaesthesia is crucial to the successful outcome of surgery of experimental animals. Rabbits have the reputation of being difficult to anaesthetise for several reasons. Signs of an-aesthesia in the rabbit are often unreliable. For example, the corneal reflex may persist after the stage of surgical anaesthesia has been reached, and rabbits are at risk of cardio-respiratory failure by the time deep anaesthesia has been achieved (Murdoch, 1969). Secondly, the thera-peutic-toxic ratio of many anaesthetic agents, particularly barbiturates, is very narrow in rab-bits, so that small increases in anaesthetic dosage may result in death (Zahav et al., 1983). Thirdly, considerable individual variation exists in responses of rabbits to anaesthetic drugs (Green, 1975b). Thus, choice of a simple and safe anaesthetic technique is essential. Inhalational anaesthesia is frequently problematic in rabbits. They object vigorously to forced inhalation of anaesthetic gases which are irritant to the respiratory tract, lead to exces-sive bronchial secretion and salivation, and may predispose to pneumonia (Ling, 1969). Reflex withdrawal and violent coughing may occur even if oxygen and nitrous oxide are used for in-duction and then gradually replaced by volatile anaesthetic agents (Green, 1975b). Inhalation anaesthesia for thoracic or abdominal surgery requires endotracheal intubation and fine control of ventilation and anaesthetic gas concentrations. Endotracheal intubation is difficult in rabbits because the glottis is small and the tongue tends to hide the unduly mobile larynx. Also, the hamuli epiglottici, which border each sagittal recessus just beyond the base of the epiglottis, move toward the midline during inspiration when the laryngeal opening is widest and elevate the covering epithelium, so that significant mucosal damage may occur if the intubation tech-nique is not precise (Schuyt and Leene, 1977). Also, inhalational anaesthesia requires special equipment (an anaesthetic circuit, laryngoscope and flow meters) and generally does not allow rapid repetitive anaesthesia of large numbers of animals (Green, 1975b). Saffan Anaesthesia

Saffan (Glaxo Australia Pty Ltd, Boronia, Victoria) is an ideal intravenous anaesthetic agent for renal transplantation procedures in rabbits. Saffan consists of alphaxolone (3a-hydroxy-5a-pregnane-ll,20-dione) and alphadolone acetate (21-acetoxy-3a-hydroxy-5a-pregnane-ll,20-dione) dissolved in polyoxethylated castor oil, to render the two pregnanedione compounds water-soluble. Child et al., (1971) reported on the cardiovascular and respiratory side effects of Saffan in a variety of animals, and found that Saffan produced virtually no res-piratory depression even in large doses. The therapeutic index (lethal dose 50: anaesthetic dose 50) (TI) of Saffan is very high at 30.6, making it a much safer drug than ketamine hydro-chloride (TI = 8.5), methohexitone sodium (7.4) and thiopentone sodium (6.9). Saffan has been withdrawn from use in humans because of a relative high incidence (1 in 10 000) of anaphylaxis, causing cardiovascular collapse and bronchospasm, associated with the solvent (Dundee and Clarke, 1980). An intermittent infusion of intravenous Saffan has been used for several hundred rabbit re-nal transplant procedures (Francis et al., 1990). No premedication is required. Each ampoule of Saffan (12 mg/ml) is diluted in Hartmann’s solution to produce a 15% solution (1.8 mg Saf-fan/ml) and drawn up into several 20 ml syringes. A single intravenous 4 ml bolus (7.2 mg Saffan) results in unconsciousness and loss of the corneal reflex within 5-10 seconds. After shaving and positioning the rabbit, two 2-4 ml boluses of 15% Saffan (3.6-7.2 mg) are given before laparotomy is undertaken. Absence of pedal withdrawal and ear pinch reflexes is con-firmed before commencing the abdominal incision. It is highly undesirable to judge the ade-quacy of anaesthesia by repeated attempts at the incision. During the surgical procedure, 1-2 ml aliquots (1.8-3.6 mg Saffan) are infused every 5-10 minutes to maintain surgical anaesthe-sia, during which a respiration rate of 15-20 breaths per minute should be observed. Develop-ment of rapid shallow respiration, tachycardia or fine tremulous movements of the head or limbs indicate that the level of anaesthesia is not deep enough and that more Saffan is re-quired. A 1-2 ml bolus (1.8-3.6 mg Saffan) is infused immediately prior to abdominal closure. At the end of the operation, the rabbit is placed on its side on a clean towel on a warming blanket and covered with a warm towel, and allowed to recover undisturbed but within view of the operator. Recovery occurs rapidly so long as the rabbit is not hypovolaemic. Corneal re-flexes are regained within 10-15 minutes and the rabbit can lift its head spontaneously or in response to sound within 15-25 minutes. Rabbits breathe room air throughout the operative procedures. It is well worthwhile going slowly with the anaesthetic, especially during induction and the early part of the operation, and ensuring that the rabbit is well anaesthetised before commenc-ing the incision, moving the intestine, incising the peritoneum and closing the abdomen, which are the most stimulating parts of the operation. Large boluses of Saffan, in attempts to quickly achieve anaesthesia, should be avoided at all times in case respiratory arrest occurs. The above method of anaesthesia has been used for all operative procedures in our rabbit renal transplant model. Analysis of the last 81 consecutive anaesthetics given for 43 renal transplants and 38 contralateral nephrectomies showed that no deaths were attributable to an-aesthesia, confirming the safety of Saffan as an anaesthetic drug in rabbits. There was wide variation in the response of rabbits to Saffan: the median (range) dosages required for renal transplantation and contralateral nephrectomy were 250.4 ^g/kg/min (99-456) and 294.0 /ig/kg/min (182-540) respectively. Euthanasia

