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Basic Science Aspects: Original Article


Thuijls, Geertje; de Haan, Jacco-Juri; Derikx, Joep P. M.; Daissormont, Isabelle; Hadfoune, M'hamed; Heineman, Erik; Buurman, Wim A.

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doi: 10.1097/SHK.0b013e31817fc310
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Hemorrhagic shock (HS) is a clinical condition characterized by insufficient tissue perfusion to meet the demand for oxygen and nutrients. This hypoperfusional state can trigger a systemic inflammatory response, sepsis, and ultimately multiorgan dysfunction syndrome (MODS) (1-3). The MODS commonly affects the intestine, liver, kidney, and lung.

The proper function of these visceral organs depends on generation and maintenance of compositionally distinct compartments by a barrier formed by epithelial cells sealed by tight junctions (TJs) (4). Tight junctions serve as a fence dividing cells into apical and basolateral domains. These TJs are anchored in the cell via the filamentous actin (F-actin) cytoskeleton. Hypoperfusion or ischemia can cause disruption of the F-actin cytoskeleton, with subsequent barrier failure. Gut wall integrity loss leads to paracellular leakage of microbial products (5-8). Hepatic barrier dysfunction leads to toxins and bile escaping into the systemic circulation (6, 9). Barrier failure in the kidney causes back leakage of tubular fluid (6, 10). These clinical consequences of epithelial TJ loss are potential triggers for an innate immune response. Derangement in the formation and function of TJ has a prominent role in liver, gut, and kidney dysfunction not only during sepsis, but also in MODS (6, 7, 11). However, HS does not result in early pulmonary TJ loss, pulmonary dysfunction, or leak of the alveolar-capillary membrane, in absence of severe associated factors, such as massive inflammation (2, 12).

The actin cytoskeleton is a dynamic structure that maintains cell shape and TJ stability. Increasing evidence implicates actin-depolymerizing factor/cofilin (AC) as a mediator of cellular actin dynamics by binding to F-actin and enhancing its severing and turnover (recycling) (13-16). Cofilin is expressed in the mouse small intestine (17). Adenosine triphosphate (ATP) depletion, as occurs in hypoperfusion, results in AC activation (dephosphorylation). Dephosphorylated AC binds F-actin and at relatively low stoichiometry to F-actin subunits, AC can sever F-actin and enhance subunit turnover. At higher stoichiometry, AC can stabilize and bundle F-actin into aggregates or rods (18, 19). As a result of low stoichiometric activation of AC, the actin cytoskeleton is disrupted, losing connections with TJ proteins and causing disassembly of TJ, a process that also is dependent on myosin II-based contractility (20). Indeed, in renal ischemia-reperfusion injury, a situation with great resemblance to HS, ATP depletion in the kidney causes activation of AC, its relocalization to the apical membrane, and consequent alterations in the apical actin cytoskeleton (15, 21). This study is aimed at unraveling the changes in cytoskeleton and TJ integrity after HS in the organs commonly affected in MODS (liver, kidney, and intestine) and to elucidate AC activity preceding and during cytoskeleton loss.



Rabbit anti-zonula occludens protein 1 (ZO-1) (61-7300) and rabbit anti-claudin 3 (34-1700) were purchased from Invitrogen (San Francisco, Calif). Mouse anti-globular actin (G-actin) (JLA20) was purchased from Developmental Studies Hybridoma Bank, University of Iowa (Ames). Mouse antiactin (C4) was purchased from MP Biomedicals (Aurora, Ohio). Oregon green-phalloidin (O7465) was purchased from Invitrogen. Rabbit anti-AC (total AC [tAC]) (rabbit 1439) and rabbit anti-phosphorylated AC (pAC) were previously characterized (22, 23). Mouse anti-β-actin (A 2228) was purchased from Sigma (St Louis, Mo). Texas red conjugated goat antirabbit antibody, Texas red conjugated rat antirabbit antibody, goat antirabbit horseradish peroxidase (HRP)-conjugated secondary antibody and rat antimouse HRP-conjugated secondary antibody were purchased from Jackson (West Grove, Pa).


