Evaluation of small intestinal damage in a rat model of 6Minutes cardiac arrest

Evaluation of small intestinal damage in a rat model of 6Minutes cardiac arrest Background: Contribution of the small intestine to systemic inflammation after cardiac arrest (CA) is poorly understood. The objective was to evaluate whether an in vivo rat model of 6 min CA is suitable to initiate intestinal ischaemia-reperfusion-injury and to evaluate histomorphological changes and inflammatory processes in the small intestinal mucosa resp. in sera. Methods: Adult male Wistar rats were subjected to CA followed by cardio-pulmonary resuscitation. Proximal jejunum and serum was collected at 6 h, 24 h, 72 h and 7 d post return of spontaneous circulation (ROSC) and from a control group. The small intestine was evaluated histomorphologically. Cytokine concentrations were measured in jejunum lysates and sera. Results: Histomorphological evaluation revealed a significant increase in mucosal damage in the jejunum at all timepoints compared to controls (p < 0.0001). In jejunal tissues, concentrations of IL-1α,IL-1β, IL-10, and TNF-α showed significant peaks at 24 h and were 1.5- to 5.7-fold higher than concentrations at 6 h and in the controls (p <0.05). In serum, a significant higher amount of cytokine was detected only for IL-1β at 24 h post-ROSC compared to controls (15.78 vs. 9.76 pg/ml). Conclusion: CA resulted in mild small intestinal tissue damage but not in systemic inflammation. A rat model of 6 min CA is not capable to comprehensively mimic a post cardiac arrest syndrome (PCAS). Whether there is a vital influence of the intestine on the PCAS still remains unclear. Keywords: Cardiac arrest, Small intestine, Ischaemia-reperfusion-injury, Systemic inflammatory response syndrome Background aggravates cell death. Later, accumulation of inflamma- Cardiac arrest (CA) results in transient systemic ischaemia tory cytokines is initiated and causes a long-lasting in- followed by reperfusion as a consequence of successful re- flammatory reaction. suscitation. First, cessation of circulation results in deple- Within 4–5 min after CA, selectively vulnerable regions + + tion of oxygen followed by ATP-dependent Na /K -pump in the brain such as the hippocampal CA1 become apop- dysfunction. Resulting in a breakdown of cellular integrity, totic and necrotic [2]. As a result, brain injury is respon- glutamate is being released intracellularly and mediates sible for the mortality of 68% of the victims of CA [3]; cerebral excitotoxicity by activation of N-methyl-D-aspar- neurocognitive long-term impairment occurs in half of ++ tate receptors. Subsequently, intracellular Ca influx and the survivors [4]. In addition to that, within hours to days, activation of caspases, phospholipases, and proteases the characteristic systemic ischaemia-reperfusion-injury lead to cellular death [1]. Secondly, subsequent reperfu- provokes a systemic inflammatory release also known sion triggers formation of free radicals, which further as sepsis-like- or post-cardiac arrest syndrome (PCAS) ending up in multiple organ failure [5, 6]. In fact, CA is a complex systemic ischaemia-reperfusion-injury with * Correspondence: Daniel.Schroeder@uk-koeln.de contribution of multiple independent tissue. However, to Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Cologne, Kerpener Str. 62, Cologne, Germany date, particular contribution of peripheral organs to the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 2 of 10 development of PCAS is not clearly understood. Given (35-45 mmHg). The inspired oxygen concentration (FiO ) that more than 30% of victims of CA show bacteremia was regulated to ensure a physiological pO . Blood gas upon presentation, the small intestine was discussed to analysis was performed using ABLFlex800 (Radiometer, be an immediate by-product of a systemic ischaemia- Germany). The cardio-pulmonary resuscitation (CPR) reperfusion-injury [7, 8]. protocol fulfils the Utstein Style guidelines for laboratory As described, the small intestine is highly susceptible CPR research [17]. The rats received an oesophageal elec- to a focal ischaemia-reperfusion-injury [9, 10]. Already trode for induction of ventricular fibrillation (12 V, 50 Hz, after 15–30 min of mesenterial occlusion, morphological 1.5 min) until the mean arterial blood pressure stayed changes such as atrophy of the villi and damage of tunica below 15 mmHg [18]. After 5.5 min of CA, rats were mucosa and tunica serosa appear [11]. Consequently, loss mechanically ventilated using 100% oxygen at 60 breaths of intestinal integrity is associated with excessive fluid loss per min. At 6 min after CA, CPR started by performing a and translocation of gut bacteria and toxins into the blood manual closed-chest cardiac massage (200 times/min) and circulation [12]. Subsequent restoration of blood flow leads an injection of 20 μg/kg epinephrine (Suprarenin, Sanofi- to an activation of molecular and cellular components of Aventis, Germany). Two min later, a single bi-phasic the innate immunity resulting in an inflammatory response shock of 2–3 J (M series, Zoll Corporation, Germany) [13]. As a result, local and systemic inflammation occurs was attempted. Epinephrine administration and biphasic and causes a multiple organ dysfunction syndrome with a shocks were repeated every 30 s. ROSC was defined as mortality rate reported between 30–90% [9, 10, 13–15]. maintenance of mean arterial blood pressure above However, due to the predominant brain damage 50 mmHg for at least 10 min. If ROSC was not achieved decisive for morbidity and mortality in survivors of CA after 6 min of CPR, resuscitation procedures were termi- the role of the small intestine in the development of sys- nated. To maintain normocapnia, ventilation rate was temic inflammation after CA was not intensively investi- adjusted and sodium bicarbonate was titrated according to gated yet. Thus, the objective was (i) to evaluate whether the blood gas analysis. Once adequate spontaneous breath- an in vivo rat model of 6 min CA is suitable to initiate an ing was observed, rats were extubated, kept singly and intestinal ischaemia-reperfusion-injury to further examine monitored every 2–4h. (ii) genesis of local and systemic inflammation. It was hypothesized that mild small intestinal damage occurs Euthanasia and tissue sampling even after short durations of CA and resuscitation. A total of 55 rats (70.5%) could not be resuscitated. All 23 successfully resuscitated and sham-operated rats sur- Methods vived the observation period and were included in the Animals and husbandry study. The number of rats used for histomorphological Seventy-eight 7 to 8 weeks old male Wistar rats (Janvier, analysis and cytokine profiling is shown in Table 1. One France) weighing 280 - 320 g were transferred into the group was used as sham-operated control-group and was animal facility 10 days before surgery and had ad libitum euthanized immediately after surgical procedures without access to standard pelleted feed (Ssniff®, V1534–703, CA and CPR. Under anesthesia, the thorax was opened Germany) and water. Rats were housed under a 12:12 h and blood samples were taken from the left ventricle. light-dark cycle at 22 °C and a relative humidity of 60%. Following coagulation for 45 min at room temperature, They were allocated randomly to 5 groups (controls, 6 h, the tubes (Eppendorf, Germany) were centrifuged for 24 h, 72 h and 7 d post-ROSC). 10 min at 2500 g and 4 °C. sera were aliquoted and stored Cardiac arrest and cardio-pulmonary resuscitation Table 1 Number of rats used for histomorphological analysis The detailed experimental protocol was previously pub- (HA) and cytokine profiling (CP) of controls (C) and at 6 h, 24 h, lished by Böttiger et al. [16]. Briefly, rats were anesthetized 72 h and 7 d post-ROSC with 3% sevoflurane and 70% nitrous oxide in oxygen. Study groups and number of rats Animals were endotracheal intubated (Braunüle-MT No. 3, Analysis C 6 h 24 h 72 h 7 d Braun, Germany) and ventilated at a rate of 60 breaths per Jejunum HA 4 3 5 4 4 min (Rodent Ventilator, Harvard Apparatus, MA, USA). A Jejunum CP 4 3 5 6 4 saline-filled polyethylene catheter was advanced via Serum CP 6 3 6 4 4 cut-down into the left femoral artery to continuously This analysis is a sub-study of an investigation aimed to pursue systemic measure mean arterial pressure (MAP, SC7000, Siemens inflammation in multiple tissues after CA. Serum cytokine profiling was Health Care GmbH, Germany). Another saline-filled conducted in n = 23 rats. Only n = 3 animals were resuscitated and included in the 6 h group. At least n = 4 animals were included in the 24 h, 72 h and 7 d polyethylene catheter was advanced via cut-down into the group. Additionally, n = 2 more sera in the control group