Euthanasia can be achieved simply by injecting 2 ml of Lethabarb into the marginal ear vein. GENERAL SURGICAL CONSIDERATIONS

Intravenous Cannulation

The marginal ear vein is suitable for intravenous access. The rabbit is wrapped firmly, al-though not tightly, in a towel so that movement is limited but respiration is not embarrassed. Better definition of the vein is achieved in dark coloured rabbits by shaving the overlying skin, and the vein is brought into prominence by placing the rabbit under a heating lamp or bathing the ear with alcohol or in warm water. The vein at the base of the ear is pinched lightly by an assistant and a 25 gauge butterfly needle is inserted into the vein close to the tip of the ear. The wings of the needle are secured to the ear with adhesive tape. Intravenous Fluids

Intravenous fluids must be given during renal transplantation and nephrectomy because rabbits have poor tolerance of fluid loss and dehydration. Fluid is lost because of bleeding and increased evaporative losses from the exposed intestine and peritoneal cavity, and normal in-take is reduced because of fasting before, during and after the anaesthetic. Fluid replacement is essential to correct these deficits. Saffan is made up as a dilute solution so that intravenous fluid is infused throughout the anaesthetic. It is assumed that rabbits miss approximately one quarter of their daily fluid intake because of the fasting and anaesthesia, and so a minimum of 70 ml of intravenous fluid is recommended. It is important to replace blood loss with an equal volume of intravenous fluid as soon as possible. Also, blood loss should be anticipated by hav-ing adequate amounts of intravenous fluids at hand and the rabbit should be infused immedi-ately before the vascular clamps are removed from the anastomosed renal vessels (see below). Intravenous fluid is given as 0.9% saline or Hartmann’s solution. It is not necessary to trans-fuse rabbits as blood loss should not be great and transfusions may directly interfere with ex-periments because of immunological effects (Francis, 1991). Shave

With the rabbit anaesthetised, the abdomen is shaved with electric hair clippers to remove as much hair as possible from the area of the proposed midline abdominal incision. Loose hairs are removed most satisfactorily by gentle suction with a hand-held vacuum cleaner. Position To compensate for heat loss from the peritoneal cavity and exposed intestines during the operation, the rabbit is placed supine on an electric warming blanket covered with a folded towel which tends to evenly disperse warmth and prevents the rabbit becoming overheated or even burnt by the warming blanket. The upper limbs and neck are extended, and care is taken to ensure that the airway is not compromised by excessive extension of the neck. The tongue should be gently drawn forward to ensure a patent airway. Sterility and Draping Attempts should be made to maintain sterility during operative procedures. The shaven area or the abdomen is cleansed with an antiseptic, such as a 70% alcohol solution. Sterile drapes or towels are placed along each side or the rabbit and a double thickness placed over the per-ineum and hind legs. A smaller drape is placed across the chest in such a way that the operator and assistant can readily assess respiratory movement and the airway. Sterile surgical rubber gloves are worn by the operator, instruments are sterilised before use, and sterile swabs and fluids are used. Rabbits are given prophylactic Penicillin 40 000 units after induction of anaes-thesia. Abdominal Incision The abdomen is opened using a long midline incision, from the xiphisternum to just above the symphysis pubis. This incision gives excellent access to both kidneys and results in mini-mal bleeding when accurately placed along the linea alba. Laterally placed vertical or oblique incisions result in damage to subcutaneous mammary glands, cause significantly more bleed-ing and reduce access to the contralateral kidney. After opening the abdominal cavity, the in-testine is placed outside the peritoneal cavity opposite to the side of interest and covered on all aspects with damp gauze so that it is kept moist. A self-retaining retractor is inserted between the root of the small intestine mesentery and the abdominal wall, taking care that the retractor blades are covered with a thickness of damp gauze so that the mesenteric vasculature is not in-jured. Abdominal Closure The operative field is checked to ensure that there is no bleeding and that all instruments, microvascular clips and swabs have been removed. Blood is removed from the peritoneal cav-ity with gauze swabs. About 5 ml of warm 0.9% saline is placed into the abdominal cavity to warm the intestine which is replaced carefully. The muscle layer of the abdominal wall is su-tured with a continuous 3/0 Prolene suture, ensuring, firstly, that the sutures are placed at least 1 cm away from the cut edge of the abdominal muscles and are inserted no more than 5 mm apart and, secondly, that the intestine is not inadvertently injured by the suture needle. The length of suture required to close the muscle layer is approximately four times the length of the incision. Suture knots at the beginning and end of the suture line are buried so that the rab-bit cannot bite them and cause the wound to open. Sutures must not be pulled tightly as the closure proceeds because this causes tissue ischaemia which results in breakdown of the suture line and abdominal evisceration. The skin is sutured with a continuous 3/0 Prolene suture. DONOR NEPHRECTOMY OPERATION

