ultrafast and whole-body cooling with total liquid ... · because total liquid ventilation (tlv)...

TissierZini, J.-L. Dubois-Randé, P. Carli, B. Vivien, J.-D. Ricard, A. Berdeaux and R. M. Chenoune, F. Lidouren, C. Adam, S. Pons, L. Darbera, P. Bruneval, B. Ghaleh, R.Favorable Neurological and Cardiac Outcomes After Cardiac Arrest in Rabbits

Ultrafast and Whole-Body Cooling With Total Liquid Ventilation Induces

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Resuscitation Science

Ultrafast and Whole-Body Cooling With Total LiquidVentilation Induces Favorable Neurological and Cardiac

Outcomes After Cardiac Arrest in RabbitsM. Chenoune, DVM, PhD; F. Lidouren, BSc; C. Adam, MD; S. Pons, PharmD, PhD; L. Darbera, MSc;

P. Bruneval, MD; B. Ghaleh, PhD; R. Zini, PhD; J.-L. Dubois-Rande, MD, PhD; P. Carli, MD, PhD;B. Vivien, MD, PhD; J.-D. Ricard, MD, PhD;

A. Berdeaux, MD, PhD; R. Tissier, DVM, PhD

Background—In animal models of cardiac arrest, the benefit afforded by hypothermia is closely linked to the rapidity ofthe decrease in body temperature after resuscitation. Because total liquid ventilation (TLV) with temperature-controlledperfluorocarbons induces a very rapid and generalized cooling, we aimed to determine whether this could limit thepost–cardiac arrest syndrome in a rabbit model. We especially focused on neurological, cardiac, pulmonary, liver andkidney dysfunctions.

Methods and Results—Anesthetized rabbits were submitted to either 5 or 10 minutes of untreated ventricular fibrillation.After cardiopulmonary resuscitation and resumption of a spontaneous circulation, the animals underwent eithernormothermic life support (control) or therapeutic hypothermia induced by TLV. The latter procedure decreasedesophageal and tympanic temperatures to 32°C to 33°C within only 10 minutes. After rewarming, the animals submittedto TLV exhibited an attenuated neurological dysfunction and decreased mortality 7 days later compared with control.The neuroprotective effect of TLV was confirmed by a significant reduction in brain histological damages. We alsoobserved limitation of myocardial necrosis, along with a decrease in troponin I release and a reduced myocardial caspase3 activity, with TLV. The beneficial effects of TLV were directly related to the rapidity of hypothermia inductionbecause neither conventional cooling (cold saline infusion plus external cooling) nor normothermic TLV elicited asimilar protection.

Conclusions—Ultrafast cooling instituted by TLV exerts potent neurological and cardiac protection in an experimentalmodel of cardiac arrest in rabbits. This could be a relevant approach to provide a global and protective hypothermiaagainst the post–cardiac arrest syndrome. (Circulation. 2011;124:901-911.)

Key Words: cardiopulmonary resuscitation � fibrillation � heart arrest � ischemia � ventilation

Institution of mild therapeutic hypothermia (32°C to 34°C)during 24 to 36 hours after resuscitation is known to

improve survival and neurological recovery in comatosesurvivors of cardiac arrest.1,2 However, experimental studiesin dogs,3,4 pigs,5,6 and rodents7,8 demonstrated that the neu-roprotection afforded by hypothermia was related to therapidity of the decrease in body temperature after resuscita-tion. When achieved rapidly, hypothermia could also bebeneficial for other organs because, for example, it can alsobe potently cardioprotective during myocardial ischemia.9–12

Accordingly, many strategies were proposed to afford such arapid hypothermia, including intravenous infusion of coldfluid13 and endovascular14 or intranasal cooling.15,16

Clinical Perspective on p 911Another strategy that can experimentally provide very

rapid and generalized cooling is liquid ventilation of the lungswith temperature-controlled perfluorocarbons.11,17–22 Theseliquids can use the lungs as heat exchangers while maintain-ing normal gas exchanges.18–20 In addition, this ventilation

Received December 29, 2010; accepted June 24, 2011.From INSERM, U955, Creteil (M.C., F.L., S.P., L.D., B.G., R.Z., J.-L.D.-R., A.B., R.T.); Universite Paris Est, Faculte de Medecine, Creteil (M.C.,

F.L., S.P., L.D., B.G., R.Z., J.-L.D.-R., A.B., R.T.); Ecole Nationale Veterinaire d’Alfort, Maisons-Alfort (M.C., F.L., S.P., L.D., B.G., R.Z., J.-L.D.-R.,A.B., R.T.); INSERM, Unite 970 and Assistance Publique, Hopitaux de Paris, Hopital Europeen Georges Pompidou, Paris (C.A., P.B.); INSERM, Unite722, UFR de Medecine Paris Diderot, Paris, and Assistance Publique, Hopitaux de Paris, Hopital Louis Mourier, Service de ReanimationMedico-chirurgicale, Colombes (J.-D.R.); and SAMU de Paris, Departement d’Anesthesie Reanimation, CHU Necker Enfants Malades, Faculte deMedecine Descartes–Paris V, Paris (P.C., B.V.), France.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.111.039388/-/DC1.

Presented in part at the American Heart Association Resuscitation Symposium, November 12–13, 2010, Chicago, IL.Correspondence to Renaud Tissier, INSERM, Unite 955, Equipe 3, Faculte de Medecine, 8 Rue du General Sarrail, 94010 Creteil Cedex, France. E-mail

[email protected]© 2011 American Heart Association, Inc.

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procedure protects lung integrity.20 Using a prototype of totalliquid ventilator that alternatively instills and removes a tidalvolume of perfluorocarbon from the lung, we were able todecrease the left atrial temperature to 32°C within only 5minutes in anesthetized rabbits.11,17,18 This decrease wasassociated with very potent protection against myocardialinfarction and subsequent contractile dysfunction in animalmodels of coronary artery occlusion.11,17,18 In a swinemodel of ventricular fibrillation, liquid ventilation alsoinduced a rapid convective cooling that further improvesthe chances for subsequent resumption of spontaneouscirculation.21,22 However, to the best of our knowledge, theeffect of hypothermic total liquid ventilation (TLV) hasnever been investigated in animal models of post– cardiacarrest dysfunction when instituted after resumption ofspontaneous circulation.

Accordingly, the main purpose of the present study was toinvestigate the long-term effect of ultrafast cooling inducedby TLV in a rabbit model of post–cardiac arrest dysfunctionafter ventricular fibrillation and resuscitation. To determinewhether hypothermic TLV properly protects through veryfast cooling, we investigated 2 additional groups submitted toconventional hypothermia (cold saline infusion plus externalcooling) or to normothermic TLV. The primary outcome wassurvival during 7 days of follow-up. The secondary outcomeswere clinical, biochemical, hemodynamic, and histologicalparameters describing neurological, cardiac, pulmonary,liver, and kidney potential dysfunctions. We also aimed toinvestigate whether ultrafast cooling can protect the heartthrough an early inhibition of cardiac cell death. The latterpoint was also critical to further support the relevance of veryfast cooling to limit subsequent dysfunction after cardiacarrest.