and 1 more serum left femoral vein for drug administration. The tidal breath- sample in the 24 h group were collected and analyzed from further experiments. ing volume was adjusted to ensure a physiological pCO In the 72 h group, n = 2 more jejunal samples were also analyzed 2 Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 3 of 10 at − 80 °C. A 2–3 cm piece of the mid jejunum, 8 cm distal The value 0 was attributed when the results were below from the pylorus, was excised and divided. One segment the detection limit. was shock-frozen in liquid nitrogen and stored at − 80 °C for the multiplex cytokine assay. The other segment was Statistical analyses fixed in 4% formalin for histomorphological studies. An a priori power analysis was performed to determine the adequate sample size for detection of TNF-α in serum 24 h after CA as primary outcome variable. (type Histomorphological analysis - Chiu grading 1error:5%(α < 0.05); type 2 error: 20% (β <0.80); medium Paraffin-embedded jejunal tissue was sectioned (4 μm) efficiency: 0.6). Animal studies reporting serum TNF-α and stained with hematoxylin and eosin (H&E) accord- concentrations after CA due to ventricular fibrillation are ing to standard protocols. The morphological integrity scarce. According to the literature, an average rise from 0 of the intestinal wall was classified by a blinded investi- to approximately 12 pg/mL [5] in humans is expected. gator using a modified protocol according to Chiu et al. Since no serum TNF-α was expected on day 0 (controls), [19]: Grade 0: normal mucosa; Grade 1: development of a variability of 0 was expected. To display a difference in a sub-epithelial space at the tips of the villi; Grade 2: serum TNF-α concentrations, we estimated a number of 4 more extended sub-epithelial space at the tips of the villi, rats per group. development of Gruenhagen’s space at the tips of the villi; One-way ANOVA and Tukey’s multiple comparison Grade 3: massive epithelial lifting down the sides of the test was performed using GraphPad Prism 6 for Windows. villi, villus necrosis; Grade 4: villi are denuded of epithelial For correlation of the duration of CA with cytokine layer; Grade 5: loss of villi, mucosal ulceration and necro- concentrations and Chiu-grades, the Pearson’scorrel- sis with invasion of the muscularis propria. To evaluate ation test was used with unilateral values (GraphPad oedema formation within the jejunal wall, the thickness of Software, USA). All data are presented as mean ± SD. A serosa, muscularis, submucosa and mucosa was measured p-value< 0.05 was considered statistically significant. using 10-fold magnifications (Olympus DP25, cellSens Standard 1.11, Olympus GmbH, Germany). Ethical statement All procedures were ethically approved by the appropriate Tissue lysates and protein extraction governmental authority (Landesamt für Natur, Umwelt Proteins were extracted by homogenizing 200 mg jejunal und Verbraucherschutz Nordrhein-Westfalen, LANUV, tissue with 500 μl RIPA buffer (150 mM NaCl, 1% Germany, AZ: 8.87–51.04.20.09.368) and were in accord- Triton-x-100, 1% Na-deoxycholate, 0.1% SDS, 50 mM ance with the German Animal Welfare Law. Animal care Tris-HCl pH 8, 10 mM EDTA) containing a proteinase- and use was performed by qualified individuals, supervised inhibitor (complete Mini, EDTA-free, Roche, Switzerland). by a veterinarian. All facilities and transportation complied Subsequently, the tissue lysates were centrifuged for with current legal requirements. The manuscript complies 15 min at 11000 g and 4 °C and supernatants were with the Animals in Research: Reporting In Vivo Experi- centrifuged for 40 min at 44,400 g and 4 °C. The clear ments (ARRIVE) guidelines [20]. Humane endpoints were supernatants were analyzed using the BCA test (Pierce, specified and part of the animal welfare application Thermo Scientific, Germany) and duplicate aliquots of according to the directive 2010/63/EU of the European 1000 μg/ml protein were measured directly. parliament. A list of specific clinical signs to determine the animal’s physiology and behavioral condition was used. Multiplex cytokine assay Specific experiment-related humane endpoints such as IL-1α, IL-1β, IL-6, IL-10 and TNF-α in jejunal tissues neurological disturbances, lameness, wound healing and IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 deficits and coma were evaluated. The animals were (p70), IL-13, interferon-γ (IFN-γ), granulocyte macrophage scored as mild, moderate or severely impaired. According colony-stimulating factor (GM-CSF) and TNF-α in serum to the results of scoring, animals were treated (analgesics, were measured in duplicates in a multiplex analyzer antibiotics), frequently examined or sacrificed (severe (Bio-Plex 200®, Bio-Rad Laboratories, USA) according impairment). According to the results of the scored to the manufacturer’s instructions. The jejunal tissue humane endpoints, animals received 50 μg/ml meloxicam lysate protein was diluted 1:2 with sample diluent contain- p.o. within the first three days after ROSC, if necessary. ing 0.5% BSA (500 μg/ml final protein concentration). Sera were thawed, centrifuged for 5 min at 10,000 g and Results 4 °C and diluted 1:4 in sample diluent. By using the Histomorphological analysis - Chiu grading median fluorescence intensity and the standard curve, the Representative images of intestinal tissues after CA absolute concentration of each cytokine (pg/ml) was revealing Chiu grades from 0 to 5 are shown in Fig. 1a-f. calculated (Bio-Plex Manager 6.1, Bio-Rad Laboratories). Controls lacked histomorphological changes in the small Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 4 of 10 Fig. 1 Histomorphological evaluation of the jejunal wall of rats after 6 min of CA using a modified Chiu scoring system (Chiu et al. 1970) [44]. Images represent the Chiu grades 0–5. a Intact mucosa and villus structures in control tissue of sham-operated rats (Chiu grade 0). b Development of a sub-epithelial space at the tips of the villi (arrow) 24 h post-ROSC (Chiu grade 1). c Development of a Gruenhagen’s space at the tip of a villus (arrow) 24 h post-ROSC (Chiu grade 2). d Villus necrosis (arrow) 7 d post-ROSC (Chiu grade 3). e Massive epithelial desquamation and villi which are denuded of epithelial layer (arrow) 7 d post-ROSC (Chiu grade 4). f Loss of villi, mucosal ulceration and necrosis 7 d post-ROSC (Chiu grade 5). a-f Hematoxilin-eosin staining, longitudinal section of 4 μm thickness (a, e, f) or cross section (b, c, d), scale bars represent 100 μm intestine. In rats with 6 min of CA, the intestinal mu- cosa revealed desquamation of the villus tips. Blunt, dome-shaped, fenestrated epithelial cells of submucosal arterioles were evident. Gruenhagen’s spaces, slight peri- vascular oedema, hydropic generation of epithelial cells and sparse pyknotic cells were identified. Chiu grades of jejunal mucosa revealed a significant time-dependent effect (Fig. 2, p < 0.0001). Tukey’s multiple comparison test showed a significant increase (12.5-fold) in mucosal damage 6 h post-ROSC (3.13 ± 0.64) compared to controls (0.25 ± 0.5, p < 0.001). Mucosal damage decreased significantly 2.1-fold at 24 h (1.48 ± 0.87) compared to 6 h post-ROSC (3.13 ± 0.64, p < 0.05). Chiu grading decreased significantly 2.2-fold at 72 h (1.4 ± 0.46) compared to 6 h post-ROSC (p < 0.05). At 7 d post-ROSC, Chiu grade increased again (3.33 ± 0.69, p < 0.05) compared to 24 h (1.48 ± 0.87) and was the highest compared to controls Fig. 2 Chiu Scoring. Chiu Scoring [Grades 0–5] in jejunum of controls and at 6 h, 24 h, 72 h and 7 d post-ROSC are expressed as mean ± SD. (0.25 ± 0.5, p < 0.0001). One-way ANOVA p < 0.0001. Tukey’smultiplecomparison testis marked Comparing the intestinal wall thickness of controls to with asterisks (*p < 0.05, ***p < 0.001, ****p < 0.0001) that at 6 h, 24 h, 72 h and 7 d post-ROSC showed Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 5 of 10 significant time-dependent changes (p < 0.0001). Tukey’s Comparison of jejunum and serum cytokine concentrations multiple comparison test showed that the muscularis The ratio of absolute cytokine concentrations in jejunum was significantly thinner at 7 d post-ROSC compared to compared to serum is shown in Table 3. Overall, signifi- controls (p < 0.05). A thinner muscularis was also ob- cant higher IL-1α and TNF-α concentrations were ob- served 6 h, 24 h and 72 h post-ROSC but this difference served in jejunal tissue compared to serum in both was not significantly decreased compared to controls. controls and CA groups at all timepoints (p <0.05). IL-1β in controls and at 24 h post-ROSC, IL-6 at 24 h post- ROSC and IL-10 at 24 h and 72 h post-ROSC were also Multiplex cytokine assay significantly higher in jejunum lysates compared to serum. Jejunal tissue