Aprotinin reduces oxidative stress induced by pneumoperitoneum in rats

Aprotinin reduces oxidative stress induced by pneumoperitoneum in rats

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Aprotinin reduces oxidative stress induced by pneumoperitoneum in rats Minas Baltatzis, MD,a,* Theodoros E. Pavlidis, MD, PhD,a Odysseas Ouroumidis, MD,a Georgios Koliakos, MD, PhD,b Christina Nikolaidou, MD,c Ioannis Venizelos, MD, PhD,c Anna Michopoulou, MD,b and Athanasios Sakantamis, MD, PhDa a

Second Propedeutical Department of Surgery, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece b Department of Biochemistry, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece c Department of Pathology, Hippocration Hospital, Thessaloniki, Greece

article info

abstract

Article history:

Background: Ischemiaereperfusion injury induced by pneumoperitoneum is a well-studied

Received 7 November 2013

entity, which increases oxidative stress during laparoscopic operations. The reported anti-

Received in revised form

inflammatory action of aprotinin was measured in a pneumoperitoneum model in rats for

17 February 2014

the first time in this study.

Accepted 20 February 2014

Materials and methods: A total of 60 male Albino Wistar rats were used in our protocol.

Available online 25 February 2014

Prolonged pneumoperitoneum (4 h) was applied, causing splanchnic ischemia and a period of reperfusion with a duration of 60 or 180 min followed. Several cytokines and markers of

Keywords:

oxidative stress were measured in liver, small intestine, and lungs to compare the apro-

Pneumoperitoneum

tinin group with the control group. Tissue inflammation was also evaluated and compared

Ischemiaereperfusion injury

between groups using a five-scaled histopathologic score.

Aprotinin

Results: In aprotinin group values of biochemical markers (tumor necrosis factor a, inter-

Oxidative stress

leukin 6, endothelin 1, C reactive protein, pro-oxidanteantioxidant balance, and carbonyl proteins) were lower in all tissues studied. Statistical significance was greater in liver and lungs (P < 0.05). Histopathologic examination revealed significant difference between control and aprotinin groups in all tissues examined. Aprotinin groups showed mild to moderate lesions, while in control groups severe to very severe inflammation was present. Aprotinin subgroup with prolonged reperfusion period (180 min) showed milder lesions in all tissues than the rest of the groups. Conclusions: Aprotinin reduced inflammatory response and oxidative stress induced by pneumoperitoneum in liver, small intestine, and lungs. ª 2014 Elsevier Inc. All rights reserved.

1.

Introduction

The effects of CO2 pneumoperitoneum in splanchnic circulation have been thoroughly examined during the last decades.

Several experimental and clinical studies have shown a reduction on vascular flow in both the portal vein (35%e84%) and the mesenteric arterial system (32%e44%), due to the increased intra-abdominal pressure caused by

* Corresponding author. Second Propedeutical Department of Surgery, Hippokration Hospital, Medical School, Aristotle University, 49 Konstantinoupoleos Street, Thessaloniki, Greece, 546 42, Tel.: þ30 231 089 2181/697 349 2863; fax: þ30 231 099 2932. E-mail address: [email protected] (M. Baltatzis). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.02.036

