Objectives: Our study aimed to determine whether antithrombin plays a synergistic role in accentuating the effects of intestinal ischemic preconditioning.

Materials and Methods: Fifty rats were randomly allocated to 5 groups (10 rats/group) as follows: sham treatment (group 1); ischemia-reperfusion (group 2); ischemic preconditioning followed by ischemia-reperfusion (group 3); antithrombin + ischemia-reper­fusion, similar to group 2 but including antithrombin administration (group 4); and antithrombin + ischemic preconditioning, similar to group 3 but including antithrombin administration (group 5). Blood samples and liver specimens were obtained for measurement of cytokines, myeloperoxidase, and malondialdehyde. Liver biopsies were examined by electron microscopy.

Results: Intestinal ischemia-reperfusion induced a remote hepatic inflammatory response as evidenced by the striking increase of proinflammatory cytokines, myeloperoxidase, and malondialdehyde. Tumor necro­sis factor-α levels in group 5 (12.48 ± 0.7 pg/mL) were significantly lower than in group 3 (13.64 ± 0.78 pg/mL; P = .014). Mean interleukin 1β was lower in group 5 (9.52 ± 0.67pg/mL) than in group 3 (11.05 ± 1.9 pg/mL; P > .99). Mean interleukin 6 was also significantly lower in group 5 (17.13 ± 0.54 pg/mL) than in group 3 (23.82 ± 1 pg/mL; P < .001). Myelo­peroxidase levels were significantly higher in group 3 (20.52 ± 2.26 U/g) than in group 5 (18.59 ± 1.03 U/g; P = .025). However, malondialdehyde levels did not significantly improve in group 5 (4.55 ± 0.46 μmol) versus group 3 (5.17 ± 0.61 μmol; P = .286). Tumor necrosis factor-α, interleukin 6, and myeloperoxidase findings show that antithrombin administration further attenuated the inflammatory response caused by ischemia-reperfusion, suggesting a synergistic effect with ischemic preconditioning. These findings were confirmed by electron microscopy.

Conclusions: The addition of antithrombin to ischemic preconditioning may act to attenuate or prevent damage from ischemia-reperfusion injury by inhi­biting the release of cytokines and neutrophil infiltration.

Key words : Antithrombin, Cytokines, Intestinal, Ischemia reperfusion injury, Preconditioning

Introduction

Intestinal ischemia-reperfusion injury (IRI) induces mucosal barrier damage and systemic inflammation through the endogenous production of reactive oxygen species, cytokines, and nitric oxide.¹ This causes remote organ injuries, which could lead to systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Hence, the gut has been referred to as “the motor of multiple organ dysfunction syndrome.”² It has been sug­gested that the liver is particularly vulnerable to the negative consequences of intestinal IRI, as the hepatic vasculature is in series with the intestinal circulation.³ However, the pathophysiology of acute liver injury is unclear, having a complicated interplay between cytokines, reactive oxygen species, and tissue-specific factors. Initiators of this pathop­hysiology are regulated by the transcript factor nuclear factor-κB.^4,5

Various methods with protective effects have been tested to ameliorate IRI. Conditioning (precon­ditioning, postconditioning, or remote conditioning) is among the therapeutic approaches used to enhance organ ischemia tolerance.⁶

Ischemia-reperfusion injury negatively impacts capillary density⁷ and increases vascular permeability.⁸ Adhesion molecules are expressed by the endothelial cells and promote platelet activation and aggregation,⁹ thus inducing the coagulation cascade and promoting the inflammatory process.^9-11 These result in cell edema and capillary stenosis, causing a “no reflow” phenomenon.⁸

The dominant role of coagulation in IRI has led to the use of anticoagulation therapy in an attempt to alleviate the severity of tissue injury. Antithrombin has shown promise in recent years and has been used in numerous studies in an attempt to improve organ outcomes after IRI.12-16

Although both ischemic preconditioning (IPC) and antithrombin are well known individually, their potential for acting synergistically has not yet been studied. The murine model of IRI used in this study was designed specifically to answer the following 2 questions. First, is there a protective effect of IPC on the liver inflammatory response to intestinal IRI? Second, does antithrombin play a synergistic role in accentuating the effects of transient intestinal IPC?

Herein, we demonstrate in an animal model that antithrombin therapy in combination with IPC can be beneficial in terms of an inflammatory response, as evidenced by measurement of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), myeloperoxidase, and malondialdehyde levels. Furthermore, we addressed the extent of ultrastructural cell damage by using electron microscopy for examination.

