In this model of acute, severe sepsis the sedatives, dexmedetomidine and midazolam, reduced early mortality. This mortality benefit was associated with reduced TNF-alpha signalling in both groups. Additionally, dexmedetomidine sedation also reduced IL-6 levels (P = 0.05) and splenic caspase-3 expression (P < 0.05) compared with benzodiazepine sedation. These two actions indicate that dexmedetomidine may show benefit models of sepsis explored at later time intervals.
Sedation induced anti-inflammatory effects
Previous preclinical studies had shown that sedation with dexmedetomidine does improve mortality from endotoxic shock in rats compared with a non-sedated group [
22]. Based upon the inflammatory and apoptosis biomarkers we would anticipate superior benefits of sedation with dexmedetomidine
vs midazolam in the acute phase of sepsis; possible reasons why this putative benefit was not borne out by the mortality data may relate to the 'hyper-aggressive' septic state that appears primarily to be TNF-α dependent (as mortality benefits were associated with reduced TNF-α levels). It is noteworthy that midazolam and dexmedetomidine reduced TNF-α levels by a similar amount although previous clinical trials have suggested that dexmedetomidine was superior to midazolam in this regard [
24]. Dexmedetomidine has also been shown to improve mortality and reduce inflammatory cytokine levels induced by CLIP in mice when dexmedetomidine was started prior to the sepsis [
23] though the dosing schedule in this study was irregular. In our study the sedatives were commenced by infusion shortly before provoking sepsis and therefore the levels were unlikely to be therapeutic as sepsis was induced.
The anti-inflammatory effects of dexmedetomidine have now been shown against endotoxin (compared with saline) [
22], in single CLIP [
23], in double CLIP (compared with midazolam; Figures
2,
3) and in critically ill humans (compared with propofol [
25] or midazolam [
24]). How dexmedetomidine induces its anti-inflammatory effect is currently unclear though it may be related to its central sympatholytic effects [
23,
30] and relative stimulation of the cholinergic anti-inflammatory pathway [
16,
17]. Inflammation also appears to alter the effects of α
2 adrenoceptor stimulation shifting them from a pro- to an anti-inflammatory effect [
35].
The effect of the sedatives on IL-6 require further consideration as IL-6 levels are predictive of mortality in septic humans [
36] and animals [
37]. Therefore, the reduction of IL-6 levels by dexmedetomidine relative to midazolam and saline may prove crucial in future studies. The achieved significance value of
P = 0.05 means the results are of borderline significance though we suspect this is due to a reduced sample size in the midazolam group. Power analysis based on our results suggests that six animals per group are required to achieve power to find a statistical difference of
P < 0.05. Therefore our study was designed with appropriate power but a loss of two animal samples in the midazolam group, leaving a sample size of four animals in that group, may have been responsible for our result that is of borderline significance. The superiority of dexmedetomidine's ability to reduce IL-6 levels has already been shown in humans [
24,
25]; however, it should be noted that dexmedetomidine was administered immediately after the septic insult in this study. This is important as the timing of anti-IL-6 therapy is critical; delays greater four hours after CLIP show no benefit in septic animals [
38].
How midazolam induces an anti-inflammatory effect is unclear but immune cells express both the peripheral benzodiazepine receptor [
39] and gamma-amino butyric acid receptors [
40] and thus at least two local targets exist for benzodiazepines. For example, midazolam suppressed lipopolysaccharide-induced TNF-α activity in macrophages, an effect that was blocked by the peripheral benzodiazepine receptor antagonist PK 11195 [
39]. Midazolam also inhibits lipopolysaccharide-induced up-regulation of cyclooxygenase 2 and inducible nitric oxide synthase in a macrophage cell line. Other markers of immune cell activation (induced by lipopolysaccharide) such as IκB-α degradation, nuclear factor-κB transcriptional activity, phosphorylation of p38 mitogen-activated protein kinase and superoxide production were also suppressed by the midazolam [
41].
