Effects of Levosimendan on Hemodynamics, Local Cerebral Blood Flow, Neuronal Injury, and Neuroinflammation After Asphyctic Cardiac Arrest in Rats Robert F. Kelm, MD1; Jürgen Wagenführer, MD1; Henrike Bauer, MSc2; Irene Schmidtmann, PhD3; Kristin Engelhard, MD, PhD1; Rüdiger R. Noppens, MD, PhD1
Objectives: Despite advances in cardiac arrest treatment, high mortality and morbidity rates after successful cardiopulmonary resuscitation are still a major clinical relevant problem. The post cardiac arrest syndrome subsumes myocardial dysfunction, impaired microcirculation, systemic inflammatory response, and neurological impairment. The calcium-sensitizer levosimendan was able to improve myocardial function and initial resuscitation success after experimental cardiac arrest/cardiopulmonary resuscitation. We hypothesized that levosimendan exerts beneficial effects on cerebral blood flow, neuronal injury, neurological outcome, and inflammation 24 hours after experimental cardiac arrest/cardiopulmonary resuscitation. Design: Laboratory animal study. Setting: University animal research laboratory. Subjects: Sixty-one male Sprague-Dawley rats. Interventions: Animals underwent asphyxial cardiac arrest/cardiopulmonary resuscitation, randomized to groups with levosimenDepartment of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. 2 Department of Neuropathology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. 3 Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. Dr. Kelm’s institution received grant support from the Johannes Gutenberg-University, Mainz, Germany (institutional grant [Mainzer Forschungsförderungsprogramm (MAIFOR)]). Dr. Wagenführer’s institution received grant support from the Johannes Gutenberg-University, Mainz, Germany (institutional grant [MAIFOR]). Dr. Schmidtmann lectured for Charles River Sulzfeld. Her institution consulted for Deutsche Stiftung Organtransplantation (Statistical Analysis and Consulting) and received grant support from Merck Serano (function as study statistician in ISS study funded by Merck Serano). Dr. Engelhard served as board member for Fresenius Kabi and lectured for Abbott GmbH. Dr. Noppens’s institution received grant support from MAIFOR institutional grant. Dr. Bauer has disclosed that he does not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000308 1
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dan treatment (bolus 12 µg/kg and infusion for 3 hr [0.3 µg/min/ kg]) or vehicle (saline 0.9% bolus and infusion for 3 hr [equivalent fluid volume]). Cardiac index, local cerebral blood flow, and hemodynamic variables were measured for 180 minutes after cardiac arrest/cardiopulmonary resuscitation. Behavioral and neurological evaluations were conducted 24 hours after cardiac arrest/cardiopulmonary resuscitation. Furthermore, neuronal injury, expressed as Fluoro-Jade B–positive cells in the hippocampal formation, cortical and hippocampal inflammatory cytokine gene expression, and blood plasma interleukin-6 values were assessed. Measurements and Main Results: Treatment with levosimendan reduced neuronal injury and improved neurological outcome after 24 hours of reperfusion and resulted in elevated cardiac index and local cerebral blood flow compared with vehicle after cardiac arrest/cardiopulmonary resuscitation. Mean arterial blood pressure was reduced during the early reperfusion period in the levosimendan group. Cortical and hippocampal inflammatory cytokine gene expression and blood plasma interleukin-6 levels were not influenced. Conclusions: Levosimendan increased cerebral blood flow after experimental cardiac arrest/cardiopulmonary resuscitation. This effect coincided with reduced neuronal injury and improved neurologic outcome. Findings seem to be independent of inflammatory effects because no effects by levosimendan on cerebral or systemic inflammation could be detected. In summary, levosimendan is a promising agent to improve neurological outcome after cardiac arrest/cardiopulmonary resuscitation. (Crit Care Med 2014; 42:e410–e419) Key Words: cardiac output; cardiopulmonary resuscitation; cerebral blood flow; cerebral ischemia; inflammation; levosimendan
T
he overall survival rate of resuscitated patients after cardiac arrest (CA) is around 10% in the United States, Europe, and Australia (1, 2). Despite obvious advances in CA treatment, neurological disorders and other June 2014 • Volume 42 • Number 6
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organ dysfunctions cause substantial mortality and morbidity after return of spontaneous circulation (ROSC) (3, 4). After CA, recognized as a whole-body ischemia, reperfusion by successful cardiopulmonary resuscitation (CPR) generates various pathologies subsumed under the post cardiac arrest syndrome (5). Temporary myocardial dysfunction, basically caused by myocardial stunning, impaired cerebrovascular microcirculation, and systemic inflammatory response have been described as the major problems (6–11). Cerebral vasoconstriction, increased vascular permeability and consecutive perivascular edema, endothelial cell swelling, increased blood viscosity, and thrombus formation resulted in the “no-reflow” phenomenon of cerebral microcirculation immediately after ROSC and the delayed hypoperfusion period up to 24 hours after CA (12–15). Delayed cerebral hypoperfusion has been considered to aggravate neuronal injury (16). In addition, similarities were found during reperfusion between the systemic inflammatory response after CA and severe sepsis (17). The calcium (Ca2+)-sensitizer levosimendan is a positive inotropic agent and is currently used for the treatment of acute decompensated heart failure (18, 19). In experimental settings, levosimendan improved initial resuscitation success, increased regional brain oxygen saturation during CPR, ameliorated myocardial function, and extended the duration of short-term postresuscitation survival to 18 hours after CA (20–22). Levosimendan improves cardiac function via Ca2+-sensitizing effects. These effects, mediated by specific interaction with the Ca2+-sensor troponin C molecule, are leading to enhanced contractility of cardiac myofilaments. Potentially vasorelaxant effects have been reported on peripheral arterial vessels mediated by adenosine triphosphate (ATP)-sensitive potassium (K+) channels (23, 24). Furthermore, a coronary vasorelaxant effect was demonstrated (25). Finally, positive effects on microvascular oxygenation in a model of septic shock and anti-inflammatory action in patients with decompensated heart failure were detected (26–28). No present data exist about potential neuroprotective effects of levosimendan after CA/CPR and a possible relationship to cerebral blood flow after CA. Therefore, the present study hypothesized an improved cerebral blood flow due to levosimendan treatment in the early reperfusion period after experimental CA/CPR, in particular in the delayed cerebral hypoperfusion period. Furthermore, the effect of levosimendan on myocardial dysfunction and neuronal survival was investigated 24 hours after CA/CPR. Because of findings about anti-inflammatory effects of levosimendan, neuroinflammation and systemic inflammation were investigated.
MATERIALS AND METHODS Subjects After approval of the governmental animal care committee (Koblenz, Germany), 61 male Sprague-Dawley rats (325–375 g, Charles River, Sulzfeld, Germany) were treated in accordance Critical Care Medicine
with international and institutional guidelines. Animals were housed in a temperature-controlled environment (22°C) under a 12:12 hours dark/light cycle and had free access to food and water. Animal Preparation All animals were fasted for 12 hours with free access to water before surgery. Experimental procedures were performed as previously described (29). Briefly, anesthesia was induced with sevoflurane 4 vol% and an Fio2 of 0.8. After confirming a deep level of anesthesia, tracheal intubation was performed and animals were mechanically ventilated. End-tidal carbon dioxide tension was controlled at values of 40 mm Hg. During surgery, anesthesia was maintained with sevoflurane 3–4 vol% and Fio2 of 0.3. To minimize pain by surgical intervention, all skin incisions were flushed with bupivacaine 0.5% for 3 minutes. The right femoral artery was catheterized using polyethylene tubes for blood sampling and continuous monitoring of mean arterial blood pressure (MAP). The right femoral vein was catheterized for IV drug administration and continuous fluid infusion of 0.3 mL/hr isotonic electrolyte solution. A third catheter was inserted into the right jugular vein, used for cold isotonic electrolyte solution injection for transpulmonary cardiac output (CO) measurement. A thermocouple microprobe was placed in this venous catheter for temperature measurement of injected fluid. Using the left femoral artery, a second thermocouple microprobe was placed into the abdominal aorta for measuring CO. Limb lead II electrocardiogram (ECG) was recorded using subcutaneous electrodes. The skull was fixed in a stereotactic frame and a small closed cranial window was created over the left hemisphere for measuring local cerebral blood flow (lCBF) using a stationary laser Doppler probe (Fine Needle Probe, ADInstruments GmbH, Spechbach, Germany). Tympanic temperature was monitored within the right auricular tube using an ear-bar thermocouple probe linked to a heating lamp maintaining pericranial temperature at 37°C. Rectal temperature was also controlled at 37°C with a biofeedback-heating pad. Experimental Protocol At the end of surgery, anesthesia was maintained at sevoflurane 2.5–3.5 vol% and Fio2 of 0.3. After 10 minutes of postsurgical stabilization, the anesthetized rats were paralyzed with 0.5 mg/kg pancuronium bromide and baseline measurements were accomplished for 15 minutes (Fig. 1). Nine minutes of asphyxia was induced by stopping the controlled ventilation, resulting in CA after a short period. CPR was initiated by starting ventilation with Fio2 of 1.0. Thirty seconds after initiation of ventilation, external chest compression (200 min–1) was conducted, a single dose of vasopressin (0.4 U/kg) was injected, and treatment according to randomization was started. ROSC had to be achieved within 3 minutes, otherwise animals were excluded. The period between MAP below 20 mm Hg and above 40 mm Hg, with ROSC, was defined as duration of CA. Heating pad and lamp were www.ccmjournal.org
