How to Never Get Sick Again the Wim Hof Iceman Method n

• Journal Listing
• Proc Natl Acad Sci U S A
• v.111(xx); 2014 May twenty
• PMC4034215

Proc Natl Acad Sci U South A. 2014 May 20; 111(twenty): 7379–7384.

Immunology

Voluntary activation of the sympathetic nervous organisation and attenuation of the innate allowed response in humans

Matthijs Kox,^{a, b, c, 1} Lucas T. van Eijk,^{a, c} Jelle Zwaag,^{a, c} Joanne van den Wildenberg,^{a, c} Fred C. One thousand. J. Sweep,^d Johannes G. van der Hoeven,^{a, c} and Peter Pickkers^{a, c}

Matthijs Kox

^aIntensive Care Medicine,

^bAnesthesiology,

^cNijmegen Institute for Infection, Inflammation and Immunity, and

Lucas T. van Eijk

^aIntensive Care Medicine,

^cNijmegen Institute for Infection, Inflammation and Immunity, and

Jelle Zwaag

^aIntensive Care Medicine,

^cNijmegen Institute for Infection, Inflammation and Immunity, and

Joanne van den Wildenberg

^aIntensive Care Medicine,

^cNijmegen Institute for Infection, Inflammation and Amnesty, and

Fred C. M. J. Sweep

^dLaboratory Medicine, Radboud University Medical Centre, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands

Johannes 1000. van der Hoeven

^aIntensive Care Medicine,

^cNijmegen Institute for Infection, Inflammation and Immunity, and

Peter Pickkers

^aIntensive Intendance Medicine,

^cNijmegen Institute for Infection, Inflammation and Immunity, and

Significance

Hitherto, both the autonomic nervous system and innate allowed system were regarded equally systems that cannot be voluntarily influenced. The nowadays study demonstrates that, through practicing techniques learned in a brusk-term training program, the sympathetic nervous organisation and immune system can indeed be voluntarily influenced. Salubrious volunteers practicing the learned techniques exhibited profound increases in the release of epinephrine, which in plow led to increased product of anti-inflammatory mediators and subsequent dampening of the proinflammatory cytokine response elicited by intravenous assistants of bacterial endotoxin. This written report could take important implications for the treatment of a diverseness of atmospheric condition associated with excessive or persistent inflammation, peculiarly autoimmune diseases in which therapies that antagonize proinflammatory cytokines have shown groovy benefit.

Keywords: LPS, cathecholamines, cortisol

Abstract

Excessive or persistent proinflammatory cytokine product plays a fundamental part in autoimmune diseases. Acute activation of the sympathetic nervous system attenuates the innate immune response. However, both the autonomic nervous organization and innate allowed system are regarded as systems that cannot be voluntarily influenced. Herein, we evaluated the effects of a training programme on the autonomic nervous organization and innate immune response. Healthy volunteers were randomized to either the intervention (north = 12) or control grouping (northward = 12). Subjects in the intervention grouping were trained for ten d in meditation (3rd eye meditation), breathing techniques (i.a., cyclic hyperventilation followed past breath retention), and exposure to cold (i.a., immersions in ice cold water). The control group was not trained. After, all subjects underwent experimental endotoxemia (i.v. assistants of 2 ng/kg Escherichia coli endotoxin). In the intervention group, practicing the learned techniques resulted in intermittent respiratory alkalosis and hypoxia resulting in profoundly increased plasma epinephrine levels. In the intervention grouping, plasma levels of the anti-inflammatory cytokine IL-10 increased more chop-chop after endotoxin administration, correlated strongly with preceding epinephrine levels, and were higher. Levels of proinflammatory mediators TNF-α, IL-6, and IL-viii were lower in the intervention group and correlated negatively with IL-10 levels. Finally, flu-like symptoms were lower in the intervention grouping. In determination, we demonstrate that voluntary activation of the sympathetic nervous organization results in epinephrine release and subsequent suppression of the innate immune response in humans in vivo. These results could have important implications for the handling of conditions associated with excessive or persistent inflammation, such equally autoimmune diseases.