This study was performed according to the guidelines of the Animal Care Committee of Maastricht University. Sprague-Dawley rats, healthy males weighing 266 to 450 g (mean, 349 g) purchased from Charles River (Maastricht, the Netherlands), were housed under controlled conditions of temperature and humidity. Before the start of the experiments, rats were fed water and chow ad libitum.

Experimental design and HS procedure

Rats were allocated to five groups (n = 6 per group) before the start of the experiments. Control rats (group 1) were sacrificed without intervention. The other rats were exposed to nonlethal HS after 18 h of fasting as previously described (24). Briefly, rats were anesthetized with isoflurane (induction 4%, maintenance 1.5%). The femoral artery was aseptically dissected and cannulated with polyethylene tubing (PE-10) containing heparinized saline (10 IU/mL). The MAP and heart rate (HR) were assessed continuously. After 30 min of acclimatization period, 2.1 mL blood/100 g of body weight was withdrawn (representing approximately 30% to 40% of the circulating volume) at a rate of 1 mL/min. Groups 2, 3, 4, and 5 were sacrificed at 15, 30, 60, and 90 min after shock, respectively. At killing, blood and tissue samples were taken.

The severity of the HS as reflected by changes in MAP, HR, and hematocrit was similar for all animals studied. Immediately after induction of shock (t = 0), mean MAP values decreased from 89 mmHg (range, 80 - 105 mmHg) to 24 mmHg (range, 20 - 32 mmHg), and the HR decreased from 397 beats per min ([bpm] range, 350 - 470 bpm) to 226 bpm (range, 160 - 270 bpm) in all shock groups. Hematocrit was reduced from 43% ± 2.0% to 35% ± 2.9% after shock at all time intervals studied (Table 1). These data are comparable to previously published data using the same HS model (25, 26).

The severity of the HS as reflected by mean changes in MAP, HR, and Ht was similar for all animals studied


Tight junction distribution and the actin cytoskeleton were examined by immunofluorescent staining of frozen sections (3 μm) for ZO-1, claudin 3, G-actin, and F-actin. Ileum, liver, and kidney sections were fixed with 4% paraformaldehyde. Nonspecific binding sites were blocked with 10% goat serum and incubated overnight at 4°C with anti-ZO-1, anti-claudin 3, or anti-G-actin. Thereafter, the sections were incubated for 45 min with Texas red conjugated goat antirabbit antibody or with Texas red conjugated rat antirabbit antibody. F-actin sections were stained with Oregon green-Phalloidin for 45 min, followed by 2 min of incubation with 4′,6-diamino-2-phenyl indole (DAPI), dehydrated in ascending ethanol series and mounted in fluorescence mounting solution (Dakocytomation, Glostrup, Denmark). The distribution of TJ and the actin cytoskeleton was recorded at a magnification of 200 times/400 times using the Metasystems Image Pro System (black and white charge-couple device camera; Metasystems, Sandhausen, Germany) mounted on a Leica DM-RE fluorescence microscope (Leica, Wetzler, Germany). All images were taken at equal time exposures after being normalized to negative control sections without primary antibody, to exclude for nonspecific binding of the secondary antibody or autofluorescence. At least 25 microscopic fields for each tissue section were examined.

Epithelial cell isolation and protein extraction

Intestinal epithelial cells of rats were isolated using a modification of a previously published method (27, 28). In short, a fresh section of intestine was inverted and washed in 4°C phosphate buffered saline (PBS) containing 50 nM of the phosphatase inhibitor calyculin A (Merck Biosciences, Nottingham, United Kingdom). Next, the tissue was transferred to Ca2+- and Mg+ -free Hank's balanced salt solution containing 30 mM EDTA and 50 nM calyculin A and incubated for 20 min at 4°C. After incubation, the tissue was transferred to a fresh tube containing Ca2+- and Mg+ -free Hank buffer salt solution with 0.3 U/mL dispase (Boehringer Mannheim, Germany) and 50 nM calyculin A. After incubation at 37°C for 20 min, epithelial cells were dislodged by scraping the epithelial surface. Isolated cells were analyzed microscopically, and only epithelial cells were observed.

