Evaluation of lung recovery after static administration of three different perfluorocarbons in pigs.

International audienceBackground: The respiratory properties of perfluorocarbons (PFC) have been widely studied for liquid ventilation inhumans and animals. Several PFC were tested but their tolerance may depend on the species. Here, the effects of asingle administration of liquid PFC into pig lungs were assessed and compared. Three different PFC having distinctevaporative and spreading coefficient properties were evaluated (Perfluorooctyl bromide [PFOB], perfluorodecalin[PFD] and perfluoro-N-octane [PFOC]).Methods: Pigs were anesthetized and submitted to mechanical ventilation. They randomly received an intra-trachealadministration of 15 ml/kg of either PFOB, PFD or PFOC with 12 h of mechanical ventilation before awakening andweaning from ventilation. A Control group was submitted to mechanical ventilation with no PFC administration. Allanimals were followed during 4 days after the initial PFC administration to investigate gas exchanges and clinicalrecovery. They were ultimately euthanized for histological analyses and assessment of PFC residual concentrationswithin the lungs using dual nuclei fluorine and hydrogen Magnetic Resonance Imaging (MRI). Sixteen animals wereincluded (4/group).Results: In the PFD group, animals tended to be hypoxemic after awakening. In PFOB and PFOC groups, blood gaseswere not significantly different from the Control group after awakening. The poor tolerance of PFD was likely related toa large amount of residual PFC, as observed using MRI in all lung samples (≈10% of lung volume). This percentage waslower in the PFOB group (≈1%) but remained significantly greater than in the Control group. In the PFOC group, thepercentage of residual PFC was not significantly different from that of the Control group (≈0.1%). Histologically, themost striking feature was an alveolar infiltration with foam macrophages, especially in the groups treated by PFD orPFOB.Conclusions: Of the three tested perfluorocarbons, PFOC offered the best tolerance in terms of lung function, gasexchanges and residuum in the lung. PFOC was rapidly cleared from the lungs and virtually disappeared after 4 dayswhereas PFOB persisted at significant levels and led to foam macrophage infiltration. PFOC could be relevant for shortterm total liquid ventilation with a rapid weaning

Study of hypothermia introduced by total liquid ventilation in cardiac and neurological dysfunction in the course of a cardiac arrest in experimental rabbits