Increased concentrations of IL-1β at 72 h and 7 d In jejunal tissue, IL-1α was altered over time with peak post-ROSC and IL-10 in controls and at 6 h and 7 d values at 24 h post-ROSC. (p < 0.01, Fig. 3a). Tukey’s post-ROSC were detected in jejunum but were not signifi- multiple comparison test identified significant elevations cantly different. IL-6 concentrations in jejunum and in IL-1α at 24 h compared to controls (1.7-fold increase, serum in controls and at 6 h, 72 h and 7 d post-ROSC and p < 0.01,) and the 6 h group (1.7-fold increase, p < 0.05). IL-1β concentrations at 6 h post-ROSC were comparable. At 72 h, IL-1α concentration decreased significantly 1.6-fold Comparing the time-dependent profiles of jejunum/serum (p < 0.01) compared to the 24 h group. No significant differ- ratio with profiles in jejunal tissue, IL-1α, IL-6, IL-10 and ence was detected between 24 h and 7 d post-ROSC TNF-α displayed a similar alteration with a peak at 24 h (Fig. 3a). IL-1β showed significant time-dependent changes post-ROSC while IL-1β peaked at 7 d post-ROSC, in (p <0.01). IL-1β peaked at 24 h compared to controls contrast to jejunal tissue at 24 h post-ROSC (Fig. 3a-e). (5.7-fold increase, p < 0.05) and 6 h (7.9-fold increase, p<0.05).At 72h,IL-1β was 2.5-fold lower than at 24 h Correlation analysis and 1.9-fold lower than at 7 d post-ROSC but differences No significant correlation between the duration of CA were not statistically different (Fig. 3b). IL-6 concentrations and cytokine concentrations in jejunum was detected showed time-dependent changes (p < 0.01). Additionally, a 6 h and 24 h post-ROSC: 6 h group: IL-1α: r = 0.43, p = significant 1.2-fold decrease was detected at 72 h compared 0.546; IL-1β: r = 0.002, p = 0.970; IL-6: r = 0.06, p = 0.848; to 24 h post-ROSC (p < 0.01, Fig. 3c). IL-10 concentrations IL-10: r = 0.44, p = 0.532; TNF-α: r = 0.07, p = 0.824. 24 h revealed significant time-dependent changes (p < 0.0001). group: IL-1α: r = 0.01, p = 0.874; IL-1β: r = 0.26, p = At 24 h, it peaked significantly compared to controls 0.492; IL-6: r = 0.86, p = 0.073; IL-10: r = 0.34, p = 0.413; (p < 0.0001) and 6 h post-ROSC (p < 0.001) resulting in TNF-α: r = 0.32, p = 0.438. However, there was a statisti- a 2.2-fold and 1.9-fold increase, respectively. Concen- cally significant correlation between the duration of CA trations at 72 h (1.6-fold, p <0.001) and 7 d (1.5-fold, and the Chiu-Grade 7 d after CA (r = 0.97, p = 0.012), p < 0.05) decreased significantly compared to that at while the other timepoints were not statistically signifi- 24 h (Fig. 3d). TNF-α concentrations showed a significant cant (6 h group: r = 0.85, p = 0.251; 24 h group: r = 0.24, time-dependent effect (p < 0.01) at 24 h post-ROSC p = 0.501; 72 h group: r = 0.21, p = 0.537). Additional in- compared to controls (1.5-fold increase, p <0.01) and formation pertaining to the duration of cardiac arrest 6 h post-ROSC (1.4-fold increase, p < 0.05). TNF-α and the different groups are shown in Additional file 1 decreased significantly at 72 h post-ROSC compared but no significant differences were found between groups. to 24 h post-ROSC (1.5-fold, p < 0.01). There were no significant changes between 72 h and 7 d post-ROSC Discussion (Fig. 3e). This study reveals three major findings: (i) mild intestinal barrier damage could be detected within 24 h in a rat model of 6 min CA. (ii) only mild local intestinal inflamma- Serum tion could be shown within 24 h after CA. (iii) a systemic Absolute concentrations of IL-2, IL-4, IL-5, IL-12 (p70), inflammation and thus a potential contribution of the small IL-13, GM-CSF and IFN-γ in serum are shown in intestine to systemic inflammation could not be simulated Table 2. Results for IL-1α,IL-1β, IL-6, IL-10 and TNF-α after6minofCAinrats. are shown in Fig. 3f-j. Serum concentrations of IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, GM-CSF, IFN-γ and TNF-α did not differ significantly between Intestinal barrier damage controls and at 6 h, 24 h, 72 h and 7 d post-ROSC. Although 6 min of transient global ischaemia and subse- IL-1β showed a significant time-dependent effect (p < quent reperfusion led to mucosal damage and decreased 0.05). At 24 h post-ROSC, IL-1β significantly increased thickness of the muscularis in the small intestine, jejunal 1.6-fold compared to controls (p < 0.05, Fig. 3g). tissue concentrations of IL-1α, IL-1β, IL-10 and TNF-α Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 6 of 10 Fig. 3 (See legend on next page.) Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 7 of 10 (See figure on previous page.) Fig. 3 Cytokine concentrations of IL-1α,IL-1β, IL-6, IL-10 and TNF-α (mean ± SD, pg/ml) in jejunum (left column) and serum (right column) in controls andat 6 h, 24 h, 72 hand 7dpost-ROSC. ThesignificanceofTukey’smultiplecomparisontest ismarkedwithasterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). a IL-1α concentrations in jejunum (one-way ANOVA p < 0.01). b IL-1β concentrations in jejunum (one-way ANOVA p < 0.01). c IL-6 concentrations in jejunum (one-way ANOVA p < 0.01). d IL-10 concentrations in jejunum (one-way ANOVA p < 0.0001). e TNF-α concentrations in jejunum (one-way ANOVA p < 0.01). f IL-1α concentrations in serum (one-way ANOVA p >0.05). g IL-1β concentrations in serum (one-way ANOVA*). h IL-6 concentrations in serum (one-way ANOVA p >0.05). i IL-10 concentrations in serum (one-way ANOVA p >0.05). j TNF-α concentrations in serum (one-way ANOVA p >0.05) were only slightly affected. This result may be due to to oxidative stress, NF-КBis up-regulatedandorches- several reasons. trates the release of a number of cytokines such as IL-1ß, First, the duration of 6 min CA in the present study that IL-2, IL-4, IL-5, IL-6 and IL-10, IL-12, IL-13 and TNF-α is sufficient to show cerebral damage [21] is inadequate to [28, 29]. Accordingly, we collected blood (sera) and tissues initiate a systemic inflammatory response, which is a clear at early stages including 6 h and 24 h post-ROSC and limitation of this study. As shown by Qian, exceeding the chose a similar cytokine profile to be investigated. How- duration of CA of 6 min may extend intestinal damage ever, in clinical studies, it is often proposed that the PCAS and influence serum cytokine concentrations. In detail, may result from a systemic inflammatory activation persist- intestinal microcirculatory blood flow was significantly ing for days [5, 30]. Therefore, cytokine concentrations in decreased accompanied by mild elevated serum con- jejunum and sera were also evaluated within the first 7 days centrations of TNF-α and IL-6 during 8 min of CA in after CA in the present study. pigs [22]. In contrast, significant inflammatory response Third, as repeatedly shown, simulation of a PCAS is following local ischaemia andreperfusion of thesmall difficult in rats and accompanied by a high failure rate intestine was shown to be initiated after a duration of of more than 50% [24, 31]. Thus, on the basis of previ- at least 15–30 min [23]. However, models of focal ischae- ous scientific findings, we conclude, that severely injured mia and reperfusion are not capable of being translated to rats that would develop a PCAS initially died during re- conditions of systemic ischaemia and reperfusion. In fact, suscitation procedures. With a survival rate of only CA leads to a complex systemic ischaemia-reperfusion-in- 29.5% in the present study, we conclude that this model jury with contribution of multiple independent tissues, can be utilized to a limited extent to reproduce a PCAS which are integrated into a complicated cascade of cell- or peripheral tissue damage, respectively. Nevertheless, death and systemic inflammation [5, 6]. ROSC rates are comparable to previous experiments Secondly, intestinal repair mechanisms seem to be performed in our group, which are able to adequately commenced immediately after CA. A normalization of determine cerebral damage [16, 18, 21, 24, 32–34]. In leukocyte-endothelial interaction as well as the wall shear this pilot study, our main objective was to evaluate per- rate was reported to be initiated within 120 min after CA, ipheral tissue potentially injured due to CA. Notably, we which is a period not mirrored in our study [24]. However, did not lose any animals after ROSC, which is advanta- reports show that cytokines such as IL-1ra, IL-6, IL-8, geous since post-ROSC mortality is known to be 38% IL-10 and TNF-α are expressed within 3 h [5, 6, 25]to 6 h within an observation period > 48 h [35]. Interestingly, [24, 26] and peak within the first 2 days after CA [5, 6]. Vognsen et al. recently showed that only 12% of animal This is in line with studies reporting a massive up-regula- studies sufficiently report outcome parameters according tion of cytokines after ischaemic brain