The isolated intestinal epithelial cells, liver samples, and kidney samples were lysed in lysis buffer containing 200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycine, 1 mM phenylmethanesulfonyl fluoride, 1 μg/mL leupeptin, and 28 μg/mL aprotinin, and centrifuged at 40,000g for 10 min at 4°C. The protein concentration of the supernatants was measured using the Bradford method (Biorad, Hercules, Calif).

Western blotting

Aliquots with equal amounts of protein determined with the Bradford method (extracts from isolated rat intestinal epithelial cells, liver, and kidney) were heated at 100°C for 5 min in sodium dodecyl sulfate sample buffer, separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membrane (Immobilin P, Millipore, Bedford, Mass). For G-actin, nonreducing Western blotting was performed. After transfer of proteins, a blocking step was performed in Tris buffered saline with 5% nonfat dry milk and 0.05% Tween. Membranes were probed using primary antibodies to ZO-1, claudin 3, tAC, pAC, actin, and β-actin in Tris buffered saline and 0.05% Tween. To confirm equal protein loading, immunoblotting was performed with β-actin. After incubation with goat antirabbit or rat antimouse HRP-conjugated secondary antibody, signal was detected by chemiluminescence on film (Pierce Biotechnology Inc, Rockland, Ill). Band intensity was quantitatively analyzed using Quantity One (Biorad).

Bacterial translocation

In all rats, mesenteric lymph nodes (MLNs), the midsection of the spleen, and segment IV of the liver were collected aseptically in 2 mL preweighed thioglycolate broth tubes (Becton Dickinson [BBL] Microbiology Europe, Maylan, France). After weighing, the tissue specimens were homogenized and subsequently transferred onto the following agar plates: Columbia III blood agar base supplemented with 5% vol/vol sheep blood (BBL) (duplicate plates) and Chocolate PolyviteX agar (BioMÉrieux, Marcy L'Etoile, France). After 48 h of incubation, the colonies were counted, adjusted to the weight of the tissue, and expressed as number of colony forming units per gram tissue.

Statistical analyses

Gaussian distribution was tested using the Kolmogorov-Smirnov test. One-way ANOVA followed by Dunnett multiple comparison test was used for between-group comparisons. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM/range.


Actin filament severing

The turnover of F-actin into G-actin after shock in ileum, liver, and kidney was investigated by immunofluorescence and Western blotting. Immunofluorescence showed increased G-actin from 15 min after induction of HS in the ileum, with simultaneous decrease in F-actin. The intensity of G-actin was still increased at 30 and 60 min after induction of shock. Rats sacrificed 90 min after induction of HS also showed an upregulation of G-actin and significant loss of F-actin compared with control rats, although less G-actin and more F-actin was observed compared with rats sacrificed 15, 30, and 60 min after HS (Fig. 1A). Western blot of G-actin showed a statistically significant increase in densitometric intensity at 15 min after shock compared with controls (*P < 0.05; Fig. 1B).

Fig. 1:
A, Immunolocalization of G-actin (red) and F-actin (green) in the ileum after HS. Nuclei are stained with DAPI (blue). Control rats showed a regular distribution of F-actin and no detectable G-actin. Rats sacrificed 15, 30, and 60 min after induction of HS showed an upregulation of G-actin and significant loss of F-actin. Rats sacrificed 90 min after induction of HS also showed an upregulation of G-actin and a significant loss of F-actin compared with control rats, although less G-actin and more F-actin was observed compared with rats sacrificed 15, 30, and 60 min after HS (original magnification ×200). B, Densitometric analysis of Western blot of G-actin in rat intestinal epithelial cells. Western blot of G-actin showed a significant increase in densitometric intensity at 15 min after shock compared with controls (*P < 0.05). β-Actin was used to confirm equal protein loading. Values are expressed as mean ± SEM.

