levo semendan

Correspondence: Hanna Alaoja Jensen, Clinical Research Center, Oulu University Hospital, P.O. Box 5000, 90014 Oulu University, Finland. Tel: � 358 40 7732183. Fax: � 358 8 315 2577. E-mail: [email protected]

(Received 14 February 2011 ; accepted 19 April 2011 )

ORIGINAL ARTICLE

Levosimendan decreases intracranial pressure after hypothermic circulatory arrest in a porcine model

HANNA JENSEN 1,2 , RIMPIL Ä INEN EIJA 1,2 , M Ä KEL Ä TUOMAS 1,2 , M Ä KEL Ä JUSSI 1,2 , YANNOPOULOS FREDRIK 1,2 , ALESTALO KIRSI 1,2 , POKELA MATTI 1,2 , KIVILUOMA KAI 1,3 , TUOMINEN HANNU 1,4 , ANTTILA VESA 1,2 & JUVONEN TATU 1,2

1 Clinical Research Center, 2 Department of Surgery, 3 Department of Anesthesiology and 4 Department of Pathology, Oulu University Hospital, University of Oulu, Finland

Abstract Objectives . Hypothermic circulatory arrest (HCA) provides an optimal operating fi eld in aortic arch surgery, but it is asso-ciated with neurological complications. Levosimendan is an inotropic agent with clinical indications for open-heart surgery. Through peripheral vasodilatation, cardiac contractility enhancement and anti-infl ammatory function it has a potential to improve cerebral protection after HCA. Design . Eighteen piglets were randomly assigned to a levosimendan group (n � 9) and a placebo group (n � 9) and underwent a 60-minute period of hypothermic circulatory arrest at 18 ° C. A levosimendan or placebo infusion (0.2 μ g/kg/min) was commenced at the onset of anesthesia and continued for 24 hours. Animals were followed for one week and their neurological recovery was assessed daily. Finally the animals were electively sacrifi ced and their brain was harvested for histopathological examination. Results . Levosimendan decreased intracranial pressure during the experiment. There were no differences between the groups in terms of hemodynamic or metabolic data, brain metab-olism, neurological recovery or histopathology of the cerebral tissue. In the levosimendan group, cardiac enzymes were slightly more elevated. Conclusions . Levosimendan decreased intracranial pressure during the experiment, but in terms of cerebral metabolism, neurological recovery and histopathology of the brain tissue levosimendan did not improve brain protection in this experimental setting.

Key words: hypothermic circulatory arrest , cerebral protection , levosimendan

Hypothermic circulatory arrest (HCA) provides an optimal operating fi eld in aortic arch surgery, but it is associated with an increase in neurological compli-cations due to global cerebral ischemia and reperfu-sion injury. Numerous studies with various strategies, pharmacological interventions and approaches have attempted to fi nd means to improve the neurological outcome of patients after surgery with HCA (1 – 3).

Levosimendan is a promising alternative inotro-pic agent with possible clinical indication for open-heart surgery (4,5). It enhances cardiac contractility by improving the response of the myofi laments to intracellular calcium (6) and opens the adenosine triphosphate (ATP) dependent potassium channels in the vascular smooth muscle cells (7), causing

peripheral arterial and venous dilatation (8). These two drug-induced actions lead to a reduction of peripheral vascular resistance and cardiac workload, and thus result in a signifi cant increase of cardiac output (9).

In addition to its hemodynamic properties, some studies have shown levosimendan to be anti-infl ammatory. The benefi cial effects of levosimendan on circulating pro-infl ammatory cytokines (IL-6) and soluble apoptosis mediators (TNF- α ) have been shown in a randomized, placebo-controlled study (10). It is also plausible to assume that the cardio-vascular effects of levosimendan can decrease periph-eral tissue hypoperfusion, leading to down-regulation of systemic cytokine production (11).

Scandinavian Cardiovascular Journal, 2011; 45: 307–315

ISSN 1401-7431 print/ISSN 1651-2006 online © 2011 Informa HealthcareDOI: 10.3109/14017431.2011.583356

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308 H. Jensen et al.

