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Effects of Tigecycline and Doxycycline on Inflammation and Hemodynamics in Porcine Endotoxemia

A Prospective, Randomized, and Placebo-Controlled Trial

von Seth, Magnus*; Sjölin, Jan; Larsson, Anders; Eriksson, Mats*; Hillered, Lars§; Lipcsey, Miklós*

doi: 10.1097/SHK.0000000000000351
Basic Science Aspects
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ABSTRACT Antibiotics might, apart from an antimicrobial effect, also exert anti-inflammatory effects. The novel antibiotic tigecycline, potentially useful in septic shock from gram-negative multiresistant bacteria, is structurally related to antibiotics with known anti-inflammatory properties. However, its anti-inflammatory effects have not been previously explored in vivo. Using a sterile integrative porcine sepsis model, we investigated the anti-inflammatory and circulatory effects of tigecycline in comparison with doxycycline and placebo. Eighteen pigs were randomized to receive tigecycline 100 mg, doxycycline 200 mg, or placebo and subjected to 6-h endotoxin infusion at 0.5 μg kg−1 h−1. Markers of inflammation, nitric oxide production, vascular permeability, hemodynamics, organ dysfunction, tissue metabolism, and acid-base parameters were monitored. Peak plasma tumor necrosis factor-α was lower in the doxycycline group (P = 0.031) but not in the tigecycline group (P = 0.86) compared with placebo, with geometric mean plasma concentrations of 16, 79, and 63 ng mL−1, respectively. Mean arterial pressure was higher 4 to 6 h in the tigecycline group, with values at 6 h of 107 ± 9 mmHg compared with the placebo and doxycycline groups (85 ± 27 mmHg and 90 ± 32 mmHg, respectively; P = 0.025). The white blood cell and the neutrophil granulocyte counts were less reduced in the doxycycline group but not in the tigecycline group at 4 to 6 h (P = 0.009 and P = 0.019, respectively). Other markers of inflammation, organ dysfunction, tissue metabolism, and acid-base parameters were unaffected by tigecycline. Consistent with known anti-inflammatory properties, doxycycline yielded decreased tumor necrosis factor-α levels. Tigecycline did not affect cytokine levels but counteracted hypotension and hypoperfusion.

*Section of Anesthesiology and Intensive Care, Department of Surgical Sciences, Sections of Infectious Diseases and Clinical Chemistry, Department of Medical Sciences, and §Section of Neurosurgery, Department of Neuroscience, Uppsala University, Uppsala, Sweden

Received 30 Sep 2014; first review completed 21 Oct 2014; accepted in final form 28 Jan 2015

Address reprint requests to Magnus von Seth, MD, Section of Anesthesiology and Intensive Care, Department of Surgical Sciences, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden. E-mail: magnus.von.seth@surgsci.uu.se.

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INTRODUCTION

Early and appropriate antibacterial treatment has been shown to decrease mortality in severe sepsis and septic shock (1). Because the causative agent usually is unknown at presentation of symptoms, broad-spectrum antibiotics or combinations of antibiotics are used in the initial treatment of septic shock to ensure that possible organisms are covered by at least one active drug. Recently, it was demonstrated that two active antibiotics resulted in an additive beneficial effect in patients with the most life-threatening infections (1–6).

In severe sepsis and septic shock, multiple organ dysfunction syndrome contributes significantly to mortality, and the search for substances that would inhibit the developing organ dysfunction has been ongoing the last two decades. Apart from controlling the trigger of sepsis by exerting antimicrobial effects, several antibiotics have been shown to exhibit some degree of anti-inflammatory activity (7–13). Synergistic antimicrobial and anti-inflammatory effects have been proposed as possible modes of action explaining the beneficial effect of the combination of two active antibiotics (3, 4).

