Biological Services Unit, The Medical School, University of Manchester, Oxford Road,. Manchester ... extracorporeal veno-venous circuit from a support animal.
A porcine ex vivo paracorporeal model of lung transplantation N. C. Wright, D. N. Hopkinson, T. E. Shaw & T. L. Hooper Biological Services Unit, The Medical School, University of Manchester, Manchester M13 9PT, UK
Oxford Road,
Summary A technique is described which allows perfusion of an isolated, ventilated pig lung with an extracorporeal veno-venous circuit from a support animal. This model is stable for up to 4 h, and avoids some of the disadvantages of alternative small animal and large animal models of lung transplantation. It may be useful in the investigation of factors affecting lung preservation and reperfusion injury. Keywords
Pig; lung; reperfusion; para corporeal
There is a rapidly increasing body of research devoted to lung transplantation and reperfusion injury, utilizing a variety of models and techniques. Models range from large animal allotransplantation to rodent ex vivo reperfusion models. All have limitations to be borne in mind when interpreting results. Allotransplantation is technically difficult and vascular anastomoses can introduce variability into results. vivo models, with an isolated lung perfused on a circuit, have a number of disadvantages. For example, a very short reperfusion period may not allow full assessment of function; ideally the oxygenating ability of the graft should be measured
and reperfusion techniques (Bhabra et a1. 1996, Hopkinson et a1. 1996). However, this model has limitations: it functions well for 2 h but then deteriorates; multiple sequential graft biopsies are not possible and serial bronchoalveolar lavage studies would be difficult. Also the rat, a small rodent, is regarded as more distant from the human in terms of anatomy and physiology than, for example, the pig. Therefore we aimed to develop an isolated, ventilated and perfused porcine lung transplantation model in which reperfusion factors could be easily controlled and function readily assessed. This model would then
with blood as the perfusate} presented to the
be used to further investigate lung graft
graft with a systemic venous oxygen tension; and the circuit should involve another animal to allow for a full pathological response to reperfusion. An ex vivo paracorporeal model of lung graftreperfusion in the rat was developed in our laboratory (Hopkinson et a1. 1995), and has proven useful for studying different storage solutions (Hopkinson et a1. 1994a,b)
preservation and reperfusion strategies.
Ex
Correspondence to: Mr T. L. Hooper, Senior Lecturer and Consultant Cardiothoracic Surgeon, Department of Cardiothoracic Surgery, Wythenshawe Hospital, Southmoor Road, Manchester M23 9LT, UK Accepted 15 July 1999
Materials and methods Twenty-two outbred female Large White pigs of conventional microbiological status, purchased from a commercial supplier ('Tweeds', Cheshiret were used. All animals were cared for in accordance with the United Kingdom Government's Animals (Scientific Proceduresl Act 1986. Animals were housed in galvanized steel pens (approximate dimensions 4mx2m, one or two animals per pen), © Laboratory Animals Ltd. Laboratory Animals .(2000) 34, 56-62
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Porcine model of lung transplantation
on softwood shavings and autoclaved straw, for an acclimatization period of one week prior to use. 'Grocare' standard pig feed was given, 490 g twice daily per animal, and water was available ad libitum. The environmental temperature was regulated between 18-22°C and relative humidity between 30-70%. Lighting was artificial, from 08:00 to 20:00 h, and ventilation was by filtered air with 15 air changes per hour. Half of the animals, weighing 18-27kg, were used for lung graft donation and half were used as support animals, weighing 38-57 kg.