MethodsThe animal instrumentation and ensuing experiments were con-ducted in accordance with French official regulations (agreementA94-046-13) after approval by the local ethics committee. Theinvestigation conformed to the Guide for the Care and Use ofLaboratory Animals published by the US National Institutesof Health.

Animal PreparationNew Zealand rabbits (3.0 to 3.5 kg) were anesthetized with zolaz-epam, tiletamine, and pentobarbital (all 20 to 30 mg/kg IV). Theywere intubated and mechanically ventilated. After administration ofpancuronium bromide (200 �g/kg IV), 2 electrodes were implantedon the chest and inserted into the esophagus for subsequent inductionof ventricular fibrillation. Rectal, esophageal, and tympanic temper-atures were monitored continuously with thermal probes (HarvardApparatus, Paris, France). Throughout the protocol, external ECGwas recorded, as well as arterial blood pressure from a catheterimplanted in the ear artery. Data were digitalized and analyzed withthe data acquisition software HEM version 3.5 (Notocord, Croissy-sur-Seine, France).

Cardiac Arrest and Cardiopulmonary ResuscitationAfter animal preparation and subsequent stabilization, ventricularfibrillation was induced by passing an alternative current (10 V, 4mA; 2 minutes) between the implanted electrodes. Mechanicalventilation was stopped at the onset of fibrillation and throughout the

subsequent period of cardiac arrest. After either 5 or 10 minutes ofuntreated fibrillation, cardiopulmonary resuscitation was started withcardiac massage (�200 bpm), electric attempts at defibrillation (5 to10 J/kg), and intravenous administration of epinephrine (15 �g/kgIV). Resumption of spontaneous circulation (ROSC) was consideredan organized cardiac rhythm associated with a mean arterial pressure�40 mm Hg for at least 1 minute. After ROSC, administration ofepinephrine was further permitted during a maximum of 7 hours ata dosage appropriately adjusted to maintain the mean arterialpressure at �80 mm Hg. Mechanical ventilation was continued untilweaning and awakening of the animals. Rabbits were subsequentlyreturned to their cage for survival follow-up. They received antibi-otics (enrofloxacine 5 mg/kg IM) for 7 days and analgesics (bu-prenorphine 30 �g/kg SC) for 3 days.

Experimental ProtocolAs shown in Figure 1, rabbits randomly underwent either 5 or 10minutes of cardiac arrest with subsequent cardiopulmonary resusci-tation. For each duration of cardiac arrest, rabbits were randomlyallocated to resuscitation under normothermic conditions (Control5�

and Control10� groups) or with hypothermia induced by TLV(H-TLV5� and H-TLV10� groups). In these last 2 groups, TLV wasstarted at the 10th minute after cardiopulmonary resuscitation (ie,after ROSC) by filling the lung with 10 mL/kg perfluorocarbon(Fluorinert 3M, Cergy, France) and then connecting the endotrachealtube to our prototype liquid ventilator (Figure I in the online-onlyData Supplement).11,17,18 This ventilator was set to a tidal volume of�7 to 10 mL/kg body weight with a respiratory rate of 6 breaths perminute. For each breath, the ventilator pumped the tidal volume ofliquids into and out of the lungs. The perfluorocarbon mixture wasbubbled with 100% O2. The temperature of the heat exchanger wasadjusted to maintain esophageal and tympanic temperatures at atarget temperature of �32°C. After 20 minutes of TLV and achieve-ment of the hypothermic target temperature, the perfluorocarbon wasevacuated from the lungs by gravity, and the endotracheal tube wasagain connected to a conventional mechanical ventilator. Hypother-mia was further maintained at 32°C during 3 hours, if necessary withcold blankets. Animals were subsequently rewarmed with infraredlights and thermal pads until weaning from conventional ventilationand awakening. Animals were housed in a closed cage enriched inO2 for 2 to 3 days to avoid hypoxic episodes. To determine whetherhypothermic TLV properly protects through very fast cooling, weinvestigated 2 randomly allocated additional groups submitted to 10minutes of cardiac arrest. The first of these groups (Saline10�) wassubmitted to 3 hours of conventional hypothermia through thecombination of cold saline administration (30 mL/kg IV, NaCl 0.9%at 4°C) and external cooling. The second additional group wassubmitted to an episode of TLV with normothermic perfluorocarbons(N-TLV10� group) to determine their proper effects.

To further investigate the effects of hypothermic TLV, addi-tional rabbits were included in the Control10� and H-TLV10�

groups. These animals were euthanized 1 hour after the cardiacarrest episode for collection of myocardial and blood samples forcaspase activity assays and measurement of circulating troponin I,respectively.

Neurological and Cardiac Dysfunction AssessmentNeurological dysfunction was evaluated daily in surviving animalswith a clinical score previously validated in rabbits,23 as shown inTable I in the online-only Data Supplement (0% to 10%�normal,100%�brain death). After 7 days of follow-up, surviving rabbitswere reanesthetized, and a pressure catheter (SciSense, London,Ontario, Canada) was introduced into the left ventricle through theright carotid artery for measurement of end-diastolic pressures andpositive and negative left ventricular rates of pressure development(dP/dtmax and dP/dtmin). These parameters were also measured in agroup of sham rabbits that were not submitted to either cardiac arrestor hypothermia.

902 Circulation August 23, 2011



Blood Chemistry and Caspase Activity AssayBlood pH and carbon dioxide and oxygen partial pressures (PCO2 andPO2, respectively) were assessed from arterial blood samples with anABL 77 series analyzer (Radiometer SA, France). Blood lactate wasdetermined on an Accutrend Plus analyzer (Roche Diagnostics,Mannheim, Germany). Liver and renal function was evaluated bymeasuring the alanine aminotransferase and creatinine concentra-tions (Prestige 24i, Tokyo-Boehi, Japan). Troponin I and creatininekinase were measured by an offsite laboratory (IDEXX Laboratories,Alfortville, France).