Tigecycline, to date, the only commercially available glycylcycline, is a chemically modified tetracycline. Tetracyclines are antibiotics with proven anti-inflammatory properties (8–10, 12, 14). Therefore, the aim of the present study was to investigate whether tigecycline exhibits anti-inflammatory properties similar to tetracycline and, if any, anti-inflammatory properties of the drugs would alter the progression into multiple organ dysfunction in sepsis with an exaggerated inflammatory response. Another nonantimicrobial effect by tigecycline that might affect organ function is its in vitro capability to inhibit nitric oxide (NO), an important molecule in sepsis-induced hypotension (15–18). An additional hypothesis was therefore that tigecycline would inhibit NO production and excretion and thereby mitigate hypotension in septic shock.

To test these hypotheses, a sterile integrative porcine intensive care model of endotoxin-induced septic shock was used. This model gives the opportunity to study the immunomodulating properties of the antibiotics without any interference of their antibacterial effect (19). To ensure that a tetracycline-like anti-inflammatory effect was demonstrable with the model used, doxycycline was used as a control substance.

Accordingly, the primary study end point was to compare the production of tumor necrosis factor-α (TNF-α) during endotoxin administration in tigecycline-treated animals with that in animals given doxycycline or saline. Effect on excretion of NO metabolites, hypotension, tissue metabolism, and organ dysfunction constituted secondary end points.

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MATERIALS AND METHODS

Subjects

The study included 18 domestic male pigs weighing between 22.3 and 29.3 kg. The animals were 9 to 11 weeks old, and all pigs were handled according to the guidelines of the Swedish National Board for Laboratory Animals and the European Convention on Animal Care. The experiment was approved by the Animal Ethics Committee of Uppsala University, Sweden.

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Inclusion criteria

The following inclusion criteria were applied: no apparent disease, arterial oxygen tension (PaO2) more than 10 kPa, and a mean pulmonary arterial pressure (MPAP) of 20 mmHg or less at baseline, which was 60 min after the completed preparatory procedure (see below).

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Anesthesia

The animals were fasted overnight but had free access to water until they were transported to the laboratory. A single intramuscular injection of 50 mg xylazine (Bayer, Leverkusen, Germany) was given to all animals as premedication before transport to the laboratory. General anesthesia was induced by injecting a mixture of 6 mg kg−1 tiletamine-zolazepam (Virbac Laboratories, Carros, France) and 2.2 mg kg−1 xylazine intramuscularly. Anesthesia was maintained with a constant rate of sodium pentobarbital (8 mg kg−1 h−1; Apoteket, Umeå, Sweden), pancuronium bromide (0.26 mg kg−1 h−1; Organon, Oss, The Netherlands), and morphine (0.48 mg kg−1 h−1; Pfizer, Sollentuna, Sweden) dissolved in 2.5% glucose solution. Ringer’s acetate was infused intravenously, resulting in a total fluid administration rate of 30 mL kg−1 h−1 during the entire experiment.

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Preparatory procedure

A bolus dose of 20-mg morphine and 100-mg ketamine hydrochloride (Pfizer) was administered intravenously before securing the airway with orotracheal intubation. The animals were mechanically ventilated throughout the experimental procedure (Servo-I; Maquet, Stockholm, Sweden) with 30% oxygen in medical air. After completion of the preparation procedure, ventilation was adjusted to yield a PaCO2 between 5.0 and 5.5 kPa and, thereafter, ventilator settings were kept constant throughout the experiment. The respiratory rate was 25 min−1, and the inspiratory-to-expiratory ratio was 1:2. A positive end-expiratory pressure of 5 cm H2O was maintained. The pigs were placed in prone position as soon as the preparation was completed.

A cervical artery was catheterized for pressure monitoring and blood sampling. A central venous line and a Swan-Ganz catheter were inserted through the external jugular vein into the superior caval vein and into the pulmonary artery, respectively. A CMA 60 catheter (30-mm membrane) 100-kd cutoff molecular weight microdialysis catheter (M Dialysis AB, Solna, Sweden) was inserted into a thigh muscle. The catheter was perfused with perfusion fluid (T1; M Dialysis AB) by a CMA 107 pump (M Dialysis AB) at a rate of 0.2 μL min−1. A bladder incision was performed with an indwelling urinary catheter introduced into the bladder. After completed preparation, a 60-min stabilization period passed before baseline values were registered and baseline blood samples collected.

