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Effects of intravenous and inhaled levosimendan in severe rodent sepsis

Intensive Care Medicine volume 35, pages 1412–1419 (2009)Cite this article

Abstract

Purpose

We aimed at comparing the effects of intravenous (i.v.) and inhaled (inh.) levosimendan (LEVO) on survival, inflammatory cytokines and the apoptotic mediator caspase-3 in a rat model of severe sepsis induced by cecal ligation and incision (CLI).

Methods

Twenty-eight anesthetized/ventilated male Sprague–Dawley rats (body weight 528 ± 20 g) underwent laparotomy. Cecal mobilisation served as control (SHAM, n = 7). In all other groups, severe sepsis was induced by CLI. No further intervention occurred in the CLI-group (n = 7). 180 min after CLI, 24 μg/kg i.v. LEVO was administered in the CLI + LEVO-IV-group (n = 7), and 24 μg/kg inh. LEVO was administered via jet nebulizer in the CLI + LEVO-INH-group (n = 7).

Results

CLI induced arterial hypotension, with i.v. and inh. LEVO attenuating blood pressure decrease over 390 min [CLI 34(31/50), CLI + LEVO-IV 82(69/131)*, CLI + LEVO-INH 78(62/85)* mmHg; median(25/75% quartile), *P < 0.05]. CLI induced metabolic acidosis. I.v. and inh. LEVO avoided arterial pH [CLI 7.18(7.16/7.2), CLI + LEVO-IV 7.27(7.24/7.31)*, CLI + LEVO-INH 7.26(7.24/7.28)*] and base excess deterioration [CLI −19(−21.8/−17.9), CLI + LEVO-IV −13(−14.8/−12)*, CLI + LEVO-INH −12.7(−14/−12.2)* mmol/l]. Overall mortality in the CLI-group was 57% compared to 0%* in both LEVO-treated groups after 390 min. LEVO administration significantly attenuated the increase in proinflammatory interleukin (IL)-1β [CLI 896(739/911), CLI + LEVO-IV 302(230/385)*, CLI + LEVO-INH 346(271/548) pg/ml] and IL-6 [CLI 35651(31413/35816), CLI + LEVO-IV 21156(18397/28026), CLI + LEVO-INH 13674(10105/24843) pg/ml] in the plasma and reduced cleaved caspase-3 expression in the spleen.

Conclusions

In a rat model of severe sepsis induced by CLI, i.v. and inh. LEVO equally attenuated arterial hypotension, metabolic acidosis and prolonged survival. Moreover, i.v. and inh. LEVO inhibited proinflammatory mediator release and reduced splenic caspase-3 expression.

Introduction

Sepsis is a leading cause of death among critically ill patients despite significant progress in intensive care therapy [1]. Severe sepsis is characterized by organ dysfunction and systemic inflammation resulting from an infectious origin [2]. Arterial hypotension despite adequate fluid resuscitation is a major problem during the course of severe sepsis. For this reason, the use of vasopressors is recommended [3]. Levosimendan (LEVO) is not a vasopressor, but a fairly new calcium sensitizer with positive inotropic and vasodilating properties [4]. In contrast to standard inotropic agents, LEVO does not increase myocardial oxygen consumption or induce ventricular arrhythmias [5]. Yet, LEVO failed to prove superior to standard vasopressor therapy in acute decompensated heart failure [6]. However, LEVO may be an alternative in the septic patient with myocardial dysfunction when standard vasopressor therapy is no longer efficacious. Fries et al. [7] showed that LEVO and norepinephrine were equally effective in restoring cardiac output in experimental sepsis, but only LEVO improved microvascular oxygenation. In endotoxemia, LEVO was shown to improve left ventricular contractility [8] and to increase intestinal blood flow [9]. While a clinical study has already shown the beneficial effects of LEVO on hemodynamics in the context of inflammation [10], data on proinflammatory cytokine release and apoptosis during severe sepsis after LEVO administration are still limited.