Le refroidissement in vivo du myocarde jusqu'à 32-34°C est une stratégie cardioprotectrice extrêmement puissante expérimentalement. La ventilation liquide totale (VLT) par des perfluorocarbones permet d'induire cette hypothermie très rapidement en utilisant le poumon comme bioéchangeur thermique, tout en maintenant des échanges gazeux normaux. L'objectif de notre travail de thèse a été d'étudier les effets de la VLT hypothermisante sur les lésions d'ischémie-reperfusion dans des modèles d'ischémie myocardique régionale et d'arrêt cardiorespiratoire chez le lapin. Dans une première étude, nous avons démontré que l'instauration per-ischémique d'un épisode de VLT hypothermisante permettait d'atteindre la température cible de 32-33°C en seulement 5 min chez des lapins chroniquement instrumentés soumis à 30 min d'occlusion coronaire. Cela permettait d'atténuer non seulement la taille d'infarctus mais également la dysfonction contractile post-infarctus. La taille d'infarctus était ainsi respectivement réduite de 89 et 71% lorsque la VLT était débutée à la 5ème ou à la 15ème min d'occlusion coronaire par rapport au groupe Témoin. Cette protection était associée à un effet bénéfique sur la fonction mitochondriale dont la capacité de rétention calcique était significativement accrue par l'hypothermie.Dans une seconde étude, nous avons montré que cet effet cardioprotecteur de la VLT hypothermisante n'était pas limité par un plateau de cardioprotection puisqu'il persistait lors d'ischémies prolongées (60 min) chez des lapins anesthésiés. Les tailles de l'infarctus et du no-reflow étaient en effet respectivement diminuées de 45 et 74% dans un groupe d'animaux soumis à une VLT hypothermisante à partir de la 5ème min d'occlusion coronaire par rapport au groupe Témoin. Lorsque la VLT était instaurée plus tardivement (20ème min d'occlusion coronaire), ces réductions restaient significatives (respectivement -30% et -60%) alors qu'un refroidissement conventionnel par application de couvertures froides n'induisait aucun bénéfice, même lorsqu'il était débuté dès la 5ème min d'occlusion coronaire.Grâce aux travaux précédents, nous avons obtenu la preuve de concept de l'effet cardioprotecteur de la VLT hypothermisante au cours d'une ischémie myocardique régionale. Cependant, la translation clinique de cette stratégie est probablement délicate dans la situation de l'infarctus du myocarde. Il nous a en revanche semblé qu'elle pourrait être également bénéfique au cours de la réanimation post-arrêt cardiaque. Pour étudier ce phénomène, nous avons mis au point un modèle expérimental d'arrêt cardiaque et de réanimation cardiopulmonaire chez des lapins soumis à un épisode de fibrillation ventriculaire. Nous avons ainsi montré que la VLT hypothermisante permettait d'augmenter significativement la survie et d'atténuer le syndrome post-arrêt cardiaque par rapport à une réanimation conventionnelle. Après 10 min d'arrêt cardiaque, la survie était par exemple respectivement de 7/10 vs 0/10 chez les animaux soumis à la VLT hypothermisante par rapport au groupe Témoin. Ces effets bénéfiques étaient directement liés à la rapidité d'induction de l'hypothermie par la VLT puisqu'une hypothermie conventionnelle par refroidissement externe et perfusion de fluides refroidis n'a pas permis d'induire de protection significative.En conclusion, la VLT hypothermisante est une approche puissamment protectrice lors d'ischémie myocardique régionale et au décours d'un arrêt cardiaque. L'évaluation plus approfondie de cette stratégie nécessite à présent le développement de dispositifs sécurisés pouvant être utilisés sur un modèle gros animal.MOTS-CLES : Ventilation liquide totale, perfluorocarbone, hypothermie, infarctus, no-reflow, arrêt cardiaque. LABORATOIRE D'ACCUEIL : Laboratoire de pharmacologie, INSERM U955 équipe 3, Ecole nationale vétérinaire d'Alfort, Faculté de médecine de Créteil, Université Paris-Est, Créteil.Cooling the ischemic myocardium to 32-34 °C is known to be potently cardioprotective. Total liquid ventilation (TLV) with perfluorocarbons can induce such an hypothermia very fastly using the lung as a heat exchanger while maintaining normal gas exchanges. The aim of this thesis was to investigate its beneficial effects in experimental models of myocardial ischemia and cardiac arrest in rabbits.In our first study, we demonstrated that the institution of hypothermic TLV allowed achieving a cardiac temperature of 32-33°C within only 5 min in chronically instrumented rabbits subjected to 30 min of coronary artery occlusion. This was associated with a significant reduction in infarct size along with a dramatic attenuation of the post-ischemic myocardial contractile dysfunction. As example, infarct size was reduced by 89 and 71% when TLV was initiated at the 5th or 15th min of coronary artery occlusion, respectively. This protection was associated with an attenuation of the mitochondrial dysfunction, as shown by an increased calcium restrain capacity.In a second study, we demonstrated that the protective effect of hypothermic TLV was not limited by a ceiling of cardioprotection since infarct size reduction persisted following prolonged ischemia (60 min) in anesthetized rabbits. Infarct and no-reflow sizes were indeed reduced by 45 and 74% in animals subjected to hypothermic TLV from the 5th min of coronary occlusion as compared to corresponding Controls, respectively. In comparison, a conventional external cooling with cold blankets did not reduce infarct size even when instituted since the 5th of ischemia.Thanks to the previous works, we obtained the proof of concept that hypothermic TLV could offer a dramatic cardioprotection during regional myocardial ischemia. However, the clinical translation of this strategy might be challenging in the clinical scenario of ST-elevation myocardial infarction. This might conversely be less challenging and also beneficial to prevent the post-cardiac arrest syndrome in patients resuscitated from cardiac arrest. In order to investigate this phenomenon, we developed an experimental model of cardiac arrest and cardiopulmonary resuscitation in rabbits submitted to an episode of ventricular fibrillation. Accordingly, we have demonstrated in this model that hypothermic TLV significantly increased survival and attenuated the post-cardiac arrest syndrome as compared to conventional resuscitation. After 10 min of cardiac arrest, survival was for example 7/10 vs 0/10 in animals subjected to hypothermic TLV vs corresponding Controls, respectively. These beneficial effects were directly related to the rapidity at inducing hypothermia with TLV since conventional hypothermia using external cooling and infusion of cold fluids failed to induce a significant protection.In conclusion, hypothermic TLV can induce a dramatic protection during regional myocardial ischemia or following cardiac arrest and resuscitation. In order to further investigate this strategy, we are now requiring a secured device that can be used in large animals.KEYWORDS: total liquid ventilation, perfluorocarbon, hypothermia, infarct, no-reflow, cardiac arrest.Laboratory: Laboratory of Pharmacology, INSERM U955 équipe 3, Ecole nationale vétérinaire d'Alfort, Faculté de médecine de Créteil, Université Paris-Est, Créteil

Rapid cooling of the heart with total liquid ventilation prevents transmural myocardial infarction following prolonged ischemia in rabbits.