Interestingly dexmedetomidine and midazolam appear to exert opposite effects on innate immunity. Dexmedetomidine appears to potentiate macrophage function and phagocytosis [
27‐
29], while, as described above, midazolam inhibits it [
39,
41,
42]. This may be related to opposing effects on p38 mitogen-activated protein kinase signaling in these cells [
41,
43]. Thus although both sedatives suppressed circulating cytokines, at a local level the effects on macrophages may have been very different. Benzodiazepine induced suppression of immunity has been noted against
Salmonella typhimurium with 15 days of diazepam treatment [
19] and
Klebsiella pneumoniae with three days of diazepam treatment
in vivo [
20]. In these settings of infection, diazepam treatment increased animal mortality. Thus longer treatment times may be needed to show impairment of immune responses by midazolam than used in this study. We consider that differing effects on innate immunity may explain why critically ill patients sedated with dexmedetomidine experienced fewer infections than those patient sedated with midazolam in a recent randomized controlled trial of 366 critically ill patients [
44]. Further studies addressing the relative effects of longer dosing schedules and different doses of the two sedatives on innate immune responses are in progress. It is interesting to note that daily interruption of sedative infusions appear to be associated with fewer infective complications [
45]; this may be related to the reduced dose of sedatives resulting in less inhibition of the immune system. Recently, deep sedation has been associated with increased mortality in the critically ill [
46] although it is unclear whether this affected immune responses. In this study we did not measure depth of sedation with electroencephalogram monitoring; however, based on recently published clinical data [
46], future studies should consider this. Nonetheless our data suggests that the sedatives are equally able to reduce mortality during the acute phase of sepsis and therefore that choice of sedative in this acute phase may not matter.
Effects of sedation on apoptosis in sepsis
Apoptotic (or programmed) cell death occurs in physiological conditions; for example, it is an important mechanism by which immune responses are controlled via activated cell death of lymphocytes. Sepsis induces apoptosis in lymphocytes, dendritic cells and enterocytes and death of these cells appear pivotal to the pathogenesis of the hypo-inflammatory phase of the condition [
2,
3]. Prevention of this apoptotic injury with inhibitors of the caspase enzymes [
47], regarded as the final executioners in apoptosis or of over expression of anti-apoptotic proteins, has been shown to improve survival in animal models of less acute sepsis.[
2,
3] Critical mediators of this septic apoptotic injury include pro-apoptotic proteins such as BAX and activated caspase-3 [
2,
3].
Both midazolam and dexmedetomidine reduced the burden of splenic caspase-3 expression indicating that they may exert some anti-apoptotic effects in the presence of severe sepsis. It is possible that in the present model, TNF-α binding stimulated the extrinsic apoptotic cascade. Thus the observed inhibition of apoptotic markers may be, in part, due to suppression of the inflammatory response. This would account for why both sedatives showed some anti-apoptotic ability. Interestingly, midazolam was only capable of reducing the 19 KDa fragment of cleaved caspase-3; why it had such an effect is currently unclear. Nonetheless, dexmedetomidine exhibited significantly superior anti-apoptotic effects, consistent with previous reports demonstrating that dexmedetomidine could prevent apoptotic injury from hypoxia and isoflurane in neurons [
26,
48]. α
2 adrenoceptor stimulation reduces pro-apoptotic proteins such as BAX and increases anti-apoptotic Bcl-2 signaling [
49], indicating activity against the intrinsic apoptotic cascade. As apoptotic mechanisms are highly conserved and therefore anti-apoptotic agents are likely to work in different tissue types we hypothesized that stimulation of α
2 adrenoceptors by dexmedetomidine may inhibit septic apoptosis. Indeed activation of AKT/protein kinase B, extracellular regulated signalling kinase and Bcl-2 improves survival in sepsis [
2,
3] and these effectors are upregulated by dexmedetomidine [
49,
50]. Therefore, the reduction in sepsis-induced splenic apoptosis is plausible (Figure
3).
The consequences of apoptosis may be more relevant in clinical sepsis and in the less acute phase of sepsis in animal models. Also, in acute severe sepsis apoptosis of cells may have a protective effect by dampening the immune response; improved mortality has been noted from endotoxic shock in animals treated with apoptotic cells [
51]. This suggests a complex and dynamic set of circumstances pertain during sepsis expressed in apoptotic and inflammatory responses that are observed at different times. Indeed corticosteroids show anti-inflammatory effects (that have correlated with increased speed of reversal of septic shock in the CORTICUS trial [
10]) but exacerbate lipopolysaccharide-induced apoptosis [
52]. However an agent, such as dexmedetomidine, that can combat both inflammation (in the early phase of sepsis) and apoptosis (in the later phase of sepsis) could have particular utility in septic patients. These data also help explain the remarkable mortality benefit we have seen in septic patients from the MENDS study [
32]. This hypothesis will need evaluation in further preclinical studies.