We hypothesized that levosimendan ’ s benefi cial hemodynamic effects create an advantageous envi-ronment for the preservation of cerebral tissue. Fur-thermore, the immunological component of the reperfusion injury following deep HCA is thought to play a major role in the development of post-operative cerebral damage. In several studies, reduced accumu-lation of leukocytes has been associated with a reduced histological infl ammatory response and ischemic brain injury (12,13). The aim of this study was to investigate whether levosimendan could improve cerebral protection after deep HCA; possible mecha-nisms being the advantageous hemodynamic condi-tions for the cerebral tissue and reduced effect of systemic infl ammatory response in the brain.

Material and methods

Experimental setting

Eighteen 6 – 7 week-old piglets were randomly assigned to undergo CPB and a 60-minute HCA at 18 ° C with a 24-hour (0.2 μ g/kg/min) infusion of levosimendan (n � 9) or placebo (n � 9) commenced at the onset of anesthesia. Cerebral metabolism and intracranial pressure were monitored during and after surgery. Surviving animals were followed for one week post-operatively and their neurological recovery was assessed daily. At the end of the exper-iment, the animals were electively sacrifi ced and their brain was harvested for histopathological examina-tion. All animals received humane care in accordance with the “Principles of Laboratory Animal Care” for-mulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources, National Research Council (pub-lished by the National Academy Press, revised in 1996). The study was approved by the Research Animal Care and Use Committee of Finland.

Anesthesia protocol

Piglets were sedated with intramuscular ketamine hydrochloride (350 mg) and midazolam (45 mg). Using thiopental for further sedation as required, the piglets were intubated with a 6 mm cuffed endotra-cheal tube and ventilated with 50% oxygen and a rate of 12 – 15 breaths per minute, to achieve an end-tidal carbon dioxide concentration in the expired air (EtCO 2 ) of 4.5 – 5.0%. After induction with fentanyl (50 μ g/kg), anesthesia was maintained by a continu-ous infusion of fentanyl (25 μ g/kg/h), midazolam (0.25 mg/kg/h) and pancuronium (0.2 mg/kg/h), as well as inhalation anesthesia of 0.5% isofl urane throughout the entire experiment, excluding the period of HCA.

Study drug administration

A continuous intravenous levosimendan or placebo infusion (0.2 μ g/kg/min) was commenced as soon as the animal was under anesthesia and continued for 24 h, excluding the time period of circulatory arrest (60 minutes). The drug and placebo were of same consistence and color and the conductors of the experiment were blinded to the protocol.

Hemodynamic monitoring

An arterial line for arterial pressure monitoring and blood sampling was placed on the left femoral artery. A pulmonary artery thermodilution catheter (Criti-Cath, 7 French [Ohmeda GmbH & Co, Erlangen, Germany]) for blood sampling, monitoring the pul-monary pressure, measuring the cardiac output, pul-monary capillary wedge pressure as well as central venous pressure and recording of the temperature of blood was introduced through the left femoral vein. A 10 French catheter was placed in the urinary blad-der to monitor urine output. Baseline measurements were recorded.

A right anterolateral thoracotomy was performed in the fourth intercostal space to expose the right atrium for CPB cannulation. The right thoracic ves-sels were ligated and cut and the pericardium was opened. After systemic heparinization (500 IU/kg), the ascending aorta was cannulated with a 16 French arterial cannula, and the right atrial appendage was cannulated with a single 24 French atrial cannula. A 12 French intracardiac sump cannula was positioned into the apex of the heart for decompression of the heart during CPB.

Cranial procedures

Two small windows (about 5 mm in diameter) were drilled over the parietal cortex. In one of them, a temperature probe (Thermocouple Temperature Catheter-Micro-Probe, Ref C8.B [GMS]) was inserted to monitor intracerebral temperature throughout the experiment. Into the same window, a pressure-monitoring catheter (Codman Micro-Sensor ICP Transducer, Codman ICP Express Mon-itor [Codman & Shurtleff, Inc, Raynham, MA]) was placed into the cerebral tissue.