Operations Donor This was a sterile procedure with full aseptic precautions. Donor pigs were anaesthetized using inhalational induction with halothane (Fluothane, ICI, UK), oxygen and nitrous oxide. Electrocardiograph leads were placed to monitor heart rate (Datascope 2000). An endotracheal tube was placed and the animals ventilated using an oxygen-nitrous oxide mixture (30:70%) at a tidal volume of 11-12ml/kg and a minute volume of 200 ml/kg (ventilator: model BM2, Blease, UK). Central venous and arterial cannulae were placed surgically in the left external jugular vein and left carotid artery respectively, for monitoring of central venous pressure and arterial pressure, and administration of fluids and drugs. Anaesthesia was converted to the intravenous route, using propofol ('Diprivan', Zeneca, UK, 20-40mg bolus, followed by infusion 4 - 12 mg/kg/h) and alfentanyl ('Rapifen', Janssen, UK, 0.5 mg bolus every 20-30min as requiredl. Cefuroxime 750mg (Zinacef, Glaxo, UK) antimicrobial prophylaxis and methylprednisolone 500 mg (Solu-Medrone, Upjohn, UKl, to facilitate flush, were given as per clinical lung transplantation practice (at least 30 min prior to flushingl. Bretylium tosylate 50 mg (IMS)was given prophylactically against ventricular arrhythmias. Muscle relaxant Pancuronium (0.1mg/kg, David Bull) was given to provide balanced anaesthesia and facilitate dissection. Following median sternotomy, pleurae and pericardium were opened. The superior and
inferior vena cavae ISVC, IVC) were isolated and encircled with ligatures. The azygos vein was identified and tied off. A pursestring suture (5/0 Prolene) was introduced into the pulmonary artery (PA). Following systemic heparinization (250iu/ kg, Multiparin, CP Pharmaceuticals, UK) an 18F gauge cannula (Polystan, Denmark) was placed and tied into the PA. The caval ligatures were tied and the heart allowed 5-6 beats to empty. The IVC was divided proximally, the left atrium (LA) incised and cold (4°C)flush solution (EuroCollins solution, Fresenius, FDR, modified with magnesium sulphate 5 mmol/l)60 ml/kg donor body weight-infused into the PA via the previously placed cannula from a height of 60 cm, over 6 - 9 min. The aorta was clamped. Chilled saline was applied topically to the lungs and cold effluent from the LA also had a topical cooling effect. Ventilation was continued during the flush at half normal rate and converted to room air. During this period a blood donation was obtained from the venous access line into a citrate-phosphate-dextrose storage bag (Baxter Healthcare, Norfolk, UKI. Following flush the heart -lung bloc was dissected free and transferred to a bowl in preservation solution and saline ice slush. The bloc was prepared for reperfusion of the left lung only. The post-caval lobe and right pulmonary veins were isolated, tied and cut. The right pulmonary artery was divided. Only the bronchial tree to the right lung remained intact. Monitoring cannulae (5 French gaugel were placed and secured in the PA and LA. Large (6.25mm) cannulae for reperfusion were placed in the PA and LA. The atrial (venousl cannula had a 'basket' tip to avoid blocking venous return. The bloc was stored partially inflated with room air in preservation solution in a medical refrigerator at 4°C for approximately 4 or 24 h. Ischaemic time was measured from beginning of flush to commencement of reperfusion.
Preparation of the support animal This was a sterile procedure. The animals were anaesthetized using inhalational Laboratory Animals (2000) 34
Wright et al.