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All animals were placed for 24 hours in an incubator tempered at 36°C (30). Experimental Groups Forty-three animals underwent asphyxial CA/CPR, randomized to a group with levosimendan (Orion Corporation, Espoo, Finland) treatment (Levo, n = 22, bolus during CPR [12 µg/kg] and infusion for 3 hr [0.3 µg/min/ kg]) or vehicle using saline 0.9% (Vehicle, n = 21, bolus during CPR and infusion for 3 hr [equivalent fluid volume]) using a motor-driven syringe pump (Fig. 2). For technical control, 10 animals were randomized in two sham-operated groups (sLevo, n = 5; sVehicle, n = 5). Sham animals were treated similar to Figure 1. Experimental procedure and measurements during baseline, asphyxia, cardiac arrest/cardiopulmonary resuscitation (CPR), and reperfusion. Blood analysis included arterial blood gases, electrolytes, pH, base CA/CPR groups, except carexcess, hematocrit, hemoglobin, blood glucose, and lactate concentration. MAP = mean arterial blood pressure, diac standstill and vasopressin ECG = electrocardiogram, HR = heart rate, lCBF = local cerebral blood flow, CI = cardiac index. application. Five rats were used discontinued during asphyxia and CPR. Controlled ventila- as naïve animals for cerebral cytokine expression and neurotion and anesthesia with sevoflurane 1.0–1.5 vol% were con- histopathologic control. tinued for 180 minutes after CA/CPR. Fio2 was reduced at fixed time points from 1.0 to baseline values (0.3) at the end Measurements of the experimental protocol. No additional vasoactive drugs MAP, ECG, heart rate (HR), lCBF, end-tidal sevoflurane conwere used in this experimental setup. After the observation centration (ETsevo), and tympanic and rectal temperatures period, all intravascular catheters were removed, the vessels were recorded continuously using the PowerLab data acquisiwere ligated, and incisions were closed. Anesthesia was distion system and LabChart 6.0 pro recording software (ADIncontinued and the animals were weaned from the ventilator. struments GmbH, Spechbach, Germany). lCBF measurements At adequate spontaneous respiration, the tube was removed. were recorded in blood perfusion units and expressed in percent of baseline values. CO was measured 10 minutes before CA (baseline), at 5, 30, 60, 90, 120, 150, and 180 minutes of the reperfusion period using the thermodilution technique (29) (Fig. 1). CO measurements were used for calculation of cardiac index (CI; CO/ body weight). CI was expressed in absolute values (mL/min/ kg) and, additionally, in percentage of baseline. Blood samples were taken (100 µL) for analysis from the right femoral artery 15 minutes before CA (baseline) and at 1, 20, 70, 120, and 180 minutes after CA/ CPR (Fig. 1). Arterial blood Figure 2. Flow diagram of the experimental groups. ROSC = return of spontaneous circulation. e412
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gases, electrolytes, pH, base excess, hematocrit, hemoglobin, and blood glucose and lactate concentrations were measured (ABL 800 Basic, Radiometer GmbH, Willich, Germany). Neurological Assessment Score Twenty-four hours after CA/CPR, animals underwent behavioral testing using a neurologic assessment score adapted for this experiment (31–33). Testing was performed in a quiet room with dimmed light by one investigator, who was unaware of randomization. Food and water intake, consciousness, breathing pattern, vibrissae movement, motoric function, and interaction with the environment were scored (13 = normal performance, no deficit; 44 = most severe deficit). Blood Samples and Neurohistopathologic Evaluation After behavioral and neurologic evaluation, the animals were deeply anesthetized with sevoflurane 5 vol% and tracheal intubated. After median sterno-laparotomy, the descending aorta was catheterized, and blood samples were taken for preparing blood plasma. After incision of the right atrium, animals were briefly perfused via the descending aorta with cold saline 0.9%. Brains were carefully removed, cryofixated in liquid nitrogen, and stored at –20°C. Coronal sections (10 µm) were prepared and stained with Fluoro-Jade B (Histo-Chem, Jefferson, AR), a fluorochrome marker for degenerating neurons (34, 35). Fluoro-Jade B–positive neurons were counted by an investigator blinded to the group allocation. The entire cornu ammonis (CA 1–4) of the hippocampal region and the CA1 region (magnification 400-fold), defined at interaural 5.7 mm and bregma –3.3 mm, were evaluated (36). Three consecutive sections were analyzed in both hemispheres, and the total mean of positive neurons was calculated.
measurements such as CI, MAP, HR, lCBF, ETsevo, physiologic variables, and tympanic and rectal temperatures within the groups were conducted with two-way repeated measurement analysis of variance (factors treatment and time) and Bonferroni posttests. Unpaired t tests were used to compare animals subjected to CA with and without levosimendan with respect to neuronal injury (Fluoro-Jade B staining), cerebral cytokine expression, CA duration, and neurological assessment score. Plasma IL-6 analysis was evaluated with Wilcoxon test. For comparison of descriptive results, shamoperated and naïve animals are displayed beside the results of CA animals. All tests were two-sided with a significance level of α equals 0.05. Statistical analysis was realized with GraphPad Prism Version 5.03 (GraphPad Software, La Jolla, CA). Data are presented as mean ± sd.
RESULTS A total of 61 animals were investigated, 43 animals randomized to the CA groups, 10 animals in the sham groups, and 5 animals in the Naive group. Three animals were excluded during the preparation period due to vessel rupture. A total of 13 animals did not achieve ROSC within 3 minutes (Levo n = 7, Vehicle n = 6) and were replaced accordingly (30%). Cardiac Arrest Duration of CA was comparable between the Levo and Vehicle groups. Total arrest time was 444 ± 36 seconds for the Levo group and 451 ± 39 seconds for the Vehicle group.
Blood Plasma Interleukin-6 Cytokine Analysis For determination of interleukin (IL)-6, the Quantikine ELISA Rat IL-6 Immunoassay Kit (R&D Systems GmbH, Wiesbaden, Germany) was used according to the manufacturer’s instructions. Plasma IL-6 cytokine values were expressed in pg/mL.
Physiological Values, End-Tidal Sevoflurane Concentration, and Temperature At baseline, all measured physiological values were within physiological ranges (Table 1). Most of the variables were affected by CA, except blood glucose and sodium concentration (data not shown). Differences were detected between the CA groups regarding K+ and lactate concentration. Blood analysis after the first minute of reperfusion showed lower K+ concentrations in the Levo group (6.5 ± 1.2 mmol/L) compared with the Vehicle group (7.3 ± 1 mmol/L, p < 0.01). Measurement of lactate concentration detected differences between the CA groups 20 minutes after reperfusion (Levo 2.8 ± 0.7 vs Vehicle 3.8 ± 0.6, p < 0.001), whereas for sham animals no differences were detected (Table 1). Measurement of ETsevo (data not shown) demonstrated no differences in anesthesia between CA groups and between sham groups. Tympanic and rectal temperatures were in physiologic ranges and did not differ between the two CA and sham groups throughout the observation period (data for tympanic temperature are given in Table 1).
Statistical Analyses Twelve to seventeen animals per group are necessary in order to demonstrate a decrease in affected neuronal cells (neuronal injury) from 35% to 10% with a power of 80% at the 5% level and sd between 20% and 25%. Sample size calculation was conducted using nQuery 5.0 Software (Statistical Solutions, Cork, Ireland). Exploratory comparisons between time-based
Cardiac Index At baseline, calculated CI values were 421 ± 42 mL/min/ kg in the Levo group and 429 ± 47 mL/min/kg in the Vehicle group (Fig. 3A). Five minutes after CA/CPR, CI decreased to 84% ± 11% of baseline values in the Vehicle group (Fig. 3B). Constant values compared to baseline were detected at the same time point in animals treated with levosimendan
Cerebral Cytokine Gene Expression Analysis For gene expression analysis, coronal sectioned tissue samples were collected. Interaural between 6.8 mm and 5.7 mm and between bregma –2.12 mm and –3.3 mm tissue of the entire hippocampal region and cortical tissue (gray and white matter) were separated and stored in tubes at –80°C. Gene expression analysis was performed with reverse transcriptase polymerase chain reaction as previously described (37, 38).
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Table 1.