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Protocol

The pigs were randomly allocated to one of three groups: tigecycline group (n = 6), doxycycline group (n = 6), or placebo group (n = 6). Randomization in blocks was performed. The tigecycline group received 100 mg tigecycline (Pfizer, Berkshire, UK), and the doxycycline group received 200 mg doxycycline (Merckle, Blaubeuren, Germany). The drugs were given as an intravenous infusion in 100 mL of normal saline immediately after the preparation was completed, that is, 1 h before the endotoxin infusion was started at baseline. The placebo group received the corresponding volume of normal saline during the same period. Endotoxemic shock was induced in all pigs by a 6-h-long continuous infusion of Escherichia coli endotoxin (E. coli O111:B4; Sigma Chemicals, St. Louis, Mo) at 0.5 μg kg−1 h−1. During the first hour of endotoxin infusion, a salvage dose of 1-mL adrenaline 1:100,000 was given if mean arterial pressure (MAP) was equal to or less than the MPAP. Arterial blood, mixed venous blood, microdialysis, and urine samples were collected at baseline and then hourly for the rest of the experiment. At the same time points, cardiac output was measured with the thermodilution method, and physiology data were registered (e.g., airway pressures from the ventilator). The pigs were terminated at the end of the experiment with an intravenous potassium chloride injection.

Derived physiologic variables were calculated from their conventional formulae.

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Biochemical analyses

Sandwich enzyme-linked immunosorbent assays (DY686 and DY690; R&D Systems, Minneapolis, Minn) were used to determine TNF-α and interleukin-6 (IL-6) in plasma. S100B was measured by CanAg S100 EIA assay (Fujirebio Diagnostics, Gothenburg, Sweden). Urinary excreted nitrate was measured using the Parameter assay (R&D Systems). Blood cell count and hemoglobin were analyzed on a CELL-DYN 4000 (Abbott, Abbott Park, Ill) and plasma creatinine (reagent 14.3600.01; Synermed International, Westfield, Ind) and bilirubin on an Architect Ci8200 analyzer (Abbott). Arterial and mixed venous blood gases were analyzed on an ABL 300 and a Hemoximeter (Radiometer, Brønhøj, Denmark).

The microdialysis samples were analyzed for glucose, lactate, pyruvate, glutamate, glycerol, and urea using the CMA 600 analyzer (M Dialysis AB). Between-assay coefficients of variation were less than 10% for all analytes.

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Statistical analysis

Sample size was calculated using data from previous experiments. To detect a difference of 10% in log-TNF-α at 1 h between the tigecycline and placebo group with a power of 0.8, an α error of 0.05, and a standard deviation of 10%, six animals in each group were required. The mean and variance of TNF-α at 1 h from this experiment corresponded to the data the power calculation was based on.

Data were tested for normal distribution by the Kolmogorov-Smirnov test.

Apart from cytokine response that typically peaks early during the experiment, the main impact of an anti-inflammatory intervention would be expected late during the experiment. Consequently, group differences in cytokine response were assessed at peak cytokine response with one-way analysis of variance (ANOVA). Responses in all other variables were assessed during the last 3 h of the experiment (4 – 6 h) with split-plot ANOVA. If this test indicated a group difference at the end of the experiment, a post hoc analysis was performed to identify which groups differed. For non-normally distributed data, Mann-Whitney U test was used to calculate between-group differences.

To verify the development of sepsis, hypoperfusion and organ dysfunction changes from baseline to the end of the experiment, 6 h, were assessed with paired t test or Wilcoxon matched-pairs test as appropriate.

