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Remote Intestinal Injury Early After Experimental Polytrauma and Hemorrhagic Shock

Wrba, Lisa; Ohmann, Julia J.; Eisele, Philipp; Chakraborty, Shinjini; Braumüller, Sonja; Braun, Christian K.; Klohs, Bettina; Schultze, Anke; von Baum, Heike; Palmer, Annette; Huber-Lang, Markus; Halbgebauer, Rebecca

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doi: 10.1097/SHK.0000000000001271



As recently published, polytrauma (PT) and hemorrhagic shock (HS) induce multiple direct effects on hemodynamics, lungs, brain, and endothelium (1–4). The detrimental effects of HS in addition to PT are also reflected in a recent clinical study (5), where HS in addition to PT was associated with higher levels of organ and damage markers. Besides the impacts from direct injury, we also found some remote injury in organs not directly exposed to the force vector, especially in the kidneys (1).

The importance of barrier molecules for the pathophysiological changes after trauma and HS is evident as the plasma concentrations of the major tight junction protein claudin-5 were increased after PT and HS and positively correlated with lactate levels in the blood (5). Similarly, levels of the tight junction protein junctional adhesion molecule 1 (JAM-1) were also increased in plasma, but decreased in lung tissues after trauma in mice, associated with elevated permeability of the blood–air barrier. Clear evidence of circulating JAM-1 proteins in plasma was also found in polytraumatized patients, related to higher acute physiology and chronic health evaluation II (APACHE II) and sepsis-related organ failure assessment (SOFA) score (6), indicating a connection between disrupted barrier function and the clinical condition of a patient. Zonula occludens protein 1 (ZO-1) is another important molecule for the function of tight junctions, as it anchors the tight junction protein occludin to cytoskeletal actin (7). There is evidence for decreased ZO-1 protein levels as well as reorganization of ZO-1 and increased permeability in murine burn injury (8) and HS (9).

The gut is known to play a central role in the development of remote organ injury. HS in particular can lead to generation of biologically active lymph, leading to priming of immune cells and finally causing remote organ damage (10–12). As recently reviewed by our group (13), traumatic brain injury and HS increase intestinal permeability and can thereby lead to translocation of pathogen-associated molecular patterns (PAMPs) of commensal bacteria and endogenous damage-associated molecular patterns (DAMPs) over the epithelial barrier, further aggravating the immune reaction. Thus, the gut significantly contributes to the posttraumatic pro-inflammatory response and may act as a central engine of multiple organ failure (14). However, little is known about the differential effects of PT and HS on alterations of intestinal permeability and inflammation in a highly standardized model of PT and HS. Therefore, this study aims to describe effects of PT and HS on remote intestinal inflammation and barrier function, especially regarding the role of tight junction protein ZO-1.


Animals and anesthesia

Animal experiments were performed according to the National Institutes of Health Guidelines for the use of laboratory animals. The study protocol was approved by the University Animal Care Committee and the Federal Authorities for Animal Research, Tübingen, Germany (Approval No. 1194).

Eight to 12 weeks old C57BL/6 mice weighing 24.5 ± 1.9 g were randomly divided into 4 groups with n = 5 to 8 animals each: PT, HS, PT combined with HS (PT+HS), and sham-treated animals (sham), which were treated exactly the same including anesthesia and catheterization, except for trauma and hemorrhage. Mice were anesthetized with 4% sevoflurane (Sevorane Abbott, Wiesbaden, Germany) in 96% oxygen. Anesthesia was continued during the whole experimental period following a preset protocol. For analgesia, the mice received 0.05 mg/kg buprenorphine (Temgesic, RB Pharmaceuticals, Slough, UK) by subcutaneous injection.

Induction of PT and HS

As described previously (15), PT was induced by application of a blunt bilateral chest trauma, closed head injury, and a distal femur fracture accompanied by soft tissue injury.

After induction of PT, catheters were inserted into the femoral artery of the uninjured left leg and the jugular vein as described previously (1) to allow continuous monitoring of the blood pressure as well as blood withdrawal, resuscitation, and administration of catecholamines. Mice in the shock groups were bled to a medium arterial blood pressure (MAP) of 30 ± 5 mmHg and kept on this level for 60 min. Afterwards, they were resuscitated with 4 times the volume drawn using a balanced electrolyte solution (Jonosteril, Fresenius Kabi, Bad Homburg, Germany). During the following 2 h observation period, mice were kept at a MAP of ≥50 mmHg by administration of norepinephrine (0.1–0.9 μg/(kgmin)) (Sanofi, Frankfurt (M), Germany), when necessary. After instrumentation and at the end of the observation period, the abdominal girth of the animals was measured.

