In an inspirational series of studies, the vagus nerve was identified as an important part of the afferent and efferent arms of neural circuits that can regulate the immune response, and the concepts of the cholinergic anti-inflammatory pathway and the inflammatory reflex were established (1–4). According to this concept, the afferent signals induced by endogenous and exogenous products of inflammatories (cytokines, eicosanoids, pathogen-, and danger-associated molecular patterns) are transmitted through the vagus nerve to the nucleus tractus solitarii, which relays the signals further to nuclei in the brain stem and hypothalamus (5). The efferent signals travel back via the vagus nerve to the celiac ganglia, in which the adrenergic splenic neurons are activated and transmit signals to the spleen (4, 6). Macrophages of the spleen are then modulated through either α7 subunit nicotinic acetylcholine receptors or β2-adrenergic receptors to down-regulate inflammatory mediator production (7–9).
Extensive experimental work demonstrated that cholinergic/vagus nerve stimulation inhibits cytokine production and activities and improves disease endpoints in a number of experimental rodent models of systemic inflammation and sepsis (10–14).
Although the body of evidence accumulated so far in favor of the cholinergic anti-inflammatory pathway is impressive, to the best of our knowledge, the therapeutic potential in sepsis/endotoxemia was only tested in short-term small animal (mice, rats) models. However, host susceptibility to pathogenic factors and the characteristics of immune, inflammatory, metabolic, and hemodynamic responses in these rodent models do not mimic human stress responses. Consequently, the translation of these experimental findings to the clinical level is limited (15).
In this study, we addressed this potential shortcoming and examined possible therapeutic effects of vagus nerve stimulation in a clinically relevant porcine model of peritonitis-induced progressive sepsis. This model closely mimics human pathophysiology, with hyperdynamic circulation, low systemic vascular resistance, and multiple organ dysfunction.
MATERIALS AND METHODS
Animal handling was performed in accordance with the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU). The experiments were approved by the Committee for Experiments on Animals of the Charles University Faculty of Medicine in Pilsen and by the Ministry of Education, Youth, and Sports of the Czech Republic (protocol number MSMT-24725/2014–5). All experiments were performed in the Laboratory of Experimental Intensive Care Medicine of the Biomedical Center at the Faculty of Medicine in Pilsen. Twenty-five domestic pigs of both sexes (18 barrows, seven sows) and of similar weight (44.5 ± 5.7 kg) were randomly divided into three groups: 1) sepsis group (eight pigs), 2) sepsis + vagus nerve stimulation group (nine pigs), and 3) control sham group (eight pigs).
Anesthesia, Instrumentation, and Experimental Protocols
Anesthesia, instrumentation, and experimental protocols were similar to those previously described (16, 17) and are provided in detail in Supplemental Materials and Methods (Supplemental Digital Content 1, http://links.lww.com/CCM/E498). In the vagus nerve stimulation group, the cervical portion of the left vagus nerve was exposed and attached to the bipolar stimulation electrode (Harvard Apparatus, Holliston, MA). The stimulation started 6 hours after the induction of peritonitis and continued until the end of the experiment. The vagus nerve was stimulated by rectangular impulses (frequency 2 Hz, amplitude 5 mA, duration 2 ms) using a constant current stimulus isolator with an integrated pulse generator (Isostim A320; WPI, Sarasota, FL).
Measurements and Analysis
The measurements and analyses were similar to those previously described (16, 17) and are provided in the Supplemental Digital Content in detail.
All animals survived during the 24 hours of resuscitated experimental sepsis. Sequential Organ Failure Assessment (SOFA) score values were significantly elevated in the septic group (Fig. 1A), although the individual responses to the infectious stimulus were heterogeneous, and two animals did not meet Sepsis-3 criteria for the development of sepsis (Supplemental Fig. 1, Supplemental Digital Content 2, http://links.lww.com/CCM/E499; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). At the start of the vagus nerve stimulation (6 hr after induction of peritonitis), there were no statistically significant differences in any measured variables between sepsis and sepsis with vagus nerve stimulation groups (data not shown). In the vagus nerve stimulation group, sepsis progression was attenuated, as documented by SOFA scores similar to control 24 hours after the induction of peritonitis (Fig. 1A). In the organ systems that were included in the SOFA score, beneficial effects of vagus nerve stimulation were found in the respiratory system (PaO2/FIO2 of 361.5 mm Hg [85–376 mm Hg] in sepsis vs 447 mm Hg [132–176.5 mm Hg] in sepsis with vagal stimulation; p < 0.05) and in the liver (serum aspartate transaminase of 2.6 μkat/L [1.4–7.9 μkat/L] in sepsis vs 1.6 μkat/L [0.5–1.1 μkat/L] in sepsis with vagal stimulation; p < 0.05). In the kidney, the vagus nerve stimulation did not affect either plasma creatinine levels (122.5 μmol/L [125–187 μmol/L] in sepsis vs 110 μmol/L [36–82 μmol/L] in sepsis with vagal stimulation; p > 0.05) or the acute kidney injury (AKI) frequency (AKI present in 4/8 pigs with sepsis vs 3/9 pigs with sepsis and vagal stimulation; p > 0.05). Platelet counts were similar in both septic groups (120 × 109/L [44–145 × 109/L] in sepsis vs 147 × 109/L [39–73 × 109/L] in sepsis with vagal stimulation; p > 0.05). The vagus nerve stimulation completely abolished the sepsis-induced rise in plasma lactate levels (Fig. 1B).
