Eur J Cardiothorac Surg 2001;19:333-338
© 2001 Elsevier Science NL
A comparative study of Euro-Collins, low potassium University of Wisconsin and cold modified blood solutions in lung preservation in acute autotransplantations in the pig
Duilio Divisia,
Pietro Montagnaa,
Olivier Jegadena,
Lorenzo Giustia,
Alberto Bertia,
Giorgio Furio Colonib,
Costante Riccib,
Philippe Mikaeloffa
a Department of Cardio-thoracic Surgery, Cardiologique Hospital, Claude Bernard-Lyon I University, 59 Boulevard Pinel, 69003 Lyon, France
b Department of Thoracic Surgery, University of Rome La Sapienza, Via le del Policlinico, 00100 Rome, Italy
Received 6 April 2000;
received in revised form 26 October 2000;
accepted 13 December 2000.
Corresponding author Tel.: +39-0861-429547; fax: +39-0861-211626.
e-mail: dudivisi{at}tin.it
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Abstract
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Objective: The aim of the study was to assess the quality of lung preservation offered by Euro-Collins solution (EC), Cold Modified Blood solution (CMB) and low potassium University of Wisconsin solution (UWLP). Method: Fifteen right lung auto-transplantations (five for each solution) in the pig (Large White) were performed after 2 h of cold ischaemic storage in physiological solution at 4°C. Right lung biopsies were performed before ischaemia and 30 min after reperfusion, for histoenzymatic, histopathological and electron microscope studies. Results: After reperfusion, significant alterations were observed in the haemodynamics with only the right lung perfused; pulmonary arteriolar resistance increased by a factor of 5 in the EC group, by a factor of 4 in the CMB group and by a factor of 1.2 in the UWLP group; the right ventricular ejection fraction fell by 60% in the EC group, by 50% in the CMB group and by 31% in the UWLP group. Haemodynamic impairment was lower in the UWLP group (P<0.05; P<0.001) as was ischaemic-reperfusion injury (P<0.05). Oedema was observed in the EC group and extensive alveolar wall damage in the CMB group. Hypoxaemia was observed in all groups but the differences in the degree of hypoxaemia were not significant. Conclusions: The authors concluded that UWLP solution was the most effective of the three in this transplant model.
Key Words: Lung transplantation • Lung preservation • Single flush perfusion
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1. Introduction
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Organ preservation is a limiting factor in lung transplants, regardless of the surgical procedure involved (monopulmonary, bipulmonary or heart–lung). Present systems in use permit 6–8 h of cold ischaemia although a degree of functional insufficiency is observed in the period immediately following the transplant. This insufficiency is due to an increase in resistance and vascular permeability resulting in oedema, necrosis, parenchymal haemorrage and structural alterations [1]. Up to 36–48 h of ischaemia have been reached in experimental situations however there is a qualitative impairment of ventilation and pulmonary perfusion as a result of the double trauma of removal/re-insertion and ischaemia/reperfusion. A number of solutions have been proposed to minimize the pathological alterations arising from ischaemia. Ideally a solution should limit the spread of interstitial oedema, inhibit intracellular oedema, acidosis and the production of free radicals and provide the metabolic substrates necessary for the renewal of energy processes at the moment of reperfusion. The Euro-Collins (EC) solution, which is of an intracellular nature, together with prostaglandins is used in a lot of clinical and experimental work [2,3]. The high concentration of K+ and absence of macromolecules would appear, in theory, to: (a) eliminate electrochemical Na+/K+ membrane gradients; (b) reduce ionic fluxes; (c) inhibit cell lesions.
