Skip main navigation

Ultrafast and Whole-Body Cooling With Total Liquid Ventilation Induces Favorable Neurological and Cardiac Outcomes After Cardiac Arrest in Rabbits

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.111.039388Circulation. 2011;124:901–911

Abstract

Background—

In animal models of cardiac arrest, the benefit afforded by hypothermia is closely linked to the rapidity of the decrease in body temperature after resuscitation. Because total liquid ventilation (TLV) with temperature-controlled perfluorocarbons induces a very rapid and generalized cooling, we aimed to determine whether this could limit the post–cardiac arrest syndrome in a rabbit model. We especially focused on neurological, cardiac, pulmonary, liver and kidney 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 either normothermic life support (control) or therapeutic hypothermia induced by TLV. The latter procedure decreased esophageal and tympanic temperatures to 32°C to 33°C within only 10 minutes. After rewarming, the animals submitted to 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 also observed limitation of myocardial necrosis, along with a decrease in troponin I release and a reduced myocardial caspase 3 activity, with TLV. The beneficial effects of TLV were directly related to the rapidity of hypothermia induction because neither conventional cooling (cold saline infusion plus external cooling) nor normothermic TLV elicited a similar protection.

Conclusions—

Ultrafast cooling instituted by TLV exerts potent neurological and cardiac protection in an experimental model of cardiac arrest in rabbits. This could be a relevant approach to provide a global and protective hypothermia against the post–cardiac arrest syndrome.

Introduction

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 comatose survivors of cardiac arrest.1,2 However, experimental studies in dogs,3,4 pigs,5,6 and rodents7,8 demonstrated that the neuroprotection afforded by hypothermia was related to the rapidity of the decrease in body temperature after resuscitation. When achieved rapidly, hypothermia could also be beneficial for other organs because, for example, it can also be potently cardioprotective during myocardial ischemia.912 Accordingly, many strategies were proposed to afford such a rapid hypothermia, including intravenous infusion of cold fluid13 and endovascular14 or intranasal cooling.15,16

Clinical Perspective on p 911

Another strategy that can experimentally provide very rapid and generalized cooling is liquid ventilation of the lungs with temperature-controlled perfluorocarbons.11,1722 These liquids can use the lungs as heat exchangers while maintaining normal gas exchanges.1820 In addition, this ventilation procedure protects lung integrity.20 Using a prototype of total liquid ventilator that alternatively instills and removes a tidal volume of perfluorocarbon from the lung, we were able to decrease the left atrial temperature to 32°C within only 5 minutes in anesthetized rabbits.11,17,18 This decrease was associated with very potent protection against myocardial infarction and subsequent contractile dysfunction in animal models of coronary artery occlusion.11,17,18 In a swine model of ventricular fibrillation, liquid ventilation also induced a rapid convective cooling that further improves the chances for subsequent resumption of spontaneous circulation.21,22 However, to the best of our knowledge, the effect of hypothermic total liquid ventilation (TLV) has never been investigated in animal models of post–cardiac arrest dysfunction when instituted after resumption of spontaneous circulation.

Accordingly, the main purpose of the present study was to investigate the long-term effect of ultrafast cooling induced by TLV in a rabbit model of post–cardiac arrest dysfunction after ventricular fibrillation and resuscitation. To determine whether hypothermic TLV properly protects through very fast cooling, we investigated 2 additional groups submitted to conventional hypothermia (cold saline infusion plus external cooling) or to normothermic TLV. The primary outcome was survival during 7 days of follow-up. The secondary outcomes were clinical, biochemical, hemodynamic, and histological parameters describing neurological, cardiac, pulmonary, liver, and kidney potential dysfunctions. We also aimed to investigate whether ultrafast cooling can protect the heart through an early inhibition of cardiac cell death. The latter point was also critical to further support the relevance of very fast cooling to limit subsequent dysfunction after cardiac arrest.

Methods

The animal instrumentation and ensuing experiments were conducted in accordance with French official regulations (agreement A94-046-13) after approval by the local ethics committee. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Animal Preparation

New Zealand rabbits (3.0 to 3.5 kg) were anesthetized with zolazepam, tiletamine, and pentobarbital (all 20 to 30 mg/kg IV). They were intubated and mechanically ventilated. After administration of pancuronium bromide (200 μg/kg IV), 2 electrodes were implanted on the chest and inserted into the esophagus for subsequent induction of ventricular fibrillation. Rectal, esophageal, and tympanic temperatures were monitored continuously with thermal probes (Harvard Apparatus, Paris, France). Throughout the protocol, external ECG was recorded, as well as arterial blood pressure from a catheter implanted in the ear artery. Data were digitalized and analyzed with the data acquisition software HEM version 3.5 (Notocord, Croissy-sur-Seine, France).