Permeability measurements using Evans blue dye

One hour before the end of the observation period, animals received 200 μL of 2% Evans blue dye (Sigma, St. Louis, MO) in phosphate buffered saline over 5 min via the venous catheter. At the end of the experiment, blood was drawn by cardiac puncture and divided. 200 μL were mixed with a bouillon for bacterial culture, the rest was anticoagulated with ethylenediaminetetraacetic acid and centrifuged for generation of plasma. Afterwards, the pulmonary circulation was flushed with 10 mL of ice-cold 0.9% NaCl. The systemic circulation was flushed with 40 mL of NaCl to remove residual dye from the vasculature. Organs were then harvested and incubated in 10% formaldehyde solution (Geyer, Renningen, Germany) for 24 h at 60°C to elute the dye. For standardization, 2 cm of the distal jejunum and 2 cm of the proximal colon with all contents were used. The dye concentration in the supernatant fluids was measured spectrophotometrically at 620 nm and normalized to the plasma concentration of the respective animal to correct for dilution effects due to resuscitation and catecholamine administration.

Bacterial culture

200 μL of blood was cultured in brain heart infusion culture medium (Oxoid, Basingstoke, UK) for 5 to 7 days at 37°C. In addition, stool samples were analyzed to determine which bacteria were present in the intestinal microbiome. Bacterial strains in blood were identified and compared to those found in the stool samples.

Histological analyses

For histological stainings, a separate set of animals (sham and PT+HS only, n = 8 per group) was used. 1 cm of the distal ileum and 1 cm of the proximal colon were removed, fixed in 3.7% formaldehyde solution (Otto Fischar, Saarbrücken, Germany), embedded in paraffin and cut in 4 μm sections.

For hematoxylin and eosin stainings, a staining kit (Morphisto, Frankfurt/M, Germany) was used according to the manufacturer's instructions.

For immunohistochemical staining, sections were deparaffinized, rehydrated in a descending alcohol series, and boiled in sodium citrate buffer (Sodium citrate dihydrate, Sigma, 3.0625 mg/mL, pH 6.0) for retrieval of antigen epitopes. Sections were then incubated in 3% H2O2 (Otto Fischar) to block the endogenous peroxidase activity. After treatment with 10% goat serum (Jackson Laboratories, Bar Harbor, ME) for 30 min and washing in tris-buffered saline, sections were incubated for 1 h with the following primary antibodies: anti-ZO-1 (rabbit polyclonal ZO-1 antibody, Thermo Fisher, Waltham, MA), anti-Toll-like receptor (TLR)-2 (Bioss, Woburn, MA), anti-TLR-4 (Thermo Fisher), and anti-C3aR (Bioss). After washing, the primary antibody was detected using horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, Danvers, MA). Afterwards, staining was developed using 3,3′-diaminobenzidine enhanced liquid substrate system (Sigma). Specimens were dehydrated in ethanol and xylene, mounted in NeoMount medium (Merck, Darmstadt, Germany), and glass covered.

For immunofluorescence, the sections were treated as described above. As a secondary antibody, Alexa488-conjugated goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch, West Grove, PA) was used for 1 h. After washing, the specimens were directly covered with Prolong Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA) for counterstaining of nuclei.

Densitometric analyses of HRP-stained sections were performed with Zeiss AxioVision M1 microscope. Therefore, sections of 8 animals per group were evaluated in a blinded fashion. Five pictures of representative regions of each specimen were taken, using the 200-fold magnification. Pictures were analyzed using the Zeiss AxioVision SE64 Rel. 4.9 Software (Zeiss, Oberkochen, Germany). Results are presented as densitometric sum (arbitrary units).

Histological scoring of ZO-1 immunofluorescence staining was also performed in a blinded fashion and with n = 8 specimens per group. The proportion of cells with visible staining at cell–cell contacts was assessed for 60 crypts and 60 villi (ileum) or 60 segments of surface epithelium between 2 crypts (colon) and assigned to one of the following classes: 0% to 25% (score point 1), >25% to 50% (score point 2), >50% to 75% (score point 3), or >75% to 100% (score point 4).

Cell culture

For cell culture experiments, the human colon carcinoma cell line T84 (ATCC, Manassas, VA) was used. Cells were grown to confluency on polycarbonate transwell filters with a pore size of 0.4 μm (Corning, Corning, NY). Cells were stimulated either from the apical or the basolateral compartment with C3a (1000 ng/mL, Complement Technology, Tyler, TX), peptidoglycan (PGN, 10 μg/mL, Sigma), PGN + C3a, lipopolysaccharide (LPS, 1000 ng/mL, Sigma), or LPS + C3a for 6 h. Afterwards, the medium from the apical and the basolateral compartment was collected, centrifuged, and stored at −80°C until analysis. n = 5 biological replicates per condition were done.