In the second window, a microdialysis catheter (CMA 70 [CMA/Microdialysis, Stockholm, Sweden]) was placed into the brain cortex for a depth of 15 mm below the dura mater. The catheter was con-nected to a 2.5 ml syringe placed into a microinfu-sion pump (CMA 106 [CMA/Microdialysis]) and perfused with Ringer solution at a rate of 0.3 μ l/min (Perfusion Fluid CNS [CMA/Microdialysis]). Sample

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Levosimendan decreases intracranial pressure after HCA 309

collection from microdialysis catheters was per-formed at set time points peri-operatively (baseline, 30 min cooling, 30 min HCA, 60 min HCA, 30 min rewarm, 1 h rewarm, 1 h 30 min rewarm, 2 h rewarm, 2 h 30 min rewarm, 3 h rewarm, 4 h rewarm, 5 h rewarm, 6 h rewarm, 7 h rewarm, 8 h rewarm). The concentrations of cerebral tissue glucose, lactate, pyruvate, glutamate, and glycerol were measured immediately after collection with a microdialysis analyzer (CMA 600 [CMA/Microdialysis]).

Cardiopulmonary bypass

After baseline recordings, a membrane oxygenator (D905 Eos [Dideco, Mirandola, Italy]) was primed with 1 l of Ringer acetate and heparin (5000 IU). Non-pulsatile CPB was initiated at a fl ow rate of 90 – 110 ml/kg/min, and the fl ow was adjusted to maintain an arterial pressure of 50 to 70 mmHg.

A 30-minute cooling period was carried out to attain a brain temperature of 18 ° C. Cooling was managed according to the pH-stat method of CPB for 25 minutes. For the last fi ve minutes preceding the circulatory arrest, the cooling strategy was switched to α -stat principles. A heat exchanger was used for core cooling.

After 30 minutes of cooling, a 60-minute period of HCA at 18 ° C was initiated and potassium chlo-ride (40 mmol) was injected towards the heart via CPB arterial cannula. Cardiac cooling with topical ice slush was maintained throughout HCA. The intracerebral temperature was controlled and main-tained at 18 ° C with ice packs placed over the head.

At the beginning of rewarming furosemide (40 mg), methylprednisolone (80 mg), mannitol (150 g), calcium bioglyconate (2.25 mmol Ca 2 � ) and lidocaine (40 mg) were administered into the pump. During 45 minutes of rewarming, the piglets were warmed to 35 ° C with a 100 ml/kg/min fl ow rate, so that the warming gradient was 10 ° C or less at all times. Warming was carried out according to the α -stat strategy. The heart was defi brillated if neces-sary at 30 ° C. The sump cannula was removed after 30 minutes of rewarming. Suffi cient ventilation was restored 10 minutes before weaning from CPB, which itself occurred at 45 minutes after the start of rewarming.

Noradrenalin was postoperatively used as required. The animals of both groups were extubated 8 h after the start of rewarming.

Biochemical data

Arterial blood gases, pH, electrolytes, serum ionized calcium, glucose and hemoglobin levels (i-STAT Analyzer; i-STAT Corporation, East Windsor, NJ)

were measured at baseline, at the end of cooling, as well as 30 min, 2 h, 4 h, and 8 h after the start of rewarming. At these time points, samples for cardiac troponin I and Creatine Kinase Isoenzyme MB (CK-MB) were also collected.

Blood transfusions

Fresh whole blood from a donor pig, drawn on the operative day, was transfused into the prime as required to maintain the hematocrit of all animals above 25% after the operation.

Follow-up

Daily neurological assessment, elective sacrifi ce and the harvesting of brains after seven days, as well as a histopathological analysis of the cerebral tissue were performed as described in detail in our previous studies (13).

Statistical analysis

Statistical analysis was performed using SPSS (SPSS, version 15.0, SPSS Inc, Chicago, IL) and SAS (ver-sion 9.1.3, SAS Institute Inc., Cary, NC) statistical software. Continuous and ordinal variables are expressed as the median and 25th and 75th percen-tiles. SAS procedure Mixed was used for repeated measurements. Since the measurement intervals were uneven, spatial exponential covariance struc-ture was defi ned in repeated statement. Complete independence was assumed across animals (by ran-dom statement). Reported p-values are as follows: p-between groups (p g ), indicates a level of difference between the groups, p-time ∗ group (p t

∗ g ), indicates

behavior between the groups over time. Either Stu-dent ’ s t-test or Mann-Whitney U-test was used to assess the distribution of variables between the study groups. Two-tailed signifi cance levels are reported. P � 0.05 was considered statistically signifi cant.

