Central sympatholytics prolong survival in experimental sepsis

Abstract

Introduction: One of the main causes of death in European and US intensive care units is sepsis. It involves a network of pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IL-6. Furthermore, there is an up regulation of transcription factors such as nuclear factor (NF) kappaB. It has previously been shown that clonidine is able to significantly reduce pro-inflammatory cytokines in surgical patients. We therefore hypothesise that the clinically used central alpha-2 agonist clonidine has the ability to improve survival in experimental sepsis by inhibiting the sympathetic tone and consequently inhibiting the pro-inflammatory cytokine release.

Methods: To investigate this therapeutic potential of clonidine in a prospective randomised laboratory investigation we used a murine model of caecal ligation and puncture (CLP) induced sepsis. Animals receiving pre-emptive injections were treated with either clonidine (5 microg/kg) or dexmedetomidine (40 microg/kg) 12 and 1 hours before the operation, as well as 1, 6 and 12 hours afterwards. Another group of animals only received clonidine (5 microg/kg) 1, 6 and 12 hours after the operation, while the pre-emptive injections were normal saline. The control groups received solvent injections at the respective time points.

Results: Pre-emptive administration of a central sympatholytic significantly reduced mortality (clonidine: p = 0.015; dexmedetomidine: p = 0.029), although postoperative administration of clonidine failed to significantly prolong survival. Furthermore pre-emptive administration of clonidine significantly attenuated the cytokine response after CLP-induced sepsis (mIL-1beta: p = 0.017; mIL-6: p < 0.0001; mTNF-alpha: p < 0.0001), preserved blood pressure control (p = 0.024) and down-regulated the binding activity of NF-kappaB. There were no changes in the pro-inflammatory cytokine response when peripheral blood was incubated with lipopolysaccharide alone compared with incubation with clonidine (10-4 M) plus LPS (p > 0.05).

Conclusions: Our results demonstrate that the pre-emptive administration of either clonidine or dexmedetomidine have the ability to successfully improve survival in experimental sepsis. Furthermore, there seems to be a connection between the central muscarinic network and the vagal cholinergic response. By down-regulating pro-inflammatory mediators sympatholytics may be a useful adjunct sedative in patients with a high risk for developing sepsis.

Figure 1

Survival after caecal ligation and puncture (CLP)-induced sepsis. Kaplan-Meier survival curves of mice after CLP-induced sepsis (t = 0). (a) Pre-emptive administration of clonidine improves survival after CLP-induced sepsis. The verum group (solid line, n = 30) received clonidine (5 μg/kg) 12 hours and 1 hour before CLP, as well as 1 hour, 6 hours and 12 hours after CLP. The control group received corresponding injections with solvent (dashed line, n = 30). Clonidine-treated animals survived significantly longer than their controls (p = 0.015, n = 60). (b) Pre-emptive administration of dexmedetomidine statistically prolongs survival after CLP-induced sepsis. The verum group (solid line, n = 40) received dexmedetomidine (40 μg/kg) 12 hours and 1 hour before CLP, as well as 1 hour, 6 hours and 12 hours after CLP. The control group received corresponding injections with solvent (dashed line, n = 40). Dexmedetomidine-treated animals survived significantly longer than their controls (p = 0.029, n = 80). (c) Post-operative administration of clonidine does not improve survival after CLP-induced sepsis. The verum group (solid line, n = 30) received clonidine (5 μg/kg) 1 hour, 6 hours and 12 hours after CLP. The control group received corresponding injections with solvent (dashed line, n = 30). There was no statistically significant difference between both groups (p = 0.228, n = 60). * indicates statistical significant differences between the groups.

Figure 2

Clonidine attenuates cytokine response after caecal ligation and puncture (CLP)-induced sepsis. Mice in the verum group (n = 10) received clonidine (5 μg/kg) 12 hours and 1 hour before CLP, as well as 1 hour, 6 hours and 12 hours after CLP. The control group (n = 10) received injections with solvent respectively. (a) Pre-emptive administration of clonidine decreases mIL-1β levels after CLP induced sepsis. Experimental sepsis led to a profound increase in mIL-1β in mouse serum 24 hours after CLP. This increase was significantly attenuated by pre-emptive clonidine injections (p = 0.017) as described above. (b) Pre-emptive administration of clonidine decreases mIL-6 levels after CLP induced sepsis. Experimental sepsis led to a profound increase in mIL-6 in mouse serum 24 hours after CLP. This increase was significantly attenuated by pre-emptive clonidine injections (p < 0.0001) as described above. (c) Pre-emptive administration of clonidine decreases mTNF-α levels after CLP-induced sepsis. Experimental sepsis led to a profound increase in TNF-α in mouse serum 24 hours after CLP. This increase was significantly attenuated by pre-emptive clonidine injections (p < 0.0001) as described above. * indicates statistical significant differences between the groups.

Figure 3

Clonidine reduces nuclear factor (NF) κB activation after caecal ligation and puncture (CLP)-induced sepsis. The figure shows NF-κB binding activity, as assessed by electrophoretic mobility shift assay (EMSA) assay in the livers of mice that underwent CLP surgery. CLP led to a strong increase in NF-κB binding activity. In contrast, in mice treated with clonidine we found no difference in NF-κB binding activity compared with mice receiving placebo injections with normal saline. Furthermore, there was no difference between mice with CLP-induced sepsis receiving clonidine and sham-operated mice. Group I 'Sham OP'; EMSA for NF-κB binding activity; liver tissue. Group II 'CLP + clonidine', clonidine therapy, EMSA for NF-κB binding activity; liver tissue. Group III: 'CLP + placebo', saline injection, EMSA for NF-κB binding activity; liver tissue

Figure 4

Whole blood incubation with lipopolysaccharide and clonidine respectively. Whole blood samples of healthy donors (n = 4) were incubated for 24 hours with either LPS alone (1 μg) or LPS (1 μg) plus clonidine (10^-4). (a) IL-1β levels after whole blood incubation with LPS and clonidine respectively. The concentration of IL-1β increased significantly after LPS administration compared with untreated controls (p = 0.0005). There was no statistically significant difference in IL-1β levels between samples treated with LPS alone and those incubated with LPS plus clonidine (10^-4; p = 0.89). (b) IL-6 levels after whole blood incubation with LPS and clonidine respectively. The concentration of IL-6 increased significantly after LPS administration compared with untreated controls (p = 0.0001). There was no statistically significant difference in IL-6 levels between samples treated with LPS alone and those incubated with LPS plus clonidine (10^-4; p = 0.88). (c) TNF-α levels after whole blood incubation with LPS and clonidine respectively. The concentration of TNF-α increased significantly after LPS administration compared with untreated controls (p = 0.025). There was no statistically significant difference in TNF-α levels between samples treated with LPS alone and those incubated with LPS plus clonidine (10^-4; p = 0.99). * indicates statistical significant differences when compared with untreated.