The innate immune system is crucial to our survival, but excessive or persistent proinflammatory cytokine product can outcome in tissue damage and organ injury, such as in autoimmune diseases. Biological therapies that antagonize proinflammatory cytokines or their receptors are very constructive and accept revolutionized the treatment of autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease (1, 2). Withal, these drugs are expensive and have serious side effects (3, four). Therefore, innovative therapies aimed at limiting inflammatory cytokine product in a more physiological way are warranted.

Astute activation of the sympathetic nervous system attenuates inflammation via activation of β2-adrenoreceptors by catecholamines, exemplified by the fact that (nor)epinephrine attenuates lipopolysaccharide (LPS)-induced TNF-α release in vitro (five, 6) and brusque-term infusion of epinephrine limits product of proinflammatory cytokines in vivo during experimental endotoxemia (i.5. assistants of LPS in healthy volunteers) (7). In addition, every bit part of a stress response, increased levels of catecholamines are often accompanied by elevations of the well-known immunosuppressive hormone cortisol [via activation of the hypothalamic–pituitary–adrenal (HPA) axis] (8, 9).

Adjacent to exogenous (i.eastward., pharmacological or electrical) modulation of the autonomic nervous system (ANS), endogenous stimulation of ANS activity may too limit the inflammatory response, but the ANS is more often than not regarded every bit a system that cannot be voluntarily influenced. However, results from a recently performed instance study on a Dutch individual, who holds several world records with regard to withstanding extreme common cold, advise otherwise (10). It was shown that this individual was able to voluntarily activate the sympathetic nervous organization through a self-developed method involving meditation, exposure to common cold, and breathing techniques. This resulted in increased catecholamine and cortisol release and a remarkably mild innate immune response during experimental endotoxemia compared with more than 100 subjects who previously underwent experimental endotoxemia. In the nowadays written report, nosotros investigated the effects of his training program (see Movie S1 for an impression) on sympathetic nervous system parameters and the innate immune response in good for you male volunteers during experimental endotoxemia in a randomized controlled mode.

Results

Baseline characteristics of subjects that underwent experimental endotoxemia in both groups were like (Table 1).

Table 1.

Discipline demographic characteristics

Parameter	Trained group, n = 12	Command group, n = 12	P value
Historic period, y	24 (19–27)	22 (nineteen–27)	0.43
Height, cm	181 (172–190)	185 (179–189)	0.30
Weight, kg	75 (58–92)	78 (65–91)	0.25
BMI, kg/grand2	23 (nineteen–26)	23 (20–27)	0.98
Hr, beats/min	60 (41–80)	61 (40–75)	0.88
MAP, mmHg	92 (82–113)	94 (78–105)	0.89

Cardiorespiratory Parameters, Temperature, and Symptoms.

In the command grouping, arterial blood gas parameters pCO₂, pO₂, pH, bicarbonate, lactate, and oxygen saturation were normal and did not substantially modify during endotoxemia (Fig. ane A–F ). In contrast, in trained individuals, practicing the learned breathing techniques resulted in an immediate and profound decrease of pCO₂ and bicarbonate, and an increase in pH (reaching up to vii.75 in individual subjects; Fig. two and Movie S2), indicating acute respiratory alkalosis, which normalized quickly afterward abeyance of the breathing techniques. Mean pO_ii remained virtually unaltered in trained subjects, whereas lactate levels were significantly elevated, but not to clinically relevant levels. A meaning decrease in oxygen saturation was observed in the trained group during practicing of the breathing techniques (Fig. 1F ). Minimum oxygen saturation levels in each cycle of hyper/hypoventilation (subsequently cessation of animate for several minutes) typically dropped to around l% in trained individuals for a brusque period (∼x s; Fig. 2 and Flick S2). Heart rate and mean arterial blood force per unit area (MAP) showed a pattern typical for endotoxemia in the control group: a gradual decrease in MAP and a compensatory rise in heart rate after LPS administration (Fig. 1 G and H ). In the trained group, heart rate increased afterwards commencing the breathing techniques and normalized earlier compared with the control group, whereas MAP decreased during the breathing techniques and thereafter followed the same pattern as in the command grouping. LPS assistants resulted in fever, with a maximum temperature increase in the control group of 1.9 ± 0.2 °C (mean ± SEM), whereas this increment was less pronounced and normalized earlier in the trained group (Fig. 1I ). Cocky-reported symptoms (nausea, headache, shivering, and musculus and dorsum pain on a six-indicate Likert calibration) peaked ane.5 h later on LPS administration in both groups, but were attenuated in the trained individuals compared with the control group (reduction of 56% in peak levels; Fig. iJ ).

