The autonomic nervous system (ANS) and the inflammatory response are intimately linked. The sympathetic nervous system can attenuate the systemic inflammatory response via activation of β2-adrenoceptors (1-3), but can also enhance the inflammatory response locally via stimulation of α2-adrenoceptors (4, 5). The parasympathetic nervous system, through the afferent vagus nerve, can sense inflammation in the periphery and relay this information to the brain, resulting in fever generation and activation of the hypothalamic-pituitary-adrenal axis, which in turn leads to cortisol release and stimulation of the sympathetic nervous system (6, 7). Furthermore, electrical stimulation of the efferent vagus nerve greatly attenuates the inflammatory response in animal models (8). These findings indicate that there is considerable crosstalk between the ANS and the inflammatory response and that the two branches of the ANS can modulate the inflammatory response.
The ANS innervates virtually every organ in the body. However, outflow of a particular branch of the ANS might be organ-specific. Heart rate variability (HRV) analysis is the only noninvasive tool available in humans to monitor activity of both branches of the ANS (9), but assesses only ANS innervation of the heart. Heart rate variability indices (both time and frequency domains) and their physiological correlates (10-13) are listed in Table 1.
Decreased HRV is associated with pathological conditions. In systemically inflamed critically ill patients, such as in sepsis, HRV is completely diminished (14-17) and inversely correlated with disease severity (15, 16), and reduced HRV is a predictor of multiorgan dysfunction syndrome and death (18, 19). These findings suggest that HRV alterations may reflect the magnitude of the inflammatory response.
In this study, we investigated the interplay between the acute inflammatory response to endotoxin and cardiac ANS activity, measured by HRV. We used the experimental human endotoxemia model, a well-characterized, standardized model of systemic inflammation widely used to study the acute inflammatory response (20). This model is not hampered by confounding factors such as sedatives, inotropes, and pressors often present in the clinical situation. It therefore provides readily interpretable data regarding the interplay between the acute inflammatory response and HRV. Because ANS-inflammatory interactions are bidirectional, we determined the relation between baseline HRV indices and the inflammatory response as well as the relation between the magnitude of the inflammatory response and HRV alterations.
MATERIALS AND METHODS
Heart rate variability was measured in a total of 40 healthy young male nonsmoking volunteers participating in two human endotoxemia studies. Data of 28 subjects were obtained from a study (Clinical Trial Register number NCT00513110) where LPS (endotoxin) was administered once; the other 12 subjects participated in a human endotoxemia study (Clinical Trial Register number NCT00783068) in which they received LPS twice (LPS visits 1 and 2) with a mean interval of 14 days (range, 11-18 days). The study protocols were approved by the Ethics Committee of the Radboud University Nijmegen Medical Centre and comply with the Declaration of Helsinki including current revisions and the Good Clinical Practice guidelines. Written informed consent was obtained from all study participants. The findings of the physical examinations, electrocardiography, and routine laboratory studies on all the volunteers before the start of the experiment showed normal results. Volunteers were not taking any prescription medications, and they were negative for hepatitis B surface antigen and HIV infection.
Human endotoxemia model
Subjects refrained from food 12 h before the start of the experiment, and caffeine- or alcohol-containing substances 24 h before the start of the experiment. The experiments were performed at the research unit of the intensive care department, with subjects in supine position. After administration of local anesthesia (lidocaine HCl 20 mg/mL), the radial artery was cannulated using a 20-gauge arterial catheter (Angiocath, Becton Dickinson, Sandy, Utah) and connected to an arterial pressure monitoring set (Edwards Lifesciences LLC, Irvine, Calif), connected to a Phillips IntelliVue MP70 monitor (Philips Medical Systems, Eindhoven, The Netherlands). A cannula was placed in the antecubital vein to permit infusion of 2.5% glucose/0.45% saline solution; subjects received a bolus of 1.5 L during 1 h before LPS infusion (prehydration), followed by 150 mL/h until 6 h after LPS infusion and 75 mL/h until the end of the experiment. US Reference Escherichia coli endotoxin (LPS derived from E. coli O:113; Clinical Center Reference Endotoxin, National Institutes of Health, Bethesda, Md) was used. Ec-5 endotoxin, supplied as a lyophilized powder, was reconstituted in 5 mL saline 0.9% for injection and vortex-mixed for at least 10 min after reconstitution. The endotoxin solution was administered as an intravenous bolus injection at a dose of 2 ng/kg of body weight. Vital signs, including heart rate (HR) and mean arterial pressure (MAP), were monitored continuously throughout the study protocol.