Cardiac Arrest and Cardiopulmonary Resuscitation

After animal preparation and subsequent stabilization, ventricular fibrillation was induced by passing an alternative current (10 V, 4 mA; 2 minutes) between the implanted electrodes. Mechanical ventilation was stopped at the onset of fibrillation and throughout the subsequent period of cardiac arrest. After either 5 or 10 minutes of untreated fibrillation, cardiopulmonary resuscitation was started with cardiac massage (≈200 bpm), electric attempts at defibrillation (5 to 10 J/kg), and intravenous administration of epinephrine (15 μg/kg IV). Resumption of spontaneous circulation (ROSC) was considered an organized cardiac rhythm associated with a mean arterial pressure >40 mm Hg for at least 1 minute. After ROSC, administration of epinephrine was further permitted during a maximum of 7 hours at a dosage appropriately adjusted to maintain the mean arterial pressure at ≈80 mm Hg. Mechanical ventilation was continued until weaning and awakening of the animals. Rabbits were subsequently returned to their cage for survival follow-up. They received antibiotics (enrofloxacine 5 mg/kg IM) for 7 days and analgesics (buprenorphine 30 μg/kg SC) for 3 days.

Experimental Protocol

As shown in Figure 1, rabbits randomly underwent either 5 or 10 minutes of cardiac arrest with subsequent cardiopulmonary resuscitation. For each duration of cardiac arrest, rabbits were randomly allocated 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 was started 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 endotracheal tube to our prototype liquid ventilator (Figure I in the online-only Data 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 per minute. For each breath, the ventilator pumped the tidal volume of liquids into and out of the lungs. The perfluorocarbon mixture was bubbled with 100% O2. The temperature of the heat exchanger was adjusted to maintain esophageal and tympanic temperatures at a target temperature of ≈32°C. After 20 minutes of TLV and achievement of the hypothermic target temperature, the perfluorocarbon was evacuated from the lungs by gravity, and the endotracheal tube was again connected to a conventional mechanical ventilator. Hypothermia was further maintained at 32°C during 3 hours, if necessary with cold blankets. Animals were subsequently rewarmed with infrared lights and thermal pads until weaning from conventional ventilation and awakening. Animals were housed in a closed cage enriched in O2 for 2 to 3 days to avoid hypoxic episodes. To determine whether hypothermic TLV properly protects through very fast cooling, we investigated 2 randomly allocated additional groups submitted to 10 minutes of cardiac arrest. The first of these groups (Saline10′) was submitted to 3 hours of conventional hypothermia through the combination of cold saline administration (30 mL/kg IV, NaCl 0.9% at 4°C) and external cooling. The second additional group was submitted to an episode of TLV with normothermic perfluorocarbons (N-TLV10′ group) to determine their proper effects.

Figure 1.

Figure 1. Experimental protocol. CA indicates cardiac arrest; TLV, total liquid ventilation; H-TLV, hypothermic TLV; N-TLV, normothermic TLV; Saline, hypothermia induced by intravenous administration of cold saline combined with external cooling; and ROSC, resumption of spontaneous circulation.

To further investigate the effects of hypothermic TLV, additional rabbits were included in the Control10′ and H-TLV10′ groups. These animals were euthanized 1 hour after the cardiac arrest episode for collection of myocardial and blood samples for caspase activity assays and measurement of circulating troponin I, respectively.

Neurological and Cardiac Dysfunction Assessment

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

Blood Chemistry and Caspase Activity Assay

Blood pH and carbon dioxide and oxygen partial pressures (Pco2 and Po2, respectively) were assessed from arterial blood samples with an ABL 77 series analyzer (Radiometer SA, France). Blood lactate was determined on an Accutrend Plus analyzer (Roche Diagnostics, Mannheim, Germany). Liver and renal function was evaluated by measuring the alanine aminotransferase and creatinine concentrations (Prestige 24i, Tokyo-Boehi, Japan). Troponin I and creatinine kinase were measured by an offsite laboratory (IDEXX Laboratories, Alfortville, France).

Caspase 3 activity was assayed from cardiac samples, as previously 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 phenylmethanesulfonyl fluoride, 5 μL/mL protease cocktail inhibitor P8340; Sigma-Aldrich, St. Louis, MO). Homogenates were centrifuged and supernatants collected. Proteins (90 μg) were incubated in caspase assay 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 of 0.2 mmol/L of the fluorogenic substrates ac-DEVD-AFC (Biomol Research Laboratories, Hamburg, Germany). Fluorescent arbitrary units were converted into picomoles per 1 mg protein per hour with a standard curve of free AFC (Biomol Research Laboratories).