Experience has shown, however, that high levels of potassium result in [4,5]: (a) the permanent depolarization of the smooth muscle cells that make up blood vessel walls; these cells are responsible for the opening of the slow Ca2+ channels and vasoconstriction; (b) an increase in intracellular K+ that causes direct cellular lesions. Vasoconstriction causes a reduction of the flux in the pulmonary microcirculation at reperfusion (no reflow phenomenon); this reduction is accentuated by the stacking of red blood cells caused by the loss of membrane elasticity and the formation of leucocyte-platelet aggregates [6,7]. Vasoconstriction is also at the origin of pulmonary arterial hypertension which together with the partial destruction of the endothelial barrier causes interstitial exudation and pulmonary oedema. These side effects have led to the development of extracellular ionic solutions such as: the low potassium dextran solution (LPD) which has an anti-thrombotic effect thanks to the dextran which adheres to red blood cells, platelets and endothelial cells [8], the Papwort which contains prostacyclin, heparin, mannitol, donor blood (approximately 500 ml), albumins [9] and the low potassium University of Wisconsin solution (UWLP) [5]. The UW solution has been used in multiorgan explants and with a degree of success in kidney, pancreas and liver transplants; it was first used in lung transplants by the Mayo clinic [10]. Many contradictory results have been published concerning preservation with UW. Kawahara et al. [11] observed a deterioration in compliance and an increase both in pulmonary vascular and airway resistances with EC when compared to the Belzer solution in a canine lung that had been isolated after 24 h of ischaemia. However, there were no significant differences between the two groups in the PO2 after reperfusion. Naka et al. [12] observed a significant degree of oedema and increase in the pulmonary vascular resistance after 24 h of ischaemia in a heart-lung transplant using UW in the dog. Originally of an intracellular nature containing lactobionate, raffinose and gluthatione to neutralize free radicals, it has since been modified by reducing the concentration of K+.
The aim of this work was to examine and compare the functional and anatomical-pathological aspects of EC, UWLP and cold modified blood solution (CMB) in lung preservation.
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2. Materials and methods
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Fifteen Large White pigs each weighing approximately 35 kg were randomly divided into three groups of five. A right lung auto-transplant was performed, the transplanted organs being flushed with one of the solutions under examination (Table 1).
2.1. Surgical procedure
The anaesthesia started with the administration of ketamine: 35 mg/kg i.m. and 2% xylazine: 3 mg/kg via a marginal vein in the ear and was continued with xylazine: 1.5 mg/kg and ketamine: 2.5 mg/kg, administered at 10 ml/h via a marginal vein in the ear and the animals intubated. Ventilation was 3.5 l/min with 60% oxygen and parameters control every 10 min. During haemodynamic and gasometric evaluations lungs were ventilated with 100% oxygen (Bird oxygenator). The right carotid and internal jugular vessels were prepared. Blood volume was maintained constant by a peripheral intravenous infusion (Elohes 5%, Plasmiun 5%), on the ground of central venous pressure, arterial pressure and heart rate.
A postero-lateral right thoracotomy was performed in the fifth intercostal space together with a transversal hemisternotomy and the fourth and fifth ribs were excised. Three milligrammes per kilogramme of heparin was administered at the beginning of the operation, to prevent vascular thrombosis during removal/reimplantation lung. Arterial pressure was monitored by means of a catheter introduced into the carotid artery. A Swan–Ganz ejection fraction volumetric TD catheter (Baxter) in the pulmonary artery allowed us to evaluate the ejection fraction of the right ventricle, whilst a catheter in the left atrium measured left atrial pressure. Dissection of the right auricle, superior vena cava, aortic arch was performed and section of Botallo's arterial ligament; the superior vena cava and aortic arch was found through the use of a ribbon. Left and right pulmonary arteries (one and two respectively), the tracheal bronchus, the main right bronchus and the right pulmonary veins were later dissected and examined.
2.2. Protection and removal of the right lung
The right pulmonary arteries (artery to upper lobe and artery right trunk) were clamped and cannulated. The right venal structures were clamped and successively incised after the administration of 10 mg/kg of PGE1 in the right pulmonary arteries. Flushing was performed with a litre of solution enriched with 50 mg/l of PGE1 at 4°C through the right pulmonary arteries at 15 mmHg pressure perfusion; the animal was ventilated with 100% FIO2. The right lung was removed after bipolar exclusion of the main and tracheal bronchi and conserved in physiological solution (for 1000 ml: NaCl 9.0 g, osmolarity 308 mOsm/l, 4.5<pH>7.0) at 4°C.
Haemodynamic measurements including systemic, pulmonary and atrial blood pressure and electrocardiogram readings were monitored during the period of cold ischaemia (2 h).