Group
Levo
Vehicle
Physiologic Variables and Tympanic Temperature Time (min)
sVehicle
Pao2 (mm Hg)
Lactate (mmol/L)
Glucose (mg/dL)
K+ (mmol/L)
Ca2+ (mmol/L)
Tympanic Temperature (°C)
Baseline
7.431 ± 0.020 40.4 ± 2.4 131.0 ± 12.1 0.78 ± 0.08 137.3 ± 13.7 3.49 ± 0.28 1.33 ± 0.04
37.1 ± 0.1
1
7.165 ± 0.018 50.5 ± 3.7 302.9 ± 33.6 7.44 ± 1.10 159.1 ± 41.1 6.54 ± 1.20 1.30 ± 0.07
36.0 ± 0.3
20
7.261 ± 0.034 48.3 ± 3.6 240.7 ± 49.7 2.81 ± 0.74 158.1 ± 31.9 2.94 ± 0.23 1.17 ± 0.08
37.0 ± 0.1
70
7.394 ± 0.029 40.6 ± 2.9 202.0 ± 38.9 1.11 ± 0.49 138.2 ± 25.8 4.01 ± 0.34 1.19 ± 0.07
37.0 ± 0.0
120
7.398 ± 0.025 40.8 ± 1.1 113.9 ± 11.8 0.89 ± 0.15 133.4 ± 17.9 3.60 ± 0.29 1.25 ± 0.07
37.0 ± 0.0
180
7.401 ± 0.022 40.2 ± 1.3 118.1 ± 16.1 0.77 ± 0.10 135.1 ± 15.2 3.29 ± 0.82 1.28 ± 0.05
37.0 ± 0.0
Baseline
7.424 ± 0.015 40.2 ± 1.7 131.5 ± 14.9 0.91 ± 0.12 139.3 ± 10.1 3.33 ± 0.27 1.32 ± 0.06
37.1 ± 0.1
7.170 ± 0.031 47.4 ± 4.5 300.9 ± 36.5 7.36 ± 0.65 128.4 ± 36.0 7.25 ± 0.99
1.28 ± 0.06
36.0 ± 0.2
20
7.242 ± 0.026 47.5 ± 3.0 247.6 ± 35.2 3.80 ± 0.59 144.7 ± 20.0 2.91 ± 0.32 1.16 ± 0.05
37.0 ± 0.1
70
7.400 ± 0.020 39.7 ± 2.3 200.6 ± 44.7 1.26 ± 0.45 147.6 ± 11.2 4.01 ± 0.39 1.19 ± 0.06
37.0 ± 0.1
120
7.406 ± 0.017 40.5 ± 1.4 117.1 ± 11.6 1.07 ± 0.35 146.1 ± 11.4 3.65 ± 0.28 1.28 ± 0.04
37.0 ± 0.0
180
7.406 ± 0.015 39.9 ± 1.4 120.2 ± 13.5 0.88 ± 0.14 144.1 ± 11.6 3.49 ± 0.27 1.29 ± 0.04
37.0 ± 0.1
Baseline
7.410 ± 0.010 41.0 ± 1.3 116.6 ± 13.3 0.82 ± 0.11 141.6 ± 10.6 3.68 ± 0.30 1.31 ± 0.06
37.0 ± 0.1
1
7.408 ± 0.020 40.5 ± 2.8 334.0 ± 80.3 0.78 ± 0.08 130.4 ± 13.5 3.50 ± 0.25 1.29 ± 0.05
36.9 ± 0.1
20
7.404 ± 0.021 41.7 ± 2.4 306.6 ± 73.3 0.70 ± 0.07 134.6 ± 13.5 3.44 ± 0.18 1.30 ± 0.05
37.0 ± 0.0
70
7.418 ± 0.004 41.3 ± 1.9 211.0 ± 50.6 0.70 ± 0.10 134.0 ± 12.1 3.34 ± 0.21 1.28 ± 0.04
37.0 ± 0.1
120
7.418 ± 0.026 41.9 ± 2.7 114.0 ± 14.9 0.86 ± 0.15 131.2 ± 11.7 3.32 ± 0.15 1.29 ± 0.06
37.0 ± 0.0
180
7.424 ± 0.018 41.0 ± 0.5 117.4 ± 17.4 0.82 ± 0.11 129.6 ± 8.0
3.36 ± 0.09 1.30 ± 0.04
37.0 ± 0.0
Baseline
7.430 ± 0.019 41.3 ± 3.2 126.4 ± 19.1 0.82 ± 0.08 147.8 ± 14.6 3.52 ± 0.47 1.33 ± 0.03
37.1 ± 0.1
1
7.438 ± 0.016 39.6 ± 2.6 371.0 ± 35.2 0.74 ± 0.09 138.8 ± 9.0
3.34 ± 0.46 1.31 ± 0.08
36.9 ± 0.1
20
7.434 ± 0.009 40.1 ± 1.8 353.4 ± 18.6 0.70 ± 0.07 136.2 ± 5.8
3.28 ± 0.36 1.30 ± 0.05
37.0 ± 0.0
70
7.440 ± 0.012 39.8 ± 1.8 248.0 ± 20.8 0.76 ± 0.09 138.4 ± 9.9
3.30 ± 0.24 1.30 ± 0.02
37.0 ± 0.0
120
7.450 ± 0.007 38.5 ± 1.7 131.2 ± 13.0 0.86 ± 0.15 132.2 ± 12.4 3.22 ± 0.18 1.27 ± 0.03
37.0 ± 0.1
180
7.446 ± 0.011 39.7 ± 1.5 132.6 ± 16.6 0.90 ± 0.16 133.4 ± 17.3 3.14 ± 0.18 1.27 ± 0.02
37.0 ± 0.0
1
sLevo
pH
Paco2 (mm Hg)
a
b
Bonferroni posttests: Levo versus Vehicle at the corresponding time: ap < 0.01, bp < 0.001. Mean ± sd.
(Levo 101% ± 10%, p < 0.001 Bonferroni posttest). Over time, a significant decrease of CI was detected in both CA groups (p < 0.0001). However, CI was higher with levosimendan compared with the vehicle group during the reperfusion period (Levo vs Vehicle absolute values: p = 0.0141; relative values: p < 0.0001). After 180 minutes of reperfusion, CI was reduced by 14% to baseline values in the Levo group and by 28% in the Vehicle group (Levo vs Vehicle: p < 0.001 Bonferroni posttest). Comparison of sham groups showed higher values in the sLevo group during treatment (sLevo vs sVehicle absolute values: p = 0.0122; relative values: p = 0.0008) (Fig. 3, A and B). Local Cerebral Blood Flow and Hemodynamic Measurements Measurement of lCBF during baseline and CA/CPR showed no differences between groups. Early after ROSC, a postischemic hyperperfusion period was detected in both CA/CPR e414
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groups. Maximum values after 9 minutes of reperfusion were measured at 297% ± 46% in the Levo group and 287% ± 51% in the Vehicle group (Fig. 4A). A hypoperfusion period of 20 minutes after CA/CPR was observed in both CA groups. However, postischemic hypoperfusion was more pronounced in the Vehicle group (Levo vs Vehicle: p = 0.0013). After 180 minutes of reperfusion, values in the Levo group exceeded baseline values (Levo 126% ± 25%) compared with Vehicle group (Vehicle 88% ± 17%, p < 0.05 Bonferroni posttest). Hemodynamic variables such as MAP and HR (data not shown) remained in physiological ranges during baseline. After ROSC, a period of increased MAP was detected in both CA groups. Maximum values were measured after 8 minutes of reperfusion (Levo 167 ± 13 mm Hg, Vehicle 166 ± 20 mm Hg) (Fig. 4B). In the consecutive observation period of 180 minutes, MAP was lower in the Levo group compared with Vehicle group (p = 0.0002). HR (data not shown) measurement identified a difference between June 2014 • Volume 42 • Number 6
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Neurohistopathology A lower number of positive cells in animals treated with levosimendan (Levo vs Vehicle: 997 ± 103 vs 1,113 ± 137 Fluoro-Jade B–positive cells, p = 0.0052) (Fig. 6A) was detected in the entire cornu ammonis of the hippocampal formation. Similarly, the neuronal injury in the CA 1 region was reduced in the Levo group (Levo vs Vehicle: 352 ± 30 vs 396 ± 47 Fluoro-Jade B–positive cells, p = 0.0098) (Fig. 6B). Cerebral Cytokine Gene Expression Analysis of IL-6, Tumor Necrosis Factor-α, and IL-1β No differences in all cytokine gene expressions were detected between CA groups (data not shown). Blood Plasma IL-6 Cytokine Analysis No differences in plasma IL-6 concentration were detected between CA groups (Levo 74.01 ± 43.00 pg/mL, Vehicle 68.72 ± 62.69 pg/mL).