Heart rate variability was measured hourly by 5-min recordings starting just before LPS administration (T = 0 or baseline) up to 8 h after LPS administration. Heart rate variability was measured in supine position and during quiet circumstances. A 3-lead electrocardiogram signal was obtained using a Medilog AR12 recorder (Huntleigh Healthcare, Cardiff, UK). R-peak position was determined at a sample rate of 4,096 Hz. Heart rate variability was analyzed using dedicated software (Medilog Darwin HRV; Huntleigh Healthcare). In each 5-min recording, QRS complexes were detected, and only normal-to-normal beat (NN) intervals were tabulated, yielding an interval tachogram. Recordings with artifacts such as premature (supra)ventricular beats or other arrhythmias comprising more than 5% of the total epoch were discarded. After linear detrending, power spectral density was determined by fast Fourier transformation of interval tachograms using the Welch method and a fast Fourier transformation width of 1,024. Very-low-frequency (VLF) power (0.0033-0.04 Hz) cannot be reliably obtained from 5-min recordings and was therefore not analyzed (13).
EDTA anticoagulated blood was collected from the arterial line and immediately centrifuged at 2,000g for 10 min at 4°C. Concentrations of TNF-α, IL-6, IL-10, and IL-1RA in plasma were measured using a simultaneous Luminex Assay according to the manufacturer's instructions (Bio-plex cytokine assay; Bio-Rad, Hercules, Calif). Samples from both LPS visits of the 12 subjects who received LPS twice were analyzed on the same day.
Calculations and statistical analysis
Based on distribution (calculated by the Shapiro-Wilk test), data are represented as medians or means as indicated. Non-normally distributed HRV indices and cytokine values were successfully log transformed. Statistical tests used are indicated in the figure legends. P < 0.05 was considered significant. Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, Ill) and GraphPad Prism 5 (GraphPad software, San Diego, Calif).
Correlation between baseline HRV and the LPS-induced cytokine response
LPS administration resulted in a typical transient inflammatory cytokine response, decreased MAP, and increased HR (Fig. 1). We did not find any significant correlations between basal HRV indices and the area under the curve (AUC; all correlations listed in Table 2; most relevant indices shown in Fig. 2) or peak levels (See Table S1, Supplemental Digital Content 1, https://links.lww.com/SHK/A72) of any of the cytokines measured.
Correlation between the LPS-induced cytokine response and HRV alterations
LPS administration resulted in characteristic HRV alterations (SDNN, LF/HF, LFnu, and HFnu depicted in Fig. 3; other indices are shown in Figure S1, Supplemental Digital Content 1, https://links.lww.com/SHK/A72), similar to earlier reports (2, 10, 21, 22). To determine whether the magnitude of the inflammatory response was correlated with the magnitude of changes in HRV indices, we correlated peak and AUC cytokine levels with maximum/minimum levels of HRV after LPS administration. No significant correlations were found (see Tables S2 and S3, Supplemental Digital Content 1, https://links.lww.com/SHK/A72). Furthermore, we correlated the peak and AUC cytokines to the increase or decrease in HRV indices (represented as slope of increase/decrease calculated between baseline and time of maximum/minimum value). Apart from one weak correlation between IL-RA and the decrease in LF power, the magnitude of the cytokine response (AUC) did not correlate to changes in HRV indices as well (Table 3); this was also true when peak cytokine levels were used for the calculations (see Table S4, Supplemental Digital Content 1, https://links.lww.com/SHK/A72).
LPS-induced HRV alterations in subjects who received LPS twice
The lack of correlation between the magnitude of the inflammatory response and HRV alterations was strengthened by HRV data obtained from subjects who received LPS twice with an interval of approximately 2 weeks. During the second visit, median peak levels of TNF-α, IL-6, IL-10, and IL-1RA were 43% (P = 0.0024), 41% (P = 0.0024), 54% (P = 0.0038), and 8% (P = 0.11) lower compared with the first visit (Fig. 4, upper left panel) because of the development of endotoxin tolerance (23). This phenomenon allowed us to investigate the relation between the inflammatory response and HRV within the same subjects. Despite the remarkably attenuated inflammatory response, LPS-induced HRV alterations were not different between both visits. The time courses of SDNN, LFnu, and HFnu during both endotoxemia visits are shown in Figure 4. Other changes in HRV indices were equally similar between both visits (see Figure S2, Supplementary Digital Content 1, https://links.lww.com/SHK/A72).
In the present study, we investigated the interplay between the LPS-induced acute inflammatory response and ANS outflow to the heart, determined by HRV in a large group of healthy male subjects who participated in human endotoxemia experiments. We did not find any correlations between basal HRV indices and the inflammatory response or between the magnitude of the inflammatory response and HRV alterations.