Histological Analyses

After 7 days of follow-up after cardiac arrest, the surviving rabbits were euthanized for pathological analyses of the heart, lung, kidney, liver, and brain. These organs were also removed and analyzed in the animals that died before the end of the follow-up. For lungs, a slice was sampled from each lobe (5 per lung). For the heart, we analyzed a midheart transversal biventricular section. For kidneys, 2 slices were studied for each organ. We used a 0 to 3 scoring system to blindly quantify the severity of each organ alteration, as shown in Tables II and III in the online-only Data Supplement (0=normal, 3=very severe lesion). The overall brain score was the mean value obtained for cortex, hippocampus, and cerebellum, as previously described.23 For lungs, we assessed 2 separate scores for cardiogenic lesions (serous edema and/or congestion) and infectious complication of bronchopneumonia. The first panels of Figure 2 and 3 illustrate typical normal and pathological aspects of the different organs.

Figure 2.

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 μm). In liver, we observed systematized clarification of hepatocytes (arrows; bar=120 μm). In lungs, lesions were congestion with serous edema (arrows in the left lung panel; bar=120 μm) or foci of bronchopneumonia (arrows in the right lung panel; bar=120 μm). B, Histological scores of alteration in kidney, liver, and lungs of rabbits from the different groups. For lungs, we assessed 2 separate scores for cardiogenic lesions and infection complications, respectively. Open circles represents 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.

Figure 3.

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 in the hippocampus (arrows; bar=30 μm), in laminar necrosis of Purkinje cells in the cerebellum (arrows, bar=30 μm), or in numerous ischemic neurons in the cortex (arrows; bar=30 μm). In the myocardium, we observed foci of cardiomyocytes necrosis (arrows, bar=120 μm). B, Histological scores of alteration in the brain and heart of rabbits from the different groups. Open circles represents 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.

Statistical Analyses

Data are expressed as mean±SEM. Hemodynamic and biochemical parameters were compared between the different groups and corresponding controls by use of 2-way ANOVA for repeated measures. Post hoc analyses were performed at each time point compared with controls by use of a Student t test with Bonferroni correction. Values were not compared between the different time points to avoid multiple comparisons. In each experimental group, neurological dysfunction and histological scores were compared with those of the corresponding control group by use of a Mann-Whitney nonparametric test. Survival curves were obtained with a Kaplan-Meier analysis and were compared with the corresponding control group through the use of a log-rank test. These last analyses took into account only the animals that achieved ROSC. Significant differences were determined at P≤0.05.

Results

Seventy rabbits were included in the present study and submitted to cardiac arrest (n=10, 10, 15, 15, 10, and 10 in the Control5′, H-TLV5′, Control10′, H-TLV10′, Saline10′, and N-TLV10′ groups, respectively). As shown in Table 1, all rabbits subjected to 5 minutes of cardiac arrest (Control5′ and H-TLV5′ groups) were successfully resuscitated, whereas only 10 of 15 were successfully resuscitated in the Control10′ and H-TLV10′ groups. This rate was 7 of 10 in the Saline10′ and N-TLV10′ groups. Regardless of the duration of cardiac arrest, the time to ROSC was not significantly different among groups for each duration of cardiac arrest.

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

nRate of Successful ResuscitationROSC, minEpinephrine Dose, μg/kg
Control5′1010/102.4±0.3207±58
H-TLV5′1010/102.3±0.3174±81
Control10′1510/154.8±0.4684±118
H-TLV10′1510/154.2±0.8128±128*
Saline10′107/103.7±0.7430±126
N-TLV10′107/103.7±0.4509±64

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

*P<0.05 versus corresponding control value.

As illustrated in Figure 4, esophageal, tympanic, and rectal temperatures were not significantly different among groups at baseline. A mild and passive decrease in body temperature was observed in the Control5′ and Control10′ groups after cardiac arrest but remained within the normothermic range. In the H-TLV groups, esophageal and tympanic temperatures decreased very rapidly after the institution of TLV. For example, tympanic temperature reached 33.3±0.5°C and 32.5±0.3°C in H-TLV5′ and H-TLV10′, respectively, within 10 minutes after the onset of the cooling protocol. In the Saline10′ group, such tympanic temperatures were achieved after ≈30 minutes. For esophageal and rectal temperatures, the time to achieve 32°C to 33°C was <5 and 20 minutes in H-TLV10′ and >45 and 60 minutes in Saline10′, respectively. In the N-TLV10′ group, body temperatures did not significantly differ from the Control10′ values throughout the experimental protocol.

Figure 4.

Figure 4. Esophageal, tympanic, and rectal temperatures in the different experimental groups. TLV indicates total liquid ventilation; H-TLV, 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; n=10 in Control5′, H-TLV5′, Control10′, and H-TLV10′; n=7 in Saline10′ and N-TLV10′.