Apoptosis, or programmed cell death, is the physiological process of eliminating abnormal cells within multicellular organisms [11]. Enhanced lymphocyte apoptosis has been identified as a cornerstone in sepsis pathophysiology, thus creating a state of immunosuppression [12]. The apoptotic signalling pathways involve the recruitment of several cysteine proteases, caspases, via cleavage which ultimately converge on the activation of caspase-3 [11]. Cleaved caspase-3 is the central apoptotic mediator and responsible for cellular degradation. To date, LEVO has been shown only to reduce myocardial apoptosis in hypertensive rats [13]; It remains unclear whether LEVO has any influence on caspase-3 activation in lymphatic tissue in severe sepsis.

LEVO is most commonly used intravenously (i.v.), but its prophylactic use via inhalation (inh.) was recently shown to improve survival and to attenuate inflammation in acute lung injury in rats [14]. In contrast, the administration of 50 μg/kg/h LEVO 100 min after the beginning of porcine endotoxemia had detrimental effects [15]. As a result, the time point and the route of administration as well as the dose of LEVO seem to play an important role. Therefore, we set out to compare the effects of i.v. and inh. LEVO––given 180 min after the induction of severe sepsis––on survival, hemodynamics, plasma levels of inflammatory cytokines and caspase-3 expression in lymphatic tissue in our recently described acute sepsis model [16].

Materials and methods

Animals and anesthesia

All animal experiments in this prospective randomized study were approved by the governmental board for the care of animal subjects (Regierungspräsidium Darmstadt, Germany) and were in accordance with the National Institute of Health guidelines (National Academy of Sciences, Washington DC, 1996). 28 male Sprague–Dawley rats (Harlan–Winkelmann, Borchen, Germany, body weight 528 ± 20 g, mean ± standard deviation) were kept on a 12 h light/dark cycle with free access to food and water. Rats were anaesthetized by intraperitoneal (i.p.) injection of pentobarbital (10 mg/kg body weight, Narcoren, Halbergmoos, Germany) and fentanyl (0.05 mg/kg, Janssen–Cilag, Neuss, Germany). Unconscious rats were tested for sufficient depth of anaesthesia by tail clamping, weighed and then placed supine on a heating pad. A tracheotomy was performed and a 13-G cannula (ID 2.0 mm, OD 2.5 mm, Abbott, Wiesbaden, Germany) was inserted endotracheally. Subsequently, rats were ventilated with a neonatal ventilator (Stephanie, Stephan, Gackenbach, Germany) using pressure-controlled ventilation: inspiratory oxygen fraction 0.21, T I/E 1:2, P max 20 cm H2O, PEEP 4 cm H2O, respiratory rate 31/min [17]. Respirator adjustments were not to be changed throughout the observation time. Arterial blood gas analyses (BGA) were obtained through a fluid-filled polyurethane catheter (ID 0.58 mm, OD 0.96 mm, SIMS Portex, Hythe, UK) in the right femoral artery. Subsequently, BGA were analysed for arterial pH, base excess (BE), and PaO2 (ABL500, Radiometer, Willich, Germany). In addition, the arterial catheter was connected to a monitor system (Sirecust, Siemens, Erlangen, Germany) for continuous recording of mean arterial blood pressure (MAP) and heart rate (HR). A similar polyurethane catheter was inserted into the right femoral vein for continuous i.v. infusion of 0.9% NaCl (12 ml/kg/h, B. Braun, Melsungen, Germany, as described elsewhere [18]), pentobarbital (0.6 mg/kg/h) and fentanyl (0.03 mg/kg/h). Body temperature was monitored by a rectally inserted probe.