We first set out to investigate the relation between baseline HRV and the inflammatory response. None of the baseline HRV indices, determined before LPS administration, correlated with the magnitude of the LPS-induced inflammatory response in our study. In contrast, weak (r values of 0.4-0.43) but significant correlations between baseline HRV indices and the LPS-induced inflammatory response were recently reported using a similar experimental protocol, resulting in HRV changes identical to our study (22). In our study, all indices of HRV except LFnu, HFnu, and pNN50 displayed a markedly skewed distribution, as did the peak and AUC cytokine levels. This is consistent with other HRV studies (24-27). Therefore, we log transformed the skewed variables, which probably explains the contrasting conclusions between the two studies. In addition, considering the well-established anti-inflammatory effects of vagus nerve stimulation (8, 28, 29), there is no plausible mechanism to explain why the reported higher levels of vagal HRV indices are correlated with elevated TNF-α levels (22). Especially, TNF-α has been extensively studied in the context of the cholinergic anti-inflammatory pathway and is greatly reduced by vagus nerve stimulation or cholinergic agonists in animals and in vitro (8, 29-33). The results from our prospective study in a relatively large group using a standardized model of inflammation suggest that there is no relation between basal cardiac ANS activity and the inflammatory response. A possible explanation is that vagus nerve innervation of the heart does not reflect outflow to other organs that play an important role in the inflammatory response. For example, a key effector organ of the cholinergic anti-inflammatory pathway is the spleen, one of the major cytokine-producing organs (34, 35). As basal vagal input to the spleen may be different from vagal input to the heart, HRV might not be an appropriate method to assess activation of the cholinergic anti-inflammatory pathway. However, because no other measures of ANS activity were determined in this study, we cannot provide a definitive answer to this question. Moreover, although we did not find correlations between HRV and plasma cytokines, it is conceivable that cardiac ANS activity is related to other measures of inflammation, such as immune cell subsets.
To further characterize the interplay between the inflammatory response and HRV, we examined the effects of inflammation on cardiac ANS outflow by correlating the magnitude of the LPS-induced inflammatory response to the magnitude of HRV alterations. Again, we could not demonstrate a relation between these parameters. The most compelling evidence for a lack of this relation was observed in subjects who received LPS on two occasions with an interval of 2 weeks. Whereas inflammatory cytokine levels were roughly halved during the second LPS administration because of the development of LPS tolerance, changes in HRV were identical to the first endotoxemia experiment, indicating that although the HRV alterations are associated with systemic inflammation, the magnitude of their change is not a measure of the extent of the inflammatory response. Our findings are corroborated by a recent human endotoxemia study investigating the effects hydrocortisone on the inflammatory response (21). Hydrocortisone treatment greatly reduced LPS-induced inflammatory cytokine levels, but did not influence the LPS-induced decrease in HRV. To our knowledge, there is only one human study demonstrating the effects of immunomodulation on HRV. In this study, epinephrine attenuated the inflammatory response associated with further reduced vagal HRV indices after LPS administration (2). This is likely due to epinephrine's sympathomimetic properties rather than through an immunomodulatory effect, especially because attenuation of the inflammatory response is anticipated to result in less reduction of HRV indices. Taken together, these findings indicate that changes in cardiac ANS outflow determined by HRV alterations do not reflect the magnitude of the inflammatory response in a controlled model of systemic inflammation, which is not confounded by factors such as sedatives, inotropes, and pressors often present in the clinical situation. In contrast, in septic patients, rather strong inverse correlations between log IL-6 levels and log LF (r = −0.76) and log HF (r = −0.53) power were found, suggesting that HRV does reflect the extent of inflammation in these patients (36). Possibly other inflammation-associated factors that are not induced during the milder human endotoxemia model account for the observed changes in HRV in septic patients, but at present, this discrepancy cannot be explained.
With regard to the validity of our data, the LPS-induced changes in HRV observed in our study are analogous to those observed in similar human endotoxemia experiments (2, 10, 21, 22). All "raw" time-domain and frequency-domain indices were severely depressed after LPS administration, with minimum values observed at 4 h after LPS. In this dynamic setting (where total power gradually decreases in time), normalized HRV indices and the LF/HF ratio probably provide more meaningful information about the relative contributions of the two branches of the ANS (13). Both LFnu and LF/HF are regarded to reflect sympathetic activity, whereas HFnu corresponds to parasympathetic activity (10, 13). After LPS administration, both LFnu and LF/HF increased; by analogy, HFnu decreased. These findings indicate that LPS administration results in cardiac sympathetic predominance, probably as a result of the stress response induced by LPS administration. In accordance, increased levels of the stress hormone cortisol and catecholamines have been reported during human endotoxemia, and interestingly, peak levels of cortisol and epinephrine were observed at 3 to 4 h after LPS administration, coinciding with maximum/minimum levels of HRV indices (21, 37, 38). Future studies are warranted to evaluate the relationship between cortisol/catecholamines and HRV after LPS administration. As opposed to the cardiac sympathetic predominance, muscle sympathetic nerve activity (measured in the peroneal nerve) was found to be suppressed after LPS administration (37). These findings strengthen the notion that autonomic outflow cannot be regarded as a general response, but appears to be organ-specific.
In conclusion, we demonstrate that there is no relation between HRV and the inflammatory response and vice versa in a standardized model of systemic inflammation. This suggests that cardiac ANS activity may not be representative for ANS outflow to other organs involved in the inflammatory response and indicates that changes in HRV are not a reliable surrogate measure for the extent of the inflammatory response.
The authors thank Marije Gordinou de Gouberville for help with HRV measurements, Trees Jansen for help with cytokine determinations, and the research nurses of the Department of Intensive Care Medicine for assistance in the conduct of human endotoxemia experiments.
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