Caspase 3 activity was assayed from cardiac samples, as previ-ously described.24 Briefly, tissues were homogenized in cold buffer(25 mmol/L HEPES, pH 7.5, 5 mmol/L MgCl2, 2 mmol/L EDTA,0.1% Triton X-100, 2 mmol/L dithiothreitol, 1 mmol/L phenylmeth-anesulfonyl fluoride, 5 �L/mL protease cocktail inhibitor P8340;Sigma-Aldrich, St. Louis, MO). Homogenates were centrifuged andsupernatants collected. Proteins (90 �g) were incubated in caspaseassay buffer (50 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl,1 mmol/L EDTA, 10 mmol/L dithiothreitol, Triton X-100 0.1%,glycerol 10%). Enzymatic reaction was started by the addition of0.2 mmol/L of the fluorogenic substrates ac-DEVD-AFC (BiomolResearch Laboratories, Hamburg, Germany). Fluorescent arbitraryunits were converted into picomoles per 1 mg protein per hour witha standard curve of free AFC (Biomol Research Laboratories).

Histological AnalysesAfter 7 days of follow-up after cardiac arrest, the surviving rabbitswere euthanized for pathological analyses of the heart, lung, kidney,liver, and brain. These organs were also removed and analyzed in theanimals that died before the end of the follow-up. For lungs, a slicewas sampled from each lobe (5 per lung). For the heart, we analyzeda midheart transversal biventricular section. For kidneys, 2 sliceswere studied for each organ. We used a 0 to 3 scoring system toblindly quantify the severity of each organ alteration, as shown inTables II and III in the online-only Data Supplement (0�normal,

3�very severe lesion). The overall brain score was the mean valueobtained for cortex, hippocampus, and cerebellum, as previouslydescribed.23 For lungs, we assessed 2 separate scores for cardio-genic lesions (serous edema and/or congestion) and infectiouscomplication of bronchopneumonia. The first panels of Figure 2and 3 illustrate typical normal and pathological aspects of thedifferent organs.

Statistical AnalysesData are expressed as mean�SEM. Hemodynamic and biochemicalparameters were compared between the different groups and corre-sponding controls by use of 2-way ANOVA for repeated measures.Post hoc analyses were performed at each time point compared withcontrols by use of a Student t test with Bonferroni correction. Valueswere not compared between the different time points to avoidmultiple comparisons. In each experimental group, neurologicaldysfunction and histological scores were compared with those of thecorresponding control group by use of a Mann-Whitney nonpara-metric test. Survival curves were obtained with a Kaplan-Meieranalysis and were compared with the corresponding control groupthrough the use of a log-rank test. These last analyses took intoaccount only the animals that achieved ROSC. Significant differ-ences were determined at P�0.05.

ResultsSeventy rabbits were included in the present study andsubmitted to cardiac arrest (n�10, 10, 15, 15, 10, and 10 inthe Control5�, H-TLV5�, Control10�, H-TLV10�, Saline10�, andN-TLV10� groups, respectively). As shown in Table 1, allrabbits subjected to 5 minutes of cardiac arrest (Control5� andH-TLV5� groups) were successfully resuscitated, whereasonly 10 of 15 were successfully resuscitated in the Control10�

Figure 1. Experimental protocol. CA indi-cates cardiac arrest; TLV, total liquid ven-tilation; H-TLV, hypothermic TLV; N-TLV,normothermic TLV; Saline, hypothermiainduced by intravenous administration ofcold saline combined with external cool-ing; and ROSC, resumption of spontane-ous circulation.

Chenoune et al Liquid Ventilation, Hypothermia, and Cardiac Arrest 903



and H-TLV10� groups. This rate was 7 of 10 in the Saline10�

and N-TLV10� groups. Regardless of the duration of cardiacarrest, the time to ROSC was not significantly differentamong groups for each duration of cardiac arrest.

As illustrated in Figure 4, esophageal, tympanic, and rectaltemperatures were not significantly different among groups atbaseline. A mild and passive decrease in body temperaturewas observed in the Control5� and Control10� groups aftercardiac arrest but remained within the normothermic range. Inthe H-TLV groups, esophageal and tympanic temperaturesdecreased very rapidly after the institution of TLV. Forexample, tympanic temperature reached 33.3�0.5°C and32.5�0.3°C in H-TLV5� and H-TLV10� , respectively, within10 minutes after the onset of the cooling protocol. In theSaline10� group, such tympanic temperatures were achievedafter �30 minutes. For esophageal and rectal temperatures,the time to achieve 32°C to 33°C was �5 and 20 minutes inH-TLV10� and �45 and 60 minutes in Saline10�, respectively.

In the N-TLV10� group, body temperatures did not signifi-cantly differ from the Control10� values throughout theexperimental protocol.

As shown in Table 2, heart rate significantly decreasedduring the hypothermic phase in hypothermic groups com-pared with corresponding controls (eg, �21%, �28%, and�31% at 60 minutes after cardiac arrest in H-TLV5�,H-TLV10�, and Saline10� versus corresponding controls, re-spectively). Mean arterial pressure was not significantlydifferent between groups throughout the experimental proto-col because epinephrine administration was used to maintaina �80 mm Hg value during 7 hours after cardiac arrest. Asshown in Table 1, the total amount of epinephrine adminis-tered throughout cardiopulmonary resuscitation, however,was significantly lower in H-TLV10� than in Control10�

(128�128 versus 684�118 �g/kg, respectively), suggestinga favorable hemodynamic effect of hypothermic TLV. Wedid not observe such a significant difference in H-TLV5�

Figure 2. A, Examples of normal or pathological histological appearances of the kidney, liver, and lungs in the total liquid ventilation(TLV) and control groups. In kidney, lesions consisted of dilated regenerative proximal tubules (arrows; bar�120 �m). In liver, weobserved systematized clarification of hepatocytes (arrows; bar�120 �m). In lungs, lesions were congestion with serous edema (arrowsin the left lung panel; bar�120 �m) or foci of bronchopneumonia (arrows in the right lung panel; bar�120 �m). B, Histological scoresof alteration in kidney, liver, and lungs of rabbits from the different groups. For lungs, we assessed 2 separate scores for cardiogeniclesions and infection complications, respectively. Open circles represents individual scores; thick line, the median value of the corre-sponding group. H-TLV indicates hypothermic TLV; N-TLV, normothermic TLV; and Saline, hypothermia induced by intravenous admin-istration of cold saline combined to external cooling.




versus Control5�, but epinephrine dosages were much lower(174�81 versus 207�58 �g/kg, respectively). In Saline10�

and N-TLV10�, epinephrine dosages were also not differentfrom the dosages in Control10�. After discontinuation of anypharmacological support (eg, at 8 hours after cardiac arrest),the lactate levels were significantly lower in H-TLV5� com-pared with Control5� (1.2�0.2 versus 4.8�1.7 mmol/L) and

in H-TLV10� compared with Control10� (3.6�0.7 versus7.0�1.7 mmol/L). Those levels were not significantly differ-ent among the Saline10� and N-TLV10� groups compared withthe Control10� group (5.9�0.7 and 7.6�0.6 versus 7.0�1.7 mmol/L).