The left kidney is preferred to the right because the left renal vein is longer and the origin of the adrenal artery reduces the available length of the right renal artery. However, either kidney may be used successfully for transplantation. Using the operating microscope, the renal artery and vein are exposed by incising horizontally the overlying peritoneum, from the renal hilum laterally to the adrenal vessels medially. The vessels are mobilised along their full length using fine scissors and microsurgical forceps to divide renal nerves and lymphatic trunks, and small swab sticks to separate vessels from adjacent fat. The vessels should not be directly touched with instruments so that injury and spasm are minimised. Spasm may be pre-vented by a few drops of topical papaverine. Care must be taken not to avulse small tributaries which should be sealed with the ophthalmic cautery. The kidney is then perfused m situ by in-jecting 10 ml of cold (6-8°C) Ross’s solution directly into the proximal part of the renal artery via a 27 gauge needle (Figure 1). It is helpful to bend the needle at two or three places along its length so that the tip is angled at approximately 90° to the long axis of the syringe. The needle is inserted into the renal artery with the point towards the kidney. As soon as the needle enters the vessel, a microvascular occlusion clamp is applied by the assistant to the artery immediately proximal to the puncture site and the perfusion fluid is injected. Uniform pallor of the kidney and appearance of dilute blood in the renal vein indicate ideal perfusion and should occur after 2-3 ml of perfusion fluid has been injected. If the kidney is mottled with areas of pallor and cyanosis, then perfusion is poor and must be improved quickly if the kidney is to be transplanted successfully. Check, firstly, that the tip of the needle is in the lumen of the renal artery and not within the arterial wall or outside the artery, and secondly, that the microvascular clamp is properly occluding the renal artery and preventing blood flow into the kidney. If the kidney is mottled initially, it is worthwhile perfusing it with up to 20 ml of Ross’s solution. If perfusion has not been ideal but most of the blood has been flushed from the kidney, it can still be used for transplantation. The kidney should feel cooler than the surrounding tissues. Figure 1 Injection ofRoss’s solution into the renal artery results in immediate blanching of the donor kidney. The perfusion needle is removed from the artery, the renal vein is clamped as far medial as possible and both vessels are transected lateral to the clamps, preserving as much length as possible with the kidney. The peritoneum overlying the medial, superior, lateral and inferior borders of the kidney is incised and the kidney is gently lifted from perinephric fat. The ureter is located at the infero-medial aspect of the kidney and is mobilised and transected so that a 2-3 cm length is included with the kidney. Excessive fat is trimmed from the kidney which is stored in Ross’s solution at 6-8°C. If only one kidney has been removed and the rabbit is to be maintained, the renal artery and vein are ligated with 3/0 silk ligatures. RENAL TRANSPLANT OPERATION

Several methods of vascular anastomosis and restoration of urinary tract continuity have been described. Technical problems have not been reported or analysed in some papers, and so it is not possible to directly compare different techniques. It is essential that technical failures are excluded from vascularised allograft experiments so that graft failure can be attributed cor-rectly to immunological destruction. The author’s preferred technique of end-to-end vascular and ureteric anastomoses (Francis, 1990) will be described first, followed by descriptions and discussion of alternative methods. Native Nephrectomy Orthotopic renal transplantation is performed preferably on the left side because of the longer length of the left renal vein. Using the operating microscope, the renal artery and vein are mobilised carefully over their entire length and occluded by microvascular clamps placed as far medially as practicable. Vascular spasm may be prevented by a few drops of topical pa-paverine. The renal vessels are transected close to the renal hilum. The proximal ureter is mo-bilised and divided as close to the kidney as possible. It is helpful to place a microvascular clamp on the ureter immediately below the point of transection to prevent the ureter retracting downwards out of view beneath the peritoneum. The peritoneum overlying the kidney is di-vided and the native kidney is removed. Vascular Anastomoses