Cardiorespiratory parameters, temperature, and symptoms during experimental endotoxemia in control and trained subjects. (A) Carbon dioxide fractional pressure (pCO₂) in arterial blood. (B) Oxygen fractional force per unit area (pO_ii) in arterial blood. (C) pH in arterial blood. (D) Bicarbonate (HCO3⁻) in arterial blood. (E) Lactate in arterial claret. (F) Oxygen saturation measured by pulse oximetry. (G) Heart rate (Hr). (H) Mean arterial pressure (MAP). (I) Temperature. (J) Score of cocky-reported symptoms. Data are expressed every bit mean ± SEM of 12 subjects per group. Gray box indicates menstruation in which the trained subjects proficient their learned animate techniques. P values between groups were calculated using repeated measures ii-way analysis of variance (ANOVA, interaction term). AU, arbitrary units; bpm, beats per minute.

Cardiorespiratory and biochemical changes during cyclic hyperventilation and breath retention in a representative bailiwick of the trained group. (A) The respiratory charge per unit alternately increased to around 20 breaths per minute (bpm) for several minutes, and then dropped to zero during voluntary jiff retentiveness. These cyclic changes in respiration resulted in profound changes in (B) oxygen saturation, (C) heart rate, and (D) hateful arterial pressure. The data depicted were sampled from the monitor every 10 s. At the end of each hyperventilation phase and breath retentiveness phase, an arterial blood sample was fatigued for arterial blood gas assay, of which the results are listed in the table below D. The cycles of hyper/hypoventilation in this particular subject can be viewed in Movie S2.

Catecholamine and Cortisol Levels.

Plasma epinephrine levels (Fig. 3A ) increased sharply 1 h subsequently LPS administration and peaked at T = 1.v h in the control group. In trained subjects, baseline epinephrine levels were significantly college compared with the control group (hateful ± SEM: ane.02 ± 0.22 vs. 0.35 ± 0.06 nmol/L, P = 0.007) (unpaired Pupil t examination). Afterward starting practicing the learned breathing techniques, epinephrine levels further increased in this group and peaked just before administration of LPS (mean ± SEM: 2.08 ± 0.37 nmol/L at T = 0 h, with individual subjects reaching up to 5.3 nmol/L) and remained elevated until cessation of the breathing techniques. In contrast to epinephrine, norepinephrine and dopamine levels remained inside the reference range throughout the experiment (Fig. 3 B and C ). Norepinephrine levels were similar between groups during the breathing catamenia, although trained subjects displayed higher levels at baseline and later on abeyance of the breathing techniques. In contrast, dopamine levels were slightly lower in trained individuals during the breathing techniques but were like betwixt groups before and afterward. There were no differences in serum levels of the stress hormone cortisol between the groups before or during the menstruation in which the trained group proficient their techniques; however, levels normalized more quickly in trained individuals (Fig. 3D ).

Plasma cathecholamine concentrations and serum cortisol concentrations during experimental endotoxemia in command and trained subjects. (A) Plasma epinephrine. (B) Plasma norepinephrine. (C) Plasma dopamine. (D) Serum cortisol. Information are expressed as mean ± SEM of 12 subjects per group. Gray box indicates period in which the trained subjects skilful their learned breathing techniques. P values betwixt groups were calculated using repeated measures 2-fashion analysis of variance (ANOVA, interaction term).

Leukocyte Counts.