As shown in Table 2, heart rate significantly decreased during the hypothermic phase in hypothermic groups compared with corresponding controls (eg, −21%, −28%, and −31% at 60 minutes after cardiac arrest in H-TLV5′, H-TLV10′, and Saline10′ versus corresponding controls, respectively). Mean arterial pressure was not significantly different between groups throughout the experimental protocol because epinephrine administration was used to maintain a ≈80 mm Hg value during 7 hours after cardiac arrest. As shown in Table 1, the total amount of epinephrine administered throughout cardiopulmonary resuscitation, however, was significantly lower in H-TLV10′ than in Control10′ (128±128 versus 684±118 μg/kg, respectively), suggesting a favorable hemodynamic effect of hypothermic TLV. We did not observe such a significant difference in H-TLV5′ versus Control5′, but epinephrine dosages were much lower (174±81 versus 207±58 μg/kg, respectively). In Saline10′ and N-TLV10′, epinephrine dosages were also not different from the dosages in Control10′. After discontinuation of any pharmacological support (eg, at 8 hours after cardiac arrest), the lactate levels were significantly lower in H-TLV5′ compared with Control5′ (1.2±0.2 versus 4.8±1.7 mmol/L) and in H-TLV10′ compared with Control10′ (3.6±0.7 versus 7.0±1.7 mmol/L). Those levels were not significantly different among the Saline10′ and N-TLV10′ groups compared with the Control10′ group (5.9±0.7 and 7.6±0.6 versus 7.0± 1.7 mmol/L).

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

Epinephrine perfusionnBaseline NoCardiopulmonary Resuscitation
Day 1 (n)
15 min Yes60 min Yes180 min Yes360 min Yes480 min No
Heart rate, bpm
    Control5′10257±11222±8221±7243±11216±7220±9234±8 (10)
    H-TLV5′10259±10202±12174±6*177±9*245±9234±8244±10 (10)
    Control10′10263±10219±6220±10198±8221±11231±13256±17 (7)
    H-TLV10′10267±8167±10*158±8*167±11208±12240±11252±7 (8)
    Saline10′7266±7200±10153±7*155±13*219±10218±10226±16 (6)
    N-TLV10′7256±13216±19207±9213±12207±9221±15240±28 (2)
Mean arterial pressure, mm Hg
    Control5′1081±383±482±383±183±480±483±4 (10)
    H-TLV5′1080±781±382±582±383±379±382±4 (10)
    Control10′1080±582±383±381±483±280±379±4 (7)
    H-TLV10′1083±481±482±381±381±480±479±6 (8)
    Saline10′780±886±689±278±582±576±683±9 (6)
    N-TLV10′778±778±578±178±478±575±788±4 (2)
Plasma creatinine concentrations, mg/L
    Control5′1010±111±110±110±1 (10)
    H-TLV5′1010±012±111±110±1 (10)
    Control10′109±113±114±211±1 (7)
    H-TLV10′1010±013±112±111±1 (8)
    Saline10′79±110±110±110±1 (6)
    N-TLV10′79±111±112±113±6 (2)
Plasma ALAT concentrations, UI/L
    Control5′1029±531±433±435±9 (10)
    H-TLV5′1025±326±243±530±6 (10)
    Control10′1044±1379±25115±3260±17 (7)
    H-TLV10′1048±365±5111±2783±14 (8)
    Saline10′732±248±4101±3062±27 (6)
    N-TLV10′731±566±1096±1394±37 (2)

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

*P<0.05 versus corresponding control value.

As shown in Figure 5, we observed severe acidosis with an increase in Pco2 and a decrease in Po2 in all groups after cardiac arrest. In H-TLV5′, Po2 was lower 15 minutes after cardiac arrest compared with Control5′. This could be expected because control animals were ventilated with oxygen, whereas TLV rabbits underwent liquid ventilation by that time. At 180 minutes, gas exchanges were conversely improved in H-TLV groups compared with controls. For example, blood pH and Po2 increased and Pco2 decreased in H-TLV10′ and Control10′, respectively. Importantly, all animals were submitted to conventional ventilation at that time point with standardized ventilation parameters. As illustrated in Figure 2, the safety of TLV for lungs was also documented by histology demonstrating cardiogenic lesions (serous edema and/or congestion) or infectious complications of bronchopneumonia to a similar extent in TLV groups and corresponding controls.

Figure 5.

Figure 5. Blood pH, Pco2, and Po2 in the different experimental groups. TLV indicates total liquid ventilation; H-TLV, 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; n=10 in Control5′, H-TLV5′, Control10′, and H-TLV10′; n=7 in Saline10′ and N-TLV10′.

As shown in Table 2, renal function was not affected after cardiac arrest in all groups because plasma creatinine levels remained within usual values. Conversely, we observed an increase in the liver enzyme alanine aminotransferase with no difference among TLV and corresponding controls. Kidney and liver lesions were mild with no difference among groups (Figure 2B).