In liver and kidney sections, F-actin cytoskeleton remained intact, and there was no G-actin detectable at the investigated time points (Fig. 2, G-actin staining not shown).

Fig. 2:
Immunolocalization of F-actin in liver and in kidney (green) after HS. Nuclei are stained with DAPI (blue). F-actin distribution did not change after HS in the period studied (original magnification ×200).

Intestinal AC activation after HS

We report the presence of both tAC and pAC in normal ileum using Western blot with antibodies against tAC and pAC. Furthermore, the effect of HS on the relative amounts of tAC and pAC was studied. Figure 3 shows the densitometric analysis of Western blot data of intestinal epithelial cell homogenates using antibodies to tAC and pAC. The total concentration of tAC in gut epithelial cells did not change significantly after HS (Fig. 3A). pAC expression was statistically significantly decreased 15, 30, and 60 min after induction of shock (by 21%, 27%, and 26% respectively, P < 0.05; Fig. 3B). This decrease in pAC values reflects AC activation.

Fig. 3:
Densitometric analysis of Western blot of tAC and pAC in rat intestinal epithelial cells. A, Western blot of tAC showed no significant attenuation in densitometric intensity after shock compared with controls. B, Western blot of pAC showed a significant attenuation in densitometric intensity at 15, 30, and 60 min after shock compared with controls (*P < 0.05). Signal intensity of the specific band from the untreated control rats was set as 100% and was compared with the after-shock values. β-Actin was used to confirm equal protein loading. Values are expressed as mean ± SEM.

Intestinal, liver, and renal TJ loss

A significant loss of the most important transmembrane TJ protein claudin 3 and the intracellular TJ protein ZO-1 from 30 min after induction of shock was observed by immunofluorescent staining of microscopic sections of the ileum (Fig. 4). This loss was quantified using Western blotting of rat epithelial cell homogenates. Statistically significant loss of claudin 3 and ZO-1 was seen from 60 min after induction of shock (Fig. 5).

Fig. 4:
Immunolocalization of ZO-1 and claudin 3 (both in red) in ileum showed a regular distribution in control rats and in animals sacrificed 15 min after induction of shock. Already at 30 min after HS, a significant loss of claudin 3 and ZO-1 was found, which persisted up to 90 min after shock. Nuclei are stained with DAPI (blue, original magnification ×200; for insert, original magnification ×400).
Fig. 5:
Densitometric analysis of Western blot of claudin 3 and ZO-1 in rat intestinal epithelial cells. A, Western blot of claudin 3 showed a significant attenuation in densitometric intensity at 60 and 90 min after shock compared with earlier time points (*P < 0.05). B, Western blot of ZO-1 showed a significant attenuation in densitometric intensity at 60 and 90 min after shock compared with earlier time points (*P < 0.05). Signal intensity of the specific band from the untreated control rats was set as 100% and was compared with the after-shock values. β-Actin was used to confirm equal protein loading. Values are expressed as mean ± SEM.

In liver and kidney sections, immunofluorescent staining showed no difference in ZO-1 and claudin 3 immunolocalization between control rats and rats sacrificed 15, 30, 60, and 90 min after induction of HS (data not shown). Also, Western blot analysis showed no significant loss of claudin 3 in the liver and kidney at any time point after induction of HS as compared with control rats (data not shown).

Intestinal permeability to bacteria

To determine whether hemorrhage leads to increased intestinal permeability, we measured bacterial translocation to distant organs (Fig. 6). As expected, cultures from tissues taken from the control group were mostly sterile. Bacterial translocation was significantly elevated in animals sacrificed 30, 60, and 90 min after induction of HS as compared with control animals (P < 0.05). Colony-forming units per gram tissue found in MLNs, spleen, and liver were 0.4 (range, 0.0-1.0), 8.4 (range, 0.0-21.4), 35.0 (range, 19.7-58.7), 50.1 (range, 21.9-75.7), 133.3 (range, 95.7-199.8) for controls rats and rats sacrificed at 15, 30, 60, and 90 min after HS, respectively. The most frequently found bacteria in the cultures were gut-derived Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Bacteria were more often found in MLNs than spleen or liver.