injury [2, 27]. Due to the Utstein Guidelines, which was a strong criterion to increase the validity of this study [35]. Table 2 Absolute levels of cytokines in serum (IL-2, IL-4, IL-5, Given the clinical phenomenon of bacteremia after CA IL-12 (p70), IL-13, GM-CSF, IFN-γ) in controls (C) and at 6 h, 24 h, [7, 8], intestinal damage seems to be conclusive even 72 h and 7d post-ROSC expressed in pg/ml (mean ± SD) after short periods of ischaemia. Congruently, our results C 6 h 24 h 72 h 7d point towards a mild local intestinal damage, which is in IL-2 0.36 ± 0.35 0.27 ± 0.06 0.20 ± 0.05 0.21 ± 0.07 0.34 ± 0.28 line with Pan et al. who reported similar Chiu scorings 24 h after CA of 6 min duration [36]. Likewise, Teschen- IL-4 0.24 ± 0.18 0.22 ± 0.09 0.17 ± 0.02 0.17 ± 0.02 0.23 ± 0.10 dorf et al. showed a 3–4-fold stronger plasma extravasa- IL-5 n.d. n.d. n.d. n.d. n.d. tion from post-capillary mesenteric venules at 120 min IL-12 (p70) n.d. n.d. n.d. n.d. n.d. after CA, which is a characteristic sign of endotoxaemia IL-13 n.d. n.d. n.d. n.d. n.d. [24]. Another factor taken into consideration is the short GM-CSF 0.20 ± 0.01 0.25 ± 0.06 0.23 ± 0.02 0.24 ± 0.03 0.23 ± 0.05 initial tissue hyperperfusion followed by a sustained hypo- IFN-γ 0.36 ± 0.07 0.47 ± 0.18 0.34 ± 0.01 0.31 ± 0.02 0.35 ± 0.05 perfusion of intestinal tissue after CA [37, 38]. This inevit- n.d. not detected ably leads to a prolonged period of relative ischaemia Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 8 of 10 Table 3 Ratio of absolute IL-1α, IL-1β, IL-6, IL-10, TNF-α levels in jejunum to absolute levels in serum in controls (C) and at 6 h, 24 h, 72 h and 7d post-ROSC (mean ± SD) C 6 h 24 h 72 h 7d **** **** **** *** **** IL-1α 15.2 ± 3.6 15.5 ± 1.7 30.4 ± 3.8 19.3 ± 4 13.9 ± 5 * ** IL-1β 1.7 ± 0.2 1 ± 0.6 6.9 ± 0.4 7.9 ± 2.9 10.2 ± 1.5 **** IL-6 1 ± 0.1 1 ± 0.04 1.3 ± 0.03 1 ± 0.02 1.1 ± 0.1 **** * IL-10 1.9 ± 0.4 1.8 ± 0.3 4.2 ± 0.6 3.6 ± 0.5 2.5 ± 0.7 **** * *** * ** TNF-α 1.5 ± 0.04 1.4 ± 0.1 2.1 ± 0.1 1.7 ± 0.2 2 ± 0.1 The level of significance is marked with asterisks: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001) where the intestine receives only 5% of cardiac output [39] pro-inflammatory cytokines such as IL-1α,IL-1β and and fosters further tissue damage. Interestingly, we found TNF-α. a significant correlation between the duration of CA and Overall, serum cytokine concentrations were not signifi- the Chiu-grade in the 7 d group. However, these results cantly altered except for a significant increase in IL-1β after should be considered with caution because significant 24 h post-ROSC. Additionally, IL-1α,IL-1β,IL-10 and mucosal damage was also shown with a shorter duration TNF-α concentrations were lower in serum than in the je- of CA in the 6 h group (Additional file 1). junum. This may indicate that the observed intestinal inflam- It must be noted that mucosal damage and cytokines mation may not be associated with systemic inflammation. were not analyzed in animals that could not be resusci- Notably, intestinal cytokine increments were in accord- tated. However, results were compared to a control group ance with morphological changes of the intestinal mucosa. serving as reference. Since achievement of ROSC is mainly Intestinal tissue damage peaked at 6 h post-ROSC and de- dependent on heart function, significant intestinal damage creased subsequently at 24 h and 72 h. Tissue repair mecha- detectable immediately after ceasing CPR was not as- nisms accompanied by increases in intestinal inflammatory sumed. As reported, mucosal damage develops within the cytokine release seem to have been initiated. Consequently, first 6 to 24 h after cardiac arrest [36] and may contribute desquamation of villus tips, development of a Gruenhagen’s to the development of the PCAS, which was the main space, hydropic generation of epithelial cells and changes in objective in this investigation. muscular layer thickness were observed. The mechanism, which induces a further increase in intestinal tissue damage 7 d post-ROSC, accompanied Cytokine expression in tissue and serum by rising cytokine concentrations of IL-1β and IL-10 at A systemic ischaemia-reperfusion-injury causes a so-called the same timepoint, requires further research. As the sterile inflammation, which is associated with influx of duration between 72 h and 7 d post-ROSC is comparably neutrophils and macrophages, leading to the production of long, it might be possible that further changes in cytokine inflammatory cytokines [40]. IL-1α, present in gastrointes- concentrations remained undetected. tinal epithelial and endothelial cells [41], acts via initiation of the inflammatory cascade and is thus a valuable param- Conclusions eter for the determination of inflammatory processes. In Six minutes of transient systemic ischaemia and reperfu- the present study, IL-1α showed a significant increase in sion resulted in mild small intestinal tissue damage but the jejunum, resulting in the highest concentrations at 24 h not in systemic inflammation. A rat model of 6 min CA post-ROSC. At thesametimepoint, IL-1α concentrations is not capable of mimicking a PCAS. Whether there is a in serum were close to detection limits. It has been re- vital influence of the intestine on the PCAS still remains ported that in vitro circulating IL-1α was released from unclear and should be investigated in further studies. endothelial cells [41] but was barely detectable in patients suffering from severe inflammation [42]. This suggests that jejunal and serum IL-1α concentrations do not necessarily Additional file match, as observed in the present study. Additional file 1: Duration of cardiac arrest in the different post-ROSC The IL-10 family acts protectively during intestinal in- groups. One-way ANOVA p < 0.05. Tukey’s multiple comparison test did flammation by induction of anti-inflammatory effects and not show any statistically significant differences between any of the groups. inhibition of pro-inflammatory cytokines such as IL-1β, (DOCX 28 kb) IL-6 and TNF-α [43]. In our study, in jejunal tissues, IL-1α,IL-1β and TNF-α increased significantly at 24 h Abbreviations post-ROSC indicating that increasing IL-10 concentra- ATP: Adenosine triphosphate; CA : Cardiac arrest; CPR: Cardio-pulmonary tions at the same time might counteract the increase of resuscitation; IL: Interleukin; NMDA: N-methyl-D-aspartate receptor; Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 9 of 10 PCAS: Post cardiac arrest syndrome; ROS: Reactive oxygen species; resuscitation after cardiac arrest as a "sepsis-like" syndrome. Circulation. ROSC: Return of spontaneous circulation; TNF-α: Tumor necrosis factor-α 2002;106(5):562–8. 6. Nolan JP, Neumar RW, Adrie C, Aibiki M, Berg RA, Bottiger BW, Callaway C, Clark RS, Geocadin RG, Jauch EC, et al. Post-cardiac arrest syndrome: Acknowledgements epidemiology, pathophysiology, treatment, and prognostication. A scientific The authors thank Irmgard Henke and Malte Heykants for their excellent statement from the international liaison committee on resuscitation; the technical assistance, Roland Galmabcher, Peter Teschendorf and Andreas American Heart Association emergency cardiovascular care committee; the Schneider for their methodological support and Laura A. Webb, M.Sc. for council on cardiovascular surgery and anesthesia; the council on proof-reading the manuscript. cardiopulmonary, perioperative, and critical care; the council on clinical cardiology; the council on stroke. Resuscitation. 2008;79(3):350–79. Funding 7. Carr GE, Yuen TC, McConville JF, Kress JP, VandenHoek TL, Hall JB, Edelson This work was supported by the intramural “Cologne-Fortune” programme, DP. Early cardiac arrest in patients hospitalized with pneumonia: a report Faculty of Medicine, University of Cologne (299/2014). Design of the study, from the American Heart Association's get with the guidelines-resuscitation collection, analysis, interpretation of data and writing were not influenced by program. Chest. 2012;141(6):1528–36. thy funding body. 8. Coba V, Jaehne AK, Suarez A, Dagher GA, Brown SC, Yang JJ, Manteuffel J, Rivers EP. The incidence and significance of bacteremia in out of hospital Availability of data and materials cardiac arrest. Resuscitation. 2014;85(2):196–202. The datasets used and/or analysed during the current study are available 9. Guschlbauer M, Feige K, Geburek F, Hoppe S, Hopster K, Propsting MJ, from the corresponding author on reasonable request. Huber K. Effects of in vivo lidocaine administration at the time of ischemia and reperfusion on in vitro contractility of equine jejunal smooth muscle. Authors’ contributions Am J Vet Res. 2011;72(11):1449–55. DS and MG were involved in planning and conduction of the experiments, 10. 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Abstract