Results

Comparison of study groups

The mean weight of the pigs was 23.2 kg (21.6 – 27.7) in the levosimendan (LV)-group and 28.7 kg (19.5 – 31.6) in the control group (p � 0.585). Each group contained nine animals. All animals survived the surgery. In the LV-group, fi ve animals lived for the entire observation period (seven days) and four animals died on the fi rst postoperative day. In the control group six animals lived for seven days, one died on the second postoperative day and two died

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on the fi rst postoperative day. Thus there was no sta-tistically signifi cant difference in survival (p � 0.730).

Temperatures

The cooling and rewarming times did not differ between the study groups and lasted for a median of 30 min and 45 min, respectively. During hypother-mic circulatory arrest, the brain temperatures were similar in both groups throughout the arrest, with a median of 18.0 ° C in the LV-group and 18.1 ° C in the control group (p g � 0.27). Intracerebral, rectal and blood temperatures throughout the experiment are summarized in Table I.

Hemodynamic data

The heart rate tended to be higher in the LV-group, but statistical signifi cance could only be observed at 4 h after hypothermic circulatory arrest (p � 0.05). There were no signifi cant differences between the groups concerning cardiac index, mean arterial pres-sure, central venous pressure, cerebral perfusion pressure and vascular resistance during the entire experiment (Table I). The groups did not differ in terms of pulmonary capillary wedge pressure (p g � 0.42) or pulmonary artery pressures (p g � 0.68). Both groups were suffi ciently ventilated and oxygen-ated throughout the experiment.

No differences were observed in hematocrit levels between the groups. The animals of the LV-group required more fl uids during the fi rst 2 h of rewarm-ing, but towards the end of the experiment there

were no statistically signifi cant differences in fl uid balance between the groups (Table I). There were no differences between the groups concerning osmolal-ity during the entire experiment (p g � 0.84).

Three pigs in the LV-group and three pigs in the control group required a short infusion of noradren-alin after the operation. The dosage needed to main-tain mean arterial pressure above 60 mmHg was 7.9 μ g/kg (2.6 – 24.8) in the LV-group and 4.7 μ g/kg (4.5 – 11.5) in the control group (p � 0.530).

Metabolic data

There were no signifi cant differences between the groups concerning oxygen delivery (p g � 0.66), oxy-gen consumption (p g � 0.9) or oxygen extraction (p g � 0.9). The venous blood glucose was temporar-ily lower in the LV-group at 30 min after the start of rewarming, which also coincided with more dilution in terms of fl uid balance in the LV-group (Tables I and II).

Intracerebral monitoring

Intracranial pressure is displayed in Figure 1. There were no statistically signifi cant differences in the microdialysis data, namely cerebral glucose (p g � 0.61), lactate (p g � 0.46), pyruvate (p g � 0.59), glutamate (p g � 0.46) or glycerol (p g � 0.94) during the entire experiment. The lactate-glucose ratio (p g � 0.16) as well as the lactate-pyruvate ratio (p g � 0.21) was similar in both groups throughout the experiment.

Figure 1. Intracranial pressure. Median and interquartile range.

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Figure 2. Cardiac enzymes; Cardiac Troponin I. Median and interquartile range.

Cardiac enzymes

Troponin I and CkMB – levels were elevated in all animals and are displayed in Figures 2 and 3.

Neurological recovery

We observed no differences in neurological recovery between the groups. The sum of behavioral scores from the animals that survived for seven days was 47 (44 – 53.5) in the LV group and 46.5 (39.75 – 51.5) in the control group (p � 0.575).