Data with normal distribution are presented as mean ± SD, unless otherwise indicated, whereas data with non-normal distribution are presented as median (range). STATISTICA software (StatSoft, Tulsa, Okla) was used to analyze the data. A value of P < 0.05 was considered significant. P values were corrected for multiple testing by using the unequal means method post hoc analyses in the ANOVA module (that inherently corrects for multiple testing).

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RESULTS

All animals fulfilled the inclusion criteria and were comparable in baseline data. All piglets developed endotoxemic shock, defined as an increase of mean pulmonary arterial pressure to twice the baseline value. One pig in the placebo group received one rescue dose of adrenaline, and one pig in the tigecycline group received three doses of adrenaline during the first hour of endotoxin infusion in accordance with the protocol. During the endotoxin infusion, there were signs of severe sepsis with decreases in pulmonary compliance, arterial pO2, and creatinine clearance indicating organ dysfunction (Table 2); increase in veno-arterial carbon dioxide tension indication hypoperfusion (Table 3); and increase in hemoglobin concentration indicating capillary leakage (Table 1). All animals survived 6 h of endotoxin infusion and were then terminated in accordance with the protocol.

Table 1

Table 1

Table 2

Table 2

Table 3

Table 3

Plasma TNF-α and plasma IL-6 demonstrated log-normal distribution. Plasma S100B levels, urine nitrate, heart rate, creatinine clearance, and muscle lactate-to-pyruvate ratio demonstrated a non-normal nonlogarithmic distribution. All other variables were normally distributed.

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Markers of inflammation and vascular permeability

Markers of inflammation and vascular permeability are shown in Figures 1 and 2 and Table 1. Plasma TNF-α peaked at 1 h in all groups (Fig. 1). Geometrical means of peak TNF-α in the tigecycline, doxycycline, and placebo groups were 79, 16, and 63 ng mL−1, respectively; corresponding to log10 concentrations of 4.9 ± 0.3, 4.2 ± 0.6, and 4.8 ± 0.3, respectively. Thus, during endotoxemia, plasma TNF-α was lower in the doxycycline group compared with the placebo group (P = 0.031) and peak concentration at 1 h only 25% of that in the placebo group. In contrast, plasma TNF-α concentrations were similar between the tigecycline group and the placebo group (P = 0.86). Plasma IL-6 did not differ between the three groups during the experiment, whereas the white blood cell count (Fig. 2) and the neutrophil granulocyte count were less reduced in the doxycycline group versus the placebo group at 4 to 6 h (P = 0.009 and P = 0.019, respectively). The platelet count decreased from 0 to 6 h, without any intergroup difference. Blood hemoglobin levels, which might serve as a surrogate parameter for capillary leakage, increased during the experiment in the doxycycline and placebo groups but not in the tigecycline group. However, there was no group difference at 4 to 6 h.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Urinary nitrate concentration, a marker of the NO activity in blood, did not change significantly across time in any group, and there were no significant differences between the groups.

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Hemodynamic parameters and organ dysfunction

Hemodynamic parameters and organ dysfunction are shown in Figures 3 and 4 and Table 2. Heart rate increased in the doxycycline and tigecycline groups but not in the placebo group. There were no intergroup differences at 4 to 6 h. In two pigs in the doxycycline group, a supraventricular tachycardia developed and persisted for the last 3 h of the experiment. The MAP decreased from baseline in the doxycycline and placebo groups but not in the tigecycline group (Fig. 3). The MAP was higher in the tigecycline group compared with the placebo group (P = 0.025). Correspondingly, systemic vascular resistance index was higher in the tigecycline group compared with the placebo group (P = 0.03). The MPAP increased from baseline in all groups during the experiment. Cardiac index decreased during the experiment, with a nadir at 3 h. Mixed venous oxygen saturation (SvO2) also demonstrated a transient decrease. During the last 3 h of the experiment, there was a group difference in SvO2 (P = 0.048). However, specific intergroup differences could not be identified in the post hoc analysis (Fig. 4). At 6 h, SvO2 was lower only in the doxycycline and placebo groups as compared with baseline. There was a decrease in PaO2 and static compliance and an increase in arterial carbon dioxide tension in all groups. No intergroup differences were noted. Creatinine clearance decreased numerically 0 to 6 h in all groups, however reaching significance only in the tigecycline group (P = 0.02). No intergroup differences in serum bilirubin at 4 to 6 h were detected during the experiment. S100B levels in plasma were generally low. There were no significant differences at 4 to 6 h.