Total leukocyte counts in both groups showed the typical endotoxemia-induced biphasic pattern with an initial leukopenia followed by leukocytosis (Fig. S1A). Leukocyte concentrations were markedly higher in trained individuals. 30 min after commencement of the breathing techniques (T = 0 h), an increment in lymphocytes was observed in trained individuals, which was non present in the control group (Fig. S1B). Concentrations of neutrophils and monocytes were similar between groups at this early time point, but were distinctly higher in the trained group at later fourth dimension points (Fig. S1 C and D).

Plasma Cytokines.

Plasma concentrations of proinflammatory cytokines TNF-α, IL-6, and IL-8, and the anti-inflammatory cytokine IL-10 all markedly increased after LPS administration in both groups (Fig. 4). However, in trained individuals, TNF-α, IL-half-dozen, and IL-8 levels were significantly attenuated, whereas the IL-ten response was greatly augmented compared with the control group (TNF-α, IL-six, and IL-8 levels 53%, 57%, and 51% lower; IL-ten levels 194% higher). Furthermore, IL-10 levels in the trained group increased sharply early on afterwards LPS assistants (at T = one h) and peaked 1 h before the superlative observed in the control group. In line with previous reports (11), plasma levels of the proinflammatory cytokine IL-1β were barely detectable during human endotoxemia. Concentrations were below the detection limit (3.9 pg/mL) in all just iv subjects (two in each grouping, showing very low concentrations (iv–6 pg/mL) at one to iii time points with no apparent kinetics over time). Concentrations of the anti-inflammatory cytokine TGF-β showed no kinetics after administration of LPS and were not different between groups (Fig. S2A). Nosotros too measured plasma concentrations of leptin, an adipokine that exerts proinflammatory activity. At baseline (T = −one h), in that location was a trend toward lower levels of leptin in the trained grouping compared with the control group (mean ± SEM: three.36 ± 0.55 vs. four.99 ± 0.74 ng/mL, P = 0.09, unpaired Student t test), which remained apparent at all subsequent time points (Fig. S2B). Leptin kinetics showed a biphasic pattern with an initial pocket-size decrease followed past a gradual increment in both groups. Notwithstanding, in that location were no differences between groups over time.

Plasma cytokine concentrations during endotoxemia in control and trained subjects. (A, C, E, and G) Median values of pro- (TNF-α, IL-half-dozen, and IL-8) and anti-inflammatory (IL-10) cytokines (northward = 12 per group). (B, D, F, and H) Median ± interquartile range of expanse nether curve (AUC) of pro- (TNF-α, IL-half dozen, and IL-eight) and anti-inflammatory (IL-10) cytokines (northward = 12 per group; unit: ×ten⁴ pg/mL·h). P values were calculated using Mann–Whitney u tests.

Correlation Analyses.

As depicted in Fig. 5A , there was a stiff positive correlation (r _s = 0.82, P = 0.001) between epinephrine levels in the trained group at T = 0 h (30 min later commencing the breathing techniques) and the early increase in IL-10 levels at T = i h, which was not nowadays in the control group (r _s = 0.xviii, P = 0.571). Furthermore, at that place were significant inverse correlations betwixt levels of the anti-inflammatory cytokine IL-ten at T = ane h and pinnacle levels of the proinflammatory mediators TNF-α (at T = i.5 h), IL-6 (at T = two h), and IL-8 (at T = two h) in the trained group (Fig. v B–D ). In the control group, no such inverse correlations between IL-10 and proinflammatory cytokines were observed. In fact, we found meaning positive correlations betwixt preceding TNF-α and IL-6 levels on the ane hand and IL-10 levels at later time points (TNF-α^{T = i} vs. IL-10^{T = 2}: r _s = 0.59, P = 0.045 and IL-6^{T = 1.5} vs. IL-ten^{T = 2}: r _southward = 0.sixty, P = 0.039).

Correlations in trained individuals. (A) Correlation between peak plasma levels of epinephrine (at T = 0 h) and plasma levels of the anti-inflammatory cytokine IL-ten at T = one h. (B) Correlation between plasma levels of the anti-inflammatory cytokine IL-10 at T = one h and peak plasma levels of the proinflammatory cytokine TNF-α (at T = 1.5 h). (C) Correlation between plasma levels of the anti-inflammatory cytokine IL-10 at T = one h and height plasma levels of the proinflammatory cytokine IL-6 (at T = 2 h). (D) Correlation between plasma levels of the anti-inflammatory cytokine IL-10 at T = i h and peak plasma levels of the proinflammatory cytokine IL-8 (at T = 2 h). R and P values were calculated using Spearman correlation.