Materials and Methods

All experiments were performed in the Experimental Laboratory at “G. Papanikolaou” Hospital. The Aristotle University Ethics Committee approved this study, and the use of anesthesia during surgery and subsequent postoperative management of animals were in accordance with the Federation of Laboratory Animal Science Associations guidelines.

Fifty male rats (Wistar rats, 2-3 mo old) weighing 250 to 300 g were purchased from the “Theageneio” Hospital Experimental Laboratory (Thessaloniki, Greece). As a standard protocol, all rats were housed in a quiet, nonstressful environment for 1 week before study. They were housed 4 per cage and given standard rat chow with free access to water. They remained in a conditioned room (21 ± 1°C) with a 12:12-hour day-night cycle and were fed ad libitum for 12 hours before surgery. For surgery, they were placed in a supine position on a heating pad, and body temperature was maintained between 36°C and 37°C. The animals had ether for induction and underwent intraperitoneal administration of a short-acting oxybarbiturate (pentobarbital, 50 mg/kg body weight) for maintenance. All efforts were made to minimize suffering.

The abdominal wall was shaved and cleansed with 10% povidone-iodine (Betadine, Seton, UK). After a midline incision, the superior mesenteric artery was identified and occluded with the use of an atraumatic microsurgical vascular clamp. Reperfusion was achieved by removing the clamp. Bowel ischemia was confirmed by loss of pulsatile flow and the pale color of the bowel, whereas reperfusion was confirmed by restoration of pulsatile flow.

Experimental design
Fifty Wistar rats were randomized into 5 study groups (10 rats/group). Death was caused by administering an overdose of pentobarbital. All rats were exsanguinated for blood sampling through cardiac puncture. Blood and liver specimens were obtained for studies. For the sham group, sham operation assessed the effects of laparotomy. The abdomen was left open for 110 minutes, in accordance with the overall time needed in study groups 3 and 5. The IRI group comprised 10 rats that had superior mesenteric artery occlusion for 30 minutes, followed by 60-minute reperfusion. The IPC group comprised 10 rats that had superior mesenteric artery occlusion for 10 minutes, followed by 10 minutes of intestinal reperfusion, and then treatment as described for the IRI group. For the antithrombin and IRI group (AT+IRI group), 10 rats had treatment similar to the IRI group but also administration of antithrombin (250 IU/kg, Atryn, LeoPharma, Athens, Greece) 5 minutes before IRI. For the antithrombin and IPC group (AT+IPC group), 10 rats had treatment similar to the IPC group but also administration of antithrombin (250 IU/kg) 5 minutes before IPC.

Blood samples and liver specimens were obtained after 60 minutes of reperfusion. Blood samples were obtained by cardiac puncture, and the collected sera were analyzed for cytokines (TNF-α, IL-1β, and IL-6). Liver specimens were divided into 2 specimens, with one stored at -70°C and tested for polymorphonuclear infiltration (myeloperoxidase) and the other stored at -70°C and tested for lipid peroxidation (malondialdehyde).

Biochemical analyses
Blood samples were centrifuged at 5000g for 5 minutes, and the collected sera were used for the measurement of cytokine concentrations.

For cytokines TNF-α, IL-1β, and IL-6, serum samples were stored at -70°C. The serum concen­trations of all 3 cytokines were measured by enzyme-linked immunosorbent assay using the KRC3011 TNF-α test kit, the KRC0011 IL-1β test kit, and the KRC0061 IL-6 test kit (all from Invitrogen Corporation, Camarillo, CA, USA). Detection limits of these assays were determined as 4 pg/mL, 3 pg/mL, and 5 pg/mL, respectively.

For measurement of polymorphonuclear infil­tration (using myeloperoxidase), liver tissue was weighed after surgery, and 0.5 g were mixed with normal saline and then immediately frozen at -70°C. The activity of myeloperoxidase in the inflamed tissue was determined spectrometrically according to Bradley and associates.¹⁷

For measurement of lipid peroxidation, we used malondialdehyde, a naturally occurring product of lipid peroxidation. Liver tissue was again weighed after surgery, and 0.5 g were mixed with normal saline and then immediately frozen at -70°C until assayed using the thiobarbituric acid reactive substances method, a well-established method for screening and monitoring lipid peroxidation¹⁸ (assay kit 1009055, Cayman Chemical Company, Ann Arbor, MI, USA).

