Jul 17, 1987 - Department of Surgery, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. 293. Summary. An inexpensive and convenient plasma ...
Laboratory Animals (1988) 22, 293-296
293
Plasma exchange technique in the unheparinized, unanaesthetized rat R. REDING*, D. J. G. WHITE, H. ff. S. DAVIES & R. Y. CALNE Department of Surgery, Addenbrooke's
Hospital, Hills Road, Cambridge CB2 2QQ, UK
Summary An inexpensive and convenient plasma exchange technique has been developed in the rat, which avoids heparin and membrane plasmapheresis technology. The use of a reliable venous vascular access on unanaesthetized, briefly restrained animals is described. Keywords: Rat; Plasma exchange; Antibody rebound
The development of a plasma exchange (PE) technique in experimental rodent models is of increasing importance for a variety of immunological studies. These include investigations concerning the feedback mechanisms involved in the antibody rebound after PE (Bystryn et al., 1970) and experimental xenografts in which rejection is mediated by preformed antibodies (Cameron et al., 1983). Plasmapheresis techniques in the rat using a continuous flow blood filter have been described (Euler et aJ., 1985), but this model requires systemic heparinization of the animal and a double vascular access. It is not satisfactory for the assessment of possible beneficial effects of PE in xenograft models, since heparin has been reported to delay hyperacute vascular rejection (Sakai & Kountz, 1976). This paper describes an alternative PE procedure developed for use in rats. It employs ·Address for correspondence: Department of Kidney Transplantation (U22), St-Luc University Clinics (UCL), 10 avo Hippocrate B-12oo Brussels, Belgium. Received 17 July 1987. Accepted 27 January 1988
a reliable and convenient vascular access on unheparinized, unanaesthetized animals and allows the removal of more than 75!Yfo of total plasma volume. Immunization with sheep red blood cells(SRBC) and quantitative determination of antibody levels provided a simple method of measuring the amount of plasma removed and the timing of post-PE antibody resynthesis (Euler et al., 1985). Materials and methods Animals and immunization protocol Colony bred female Sprague Dawley rats (3-4 months old, 250-350 g body weight) were maintained under a conventional husbandry system and were used throughout these experiments, and as erythrocyte donors for substitution solutions. A fixed formula diet in the form of pellets with a calculated analysis of 17' 4!Yfo crude protein was provided ad libitum (Labsure Animal Foods, Poole, Dorset, UK). Fresh water was supplied ad libitum in standard 500 ml water bottles. The animals were immunized by intraperitoneal injection of 107 SRBC at 28, 21 and 14 days before PE (i.e. day -28, - 21 and -14). Vascular access Chronic inferior vena cava (IVC) catheterization was carried out under diethyl ether anaesthesia. A polyethylene tubing (0' 38 mm i.d., 1·09 mm o.d., PE 20, Clay Adams, Becton Dickinson Benelux, Aalst, Belgium) was cut straight at the tip and inserted through a femoral vein into IVC. The catheter was positioned to provide a venous reflux whatever the position of the homolateral leg. It was then secured by a 3/0 psoas muscle
294
Reding, White, Davies & Caine
INFERIOR VENA CAVA
SILICONE RUBBER
TUNNElLED TO THE NECK
Fig. 1. The catheler is shown at the Insertion point in the femoral vein, secured by a stitch tied on silicone rubber and fixed to the psoas muscle.
stitch tied on a silicone rubber circumferencial segment (port-A-Cath, Pharmacia Belga, Brussels, Belgium) to avoid any slipping (Fig. I). A spinal needle (1' 20 mm i.d.) was inserted through the skin of the nape of the neck and tunnelled to the femoral site. The cannula was threaded through, the needle was removed and the catheter was secured to the skin using a 3/0 stitch on another silicone rubber segment. While not connected to a needle and a syringe for PE or blood sampling, the external tip was covered with an end ligated silicone rubber top (Fig. 2). The surgical procedure was performed under clean, but not sterile, conditions. No antibiotics were given. Plasma exchange procedure A two-stage PE was performed on restrained, unanaesthetized rats. Ether anaesthetic induction was used to settle the animal in a restraining cage (Clinical School Workshops, Addenbrooke's Hospital, Cambridge, UK). No subsequent analgesia was used. After recovery, a total of 30-35 ml of blood (i.e. 1O-15