The donor kidney is removed from the cold Ross’s solution, wrapped in a small moist gauze swab and orientated so that the ureter points inferiorly. The kidney is placed in the vacant re-nal bed so that the ends of the donor and recipient vessels can be brought together without ten-sion. A short strip of coloured plastic is placed beneath the vessels to aid tissue definition and accurate placement of the fine 9/0 Prolene vascular sutures. The arterial anastomosis is per-formed first because the artery has greater intrinsic strength and is less likely to be torn if the kidney is inadvertently moved or disturbed after the anastomosis has been started. Adjacent ends of the donor and recipient vessels are positioned so that kinking will not occur after anas-tomosis, trimmed to appropriate length and cleared of adventitia. Any clot in the donor or re-cipient vessel is very gently milked or teased from the vessel with microvascular forceps. An end-to-end anastomosis is fashioned by inserting 9/0 nylon sutures at the upper and lower bor-ders of the adjacent ends of the donor and recipient arteries, and tying each suture to bring the ends of the two arteries in apposition. The short end of each of the two sutures is held with microsurgical forceps by the assistant while the operator inserts a continuous suture line through the full thickness of each artery, taking small frequent bites. Approximately 4-6 bites are used to complete the anterior suture line, whereupon the suture is tied to the short end of the other nylon suture. The half-completed arterial anastomosis is then rotated through 180° by passing one of the short ends beneath the vessels, so that the posterior half of the anasto-mosis is brought into view and can be completed using the suture which has not been used for the anterior wall. On completion, the anastomosed vessel is rotated back through 180° so that it is no longer twisted. The end-to-end venous anastomosis is completed in a similar manner (Figures 2, 3 and 4). Great care must be taken not to injure the vein wall which is extremely thin. Small tears occa-sionally develop where sutures pass through the vein, but these can be managed by including the tear in the next stitch. Tears need to be sutured separately if they are large. However, if su-turing reduces the venous lumen, the anastomosis should be resected and done again. It is im-portant to use sufficient length of suture in the anastomosis to allow the vein to distend after the kidney has been revascularised, otherwise a venous stenosis will occur and the vein may thrombose. Stenosis at the venous anastomosis can be prevented by leaving 2-3 mm of suture before the knot and then not snugging the knot down onto the vein wall on completion of the anterior and posterior suture lines. The rabbit is infused with a bolus of 30-40 ml of intravenous fluid (see above) immediately before removing the microvascular clamps from the anastomosed vessels to expand the blood volume and encourage a diuresis. The venous clamp is removed first and blood should flow retrogradely across the anastomosis. Any large venous leak should be dealt with at this stage. The kidney rapidly regains its normal colour after release of the arterial clamp if the anasto-moses are satisfactory. Haemorrhage from either anastomosis, if it occurs, can nearly always be controlled by covering the anastomosis with three 3х10 mm strips of Gelfoam placed longi-tudinally along the vessel, together with gentle pressure with a large swab stick. In a personal series of 179 renal allografts using the above technique (Francis et ai, 1990), there were eight (4.4%) vascular complications detected within three days of transplantation: three cases each of renal vein and renal artery thrombosis and two of allograft thrombosis. Twisting or kinking of the vessels caused all cases of arterial thrombosis and two cases of ve-nous thrombosis. Dehydration and presumed hypotension was thought to have led to the other venous thrombosis. Hyperacute rejection was the most likely cause of allograft thromboses as the main vessels were patent and the rabbits had been presensitised by blood transfusion (Francis et aL, 1987). Ureteric Anastomosis