Histopathology

There were no signifi cant differences whatsoever between the groups concerning histopathology fi nd-ings. Total histopathological score of all animals was 9.5 (5.0 – 11.0) in the LV-group and 4.0 (3.5 – 6.5) in the control group (p � 0.119). When only animals that survived the entire seven-day follow-up period were taken into account, the total score was 8.0 (3.0 – 13.5) in the LV-group and 4.0 (2.8 – 5.8) in the con-trol group (p � 0.267).

Discussion

In our study with a chronic porcine model, a 24-hour infusion of levosimendan without a loading dose did not improve cerebral protection in association with HCA. It lowered the intracranial pressure during the experiment, but no differences in edema were observed in the histopathology one week after the operation. In terms of microdialysis data, neuro-logical recovery and histopathology there was no improvement of brain protection compared to the placebo infusion. Due to the laborious regime of a chronic large animal model as well as ethical

considerations, the number of animals was small and limitations of the study include the risk of a statisti-cal Type II error.

Levosimendan was initially targeted to patients suffering from acute decompensated heart failure (ADHF). In the SURVIVE trial levosimendan dem-onstrated benefi cial short-term effects in patients suffering from ADHF, but did not signifi cantly reduce all-cause mortality at 180 days compared to dobutamine (14). In addition to the on-going heart failure discussion, other uses of levosimendan are actively investigated. So far results have been pub-lished of levosimendan in association with cardio-genic shock (15), myocardial infarction (16) and different settings of cardiac surgery (17).

Lack of difference in heart rate and cardiac index in our study were not altogether surprising, as the loading dose was omitted. In our pilot studies the loading dose induced a clear identifi able tachycardic reaction in the animals receiving levosimendan and thus to ensure complete blinding of the study we opted to use a continuous infusion without a loading dose. Due to the vasodilatory effects of levosimen-dan, the animals of the LV-group required more fl uid during the fi rst 2 h of rewarming. The more positive fl uid balance probably provides an explanation for lesser blood glucose in the LV-group at 30 min after the start of rewarming and clarifi es the slightly slower trend of rewarming in terms of blood temperature in the LV-group, as well as the consequently higher par-tial pressure of arterial oxygen at 30 min of rewarm-ing (Tables I and II). It could be speculated whether these slight differences at the initial stages of rewarm-ing, stemming from the vasodilatory effects of levo-simendan, could have temporarily improved cerebral protection refl ected in the lower intracranial pressure of the LV-group after the operation. Levosimendan did not decrease the need for other inotropes in our

Figure 3. Cardiac enzymes; Creatine Kinase Isoenzyme MB. Median and interquartile range.

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study, a similar number of animals required nora-drenalin after the operation in both groups.

Cardiac enzymes tended to be more elevated in the LV-group. While it is plausible that a 60-minute hypothermic circulatory arrest introduces such a strain to the myocardium that calcium-sensitization can cease to be benefi cial and even becomes adverse, other possible explanations may exist. In earlier stud-ies of levosimendan ’ s effects on ischemic myocar-dium, the results have been encouraging in clinical trials with human patients (18) as well as in experi-mental models with rabbits (19) and dogs (20). Similar studies with pigs, however, have frequently led to more disappointing results in terms of myocardial infarction size (21), arrhythmias (22) and contractile function (23). The possibility of a detrimental effect of levosimendan on the porcine myocardium has to be considered.

We conclude that in this experimental setting levo-simendan decreased intracranial pressure during the experiment, but it did not demonstrate persistent neu-roprotective effects in an operation requiring the use of deep hypothermic circulatory arrest.

Acknowledgements

This study was supported by grants from the Oulu University Hospital, the Finnish Foundation for Car-diovascular Research and the Sigrid Juselius Founda-tion. The authors would like to express their deepest gratitude to anesthesiologist Vilho Vainionp ä ä , MD, PhD , biostatistician Pasi Ohtonen, MSc, and Seija Selj ä nper ä , RN, for their expert guidance and assis-tance in this study. We are also grateful to Jouko Levi-joki, of Orion Pharma, who provided the study drug. This paper was presented at the Annual Meeting of The Scandinavian Society for Cardiothoracic Surgery in Geilo, Norway, February 2008.

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

References

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