Discussion

Herein, we show that a brusk-term grooming program and practicing breathing techniques learned during this training program results in release of epinephrine, induction of early anti-inflammatory IL-10 product, and consequently attenuation of the proinflammatory innate immune response during experimental human endotoxemia. Too, trained individuals experienced fewer endotoxemia-associated flu-like symptoms, and a more swift normalization of fever and cortisol levels, which are likely the result of the attenuated proinflammatory response.

This study demonstrates that the in vivo innate immune response can exist voluntarily influenced in a nonpharmacological manner through voluntary activation of the sympathetic nervous system. In accordance with the information of our command grouping, man endotoxemia in itself has been shown previously to event in increased levels of epinephrine (12). Nevertheless, in trained individuals epinephrine levels were already greatly increased 30 min after starting time of practicing the breathing techniques, before LPS administration. Epinephrine levels in trained individuals were fifty-fifty higher than those reported in a recent study in which astute stress elicited by a bungee jump was found to suppress cytokine product by leukocytes ex vivo stimulated with LPS (13). As norepinephrine, dopamine, and cortisol levels were non increased in the training grouping, it appears that the techniques predominantly result in stimulation of the sympathetic input to the adrenal medulla, considering this is the nigh abundant source of epinephrine in the body and epinephrine-producing chromaffin cells in the adrenal medulla are much more abundant than those producing norepinephrine (xiv).

The observed potentiating effects on anti-inflammatory IL-10 production as well as the attenuation of proinflammatory cytokine levels are in agreement with a previously performed study, where epinephrine was i.five. administered before LPS in healthy volunteers and resulted in early and increased IL-10 production (vii), and with studies showing that pretreatment with IL-10 results in attenuation of the proinflammatory response in healthy volunteers (15, 16). In the training group, strong inverse correlations between IL-10 levels at an early fourth dimension point and later-occurring acme levels of the proinflammatory mediators were found, whereas in the control group the reverse was found: positive correlations between preceding levels of proinflammatory mediators with the later-occurring peak levels of IL-10. These findings bespeak that the proinflammatory response drives IL-10 production in the control group, whereas the epinephrine-induced early on increase in IL-ten product inhibits proinflammation in the trained group. The early increases in lymphocytes and subsequent higher concentrations of circulating neutrophils in the training grouping compared with the control group can also be attributed to the elevated epinephrine levels found in trained individuals, every bit catecholamines induce leukocytosis characterized by an initial lymphocytosis followed past an increase of other subpopulations (17). Furthermore, like changes in leukocyte counts were previously observed during voluntary hyperventilation (eighteen). Our study is limited by the fact that we did non measure specific leukocyte subtypes such as CD3, CD4, and CD8 numbers also equally B cells, dendritic cells, and natural killer (NK) cells, some of which have been shown to exist specifically altered by catecholamines and/or stress (xix, xx).

It appears that mainly the breathing techniques used by the trained individuals account for the increase in epinephrine and subsequent attenuation of the inflammatory response. A limitation of our study pattern is that information technology does not allow the identification of the particular component of the skilful breathing exercises that results in increased epinephrine levels. Furthermore, the effect of the length of the training and the length of propensity for altered responses afterward training has notwithstanding to exist determined. All the same, the furnishings on epinephrine are likely a event of both the hyperventilation phase and hypoxia due to breath retentiveness, as both have been demonstrated to increase epinephrine levels (eighteen, 21–24). The hyperventilation-induced increase in epinephrine was shown to be dependent on decreased levels of bicarbonate, equally hyperventilation combined with bicarbonate infusion (resulting in hypocapnia and alkalosis, but normal bicarbonate levels) nullified epinephrine increase (24). In concordance, in the present study, bicarbonate levels were significantly lower in the trained subjects during practicing of the breathing techniques compared with control subjects. The attenuated cytokine response is unlikely to be a direct result from low pCO₂ and high pH levels because hypocapnic alkalosis, equally opposed to hypercapnic acidosis (25), is not associated with anti-inflammatory effects. Therefore, epinephrine is the near probable intermediate gene (seven). Even so, it cannot exist ruled out that other elements of the training, apart from practicing the breathing exercises, ultimately affected the LPS-induced innate immune response. For case, the exposition to extreme cold and subsequent rewarming during the training sessions might have resulted ischemic preconditioning and/or release of danger associated molecular patterns (DAMPs), which could result in a tolerant country toward a subsequent LPS claiming.