As shown in Figure 5, we observed severe acidosis with anincrease in PCO2 and a decrease in PO2 in all groups aftercardiac arrest. In H-TLV5�, PO2 was lower 15 minutes aftercardiac arrest compared with Control5�. This could be ex-pected because control animals were ventilated with oxygen,whereas TLV rabbits underwent liquid ventilation by thattime. At 180 minutes, gas exchanges were conversely im-proved in H-TLV groups compared with controls. For exam-ple, blood pH and PO2 increased and PCO2 decreased inH-TLV10� and Control10�, respectively. Importantly, all ani-mals were submitted to conventional ventilation at that timepoint with standardized ventilation parameters. As illustratedin Figure 2, the safety of TLV for lungs was also documentedby histology demonstrating cardiogenic lesions (serousedema and/or congestion) or infectious complications ofbronchopneumonia to a similar extent in TLV groups andcorresponding controls.

As shown in Table 2, renal function was not affected aftercardiac arrest in all groups because plasma creatinine levelsremained within usual values. Conversely, we observed anincrease in the liver enzyme alanine aminotransferase with no

Figure 3. A, Examples of normal or pathological histological appearances of the brain and the heart in the total liquid ventilation(TLV) and control groups, respectively. In brain, ischemic disorders consisted in ischemic pyramidal cells with pycnotic nucleus inthe hippocampus (arrows; bar�30 �m), in laminar necrosis of Purkinje cells in the cerebellum (arrows, bar�30 �m), or in numer-ous ischemic neurons in the cortex (arrows; bar�30 �m). In the myocardium, we observed foci of cardiomyocytes necrosis(arrows, bar�120 �m). B, Histological scores of alteration in the brain and heart of rabbits from the different groups. Open circlesrepresents individual scores; thick line, the median value of the corresponding group. H-TLV indicates hypothermic TLV; N-TLV,normothermic TLV; and Saline, hypothermia induced by intravenous administration of cold saline combined to external cooling.*P�0.05 vs corresponding control.

Table 1. Group Characteristics During CardiopulmonaryResuscitation, Including the Rate of Successful Resuscitation,Time to Resumption of Spontaneous Circulation, and TotalAmount of Epinephrine Administered Throughout the Protocol

n

Rate ofSuccessful

ResuscitationROSC,min

EpinephrineDose, �g/kg

Control5� 10 10/10 2.4�0.3 207�58

H-TLV5� 10 10/10 2.3�0.3 174�81

Control10� 15 10/15 4.8�0.4 684�118

H-TLV10� 15 10/15 4.2�0.8 128�128*

Saline10� 10 7/10 3.7�0.7 430�126

N-TLV10� 10 7/10 3.7�0.4 509�64

ROSC indicates resumption of spontaneous circulation; TLV, total liquidventilation; H-TLV, hypothermic TLV; N-TLV, normothermic TLV; and Saline,hypothermia induced by intravenous administration of cold saline combinedwith external cooling.

*P�0.05 versus corresponding control value.




difference among TLV and corresponding controls. Kidneyand liver lesions were mild with no difference among groups(Figure 2B).

As illustrated in Figure 6, neurological dysfunction wassignificantly attenuated in the H-TLV groups compared withcontrols. This difference was significant as early as thesecond day after cardiac arrest in H-TLV5� compared withControl5� (Figure 6A), whereas this was observed within 24hours of follow-up in H-TLV10� compared with Control10�

(Figure 6B). In Saline10�, a transient improvement in neuro-logical recovery was observed at day 1, but this was no longersignificant at day 2 after cardiac arrest. As illustrated inFigure 3B, the neuroprotective effect of hypothermic TLVwas further demonstrated by a significant decrease in theseverity of the ischemic disorders in the brain in the H-TLV5�

and H-TLV10� compared with the Control5� and Control10�

groups, respectively. Conversely, no protection was seen inSaline10� and N-TLV10� compared with Control10�.

A significant difference in survival was also shown be-tween the H-TLV groups and corresponding controls, asillustrated in Figure 6C and 6D. At the end of the follow-up,9 of 10 and 7 of 10 rabbits survived in the H-TLV5� andH-TLV10� groups compared with 5 of 10 and 0 of 10 in theControl5� and Control10� groups, respectively. Conversely,survival was not significantly improved in Saline10� andN-TLV10� compared with Control10�.

As illustrated in Figure 3B, myocardial foci of necrosiswere less frequent in H-TLV10� compared with Control10�,demonstrating a cardioprotective effect of hypothermic TLV.Conversely, no difference was seen between the Saline10� and

Figure 4. Esophageal, tympanic, and rec-tal temperatures in the different experi-mental groups. TLV indicates total liquidventilation; H-TLV, hypothermic TLV;N-TLV, normothermic TLV; and Saline,hypothermia induced by intravenousadministration of cold saline combined toexternal cooling. *P�0.05 vs correspond-ing control; n�10 in Control5�, H-TLV5�,Control10�, and H-TLV10�; n�7 in Saline10�

and N-TLV10�.




N-TLV10� groups and the Control10� group. In survivinganimals, the functional myocardial sequels of cardiac arrestwere also evaluated after 7 days of follow-up. As shown inTable IV in the online-only Data Supplement, mean bloodpressure and heart rate in the conscious state were notdifferent among groups compared with a sham group. Afteranesthesia, end-diastolic left ventricular pressure, dP/dtmax,and dP/dtmin were also not different between groups, sug-gesting that there were no major functional myocardialalterations in those surviving animals.

To further explore the cardioprotective effect of hypother-mic TLV, 8 additional rabbits were included in the Control10�

and H-TLV10� groups for a surrogate study dedicated to thecaspase activity assays and measurement of troponin I levels.As shown in Figure II in the online-only Data Supplement,

troponin I measured 60 minutes after cardiac arrest wassignificantly decreased in H-TLV10� compared with Con-trol10� (1.3�0.3 versus 70.7�30.4 ng/mL, respectively). Thecardioprotective effect of hypothermic TLV was also sup-ported by a decrease in caspase 3 activity compared withcontrol (6.2�1.2 versus 10.0�1.2 pmol per 1 mg protein perhour, respectively).