Surgical procedure and experimental protocol

Rats were randomly assigned to four groups of n = 7 rats each before any experimental procedure was started. After establishment of sufficient anaesthesia and arterial baseline BGA, a 2 cm long midline laparotomy was performed in all rats. The cecum was carefully exteriorized in all animals by means of cotton sticks which had been moistened in 0.9% NaCl solution before. In the control group, the cecum was replaced into the abdomen after gentle manipulation (SHAM). In the three other groups, acute severe sepsis was established by CLI as described in detail elsewhere [16]. Briefly, the cecum and the mesenteric blood vessels were ligated below the ileocecal valve to exclude bowel obstruction. Subsequently, the ligated cecum was opened through a 1.5 cm blade incision on the anti-mesenteric side. The cecum was then carefully replaced into the abdomen. All surgical procedures were performed by the same investigator in order to minimize variability. In all groups, 2 ml/kg of 0.9% NaCl solution was given i.p. as fluid resuscitation before the abdominal wall was closed in two layers. MAP, HR, arterial BE and pH were measured hourly for 390 min. At 180 min after the surgical procedure, 0.5 ml of 0.9% NaCl was given i.v. in the SHAM-group and in the CLI-group. At the same time, 0.9% NaCl aerosol was administered to the animals of these two groups via a special jet nebulizer for use in rodents [19]. Briefly, this device produces particles with a median aerodynamic size distribution of 2.0 μm (geometric standard deviation 1.6) and an intrapulmonary deposition fraction of 3.8 ± 1.3% [19]. In the CLI + LEVO-IV-group, 0.9% NaCl aerosol was administered via the jet nebulizer, and 24 μg/kg i.v. LEVO (Simdax, Orion Pharma, Espoo, Finland) was given. In the CLI + LEVO-INH-group, 0.5 ml of 0.9% NaCl was given i.v., and 24 μg/kg intrapulmonary LEVO was administered via jet nebulizer. All animals that had survived the entire observation time were exsanguinated and heparinized whole blood samples (Heparin–Natrium, Ratiopharm, Ulm, Germany) were obtained. For protein analysis, the spleens of all animals that had survived the entire observation time were rinsed with ice-cold phosphate buffered saline (PBS, Invitrogen, Karlsruhe, Germany) and immediately stored at −80°C for further analysis.

Plasma samples, ELISA assay, and Western blotting

Plasma was separated from blood cells by centrifugation at 1,700 rpm for 10 min and stored at −80°C for further analysis. Plasma levels of interleukin (IL-)1β and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Wiesbaden, Germany) according to manufacturer’s instructions. Frozen spleens were lysated in buffer containing 1% Triton X-100, 25 mM Tris/HCl (pH = 7.4), 150 mM NaCl, 1 mM CaCl2 and protease inhibitors (Roche Diagnostics, Mannheim, Germany) DTT (1 mM), NaF (20 mM), Na3VaO4 (1 mM), PMSF (1 mM) as described before [20]. Protein content was quantified using the Bradford assay. After sodium dodecyl sulphate gel (15%) electrophoresis and 90 min semi-dry electroblotting of 100 μg spleen lysate, nitrocellulose membranes were blocked for 1 h at room temperature (RT) with 10% skim milk/Tris buffered saline with Tween (TBST, Merck, Darmstadt, Germany). Membranes were then incubated with rabbit anti-cleaved-caspase-3-antibody (#9661, Cell Signaling, Beverly, MA, USA, 1:1,000 in 5% bovine serum albumin/TBST, AppliChem, Darmstadt, Germany) and with mouse anti-β-Actin-antibody (#A5441, Sigma, Deisenhofen, Germany, 1:10,000 in 10% skim milk/TBST) at 4°C overnight. Membranes were incubated with goat secondary antibodies against rabbit (170–6515 Biorad, Hercules, CA, USA) or mouse (170–6516, Biorad, Hercules, CA, USA), respectively (both 1:10,000 in 2.5% skim milk/TBST), for 1 h at RT before incubation with chemiluminescence solution (ECL+, Amersham Biosciences, Buckinghamshire, United Kingdom) for 60 s. Membranes were then exposed to films (Hyperfilm ECL, GE Healthcare, Little Chalfont, United Kingdom).

Statistics

Statistical analysis between the groups CLI, CLI + LEVO-IV and CLI + LEVO-INH was performed with SigmaStat 3.1 (Systat Software, San Jose, CA, USA). Data in the table are expressed as median (25% quartile/75% quartile). Differences were compared using Kruskal–Wallis analysis of variance (ANOVA) on ranks with Dunn’s post-hoc test of all pairwise multiple comparison procedures. Additionally, the groups CLI, CLI + LEVO-IV and CLI + LEVO-INH were tested for statistical differences in MAP, HR, arterial BE and pH by repeated measurements using ANOVA (Friedman-ANOVA) with Dunn’s post-hoc test of all pairwise multiple comparison procedures in order to ensure that these three groups developed similarly until the start of LEVO treatment in the CLI + LEVO-IV- and the CLI + LEVO-INH-groups at 180 min after the surgical procedure. In order to avoid any statistical bias, only animals having survived the entire observation time of 390 min were included in the statistical analyses. Figure 2 shows scatter plots indicating the median as a horizontal bar. Survival differences between the groups CLI, CLI + LEVO-IV and CLI + LEVO-INH were analysed using the Kaplan–Meier log-rank test with Bonferroni correction. Differences between the groups were considered significant at P < 0.05.