It remains to be determined whether the results of this study using an acute model of inflammation in salubrious volunteers can be extrapolated to patients with chronic autoimmune diseases. For example, chronic stress might exist harmful in these atmospheric condition due to induction of proinflammatory mediators (26), whereas bouts of short-term stress, similar to the effects of the training intervention described in this study, may be benign due to immunosuppressive effects (26). Of interest, the in vivo anti-inflammatory potential in humans of biologics currently used in the treatment of rheumatoid arthritis was showtime established in proof-of-principle human endotoxemia studies (27, 28), illustrating the relevance of the model to investigate novel therapies for this blazon of affliction.

In determination, the present proof-of-principle study demonstrates that the sympathetic nervous system and immune system can be voluntarily influenced through practicing techniques that are relatively easy to larn inside a short fourth dimension frame. Information technology therefore could have important implications for the treatment of a variety of weather associated with excessive or persistent inflammation, especially auto-immune diseases.

Materials and Methods

Subjects.

This parallel randomized controlled written report was registered at ClinicalTrials.gov equally {"type":"clinical-trial","attrs":{"text":"NCT01835457","term_id":"NCT01835457"}}NCT01835457. After approval by the local ideals committee of the Radboud University Nijmegen Medical Centre (CMO 2012/455), 30 healthy, nonsmoking, Dutch male volunteers were included in the trial. All subjects provided written informed consent and experiments were in accordance with the Declaration of Helsinki, including electric current revisions, and Good Clinical Practise guidelines. Subjects were screened before the beginning of the experiment and had a normal concrete examination, electrocardiography, and routine laboratory values. Exclusion criteria were: febrile disease during the 2 wk before the endotoxemia experiment, taking any prescription medication, history of spontaneous vagal collapse, practicing or feel with whatever kind of meditation, or participation in a previous trial where LPS was administered. The subjects were randomly allocated to the trained group (n = 18) or the control group (n = 12) past the opening of a sealed envelope prepared by a research nurse non involved in the report. Later on having fulfilled the training program, 12 of the 18 trained subjects were randomly assigned to participate in the experimental endotoxemia experiments (further explained in Study Pattern and Preparation Procedure below).

Three subjects in the control group that underwent endotoxemia on the aforementioned solar day and received LPS from the same ampoule were excluded from the trial and replaced. Their symptoms, temperature rise, hemodynamic response, and cytokine response were inconsistent with having received an adequate dose of 2 ng/kg LPS. Batchwise determination of cytokine levels revealed exceptionally low levels in all 3 subjects: Their summit cytokine response (TNF-α and IL-half-dozen) was less than half of that of the lowest recorded in a accomplice of 112 salubrious male subjects that previously underwent experimental endotoxemia (x) and peaked at atypical time points (subject 1, TNF-α = 39 pg/mL at four h later on LPS administration and IL-six = 27 pg/mL at 4 h subsequently LPS assistants; field of study 2, TNF-α = 32 pg/mL at 3 h after LPS administration and IL-6 = 31 pg/mL at 3 h after LPS administration; and subject 3, TNF-α = ix pg/mL at ii h after LPS administration and IL-6 = seven pg/mL at three h after LPS administration). Therefore, a endotoxin dose administration error was assumed and the subjects were replaced.

Study Design and Training Process.