DiscussionThe present study provides proof of concept that ultrafastwhole-body cooling with hypothermic TLV limits the post–cardiac arrest syndrome when instituted after ROSC in arabbit model of ventricular fibrillation. Interestingly, weobserved potent neuroprotection and cardioprotection with

Table 2. Mean Arterial Pressure, Heart Rate, and Plasma Creatinine and Alanine Aminotransferase Concentrations Throughout theExperimental Protocol in the Different Groups

Epinephrine perfusion nBaseline

No

Cardiopulmonary Resuscitation

Day 1 (n)15 min

Yes60 min

Yes180 min

Yes360 min

Yes480 min

No

Heart rate, bpm

Control5� 10 257�11 222�8 221�7 243�11 216�7 220�9 234�8 (10)

H-TLV5� 10 259�10 202�12 174�6* 177�9* 245�9 234�8 244�10 (10)

Control10� 10 263�10 219�6 220�10 198�8 221�11 231�13 256�17 (7)

H-TLV10� 10 267�8 167�10* 158�8* 167�11 208�12 240�11 252�7 (8)

Saline10� 7 266�7 200�10 153�7* 155�13* 219�10 218�10 226�16 (6)

N-TLV10� 7 256�13 216�19 207�9 213�12 207�9 221�15 240�28 (2)

Mean arterial pressure,mm Hg

Control5� 10 81�3 83�4 82�3 83�1 83�4 80�4 83�4 (10)

H-TLV5� 10 80�7 81�3 82�5 82�3 83�3 79�3 82�4 (10)

Control10� 10 80�5 82�3 83�3 81�4 83�2 80�3 79�4 (7)

H-TLV10� 10 83�4 81�4 82�3 81�3 81�4 80�4 79�6 (8)

Saline10� 7 80�8 86�6 89�2 78�5 82�5 76�6 83�9 (6)

N-TLV10� 7 78�7 78�5 78�1 78�4 78�5 75�7 88�4 (2)

Plasma creatinineconcentrations, mg/L

Control5� 10 10�1 11�1 . . . 10�1 . . . . . . 10�1 (10)

H-TLV5� 10 10�0 12�1 . . . 11�1 . . . . . . 10�1 (10)

Control10� 10 9�1 13�1 . . . 14�2 . . . . . . 11�1 (7)

H-TLV10� 10 10�0 13�1 . . . 12�1 . . . . . . 11�1 (8)

Saline10� 7 9�1 10�1 . . . 10�1 . . . . . . 10�1 (6)

N-TLV10� 7 9�1 11�1 . . . 12�1 . . . . . . 13�6 (2)

Plasma ALATconcentrations, UI/L

Control5� 10 29�5 31�4 . . . 33�4 . . . . . . 35�9 (10)

H-TLV5� 10 25�3 26�2 . . . 43�5 . . . . . . 30�6 (10)

Control10� 10 44�13 79�25 . . . 115�32 . . . . . . 60�17 (7)

H-TLV10� 10 48�3 65�5 . . . 111�27 . . . . . . 83�14 (8)

Saline10� 7 32�2 48�4 . . . 101�30 . . . . . . 62�27 (6)

N-TLV10� 7 31�5 66�10 . . . 96�13 . . . . . . 94�37 (2)

TLV indicates total liquid ventilation; H-TLV, hypothermic TLV; N-TLV, normothermic TLV; Saline, hypothermia induced by intravenous administration of cold salinecombined with external cooling; and ALAT, alanine aminotransferase.

*P�0.05 versus corresponding control value.




hypothermic TLV, which remains a safe procedure for thelungs. Because we used only 3 hours of hypothermia, ourfinding also suggested that very early hypothermia afterROSC does not need to be prolonged to produce a strongclinical benefit. Importantly, this benefit was directly relatedto cooling rapidity with TLV because conventional coolingwith cold saline and external blankets was not significantlyprotective in similar conditions. Proper effects of the perfluo-rocarbon are unlikely because of the lack of protection withnormothermic TLV.

Our first finding is the rapidity of TLV-induced coolingbecause esophageal and brain temperatures reached �32°C to33°C within only 10 minutes. In comparison, a conventionalhypothermic protocol (cold saline infusion plus externalcooling) requires �30 and 45 minutes to similarly reducethese temperatures. The rapid cooling elicited by TLV wasrelated directly to the tidal exchange of the liquid becausesimple repetitive pulmonary lavage with a 4°C perfluorocar-bon requires �60 minutes to decrease the tympanic temper-ature to 32°C in the same species.20 In large animals,hypothermic TLV was also reported to provide very fastcooling and to reduce the pulmonary artery temperature to32°C within 9 to 10 minutes when instituted intra-arrest in aventricular fibrillation model in swine.22

Importantly, the rapid hypothermia elicited by TLV wasassociated with potent neurological protection and an in-crease in survival rate compared with control conditions.Animal studies have indicated that the neuroprotective effectof hypothermia is time dependent and that a large part of theprotection is lost when cooling is delayed.25 For example, in

a canine model of cardiac arrest, the neurological protectionwas lost after only 15 minutes of delay before the onset ofhypothermia after ROSC.25 In the present study, we observeda very potent benefit of hypothermia when achievedrapidly after ROSC with TLV, whereas conventionalhypothermia was not significantly protective. Recent ex-periments have also shown that hypothermia started beforeROSC (eg, intra-arrest hypothermia) can provide an addi-tional benefit,7,8 but it might be difficult to translate thisconcept into human clinical practice. All these findingsdemonstrate that most of the possible benefits of hypother-mia can be lost within minutes after ROSC, furthersupporting the need of devices eliciting ultrafast coolingsuch as TLV in the present study.

Importantly, the benefit of hypothermic TLV observedin our conditions was produced by a short hypothermicepisode (3 hours), whereas the current recommendation inhumans is to maintain hypothermia for 24 to 36 hours.1,2

We choose this short duration because previous experi-ments have shown that when hypothermia is achieved veryearly, it does not need to be prolonged to provide aneffective neuroprotection, eg, in a gerbil model of globalischemia.26 Mice studies also noted that 1 hour of coolingafter ROSC was sufficient to generate significant clinicalbenefit.7,8 When the severity of the ischemic insult in-creases or when the onset of cooling is delayed, it isconversely well established that prolonging hypothermia iscritical for achieving a maximal neurological protec-tion.27,28 For example, prolonged cooling provided endur-ing behavioral and histological protection in rats submitted

Figure 5. Blood pH, PCO2, and PO2 in the differentexperimental groups. TLV indicates total liquidventilation; H-TLV, hypothermic TLV; N-TLV, nor-mothermic TLV; and Saline, hypothermia inducedby intravenous administration of cold saline com-bined to external cooling. *P�0.05 vs correspond-ing control; n�10 in Control5�, H-TLV5�, Control10�,and H-TLV10�; n�7 in Saline10� and N-TLV10�.




to permanent middle cerebral artery occlusion, even whendelayed after the onset of ischemia.27

Another important beneficial effect of hypothermic TLVis the cardioprotection observed here like that previouslyshown in animal models of coronary artery occlu-sion.11,17,18 This was especially observed after 10 minutesof cardiac arrest because myocardial lesions were minor inthe groups submitted to only 5 minutes of cardiac arrest.This was evidenced by limited myocardial necrosis andpreserved myocardial functional performance in survivingrabbits. Cardioprotection was also observed very earlyafter cardiac arrest because troponin I release and caspase3 activity were significantly decreased within 60 minutesafter resuscitation in H-TLV10� compared with Control10�.In animal models of focal myocardial ischemia, the win-dow of protection with hypothermia is virtually limited tothe ischemic phase, whereas cooling at reperfusion isineffective at reducing infarct size in most experimentalstudies.12 In the present study, hypothermia was institutedafter global reperfusion (ROSC), but it is reasonable tospeculate that the myocardium remains momentarily andpartially ischemic even after ROSC. This can explain thatvery rapid cooling with TLV can still provide a beneficialeffect even if instituted after ROSC and systemic reperfu-sion. Improved postresuscitation myocardial function has

also be observed with intra-arrest rapid head cooling.29

Generalized hypothermia could even potentially affordprotection of the liver and/or kidney.30 Because theseorgans were mildly altered in control conditions in thepresent study, we were not able to show any differencewith hypothermic TLV.