Maintenance of the mean arterial pressure at a level above 65 mm Hg (Supplemental Table 1, Supplemental Digital Content 3, http://links.lww.com/CCM/E500), which is required in most septic group animals (in 6/8 pigs with sepsis vs 6/9 pigs with sepsis and vagal stimulation), was achieved through the administration of norepinephrine (Fig. 1C). The total dose of norepinephrine was significantly lower in the vagus nerve stimulation group (Fig. 1C), although the time to the first administration of norepinephrine was similar for both septic groups (not shown). Mean infusion rate showed a tendency to decrease in vagus nerve stimulation group, the difference; however, did not reach statistical significance (Supplemental Fig. 1, Supplemental Digital Content 2, http://links.lww.com/CCM/E499; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). Furthermore, vagus nerve stimulation was associated with lower volumes of resuscitation fluid, despite the similar diuresis conditions and plasmatic hemoglobin concentrations between the septic groups (Fig. 1D).
Sepsis was associated with hyperdynamic circulation with increased cardiac output (Fig. 2A). Elevated heart rate (Supplemental Fig. 2, Supplemental Digital Content 4, http://links.lww.com/CCM/E501; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505) was accompanied with decreased stroke volume (Supplemental Fig. 2, Supplemental Digital Content 4, http://links.lww.com/CCM/E501; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505) and peripheral vasodilation (Fig. 2B). Vagus nerve stimulation partially reversed these changes and prevented the cardiac output increase, predominantly by heart rate reduction (Supplemental Fig. 2, Supplemental Digital Content 4, http://links.lww.com/CCM/E500; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). Heart rate variability analysis revealed a significant increase in the low frequency to high frequency band ratio in septic animals, which was completely abolished by vagus nerve stimulation (Fig. 2C).
Blood pressures, blood gases, and acid-base balance variables were not improved by vagus nerve stimulation (Supplemental Table 1, Supplemental Digital Content 3, http://links.lww.com/CCM/E500). In all groups, normoglycemia was maintained using 10% glucose infusion (Supplemental Table 1, Supplemental Digital Content 3, http://links.lww.com/CCM/E500). Body temperature was increased in sepsis, and vagus nerve stimulation did not affect it (Supplemental Table 1, Supplemental Digital Content 3, http://links.lww.com/CCM/E500). Arterial 8-Isoprostane levels were similar in all groups (Supplemental Table 1, Supplemental Digital Content 3, http://links.lww.com/CCM/E500). In multicellular cardiac preparations (trabeculae), the action potential duration at 90% repolarization was not significantly influenced neither by sepsis nor by vagus nerve stimulation (Supplemental Fig. 3, Supplemental Digital Content 5, http://links.lww.com/CCM/E502; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505).
The contraction force was reduced during sepsis, and vagus nerve stimulation completely normalized the contraction force (Fig. 3A). Similarly, in isolated cardiac myocytes, sarcomeric shortening was inhibited by sepsis at low stimulation frequencies and was completely normalized by vagus nerve stimulation (Fig. 3B). In contrast, the calcium transient amplitude, which was also reduced in sepsis, was not affected by vagus nerve stimulation (Fig. 3C). On the other hand, vagus nerve stimulation was associated with elevated baseline intracellular calcium levels (Fig. 3D).
Both oxygen delivery and oxygen consumption increased during sepsis, and the vagus nerve stimulation partially suppressed these sepsis-induced changes (Fig. 4, A and B; and Supplemental Fig. 4, Supplemental Digital Content 6, http://links.lww.com/CCM/E503; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). The mitochondrial function of the heart was analyzed with ultrasensitive oxygraphy, which revealed a significant inhibition of Complex II and Complex IV activities in sepsis (Fig. 4, C and D; and Supplemental Fig. 4, Supplemental Digital Content 6, http://links.lww.com/CCM/E503—legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). Vagus nerve stimulation completely prevented the suppression of Complex II and Complex IV activities (Fig. 4, C and D).