2.3. Right lung autotransplant
The following steps were followed for the re-insertion of the lung:
- Anastomosis of the tracheal bronchus by means of a continuous suture with prolene 7/0
- Anastomosis of the principal bronchus by means of a continuous suture along the membranous tunic and individual stitches through the cartilage with prolene 5/0
- Separate anastomosis by means of a continuous suture with prolene 7/0 of the pulmonary arteries to upper lobe and right trunk, respectively
- Separate anastomosis by means of two continuous hemisutures with prolene 7/0 of
- the trunk the pulmonary veins to upper and middle lobes;
- the pulmonary vein to middle lobe;
- the trunk the pulmonary veins to accessory and lower lobes.
Reperfusion was achieved by progressive de-clamping of the right pulmonary arteries and veins. The lung was ventilated at low pressure and volume and 20 mg of Furosemide and 40 mg of methyl-prednisolone administered to prevent oedema.
A right pneumonectomy was performed 2 h after the transplant and the animal electively killed.
2.4. Functionality of the transplanted lung
Before ischaemia and 1 h after reperfusion arterial blood gas and haemodynamic measurements were taken with: both lungs perfused and individual lungs the other being excluded by clamping of the controlateral pulmonary artery throughout the reperfusion period. The measurements were taken after haemodynamic stability had been achieved, with 100% oxygen ventilation. The Swan–Ganz probe was positioned in the unclamped pulmonary artery; cardiac output and the ejection fraction of the right ventricle were measured by thermodilution (REF-1 computer, Baxter). Arteriolar pulmonary resistance was calculated on the basis of the formula: average arterial pulmonary pressure-average left atrial pressure/cardiac output.
2.5. Histological studies
A pulmonary biopsy was performed on the right upper lobe before ischaemia and 30 min after reperfusion. Histoenzymatic tests (ATPase and phosphatase activity) and histopathological exams (hematin, eosin and safran staining) were performed on each sample and lesions classified from 0 to 4. Specimens were also examined under the electron microscope and lesions classified as present or absent.
2.6. Statistical analysis
The software package SPSS/PC+ version 3.1 (SPSS Inc. Chicago, IL) was used for the statistical analysis. All Data were expressed as the mean and were analyzed by ANOVA and the Bonferroni/Dann method was used for multiple comparisons when a significant difference was observed. P<0.05 was accepted as indicating a statistical significance.
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3. Results
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No significant differences existed between the three groups as far as the weight of the animals (35±2 kg), warm ischaemic times (80±2 min) and the length of the operation (8±0.3 h) were concerned. The results for each group were analyzed on the basis of the evaluation criteria and then compared.
3.1. Haemodynamic studies
The results concern the physio-pathological alterations in pulmonary arteriolar resistance (PAR), the right ventricle ejection fraction (RVEF) and variations in blood gas analyses (Table 2).
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Table 2. Haemodynamic and blood gas data (mean) taken before ischaemia and 1 h after reperfusion of both lungs (2L), the left lung (LL) and the right lung (RL)a
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No significant differences were observed between the three groups before ischaemia. After reperfusion of both lungs or the left lung, a reduction of the RVEF and an increase in PAR was observed, though neither of these results was relevant. When the right lung was perfused, alterations were observed in the haemodynamics when compared to pre-ischaemic data: PAR increased by 5 units in the EC group, by 4 units in the CMB group and by 1.2 units in the UWLP group. Likewise RVEF fell by 60% in the EC group, by 50% in the CMB group and by 31% in the UWLP group. A significant degree of hypoxaemia was observed in all groups but there were no significant differences between groups.
3.2. Histological studies
Analysis of the pre-ischaemic pulmonary biopsies did not reveal tissue lesions or variations in normal enzyme activity. Thirty minutes after reperfusion the trauma of ischaemia-reperfusion resulted in oedema type lesions, congestion and exudation; the extent of the damage was greatest in the EC group followed by the CMB group (Table 3). On the contrary esocytosis is less in the EC group compared to CMB group. ATPase activity fell exclusively in the EC and the CMB groups; a good level was also conserved in the UWLP group. Acid phosphatase activity remained constant in all three groups. Alterations in sectall cells, pneumocytes and interalveolar sects were observed only in the EC and CMB groups. In both groups widespread oedema and a significant degree of leucocyte infiltration was remarked. These anatomical-pathological aspects were less important in the UWLP group.