An end-to-end ureteric anastomosis is commenced after the kidney has been revascularised and bleeding has been controlled. Peritoneum overlying the upper 10-15 mm of the recipient ureter is divided and the short section of ureter held by the microvascular clamp is excised. Blood clot often accumulates in the ureteric lumen and is removed carefully with microvascu-lar forceps. A ureteric stent greatly facilitates accurate suturing of adjacent ends of the donor and recipient ureters and ensures lumenal patency at least until the stent passes distally. The stent is fashioned by cutting a 1.5 cm length of polyethylene tubing with both ends bevelled at 45°, and it is inserted half-way into the recipient ureter and held in place by the assistant. The donor ureter may need to be reduced in length so that it does not become kinked after anasto-mosis. It is important that there is no tension at the ureteric anastomosis because this will re-sult in subsequent ischaemia and leakage. The donor ureter is passed over the stent, and the two adjacent ureteric ends are held in apposition by the assistant while the operator places 5-6 interrupted sutures of 8/0 silk to complete the anastomosis. The peri-ureteric tissue must be preserved to ensure an adequate blood supply. The full thickness of the ureteric wall must be included in each suture. Sutures must not be tied tightly, otherwise ureteric ischaemia will re-sult. After completion, the stent should be able to move freely across the anastomosis (Figure 5). The kidney usually does not require suturing to the posterior abdominal wall, although this may be necessary in very small rabbits if the kidney tends to roll medially when the abdomen is closed. The final appearance of the transplanted kidney is shown in Figure 6. The abdominal wound is sutured as above. Dilatation of the renal pelvis and ureter was observed in up to one third of transplants in a series of 179 renal allografts (Francis et al., 1990). Ureteric obstruction occurred in 12 cases (6.7%) and was due to oedema or stricture in 9 ALTERNATIVE TECHNIQUES OF RENAL TRANSPLANTATION IN THE RABBIT

Vascular Anastomoses

Direct end-to’end anastomosis Klassen and Milgrom (1969) performed heterotopic renal allografts in the neck using the common carotid artery and jugular vein. A similar technique was used by Nathan et al. (1961) who retrieved the donor kidney in continuity with a segment of the aorta and IVC, and anas-tomosed the cephalad ends of these vessels to the ends of the carotid artery and jugular vein. McDowall et al. (1973) and Owen et ol. (1968) performed end-to-end arterial anastomoses with interrupted sutures, while Holter et ol. (1972) used continuous sutures. The only reported technical complications are those of Holter et ai. (1972) who had an overall thrombosis rate or 10% in 23 recipients. Klassen and Milgrom (1969), Holter et al. (1972), and McDowall et al. (1973) described end-to-end venous anastomoses. Cuff technique of end’to’end anastomosis Heron (1970) described a technique of end-to-end anastomosis in which the end portion of the recipient renal artery or vein was passed through a 7 mm segment of 1.5 mm diameter polyethylene tube so that about 3 mm of the vessel protruded from the tube; the protruding end of the vessel was then drawn back over the cuff to partly cover it. The end of the donor vessel was dilated and brought over the cuff and everted part of the recipient vessel. The anastomosis was held in place by a circumferential ligature. In a series of 32 renal allografts, Heron (1970) had two venous thromboses (6%) and six arterial thromboses (19%) using this technique. The cuff technique is said to be quick to perform and has been used widely, especially in early se-ries when non-thrombogenic vascular sutures were not generally available (Ueno et al., 1972; Andersen et al., 1973; Green, 1973; Dunn and Randal. 1974; Beyer and Friedman, 1975; Green, 1975a; Dunn, 1976; Green and Allison, 1978). Dunn (1976) used the cuff technique for arterial anastomoses and reported no complications in the last 100 of a series of 400 allografts. End’to’side anastomosis In a series oforthotopic renal transplants, Klassen and Milgrom (1969) described end-to-side anastomosis of the renal artery and vein to the abdominal aorta and IVC respectively. Lund (1970), in a series of 180 renal allografts, had a 2% incidence of renal artery or aortic thrombosis and a 15% incidence of renal vein thrombosis after end-to-side anastomoses of the renal artery and vein with the abdominal aorta and IVC respectively. Ten per cent of rabbits in this series developed post-operative flaccid paralysis of the hind legs, presumably due to is-chaemia of the spinal cord or direct nerve damage resulting from the extensive aortic and caval mobilisation required for the end-to-side technique. Jacobsen (1978) reported only two cases of venous thrombosis (one of which was due to inaccurate suturing) and two cases of ar-terial thrombosis in 100 renal allografts. There was one case of hind limb paralysis. Dunn (1976) used an end-to-side renal vein to IVC technique in 100 allografts without complica-tions. Enbloc vascular anastomosis Bayuk and Schmidlapp (1967) described a technique of removing both kidneys en bloc with segments of donor aorta and IVC and, after transecting the recipient’s aorta and IVC below the renal vessels, inserted the en bloc preparation and restored vascular continuity using the cuff method of Heron (1970). This technique, which was successful in Bayuk’s series of 11 trans-plants, involves mobilisation of the aorta and IVC, and requires that each donor supplies only one recipient as the recipient receives both kidneys. Stented vascular anastomosis Owen (1968) described a complicated method of venous anastomosis which involved divid-ing and ligating the caudal IVC below the level of the renal vessels and passing a polyethylene stent up the cephalad part of the recipient IVC a^id into the left renal vein. The recipient left kidney was removed, leaving a length ^f stent protruding from the renal vein, which was then advanced into the donor renal vein. After suturing the venous anastomosis, the stent was re-moved from the IVC which was ligated at the exit site. The results of this technique were not discussed. Restoration of urinary continuity The stented end-to-end sutured ureteric anastomosis is the simplest method of reestablishing urinary continuity and has produced acceptable results (Francis et al., 1990). Holter et al. (1972) performed end-to-end ureteric anastomoses over a short internal polyeth-ylene stent and held adjacent ends of donor and recipient in apposition with two circumferen-tial ligatures placed around each ureter over the stent. It was claimed that this method was quicker than uretero-neocystostomy and was ‘virtually leakproof (Holter et aL, 1972). Jacobsen (1978) also used this technique in 12 rabbits and 10 (83%) failed within the first 10 days because of ureteric obstruction. Use of an internal stent appears to contribute significantly to the success of end-to-end ureteric anastomoses. Green (1973) reported a 90% failure rate in ten rabbits undergoing end-to-end ureteric anastomosis without stenting, and six ureteric obstructions and three urinary leaks developed. McDowall et al. (1973) used the end-to-end method without a stent in 24 transplants but did not comment on the results. Lund (1970) found that occlusion of the ureter occurred invariably within 1-2 days of end-to-end ureteric anastomosis without an internal stent. The donor ureter may be implanted into the bladder (‘uretero-neocystostomy’), either di-rectly (Bayuk and Schmidlapp, 1967; Owen et aL, 1968; Klassen and Milgrom. 1969; Lund, 1970; Ueno et aL, 1972; Dunn, 1976; Jacobsen, 1978) or with a small patch of donor bladder (Heron, 1970; Beyer and Friedman, 1975). Few of these studies provide data on technical re-sults. Dunn (1976), in a series of 100 renal allografts, sutured the ureter into the bladder with a single stitch and then sutured the full thickness of the bladder wall around the lowermost ex-travesical part of the ureter to create an anti-reflux tunnel. About 20% of transplants showed some evidence of ureteric obstruction and two failed because of obstruction due to blood clot in the ureter. Urinary fistulas occurred in four rabbits because the ureter became detached from the bladder. Jacobsen (1978) used this technique but found that within a month of trans-plantation 10 of 100 allografts became obstructed secondary to fibrous stricturing at the lower end of the ureter. CONTRALATERAL NEPHRECTOMY