58
induction. Intubation, ventilation and placement of central venous and arterial cannulae were as above. Anaesthesia was maintained using an intravenous regimen, with pentobarbitone (bolus then infusion 10-15 mg/ kg/h, adjusted according to heart rate and blood pressure) and alfentanyl (0.5-1.0 mg bolus every 20-30 min as required). Methylprednisolone 500 mg and pancuronium 2 ml were given. The right external jugular vein was exposed surgically and cannulated with a 16 French pulmonary artery vent cannula (DLP, Medtronic, UK). The right femoral vein was exposed and cannulated with a 21 F gauge femoral cannula (Medtronic, UK) advanced into the rve. The animal was fully heparinized (Pump-Hep, Leo Pharmaceuticals, UK, initial bolus 10000U) to maintain the activated clotting time (Hemochron) at 300500 s. The femoral cannula was attached to the afferent limb and the jugular cannula to
lated lung drained into a reservoir (Capiox venous cardiotomy reservoir, Terumo, UK) and was returned via a roller pump (Sarns, Ann Arbor) and heat exchanger (CSC 14 cardioplegia heat exchanger, Sorin Biomedica) to the jugular vein of the support animal. Pump rate was adjusted to maintain a constant level in the reservoir. The isolated left lung was ventilated by a separate ventilator (Large Animal respirator model 613, Harvard Apparatus, South Nattick, Massachusetts, USA)-tidal volume 6-8 ml/kg donor body weight, rate IS/min-with room air. During reperfusion, the lung graft was immersed in warm saline in a custom made water-jacketed tank with an internal temperature of 36°C. A water heater-pump (Sarns, Ann Arbor) provided countercurrent heating to the circuit heat exchanger and the lung tank. Reperfusion was continued for 4h.
(ECe).
performed using 20% pentobarbitone solution (Euthatal, Merial Animal Health, Harlow, UK)
the efferent limb of the extracorporeal circuit
Extracorporeal circulation (ECC) (see Fig 1) The circuit was constructed from 6.25 mm internal diameter PVC tubing and primed with colloid solution IGelofusine, Braun Medical, UK). Blood was drained by gravity via the femoral cannula, from a height of 40 em into the donor lung. This provided a physiological PA pressure of 18-20mmHg in normal lungs. A flow probe 112N,Transonic Systems, New York, USA) was sited in the afferent limb. Venous outflow from the iso-
SUPPORT ANIMAL
Flow probe Ventilator
Fig 1
The reperfusion
circuit
Laboratory Animals (2000) 34
At the end of the procedure euthanasia was,
Constants and measurements The support animal was monitored closely with continuous measurement of temperature (rectal probe), haemodynamics (heart rate, blood pressure and central venous pressurel and blood gases. A warming blanket and heat exchanger ensured constant temperature of the support animal. Ventilation was adjusted and fluid and blood replacement provided to maintain an afferent P02 to the isolated lung of 40 mmHg and haematocrit between 0.22 and 0.25 (Ames Microspin, Bayer, Germany). Blood samples from the afferent and efferent limbs of the circuit were analysed for: pH, partial pressures of oxygen and carbon dioxide, bicarbonate concentration, base excess and oxygen saturation (model 278, Ciba Corning, UK). A pressure transducer (ViggoSpectramed) was connected to the ventilator circuit for continuous monitoring of airway pressure and further pressure transducers (ViggoSpectramed) attached to the SF PA and LA monitoring cannulae
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Porcine model of lung transplantation
allowed continuous measurement of vascular pressures. All haemodynamic data were recorded on a personal computer via a data acquisition system (DATAQ, Ohio, USA). Groups were compared using repeated measures analysis of variance and differences considered significant if P < 0.05.
Oxygenating Ability vs Time 90 80 70 ~
60
~ 50 ;;
li!
40 30 20 10
o
Results Group A (n = 6) represent grafts stored for 4 h and Group B In = 5) grafts stored for 24h. Animal weights and timing of preparations are given in Table I, as mean lstandard deviation). Flush times were comparable between the groups. Time from refrigerator to reperfusion on the circuit was always less than 10min. Total ischaemic time was from beginning of flush to beginning of reperfusion. Support animal parameters were well maintained: temperature 37.5 -38.5°C, mean arterial pressure 80 - 120mmHg, and stable blood acid-base balance. Haematocrit was maintained in the range 0.22-0.26. Measurements of isolated lung function are presented graphically in Figs 2-5. Data are plotted as means with 95% confidence intervals. Afferent P02 was stable at approximately 40 mmHg and similar for the two groups; however there were slight differences between the groups, therefore 'oxygenating capacity' calculated as efferent P02-afferent P02 difference is presented (Fig 2). Group B performed considerably less well than Group A (P
