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

BACKGROUND: The respiratory properties of perfluorocarbons (PFC) have been widely studied for liquid ventilation in humans and animals. Several PFC were tested but their tolerance may depend on the species. Here, the effects of a single administration of liquid PFC into pig lungs were assessed and compared. Three different PFC having distinct evaporative 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-tracheal administration of 15 ml/kg of either PFOB, PFD or PFOC with 12 h of mechanical ventilation before awakening and weaning from ventilation. A Control group was submitted to mechanical ventilation with no PFC administration. All animals were followed during 4 days after the initial PFC administration to investigate gas exchanges and clinical recovery. They were ultimately euthanized for histological analyses and assessment of PFC residual concentrations within the lungs using dual nuclei fluorine and hydrogen Magnetic Resonance Imaging (MRI). Sixteen animals were included (4/group). RESULTS: In the PFD group, animals tended to be hypoxemic after awakening. In PFOB and PFOC groups, blood gases were not significantly different from the Control group after awakening. The poor tolerance of PFD was likely related to a large amount of residual PFC, as observed using MRI in all lung samples (≈10% of lung volume). This percentage was lower in the PFOB group (≈1%) but remained significantly greater than in the Control group. In the PFOC group, the percentage of residual PFC was not significantly different from that of the Control group (≈0.1%). Histologically, the most striking feature was an alveolar infiltration with foam macrophages, especially in the groups treated by PFD or PFOB. CONCLUSIONS: Of the three tested perfluorocarbons, PFOC offered the best tolerance in terms of lung function, gas exchanges and residuum in the lung. PFOC was rapidly cleared from the lungs and virtually disappeared after 4 days whereas PFOB persisted at significant levels and led to foam macrophage infiltration. PFOC could be relevant for short term total liquid ventilation with a rapid weaning.

Comparative effect of hypothermia and adrenaline during cardiopulmonary resuscitation in rabbits.

BACKGROUND: 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.

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

BACKGROUND: 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-ß-(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 ß2-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-1ß, 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.

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

OBJECTIVES: 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.

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

BACKGROUND: 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.

Adenosine and opioid receptors do not trigger the cardioprotective effect of mild hypothermia.

Mild hypothermia (32°C-34°C) exerts a potent cardioprotection in animal models of myocardial infarction. Recently, it has been proposed that this beneficial effect is related to survival signaling. We, therefore, hypothesized that the well-known cardioprotective pathways dependent on adenosine and/or opioid receptors could be the trigger of hypothermia-induced salvage. Open-chest rabbits were accordingly exposed to 30 minutes of coronary artery occlusion (CAO) under normothermic (NT) or hypothermic ([HT] 32°C) conditions. In the latter, hypothermia was induced by total liquid ventilation with temperature-controlled perfluorocarbons in order to effect ultrafast cooling and to accurately control cardiac temperature. After 4 hours of reperfusion, infarct and no-reflow zone sizes were assessed and quantified as a percentage of the risk zone. In animals experiencing HT ischemia, the infarct size was dramatically reduced as compared to NT animals (9% ± 3% vs 55% ± 2% of the risk zone, respectively). Importantly, administration of opioid and adenosine receptor antagonists (naloxone [6 mg/kg iv] and 8-(p-sulfophenyl) theophylline [20 mg/kg iv], respectively) did not alter the infarct size or affect the cardioprotective effect of hypothermia. Doses of these 2 antagonists were appropriately chosen since they blunted infarct size reduction induced by selective opioid or adenosine receptor stimulation with morphine (0.3 mg/kg iv) or N (6)-cyclopentyladenosine ([CPA] 100 µg/kg iv), respectively. Therefore, the cardioprotective effect of mild hypothermia is not triggered by either opioid or adenosine receptor activation, suggesting the involvement of other cardioprotective pathways.

The small chill: mild hypothermia for cardioprotection?