Contralateral nephrectomy is required so that the rabbit depends entirely on the allograft for renal function and may be undertaken immediately after transplantation or at a subsequent op-eration. Delayed contralateral nephrectomy (Francis et al., 1987) has the advantage of allow-ing the allograft time to recover from ischaemic damage while not being essential for renal function. Secondly, the allograft can be assessed at some time after transplantation and, if a technical problem has developed, it can be corrected or the animal can be withdrawn from the experiment, thereby eliminating early technical failure as a source of experimental error. Thirdly, transplant biopsy can be performed under direct vision, thereby avoiding the hazards of percutaneous biopsy (Francis et ai, 1990). The time (mean ± 1 SD) taken from induction of anaesthesia until completion of abdominal closure in the author’s series of 179 contralateral nephrectomies was 30.4 ± 8.6 minutes. POSTOPERATIVE MANAGEMENT

Before the rabbit has recovered fully from the anaesthetic, analgesia is administered as Temgesic 45 JUg/kg by intramuscular injection. The rabbit is returned to the cage after recov-ery and is allowed food and water. Regular inspection should take place, checking for distress, wound complications and signs of ill health. Temgesic can be administered every 6-8 hours for the first 2 days. Assessment of the Transplant The transplanted kidney can be assessed crudely by palpation. Transient ischaemic damage and rejection cause the graft to enlarge to a slight degree, but minor swelling may not be de-tectable in the conscious rabbit. A grossly enlarged kidney is usually obvious on palpation and signifies either ureteric obstruction or venous infarction. Collection and accurate measurement of urine volume are difficult and reflect factors in addition to renal function. Measurement of serum creatinine concentration after contralateral nephrectomy is the best means of assessing transplant function. Open Renal Biopsy