Fig. 6:
Bacterial translocation to MLNs, spleen, and liver was significantly elevated 30, 60, and 90 min after HS as compared with control animals (* P < 0.05). Values are expressed as mean ± SEM.


This study was aimed at unraveling the changes in TJ integrity after HS in the organs commonly affected in MODS (liver, kidney, and intestine) and to elucidate the events preceding TJ loss. Intestinal severing of the F-actin cytoskeleton, which intactness is necessary for TJ integrity, was observed very early after shock, starting as soon as 15 min after the onset of shock. In contrast, F-actin cytoskeleton disruption in liver and kidney was not seen at any investigated time point, for example, up to 90 min after shock.

In the current study, we found that both tAC and pAC are present in intestinal epithelial cells under physiological conditions. Phosphorylated AC decreased statistically significantly 15 min after induction of shock, which is expected to be responsible for actin cytoskeleton severing. Consequently, the interaction of TJ proteins with F-actin was disrupted leading to TJ loss. The AC binding to F-actin causes a twist in F-actin that destabilizes the filament. Furthermore, this twist in F-actin eliminates the binding sites for phalloidin such that AC-saturated filaments are not stained with fluorescent phalloidin (29). However, the increased staining of G-actin showed disruption of the actin filaments into monomers.

Tight junction protein loss was seen from 30 min after shock. The immunofluorescence data indicate that the presence of claudin 3 and ZO-1 at the side of TJs is strongly reduced from 30 min after HS. These proteins however do not immediately disappear but rather dislodge and finally end up in the lysosomal compartments (30, 31). This results in a spreading of the proteins over the cytoplasm, which reduces the local concentration and thus detection by immunofluorescence. The Western blot data clearly show that the TJ proteins claudin 3 and ZO-1 in intestinal epithelial cells are diminished from 60 min after shock. No TJ loss in liver and kidney was seen at any investigated time point. Bacterial translocation started as soon as 30 min after shock and increased markedly over time. This occurrence of bacterial translocation characterizes the functional intestinal barrier loss. Taken together, HS-induced activation of AC, cytoskeleton severing, and TJ loss was restricted to the intestine and was followed by bacterial translocation.

Our data are consistent with the work of Molitoris group, showing presence of both AC and pAC in the kidney under physiological conditions, and AC activation and actin cytoskeleton degradation after ATP depletion caused by ischemia (15). Changes in actin cytoskeleton structure contribute to TJ integrity loss (27, 32, 33).

Besides AC-mediated TJ loss by cytoskeleton filament severing and depolymerization after ATP depletion, myosin light chain (MLC) is described as regulator of actin dynamics in the enterocyte (33, 34). Phosphorylation of MLC by MLC kinase leads to contraction of the actin cytoskeleton and opening of the TJ in the intestine. Tight junction permeability is regulated by phosphorylated MLC by myosin ATPase-mediated contraction of the peri-junctional actomyosin ring and subsequent physical tension on the TJ (35, 36). This mechanism is suggested to be responsible for TJ integrity loss in inflammatory bowel disease (37). In summary, actin filament reorganization and stabilization is regulated by AC or MLC, with TJ loss caused by either AC after energy depletion or MLC in an energy-rich environment.

In conclusion, this study shows that HS results in intestinal AC activation, actin depolymerization, TJ loss, and bacterial translocation very early after the onset of shock, whereas in kidney and liver, no actin cytoskeleton disruption and TJ loss are observed.


The authors thank Carolien Boeckx for her excellent technical assistance. The authors also thank J. R. Bamburg for providing the antibodies to AC and for his critical appraisal of our manuscript.


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Actin cytoskeleton severing; systemic inflammation; actin-depolymerizing factor/cofilin; liver barrier; kidney barrier; gut barrier

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