Abstract :
[fr] Nous avons voulu améliorer les connaissances actuelles des mécanismes entraînant la non-fonction des greffons pulmonaires après conservation hypothermique et transplantation, par l’étude de la fonction respiratoire mitochondriale après ischémie froide et reperfusion. Nous avons également voulu étudier les différences de conservation après rinçage avec les deux solutions de préservation les plus employées en tranplantation pulmonaire clinique, les solutions d’Euro-Collins et “University of Wisconsin”. Nous avons enfin voulu étudier la fonction respiratoire mitochondriale après ischémie normothermique, afin d’évaluer la possibilité de prélever des greffons pulmonaires chez des donneurs d’organes à coeur non battant.
Nous avons donc développé un modèle porcin de prélèvement, d'ischémie froide et de reperfusion pulmonaire normothermique in vitro, modèle relativement simple et reproductible, et qui permet d'objectiver le développement d'une non-fonction du greffon pulmonaire. Dans ce modèle, nous avons pu montrer qu’après 24 heures d’ischémie froide, les mitochondries montrent une altération modérée des oxydoréductases mais sans atteinte de l’efficacité de la phosphorylation oxydative, atteinte qui a pu être démontrée après 48 heures d’ischémie froide. Après reperfusion, l’atteinte mitochondriale est plus profonde, avec diminution de l’efficacité de la phosphorylation oxydative. Dans ce modèle d’ischémie froide et de reperfusion normothermique, nous n'avons pas pu observer de différence significative entre les poumons rincés avec la solution d’Euro-Collins ou “University of Wisconsin”, et ce tant au niveau de paramètres physiologiques pulmonaires qu’au niveau de la fonction respiratoire mitochondriale.
Nous avons développé un modèle d’ischémie normothermique dans lequel 30 minutes d'ischémie chaude n'altèrent pas la fonction respiratoire des mitochondries pulmonaires. Par contre 45 minutes semblent provoquer des lésions mitochondriales sévères. Cette tolérance de 30 minutes d'ischémie chaude pourrait permettre l'utilisation des greffons pulmonaires prélevés chez des donneurs d’organes à coeur non battant, du point de vue bioénergétique du moins.
[en] The lungs are organs whose sensitivity to ischaemia and reperfusion is well known. In a rabbit model of lung ischaemia, we showed that the cold ischaemia longer than 6 hours is accompanied by a significant reduction in tissue contents in vitamins E and C, two important protectors against the lesions appearing at the time of ischaemia and the reperfusion (Pincemail 1999). Moreover, lungs are different from the other transplanted organs by the importance of a fragile structure, the alveole, zone of exchange between the alveolar air and the capillary blood. It was shown that among the alveolar cells, the type II pneumatocytes, secreting the surfactant, is of primary importance for the post-transplant function. It was shown that the quality of surfactant decreases after conservation and reperfusion of the lungs, and that, in vitro, the effectiveness of surfactant continuously decreases with the prolongation of the duration of ischaemia (Erasmus 1994). Moreover the administration of surfactant before the pulmonary reperfusion improves the postoperative function of the grafts in the rat (Erasmus 1996). It is known that, if all the pulmonary cells contain mitochondria, more than 50% of the mitochondria isolated from lungs come from type II pneumatocytes (Fisher, 1976). We chose to study the mitochondrial respiratory function of these important alveolar cells for the pulmonary function after ischaemia and reperfusion.
In this work, we developed a porcine model of ischaemia (hypo- and/or normothermic) and of normothermic reperfusion. This reperfusion was accompanied by a postoperative non-function, objectified by aerodynamic and hemodynamic parameters, as by the appearance of pulmonary oedema. This non-function was observed after 24 hours a hypothermic ischaemia, which is incompatible with a normal function of the pulmonary grafts in clinical transplantation. The reperfusion with a solution of Krebs-Henseleit bicarbonate remove any immunological artefact and any influence of the circulating blood cells in the pulmonary lesions appearing at the time of the reperfusion. On the other hand, this solution is different from blood to a significant degree, by not containing protectors against the production of free radicals at reperfusion. It is thus possible that our model exacerbates this production of free radicals, more especially as the lung is a tissue particularly rich in polymorphonuclear cells.
In this model, we could show that after 24 hours of cold ischaemia the mitochondria underwent a moderate deterioration of the oxidoreductases but without decrease in the effectiveness of oxidative phosphorylation, decrease that could be demonstrated after 48 hours of cold ischaemia. These lesions are comparable to the mitochondrial lesions that we had observed after cold ischaemia of rabbit kidney (Willet 1995). After reperfusion, the mitochondrial lesions are more severe, with a decrease in the effectiveness of oxidative phosphorylation. Concerning normothermic ischaemia, the first 30 minutes did not cause significant mitochondrial lesions. These results at least corroborate the literature data on the relative good pulmonary tolerance to normothermic ischaemia, explained in theory by the persistence of oxygen in the airways and thus of the persistence of aerobic metabolism in spite of the circulatory arrest. For the other organs, the circulatory arrest implies anoxia, that is not really the case of lung, as oxygen is present in airways. Pulmonary transplantation could thus profit from an increase in graft pool available by harvesting lung graft from non-heart beating donors. After 45 minutes of normothermic ischaemia, mitochondrial oxidative phosphorylation dysfunction appears, related to a significant deterioration in the ATP synthase function. These results confirm that the cellular metabolism is then disturbed by the appearance of cellular anoxia because of the progressive consumption of oxygen present in the alveoles, or of the substrates necessary to the cellular metabolism.
The description and the discussion of the importance of these mitochondrial alterations in the genesis of lung graft dysfunction after transplantation must be integrated with the very broad framework of the disturbances appearing at the time of tissue ischaemia and reperfusion. From our study it comes out that hypothermia at 4°C protects effectively the pulmonary mitochondrial function since significant deteriorations do not appear before 24 hours of hypothermic ischaemia. To determine if mitochondrial deteriorations appearing after normothermic reperfusion are the cause or the consequence of the non-function of the lung appears difficult. On the other hand the lesions appearing after 45 minutes of normothermic ischaemia deserve in an unquestionable way a later study, with evaluation of the mitochondrial function after circulatory arrest in normothermy (30 and 45 minutes) and normothermic reperfusion, and with evaluation of the mitochondrial function after 30 and 45 minutes (or more) of circulatory arrest normothermic but continuation of pulmonary ventilation, or conservation of the lungs in inflation with air or pure oxygen. Our mitochondrial data should also be compared with a study of the pulmonary function by a model of transplantation with survival of the receiver pig.