Under general anaesthesia, the abdominal wound is opened by removing the 3/0 Prolene su-tures. The intestine is placed to one side and the self-retaining retractor is inserted as before. The transplanted kidney and ureter are inspected and, if there is no technical complication, a biopsy is performed with a Trucut needle. The shaft of the needle is opened, exposing the re-cessed section, which is placed against the convex surface of the kidney. The needle is then closed so that a sliver of kidney is removed in the recessed section. A second pass may be made if the first is thought to be inadequate. A 3 X 10 mm strip of gelfoam is placed in the groove made by the biopsy needle and gentle pressure is applied for 3-4 minutes with a large swab stick to achieve haemostasis. The abdomen is closed as above. POTENTIAL TECHNICAL COMPLICATIONS OF RENAL TRANSPLANTATION

Wound Dehiscence

Abdominal wound dehiscence may be partial (involving either the skin or muscle layer) or complete (involving both layers). Dehiscence of the wound is due to either tissue necrosis be-cause of poor suturing technique, wound infection, knots coming undone, or breakage of su-tures. Skin dehiscence is of least significance and can be left alone if small but otherwise is best treated by resuture. Early failure of the deep muscle layer with the skin remaining intact, and dehiscence of both layers with evisceration, must be treated by resuture under anaesthesia as soon as possible if the rabbit is to survive. Antibiotics must also be given. Renal Artery Thrombosis Thrombosis of the renal artery is due to kinking or angulation of the donor or recipient re-nal arteries, constriction at the anastomotic site, or failure to suture the full thickness of each artery causing an intimal flap to occlude the arterial lumen. The kidney appears mottled and white or light brown in colour, and may be slightly smaller in size. It is irreparably damaged within minutes of the thrombosis. Renal Vein Thrombosis Renal vein thrombosis is due to constriction or twisting of the renal veins and is more likely to occur if the donor or recipient vein is torn during anastomosis or if the rabbit is dehydrated or hypotensive. The kidney becomes grossly enlarged and is dark blue or black in colour and may even rupture. Clot is readily visible in the renal vein. The kidney cannot be salvaged. Ureteric Obstruction Obstruction at the ureteric anastomosis, due to poor surgical technique, ureteric oedema, blood clot, or impaction of a blocked stent, causes the donor ureter and renal pelvis to dilate and the kidney to become enlarged and tense within a few days. If obstruction is suspected, a 19 gauge needle is passed from the lateral convexity of the kidney into the renal pelvis and the diagnosis is confirmed if urine flows from the needle under pressure. The needle is then re-moved and a 10 cm length of polyethylene stent tubing passed along the needle track through the renal parenchyma, into the renal pelvis and advanced into the ureter and through the anas-tomosis if possible. This may be enough to remove a blood clot or dilate a strictured anasto-mosis. A short segment of this nephrostomy tube may be cut and manipulated back across the anastomosis if the original stent has passed on into the bladder. Alternatively, the ureter may be opened through a short incision below the anastomosis and a stent passed up and left across the anastomosis, and then the ureter is closed. Ureteric Leakage A urinary fistula at the ureteric anastomosis is due to ureteric ischaemia, tearing of the ureter during the anastomosis or poor surgical technique. Urine irritates the peritoneum which often becomes infected, and so the rabbit is usually sick. It is unlikely that the fistula can be repaired but careful suture of the ureter over a stent may be worthwhile. CONCLUSIONS Rabbits have distinctive characteristics which make them suitable for models in experimen-tal surgery. Safe anaesthesia and adequate intravenous fluid infusion are critical to the success of surgical procedures. The described methods of renal donation and transplantation are straightforward and can be learnt readily. End-to-end anastomosis of renal vessels can be per-formed without mobilisation of the aorta or IVC, a procedure which is tolerated poorly by rab-bits. End-to-end uretero-ureteric anastomosis with an internal stent is relatively easy and does not involve operation on the recipient bladder. Delayed contralateral nephrectomy allows the transplanted kidney to be inspected and biopsied, technical problems to be repaired and tech-nical failures to be excluded. REFERENCES 1. Adams, С. Е. (1987) The laboratory rabbit. In The UFAW Handbook on the Care and Management of Laboratory Animals, Т. В. Poole (ed.), pp. 415-435. Harlow, England: Long-man Scientific and Technical. 