Enzyme-linked immunosorbent assay

Measurement of cytokines in cell culture media and measurement of intestinal fatty acid binding protein (i-FABP) in the plasma of mice was performed by sandwich enzyme-linked immunosorbent assay (ELISA). The following ELISA kits were used following the manufacturers’ instructions: Human interleukin (IL)-8 ELISA kit (R&D, Minneapolis, MN), Human TJP-1/ZO-1 ELISA Kit (LifeSpan Biosciences, Seattle, WA), and murine i-FABP ELISA kit (LifeSpan Biosciences).


Statistical analysis was performed with Sigma Plot software (Edition 11.0; Systat Software, Erkrath, Germany). Results are presented as mean ± SEM. For detection of differences between group means, one-way analysis of variance (ANOVA) testing was used for multiple group comparisons. Before testing, data sets were analyzed for distribution pattern and equality of variance. In case of parametric distribution, the Holm–Sidak post-hoc test was used, in case of nonparametric distribution, Kruskal–Wallis ANOVA on ranks followed by Dunnett's method was performed. For comparison of 2 groups, the unpaired t-test was used for parametric and the Mann–Whitney–Rank sum test for nonparametric distribution. For comparison of the amounts of score points, a chi-square test was carried out. Values of p < 0.05 were considered statistically significant for all test procedures.


Increased intestinal permeability after PT+HS

Mice subjected to PT or HS showed an increase in abdominal girth during the experimental period, which was significantly enhanced if PT and HS were combined (Fig. 1A). This increase was reflected by a dilated and fluid-filled intestine in the experimental groups, indicating extravasation of fluid and leakage through the epithelial barrier (Fig. 1B). The permeability index, calculated as tissue Evans blue dye concentration normalized to the respective plasma concentration, was increased in the HS group, but not in the PT or the PT+HS group. These early changes reached significance in the jejunum (Fig. 1C) and showed the same trend in the colon (Fig. 1D).

Fig. 1:
Increased intestinal permeability after trauma and hemorrhagic shock.

Effects of PT+HS on intestinal injury

As a systemic marker for intestinal injury, plasma levels of i-FABP were measured. There was a tendency to higher levels in all experimental groups early after injury compared to sham animals (Fig. 2A). Evaluation of hematoxylin and eosin-stained sections showed some detachment of the ileal epithelium in 3/8 sham and 8/8 PT+HS animals. In the colon, epithelial damage was visible in 4/8 sham and 7/8 PT+HS mice (Fig. 2B). Blood cultures were positive for bacteria only in 1 HS animal. Differentiation of bacterial strains showed Staphylococcus aureus, coagulase-negative staphylococci, and Corynebacteriaceae in the blood of this animal (Fig. 2C). Of note, S. aureus and Corynebacteriaceae were not found in the stool sample. In order to exclude effects of the Evans blue dye on bacterial growth, we tested the sensitivity of several bacterial strains to Evans blue, which had no inhibitory effect on bacterial proliferation.

Fig. 2:
Intestinal damage following polytrauma and hemorrhagic shock.

Decreased ZO-1 protein levels in intestinal epithelium after PT+HS

Intestinal epithelial cells in ileum and colon showed reduced ZO-1 staining at cell–cell contacts early after PT+HS (Fig. 3B and D) compared to sham conditions (Fig. 3A and C). This was reflected by lower score values (representing the percentage of epithelial cells with visible ZO-1 staining at cell–cell contacts) in the PT+HS group compared to sham animals in the ileum (Fig. 3E) as well as in the colon (Fig. 3F). Densitometric measurement of ZO-1 in HRP-stained sections also showed a decrease in ZO-1 protein expression (Fig. 3G and H). In an in-vitro model, apical stimulation of human T84 cells did not reduce expression of ZO-1 within the cells, whereas in contrast basolateral stimulation with of TLRs and C3aR resulted in a decreased concentration of ZO-1 (Supplemental Fig. 1a and b,

Fig. 3:
Reduced ZO-1 protein expression after polytrauma and hemorrhagic shock.

Influence of PAMPs and DAMPs on intestinal secretion of inflammatory mediators

C3aR, TLR2, and TLR4 were all expressed in the ileum and colon of mice (Fig. 4A). No significant early changes in expression levels were found between sham and PT+HS animals (Fig. 4B). In an in-vitro model, apical stimulation of T84 cells with C3a and LPS did also not affect cytokine secretion. In contrast, apical stimulation of TLR2 with PGN resulted in increased secretion of the chemokine IL-8 to the apical (Supplemental Fig. 1c, as well as to the basolateral compartment (Supplemental Fig. 1d, A similar pattern, albeit to a lesser extent was found for IL-6. Stimulation with PGN from the basolateral side also led to a slight increase in IL-8 secretion to the basolateral, but not to the apical compartment (data not shown).