The written report was sequentially conducted in two identical blocks, each consisting of 9 subjects in the trained grouping (of which half dozen finally participated in the endotoxemia experiments, further explained beneath) and 6 subjects in the control grouping. This design was chosen to minimize the bias due to differences in the interval betwixt the end of the training period and the endotoxemia experiments. Equally the aim of our study was to investigate the effects of the training intervention on the innate immune response in a standardized model of systemic inflammation, we did not assess the furnishings of the training intervention on immune organisation parameters in the absenteeism of endotoxemia. A schematic overview of the study blueprint (one block) is depicted in Fig. S3. The trained grouping was trained past Dutch private Wim Hof and three trainers who previously received an instructor form by Wim Hof to become a trainer. A medical doctor of the study team (L.T.5.Due east.) and the principal investigator (M.K.) were present during all grooming sessions (in Poland and in Holland), and during the experimental endotoxemia experiments. The start iv d of the training plan took identify in Poland and were most intensive. The program consisted of three main elements: meditation, exposure to common cold, and breathing techniques (see Movie S1 for an impression of the training program).

• i)

  Meditation, and then-called "third eye meditation," a form of meditation including visualizations aimed at total relaxation.

• ii)

  During the training, subjects voluntarily exposed themselves to cold in several means: standing in the snow barefoot for up to xxx min and lying bare chested in the snowfall for 20 min; daily dipping/swimming in water ice-common cold h2o (0–1 °C) for up to several minutes (including complete submersions); and hiking upwards a snowy mount (meridian: ane,590 thousand) blank chested, wearing zip but shorts and shoes at temperatures ranging from −5 to −12 °C (air current chill: −12 to −27 °C).

• 3)

  Breathing techniques, consisting of two exercises. In the commencement exercise subjects were asked to hyperventilate for an average of thirty breaths. Afterward, the subjects exhaled and held their jiff for ∼2–3 min ("retention phase"). The duration of jiff retention was entirely at the discretion of the bailiwick himself. Breath memory was followed by a deep inhalation jiff, that was held for 10 south. Afterward a new cycle of hyper/hypoventilation began. The 2d exercise consisted of deep inhalations and exhalations in which every inhalation and exhalation was followed by jiff holding for 10 s, during which the subject tightened all his body muscles. These two breathing exercises were as well performed during the endotoxemia experiments. Additional chemical element of the grooming programme consisted of strength exercises (due east.yard., push button-ups and yoga balance techniques).

After returning from Poland, the subjects practiced the techniques they learned daily by themselves at home (two–3 h/d; cold exposure was accomplished through taking cold showers) until the endotoxemia experiment twenty-four hours (five–nine d later). In add-on, a final group training took place and at the end of this twenty-four hour period, vi of the nine trained subjects (in each cake) were randomly selected for participation in the endotoxemia experiments, using the sealed envelope method. This selection was performed to let for field of study replacement in instance of an adverse event or affliction in i of the trained subjects selected for the endotoxemia experiments. The selected subjects practiced in a final training session led past Wim Hof on the twenty-four hour period before the endotoxemia experiment day. Wim Hof was present to coach the subjects during the endotoxemia experiment days during the 3 h that the subjects in the trained group practiced the learned techniques. The command group did non undergo whatsoever training procedures throughout the study flow.

Experimental Human Endotoxemia.