International audienceSTUDY AIM: Total liquid ventilation (TLV) with cooled perfluorocarbons has been demonstrated to induce an ultrafast cardioprotective cooling in rabbits. However, it remains unknown whether this technically challenging strategy would be actually more potent than a conventional external cooling after a prolonged ischemia inducing transmural myocardial infarction. METHODS: Anesthetized rabbits were randomly submitted to 60min of coronary artery occlusion (CAO) under normothermic conditions (Control group, n=7) or with cooling started at the 5th min of CAO (target left atrial temperature: 32 degrees C). Cooling procedures were either external cooling using cold blankets (EC group, n=7) or ultrafast cooling initiated by 20min of TLV (TLV group, n=6). An additional group underwent a similar ultrafast cooling protocol started at the 20th min of CAO (TLV(delayed) group, n=6). After reperfusion, all hypothermic animals were rewarmed and infarct size was assessed after 4h. RESULTS: In the EC group, the target temperature was reached only at 60min of CAO whereas this time-interval was dramatically reduced to 15 and 25min of CAO in TLV and TLV(delayed), respectively. Infarct sizes were significantly reduced in TLV and TLV(delayed) but not in EC groups as compared to Control (45+/-18%, 58+/-5%, 78+/-10% and 82+/-7% of the risk zone, respectively). Similar significant differences were observed for the sizes of the no-reflow zones (15+/-9%, 23+/-8%, 49+/-11% and 58+/-13% of the risk zone, respectively). CONCLUSION: Cooling induced by TLV afforded a potent cardioprotection and prevented transmural infarction following prolonged and severe ischemia, even when started later than a surface cooling in rabbits

Mild hypothermia reduces per-ischemic reactive oxygen species production and preserves mitochondrial respiratory complexes.: Mild hypothermia, cardioprotection and mitochondria

International audienceBACKGROUND: Mitochondrial dysfunction is critical following ischemic disorders. Our goal was to determine whether mild hypothermia could limit this dysfunction through per-ischemic inhibition of reactive oxygen species (ROS) generation. METHODS: First, ROS production was evaluated during simulated ischemia in an vitro model of isolated rat cardiomyocytes at hypothermic (32°C) vs. normothermic (38°C) temperatures. Second, we deciphered the direct effect of hypothermia on mitochondrial respiration and ROS production in oxygenated mitochondria isolated from rabbit hearts. Third, we investigated these parameters in cardiac mitochondria extracted after 30-min of coronary artery occlusion (CAO) under normothermic conditions (CAO-N) or with hypothermia induced by liquid ventilation (CAO-H; target temperature: 32°C). RESULTS: In isolated rat cardiomyocytes, per-ischemic ROS generation was dramatically decreased at 32 vs. 38°C (e.g., -55±8% after 140min of hypoxia). In oxygenated mitochondria isolated from intact rabbit hearts, hypothermia also improved respiratory control ratio (+22±3%) and reduced H2O2 production (-41±1%). Decreased oxidative stress was further observed in rabbit hearts submitted to hypothermic vs. normothermic ischemia (CAO-H vs. CAO-N), using thiobarbituric acid-reactive substances as a marker. This was accompanied by a preservation of the respiratory control ratio as well as the activity of complexes I, II and III in cardiac mitochondria. CONCLUSION: The cardioprotective effect of mild hypothermia involves a direct effect on per-ischemic ROS generation and results in preservation of mitochondrial function. This might explain why the benefit afforded by hypothermia during regional myocardial ischemia depends on how fast it is instituted during the ischemic process

Comparative effect of hypothermia and adrenaline during cardiopulmonary resuscitation in rabbits.: Intra-arrest hypothermia and cold saline infusion

International audienceBACKGROUND: Therapeutic hypothermia was shown to facilitate resumption of spontaneous circulation when instituted during cardiac arrest. Here, we investigated whether it directly improved the chance of successful resuscitation independently of adrenaline administration in rabbits. We further evaluated the direct effect of hypothermia on vascular function in vitro. METHODS: In a first set of experiments, four groups of anesthetized rabbits were submitted to 15 min of cardiac arrest and subsequent cardiopulmonary resuscitation (CPR). The "control" group underwent CPR with only cardiac massage and defibrillation attempts. Two other groups received cold or normothermic saline infusion during CPR (20 mL/kg of NaCl 0.9% at 4°C or 38°C, respectively). In a last group, the animals received adrenaline (15 µg/kg intravenously) during CPR. In a second set of experiments, we evaluated at 32°C vs. 38°C the vascular function of aortic rings withdrawn from healthy rabbits or after cardiac arrest. RESULTS: In the first set of experiments, cardiac massage efficiency was improved by adrenaline but neither by hypothermic nor normothermic saline administration. Resumption of spontaneous circulation was observed in five of eight animals after adrenaline as compared with none of eight in other groups. Defibrillation rates were conversely similar among groups (7/8 or 8/8). In the second set of experiments, in vitro hypothermia (32°C) was not able to prevent the dramatic alteration of vascular function observed after cardiac arrest. It also did not directly modify vasocontractile or the vasodilating functions in healthy conditions. CONCLUSION: In rabbits, hypothermia did not exert a direct hemodynamic or vascular effect that might explain its beneficial effect during CPR