Background: Contribution of the small intestine to systemic inflammation after cardiac arrest (CA) is poorly understood. The objective was to evaluate whether an in vivo rat model of 6 min CA is suitable to initiate intestinal ischaemia-reperfusion-injury and to evaluate histomorphological changes and inflammatory processes in the small intestinal mucosa resp. in sera. Methods: Adult male Wistar rats were subjected to CA followed by cardio-pulmonary resuscitation. Proximal jejunum and serum was collected at 6 h, 24 h, 72 h and 7 d post return of spontaneous circulation (ROSC) and from a control group. The small intestine was evaluated histomorphologically. Cytokine concentrations were measured in jejunum lysates and sera. Results: Histomorphological evaluation revealed a significant increase in mucosal damage in the jejunum at all timepoints compared to controls (p < 0.0001). In jejunal tissues, concentrations of IL-1α,IL-1β, IL-10, and TNF-α showed significant peaks at 24 h and were 1.5- to 5.7-fold higher than concentrations at 6 h and in the controls (p <0.05). In serum, a significant higher amount of cytokine was detected only for IL-1β at 24 h post-ROSC compared to controls (15.78 vs. 9.76 pg/ml). Conclusion: CA resulted in mild small intestinal tissue damage but not in systemic inflammation. A rat model of 6 min CA is not capable to comprehensively mimic a post cardiac arrest syndrome (PCAS). Whether there is a vital influence of the intestine on the PCAS still remains unclear. Keywords: Cardiac arrest, Small intestine, Ischaemia-reperfusion-injury, Systemic inflammatory response syndrome Background aggravates cell death. Later, accumulation of inflamma- Cardiac arrest (CA) results in transient systemic ischaemia tory cytokines is initiated and causes a long-lasting in- followed by reperfusion as a consequence of successful re- flammatory reaction. suscitation. First, cessation of circulation results in deple- Within 4–5 min after CA, selectively vulnerable regions + + tion of oxygen followed by ATP-dependent Na /K -pump in the brain such as the hippocampal CA1 become apop- dysfunction. Resulting in a breakdown of cellular integrity, totic and necrotic [2]. As a result, brain injury is respon- glutamate is being released intracellularly and mediates sible for the mortality of 68% of the victims of CA [3]; cerebral excitotoxicity by activation of N-methyl-D-aspar- neurocognitive long-term impairment occurs in half of ++ tate receptors. Subsequently, intracellular Ca influx and the survivors [4]. In addition to that, within hours to days, activation of caspases, phospholipases, and proteases the characteristic systemic ischaemia-reperfusion-injury lead to cellular death [1]. Secondly, subsequent reperfu- provokes a systemic inflammatory release also known sion triggers formation of free radicals, which further as sepsis-like- or post-cardiac arrest syndrome (PCAS) ending up in multiple organ failure [5, 6]. In fact, CA is a complex systemic ischaemia-reperfusion-injury with * Correspondence: Daniel.Schroeder@uk-koeln.de contribution of multiple independent tissue. However, to Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Cologne, Kerpener Str. 62, Cologne, Germany date, particular contribution of peripheral organs to the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 2 of 10 development of PCAS is not clearly understood. Given (35-45 mmHg). The inspired oxygen concentration (FiO ) that more than 30% of victims of CA show bacteremia was regulated to ensure a physiological pO . Blood gas upon presentation, the small intestine was discussed to analysis was performed using ABLFlex800 (Radiometer, be an immediate by-product of a systemic ischaemia- Germany). The cardio-pulmonary resuscitation (CPR) reperfusion-injury [7, 8]. protocol fulfils the Utstein Style guidelines for laboratory As described, the small intestine is highly susceptible CPR research [17]. The rats received an oesophageal elec- to a focal ischaemia-reperfusion-injury [9, 10]. Already trode for induction of ventricular fibrillation (12 V, 50 Hz, after 15–30 min of mesenterial occlusion, morphological 1.5 min) until the mean arterial blood pressure stayed changes such as atrophy of the villi and damage of tunica below 15 mmHg [18]. After 5.5 min of CA, rats were mucosa and tunica serosa appear [11]. Consequently, loss mechanically ventilated using 100% oxygen at 60 breaths of intestinal integrity is associated with excessive fluid loss per min. At 6 min after CA, CPR started by performing a and translocation of gut bacteria and toxins into the blood manual closed-chest cardiac massage (200 times/min) and circulation [12]. Subsequent restoration of blood flow leads an injection of 20 μg/kg epinephrine (Suprarenin, Sanofi- to an activation of molecular and cellular components of Aventis, Germany). Two min later, a single bi-phasic the innate immunity resulting in an inflammatory response shock of 2–3 J (M series, Zoll Corporation, Germany) [13]. As a result, local and systemic inflammation occurs was attempted. Epinephrine administration and biphasic and causes a multiple organ dysfunction syndrome with a shocks were repeated every 30 s. ROSC was defined as mortality rate reported between 30–90% [9, 10, 13–15]. maintenance of mean arterial blood pressure above However, due to the predominant brain damage 50 mmHg for at least 10 min. If ROSC was not achieved decisive for morbidity and mortality in survivors of CA after 6 min of CPR, resuscitation procedures were termi- the role of the small intestine in the development of sys- nated. To maintain normocapnia, ventilation rate was temic inflammation after CA was not intensively investi- adjusted and sodium bicarbonate was titrated according to gated yet. Thus, the objective was (i) to evaluate whether the blood gas analysis. Once adequate spontaneous breath- an in vivo rat model of 6 min CA is suitable to initiate an ing was observed, rats were extubated, kept singly and intestinal ischaemia-reperfusion-injury to further examine monitored every 2–4h. (ii) genesis of local and systemic inflammation. It was hypothesized that mild small intestinal damage occurs Euthanasia and tissue sampling even after short durations of CA and resuscitation. A total of 55 rats (70.5%) could not be resuscitated. All 23 successfully resuscitated and sham-operated rats sur- Methods vived the observation period and were included in the Animals and husbandry study. The number of rats used for histomorphological Seventy-eight 7 to 8 weeks old male Wistar rats (Janvier, analysis and cytokine profiling is shown in Table 1. One France) weighing 280 - 320 g were transferred into the group was used as sham-operated control-group and was animal facility 10 days before surgery and had ad libitum euthanized immediately after surgical procedures without access to standard pelleted feed (Ssniff®, V1534–703, CA and CPR. Under anesthesia, the thorax was opened Germany) and water. Rats were housed under a 12:12 h and blood samples were taken from the left ventricle. light-dark cycle at 22 °C and a relative humidity of 60%. Following coagulation for 45 min at room temperature, They were allocated randomly to 5 groups (controls, 6 h, the tubes (Eppendorf, Germany) were centrifuged for 24 h, 72 h and 7 d post-ROSC). 10 min at 2500 g and 4 °C. sera were aliquoted and stored Cardiac arrest and cardio-pulmonary resuscitation Table 1 Number of rats used for histomorphological analysis The detailed experimental protocol was previously pub- (HA) and cytokine profiling (CP) of controls (C) and at 6 h, 24 h, lished by Böttiger et al. [16]. Briefly, rats were anesthetized 72 h and 7 d post-ROSC with 3% sevoflurane and 70% nitrous oxide in oxygen. Study groups and number of rats Animals were endotracheal intubated (Braunüle-MT No. 3, Analysis C 6 h 24 h 72 h 7 d Braun, Germany) and ventilated at a rate of 60 breaths per Jejunum HA 4 3 5 4 4 min (Rodent Ventilator, Harvard Apparatus, MA, USA). A Jejunum CP 4 3 5 6 4 saline-filled polyethylene catheter was advanced via Serum CP 6 3 6 4 4 cut-down into the left femoral artery to continuously This analysis is a sub-study of an investigation aimed to pursue systemic measure mean arterial pressure (MAP, SC7000, Siemens inflammation in multiple tissues after CA. Serum cytokine profiling was Health Care GmbH, Germany). Another saline-filled conducted in n = 23 rats. Only n = 3 animals were resuscitated and included in the 6 h group. At least n = 4 animals were included in the 24 h, 72 h and 7 d polyethylene catheter was advanced via cut-down into the group. Additionally, n = 2 more sera in the control group and 1 more serum left femoral vein for drug administration. The tidal breath- sample in the 24 h group were collected and analyzed