DISCUSSION The present study demonstrated that levosimendan increases lCBF during hypoperfusion after experimental Figure 3. A, Cardiac index (CI) in mL/min/kg; Levo versus Vehicle: *p = 0.0141; sLevo versus sVehicle: CA/CPR, although diminished # p = 0.0122. B, CI in percent of baseline; Levo versus Vehicle: ***p < 0.0001; sLevo versus sVehicle: **p = 0.0008. Bonferroni posttests data not shown. Mean ± sd. CPR = cardiopulmonary resuscitation. MAP values were detected in animals treated with levosiLevo and Vehicle groups, with higher values in the Levo group mendan. Furthermore, levosimendan treatment reduced neu(p = 0.0459). Comparison of MAP and HR (data not shown) ronal injury and improved neurological performance after 24 between the sham groups detected no differences. hours following CA/CPR. There were no inhibitory effects of levosimendan on cerebral or systemic inflammatory response. Survival We confirmed previous results of improved cardiac function Two animals did not survive the observation period of 24 in the reperfusion period after CA/CPR due to levosimendan hours in each of the CA groups. All animals of the sham groups treatment. survived. Positive inotropic action due to Ca2+-sensitizing and phosphodiesterase-inhibitory effects in cardiac myofilaments, Neurological Assessment Score increased HR, and activation of ATP-sensitive K+-channels of Twenty-four hours after CA/CPR, the behavioral and neuro- smooth vessel muscle cells for vasodilatation are the mechalogical evaluation showed a better performance of animals nisms of levosimendan (39). No data exist about cerebral treated with levosimendan compared with Vehicle group (Levo vasodilating action due to levosimendan, but previous studvs Vehicle: 18.7 ± 2.2 vs 21.2 ± 2.8, p = 0.0208) (Fig. 5). Sham ies showed systemic and coronary vasodilatatory effects (18, animals were not affected by experimental procedures. 25). Furthermore, ATP-sensitive K+-channels with vasodilating Critical Care Medicine
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after successful CPR, that is, no-reflow or low-reflow occurs, followed by temporary hyperperfusion and longer lasting transient hypoperfusion (43). In the present study, lCBF was measured in areas of the somatosensory cortex. Therefore, the plausibility of lCBF measurements is confirmed due to the characteristic course of pathological cerebral perfusion after global ischemia. Using laser-Doppler flowmetry, a well-validated and accepted method to measure changes in cerebral perfusion over time (44), in the present study, a higher lCBF and improved microcirculation during the hypoperfusion period of reperfusion were detected, although MAP was decreased in animals treated with levosimendan. These findings are in conflict with the idea of a disturbed autoregulation of cerebral blood flow after global cerebral ischemia due to CA, which would consequently result in impaired lCBF. An absent or right-sided shift of autoregulation was previously described in the acute phase after CA, and for this reason, higher levels of MAP in the reperfusion period were recommended (45). Furthermore, after experimental CA/CPR in dogs, an enormous increase of MAP was Figure 4. A, Local cerebral blood flow (lCBF) in the somatosensory cortex expressed as relative perfusion (%) to baseline values; Levo versus Vehicle: **p = 0.0013; sLevo versus sVehicle: not significant. B, Mean associated with a better neuroarterial blood pressure (MAP) expressed in mm Hg during the experimental procedure, Levo versus Vehicle: logical outcome (46). In part, ***p = 0.0002; sLevo versus sVehicle: not significant. Bonferroni posttests data not shown. Mean ± sd. the findings of the present CPR = cardiopulmonary resuscitation. study could be associated with effects after activation were verified in smooth muscle and increased CI and vasodilating effects of levosimendan, leading endothelial cells of brain vessels (40–42). Therefore, this vaso- to an enhanced tissue microcirculation in the absence of an dilating effect of levosimendan, as an activator of ATP-sensitive increased blood pressure. In general, comparing experimental K+-channels, possibly counteract to the pathological vasocon- results from different CA models should be made with care to striction in the hypoperfusion period after global cerebral ischdifferences in terms of generated CA (e.g., ventricular fibrillaemia by CA. tion) and animal species. In the present study, levosimendan treatment improved Myocardial dysfunction was detected in both CA groups in lCBF, especially during transient hypoperfusion after CA/CPR. the reperfusion period in the present study, using the transpulIt is generally accepted that after global ischemia, CBF shows monary thermodilution technique for CI measurement. This characteristically pathological changes over time. Immediately is in agreement with previous publications (8). Levosimendan e416
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Figure 5. Neurological assessment score (13 = normal performance, no deficit; 44 = most severe deficit) 24 hr after cardiac arrest and cardiopulmonary resuscitation; Levo versus Vehicle: *p = 0.0208. Mean ± sd.
ameliorated cardiac function compared with vehicle-treated animals after CA/CPR. Neuronal injury was reduced in animals treated with levosimendan. The number of Fluoro-Jade B–positive cells, a staining method for degenerating neurons, was decreased in the hippocampal formation, precisely the entire cornu ammonis and a specific sector (CA 1), known to be highly sensitive to ischemic conditions. After global cerebral ischemia, it could be of great significance to improve microcirculation in these sensitive brain regions. The fact that levosimendan improved perfusion via increased CI and vasodilatation could contribute to reduced neuronal injury in the hippocampal formation. In an in vitro model of traumatic brain injury, levosimendan showed direct neuroprotective properties (47). However, findings in dogs indicated that levosimendan does not substantially penetrate the bloodbrain barrier in the healthy brain (48). A recent study using a bilateral common artery clamping and hypoxic ventilation for induction of cerebral ischemia indicated that only a low brain tissue concentration of levosimendan compared to serum concentrations can be achieved (49). No effects of levosimendan treatment on time course of lactate, glucose, pyruvate, and glutamate concentrations after transient ischemia were shown. It seems unlikely that direct neuroprotective effects of levosimendan contributed to our results. Neuronal injury was reduced, and neurological performance 24 hours after resuscitation was improved in animals treated with levosimendan. Compared with sham-operated animals, a severe neurological impairment was detected in animals after CA/CPR. Because of the total standstill of perfusion as a whole-body ischemia, CA models generate more severe neurological disorders compared with other models of cerebral ischemia, for example, four-vessel occlusion or focal cerebral ischemia models. Despite the severe and complex impairments in this model of CA/CPR, levosimendan treatment was able to show beneficial effects. Critical Care Medicine
Figure 6. Neuronal injury expressed as Fluoro-Jade B–positive cells 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Entire cornu ammonis (CA) of the hippocampal formation; Levo versus Vehicle: **p = 0.0052. B, CA 1 of the hippocampal formation; Levo versus Vehicle: **p = 0.0098. Mean ± sd.
Although immunological effects by levosimendan were described in several publications, no effects on neuroinflammation and systemic inflammatory response were detected in the present study. Immunological effects of levosimendan including reduced cytokine levels in patients with decompensated heart failure, reduced leukocyte reactive oxygen species release in patients with acute heart failure and septic shock, and lower pulmonary IL-6 levels in a porcine model of septic shock have been described (27, 28, 50–52). It seems likely that levosimendan did not penetrate the blood-brain barrier in sufficient quantity and, therefore, was not able to exert anti-inflammatory properties in the brain tissue in this study. However, it remains unclear why general systemic inflammatory response after CA/ CPR was not influenced by levosimendan.
CONCLUSIONS The present study demonstrated the neuroprotective effect of levosimendan by reducing myocardial dysfunction and improving cortical cerebral blood flow after experimental asphyctic CA and CPR. Recently described anti-inflammatory www.ccmjournal.org
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effects of levosimendan are unlikely to contribute to effects observed in this study. Levosimendan seems to be a potential drug in the treatment of the post cardiac arrest syndrome due to its positive effect on CI, cerebral blood flow, and neuronal injury.
ACKNOWLEDGMENTS We thank Frida Kornes, Dana Pieter, and Magdeleine Herkt for their excellent technical assistance. Data presented in this article are part of a doctoral thesis by Jürgen Wagenführer to the Medical Faculty of the Johannes Gutenberg-University, Mainz, Germany.