Fig. 4:
Expression of receptors for inflammatory mediators on the intestinal epithelium.


Dysfunction of the gut–blood barrier is an important factor for development of remote organ injury and multiple organ failure after trauma and HS (16, 17). Though enhanced epithelial permeability might lead to translocation of bacteria, this was mainly shown in rodent models (18, 19), but not in human patients (20). We found positive blood cultures only in 1 animal in our experimental HS group. This might be due to the very early time point after shock, the 1 positive sample might also be result of a contamination. Although direct bacterial translocation to systemic circulation was not found, remote organ damage may also be caused by biologically active mesenteric lymph which is generated after HS as a result of increased intestinal permeability and activation of immune cells (11). Trauma and HS may also lead to destruction of the intestinal mucus layer (21), thereby enabling bacteria to stimulate TLRs beyond the physiological level. Disruption of the epithelial barrier allows commensals and pathogens to stimulate TLRs on intestinal epithelial cells also from the basolateral side. TLR activation induces pro-inflammatory signaling in epithelial cells (22–24). Activation of TLR2 by PGN resulted in enhanced secretion of the pro-inflammatory cytokines IL-8 and IL-6. In contrast to enhanced secretion of pro-inflammatory cytokines in a model of inflammatory bowel disease (25), additional stimulation by C3a which is systemically released in the setting of PT and HS (26) did not show additive effects in our model. However, no immune cells were present in the present in-vitro study. As C3a acts as a chemoattractant and activates immune cells (27), there might be an additional effect of C3a in their presence and further investigation is required here. Stimulation of T84 cells with the TLR4-agonist LPS with or without C3a did not result in enhanced secretion of IL-8; this might be due to the low levels of TLR4 and especially the coreceptor MD-2 expressed by T84 cells as a protection mechanism against permanent stimulation by commensal bacteria (28). Besides from a direct inflammatory response of the epithelium, opening or disruption of tight junctions is an important mechanism in gut-derived multiple organ failure. Increased permeability allows gut contents such as serine proteases (16) and other DAMPs and PAMPs to induce an immunological response and thereby may contribute to remote organ damage and worsen clinical outcome (11, 29).

ZO-1 is an important protein in the tight junction complex, as it anchors the transmembrane protein occludin to the cytoskeletal actin (7). After PT+HS, we found decreased levels of ZO-1 in ileal and colonic epithelium, especially at the cell–cell contacts. This could also be shown in an in-vitro model, where stimulation of basolateral pattern recognition receptors with DAMPs and PAMPs diminished ZO-1-expression. These results are in accordance with previous studies in animal models of burn injury (8) and HS (9). In context of HS, decreased levels of ZO-1 and also cytoskeletal actin in the intestinal epithelium were shown (30). Of note, the latter study did not include a phase of reperfusion after shock, as it is present in clinical reality. As it has been reported that reperfusion might cause more damage through generation of reactive oxygen species (31), we included a reperfusion protocol in our present study. In another study, the decrease in ZO-1 levels was shown to be diminished by inhibition of histone deacetylase 6 (9) or phosphodiesterase (8), showing possible interventional targets. As plasma levels of ZO-1 are correlated to severity of sepsis, SOFA and APACHE II-score values (32), ZO-1 might be an interesting monitoring parameter also after trauma, as well as a therapeutic target in critically ill trauma patients. The increased permeability for Evans blue dye seen in the HS group in our study may reflect the loss of ZO-1. Although no increased permeability to Evans blue dye was found in the colon of the PT+HS animals, all experimental groups and especially the PT+HS group showed an increase in abdominal girth and accumulation of fluid within the intestine, likely reflecting increased permeability of the endothelium as well as the intestinal epithelial tight junctions not only after HS but also after hemodynamic stable and instable PT.

Plasma i-FABP levels were shown to be a sensitive and specific marker for intestinal injury in trauma patients (33). Elevated i-FABP levels were found in patients after trauma, especially in those with abdominal injury (33, 34) and were correlated with the severity of injury and clinical outcome (34). Animals in our model showed increased plasma i-FABP levels despite the lack of a direct force vector on the abdomen, indicating early remote intestinal injury. This injury was also reflected by some histologically visible detachment of the epithelium as a marker for intestinal damage (35).

Taken together, we showed that PT and HS increase intestinal permeability, despite the lack of a direct trauma impact on the abdomen. This elevated permeability may be due to decreased levels of ZO-1 in the epithelial cell–cell junctions and was reflected by some early increase in plasma levels of i-FABP and histological damage. As intestinal permeability is known to contribute to organ dysfunction after trauma and hemorrhage, further studies are required to show whether restoration of ZO-1 after trauma is possible and beneficial for the clinical outcome.


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Barrier dysfunction; hemorrhagic shock; intestinal permeability; polytrauma; zonula occludens protein 1

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