Results

Survival and hemodynamics

Within 390 min of observation time, overall mortality in the CLI-group was 57%. In the CLI + LEVO-IV-group and in the CLI + LEVO-INH-group, all animals survived the observation period (0% mortality in both LEVO-treated groups, Fig. 1).

Fig. 1
figure 1

Survival rate (%) of rats within 390 min after laparotomy (SHAM, n = 7, solid line), additional cecal ligation and incision (CLI, n = 7, short dash line), CLI with 24 μg/kg body weight levosimendan i.v. (CLI + LEVO-IV, n = 7, long dash line) or CLI with 24 μg/kg inhalative levosimendan (CLI + LEVO-INH, n = 7, medium dash line). Kaplan–Meier log-rank test with Bonferroni correction; * P < 0.05 versus CLI

Full size image

Starting at baseline until 150 min after sepsis induction, all groups did not differ in MAP, HR, BE and pH.

Within the observation period, the CLI-group suffered from a progressive decline of MAP which resulted in severe arterial hypotension. Although MAP also declined progressively in both treatment groups, administration of LEVO significantly attenuated arterial hypotension (Table 1). First significant changes in MAP between the CLI- and both LEVO-treated groups were observed 270 min after sepsis induction (Table 1). No significant changes in MAP between both LEVO-treated groups were detected. Throughout the entire observation time, no significant changes in HR between the three groups were observed (Table 1).

Table 1 Systemic hemodynamic and blood gas analysis parameters
Full size table

The CLI-group suffered from a progressive decline of arterial pH and arterial BE which resulted in severe metabolic acidosis. Although arterial pH and BE also declined progressively in both treatment groups, administration of LEVO significantly attenuated metabolic acidosis (Table 1). While significant changes in arterial pH between the CLI-group and the CLI + LEVO-IV-group were observed as early as 270 min after sepsis induction, first significant changes in arterial pH between the CLI- and the CLI + LEVO-INH-group were observed 330 min after sepsis induction (Table 1). We could not detect any significant changes in arterial pH between both LEVO-treated groups. First significant changes in arterial BE between the CLI-group and the two LEVO-treated groups were observed as early as 210 min after the surgical procedure (Table 1). There was no significant difference in arterial BE between the CLI + LEVO-IV- and the CLI + LEVO-INH-group.

No significant differences in body temperature, tidal volume and additional blood gas parameters (PaO2, PaCO2) were observed between the three groups (data not shown).

Cytokine plasma levels

CLI increased plasma levels of both IL-1β [896(739/911) pg/ml] and IL-6 [35651(31413/35816) pg/ml], respectively (Fig. 2). In the CLI + LEVO-IV-group, plasma IL-1β levels [302(230/385) pg/ml] were significantly reduced compared to CLI. IL-6 plasma levels [21156(18397/28026) pg/ml] were also markedly reduced compared to CLI which did not reach statistical significance (Fig. 2). In the CLI + LEVO-INH-group, plasma IL-6 levels [13674(10105/24843) pg/ml] were significantly reduced compared to CLI. IL-1β plasma levels [346(271/548) pg/ml] were also markedly reduced compared to CLI which did not reach statistical significance (Fig. 2). There was no significant difference in plasma levels of either IL-1β or IL-6 between the CLI + LEVO-IV- and the CLI + LEVO-INH-group.

Fig. 2
figure 2

Scatter plot (median is indicated as horizontal bar) showing Interleukin (IL)-1β (a) and IL-6 (b) plasma levels (pg/ml) at 390 min after laparotomy (SHAM, boxes, n = 7), additional cecal ligation and incision (CLI, circles, n = 3), CLI and application of 24 μg/kg body weight levosimendan i.v. (CLI + LEVO-IV, triangles, n = 7), CLI and application of 24 μg/kg body weight inhalative levosimendan (CLI + LEVO-INH, diamonds, n = 7). Kruskal–Wallis analysis of variance on ranks with Dunn’s post-hoc test of all pairwise multiple comparison procedures; * P < 0.05 vs. CLI

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Cleaved caspase-3 expression

In spleen lysates of SHAM-animals, only small amounts of cleaved caspase-3 were detectable (Fig. 3, columns 1 + 2). Severe sepsis (CLI) resulted in a enhanced expression of cleaved caspase-3 (Fig. 3, columns 3 + 4). There was a reduction of cleaved caspase-3 expression in the CLI + LEVO-IV- and in the CLI + LEVO-INH-group compared to the CLI-group (Fig. 3, columns 5 + 6 and 7 + 8). There was no obvious difference in cleaved caspase-3 expression between the CLI + LEVO-IV- and the CLI + LEVO-INH-group.