As illustrated in Figure 6, neurological dysfunction was significantly attenuated in the H-TLV groups compared with controls. This difference was significant as early as the second day after cardiac arrest in H-TLV5′ compared with Control5′ (Figure 6A), whereas this was observed within 24 hours of follow-up in H-TLV10′ compared with Control10′ (Figure 6B). In Saline10′, a transient improvement in neurological recovery was observed at day 1, but this was no longer significant at day 2 after cardiac arrest. As illustrated in Figure 3B, the neuroprotective effect of hypothermic TLV was further demonstrated by a significant decrease in the severity 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 in Saline10′ and N-TLV10′ compared with Control10′.

Figure 6.

Figure 6. A and B, Neurological dysfunction scores at days 1, 2, and 7 after resuscitation in the different experimental groups submitted to 5 or 10 minutes of cardiac arrest, respectively. Open circles represent individual scores; thick line, the median value of the corresponding group. Only animals achieving resumption of spontaneous circulation were included. C and D, Kaplan-Meier survival curves in the different experimental groups submitted to 5 or 10 minutes of cardiac arrest, respectively. Only animals achieving resumption of spontaneous circulation were included. TLV indicates total liquid ventilation; H-TLV, 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.

A significant difference in survival was also shown between the H-TLV groups and corresponding controls, as illustrated 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′ and H-TLV10′ groups compared with 5 of 10 and 0 of 10 in the Control5′ and Control10′ groups, respectively. Conversely, survival was not significantly improved in Saline10′ and N-TLV10′ compared with Control10′.

As illustrated in Figure 3B, myocardial foci of necrosis were less frequent in H-TLV10′ compared with Control10′, demonstrating a cardioprotective effect of hypothermic TLV. Conversely, no difference was seen between the Saline10′ and N-TLV10′ groups and the Control10′ group. In surviving animals, the functional myocardial sequels of cardiac arrest were also evaluated after 7 days of follow-up. As shown in Table IV in the online-only Data Supplement, mean blood pressure and heart rate in the conscious state were not different among groups compared with a sham group. After anesthesia, end-diastolic left ventricular pressure, dP/dtmax, and dP/dtmin were also not different between groups, suggesting that there were no major functional myocardial alterations in those surviving animals.

To further explore the cardioprotective effect of hypothermic TLV, 8 additional rabbits were included in the Control10′ and H-TLV10′ groups for a surrogate study dedicated to the caspase 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 was significantly decreased in H-TLV10′ compared with Control10′ (1.3±0.3 versus 70.7±30.4 ng/mL, respectively). The cardioprotective effect of hypothermic TLV was also supported by a decrease in caspase 3 activity compared with control (6.2±1.2 versus 10.0±1.2 pmol per 1 mg protein per hour, respectively).

Discussion

The present study provides proof of concept that ultrafast whole-body cooling with hypothermic TLV limits the post–cardiac arrest syndrome when instituted after ROSC in a rabbit model of ventricular fibrillation. Interestingly, we observed potent neuroprotection and cardioprotection with hypothermic TLV, which remains a safe procedure for the lungs. Because we used only 3 hours of hypothermia, our finding also suggested that very early hypothermia after ROSC does not need to be prolonged to produce a strong clinical benefit. Importantly, this benefit was directly related to cooling rapidity with TLV because conventional cooling with cold saline and external blankets was not significantly protective in similar conditions. Proper effects of the perfluorocarbon are unlikely because of the lack of protection with normothermic TLV.

Our first finding is the rapidity of TLV-induced cooling because esophageal and brain temperatures reached ≈32°C to 33°C within only 10 minutes. In comparison, a conventional hypothermic protocol (cold saline infusion plus external cooling) requires ≈30 and 45 minutes to similarly reduce these temperatures. The rapid cooling elicited by TLV was related directly to the tidal exchange of the liquid because simple repetitive pulmonary lavage with a 4°C perfluorocarbon requires >60 minutes to decrease the tympanic temperature to 32°C in the same species.20 In large animals, hypothermic TLV was also reported to provide very fast cooling and to reduce the pulmonary artery temperature to 32°C within 9 to 10 minutes when instituted intra-arrest in a ventricular fibrillation model in swine.22

Importantly, the rapid hypothermia elicited by TLV was associated with potent neurological protection and an increase in survival rate compared with control conditions. Animal studies have indicated that the neuroprotective effect of hypothermia is time dependent and that a large part of the protection is lost when cooling is delayed.25 For example, in a canine model of cardiac arrest, the neurological protection was lost after only 15 minutes of delay before the onset of hypothermia after ROSC.25 In the present study, we observed a very potent benefit of hypothermia when achieved rapidly after ROSC with TLV, whereas conventional hypothermia was not significantly protective. Recent experiments have also shown that hypothermia started before ROSC (eg, intra-arrest hypothermia) can provide an additional benefit,7,8 but it might be difficult to translate this concept into human clinical practice. All these findings demonstrate that most of the possible benefits of hypothermia can be lost within minutes after ROSC, further supporting the need of devices eliciting ultrafast cooling such as TLV in the present study.