The WBC counts were reduced during sepsis, and vagus nerve stimulation did not affect this reduction (Fig. 5A). The relative neutrophil and lymphocyte counts were not affected by sepsis nor vagus nerve stimulation (not shown). The relative CD14 monocyte counts were reduced in sepsis, and vagus nerve stimulation did not counteract this effect (Supplemental Fig. 5, Supplemental Digital Content 7, http://links.lww.com/CCM/E504; legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). The relative counts of activated CD14/swine leukocyte antigen-DR (SLA-DR) monocytes were reduced in sepsis and normalized upon vagus nerve stimulation (Fig. 5B). The plasma levels of pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)–6, and IL-8 significantly increased in sepsis regardless of vagus nerve stimulation (Fig. 5C; and Supplemental Fig. 5, Supplemental Digital Content 7, http://links.lww.com/CCM/E504—legend, Supplemental Digital Content 8, http://links.lww.com/CCM/E505). The plasma levels of anti-inflammatory cytokine IL-10 were below the detection limit (3 pg/mL) in all pigs.
In this study, the concept of the cholinergic anti-inflammatory pathway was tested. To the best of our knowledge, this was the first time this pathway was tested in a large animal model of sepsis, and the positive results indicate the significant translational potential of vagus nerve stimulation and promising clinical implications. In resuscitated porcine progressive sepsis, the vagus nerve stimulation was associated with a number of beneficial effects, especially with respect to the cardiovascular system and energy metabolism, and consequently, vagus nerve stimulation significantly attenuated multiple organ dysfunction and reduced vasopressor requirements. The vagus nerve stimulation partially or completely prevented the development of hyperdynamic circulation, cellular myocardial depression, hyperlactatemia, the shift in the sympathovagal balance toward sympathetic dominance, and cardiac mitochondrial dysfunction and reduced the number of activated monocytes. The data suggest a significant therapeutic potential of vagus nerve stimulation for conditions related to sepsis and septic shock in a large animal model.
Septic hyperdynamic circulation was inhibited by the vagus nerve stimulation; only a slight increase in cardiac output and maintained stroke volume were observed in stimulated animals. On the contrary, in septic animals, high drive tachycardia that led to decreased stroke volume was observed. The hypothesis that decreasing the sympathetic effect on the heart may be beneficial in patients with septic shock has been already stated and tested by the use of beta-blockers (18) or ivabradine (19). A direct central parasympathetic effect induced by vagal stimulation might provide another way to decrease demands on the cardiovascular system and protect the heart. The dominant mode of action of vagus nerve stimulation, however, remains unclear. Low frequency (1–10 Hz) stimulation should preferentially activate vagal efferents (20), which suggests a direct cardiac effect of vagus nerve stimulation in our experimental conditions. Analysis of heart rate variability also showed a parasympathetic shift of the sympathovagal balance after vagus nerve stimulation; however, a central autonomic effect due to afferent stimulation cannot be excluded. Since the vagus nerve contains both afferent and efferent fibers (afferent fibers representing over 80% of the nerve ), it is virtually impossible to distinguish afferent and efferent mechanisms of vagus nerve stimulation, which probably influence each other in a complex reflexive manner (22). Besides, the other observed effects of vagal stimulation (decrease in oxygen consumption, retained systemic vascular resistance, etc.) are difficult to adjudge to cardiac effects only.
Interestingly, pigs with comparable cardiac preload that were treated with vagal nerve stimulation required lower amounts of resuscitation fluid and less norepinephrine doses to achieve the hemodynamic targets. Taken together, we speculate that vagal nerve stimulation improves vasomotoricity and ameliorates the sepsis-induced vascular barrier dysfunction that is associated with microvascular fluid leakage. Although the exact mechanism cannot be inferred from our study, our results suggest that the basis for improved vascular functions may be attributable to the restoration of cellular energy metabolism.
In this study, cardiac mitochondrial respiration was impaired in sepsis and normalized by vagus nerve stimulation. Although impaired mitochondrial oxygen consumption is frequently mentioned as a crucial factor that contributes to the pathogenesis of (multiple)organ dysfunction (23), studies performed in various rodent models of sepsis showed unchanged, decreased, or even increased mitochondrial respiration in the heart (24). Rare data on porcine fecal peritonitis models did not show any significant differences in mitochondrial oxygen consumption between control and vigorously resuscitated (including antibiotics) piglet groups (25). It is well known that factors such a particular sepsis models, animal species, disease severity and phase, and the experimental set-up can substantially impact variability of experimental results (26). Our study shows that sepsis-induced myocardial depression was associated with mitochondrial respiratory dysfunction, which corresponds well with the decreased oxygen extraction ratio and together suggests that in this porcine model, the mitochondrial dysfunction is not limited to cardiac tissue. To prove this hypothesis, however, mitochondrial studies in other than cardiac tissues will be necessary. Furthermore, the fact that in sepsis the oxygen delivery increased more than oxygen consumption suggests that mitochondrial dysfunction might be the primary organ dysfunction event. In this study, the suppression of mitochondrial respiration was dominantly due to the inhibition of Complex II and Complex IV. In contrast to studies in rodents (27), the activity of Complex I was not affected.