Fig. 3
figure 3

Cleaved caspase-3 expression in rat splenic lysates; β-actin expression as control; two representative animals per group; SHAM laparotomy; CLI cecal ligation and incision; CLI + LEVO-IV cecal ligation and incision and application of 24 μg/kg body weight levosimendan i.v.; CLI + LEVO-INH cecal ligation and incision and application of 24 μg/kg body weight inhalative levosimendan

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Discussion

Our recently established rodent sepsis model of CLI is characterized by arterial hypotension and severe metabolic acidosis during a total observation time of 390 min [16]. During the course of this severe sepsis, the administration of i.v. or inh. LEVO conserves MAP and improves arterial pH and BE resulting in prolonged survival of the LEVO-treated animals. I.v. and inh. LEVO––administered 180 min after the sepsis-inducing surgical procedure––are capable of inhibiting the increase of proinflammatory cytokines IL-1β and IL-6 in the plasma of septic animals. In addition, both methods of LEVO administration reduce the expression of cleaved caspase-3 in the spleen.

LEVO belongs to the novel class of calcium sensitizers and is currently approved in Europe for the use in acute decompensated heart failure since LEVO increases cardiac output without deleterious effects on myocardial oxygen consumption [5]. Targeting persistent arterial hypotension with subsequent metabolic acidosis is crucial in the treatment of severe sepsis [3]. In this context, LEVO may represent an interesting drug since it shows positive inotropic and vasodilating properties by increasing myocardial sensitivity for calcium [21], by opening adenosine triphosphate-dependent potassium channels [22] and––in higher concentrations––by inhibiting phosphodiesterase-III [23]. The standard method of LEVO administration remains the i.v. route. Yet, the present study shows, for the first time, beneficial effects of nebulized LEVO in experimental sepsis. By comparing i.v. to inh. LEVO, we have shown that both routes of administration are equally effective in prolonging survival and in attenuating the decrease in arterial blood pressure without obvious deleterious side effects. While i.v. LEVO shows a tendency to preserve MAP and to inhibit the increase in plasma levels of proinflammatory IL-1β more efficiently than inh. LEVO, no statistically significant difference between both LEVO-treated groups was observed in our study. Although the bolus injection of LEVO has been abandoned in many intensive care units in favour of the continuous i.v. infusion [24], an interesting observation in the present study is the fact that apparently a single dose of either i.v. or inh. LEVO has the capability of prolonging survival for more than three hours after the onset of severe sepsis. The elimination half-life of LEVO is approximately one hour. Yet, the active LEVO metabolite OR-1896 has a half-life of 70–80 h [25]. Although we are not aware of any data showing OR-1896 detection at 390 min past LEVO infusion/inhalation, we may hypothesize that OR-1896 could potentially account for the effectiveness of LEVO as observed in our study. Unfortunately, we were not able to analyse plasma levels for effective concentrations of this compound after both methods of administration. However, the data show that an intrapulmonary deposit of LEVO can prove as effective as plasma bound LEVO. Therefore, we may speculate that administration via aerosol will indeed create considerable plasma levels of LEVO. In the present study, the administration of i.v. or inh. LEVO attenuated arterial hypotension and avoided deterioration of metabolic acidosis, two key features of severe sepsis [26]. LEVO is known for its positive inotropic and vasodilating properties. Vasodilation is a frequent adverse event after bolus administration of LEVO and may possibly aggravate organ dysfunction [24]. However, the attenuation of both arterial hypotension and metabolic acidosis after LEVO administration shows that the bolus administration in the particular context of CLI-induced severe sepsis does not cause obvious deleterious side effects. This could be due to the fact that the positive inotropic functions of LEVO may outweigh the vasodilating component of the pharmacologic profile of LEVO. We must acknowledge, though, that there is still considerable hypoperfusion despite LEVO administration since both treatment groups feature near normal arterial pH and quite low BE which usually results from lactate production associated with hypoperfusion.