Importantly, the benefit of hypothermic TLV observed in our conditions was produced by a short hypothermic episode (3 hours), whereas the current recommendation in humans is to maintain hypothermia for 24 to 36 hours.1,2 We choose this short duration because previous experiments have shown that when hypothermia is achieved very early, it does not need to be prolonged to provide an effective neuroprotection, eg, in a gerbil model of global ischemia.26 Mice studies also noted that 1 hour of cooling after ROSC was sufficient to generate significant clinical benefit.7,8 When the severity of the ischemic insult increases or when the onset of cooling is delayed, it is conversely well established that prolonging hypothermia is critical for achieving a maximal neurological protection.27,28 For example, prolonged cooling provided enduring behavioral and histological protection in rats submitted to permanent middle cerebral artery occlusion, even when delayed after the onset of ischemia.27

Another important beneficial effect of hypothermic TLV is the cardioprotection observed here like that previously shown in animal models of coronary artery occlusion.11,17,18 This was especially observed after 10 minutes of cardiac arrest because myocardial lesions were minor in the groups submitted to only 5 minutes of cardiac arrest. This was evidenced by limited myocardial necrosis and preserved myocardial functional performance in surviving rabbits. Cardioprotection was also observed very early after cardiac arrest because troponin I release and caspase 3 activity were significantly decreased within 60 minutes after resuscitation in H-TLV10′ compared with Control10′. In animal models of focal myocardial ischemia, the window of protection with hypothermia is virtually limited to the ischemic phase, whereas cooling at reperfusion is ineffective at reducing infarct size in most experimental studies.12 In the present study, hypothermia was instituted after global reperfusion (ROSC), but it is reasonable to speculate that the myocardium remains momentarily and partially ischemic even after ROSC. This can explain that very rapid cooling with TLV can still provide a beneficial effect even if instituted after ROSC and systemic reperfusion. Improved postresuscitation myocardial function has also be observed with intra-arrest rapid head cooling.29 Generalized hypothermia could even potentially afford protection of the liver and/or kidney.30 Because these organs were mildly altered in control conditions in the present study, we were not able to show any difference with hypothermic TLV.

Importantly, TLV was a safe procedure for the lungs. We observed improved gas exchanges using standardized ventilatory parameters in TLV compared with control groups 3 hours after cardiac arrest. After weaning from ventilation, however, animals were maintained in a cage enriched in oxygen to avoid hypoxic episodes.11 In pigs, intra-arrest liquid ventilation was demonstrated to alter lung function because activation of pulmonary macrophages might alter gas exchanges after resumption of conventional ventilation.21,22 In our study, the tolerance of TLV was shown by histological examinations, and this is supported by several reports from the literature demonstrating that liquid ventilation can protect the lungs.19,20 Several prototypes of liquid ventilator have been developed, and the clinical translation of this concept might accordingly be feasible when those devices are available for clinical use.31 To date, the current prototypes are developed mostly for pediatric use,31 and accordingly, the translation of TLV-induced hypothermia would be possible first in newborns presenting global ischemia. Further developments might also ultimately permit a translation to adult patients.

Our study has several limitations. First, neurological dysfunctions were assessed on the basis of clinical and histological parameters. Other more functional tests or imaging would also be important. Second, histological analyses were performed in all animals, regardless of their survival time. This would have led to an underestimation of the histological scores in some animals that died very early after cardiac arrest. However, because the lower scores were observed in the group that lived for the longer time (H-TLV10′), this limitation should not affect our conclusions.

Conclusions

Ultrafast cooling instituted by hypothermic TLV limits the post–cardiac arrest dysfunction with associated neuroprotective and cardioprotective effects. Importantly, TLV was a safe procedure for the lungs in our experimental conditions. The beneficial effects of hypothermic TLV were probably directly related to the rapidity of the temperature decrease because myocardial cell death inhibition was seen even very early after resuscitation.

Acknowledgments

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

Sources of Funding

This study was supported by grant TLV-CARDAREST (R10028JS) from INSERM and ITMO Technologies pour la Santé and grant ET7-460 from the Fondation de l'Avenir. Dr Chenoune was supported by a grant from the Groupe de Reflexion sur la Recherche Cardiovasculaire and by a Poste d'accueil INSERM 2009. Dr Tissier was also a recipient of a Contrat d'Interface INSERM-ENV (2010) and of a grant from the Société Frana̧ise de Cardiologie (Edouard Corraboeuf grant, 2010).

Disclosures

None.