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Cardiovascular Care Committee; the Council on Cardiovascular Surgery and Anesthesia; the Council on Cardiopulmonary, Perioperative, and Critical Care; the Council on Clinical Cardiology; the Council on Stroke. Resuscitation 2008; 79:350–379 18. Pagel PS, Harkin CP, Hettrick DA, et al: Levosimendan (OR-1259), a myofilament calcium sensitizer, enhances myocardial contractility but does not alter isovolumic relaxation in conscious and anesthetized dogs. Anesthesiology 1994; 81:974–987 19. Delaney A, Bradford C, McCaffrey J, et al: Levosimendan for the treatment of acute severe heart failure: A meta-analysis of randomised controlled trials. Int J Cardiol 2010; 138:281–289 20. Koudouna E, Xanthos T, Bassiakou E, et al: Levosimendan improves the initial outcome of cardiopulmonary resuscitation in a swine model of cardiac arrest. Acta Anaesthesiol Scand 2007; 51:1123–1129 21. Huang L, Weil MH, Tang W, et al: Comparison between dobutamine and levosimendan for management of postresuscitation myocardial dysfunction. Crit Care Med 2005; 33:487–491 22. Huang L, Weil MH, Sun S, et al: Levosimendan improves postresuscitation outcomes in a rat model of CPR. J Lab Clin Med 2005; 146:256–261 23. Yokoshiki H, Katsube Y, Sunagawa M, et al: Levosimendan, a novel Ca2+ sensitizer, activates the glibenclamide-sensitive K+ channel in rat arterial myocytes. Eur J Pharmacol 1997; 333:249–259 24. Yildiz O: Vasodilating mechanisms of levosimendan: Involvement of K+ channels. J Pharmacol Sci 2007; 104:1–5 25. Gruhn N, Nielsen-Kudsk JE, Theilgaard S, et al: Coronary vasorelaxant effect of levosimendan, a new inodilator with calcium-sensitizing properties. J Cardiovasc Pharmacol 1998; 31:741–749 26. Fries M, Ince C, Rossaint R, et al: Levosimendan but not norepinephrine improves microvascular oxygenation during experimental septic shock. Crit Care Med 2008; 36:1886–1891 27. Parissis JT, Adamopoulos S, Antoniades C, et al: Effects of levosimendan on circulating pro-inflammatory cytokines and soluble apoptosis mediators in patients with decompensated advanced heart failure. Am J Cardiol 2004; 93:1309–1312 28. Adamopoulos S, Parissis JT, Iliodromitis EK, et al: Effects of levosimendan versus dobutamine on inflammatory and apoptotic pathways in acutely decompensated chronic heart failure. Am J Cardiol 2006; 98:102–106 29. Kelm RF, Wagenführer J, Schmidtmann I, et al: Transpulmonary cardiac output measurement in a rat model of cardiac arrest and CPR: Impact of vascular access. Resuscitation 2010; 81:248–254 30. Hickey RW, Ferimer H, Alexander HL, et al: Delayed, spontaneous hypothermia reduces neuronal damage after asphyxial cardiac arrest in rats. Crit Care Med 2000; 28:3511–3516 31. Hendrickx HH, Safar P, Miller A: Delayed recovery of behavior after anesthesia in rats. Resuscitation 1984; 12:213–221 32. Katz L, Ebmeyer U, Safar P, et al: Outcome model of asphyxial cardiac arrest in rats. J Cereb Blood Flow Metab 1995; 15:1032–1039 33. Noppens RR, Kelm RF, Lindemann R, et al: Effects of a singledose hypertonic saline hydroxyethyl starch on cerebral blood flow, long-term outcome, neurogenesis, and neuronal survival after cardiac arrest and cardiopulmonary resuscitation in rats. Crit Care Med 2012; 40:2149–2156 34. Schmued LC, Hopkins KJ: Fluoro-Jade B: A high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 2000; 874:123–130 35. Schmued LC, Albertson C, Slikker W Jr: Fluoro-Jade: A novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 1997; 751:37–46 36. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates. Compact Third Edition. San Diego, Academic Press, 1997 37. Thal SC, Wyschkon S, Pieter D, et al: Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice. J Neurotrauma 2008; 25:785–794 38. Langnaese K, John R, Schweizer H, et al: Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model. BMC Mol Biol 2008; 9:53 39. Papp Z, Édes I, Fruhwald S, et al: Levosimendan: Molecular mechanisms and clinical implications: Consensus of experts on June 2014 • Volume 42 • Number 6
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47. Roehl AB, Hein M, Loetscher PD, et al: Neuroprotective properties of levosimendan in an in vitro model of traumatic brain injury. BMC Neurol 2010; 10:97 48. Antila S, Huuskonen H, Nevalainen T, et al: Site dependent bioavailability and metabolism of levosimendan in dogs. Eur J Pharm Sci 1999; 9:85–91 49. Roehl AB, Zoremba N, Kipp M, et al: The effects of levosimendan on brain metabolism during initial recovery from global transient ischaemia/hypoxia. BMC Neurol 2012; 12:81 50. Avgeropoulou C, Andreadou I, Markantonis-Kyroudis S, et al: The Ca2+-sensitizer levosimendan improves oxidative damage, BNP and pro-inflammatory cytokine levels in patients with advanced decompensated heart failure in comparison to dobutamine. Eur J Heart Fail 2005; 7:882–887 51. Hasslacher J, Bijuklic K, Bertocchi C, et al: Levosimendan inhibits release of reactive oxygen species in polymorphonuclear leukocytes in vitro and in patients with acute heart failure and septic shock: A prospective observational study. Crit Care 2011; 15:R166 52. Ji M, Li R, Li GM, et al: Effects of combined levosimendan and vasopressin on pulmonary function in porcine septic shock. Inflammation 2012; 35:871–880
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Objectives: Despite advances in cardiac arrest treatment, high mortality and morbidity rates after successful cardiopulmonary resuscitation are still a major clinical relevant problem. The post cardiac arrest syndrome subsumes myocardial dysfunction, impaired microcirculation, systemic inflammatory response, and neurological impairment. The calcium-sensitizer levosimendan was able to improve myocardial function and initial resuscitation success after experimental cardiac arrest/cardiopulmonary resuscitation. We hypothesized that levosimendan exerts beneficial effects on cerebral blood flow, neuronal injury, neurological outcome, and inflammation 24 hours after experimental cardiac arrest/cardiopulmonary resuscitation. Design: Laboratory animal study. Setting: University animal research laboratory. Subjects: Sixty-one male Sprague-Dawley rats. Interventions: Animals underwent asphyxial cardiac arrest/cardiopulmonary resuscitation, randomized to groups with levosimenDepartment of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. 2 Department of Neuropathology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. 3 Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. Dr. Kelm’s institution received grant support from the Johannes Gutenberg-University, Mainz, Germany (institutional grant [Mainzer Forschungsförderungsprogramm (MAIFOR)]). Dr. Wagenführer’s institution received grant support from the Johannes Gutenberg-University, Mainz, Germany (institutional grant [MAIFOR]). Dr. Schmidtmann lectured for Charles River Sulzfeld. Her institution consulted for Deutsche Stiftung Organtransplantation (Statistical Analysis and Consulting) and received grant support from Merck Serano (function as study statistician in ISS study funded by Merck Serano). Dr. Engelhard served as board member for Fresenius Kabi and lectured for Abbott GmbH. Dr. Noppens’s institution received grant support from MAIFOR institutional grant. Dr. Bauer has disclosed that he does not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000308 1
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dan treatment (bolus 12 µg/kg and infusion for 3 hr [0.3 µg/min/ kg]) or vehicle (saline 0.9% bolus and infusion for 3 hr [equivalent fluid volume]). Cardiac index, local cerebral blood flow, and hemodynamic variables were measured for 180 minutes after cardiac arrest/cardiopulmonary resuscitation. Behavioral and neurological evaluations were conducted 24 hours after cardiac arrest/cardiopulmonary resuscitation. Furthermore, neuronal injury, expressed as Fluoro-Jade B–positive cells in the hippocampal formation, cortical and hippocampal inflammatory cytokine gene expression, and blood plasma interleukin-6 values were assessed. Measurements and Main Results: Treatment with levosimendan reduced neuronal injury and improved neurological outcome after 24 hours of reperfusion and resulted in elevated cardiac index and local cerebral blood flow compared with vehicle after cardiac arrest/cardiopulmonary resuscitation. Mean arterial blood pressure was reduced during the early reperfusion period in the levosimendan group. Cortical and hippocampal inflammatory cytokine gene expression and blood plasma interleukin-6 levels were not influenced. Conclusions: Levosimendan increased cerebral blood flow after experimental cardiac arrest/cardiopulmonary resuscitation. This effect coincided with reduced neuronal injury and improved neurologic outcome. Findings seem to be independent of inflammatory effects because no effects by levosimendan on cerebral or systemic inflammation could be detected. In summary, levosimendan is a promising agent to improve neurological outcome after cardiac arrest/cardiopulmonary resuscitation. (Crit Care Med 2014; 42:e410–e419) Key Words: cardiac output; cardiopulmonary resuscitation; cerebral blood flow; cerebral ischemia; inflammation; levosimendan
T
he overall survival rate of resuscitated patients after cardiac arrest (CA) is around 10% in the United States, Europe, and Australia (1, 2). Despite obvious advances in CA treatment, neurological disorders and other June 2014 • Volume 42 • Number 6
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organ dysfunctions cause substantial mortality and morbidity after return of spontaneous circulation (ROSC) (3, 4). After CA, recognized as a whole-body ischemia, reperfusion by successful cardiopulmonary resuscitation (CPR) generates various pathologies subsumed under the post cardiac arrest syndrome (5). Temporary myocardial dysfunction, basically caused by myocardial stunning, impaired cerebrovascular microcirculation, and systemic inflammatory response have been described as the major problems (6–11). Cerebral vasoconstriction, increased vascular permeability and consecutive perivascular edema, endothelial cell swelling, increased blood viscosity, and thrombus formation resulted in the “no-reflow” phenomenon of cerebral microcirculation immediately after ROSC and the delayed hypoperfusion period up to 24 hours after CA (12–15). Delayed cerebral hypoperfusion has been considered to aggravate neuronal injury (16). In addition, similarities were found during reperfusion between the systemic inflammatory response after CA and severe sepsis (17). The calcium (Ca2+)-sensitizer levosimendan is a positive inotropic agent and is currently used for the treatment of acute decompensated heart failure (18, 19). In experimental settings, levosimendan improved initial resuscitation success, increased regional brain oxygen saturation during CPR, ameliorated myocardial function, and extended the duration of short-term postresuscitation survival to 18 hours after CA (20–22). Levosimendan improves cardiac function via Ca2+-sensitizing effects. These effects, mediated by specific interaction with the Ca2+-sensor troponin C molecule, are leading to enhanced contractility of cardiac myofilaments. Potentially vasorelaxant effects have been reported on peripheral arterial vessels mediated by adenosine triphosphate (ATP)-sensitive potassium (K+) channels (23, 24). Furthermore, a coronary vasorelaxant effect was demonstrated (25). Finally, positive effects on microvascular oxygenation in a model of septic shock and anti-inflammatory action in patients with decompensated heart failure were detected (26–28). No present data exist about potential neuroprotective effects of levosimendan after CA/CPR and a possible relationship to cerebral blood flow after CA. Therefore, the present study hypothesized an improved cerebral blood flow due to levosimendan treatment in the early reperfusion period after experimental CA/CPR, in particular in the delayed cerebral hypoperfusion period. Furthermore, the effect of levosimendan on myocardial dysfunction and neuronal survival was investigated 24 hours after CA/CPR. Because of findings about anti-inflammatory effects of levosimendan, neuroinflammation and systemic inflammation were investigated.