Reducing the heart's temperature by 2-5°C is a potent cardioprotective treatment in animal models of coronary artery occlusion. The anti-infarct benefit depends upon the target temperature and the time at which cooling is instituted. Protection primarily results from cooling during the ischaemic period, whereas cooling during reperfusion or beyond offers little protection. In animal studies, protection is proportional to both the depth and duration of cooling. An optimal cooling protocol must appreciably shorten the normothermic ischaemic time to effectively salvage myocardium. Patients presenting with acute myocardial infarction could be candidates for mild hypothermia since the current door-to-balloon time is typically 90 min. But they would have to be cooled quickly shortly after their arrival. Several strategies have been proposed for ultra-fast cooling, but most like liquid ventilation and pericardial perfusion are too invasive. More feasible strategies might include cutaneous cooling, peritoneal lavage with cold solutions, and endovascular cooling with intravenous thermodes. This last option has been investigated clinically, but the results have been disappointing possibly because the devices lacked capacity to cool the patient quickly or cooling was not implemented soon enough. The mechanism of hypothermia's protection has been assumed to be energy conservation. However, whereas deep hypothermia clearly preserves ATP, mild hypothermia has only a modest effect on ATP depletion during ischaemia. Some evidence suggests that intracellular signalling pathways might be responsible for the protection. It is unknown how cooling could trigger these pathways, but, if true, then it might be possible to duplicate cooling's protection pharmacologically.

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

STUDY 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.

The ceiling effect of pharmacological postconditioning with the phytoestrogen genistein is reversed by the GSK3beta inhibitor SB 216763 [3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] through mitochondrial ATP-dependent potassium channel opening.

In the present study, we investigated the efficacy of pharmacological postconditioning induced by 17beta-estradiol and the phytoestrogen, genistein, against myocardial infarction induced by increasing durations of coronary artery occlusion (CAO). Anesthetized rabbits underwent either 20-min (protocol A) or 30-min (protocol B) CAO, followed by 4 h of coronary artery reperfusion (CAR). Before CAR, they randomly received an intravenous injection of either vehicle (control), 100 or 1000 microg/kg genistein (Geni(100) and Geni(1000), respectively), or 100 microg/kg 17beta-estradiol (17beta-E(100)). In protocol A, infarct size was significantly reduced in Geni(100) (n = 6), Geni(1000) (n = 6), and 17beta-E(100) (n = 6) versus control (n = 9) (6 +/- 2, 15 +/- 4, and 11 +/- 3 versus 35 +/- 5%, respectively). In protocol B, none of these drugs reduced infarct size versus control. Western blots demonstrated an increase of Akt phosphorylation in the Geni(100) and 17beta-E(100) hearts submitted to 20-min CAO but not to 30-min CAO. The selective GSK3beta inhibitor SB 216763 (0.2 mg/kg) [3-(2,4)-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] did not exhibit cardioprotection at this dose, but its administration restored the cardioprotective effect of genistein and 17beta-estradiol with 30-min CAO. Administration of 5-hydroxydecanoate (5 mg/kg) abolished the cardioprotective effects of Geni(100) and 17beta-E(100) alone with 20-min CAO and also those observed when combined to SB 216763 with 30-min CAO. Thus, pharmacological postconditioning with genistein and 17beta-estradiol is limited by a "ceiling effect of protection" along with a loss of Akt phosphorylation. However, this ceiling effect is reversed by concomitant inhibition of GSK3beta by SB 216763 through opening of mitochondrial ATP-dependent potassium channels.

Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction.

AIMS: We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion. METHODS AND RESULTS: Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV(15') groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32-34 degrees C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (15.1 +/- 3.3%, 16.4 +/- 2.3%, 1.8 +/- 0.6%, and 1.1 +/- 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (4 +/- 1%, 11 +/- 5%, 39 +/- 2%, and 42 +/- 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 +/- 12 vs. 86 +/- 12 microM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control. CONCLUSION: Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.