Footnotes

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, Unité 955, Equipe 3, Faculté de Médecine, 8 Rue du Général Sarrail, 94010 Créteil Cedex, France
. E-mail

References

  • 1. Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, Smith K. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med. 2002; 346:557–563.CrossrefMedlineGoogle Scholar
  • 2. Hypothermia After Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med. 2002; 346:549–556.CrossrefMedlineGoogle Scholar
  • 3. Nozari A, Safar P, Stezoski SW, Wu X, Henchir J, Radovsky A, Hanson K, Klein E, Kochanek PM, Tisherman SA. Mild hypothermia during prolonged cardiopulmonary cerebral resuscitation increases conscious survival in dogs. Crit Care Med. 2004; 32:2110–2116.CrossrefMedlineGoogle Scholar
  • 4. Nozari A, Safar P, Stezoski SW, Wu X, Kostelnik S, Radovsky A, Tisherman S, Kochanek PM. Critical time window for intra-arrest cooling with cold saline flush in a dog model of cardiopulmonary resuscitation. Circulation. 2006; 113:2690–2696.LinkGoogle Scholar
  • 5. Guan J, Barbut D, Wang H, Li Y, Tsai MS, Sun S, Inderbitzen B, Weil MH, Tang W. A comparison between head cooling begun during cardiopulmonary resuscitation and surface cooling after resuscitation in a pig model of cardiac arrest. Crit Care Med. 2008; 36:S428–S433.CrossrefMedlineGoogle Scholar
  • 6. Tsai MS, Barbut D, Tang W, Wang H, Guan J, Wang T, Sun S, Inderbitzen B, Weil MH. Rapid head cooling initiated coincident with cardiopulmonary resuscitation improves success of defibrillation and post-resuscitation myocardial function in a porcine model of prolonged cardiac arrest. J Am Coll Cardiol. 2008; 51:1988–1990.CrossrefMedlineGoogle Scholar
  • 7. Zhao D, Abella BS, Beiser DG, Alvarado JP, Wang H, Hamann KJ, Hoek TL, Becker LB. Intra-arrest cooling with delayed reperfusion yields higher survival than earlier normothermic resuscitation in a mouse model of cardiac arrest. Resuscitation. 2008; 77:242–249.CrossrefMedlineGoogle Scholar
  • 8. Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL, Becker LB. Intra-arrest cooling improves outcomes in a murine cardiac arrest model. Circulation. 2004; 109:2786–2791.LinkGoogle Scholar
  • 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.CrossrefMedlineGoogle Scholar
  • 10. Miki T, Swafford AN, Cohen MV, Downey JM. Second window of protection against infarction in conscious rabbits: real or artifactual. J Mol Cell Cardiol. 1999; 31:809–816.CrossrefMedlineGoogle Scholar
  • 11. Tissier R, Couvreur N, Ghaleh B, Bruneval P, Lidouren F, Morin D, Zini R, 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 mitochondrial damage and left ventricular dysfunction. Cardiovasc Res. 2009; 83:345–353.CrossrefMedlineGoogle Scholar
  • 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.CrossrefMedlineGoogle Scholar
  • 13. Larsson IM, Wallin E, Rubertsson S. Cold saline infusion and ice packs alone are effective in inducing and maintaining therapeutic hypothermia after cardiac arrest. Resuscitation. 2010; 81:15–19.CrossrefMedlineGoogle Scholar
  • 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 endovascular cooling during primary percutaneous coronary intervention for acute myocardial infarction. J Am Coll Cardiol. 2002; 40:1928–1934.CrossrefMedlineGoogle Scholar
  • 15. Yu T, Barbut D, Ristagno G, Cho JH, Sun S, Li Y, Weil MH, Tang W. Survival and neurological outcomes after nasopharyngeal cooling or peripheral vein cold saline infusion initiated during cardiopulmonary resuscitation in a porcine model of prolonged cardiac arrest. Crit Care Med. 2010; 38:916–921.CrossrefMedlineGoogle Scholar
  • 16. Boller M, Lampe JW, Katz JM, Barbut D, Becker LB. Feasibility of intra-arrest hypothermia induction: a novel nasopharyngeal approach achieves preferential brain cooling. Resuscitation. 2010; 81:1025–1030.CrossrefMedlineGoogle Scholar
  • 17. Chenoune M, Lidouren F, Ghaleh B, Couvreur N, Dubois-Rande J-L, Berdeaux A, Tissier R. Rapid cooling of the heart with total liquid ventilation prevents transmural myocardial infarction following prolonged ischemia in rabbits. Resuscitation. 2010; 81:359–362.CrossrefMedlineGoogle Scholar
  • 18. Tissier R, Hamanaka K, Kuno A, Parker JC, Cohen MV, Downey JM. Total liquid ventilation provides ultra-fast cardioprotective cooling. J Am Coll Cardiol. 2007; 49:601–605.CrossrefMedlineGoogle Scholar
  • 19. Wolfson MR, Shaffer TH. Pulmonary applications of perfluorochemical liquids: ventilation and beyond. Paediatr Respir Rev. 2005; 6:117–127.CrossrefMedlineGoogle Scholar