Electron microscopy
Liver specimens of 1 mm3 were fixed in a phosphate-buffered saline solution, pH 4, containing 3% glutaraldehyde for 2 hours. The specimens were fixed in osmium tetroxide and then stained with 2% aqueous uranyl acetate for 14 to 16 hours. The specimens were then subjected to dehydration through 30%, 50%, 70%, 90%, and 100% alcohol and embedded in epoxy resin (EPON 812). Semithin sections (1.5-3 µm) were first examined under light microscopy and then processed for electron microscopy. Sections of 70 nm were stained, followed by Reynold lead citrate, and viewed with JEOL transmission electron microscope 2000 FX 11 (Tokyo, Japan) at 80 kV. The parameters of cell injury were examined based on previous reports.^19-21

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 20.0, IBM Corporation, Armonk, NY, USA). The Kolmogorov-Smirnov test was used for normality distribution assessment, and all normally distributed data are expressed as means ± standard deviation. One-way analysis of variance was used for multiple comparisons, followed by Bonferroni post hoc analysis to compare data between groups. P< .05 was considered statistically significant.

Results

The median and first and third quartile levels for TNF-α, IL-1β, IL-6, myeloperoxidase, and malondialdehyde levels for the 5 groups are depicted in the box plots shown in Figures 1 to 5.

Tumor necrosis factor-α
The IRI group had the highest mean TNF-α levels (17.05 ± 1 pg/mL), which reached statistical significance (P < .05) versus all other groups. Levels in the AT+IPC group (12.48 ± 0.7 pg/mL) were significantly lower than in the IPC group (13.64 ± 0.78 pg/mL; P = .014) and the AT+IRI group (15.78 ± 0.85 pg/mL; P < .001). The lowest TNF-α value was observed in the sham group (5.3 ± 0.3 pg/mL), and this was statistically significant versus all other groups (P < .001) (Figure 1).

Interleukin 1β
Interleukin 1β levels were significantly lower in the IPC and the AT+IPC groups than in the IRI and the AT+IRI groups (P < .05). Levels were significantly lower in the AT+IRI group (21.79 ± 1.51 pg/mL) than in the IRI group (26.06 ± 5 pg/mL; P = .006). The mean IL-1β level was lower in the AT+IPC group (9.52 ± 0.67 pg/mL) than in the IPC group (11.05 ± 1.9 pg/mL), although this was not statistically significant (P > .99). The lowest IL-1β values were observed in the sham group (6.13 ± 1.33 pg/mL), which was statistically significant versus the IRI, IPC, and AT+IRI groups (P < .05) and almost reached statistical significance for the AT+IPC group (P = .051) (Figure 2).

Interleukin 6
Interleukin 6 levels were significantly lower in the IPC and the AT+IPC groups than in the IRI and the AT+IRI groups (P < .05). Interleukin 6 levels in the AT+IRI group (26.09 ± 1.15 pg/mL) were significantly lower than in the IRI group (27.7 ± 1.24 pg/mL; P = .005). The mean IL-6 level was lower in the AT+IPC group (17.13 ± 0.54 pg/mL) than in the IPC group (23.82 ± 1 pg/mL), and this was statistically significant (P < .001). The lowest IL-6 value was observed in the sham group (6.95 ± 0.65 pg/mL), and this was statistically significant compared with values shown in other groups (P < .001) (Figure 3).

Myeloperoxidase
The IRI group had the highest myeloperoxidase level (24.82 ± 1.24 U/g) compared with all the other groups (P < .001). The IPC group (20.52 ± 2.26 U/g) showed significantly higher levels than the AT+IPC group (18.59 ± 1.03 U/g, P = .025). The lowest myeloperoxidase value was observed in the sham group (17.41 ± 0.85 U/g), which was statistically significant compared with the IRI, IPC, and AT+IRI groups (P < .001). However, this was not statistically significant compared with the AT+IPC group (P = .56). There were no significant differences between the IPC (20.52 ± 2.26 U/g) and the AT+IRI groups (22.12 ± 0.79 U/g; P = .105) (Figure 4).

Malondialdehyde
The IRI group showed the highest mean malon­dialdehyde levels (6.84 ± 0.87 μmol) compared with all other groups (P < .001). There was no significant improvement in malondialdehyde levels in the AT+IPC group (4.55 ± 0.46 μmol) compared with the IPC group (5.17 ± 0.61 μmol; P = .286). Levels in the AT+IRI group (5.57 ± 0.34 μmol) were significantly lower than in the IRI group (P < .001). However, there was no significant difference compared with the IPC group (P > .99). The lowest malondialdehyde value was observed in the sham group (4.61 ± 0.6 μmol), which was statistically significant compared with the IRI group (P < .001). However, this was not statistically significant compared with the IPC (P = 0.461) and the AT+IPC groups (P > 0.99) (Figure 5).