MATERIALS AND METHODS Subjects After approval of the governmental animal care committee (Koblenz, Germany), 61 male Sprague-Dawley rats (325–375 g, Charles River, Sulzfeld, Germany) were treated in accordance Critical Care Medicine
with international and institutional guidelines. Animals were housed in a temperature-controlled environment (22°C) under a 12:12 hours dark/light cycle and had free access to food and water. Animal Preparation All animals were fasted for 12 hours with free access to water before surgery. Experimental procedures were performed as previously described (29). Briefly, anesthesia was induced with sevoflurane 4 vol% and an Fio2 of 0.8. After confirming a deep level of anesthesia, tracheal intubation was performed and animals were mechanically ventilated. End-tidal carbon dioxide tension was controlled at values of 40 mm Hg. During surgery, anesthesia was maintained with sevoflurane 3–4 vol% and Fio2 of 0.3. To minimize pain by surgical intervention, all skin incisions were flushed with bupivacaine 0.5% for 3 minutes. The right femoral artery was catheterized using polyethylene tubes for blood sampling and continuous monitoring of mean arterial blood pressure (MAP). The right femoral vein was catheterized for IV drug administration and continuous fluid infusion of 0.3 mL/hr isotonic electrolyte solution. A third catheter was inserted into the right jugular vein, used for cold isotonic electrolyte solution injection for transpulmonary cardiac output (CO) measurement. A thermocouple microprobe was placed in this venous catheter for temperature measurement of injected fluid. Using the left femoral artery, a second thermocouple microprobe was placed into the abdominal aorta for measuring CO. Limb lead II electrocardiogram (ECG) was recorded using subcutaneous electrodes. The skull was fixed in a stereotactic frame and a small closed cranial window was created over the left hemisphere for measuring local cerebral blood flow (lCBF) using a stationary laser Doppler probe (Fine Needle Probe, ADInstruments GmbH, Spechbach, Germany). Tympanic temperature was monitored within the right auricular tube using an ear-bar thermocouple probe linked to a heating lamp maintaining pericranial temperature at 37°C. Rectal temperature was also controlled at 37°C with a biofeedback-heating pad. Experimental Protocol At the end of surgery, anesthesia was maintained at sevoflurane 2.5–3.5 vol% and Fio2 of 0.3. After 10 minutes of postsurgical stabilization, the anesthetized rats were paralyzed with 0.5 mg/kg pancuronium bromide and baseline measurements were accomplished for 15 minutes (Fig. 1). Nine minutes of asphyxia was induced by stopping the controlled ventilation, resulting in CA after a short period. CPR was initiated by starting ventilation with Fio2 of 1.0. Thirty seconds after initiation of ventilation, external chest compression (200 min–1) was conducted, a single dose of vasopressin (0.4 U/kg) was injected, and treatment according to randomization was started. ROSC had to be achieved within 3 minutes, otherwise animals were excluded. The period between MAP below 20 mm Hg and above 40 mm Hg, with ROSC, was defined as duration of CA. Heating pad and lamp were www.ccmjournal.org
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All animals were placed for 24 hours in an incubator tempered at 36°C (30). Experimental Groups Forty-three animals underwent asphyxial CA/CPR, randomized to a group with levosimendan (Orion Corporation, Espoo, Finland) treatment (Levo, n = 22, bolus during CPR [12 µg/kg] and infusion for 3 hr [0.3 µg/min/ kg]) or vehicle using saline 0.9% (Vehicle, n = 21, bolus during CPR and infusion for 3 hr [equivalent fluid volume]) using a motor-driven syringe pump (Fig. 2). For technical control, 10 animals were randomized in two sham-operated groups (sLevo, n = 5; sVehicle, n = 5). Sham animals were treated similar to Figure 1. Experimental procedure and measurements during baseline, asphyxia, cardiac arrest/cardiopulmonary resuscitation (CPR), and reperfusion. Blood analysis included arterial blood gases, electrolytes, pH, base CA/CPR groups, except carexcess, hematocrit, hemoglobin, blood glucose, and lactate concentration. MAP = mean arterial blood pressure, diac standstill and vasopressin ECG = electrocardiogram, HR = heart rate, lCBF = local cerebral blood flow, CI = cardiac index. application. Five rats were used discontinued during asphyxia and CPR. Controlled ventila- as naïve animals for cerebral cytokine expression and neurotion and anesthesia with sevoflurane 1.0–1.5 vol% were con- histopathologic control. tinued for 180 minutes after CA/CPR. Fio2 was reduced at fixed time points from 1.0 to baseline values (0.3) at the end Measurements of the experimental protocol. No additional vasoactive drugs MAP, ECG, heart rate (HR), lCBF, end-tidal sevoflurane conwere used in this experimental setup. After the observation centration (ETsevo), and tympanic and rectal temperatures period, all intravascular catheters were removed, the vessels were recorded continuously using the PowerLab data acquisiwere ligated, and incisions were closed. Anesthesia was distion system and LabChart 6.0 pro recording software (ADIncontinued and the animals were weaned from the ventilator. struments GmbH, Spechbach, Germany). lCBF measurements At adequate spontaneous respiration, the tube was removed. were recorded in blood perfusion units and expressed in percent of baseline values. CO was measured 10 minutes before CA (baseline), at 5, 30, 60, 90, 120, 150, and 180 minutes of the reperfusion period using the thermodilution technique (29) (Fig. 1). CO measurements were used for calculation of cardiac index (CI; CO/ body weight). CI was expressed in absolute values (mL/min/ kg) and, additionally, in percentage of baseline. Blood samples were taken (100 µL) for analysis from the right femoral artery 15 minutes before CA (baseline) and at 1, 20, 70, 120, and 180 minutes after CA/ CPR (Fig. 1). Arterial blood Figure 2. Flow diagram of the experimental groups. ROSC = return of spontaneous circulation. e412
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gases, electrolytes, pH, base excess, hematocrit, hemoglobin, and blood glucose and lactate concentrations were measured (ABL 800 Basic, Radiometer GmbH, Willich, Germany). Neurological Assessment Score Twenty-four hours after CA/CPR, animals underwent behavioral testing using a neurologic assessment score adapted for this experiment (31–33). Testing was performed in a quiet room with dimmed light by one investigator, who was unaware of randomization. Food and water intake, consciousness, breathing pattern, vibrissae movement, motoric function, and interaction with the environment were scored (13 = normal performance, no deficit; 44 = most severe deficit). Blood Samples and Neurohistopathologic Evaluation After behavioral and neurologic evaluation, the animals were deeply anesthetized with sevoflurane 5 vol% and tracheal intubated. After median sterno-laparotomy, the descending aorta was catheterized, and blood samples were taken for preparing blood plasma. After incision of the right atrium, animals were briefly perfused via the descending aorta with cold saline 0.9%. Brains were carefully removed, cryofixated in liquid nitrogen, and stored at –20°C. Coronal sections (10 µm) were prepared and stained with Fluoro-Jade B (Histo-Chem, Jefferson, AR), a fluorochrome marker for degenerating neurons (34, 35). Fluoro-Jade B–positive neurons were counted by an investigator blinded to the group allocation. The entire cornu ammonis (CA 1–4) of the hippocampal region and the CA1 region (magnification 400-fold), defined at interaural 5.7 mm and bregma –3.3 mm, were evaluated (36). Three consecutive sections were analyzed in both hemispheres, and the total mean of positive neurons was calculated.
measurements such as CI, MAP, HR, lCBF, ETsevo, physiologic variables, and tympanic and rectal temperatures within the groups were conducted with two-way repeated measurement analysis of variance (factors treatment and time) and Bonferroni posttests. Unpaired t tests were used to compare animals subjected to CA with and without levosimendan with respect to neuronal injury (Fluoro-Jade B staining), cerebral cytokine expression, CA duration, and neurological assessment score. Plasma IL-6 analysis was evaluated with Wilcoxon test. For comparison of descriptive results, shamoperated and naïve animals are displayed beside the results of CA animals. All tests were two-sided with a significance level of α equals 0.05. Statistical analysis was realized with GraphPad Prism Version 5.03 (GraphPad Software, La Jolla, CA). Data are presented as mean ± sd.
RESULTS A total of 61 animals were investigated, 43 animals randomized to the CA groups, 10 animals in the sham groups, and 5 animals in the Naive group. Three animals were excluded during the preparation period due to vessel rupture. A total of 13 animals did not achieve ROSC within 3 minutes (Levo n = 7, Vehicle n = 6) and were replaced accordingly (30%). Cardiac Arrest Duration of CA was comparable between the Levo and Vehicle groups. Total arrest time was 444 ± 36 seconds for the Levo group and 451 ± 39 seconds for the Vehicle group.
Blood Plasma Interleukin-6 Cytokine Analysis For determination of interleukin (IL)-6, the Quantikine ELISA Rat IL-6 Immunoassay Kit (R&D Systems GmbH, Wiesbaden, Germany) was used according to the manufacturer’s instructions. Plasma IL-6 cytokine values were expressed in pg/mL.