from further experiments. ing volume was adjusted to ensure a physiological pCO In the 72 h group, n = 2 more jejunal samples were also analyzed 2 Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 3 of 10 at − 80 °C. A 2–3 cm piece of the mid jejunum, 8 cm distal The value 0 was attributed when the results were below from the pylorus, was excised and divided. One segment the detection limit. was shock-frozen in liquid nitrogen and stored at − 80 °C for the multiplex cytokine assay. The other segment was Statistical analyses fixed in 4% formalin for histomorphological studies. An a priori power analysis was performed to determine the adequate sample size for detection of TNF-α in serum 24 h after CA as primary outcome variable. (type Histomorphological analysis - Chiu grading 1error:5%(α < 0.05); type 2 error: 20% (β <0.80); medium Paraffin-embedded jejunal tissue was sectioned (4 μm) efficiency: 0.6). Animal studies reporting serum TNF-α and stained with hematoxylin and eosin (H&E) accord- concentrations after CA due to ventricular fibrillation are ing to standard protocols. The morphological integrity scarce. According to the literature, an average rise from 0 of the intestinal wall was classified by a blinded investi- to approximately 12 pg/mL [5] in humans is expected. gator using a modified protocol according to Chiu et al. Since no serum TNF-α was expected on day 0 (controls), [19]: Grade 0: normal mucosa; Grade 1: development of a variability of 0 was expected. To display a difference in a sub-epithelial space at the tips of the villi; Grade 2: serum TNF-α concentrations, we estimated a number of 4 more extended sub-epithelial space at the tips of the villi, rats per group. development of Gruenhagen’s space at the tips of the villi; One-way ANOVA and Tukey’s multiple comparison Grade 3: massive epithelial lifting down the sides of the test was performed using GraphPad Prism 6 for Windows. villi, villus necrosis; Grade 4: villi are denuded of epithelial For correlation of the duration of CA with cytokine layer; Grade 5: loss of villi, mucosal ulceration and necro- concentrations and Chiu-grades, the Pearson’scorrel- sis with invasion of the muscularis propria. To evaluate ation test was used with unilateral values (GraphPad oedema formation within the jejunal wall, the thickness of Software, USA). All data are presented as mean ± SD. A serosa, muscularis, submucosa and mucosa was measured p-value< 0.05 was considered statistically significant. using 10-fold magnifications (Olympus DP25, cellSens Standard 1.11, Olympus GmbH, Germany). Ethical statement All procedures were ethically approved by the appropriate Tissue lysates and protein extraction governmental authority (Landesamt für Natur, Umwelt Proteins were extracted by homogenizing 200 mg jejunal und Verbraucherschutz Nordrhein-Westfalen, LANUV, tissue with 500 μl RIPA buffer (150 mM NaCl, 1% Germany, AZ: 8.87–51.04.20.09.368) and were in accord- Triton-x-100, 1% Na-deoxycholate, 0.1% SDS, 50 mM ance with the German Animal Welfare Law. Animal care Tris-HCl pH 8, 10 mM EDTA) containing a proteinase- and use was performed by qualified individuals, supervised inhibitor (complete Mini, EDTA-free, Roche, Switzerland). by a veterinarian. All facilities and transportation complied Subsequently, the tissue lysates were centrifuged for with current legal requirements. The manuscript complies 15 min at 11000 g and 4 °C and supernatants were with the Animals in Research: Reporting In Vivo Experi- centrifuged for 40 min at 44,400 g and 4 °C. The clear ments (ARRIVE) guidelines [20]. Humane endpoints were supernatants were analyzed using the BCA test (Pierce, specified and part of the animal welfare application Thermo Scientific, Germany) and duplicate aliquots of according to the directive 2010/63/EU of the European 1000 μg/ml protein were measured directly. parliament. A list of specific clinical signs to determine the animal’s physiology and behavioral condition was used. Multiplex cytokine assay Specific experiment-related humane endpoints such as IL-1α, IL-1β, IL-6, IL-10 and TNF-α in jejunal tissues neurological disturbances, lameness, wound healing and IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 deficits and coma were evaluated. The animals were (p70), IL-13, interferon-γ (IFN-γ), granulocyte macrophage scored as mild, moderate or severely impaired. According colony-stimulating factor (GM-CSF) and TNF-α in serum to the results of scoring, animals were treated (analgesics, were measured in duplicates in a multiplex analyzer antibiotics), frequently examined or sacrificed (severe (Bio-Plex 200®, Bio-Rad Laboratories, USA) according impairment). According to the results of the scored to the manufacturer’s instructions. The jejunal tissue humane endpoints, animals received 50 μg/ml meloxicam lysate protein was diluted 1:2 with sample diluent contain- p.o. within the first three days after ROSC, if necessary. ing 0.5% BSA (500 μg/ml final protein concentration). Sera were thawed, centrifuged for 5 min at 10,000 g and Results 4 °C and diluted 1:4 in sample diluent. By using the Histomorphological analysis - Chiu grading median fluorescence intensity and the standard curve, the Representative images of intestinal tissues after CA absolute concentration of each cytokine (pg/ml) was revealing Chiu grades from 0 to 5 are shown in Fig. 1a-f. calculated (Bio-Plex Manager 6.1, Bio-Rad Laboratories). Controls lacked histomorphological changes in the small Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 4 of 10 Fig. 1 Histomorphological evaluation of the jejunal wall of rats after 6 min of CA using a modified Chiu scoring system (Chiu et al. 1970) [44]. Images represent the Chiu grades 0–5. a Intact mucosa and villus structures in control tissue of sham-operated rats (Chiu grade 0). b Development of a sub-epithelial space at the tips of the villi (arrow) 24 h post-ROSC (Chiu grade 1). c Development of a Gruenhagen’s space at the tip of a villus (arrow) 24 h post-ROSC (Chiu grade 2). d Villus necrosis (arrow) 7 d post-ROSC (Chiu grade 3). e Massive epithelial desquamation and villi which are denuded of epithelial layer (arrow) 7 d post-ROSC (Chiu grade 4). f Loss of villi, mucosal ulceration and necrosis 7 d post-ROSC (Chiu grade 5). a-f Hematoxilin-eosin staining, longitudinal section of 4 μm thickness (a, e, f) or cross section (b, c, d), scale bars represent 100 μm intestine. In rats with 6 min of CA, the intestinal mu- cosa revealed desquamation of the villus tips. Blunt, dome-shaped, fenestrated epithelial cells of submucosal arterioles were evident. Gruenhagen’s spaces, slight peri- vascular oedema, hydropic generation of epithelial cells and sparse pyknotic cells were identified. Chiu grades of jejunal mucosa revealed a significant time-dependent effect (Fig. 2, p < 0.0001). Tukey’s multiple comparison test showed a significant increase (12.5-fold) in mucosal damage 6 h post-ROSC (3.13 ± 0.64) compared to controls (0.25 ± 0.5, p < 0.001). Mucosal damage decreased significantly 2.1-fold at 24 h (1.48 ± 0.87) compared to 6 h post-ROSC (3.13 ± 0.64, p < 0.05). Chiu grading decreased significantly 2.2-fold at 72 h (1.4 ± 0.46) compared to 6 h post-ROSC (p < 0.05). At 7 d post-ROSC, Chiu grade increased again (3.33 ± 0.69, p < 0.05) compared to 24 h (1.48 ± 0.87) and was the highest compared to controls Fig. 2 Chiu Scoring. Chiu Scoring [Grades 0–5] in jejunum of controls and at 6 h, 24 h, 72 h and 7 d post-ROSC are expressed as mean ± SD. (0.25 ± 0.5, p < 0.0001). One-way ANOVA p < 0.0001. Tukey’smultiplecomparison testis marked Comparing the intestinal wall thickness of controls to with asterisks (*p < 0.05, ***p < 0.001, ****p < 0.0001) that at 6 h, 24 h, 72 h and 7 d post-ROSC showed Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 5 of 10 significant time-dependent changes (p < 0.0001). Tukey’s Comparison of jejunum and serum cytokine concentrations multiple comparison test showed that the muscularis The ratio of absolute cytokine concentrations in jejunum was significantly thinner at 7 d post-ROSC compared to compared to serum is shown in Table 3. Overall, signifi- controls (p < 0.05). A thinner muscularis was also ob- cant higher IL-1α and TNF-α concentrations were ob- served 6 h, 24 h and 72 h post-ROSC but this difference served in jejunal tissue compared to serum in both was not significantly decreased compared to controls. controls and CA groups at all timepoints (p <0.05). IL-1β in controls and at 24 h post-ROSC, IL-6 at 24 h post- ROSC and IL-10 at 24 h and 72 h post-ROSC were also Multiplex cytokine assay significantly higher in jejunum lysates compared to serum. Jejunal tissue Increased concentrations of IL-1β at 72 h and 7 d In jejunal tissue, IL-1α was altered over time with peak post-ROSC and IL-10 in