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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Acid-base parameters and tissue metabolism

Acid-base parameters and tissue metabolism are shown in Table 3. During the experiment, pH decreased in the doxycycline and placebo groups but not in the tigecycline group. Base excess decreased in the placebo group but not in the treatment groups. The veno-arterial carbon dioxide tension difference peaked at 3 h. There was a group difference 4 to 6 h with split-plot ANOVA, but post hoc analysis could not identify any significant differences when comparing with placebo. There were no intergroup differences in plasma lactate or in the microdialysis variables, such as muscle lactate, muscle lactate-to-pyruvate ratio, and muscle glycerol levels.

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DISCUSSION

Previous studies

Tetracyclines and related compounds may have anti-inflammatory properties via several different molecular pathways. At the cellular level, in neutrophil granulocytes, the effect on Ca2+ influx and interference with the production of reactive oxygen species have been proposed as plausible mechanisms of the anti-inflammatory effect. Moreover, tetracyclines and related compounds are potent inhibitors of matrix metalloproteases and the release of TNF-α into the bloodstream is dependent on cleavage of a membrane bound pro-TNF dimer into a soluble form by matrix metalloproteinase-9 (8–10, 20). Despite these broad effects on the inflammatory pathways, relatively little data are available on the effects of tetracyclines and related compounds on organ failure. One study reported decreased mortality in endotoxemic rats after administration of tetracycline base and doxycycline (10), whereas another study reported prevention of hypotension and preservation of lung function in pigs with bowel ischemia and fecal peritonitis after treatment with tetracycline (12).

The role of the doxycycline arm in the present study was to demonstrate that tetracycline-like substances inhibited the inflammatory response to endotoxemia in the model used. In contrast to doxycycline, tigecycline did not attenuate the inflammatory response as measured by cytokine release.

The glycylcyclines are chemically modified tetracyclines. Tigecycline, to date, the only commercially available glycylcycline, is structurally derived from minocycline by adding a side chain to the carbon ring of the tetracycline backbone, yielding an extended antimicrobial spectrum (21). How this modification preserves the anti-inflammatory properties is not completely explored. Previously published data on the effect of tigecycline on TNF-α release are conflicting. Mice treated with tigecycline subcutaneously showed a reduction in the expression of TNF-α in a mycoplasma lower airway infection model; however, the difference did not occur until after 3 days of treatment (22). Other studies demonstrated no effect of tigecycline on TNF-α release from endotoxin-stimulated human leukocytes (23, 24). In what way tigecycline inhibits release of TNF-α in a large animal model has not been studied previously. Thus, the finding of our study that tigecycline did not decrease TNF-α liberation in the circulation in vivo is consistent with previous ex vivo studies, and it may be speculated that the effect seen in the mycoplasma study might be the result of the antimicrobial effect of tigecycline and not an anti-inflammatory effect per se.