During sepsis, maintaining adequate MAP is crucial [27]. Recent clinical findings [28] support the current guidelines endorsing a MAP >65 mmHg in severe sepsis [3]. In accordance with these data, we also show in our short term sepsis model that a higher MAP after LEVO administration prolongs survival.

We present evidence that elevated plasma levels of IL-1β and IL-6 were effectively inhibited by i.v. and inh. LEVO. Data on LEVO action on proinflammatory cytokine levels in the context of experimental sepsis or endotoxemia are still limited [29]. Due to the lack of a comparative positive inotropic agent (e.g. dobutamine) in our study, we cannot conclude that LEVO has indeed potent antiinflammatory properties. In fact, we must consider that our observations might be the result of a higher MAP after LEVO administration. Future research will be necessary to address this interesting issue.

Enhanced expression of cleaved caspase-3 in the spleens of septic animals was suppressed by i.v. and inh. LEVO in the present study. Caspase-3 is the central apoptotic mediator and responsible for cellular degradation during the process of programmed cell death [11]. Enhanced apoptosis in the spleens of septic mice has been reported before [30], and preventing lymphocyte apoptosis during the course of sepsis may represent a way of effectively treating this disorder [31]. In addition, inhibiting caspase-3 in lymphocytes has been shown to improve survival in septic mice [32]. We do acknowledge that the beneficial effects of LEVO as presented in our short term model of severe sepsis are most likely not due to a possible caspase-3 inhibiting effect of LEVO, but rather due to its positive inotropic properties. However, investigations by Oberbeck et al. [33–35] showed that standard vasopressors (e.g. epinephrine, dopamine, dopexamine) induce lymphocyte apoptosis in murine sepsis. This may potentially increase the possible future value of LEVO in the treatment of septic arterial hypotension when the recommended vasopressor therapy is no longer sufficient.

For the present study, we chose a relatively short observation time and a moderate rate of crystalloid fluid administration (12 ml/kg/h). Therefore, we cannot comment on long-term survival or on what might have happened if aggressive fluid resuscitation and additional vasopressor therapy––as encouraged by the current guidelines [3]––had been allowed in our study. Instead, our aim was to characterize the short term effects of LEVO administration in acute severe sepsis. Taken together, our data provide additional pieces of evidence in favour of possible beneficial effects of LEVO in the context of severe sepsis. Consequently, future comparative investigations including standard vasopressor therapy will have to be conducted in order to clarify the immunmodulatory and antiapoptotic potential of LEVO during experimental sepsis.

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Acknowledgments

The authors thank Hanns Ackermann, Ph.D. and Gerald Morawe, Ph.D. for helpful advice on the statistical analyses. Moreover, we thank Elke Müller, Ph.D., Andreas Linke, Ph.D. and Konstantinos Goulis for expert technical assistance. The study was performed at the Hospital of the Johann Wolfgang Goethe-University Frankfurt/Main, Germany, and supported by departmental funding.

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Affiliations

  1. Department of Anaesthesiology, Hospital of the Ludwig-Maximilians-University Munich, Marchioninistr. 15, 81377, Munich, Germany

    Patrick Scheiermann, Sandra Hoegl, Bernhard Zwissler & Kim A. Boost

  2. Department of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany

    Devan Ahluwalia, Andrea Dolfen, Marc Revermann & Christian Hofstetter

  3. Pharmazentrum/ZAFES, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany

    Heiko Muhl

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  1. Patrick Scheiermann
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  2. Devan Ahluwalia
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Correspondence to Patrick Scheiermann.

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Scheiermann, P., Ahluwalia, D., Hoegl, S. et al. Effects of intravenous and inhaled levosimendan in severe rodent sepsis. Intensive Care Med 35, 1412–1419 (2009). https://doi.org/10.1007/s00134-009-1481-9

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  • DOI: https://doi.org/10.1007/s00134-009-1481-9

Keywords

  • Levosimendan
  • Cecal ligation and incision
  • Severe sepsis
  • Cytokines
  • Apoptosis
  • Rat