controls and at 6 h and 7 d values at 24 h post-ROSC. (p < 0.01, Fig. 3a). Tukey’s post-ROSC were detected in jejunum but were not signifi- multiple comparison test identified significant elevations cantly different. IL-6 concentrations in jejunum and in IL-1α at 24 h compared to controls (1.7-fold increase, serum in controls and at 6 h, 72 h and 7 d post-ROSC and p < 0.01,) and the 6 h group (1.7-fold increase, p < 0.05). IL-1β concentrations at 6 h post-ROSC were comparable. At 72 h, IL-1α concentration decreased significantly 1.6-fold Comparing the time-dependent profiles of jejunum/serum (p < 0.01) compared to the 24 h group. No significant differ- ratio with profiles in jejunal tissue, IL-1α, IL-6, IL-10 and ence was detected between 24 h and 7 d post-ROSC TNF-α displayed a similar alteration with a peak at 24 h (Fig. 3a). IL-1β showed significant time-dependent changes post-ROSC while IL-1β peaked at 7 d post-ROSC, in (p <0.01). IL-1β peaked at 24 h compared to controls contrast to jejunal tissue at 24 h post-ROSC (Fig. 3a-e). (5.7-fold increase, p < 0.05) and 6 h (7.9-fold increase, p<0.05).At 72h,IL-1β was 2.5-fold lower than at 24 h Correlation analysis and 1.9-fold lower than at 7 d post-ROSC but differences No significant correlation between the duration of CA were not statistically different (Fig. 3b). IL-6 concentrations and cytokine concentrations in jejunum was detected showed time-dependent changes (p < 0.01). Additionally, a 6 h and 24 h post-ROSC: 6 h group: IL-1α: r = 0.43, p = significant 1.2-fold decrease was detected at 72 h compared 0.546; IL-1β: r = 0.002, p = 0.970; IL-6: r = 0.06, p = 0.848; to 24 h post-ROSC (p < 0.01, Fig. 3c). IL-10 concentrations IL-10: r = 0.44, p = 0.532; TNF-α: r = 0.07, p = 0.824. 24 h revealed significant time-dependent changes (p < 0.0001). group: IL-1α: r = 0.01, p = 0.874; IL-1β: r = 0.26, p = At 24 h, it peaked significantly compared to controls 0.492; IL-6: r = 0.86, p = 0.073; IL-10: r = 0.34, p = 0.413; (p < 0.0001) and 6 h post-ROSC (p < 0.001) resulting in TNF-α: r = 0.32, p = 0.438. However, there was a statisti- a 2.2-fold and 1.9-fold increase, respectively. Concen- cally significant correlation between the duration of CA trations at 72 h (1.6-fold, p <0.001) and 7 d (1.5-fold, and the Chiu-Grade 7 d after CA (r = 0.97, p = 0.012), p < 0.05) decreased significantly compared to that at while the other timepoints were not statistically signifi- 24 h (Fig. 3d). TNF-α concentrations showed a significant cant (6 h group: r = 0.85, p = 0.251; 24 h group: r = 0.24, time-dependent effect (p < 0.01) at 24 h post-ROSC p = 0.501; 72 h group: r = 0.21, p = 0.537). Additional in- compared to controls (1.5-fold increase, p <0.01) and formation pertaining to the duration of cardiac arrest 6 h post-ROSC (1.4-fold increase, p < 0.05). TNF-α and the different groups are shown in Additional file 1 decreased significantly at 72 h post-ROSC compared but no significant differences were found between groups. to 24 h post-ROSC (1.5-fold, p < 0.01). There were no significant changes between 72 h and 7 d post-ROSC Discussion (Fig. 3e). This study reveals three major findings: (i) mild intestinal barrier damage could be detected within 24 h in a rat model of 6 min CA. (ii) only mild local intestinal inflamma- Serum tion could be shown within 24 h after CA. (iii) a systemic Absolute concentrations of IL-2, IL-4, IL-5, IL-12 (p70), inflammation and thus a potential contribution of the small IL-13, GM-CSF and IFN-γ in serum are shown in intestine to systemic inflammation could not be simulated Table 2. Results for IL-1α,IL-1β, IL-6, IL-10 and TNF-α after6minofCAinrats. are shown in Fig. 3f-j. Serum concentrations of IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, GM-CSF, IFN-γ and TNF-α did not differ significantly between Intestinal barrier damage controls and at 6 h, 24 h, 72 h and 7 d post-ROSC. Although 6 min of transient global ischaemia and subse- IL-1β showed a significant time-dependent effect (p < quent reperfusion led to mucosal damage and decreased 0.05). At 24 h post-ROSC, IL-1β significantly increased thickness of the muscularis in the small intestine, jejunal 1.6-fold compared to controls (p < 0.05, Fig. 3g). tissue concentrations of IL-1α, IL-1β, IL-10 and TNF-α Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 6 of 10 Fig. 3 (See legend on next page.) Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 7 of 10 (See figure on previous page.) Fig. 3 Cytokine concentrations of IL-1α,IL-1β, IL-6, IL-10 and TNF-α (mean ± SD, pg/ml) in jejunum (left column) and serum (right column) in controls andat 6 h, 24 h, 72 hand 7dpost-ROSC. ThesignificanceofTukey’smultiplecomparisontest ismarkedwithasterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). a IL-1α concentrations in jejunum (one-way ANOVA p < 0.01). b IL-1β concentrations in jejunum (one-way ANOVA p < 0.01). c IL-6 concentrations in jejunum (one-way ANOVA p < 0.01). d IL-10 concentrations in jejunum (one-way ANOVA p < 0.0001). e TNF-α concentrations in jejunum (one-way ANOVA p < 0.01). f IL-1α concentrations in serum (one-way ANOVA p >0.05). g IL-1β concentrations in serum (one-way ANOVA*). h IL-6 concentrations in serum (one-way ANOVA p >0.05). i IL-10 concentrations in serum (one-way ANOVA p >0.05). j TNF-α concentrations in serum (one-way ANOVA p >0.05) were only slightly affected. This result may be due to to oxidative stress, NF-КBis up-regulatedandorches- several reasons. trates the release of a number of cytokines such as IL-1ß, First, the duration of 6 min CA in the present study that IL-2, IL-4, IL-5, IL-6 and IL-10, IL-12, IL-13 and TNF-α is sufficient to show cerebral damage [21] is inadequate to [28, 29]. Accordingly, we collected blood (sera) and tissues initiate a systemic inflammatory response, which is a clear at early stages including 6 h and 24 h post-ROSC and limitation of this study. As shown by Qian, exceeding the chose a similar cytokine profile to be investigated. How- duration of CA of 6 min may extend intestinal damage ever, in clinical studies, it is often proposed that the PCAS and influence serum cytokine concentrations. In detail, may result from a systemic inflammatory activation persist- intestinal microcirculatory blood flow was significantly ing for days [5, 30]. Therefore, cytokine concentrations in decreased accompanied by mild elevated serum con- jejunum and sera were also evaluated within the first 7 days centrations of TNF-α and IL-6 during 8 min of CA in after CA in the present study. pigs [22]. In contrast, significant inflammatory response Third, as repeatedly shown, simulation of a PCAS is following local ischaemia andreperfusion of thesmall difficult in rats and accompanied by a high failure rate intestine was shown to be initiated after a duration of of more than 50% [24, 31]. Thus, on the basis of previ- at least 15–30 min [23]. However, models of focal ischae- ous scientific findings, we conclude, that severely injured mia and reperfusion are not capable of being translated to rats that would develop a PCAS initially died during re- conditions of systemic ischaemia and reperfusion. In fact, suscitation procedures. With a survival rate of only CA leads to a complex systemic ischaemia-reperfusion-in- 29.5% in the present study, we conclude that this model jury with contribution of multiple independent tissues, can be utilized to a limited extent to reproduce a PCAS which are integrated into a complicated cascade of cell- or peripheral tissue damage, respectively. Nevertheless, death and systemic inflammation [5, 6]. ROSC rates are comparable to previous experiments Secondly, intestinal repair mechanisms seem to be performed in our group, which are able to adequately commenced immediately after CA. A normalization of determine cerebral damage [16, 18, 21, 24, 32–34]. In leukocyte-endothelial interaction as well as the wall shear this pilot study, our main objective was to evaluate per- rate was reported to be initiated within 120 min after CA, ipheral tissue potentially injured due to CA. Notably, we which is a period not mirrored in our study [24]. However, did not lose any animals after ROSC, which is advanta- reports show that cytokines such as IL-1ra, IL-6, IL-8, geous since post-ROSC mortality is known to be 38% IL-10 and TNF-α are expressed within 3 h [5, 6, 25]to 6 h within an observation period > 48 h [35]. Interestingly, [24, 26] and peak within the first 2 days after CA [5, 6]. Vognsen et al. recently showed that only 12% of animal This is in line with studies reporting a massive up-regula- studies sufficiently report outcome parameters according tion of cytokines after ischaemic brain injury [2, 27]. Due to the Utstein Guidelines, which was a strong criterion to increase the validity of this study [35]. Table 2 