Importantly, TLV was a safe procedure for the lungs. Weobserved improved gas exchanges using standardized venti-latory parameters in TLV compared with control groups 3hours after cardiac arrest. After weaning from ventilation,however, animals were maintained in a cage enriched inoxygen to avoid hypoxic episodes.11 In pigs, intra-arrestliquid ventilation was demonstrated to alter lung functionbecause activation of pulmonary macrophages might alter gasexchanges after resumption of conventional ventilation.21,22

In our study, the tolerance of TLV was shown by histologicalexaminations, and this is supported by several reports fromthe literature demonstrating that liquid ventilation can protectthe lungs.19,20 Several prototypes of liquid ventilator havebeen developed, and the clinical translation of this conceptmight accordingly be feasible when those devices are avail-able for clinical use.31 To date, the current prototypes aredeveloped mostly for pediatric use,31 and accordingly, thetranslation of TLV-induced hypothermia would be possiblefirst in newborns presenting global ischemia. Further devel-

Figure 6. A and B, Neurological dysfunction scores at days 1, 2, and 7 after resuscitation in the different experimental groups submit-ted to 5 or 10 minutes of cardiac arrest, respectively. Open circles represent individual scores; thick line, the median value of the corre-sponding group. Only animals achieving resumption of spontaneous circulation were included. C and D, Kaplan-Meier survival curvesin the different experimental groups submitted to 5 or 10 minutes of cardiac arrest, respectively. Only animals achieving resumption ofspontaneous circulation were included. TLV indicates total liquid ventilation; H-TLV, hypothermic TLV; N-TLV, normothermic TLV; andSaline, hypothermia induced by intravenous administration of cold saline combined to external cooling. *P�0.05 vs correspondingcontrol.




opments might also ultimately permit a translation to adultpatients.

Our study has several limitations. First, neurological dys-functions were assessed on the basis of clinical and histolog-ical parameters. Other more functional tests or imaging wouldalso be important. Second, histological analyses were per-formed in all animals, regardless of their survival time. Thiswould have led to an underestimation of the histologicalscores in some animals that died very early after cardiacarrest. However, because the lower scores were observed inthe group that lived for the longer time (H-TLV10�), thislimitation should not affect our conclusions.

ConclusionsUltrafast cooling instituted by hypothermic TLV limits thepost–cardiac arrest dysfunction with associated neuroprotec-tive and cardioprotective effects. Importantly, TLV was asafe procedure for the lungs in our experimental conditions.The beneficial effects of hypothermic TLV were probablydirectly related to the rapidity of the temperature decreasebecause myocardial cell death inhibition was seen even veryearly after resuscitation.

AcknowledgmentsWe are indebted to Drs J.M. Downey, M.V. Cohen, and J.C. Parkerfor their insightful comments and creative ideas at the beginning ofthese investigations. We are also greatly indebted to Professor J.Grassi (ITMO Technologies pour la Sante) and Dr C. Cans(INSERM-transfert) for their important support and advice. Wethank Sandrine Bonizec for her excellent administrative support andthe central laboratory of the National Veterinary School of Alfort,which performed the biochemical analyses of the kidney and liverblood parameters.

Sources of FundingThis study was supported by grant TLV-CARDAREST (R10028JS)from INSERM and ITMO Technologies pour la Sante and grantET7-460 from the Fondation de l’Avenir. Dr Chenoune was sup-ported by a grant from the Groupe de Reflexion sur la RechercheCardiovasculaire and by a Poste d’accueil INSERM 2009. Dr Tissierwas also a recipient of a Contrat d’Interface INSERM-ENV (2010)and of a grant from the Societe Franaise de Cardiologie (EdouardCorraboeuf grant, 2010).

DisclosuresNone.

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Smith K. Treatment of comatose survivors of out-of-hospital cardiacarrest with induced hypothermia. N Engl J Med. 2002;346:557–563.

2. Hypothermia After Cardiac Arrest Study Group. Mild therapeutic hypo-thermia to improve the neurologic outcome after cardiac arrest. N EnglJ Med. 2002;346:549–556.

3. Nozari A, Safar P, Stezoski SW, Wu X, Henchir J, Radovsky A, HansonK, Klein E, Kochanek PM, Tisherman SA. Mild hypothermia duringprolonged cardiopulmonary cerebral resuscitation increases conscioussurvival in dogs. Crit Care Med. 2004;32:2110–2116.

4. Nozari A, Safar P, Stezoski SW, Wu X, Kostelnik S, Radovsky A,Tisherman S, Kochanek PM. Critical time window for intra-arrest coolingwith cold saline flush in a dog model of cardiopulmonary resuscitation.Circulation. 2006;113:2690–2696.

5. Guan J, Barbut D, Wang H, Li Y, Tsai MS, Sun S, Inderbitzen B, WeilMH, Tang W. A comparison between head cooling begun during cardio-pulmonary resuscitation and surface cooling after resuscitation in a pigmodel of cardiac arrest. Crit Care Med. 2008;36:S428–S433.

6. Tsai MS, Barbut D, Tang W, Wang H, Guan J, Wang T, Sun S, Inder-bitzen B, Weil MH. Rapid head cooling initiated coincident with cardio-pulmonary resuscitation improves success of defibrillation and post-resuscitation myocardial function in a porcine model of prolonged cardiacarrest. J Am Coll Cardiol. 2008;51:1988–1990.

7. Zhao D, Abella BS, Beiser DG, Alvarado JP, Wang H, Hamann KJ, HoekTL, Becker LB. Intra-arrest cooling with delayed reperfusion yieldshigher survival than earlier normothermic resuscitation in a mouse modelof cardiac arrest. Resuscitation. 2008;77:242–249.

8. Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL, BeckerLB. Intra-arrest cooling improves outcomes in a murine cardiac arrestmodel. Circulation. 2004;109:2786–2791.