The impact of vagus nerve stimulation on mitochondrial oxygen consumption by cardiac tissue has not been studied. However, some data indicate that cardioprotective effects of vagus nerve stimulation might be at least partly mediated by improved mitochondrial functions (28). For example, the beneficial effects of vagus nerve stimulation on mitochondria have been documented in a porcine model of myocardial ischemia, where the vagus nerve stimulation attenuated cardiac mitochondrial reactive oxygen species production, depolarization, and swelling (29). A recently-published study suggested that α7-nicotinic acetylcholine receptor could be involved in the protection against lipopolysaccharide-induced sepsis myocardial injury and apoptosis in mice (30). The present data indicate that vagus nerve stimulation improves mitochondrial respiration and cardiac metabolism.
The peritonitis-induced sepsis porcine model was associated with myocardial depression, as evidenced by decreased stroke volume, reduced contraction force of cardiac trabeculae, and reduced sarcomeric shortening in isolated cardiac myocytes. The vagus nerve stimulation completely reversed all of these manifestations of septic myocardial depression. In septic cardiac myocytes, the reduction of sarcomeric shortening was associated with decreased calcium transient. The vagus nerve stimulation did not affect the amplitude of calcium transient but led to elevated baseline intracellular calcium levels, which could contribute to the normalization of cardiac contractile forces. Furthermore, higher intracellular calcium levels might contribute to the improved mitochondrial function that was observed upon vagus nerve stimulation. The cardiac sarcoplasmic reticulum and mitochondria form a mitochondrial calcium microdomain (31), and mitochondrial calcium uptake stimulates the tricarboxylic acid cycle and adenosine triphosphate production (32).
Among the multiple beneficial effects of vagus nerve stimulation, it is difficult to determine the primary effect. The lack of effect on the plasma levels of cytokines argues against the dominant role of upstream anti-inflammatory mechanisms, although the reversal of the reduced CD14/SLA-DR monocyte count might be of importance. The reduction of CD14/SLA-DR monocytes in sepsis probably reflects the transition of steady-state monocytes to inflammatory monocytes that lack SLA-DR (33). Although the vagus nerve stimulation suppressed this effect of sepsis on monocyte phenotype, it did not prevent an increased production of inflammatory mediators. The absence of an effect of vagus nerve stimulation on cytokine production in porcine progressive sepsis stands in stark contrast to the cytokine inhibition that was observed in rodent sepsis models (10) and suggests that significant species differences in cholinergic anti-inflammatory mechanisms exist.
The translation of vagus nerve stimulation to the clinical level should be feasible since vagus nerve stimulation has already been approved by regulatory bodies of the United States, European Union, and Canada as a treatment for epilepsy and depression. Furthermore, vagus nerve stimulation is considered a safe technique, and few side effects are observed at the low frequencies that are classically used in inflammatory conditions (20). An implantable device for direct vagus nerve stimulation that includes spiral electrodes and a battery-powered pulse generator is commercially available. As an alternative noninvasive option, transcutaneous vagus nerve stimulation has been demonstrated to be a safe and well-tolerated method in patients with pharmacoresistant epilepsy (34).
Vagus nerve stimulation was initiated 6 hours after the induction of peritonitis when the sepsis was not fully developed. The efficacy of the vagus nerve stimulation when applied in later stages of sepsis progression or during septic shock remains to be examined.
Our experimental design and subsequent in vitro analysis of tissues and cells precluded the analysis of mortality and/or recovery from the disease. Future studies will be needed to determine the potential mortality benefit for this therapeutic option.
Different norepinephrine requirements might contribute to the results reported and complicate their interpretation. Although norepinephrine was administered secondarily in response to a drop in blood pressure, possible contributions of norepinephrine to sympathovagal balance through various complex feedback loops cannot be excluded and will require further clarification.
The effects of sepsis and of vagus nerve stimulation were only studied in the first 24 hours of sepsis, and therefore, the potential long-term implications of the disease and of the intervention remain unclear. To reach the stage of refractory septic shock within 24 hours from induction of peritonitis, antibiotic therapy must be omitted, which might represent a potential shortcoming of the model clinical relevance. Possible interactions of the antibiotic therapy and vagus nerve stimulation represent another important question, which will warrant further investigation.
In a clinically-relevant porcine model of sepsis and septic shock, vagus nerve stimulation was associated with a number of beneficial effects that resulted in significantly attenuated multiple organ dysfunction and reduced vasopressor and fluid resuscitation requirements. Vagus nerve stimulation might provide a significant therapeutic potential in a large animal model of progressive septic shock.
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