Absolute levels of cytokines in serum (IL-2, IL-4, IL-5, Given the clinical phenomenon of bacteremia after CA IL-12 (p70), IL-13, GM-CSF, IFN-γ) in controls (C) and at 6 h, 24 h, [7, 8], intestinal damage seems to be conclusive even 72 h and 7d post-ROSC expressed in pg/ml (mean ± SD) after short periods of ischaemia. Congruently, our results C 6 h 24 h 72 h 7d point towards a mild local intestinal damage, which is in IL-2 0.36 ± 0.35 0.27 ± 0.06 0.20 ± 0.05 0.21 ± 0.07 0.34 ± 0.28 line with Pan et al. who reported similar Chiu scorings 24 h after CA of 6 min duration [36]. Likewise, Teschen- IL-4 0.24 ± 0.18 0.22 ± 0.09 0.17 ± 0.02 0.17 ± 0.02 0.23 ± 0.10 dorf et al. showed a 3–4-fold stronger plasma extravasa- IL-5 n.d. n.d. n.d. n.d. n.d. tion from post-capillary mesenteric venules at 120 min IL-12 (p70) n.d. n.d. n.d. n.d. n.d. after CA, which is a characteristic sign of endotoxaemia IL-13 n.d. n.d. n.d. n.d. n.d. [24]. Another factor taken into consideration is the short GM-CSF 0.20 ± 0.01 0.25 ± 0.06 0.23 ± 0.02 0.24 ± 0.03 0.23 ± 0.05 initial tissue hyperperfusion followed by a sustained hypo- IFN-γ 0.36 ± 0.07 0.47 ± 0.18 0.34 ± 0.01 0.31 ± 0.02 0.35 ± 0.05 perfusion of intestinal tissue after CA [37, 38]. This inevit- n.d. not detected ably leads to a prolonged period of relative ischaemia Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 8 of 10 Table 3 Ratio of absolute IL-1α, IL-1β, IL-6, IL-10, TNF-α levels in jejunum to absolute levels in serum in controls (C) and at 6 h, 24 h, 72 h and 7d post-ROSC (mean ± SD) C 6 h 24 h 72 h 7d **** **** **** *** **** IL-1α 15.2 ± 3.6 15.5 ± 1.7 30.4 ± 3.8 19.3 ± 4 13.9 ± 5 * ** IL-1β 1.7 ± 0.2 1 ± 0.6 6.9 ± 0.4 7.9 ± 2.9 10.2 ± 1.5 **** IL-6 1 ± 0.1 1 ± 0.04 1.3 ± 0.03 1 ± 0.02 1.1 ± 0.1 **** * IL-10 1.9 ± 0.4 1.8 ± 0.3 4.2 ± 0.6 3.6 ± 0.5 2.5 ± 0.7 **** * *** * ** TNF-α 1.5 ± 0.04 1.4 ± 0.1 2.1 ± 0.1 1.7 ± 0.2 2 ± 0.1 The level of significance is marked with asterisks: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001) where the intestine receives only 5% of cardiac output [39] pro-inflammatory cytokines such as IL-1α,IL-1β and and fosters further tissue damage. Interestingly, we found TNF-α. a significant correlation between the duration of CA and Overall, serum cytokine concentrations were not signifi- the Chiu-grade in the 7 d group. However, these results cantly altered except for a significant increase in IL-1β after should be considered with caution because significant 24 h post-ROSC. Additionally, IL-1α,IL-1β,IL-10 and mucosal damage was also shown with a shorter duration TNF-α concentrations were lower in serum than in the je- of CA in the 6 h group (Additional file 1). junum. This may indicate that the observed intestinal inflam- It must be noted that mucosal damage and cytokines mation may not be associated with systemic inflammation. were not analyzed in animals that could not be resusci- Notably, intestinal cytokine increments were in accord- tated. However, results were compared to a control group ance with morphological changes of the intestinal mucosa. serving as reference. Since achievement of ROSC is mainly Intestinal tissue damage peaked at 6 h post-ROSC and de- dependent on heart function, significant intestinal damage creased subsequently at 24 h and 72 h. Tissue repair mecha- detectable immediately after ceasing CPR was not as- nisms accompanied by increases in intestinal inflammatory sumed. As reported, mucosal damage develops within the cytokine release seem to have been initiated. Consequently, first 6 to 24 h after cardiac arrest [36] and may contribute desquamation of villus tips, development of a Gruenhagen’s to the development of the PCAS, which was the main space, hydropic generation of epithelial cells and changes in objective in this investigation. muscular layer thickness were observed. The mechanism, which induces a further increase in intestinal tissue damage 7 d post-ROSC, accompanied Cytokine expression in tissue and serum by rising cytokine concentrations of IL-1β and IL-10 at A systemic ischaemia-reperfusion-injury causes a so-called the same timepoint, requires further research. As the sterile inflammation, which is associated with influx of duration between 72 h and 7 d post-ROSC is comparably neutrophils and macrophages, leading to the production of long, it might be possible that further changes in cytokine inflammatory cytokines [40]. IL-1α, present in gastrointes- concentrations remained undetected. tinal epithelial and endothelial cells [41], acts via initiation of the inflammatory cascade and is thus a valuable param- Conclusions eter for the determination of inflammatory processes. In Six minutes of transient systemic ischaemia and reperfu- the present study, IL-1α showed a significant increase in sion resulted in mild small intestinal tissue damage but the jejunum, resulting in the highest concentrations at 24 h not in systemic inflammation. A rat model of 6 min CA post-ROSC. At thesametimepoint, IL-1α concentrations is not capable of mimicking a PCAS. Whether there is a in serum were close to detection limits. It has been re- vital influence of the intestine on the PCAS still remains ported that in vitro circulating IL-1α was released from unclear and should be investigated in further studies. endothelial cells [41] but was barely detectable in patients suffering from severe inflammation [42]. This suggests that jejunal and serum IL-1α concentrations do not necessarily Additional file match, as observed in the present study. Additional file 1: Duration of cardiac arrest in the different post-ROSC The IL-10 family acts protectively during intestinal in- groups. One-way ANOVA p < 0.05. Tukey’s multiple comparison test did flammation by induction of anti-inflammatory effects and not show any statistically significant differences between any of the groups. inhibition of pro-inflammatory cytokines such as IL-1β, (DOCX 28 kb) IL-6 and TNF-α [43]. In our study, in jejunal tissues, IL-1α,IL-1β and TNF-α increased significantly at 24 h Abbreviations post-ROSC indicating that increasing IL-10 concentra- ATP: Adenosine triphosphate; CA : Cardiac arrest; CPR: Cardio-pulmonary tions at the same time might counteract the increase of resuscitation; IL: Interleukin; NMDA: N-methyl-D-aspartate receptor; Schroeder et al. BMC Anesthesiology (2018) 18:61 Page 9 of 10 PCAS: Post cardiac arrest syndrome; ROS: Reactive oxygen species; resuscitation after cardiac arrest as a "sepsis-like" syndrome. Circulation. ROSC: Return of spontaneous circulation; TNF-α: Tumor necrosis factor-α 2002;106(5):562–8. 6. Nolan JP, Neumar RW, Adrie C, Aibiki M, Berg RA, Bottiger BW, Callaway C, Clark RS, Geocadin RG, Jauch EC, et al. Post-cardiac arrest syndrome: Acknowledgements epidemiology, pathophysiology, treatment, and prognostication. A scientific The authors thank Irmgard Henke and Malte Heykants for their excellent statement from the international liaison committee on resuscitation; the technical assistance, Roland Galmabcher, Peter Teschendorf and Andreas American Heart Association emergency cardiovascular care committee; the Schneider for their methodological support and Laura A. Webb, M.Sc. for council on cardiovascular surgery and anesthesia; the council on proof-reading the manuscript. cardiopulmonary, perioperative, and critical care; the council on clinical cardiology; the council on stroke. Resuscitation. 2008;79(3):350–79. Funding 7. Carr GE, Yuen TC, McConville JF, Kress JP, VandenHoek TL, Hall JB, Edelson This work was supported by the intramural “Cologne-Fortune” programme, DP. Early cardiac arrest in patients hospitalized with pneumonia: a report Faculty of Medicine, University of Cologne (299/2014). Design of the study, from the American Heart Association's get with the guidelines-resuscitation collection, analysis, interpretation of data and writing were not influenced by program. Chest. 2012;141(6):1528–36. thy funding body. 8. Coba V, Jaehne AK, Suarez A, Dagher GA, Brown SC, Yang JJ, Manteuffel J, Rivers EP. The incidence and significance of bacteremia in out of hospital Availability of data and materials cardiac arrest. Resuscitation. 2014;85(2):196–202. The datasets used and/or analysed during the current study are available 9. Guschlbauer M, Feige K, Geburek F, Hoppe S, Hopster K, Propsting MJ, from the corresponding author on reasonable request. Huber K. Effects of in vivo lidocaine administration at the time of ischemia and reperfusion on in vitro contractility of equine jejunal smooth muscle. Authors’ contributions Am J Vet Res. 2011;72(11):1449–55. DS and MG were involved in planning and conduction of the experiments, 10. 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BMC AnesthesiologySpringer Journals

Published: Jun 5, 2018

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