Ultrastructural studies
Our microscopic examination method (random and adjacent areas; Figures 6-8) showed that hepatocytes seem to be better protected with the combination of antithrombin and IPC. The IRI group showed severe mitochondrial damage, endoplasmic reticulum dilatation, cytosolic vacuole formation, phagolyso­somal formation, and lipid droplet formation. The main ultrastructural features exhibited by the IPC and the AT+IRI groups consisted of pleomorphic undamaged mitochondria, endoplasmic reticulum dilatation, phagolysosomal formation, and lipid droplet formation. Nevertheless, the damages seemed to be more pronounced in the AT+IRI group. The AT+IPC group showed pleomorphic undam­aged mitochondria, endoplasmic reticulum dilatation, phagolysosomal formation, and lipid droplet formation.

Discussion

This is the first study to explore and demonstrate a synergistic effect of antithrombin and IPC on reducing the level of IRI reflected in the liver in a murine model of IRI.

Intestinal IRI induced a remote hepatic inflammatory response, as evidenced by the striking increase in expression of the cytokines TNF-α, IL-1β, and IL-6 and also myeloperoxidase and malon­dialdehyde in the liver. This study demonstrates that intestinal IPC reduced this inflammatory response induced in the liver by intestinal IRI. The exact mechanism by which IPC confers protection in the intestine is unclear. Several mediators have been advocated to play a crucial role in this protective phenomenon, including adenosine,²² nitric oxide,²³ oxidative stress,²⁴ heme oxygenase one,²⁵ and antiapoptosis.²⁶

Cytokines are known to exert their effects in distant organs; therefore, their local increase follo­wing IRI is the beginning for systemic IRI. In this experiment, intestinal IRI significantly increased the levels of the proinflammatory cytokines TNF-α, IL-6, and IL-1β, which have been shown to increase in several models of IRI. Myeloperoxidase and malon­dialdehyde activity levels in liver specimens were also signi­ficantly increased in the IRI group. In light of these findings, it is evident that inflammatory mediators produced after intestinal IRI circulate on reperfusion and induce inflammatory responses in the liver.

Our results confirm previous studies on the beneficial effect of IPC on IRI.5 The groups that underwent IPC have significantly reduced TNF-α and IL-6 in the blood. In addition, myeloperoxidase as an index of polymorphonuclear accumulation was significantly less in the IPC groups and especially in the groups treated with AT, in agreement with similar studies.²⁷

Tumor necrosis factor α is the central component of the proinflammatory cytokine cascade in liver IRI and is a crucial effecter of remote organ damage after hepatic IRI.^28,29 It has been reported that TNF-α elevation was responsible for pulmonary damage after hepatic IRI.³⁰ Yin and associates and Ji and associates have shown that IPC prevents TNF-α elevation after IRI.^5,31 Our study concurs with published data and shows that TNF-α levels after 30 minutes of ischemia and 60 minutes of reperfusion were lower in animals pretreated with IPC. Levels of TNF-α were decreased by brief IPC, and this con­firms the role of TNF-α as one of the mediators responsible for the remote effects of IPC. Administration of antithrombin attenuated further the inflammatory response, suggesting a synergistic effect with IPC.

The activation of Kupffer cells in the acute phase after liver IRI results in the production of the early response cytokine IL-1. Kato and associates were the first to provide important information regarding the role of IL-1 in the inflammatory injury induced by hepatic IRI.³² Their data indicated that IL-1 functions to induce nuclear factor-κB activation and expression of CXC chemokines during the later phases of this inflammatory response. These effects augmented the recruitment of neutrophils to the hepatic parenchyma but did not significantly alter the extent of hepatocellular injury. It was therefore suggested that IL-1 is not a primary mediator of this response and has limited significance in the inflammatory injury to the liver. These findings were similar to those in a murine model of renal IRI.³³ Recently, in a swine lung autotransplant model, Huerta and associates³⁴ showed that IPC prevented liver injury induced by lung IRI through the reduction of proinflammatory cytokines (TNF-α, IL-1) and hepatocyte apoptosis. Our study concurs with the Huerta group, as we found IL-1β to be significantly improved in the IPC group compared with the IRI group. Ischemic preconditioning and antithrombin were not found to be significantly synergistic. However, the administration of antithrombin in the IRI group resulted in ameliorating the injury induced by ischemia-reperfusion (P = .005). It should be noted that the IL-1β levels in the AT+IPC group were not significantly higher than those in the sham group.