Subjects refrained from caffeine- or alcohol-containing substances 24 h before the start of the experiment, and food 10 h before the start of the endotoxemia experiment. The experiments were performed at the research unit of the intensive care department. The procedures on the endotoxemia experiment 24-hour interval are depicted in Fig. S4. Purified lipopolysaccharide (LPS, Usa Standard Reference Endotoxin Escherichia coli O:113) obtained from the Pharmaceutical Development Section of the National Institutes of Health, supplied as a lyophilized powder, was reconstituted in 5 mL saline 0.9% for injection and vortex mixed for at least xx min afterward reconstitution. The LPS solution was administered as an i.v. bolus injection at a dose of 2 ng/kg body weight in 1 min at T = 0 h. A cannula was placed in an antecubital vein to permit infusion of 0.ix% NaCl solution; the subjects received 1.v 50 0.9% NaCl during 1 h starting 1 h before endotoxin infusion (prehydration) every bit part of our standard endotoxemia protocol (29), followed by 150 mL/h until 6 h after endotoxin infusion and 75 mL/h until the stop of the experiment. The radial avenue was cannulated using a xx-approximate arterial catheter (Angiocath; Becton Dickinson) and connected to an arterial pressure monitoring ready (Edwards Lifesciences) to allow the continuous monitoring of blood pressure and blood sampling. Eye rate (three-lead electrocardiogram), blood pressure, respiratory charge per unit, and oxygen saturation (pulse oximetry) information were recorded from a Philips MP50 patient monitor every 30 s by a custom in-house–developed data recording system, starting i h before administration of LPS until belch from the intensive intendance unit eight h later LPS administration. Body temperature was measured using an infrared tympanic thermometer (FirstTemp Genius 2; Sherwood Medical). LPS-induced flu-like symptoms (headache, nausea, shivering, muscle and dorsum pain) were scored every 30 min on a six-point Likert scale (0 = no symptoms, 5 = worst ever experienced), resulting in a full score of 0–25.

Thirty minutes before LPS assistants (T = −0.5 h), subjects in the trained group started the first breathing technique (hyper/hypoventilation cycles, see Movie S2) until T = i h, followed by the second animate technique (deep inhalation and exhalation in combination with tightening muscles) until T = 2.5 h. Later on, the subjects stopped practicing all of the techniques. The control group did not exercise whatever techniques throughout the endotoxemia experiment day.

Blood Gas Parameters.

Blood gas parameters were analyzed in lithium heparin anticoagulated arterial claret using CG4+ cartridges and a point-of-care i-STAT claret gas analyzer (Abbott).

Catecholamines.

Blood was collected into chilled lithium-heparin tubes and were immediately placed on ice and centrifuged at 2,000 × k for 10 min at 4 °C after which plasma was stored at −lxxx °C until analysis. Plasma norepinephrine, epinephrine, and dopamine concentrations were measured using routine analysis methods likewise used for patient samples (HPLCy with fluorometric detection, as described previously) (30).

Cortisol.

Blood was collected in serum-separating tubes and was allowed to clot at room temperature for a minimum of xxx min. Subsequently, samples were centrifuged at 2,000 × k for ten min at iv °C, after which serum was stored at −80 °C until analysis. Cortisol levels were determined using a routine analysis method also used for patient samples (electrochemiluminescent immunoassay on a Modular Analytics E170 (Roche Diagnostics).

Leukocyte Counts and Differentiation.

Analysis of leukocyte counts and differentiation was performed in EDTA anticoagulated claret using routine analysis methods also used for patient samples (flow cytometric assay on a Sysmex XE-5000.

Plasma Cytokines.

EDTA anticoagulated blood was centrifuged immediately at 2,000 × g for 10 min at 4 °C subsequently which plasma was stored at −80 °C until analysis. Concentrations of TNF-α, IL-6, IL-8, and IL-10 were measured using a simultaneous Luminex assay according to the manufacturer's instructions (Milliplex; Millipore). IL-1β, TGF-β, and leptin were measured using ELISAs according to the manufacturer's instructions (IL-1β and TGF-β, Quantikine and leptin, Duoset; both R&D Systems).

Calculations and Statistical Analysis.

Data are represented as median and interquartile range/range or mean and SEM based on their distribution (calculated by the Shapiro–Wilk examination). Statistical tests used are indicated in the figure/table legends or text. Spearman's correlation was used. A P value of <0.05 was considered statistically meaning. Statistical calculations were performed using Graphpad Prism version 5.0 (GraphPad Software).

Supplementary Material

Acknowledgments

The authors thank K. Mostard, K. Pijper, D. Bernard, and E. Hof for aid during the preparation sessions; the Radboud University Nijmegen Sports Centre for providing space for training days in The Netherlands; and the inquiry nurses of the Radboud Academy Medical Centre Intensive Care Unit for help during the endotoxemia experiments. This study was supported past a Serendipity Grant from Reumafonds (world wide web.reumafonds.nl).

Footnotes

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034215/

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