Nitric oxide is an important mediator of hypotension and capillary leakage in septic shock (15, 17). A tendency to a greater overall hemodynamic stability in the tigecycline group was reflected by less reduction in MAP, SvO2, and base excess. The reduced increase in blood hemoglobin in the tigecycline group could have also been caused by a diminished NO-mediated capillary leakage. Inhibition of the production of NO could thus be one of the candidate pathways that mediate the effect of tigecycline on blood pressure. Doxycycline and tigecycline have recently been subjected to a proteomics study to investigate their anti-inflammatory effect, and both were shown to prevent the production of NO, with tigecycline being the more potent inhibitor (18). However, NO is a short-lived molecule with a plasma half-life in the span of 20 s. Its metabolites, nitrite and nitrate, are excreted in urine and can be analyzed and used as an indirect parameter of NO production. Measuring NO metabolites in urine is associated with several sources of error. Nitric oxide production has several feedback loops, and NO may be produced in tissues other than inflammatory cells. Furthermore, renal excretion of NO metabolites is dependent on glomerular excretion and tubular reabsorption that may be affected by the sepsis-induced inflammatory response and circulatory changes (16). In this study, we were not able to detect any significant difference in the urinary nitrite/nitrate excretion. Further studies, where NO is measured directly in serum, can elucidate whether NO-induced hypotension is affected by tigecycline inhibition of NO synthesis. The consequences of the reduced hypotension are not known but, in the present study, the increased hemodynamic stability in the tigecycline group did not translate into improved organ function or tissue metabolism in the muscle.

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Limitations and strengths

A limitation of this model of porcine endotoxemia is the short observation period of 6 h. It cannot be excluded that the changes in circulatory variables seen at 6 h in this study could have had a significant effect on organ dysfunction with a longer observation period. Nor can a late anti-inflammatory effect of tigecycline be excluded.

In our study, all piglets developed hypodynamic shock, as opposed to the hyperdynamic circulatory state often encountered in adult sepsis resuscitated with fluids and vasopressors. These piglets, however, were not given vasopressors except for single bolus doses of adrenaline during the first hour, and fluids were not given to target normotension. Furthermore, our animals are young and the sepsis induced is similar to the hypodynamic sepsis often encountered in pediatric patients (25).

This is an experimental animal study, and results from animal studies should always be extrapolated to humans with caution. However, in terms of medical research, pigs have the greatest similarities to humans among nonprimate species (26). In addition, the size of the pig facilitates the utilization of human intensive care equipment, which increases the clinical relevance of the model because sedation, mechanical ventilation, and vasopressors affect the innate immune response in these intensive care patients at risk for infections with multidrug-resistant bacteria, where treatment with tigecycline might be an option (27).

A very limited amount of research has been made on the pharmacokinetics of tigecycline and doxycycline in the pig. A doxycycline dose of 200 mg to young pigs results in plasma concentrations similar to those seen in humans (28). Elimination rate is somewhat quicker in the pig but is of limited importance in this short-term endotoxin model. To our knowledge, there are up to now no published data on tigecycline pharmacokinetics in the pig. However, given that these juvenile pigs were given the human adult loading dose of tigecycline, it is less likely that therapeutic antimicrobial levels were not reached.

The strength of this nonbacterial model is its ability to study the effect of antibiotic drugs in a sepsis-like condition with multiple organ dysfunction syndrome without the bias of their antimicrobial effects, given that alimental bacterial translocation does not significantly contribute to the deterioration seen during the experiment. Another strength is the addition of a group treated with doxycycline, an antimicrobial agent known to inhibit TNF-α increase in plasma, to confirm that the anti-inflammatory effects of tetracyclines could be demonstrated with the model chosen.

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Key findings

In this animal study, doxycycline, but not tigecycline, yielded lower TNF-α levels after endotoxemia. Anti-inflammatory effects with less reduction on white blood cell count and neutrophil granulocyte count could be seen in the doxycycline group in contrast to the tigecycline group. No other effect on the progression into multiple organ failure was noted.

Tigecycline counteracted the development of hypotension and decrease of systemic vascular resistance. Moreover, mixed venous saturation, capillary leakage, veno-arterial carbon dioxide tension difference, base excess, and pH did not deteriorate in tigecycline-treated animals, as in the two other groups. In our study, this could not be attributed to differences in NO production.

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ACKNOWLEDGMENTS

The authors thank Charina Brännström, Inger Ståhl-Mylliaho, Anders Nordgren, and Monika Hall for excellent technical assistance.

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Keywords:

Animal models; sepsis; endotoxins; tigecycline; doxycycline; tumor necrosis factor-α; swine; nitric oxide

© 2015 by the Shock Society