Physiological Values, End-Tidal Sevoflurane Concentration, and Temperature At baseline, all measured physiological values were within physiological ranges (Table 1). Most of the variables were affected by CA, except blood glucose and sodium concentration (data not shown). Differences were detected between the CA groups regarding K+ and lactate concentration. Blood analysis after the first minute of reperfusion showed lower K+ concentrations in the Levo group (6.5 ± 1.2 mmol/L) compared with the Vehicle group (7.3 ± 1 mmol/L, p < 0.01). Measurement of lactate concentration detected differences between the CA groups 20 minutes after reperfusion (Levo 2.8 ± 0.7 vs Vehicle 3.8 ± 0.6, p < 0.001), whereas for sham animals no differences were detected (Table 1). Measurement of ETsevo (data not shown) demonstrated no differences in anesthesia between CA groups and between sham groups. Tympanic and rectal temperatures were in physiologic ranges and did not differ between the two CA and sham groups throughout the observation period (data for tympanic temperature are given in Table 1).
Statistical Analyses Twelve to seventeen animals per group are necessary in order to demonstrate a decrease in affected neuronal cells (neuronal injury) from 35% to 10% with a power of 80% at the 5% level and sd between 20% and 25%. Sample size calculation was conducted using nQuery 5.0 Software (Statistical Solutions, Cork, Ireland). Exploratory comparisons between time-based
Cardiac Index At baseline, calculated CI values were 421 ± 42 mL/min/ kg in the Levo group and 429 ± 47 mL/min/kg in the Vehicle group (Fig. 3A). Five minutes after CA/CPR, CI decreased to 84% ± 11% of baseline values in the Vehicle group (Fig. 3B). Constant values compared to baseline were detected at the same time point in animals treated with levosimendan
Cerebral Cytokine Gene Expression Analysis For gene expression analysis, coronal sectioned tissue samples were collected. Interaural between 6.8 mm and 5.7 mm and between bregma –2.12 mm and –3.3 mm tissue of the entire hippocampal region and cortical tissue (gray and white matter) were separated and stored in tubes at –80°C. Gene expression analysis was performed with reverse transcriptase polymerase chain reaction as previously described (37, 38).
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Table 1.
Group
Levo
Vehicle
Physiologic Variables and Tympanic Temperature Time (min)
sVehicle
Pao2 (mm Hg)
Lactate (mmol/L)
Glucose (mg/dL)
K+ (mmol/L)
Ca2+ (mmol/L)
Tympanic Temperature (°C)
Baseline
7.431 ± 0.020 40.4 ± 2.4 131.0 ± 12.1 0.78 ± 0.08 137.3 ± 13.7 3.49 ± 0.28 1.33 ± 0.04
37.1 ± 0.1
1
7.165 ± 0.018 50.5 ± 3.7 302.9 ± 33.6 7.44 ± 1.10 159.1 ± 41.1 6.54 ± 1.20 1.30 ± 0.07
36.0 ± 0.3
20
7.261 ± 0.034 48.3 ± 3.6 240.7 ± 49.7 2.81 ± 0.74 158.1 ± 31.9 2.94 ± 0.23 1.17 ± 0.08
37.0 ± 0.1
70
7.394 ± 0.029 40.6 ± 2.9 202.0 ± 38.9 1.11 ± 0.49 138.2 ± 25.8 4.01 ± 0.34 1.19 ± 0.07
37.0 ± 0.0
120
7.398 ± 0.025 40.8 ± 1.1 113.9 ± 11.8 0.89 ± 0.15 133.4 ± 17.9 3.60 ± 0.29 1.25 ± 0.07
37.0 ± 0.0
180
7.401 ± 0.022 40.2 ± 1.3 118.1 ± 16.1 0.77 ± 0.10 135.1 ± 15.2 3.29 ± 0.82 1.28 ± 0.05
37.0 ± 0.0
Baseline
7.424 ± 0.015 40.2 ± 1.7 131.5 ± 14.9 0.91 ± 0.12 139.3 ± 10.1 3.33 ± 0.27 1.32 ± 0.06
37.1 ± 0.1
7.170 ± 0.031 47.4 ± 4.5 300.9 ± 36.5 7.36 ± 0.65 128.4 ± 36.0 7.25 ± 0.99
1.28 ± 0.06
36.0 ± 0.2
20
7.242 ± 0.026 47.5 ± 3.0 247.6 ± 35.2 3.80 ± 0.59 144.7 ± 20.0 2.91 ± 0.32 1.16 ± 0.05
37.0 ± 0.1
70
7.400 ± 0.020 39.7 ± 2.3 200.6 ± 44.7 1.26 ± 0.45 147.6 ± 11.2 4.01 ± 0.39 1.19 ± 0.06
37.0 ± 0.1
120
7.406 ± 0.017 40.5 ± 1.4 117.1 ± 11.6 1.07 ± 0.35 146.1 ± 11.4 3.65 ± 0.28 1.28 ± 0.04
37.0 ± 0.0
180
7.406 ± 0.015 39.9 ± 1.4 120.2 ± 13.5 0.88 ± 0.14 144.1 ± 11.6 3.49 ± 0.27 1.29 ± 0.04
37.0 ± 0.1
Baseline
7.410 ± 0.010 41.0 ± 1.3 116.6 ± 13.3 0.82 ± 0.11 141.6 ± 10.6 3.68 ± 0.30 1.31 ± 0.06
37.0 ± 0.1
1
7.408 ± 0.020 40.5 ± 2.8 334.0 ± 80.3 0.78 ± 0.08 130.4 ± 13.5 3.50 ± 0.25 1.29 ± 0.05
36.9 ± 0.1
20
7.404 ± 0.021 41.7 ± 2.4 306.6 ± 73.3 0.70 ± 0.07 134.6 ± 13.5 3.44 ± 0.18 1.30 ± 0.05
37.0 ± 0.0
70
7.418 ± 0.004 41.3 ± 1.9 211.0 ± 50.6 0.70 ± 0.10 134.0 ± 12.1 3.34 ± 0.21 1.28 ± 0.04
37.0 ± 0.1
120
7.418 ± 0.026 41.9 ± 2.7 114.0 ± 14.9 0.86 ± 0.15 131.2 ± 11.7 3.32 ± 0.15 1.29 ± 0.06
37.0 ± 0.0
180
7.424 ± 0.018 41.0 ± 0.5 117.4 ± 17.4 0.82 ± 0.11 129.6 ± 8.0
3.36 ± 0.09 1.30 ± 0.04
37.0 ± 0.0
Baseline
7.430 ± 0.019 41.3 ± 3.2 126.4 ± 19.1 0.82 ± 0.08 147.8 ± 14.6 3.52 ± 0.47 1.33 ± 0.03
37.1 ± 0.1
1
7.438 ± 0.016 39.6 ± 2.6 371.0 ± 35.2 0.74 ± 0.09 138.8 ± 9.0
3.34 ± 0.46 1.31 ± 0.08
36.9 ± 0.1
20
7.434 ± 0.009 40.1 ± 1.8 353.4 ± 18.6 0.70 ± 0.07 136.2 ± 5.8
3.28 ± 0.36 1.30 ± 0.05
37.0 ± 0.0
70
7.440 ± 0.012 39.8 ± 1.8 248.0 ± 20.8 0.76 ± 0.09 138.4 ± 9.9
3.30 ± 0.24 1.30 ± 0.02
37.0 ± 0.0
120
7.450 ± 0.007 38.5 ± 1.7 131.2 ± 13.0 0.86 ± 0.15 132.2 ± 12.4 3.22 ± 0.18 1.27 ± 0.03
37.0 ± 0.1
180
7.446 ± 0.011 39.7 ± 1.5 132.6 ± 16.6 0.90 ± 0.16 133.4 ± 17.3 3.14 ± 0.18 1.27 ± 0.02
37.0 ± 0.0
1
sLevo
pH
Paco2 (mm Hg)
a
b
Bonferroni posttests: Levo versus Vehicle at the corresponding time: ap < 0.01, bp < 0.001. Mean ± sd.
(Levo 101% ± 10%, p < 0.001 Bonferroni posttest). Over time, a significant decrease of CI was detected in both CA groups (p < 0.0001). However, CI was higher with levosimendan compared with the vehicle group during the reperfusion period (Levo vs Vehicle absolute values: p = 0.0141; relative values: p < 0.0001). After 180 minutes of reperfusion, CI was reduced by 14% to baseline values in the Levo group and by 28% in the Vehicle group (Levo vs Vehicle: p < 0.001 Bonferroni posttest). Comparison of sham groups showed higher values in the sLevo group during treatment (sLevo vs sVehicle absolute values: p = 0.0122; relative values: p = 0.0008) (Fig. 3, A and B). Local Cerebral Blood Flow and Hemodynamic Measurements Measurement of lCBF during baseline and CA/CPR showed no differences between groups. Early after ROSC, a postischemic hyperperfusion period was detected in both CA/CPR e414
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groups. Maximum values after 9 minutes of reperfusion were measured at 297% ± 46% in the Levo group and 287% ± 51% in the Vehicle group (Fig. 4A). A hypoperfusion period of 20 minutes after CA/CPR was observed in both CA groups. However, postischemic hypoperfusion was more pronounced in the Vehicle group (Levo vs Vehicle: p = 0.0013). After 180 minutes of reperfusion, values in the Levo group exceeded baseline values (Levo 126% ± 25%) compared with Vehicle group (Vehicle 88% ± 17%, p < 0.05 Bonferroni posttest). Hemodynamic variables such as MAP and HR (data not shown) remained in physiological ranges during baseline. After ROSC, a period of increased MAP was detected in both CA groups. Maximum values were measured after 8 minutes of reperfusion (Levo 167 ± 13 mm Hg, Vehicle 166 ± 20 mm Hg) (Fig. 4B). In the consecutive observation period of 180 minutes, MAP was lower in the Levo group compared with Vehicle group (p = 0.0002). HR (data not shown) measurement identified a difference between June 2014 • Volume 42 • Number 6
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Neurohistopathology A lower number of positive cells in animals treated with levosimendan (Levo vs Vehicle: 997 ± 103 vs 1,113 ± 137 Fluoro-Jade B–positive cells, p = 0.0052) (Fig. 6A) was detected in the entire cornu ammonis of the hippocampal formation. Similarly, the neuronal injury in the CA 1 region was reduced in the Levo group (Levo vs Vehicle: 352 ± 30 vs 396 ± 47 Fluoro-Jade B–positive cells, p = 0.0098) (Fig. 6B). Cerebral Cytokine Gene Expression Analysis of IL-6, Tumor Necrosis Factor-α, and IL-1β No differences in all cytokine gene expressions were detected between CA groups (data not shown). Blood Plasma IL-6 Cytokine Analysis No differences in plasma IL-6 concentration were detected between CA groups (Levo 74.01 ± 43.00 pg/mL, Vehicle 68.72 ± 62.69 pg/mL).