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pneumoperitoneum [1e3]. The extent of this reduction is proportional to the intra-abdominal pressure, the duration of pneumoperitoneum, and it also depends on the gas used [4,5]. According to a recent animal study, prolonged pneumoperitoneum (180 min) increased ischemiaereperfusion injury and oxidative stress when compared with 60 min pneumoperitoneum [6]. Oxidative stress is defined as the imbalance between oxidants and antioxidants in favor of the former. This imbalance is induced by either overproduction of reactive oxygen species (ROS) or deficiency-malfunction of the scavenging systems (superoxide dismutases, catalases, and glutathione peroxidases). Abundance of ROS causes oxidization of membrane phospholipids, proteins, and DNA, resulting in cellular necrosis and organ dysfunction. Organ damage from oxidative stress, induced by pneumoperitoneum is not limited to the abdominal cavity. The so-called “remote” ischemiae reperfusion injury also affects extra-abdominal organs, especially the lungs [7e10]. A systematic review of oxidative stress associated with pneumoperitoneum was presented in 2009 [11]. The authors analyzed data from 73 published relevant articles (experimental and clinical studies, case reports, and reviews) stating that there is sufficient evidence that pneumoperitoneum induces oxidative stress. In the great majority of these studies, biochemical markers of oxidative stress and histologic evaluation of tissue injury were used as sources of evidence. The review concluded that further research is required to evaluate the extent of this phenomenon in the clinical field. In another recent clinical study, plasma malondialdehyde levels increased and gastric mucosa pH decreased after prolonged pneumoperitoneum for robotic-assisted prostatectomy in ASA II and III patients (American Society of Anesthesiologists physical status classification system) [12]. Although hemodynamic effects and increased oxidative stress during pneumoperitoneum are transient phenomena and have probably no clinical impact on young and healthy patients, they could be potentially hazardous in the elderly, obese, and ASA III and IV patients [13e15]. Agents that reduce oxidative stress could be beneficial, especially for these subgroups of patients. Aprotinin, a serine protease inhibitor, is one of the substances tested for their efficacy to reduce ischemiae reperfusion injury and oxidative stress. Our study is the first to evaluate this drug in a pneumoperitoneum model in rats. For almost two decades, aprotinin has been widely used for its antifibrinolytic action, which results in the decrease of bleeding and transfusion rate during major thoracic and abdominal operations. In 2008, BART trial (Blood conservation using Antifibrinolytics in a Randomized Trial) showed higher mortality rates between patients taking aprotinin compared with patients receiving the newer antifibrinolytic agents [16]. Aprotinin withdrawal was consequently decided at the same year. However, more recent trials and meta-analyses raised a question about the validity of BART trial conclusions [17e19]. Health Canada published a safety review on aprotinin in 2011, which concluded that the benefit of using aprotinin in noncomplex cardiac surgery might outweigh the risk [20]. As a result, aprotinin was available again in Canada for restricted use in isolated coronary bypass graft surgery. Moreover, the European Medicines Agency also recommended lifting the

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suspension of aprotinin in 2012, after publishing a review on the risks and benefits of antifibrinolytic drugs [21]. Taking into consideration that withdrawal of aprotinin is probably history, further research about its anti-inflammatory and antioxidant action seems reasonable. Several studies have shown that aprotinin inhibits adhesion of leucocytes in the vascular endothelium and their migration in the interstitial space. Possible mechanisms involved are: (a) inhibition of adhesion molecules such as P-selectin and CD11b, (b) inhibition of the interleukin (IL) 8-, metalloproteinase-2-, and platelet activating factor-induced neutrophil diapedesis, and (c) inhibition of elastase and cathepsin, which also play a critical role in leukocyte migration [22e25]. Furthermore, aprotinin is reported to reduce neutrophil D-phospholipase and myeloperoxidase secretion and therefore to decrease cellular damage [26,27]. The effect of administration of aprotinin on suppression of proinflammatory cytokines and ROS production has been evaluated in several studies [27e30], which have provided strong evidence for the antiinflammatory action of the drug. In the present study, we evaluated the anti-inflammatory properties of aprotinin in a rat model of splanchnic ischemiaereperfusion injury caused by prolonged pneumoperitoneum.

2.

Material and methods

2.1.