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4. Discussion
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Flush perfusion of the pulmonary artery followed by cold static storage is one of the most common methods of organ preservation. Experimental work has shown that the addition of the prostaglandine E1 or prostacycline I2 gives good results in terms of preservation. These molecules produce bronchodilation, vasodilation, reduce post-ischaemic vasoconstriction and the no reflow phenomenon, modulate leucocyte and platelet activity and stabilize lysosomal membranes and hence protect cells [13]. Xiong et al. [14] noticed a smaller increase in the filtration coefficient (Kfc) and pulmonary resistance with Wallwork (WA) after 4–6 h of ischaemia when compared to EC, LPD and UW. This effect is pronounced if prostacycline and L-arginine (precursor of nitric oxide) are present. Hooper et al. [15] found that the use of Iloprost, a synthetic prostacycline analogue (20 ng/kg per min i.v. and 20 µg/l associated with EC), for 6 h of ischaemia in the dog, did not produce effective results in terms of the quality of lung preservation, distribution of the liquid used for flushing the lung, post-transplant functionality and the degree of oedema. The authors suggested that the stockage time was insufficient to produce an improvement and the corticosteroid treatment might have masked the positive effects of Iloprost. The CMB solution satisfies the physiological criteria of preservation given that the blood used in the solution is taken from the donor during the explant. However, we have observed a certain degree of oedema at reperfusion during lung and heart and lung transplants when using this solution, secondary to alterations of the alveolar-capillary membrane [16]. Miyoshi et al. [5] demonstrated that low potassium UW provides better lung preservation than the original solution and that lactobionate and raffinose were superfluous. Oka et al. [17] observed less oedema, a lower average pulmonary arterial pressure and an increase in gaseous exchange values when using modified UW and LPD rather than the original UW solution and EC. Much work in this field has focused on haemodynamic and blood gas analyses; our study also took into consideration the impact of the solutions on histological components of the transplanted organ. Furthermore, we chose auto-transplants to avoid complications arising from rejection phenomena which could interfere with the preservation methodology. The increase in PAR and the fall in RVEF, measured after reperfusion of only the right lung, can be correlated to an obstacle at the alveolar–capillary membrane. Variations in pulmonary function are linked to metabolic and histological modifications of the parenchyma during ischaemia and reperfusion. Alterations in enzyme activity (a fall in ATPase activity and an increase in acid phosphatase activity) represent the organic response to anoxia, although forced insufflation (FIO2 100%) allows for a certain degree of aerobiosis with the production of ATP during ischaemia. The rupture of pneumocytes, oedema and intra-alveolar exudation with the destruction of the alveolar-capillary membrane are the consequences of anoxia shock syndrome [18,19]. However, these lesions were modest, topographically and morphologically speaking, when correlated to the pulmonary mass and indicative of a satisfactory preservation. No perivascular haemorraging or dimorphism of endothelial cells was observed. However, a widespread leucocyte infiltration was observed in the EC and CMB groups compared to the UWLP group. Current experimental reperfusion models using leucocyte-free blood [20] or neutrophil inhibitors [21] aim to eliminate secondary effects caused by leucocyte activation. Comparative analyses of the three solutions revealed little difference in blood gas measurements while important differences in haemodynamic and histopathological data. The hypoxemia observed in all groups after reperfusion is mainly due to the haemodilution of the animal during the operation.
In conclusion, in this study the EC and CMB solutions give similar results as far as the quality of lung preservation is concerned. A significant degree of oedema is observed with the EC solution whilst the CMB solution determines severe alterations in the alveolar–capillary membrane; a significant leucocyte infiltration was observed in both groups. The low potassium Wisconsin solution appears to be the most efficient of the three; the histopathological lesions observed were less serious and the functionality of the transplanted lung was satisfactory. Improvements in the quality of lung preservation might be achieved by introducing other molecular groups such as scavengers and/or the anti-platelet activating factor.
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Abstract
1. Introduction