Hypothermic liquid ventilation prevents early hemodynamic dysfunction and cardiovascular mortality after coronary artery occlusion complicated by cardiac arrest in rabbits.

International audienceOBJECTIVES: Ultrafast and whole-body cooling can be induced by total liquid ventilation with temperature-controlled perfluorocarbons. Our goal was to determine whether this can afford maximal cardio- and neuroprotections through cooling rapidity when coronary occlusion is complicated by cardiac arrest. DESIGN: Prospective, randomized animal study. SETTING: Academic research laboratory. SUBJECTS: Male New Zealand rabbits. INTERVENTIONS: Chronically instrumented rabbits were submitted to coronary artery occlusion and ventricular fibrillation. After 8 minutes of cardiac arrest, animals were resuscitated and submitted to a normothermic follow-up (control group) or to 3 hours of mild hypothermia induced by total liquid ventilation (total liquid ventilation group) or by combination of cold saline infusion and cold blankets application (saline group). Coronary reperfusion was permitted 40 minutes after the onset of occlusion. After awakening, rabbits were followed up during 7 days. MEASUREMENTS AND MAIN RESULTS: Ten animals were resuscitated in each group. In the control group, all animals secondarily died of cardiac/respiratory failure (8 of 10) or neurological dysfunction (2 of 10). In the saline group, the target temperature of 32°C was achieved within 30-45 minutes after cooling initiation. This slightly reduced infarct size versus control (41% ± 16% vs 54% ± 8% of risk zone, respectively; p < 0.05) but failed to significantly improve cardiac output, neurological recovery, and survival rate (three survivors, six death from cardiac/respiratory failure, and one from neurological dysfunction). Conversely, the 32°C temperature was achieved within 5-10 minutes in the total liquid ventilation group. This led to a dramatic reduction in infarct size (13% ± 4%; p < 0.05 vs other groups) and improvements in cardiac output, neurological recovery, and survival (eight survivors, two deaths from cardiac/respiratory failure). CONCLUSIONS: Achieving hypothermia rapidly is critical to improve the cardiovascular outcome after cardiac arrest with underlying myocardial infarction

Kidney protection by hypothermic total liquid ventilation after cardiac arrest in rabbits.: renal protection and hypothermic liquid ventilation

International audienceBACKGROUND: Total liquid ventilation (TLV) with perfluorocarbons has been shown to induce rapid protective cooling in animal models of myocardial ischemia and cardiac arrest, with improved neurological and cardiovascular outcomes after resuscitation. In this study, the authors hypothesized that hypothermic TLV can also limit kidney injury after cardiac arrest. METHODS: Anesthetized rabbits were submitted to 15 min of untreated ventricular fibrillation. After resuscitation, three groups of eight rabbits each were studied such as (1) life support plus hypothermia (32°-33 °C) induced by cold TLV (TLV group), (2) life support without hypothermia (control group), and (3) Sham group (no cardiac arrest). Life support was continued for 6 h before euthanasia and kidney removal. RESULTS: Time to target esophageal temperature was less than 5 min in the TLV group. Hypothermia was accompanied by preserved renal function in the TLV group as compared with control group regarding numerous markers including creatinine blood levels (12 ± 1 vs. 16 ± 2 mg/l, respectively; mean ± SEM), urinary N-acetyl-β-(D)-glucosaminidase (1.70 ± 0.11 vs. 3.07 ± 0.10 U/mol of creatinine), γ-glutamyltransferase (8.36 ± 0.29 vs. 12.96 ± 0.44 U/mol of creatinine), or β2-microglobulin (0.44 ± 0.01 vs. 1.12 ± 0.04 U/mol of creatinine). Kidney lesions evaluated by electron microscopy and conventional histology were also attenuated in TLV versus control groups. The renal-protective effect of TLV was not related to differences in delayed inflammatory or immune renal responses because transcriptions of, for example, interferon-γ, tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, and vascular endothelial growth factor were similarly altered in TLV and control versus Sham. CONCLUSION: Ultrafast cooling with TLV is renal protective after cardiac arrest and resuscitation, which could increase kidney availability for organ donation