9. Hale SL, Dae MW, Kloner RA. Hypothermia during reperfusion limits‘no-reflow’ injury in a rabbit model of acute myocardial infarction.Cardiovasc Res. 2003;59:715–722.

10. Miki T, Swafford AN, Cohen MV, Downey JM. Second window ofprotection against infarction in conscious rabbits: real or artifactual. J MolCell Cardiol. 1999;31:809–816.

11. Tissier R, Couvreur N, Ghaleh B, Bruneval P, Lidouren F, Morin D, ZiniR, Bize A, Chenoune M, Belair MF, Mandet C, Douheret M,Dubois-Rande JL, Parker JC, Cohen MV, Downey JM, Berdeaux A.Rapid cooling preserves the ischaemic myocardium against mitochondrialdamage and left ventricular dysfunction. Cardiovasc Res. 2009;83:345–353.

12. Tissier R, Chenoune M, Ghaleh B, Cohen MV, Downey JM, Berdeaux A.The small chill: mild hypothermia for cardioprotection? Cardiovasc Res.2010;88:406–414.

13. Larsson IM, Wallin E, Rubertsson S. Cold saline infusion and ice packsalone are effective in inducing and maintaining therapeutic hypothermiaafter cardiac arrest. Resuscitation. 2010;81:15–19.

14. Dixon SR, Whitbourn RJ, Dae MW, Grube E, Sherman W, Schaer GL,Jenkins JS, Baim DS, Gibbons RJ, Kuntz RE, Popma JJ, Nguyen TT,O’Neill WW. Induction of mild systemic hypothermia with endovascularcooling during primary percutaneous coronary intervention for acutemyocardial infarction. J Am Coll Cardiol. 2002;40:1928–1934.

15. Yu T, Barbut D, Ristagno G, Cho JH, Sun S, Li Y, Weil MH, Tang W.Survival and neurological outcomes after nasopharyngeal cooling orperipheral vein cold saline infusion initiated during cardiopulmonaryresuscitation in a porcine model of prolonged cardiac arrest. Crit CareMed. 2010;38:916–921.

16. Boller M, Lampe JW, Katz JM, Barbut D, Becker LB. Feasibility ofintra-arrest hypothermia induction: a novel nasopharyngeal approachachieves preferential brain cooling. Resuscitation. 2010;81:1025–1030.

17. Chenoune M, Lidouren F, Ghaleh B, Couvreur N, Dubois-Rande J-L,Berdeaux A, Tissier R. Rapid cooling of the heart with total liquidventilation prevents transmural myocardial infarction following pro-longed ischemia in rabbits. Resuscitation. 2010;81:359–362.

18. Tissier R, Hamanaka K, Kuno A, Parker JC, Cohen MV, Downey JM.Total liquid ventilation provides ultra-fast cardioprotective cooling. J AmColl Cardiol. 2007;49:601–605.

19. Wolfson MR, Shaffer TH. Pulmonary applications of perfluorochemicalliquids: ventilation and beyond. Paediatr Respir Rev. 2005;6:117–127.

20. Yang SS, Jeng MJ, McShane R, Chen CY, Wolfson MR, Shaffer TH.Cold perfluorochemical-induced hypothermia protects lung integrity innormal rabbits. Biol Neonate. 2005;87:60–65.

21. Riter HG, Brooks LA, Pretorius AM, Ackermann LW, Kerber RE.Intra-arrest hypothermia: both cold liquid ventilation with perfluoro-carbons and cold intravenous saline rapidly achieve hypothermia, butonly cold liquid ventilation improves resumption of spontaneous cir-culation. Resuscitation. 2009;80:561–566.

22. Staffey KS, Dendi R, Brooks LA, Pretorius AM, Ackermann LW, ZambaKD, Dickson E, Kerber RE. Liquid ventilation with perfluorocarbonsfacilitates resumption of spontaneous circulation in a swine cardiac arrestmodel. Resuscitation. 2008;78:77–84.

23. Baker AJ, Zornow MH, Grafe MR, Scheller MS, Skilling SR, SmullinDH, Larson AA. Hypothermia prevents ischemia-induced increases inhippocampal glycine concentrations in rabbits. Stroke. 1991;22:666–673.

24. Souktani R, Pons S, Guegan C, Bouhidel O, Bruneval P, Zini R, MandetC, Onteniente B, Berdeaux A, Ghaleh B. Cardioprotection against myo-cardial infarction with PTD-BIR3/RING, a XIAP mimicking protein.J Mol Cell Cardiol. 2009;46:713–718.




25. Kuboyama K, Safar P, Radovsky A, Tisherman SA, Stezoski SW,Alexander H. Delay in cooling negates the beneficial effect of mildresuscitative cerebral hypothermia after cardiac arrest in dogs: a pro-spective, randomized study. Crit Care Med. 1993;21:1348–1358.

26. Carroll M, Beek O. Protection against hippocampal CA1 cell loss bypost-ischemic hypothermia is dependent on delay of initiation andduration. Metab Brain Dis. 1992;7:45–50.

27. Clark DL, Penner M, Wowk S, Orellana-Jordan I, Colbourne F.Treatments (12 and 48 h) with systemic and brain-selective hypothermiatechniques after permanent focal cerebral ischemia in rat. Exp Neurol.2009;220:391–399.

28. Wu X, Drabek T, Kochanek PM, Henchir J, Stezoski SW, Stezoski J,Cochran K, Garman R, Tisherman SA. Induction of profound hypo-thermia for emergency preservation and resuscitation allows intact

survival after cardiac arrest resulting from prolonged lethal hemorrhageand trauma in dogs. Circulation. 2006;113:1974–1982.

29. Tsai MS, Barbut D, Wang H, Guan J, Sun S, Inderbitzen B, Weil MH,Tang W. Intra-arrest rapid head cooling improves postresuscitation myo-cardial function in comparison with delayed postresuscitation surfacecooling. Crit Care Med. 2008;36:S434–S439.

30. Kang J, Albadawi H, Casey PJ, Abbruzzese TA, Patel VI, Yoo HJ,Cambria RP, Watkins MT. The effects of systemic hypothermia on amurine model of thoracic aortic ischemia reperfusion. J Vasc Surg.2010;52:435–443.

31. Robert R, Micheau P, Avoine O, Beaudry B, Beaulieu A, Walti H. Aregulator for pressure-controlled total-liquid ventilation. IEEE TransBiomed Eng. 2010;57:2267–2276.