Previous reports suggested that increased IL-6 levels might predict acute bowel ischemia and call for urgent operation.³⁵ Zaki and associates³⁶ have shown in a rat hepatic IRI model that induction of IRI significantly increased levels of IL-6 compared with the sham-operated group. In our study, IL-6 was significantly increased during IRI. Ischemic preconditioning significantly decreased the IL-6 levels, and the administration of antithrombin attenuated even further the inflammatory response in all relevant groups. Therefore, IPC and antithrombin proved synergistic.

It has been shown previously that myelo­peroxidase activity is a reliable index of neutrophil infiltration.³⁷ Myeloperoxidase activity in intestinal tissue increased 3-fold after 60-minute intestinal ischemia and 8- to 9-fold after 60-minute reperfusion.³⁸ Ozden and associates²⁷ showed that mucosal myeloperoxidase activity was increased in the control group, confirming postreperfusion neutrophil infiltration compared with that shown in the sham group. Reduced myeloperoxidase activity in the antithrombin groups showed that antithrombin inhibited polymorphonuclear infiltration in the reperfused intestine. Ostrovsky and associates³⁹ have shown that both pretreatment and posttreatment with antithrombin reduced neutrophil rolling and adhesion. Recently, Ji and associates showed that intestinal IPC significantly decreased intestinal myeloperoxidase levels.⁵ Our study is consistent with published data in which antithrombin de­creased myeloperoxidase activity; in addition, it was proven synergistic with IPC. The fact that the myeloperoxidase levels in the AT+IPC group were not significantly higher than in the sham group stresses the decisive role of antithrombin in dampening the IRI. This is also proven when the IPC and the AT+IRI groups were compared (P = .105); the administration of antithrombin to the IRI group achieved myeloperoxidase levels similar to the IPC group.

A significant increase in malondialdehyde has been observed in various organs after IRI,³⁷ concurring with the results from our study. Our study agrees with previous experiments that IPC was beneficial for reduced levels of malondialdehyde,36 and also groups treated with antithrombin were found to have decreased levels of malondialdehyde, indicating that lipid peroxidation was inhibited by antithrombin. As with myeloperoxidase, the fact that malondialdehyde levels in the AT+IPC group were not significantly higher than in the sham group could depict important roles for IPC and antithrombin in ameliorating IRI. However, IPC and antithrombin were not found to be significantly synergistic (P = .286).

Previous studies explored the effect of intestinal IPC on IRI using hematoxylin eosin staining⁵; here, we evaluated the potential beneficial roles of IPC and antithrombin by electron microscopy (Figures 6-8). As expected, the IRI group showed the most extensive damage, whereas the AT+IPC group showed the least pronounced damage. The IPC and the AT+IRI groups showed similar degrees of injury. Nevertheless, the damages seemed to be more pronounced in the AT+IRI group, suggesting a beneficial and synergistic role of IPC and antithrombin.

Of note, our study demonstrated that the antico­agulation effects of antithrombin act synergistically with IPC, which has been shown to prevent or attenuate the harmful process triggered by reper­fused ischemic tissues. Ischemic precon­ditioning dampens the inflammatory process and the as­sociated “no reflow” phenomenon, as a result of cell edema and capillary stenosis; in addition, antithrombin facilitates better blood flow to the reperfused tissues. The implications associated with these findings are significant because they could be utilized in IRI scenarios (eg, transplant and liver resection).

This invasive IPC rat model described herein is primarily to be used as a well-controlled, robust experimental model to investigate the mechanisms underlying the protection of IPC combined with antithrombin. Although the hepatic inflammatory responses seemed to be preferentially enhanced by intestinal reperfusion, increases in TNF-α, IL-1β, IL-6, myeloperoxidase, and malondialdehyde may also be induced by both surgical stress and remote IRI injury. However, IRI injury and surgical stress inevitably occur in conjunction with these procedures, and there is no conclusive way to discern their respective impact on liver response.

Conclusions

This is the first study to investigate a synergistic protective effect of IPC and antithrombin in an IRI model. Our experimental study shows that intestinal IPC has a beneficial remote effect on liver injury caused by intestinal IRI. The addition of anti­thrombin before IPC might attenuate or prevent liver damage from intestinal IRI presumably by inhibiting the “no reflow” phenomenon, which is a result of the associated coagulopathy after IRI. Increased levels of TNF-α, IL-6, and myelo­peroxidase confirmed a significant synergistic effect of IPC and antithrombin, thus suggesting a beneficial role of antithrombin in IRI when combined with IPC.

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