  • 20. Yang SS, Jeng MJ, McShane R, Chen CY, Wolfson MR, Shaffer TH. Cold perfluorochemical-induced hypothermia protects lung integrity in normal rabbits. Biol Neonate. 2005; 87:60–65.CrossrefMedlineGoogle Scholar
  • 21. Riter HG, Brooks LA, Pretorius AM, Ackermann LW, Kerber RE. Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation. Resuscitation. 2009; 80:561–566.CrossrefMedlineGoogle Scholar
  • 22. Staffey KS, Dendi R, Brooks LA, Pretorius AM, Ackermann LW, Zamba KD, Dickson E, Kerber RE. Liquid ventilation with perfluorocarbons facilitates resumption of spontaneous circulation in a swine cardiac arrest model. Resuscitation. 2008; 78:77–84.CrossrefMedlineGoogle Scholar
  • 23. Baker AJ, Zornow MH, Grafe MR, Scheller MS, Skilling SR, Smullin DH, Larson AA. Hypothermia prevents ischemia-induced increases in hippocampal glycine concentrations in rabbits. Stroke. 1991; 22:666–673.LinkGoogle Scholar
  • 24. Souktani R, Pons S, Guegan C, Bouhidel O, Bruneval P, Zini R, Mandet C, Onteniente B, Berdeaux A, Ghaleh B. Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein. J Mol Cell Cardiol. 2009; 46:713–718.CrossrefMedlineGoogle Scholar
  • 25. Kuboyama K, Safar P, Radovsky A, Tisherman SA, Stezoski SW, Alexander H. Delay in cooling negates the beneficial effect of mild resuscitative cerebral hypothermia after cardiac arrest in dogs: a prospective, randomized study. Crit Care Med. 1993; 21:1348–1358.CrossrefMedlineGoogle Scholar
  • 26. Carroll M, Beek O. Protection against hippocampal CA1 cell loss by post-ischemic hypothermia is dependent on delay of initiation and duration. Metab Brain Dis. 1992; 7:45–50.CrossrefMedlineGoogle Scholar
  • 27. Clark DL, Penner M, Wowk S, Orellana-Jordan I, Colbourne F. Treatments (12 and 48 h) with systemic and brain-selective hypothermia techniques after permanent focal cerebral ischemia in rat. Exp Neurol. 2009; 220:391–399.CrossrefMedlineGoogle Scholar
  • 28. Wu X, Drabek T, Kochanek PM, Henchir J, Stezoski SW, Stezoski J, Cochran K, Garman R, Tisherman SA. Induction of profound hypothermia for emergency preservation and resuscitation allows intact survival after cardiac arrest resulting from prolonged lethal hemorrhage and trauma in dogs. Circulation. 2006; 113:1974–1982.LinkGoogle Scholar
  • 29. Tsai MS, Barbut D, Wang H, Guan J, Sun S, Inderbitzen B, Weil MH, Tang W. Intra-arrest rapid head cooling improves postresuscitation myocardial function in comparison with delayed postresuscitation surface cooling. Crit Care Med. 2008; 36:S434–S439.CrossrefMedlineGoogle Scholar
  • 30. Kang J, Albadawi H, Casey PJ, Abbruzzese TA, Patel VI, Yoo HJ, Cambria RP, Watkins MT. The effects of systemic hypothermia on a murine model of thoracic aortic ischemia reperfusion. J Vasc Surg. 2010; 52:435–443.CrossrefMedlineGoogle Scholar
  • 31. Robert R, Micheau P, Avoine O, Beaudry B, Beaulieu A, Walti H. A regulator for pressure-controlled total-liquid ventilation. IEEE Trans Biomed Eng. 2010; 57:2267–2276.CrossrefMedlineGoogle Scholar

Clinical Perspective

Mild therapeutic hypothermia is known to improve survival and neurological recovery in patients resuscitated from cardiac arrest. However, previous experimental studies demonstrated that the benefit afforded by hypothermia was closely linked to the rapidity of the decrease in body temperature after resuscitation. The present article investigates an original approach offering 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 strategy potently limits the post–cardiac arrest syndrome when instituted after resumption of spontaneous circulation in a rabbit model of cardiac arrest. The protection was evidenced in terms of survival and neurological and cardiac sequels. The benefit was directly related to cooling rapidity because a conventional cooling with infusion of cold saline and external blankets was not significantly protective in similar conditions. Proper effects of the perfluorocarbon are also unlikely because normothermic total liquid ventilation was not protective. These results offer promising perspectives for the induction 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 feasible when they are available for clinical use. The current prototypes are developed primarily for pediatric use, and the translation of hypothermic total liquid ventilation would be possible first in newborns presenting global ischemia. The current development of devices devoted to liquid ventilation in adult patients will further expand the possible applications of this original approach.

eLetters(0)

eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.