CLINICAL PERSPECTIVEMild therapeutic hypothermia is known to improve survival and neurological recovery in patients resuscitated from cardiacarrest. However, previous experimental studies demonstrated that the benefit afforded by hypothermia was closely linkedto the rapidity of the decrease in body temperature after resuscitation. The present article investigates an original approachoffering a very rapid and generalized cooling using total liquid ventilation with temperature-controlled perfluorocarbons.These liquids can use the lungs as heat exchangers while maintaining normal gas exchanges. We showed that this strategypotently limits the post–cardiac arrest syndrome when instituted after resumption of spontaneous circulation in a rabbitmodel of cardiac arrest. The protection was evidenced in terms of survival and neurological and cardiac sequels. Thebenefit was directly related to cooling rapidity because a conventional cooling with infusion of cold saline and externalblankets was not significantly protective in similar conditions. Proper effects of the perfluorocarbon are also unlikelybecause normothermic total liquid ventilation was not protective. These results offer promising perspectives for theinduction of a neuroprotective and cardioprotective rapid cooling using total liquid ventilation in resuscitated patients.Several prototypes of liquid ventilator have been developed, and the clinical translation of this concept will be feasiblewhen they are available for clinical use. The current prototypes are developed primarily for pediatric use, and the translationof hypothermic total liquid ventilation would be possible first in newborns presenting global ischemia. The currentdevelopment of devices devoted to liquid ventilation in adult patients will further expand the possible applications of thisoriginal approach.




SUPPLEMENTAL MATERIAL

Supplemental Table 1: Rabbit neurological deficit grading scale (from ref 23)

Maximum score

Level of conciousness

Normal 0 Clouded 5 Stuporous 10 Comatose 25 25 Respiration Normal 0 Abnormal 5 5 Cranial nervs Vision 1 Light reflex 1 Oculocephalic 1 Corneal 1 Facial sensation 1 Auditory 1 Gag reflex 1 7 Motor and sensory function Flexor response to pain (Front) 2 Flexor response to pain (Rear) 2 Righting reflex 10 14 Gait Normal 0 Minimal ataxia 5 Moderate ataxia 10 Able to stand 15 Unable to stend 20 No purposeful movement 25 25 Behavior Grooming 4 Eating/drinking 10 Exploring 10 24 Total 100



2

Supplemental Table 2: Histological cerebral lesion severity grading scale (from ref 23)

Score

Neocortex Normal 0 Rare ischemic neurons (<10%) 1 Frequent ischemic neurons (10-50%) 2 Majority of neurons ischemic (>50%) 3 Hippocampus Normal 0 Rare ischemic pyramidal or granule cells 1 Focal ischemic damage 2 Severe, diffuse ischemic damage 3 Cerebellum Normal 0 Rare ischemic Purkinje cells 1 10-50% ischemic Purkinje cells 2 >50% ischemic Purkinje cells 3



3

Supplemental Table 3: Histological lesion severity grading scale for the kidney, liver, heart and

lung. For this last organ, we assessed two separate scores for the cardiogenic lesions and the

infectious complication, respectively.

Score

Kidney

Normal 0 Dilated regenerative proximal tubule 1 Focal scar fibrosis 2 Extensive scar fibrosis 3 Liver Normal 0 Limited clarification of hepatocytes 1 Moderate clarification of hepatocytes 2 Extensive clarification of hepatocytes 3 Heart Normal 0 Very rare foci of cardiomyocyte necrosis 1 Rare foci of cardiomyocyte necrosis 2 Frequent foci of cardiomyocyte necrosis 3 Lung (cardiogenic lesion) Normal 0 Limited congestion and/or serous edema 1 Moderate congestion and/or serous edema 2 Extended congestion and/or serous edema 3 Lung (infection) Normal 0 Limited foci of bronchopneumia 1 Moderate foci of bronchopneumia 2 Extended foci of bronchopneumia 3



4

Supplemental Table 4 : Hemodynamic parameters in surviving rabbits after 7 days following cardiac

arrest.

n Conscious state Anesthesia

Heart rate (beat/min) Sham 5 254±19 282±9 Control 5' 5 219±14 261±17 H-TLV 5' 9 258±18 261±18 Control 10' 0 - - H-TLV 10' 7 230±14 273±34 Saline 10' 2 247±2 246±20 N-TLV 10' 1 302 *

Mean arterial pressure (mmHg) Sham 5 81±8 82±4 Control 5' 5 98±11 83±5 H-TLV 5' 9 100±2 93±7 Control 10' 0 - - H-TLV 10' 7 94±6 94±10 Saline 10' 2 89±11 81±15 N-TLV 10' 1 90 *

End diastolic left ventricular pressure (mmHg) Sham 5 - 3.6±0.6 Control 5' 5 - 3.3±0.8 H-TLV 5' 9 - 3.8±1.1 Control 10' 0 - - H-TLV 10' 7 - 2.7±0.5 Saline 10' 2 - 6.0±1.6 N-TLV 10' 1 - *

dP/dt max (mmHg/s) Sham 5 - 7182±1313 Control 5' 5 - 7455±927 H-TLV 5' 9 - 7132±1157 Control 10' 0 - - H-TLV 10' 7 - 6518±1958 Saline 10' 2 - 4810±1169 N-TLV 10' 1 - *

dP/dt min (mmHg/s) Sham 5 - -5364±914 Control 5' 5 - -6156±1251 H-TLV 5' 9 - -6107±758 Control 10' 0 - - H-TLV 10' 7 - -6323±1426 Saline 10' 2 - -4939±2772 N-TLV 10' 1 - *

TLV, total liquid ventilation; H-TLV, hypothermic TLV; N-TLV, normothermic TLV; Saline,

hypothermia induced by intravenous administration of cold saline combined to external cooling; dP/dt

max, maximal positive left ventricular rate of pressure development; dP/dt min, maximal negative left



5

ventricular rate of pressure development; *, data not available since the animal died during

anesthesia.



6

Supplemental Figure 1: Schematic representation of the prototype of liquid ventilator.

Inflow pump

Outflow pump

Inflow/Outflowsolenoid valves

Vacuumcontrol

O2

Perfluorocarbonsreservoir

Liquid ventilator

Heat exchanger

Chest

Targettemperaturewater bath

Electroniccontrol

Inflow pump

Outflow pump

Inflow/Outflowsolenoid valves

Vacuumcontrol

O2

Perfluorocarbonsreservoir

Liquid ventilator

Heat exchanger

Chest

Targettemperaturewater bath

Electroniccontrol



7

Supplemental Figure 2: Troponin I blood levels (left panel) and myocardial caspase 3 activity

(right panel) in rabbits submitted to 10 min of cardiac arrest under control conditions or with

hypothermic liquid ventilation after resumption of spontaneous circulation (Control10’ and H-TLV10’,

respectively). Caspase 3 activity assays were performed in myocardial samples withdrawn 60

min after the cardiac arrest episode.

* p<0.05 vs corresponding control; n=8 in each experimental group; H-TLV, hypothermic total

liquid ventilation.

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Baseline T=60 min aftercardiac arrest

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ultrafast and whole-body cooling with total liquid ... · because total liquid ventilation (tlv)...

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