2. Andersen, 0. S., Tissot, R. G., Cohen, C. and Jonasson, 0. (1973) Platelet survival test. An accurate prediction ofhyperacute rejection of renal allografts in rabbits. Transplantation, 15, 105-110. 3. Bayuk, J. M. and Schmidlapp, C. J. (1967) Kidney transplantation in the rabbit. A method for the study of transplantation immunology. Investigative Urology, 4,378-388. 4. Beyer, M. M. and Friedman, Е. A. (1975) Prolonged survival of rabbit skin and kidney al-lografts following donor treatment with cytosine arabinoside. Transplantation, 19, 60-63. 5. Child, К. J., Currie, J. P., Davis, В., Dodds, H. G., Pearce, D. R. and Twissell. D. J. (1971) The pharmacological properties in animals ofCT1341— A new steroid anaesthetic agent. Brit-ish Journal of Anaesthesia, 43, 2-13. 6. Dundee, J. W. and Clarke, R.S.J. (1980) Non-inhalational anaesthetics. In General Anaes-thesia, T. C. Gray, J. Е Nunn and J. Е. Utting (eds), 4th edn, p. 253. London: Butterworth and Company (Publishers) Ltd. 7. Dunn, D. С. (1976) Orthotopic renal transplantation in the rabbit Transplantation, 22, 427-433. Dunn, D. С. and Randal, G. К (1974) Prolonged allograft survival following spontaneous recovery from early rejection. Transplantation, 17,306-312. 8. Francis, D. M. A. (1991) Relationship between blood transfusion and tumour behaviour. British Journal of Surgery, 78, 1420- 1428. 9. Francis, D. M A , Masendycz, P. J., Dumble, L. J and Clunie, G. J. A. (1987) Enhancement of renal allografts by simultaneous cyclosponne and donor-specific blood transfusion. Trans-plantation Proceedings, 19,1464-1466. 10. Francis, D. M. A , Millar, R J.. Dumble, L. J. and Clunie, G. J. A (1990) Model oforthotopic renal transplantation in the rabbit. Australian and New Zealand Journal of Surgery, 60,45- 49. 11. Green, С J (1973) Rabbit renal autografts as an organ preservation model Laboratory Ani-mals, 7,1-11. Green, C.J. (1975a) Immunosuppression in rabbits: skin and kidney allografts. Laboratory Animals, 9, 223-232. 12. Green, С. J. (1975b) Neuroleptanalgesic drug combinations in the anaesthetic management of small laboratory animals. Laboratory Animals, 9, 161-178. 13. Green, C.J. and Allison, A. C. (1978) Extensive prolongation of rabbit kidney allograft survival after short-term cyclosponn—A treatment. Lancet, i, 1182-1184. 14. Harkness, J. E and Wagner, J. E. (1989) The Biology and Medicine of Rabbits and Ro-dents, 3rd edn, pp. 9-19. Philadelphia: Lea and Febiger. Heron, I (1970) Kidney transplantation in the rabbit. Acta Pathologica Microbiohgica 15. Scandinavia, 78,90-95. Hinton, M. (1981) Kidney disease in the rabbit: a histological sur-vey. Laboratory Animals, 15, 263-265 16. Hinton, M., Jones, D R. E and Festing, M. F. W (1982) Haematological findings in the healthy and diseased rabbits, a multivanate analysis. Laboratory Animals, 16, 123-129. 17. Hobbs, J В and Cliff, W.J. (1973) A study of allograft kidney rejection occurring simulta-neously in the whole organ and ear chamber grafts in the rabbit. Journal of Experimental Medicine, 137, 776-798 18. Holter, A.R., McKeam, T.J, Neu, M. R., Fitch, FW and Stuart, P.P. (1972) Renal transplan-tation in the rabbit I. Development of a model for the study ofhyperacute rejection and immu-nological enhancement. Transplantation, 13, 244-249. 19. Jackson, S, Chused, T. M, Wilkmson, J. M.. Leiserson, W M. and Kmdt, T.J (1983) Differ-entiation antigens identify subpopulations of rabbit T and В lymphocytes. Definition by flow cytometry Journal of Experimental Medicine, 157, 34-46. 20. Jacobsen, I. A. (1978) Renal transplantation in the rabbit: a model for preservation studies Laboratory Animals, 12, 63-70. 21. Kent, G. С (1987) Comparative Anatomy of the Vertebrates, 2nd edn, pp. 420-468 St Louis: Times Mirror/Mosby College Publishing. Klassen, J and Milgrom, F. (1969) The role ofhumoral antibodies in the rejection of renal homografts by rabbits. Transplantation, 8, 566-575. 22. Knight, К. L, Leary, A. L. and Tissot, R G. (1980) Identification of two la-like alloantigens on rabbit В lymphocytes. Immunogenetics, 10, 443—453. 23. Koene, R. A. P., Gerlag, P. G. G., Hagemann, J. F. H. M., van Haelst, U. J. G. and Wijde-veld, P. G. A. B.
(1973) Hyperacute rejection of skin allografts in the mouse by the admini-stration ofalloantibody and rabbit complement. Journal of immunology, 111, 520-526 24. Lancki, D W, Tissot, R. G. and Cohen, С (1979) Histocompatibility in the rabbit Trans-plantation, 27, 79-86.

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