DISCUSSION The present study demonstrated that levosimendan increases lCBF during hypoperfusion after experimental Figure 3. A, Cardiac index (CI) in mL/min/kg; Levo versus Vehicle: *p = 0.0141; sLevo versus sVehicle: CA/CPR, although diminished # p = 0.0122. B, CI in percent of baseline; Levo versus Vehicle: ***p < 0.0001; sLevo versus sVehicle: **p = 0.0008. Bonferroni posttests data not shown. Mean ± sd. CPR = cardiopulmonary resuscitation. MAP values were detected in animals treated with levosiLevo and Vehicle groups, with higher values in the Levo group mendan. Furthermore, levosimendan treatment reduced neu(p = 0.0459). Comparison of MAP and HR (data not shown) ronal injury and improved neurological performance after 24 between the sham groups detected no differences. hours following CA/CPR. There were no inhibitory effects of levosimendan on cerebral or systemic inflammatory response. Survival We confirmed previous results of improved cardiac function Two animals did not survive the observation period of 24 in the reperfusion period after CA/CPR due to levosimendan hours in each of the CA groups. All animals of the sham groups treatment. survived. Positive inotropic action due to Ca2+-sensitizing and phosphodiesterase-inhibitory effects in cardiac myofilaments, Neurological Assessment Score increased HR, and activation of ATP-sensitive K+-channels of Twenty-four hours after CA/CPR, the behavioral and neuro- smooth vessel muscle cells for vasodilatation are the mechalogical evaluation showed a better performance of animals nisms of levosimendan (39). No data exist about cerebral treated with levosimendan compared with Vehicle group (Levo vasodilating action due to levosimendan, but previous studvs Vehicle: 18.7 ± 2.2 vs 21.2 ± 2.8, p = 0.0208) (Fig. 5). Sham ies showed systemic and coronary vasodilatatory effects (18, animals were not affected by experimental procedures. 25). Furthermore, ATP-sensitive K+-channels with vasodilating Critical Care Medicine
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after successful CPR, that is, no-reflow or low-reflow occurs, followed by temporary hyperperfusion and longer lasting transient hypoperfusion (43). In the present study, lCBF was measured in areas of the somatosensory cortex. Therefore, the plausibility of lCBF measurements is confirmed due to the characteristic course of pathological cerebral perfusion after global ischemia. Using laser-Doppler flowmetry, a well-validated and accepted method to measure changes in cerebral perfusion over time (44), in the present study, a higher lCBF and improved microcirculation during the hypoperfusion period of reperfusion were detected, although MAP was decreased in animals treated with levosimendan. These findings are in conflict with the idea of a disturbed autoregulation of cerebral blood flow after global cerebral ischemia due to CA, which would consequently result in impaired lCBF. An absent or right-sided shift of autoregulation was previously described in the acute phase after CA, and for this reason, higher levels of MAP in the reperfusion period were recommended (45). Furthermore, after experimental CA/CPR in dogs, an enormous increase of MAP was Figure 4. A, Local cerebral blood flow (lCBF) in the somatosensory cortex expressed as relative perfusion (%) to baseline values; Levo versus Vehicle: **p = 0.0013; sLevo versus sVehicle: not significant. B, Mean associated with a better neuroarterial blood pressure (MAP) expressed in mm Hg during the experimental procedure, Levo versus Vehicle: logical outcome (46). In part, ***p = 0.0002; sLevo versus sVehicle: not significant. Bonferroni posttests data not shown. Mean ± sd. the findings of the present CPR = cardiopulmonary resuscitation. study could be associated with effects after activation were verified in smooth muscle and increased CI and vasodilating effects of levosimendan, leading endothelial cells of brain vessels (40–42). Therefore, this vaso- to an enhanced tissue microcirculation in the absence of an dilating effect of levosimendan, as an activator of ATP-sensitive increased blood pressure. In general, comparing experimental K+-channels, possibly counteract to the pathological vasocon- results from different CA models should be made with care to striction in the hypoperfusion period after global cerebral ischdifferences in terms of generated CA (e.g., ventricular fibrillaemia by CA. tion) and animal species. In the present study, levosimendan treatment improved Myocardial dysfunction was detected in both CA groups in lCBF, especially during transient hypoperfusion after CA/CPR. the reperfusion period in the present study, using the transpulIt is generally accepted that after global ischemia, CBF shows monary thermodilution technique for CI measurement. This characteristically pathological changes over time. Immediately is in agreement with previous publications (8). Levosimendan e416
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Figure 5. Neurological assessment score (13 = normal performance, no deficit; 44 = most severe deficit) 24 hr after cardiac arrest and cardiopulmonary resuscitation; Levo versus Vehicle: *p = 0.0208. Mean ± sd.
ameliorated cardiac function compared with vehicle-treated animals after CA/CPR. Neuronal injury was reduced in animals treated with levosimendan. The number of Fluoro-Jade B–positive cells, a staining method for degenerating neurons, was decreased in the hippocampal formation, precisely the entire cornu ammonis and a specific sector (CA 1), known to be highly sensitive to ischemic conditions. After global cerebral ischemia, it could be of great significance to improve microcirculation in these sensitive brain regions. The fact that levosimendan improved perfusion via increased CI and vasodilatation could contribute to reduced neuronal injury in the hippocampal formation. In an in vitro model of traumatic brain injury, levosimendan showed direct neuroprotective properties (47). However, findings in dogs indicated that levosimendan does not substantially penetrate the bloodbrain barrier in the healthy brain (48). A recent study using a bilateral common artery clamping and hypoxic ventilation for induction of cerebral ischemia indicated that only a low brain tissue concentration of levosimendan compared to serum concentrations can be achieved (49). No effects of levosimendan treatment on time course of lactate, glucose, pyruvate, and glutamate concentrations after transient ischemia were shown. It seems unlikely that direct neuroprotective effects of levosimendan contributed to our results. Neuronal injury was reduced, and neurological performance 24 hours after resuscitation was improved in animals treated with levosimendan. Compared with sham-operated animals, a severe neurological impairment was detected in animals after CA/CPR. Because of the total standstill of perfusion as a whole-body ischemia, CA models generate more severe neurological disorders compared with other models of cerebral ischemia, for example, four-vessel occlusion or focal cerebral ischemia models. Despite the severe and complex impairments in this model of CA/CPR, levosimendan treatment was able to show beneficial effects. Critical Care Medicine
Figure 6. Neuronal injury expressed as Fluoro-Jade B–positive cells 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Entire cornu ammonis (CA) of the hippocampal formation; Levo versus Vehicle: **p = 0.0052. B, CA 1 of the hippocampal formation; Levo versus Vehicle: **p = 0.0098. Mean ± sd.
Although immunological effects by levosimendan were described in several publications, no effects on neuroinflammation and systemic inflammatory response were detected in the present study. Immunological effects of levosimendan including reduced cytokine levels in patients with decompensated heart failure, reduced leukocyte reactive oxygen species release in patients with acute heart failure and septic shock, and lower pulmonary IL-6 levels in a porcine model of septic shock have been described (27, 28, 50–52). It seems likely that levosimendan did not penetrate the blood-brain barrier in sufficient quantity and, therefore, was not able to exert anti-inflammatory properties in the brain tissue in this study. However, it remains unclear why general systemic inflammatory response after CA/ CPR was not influenced by levosimendan.
CONCLUSIONS The present study demonstrated the neuroprotective effect of levosimendan by reducing myocardial dysfunction and improving cortical cerebral blood flow after experimental asphyctic CA and CPR. Recently described anti-inflammatory www.ccmjournal.org
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effects of levosimendan are unlikely to contribute to effects observed in this study. Levosimendan seems to be a potential drug in the treatment of the post cardiac arrest syndrome due to its positive effect on CI, cerebral blood flow, and neuronal injury.
ACKNOWLEDGMENTS We thank Frida Kornes, Dana Pieter, and Magdeleine Herkt for their excellent technical assistance. Data presented in this article are part of a doctoral thesis by Jürgen Wagenführer to the Medical Faculty of the Johannes Gutenberg-University, Mainz, Germany.
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