Experimental protocol

Sixty 3- to 4-mo-old male Albino Wistar rats, with weights ranging between 250 and 350 g, were used in this study. The experiment was performed in the Laboratory of Scientific Research and Experimental Surgery of our University Department of Surgery (license no. EL54BIO17). The animals lived in a stable environment of 20 Ce22 C and 12-h lightedark cycles. European Union ethical directive for treating laboratory animals (86/609EU) was strictly followed during experimentation. Rats were randomly divided into three groups of 20 members each (sham group, C: control group, AP: aprotinin group). C and AP groups were subdivided into two subgroups of 10 members each, according to the duration of reperfusion period (60 or 180 min). Subgroups were named C60, C180, AP60, and AP180, respectively. The rats were all anesthetized initially with ether and secondly with 50 mg/kg of ketamine (Ketalar) given intraperitoneally (i.p.). Additional lower doses of ketamine were administered i.p. until the end of pneumoperitoneum to maintain anesthesia. The anesthetized rats were secured in a supine position on a special table and their abdomen was shaved and sterilized with 10% povidoneeiodine solution. A 0.5 cm midline skin incision was performed and a Veress needle was gently inserted in the peritoneal cavity and fixed with a purse string suture. In sham group, no other intervention was performed, whereas in the C and AP groups, Veress needle was connected with a CO2 insufflator (Storz, Tubingen, Germany). Constant 12 mm Hg pneumoperitoneum was maintained for 4 h in both groups and their subgroups. In the AP group, a loading aprotinin dose of 28000 KIU/kg

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(sc-3595, Santa Cruz Biotechnology, Dallas, TX) was given i.p., straight after the onset of pneumoperitoneum, followed by lower maintenance doses (7500 KIU/kg), which were administered per hour until the termination of insufflation. As analyzed in detail in the “Discussion” section, dosage was selected to match clinical practice by adjusting it to the animals’ weight. To avoid increasing fluid volume in the aprotinin group alone, which would lead to a biased experiment, equal amounts of normal saline 0.9% were administered to the animals belonging to the control group (at the same time points of aprotinin administration). Four hours later, pneumoperitoneum was discontinued, Veress needle was removed, and the abdominal wound was closed with 3-0 Nylon suture. Splanchnic reperfusion period lasted 60 or 180 min (depending on the subgroup), during which no other intervention was performed. At the end of reperfusion, a midline laparotomy and sternotomy were performed under anesthesia. Liver, terminal ileum, and lung samples were obtained for biochemical tests, weighed, mixed with 1 mL normal saline, and stored at 80 C. In addition, samples of the same organs were taken for histopathologic examination and stored in formalin. Animals’ heart was punctured and 4e5 mL of blood was collected and centrifuged to use plasma for measurements. Rats surviving sampling were euthanized by intracardiac injection of thiopental.

2.2.

Biochemical analysis

All tissue samples obtained for biochemical tests were homogenized by a Heidolph Elektro KG Type E60 homogenizer (Kelheim, Germany) at 4 C. The homogenates were then centrifuged at 1600g at 4 C for 10 min and the supernatant was used for measurements. Two of the most reliable markers of oxidative stress were measured in tissue and blood samples: pro-oxidanteantioxidant balance (PAB) and carbonyl proteins. The modified assay by Koliakos et al. was used for PAB evaluation [31]. According to that method, the balance of oxidants and antioxidants can be measured using 3,30 ,5,50 -Tetramethylbenzidine (TMB) and by performing two different kinds of reactions simultaneously: one enzymatic reaction during which the chromogen TMB is oxidized to a color cation by peroxides and a chemical reaction during which the TMB cation is reduced to a colorless compound by antioxidants. Carbonyl proteins formation is attributed to ROS, which modify amino acid chains, resulting in the generation of free carbonyls, which are absent on nonoxidized proteins. The most significant advantage of carbonyl proteins as a marker of oxidative stress is their early formation and stability. The assay used for our measurements is described by Alamdari et al. [32]. In addition, tumor necrosis factor a (TNF-a) and IL-6 were evaluated in liver and intestinal tissues using enzyme-linked immunosorbent assays (ELISA). Commercial ELISA kits were used for measurements: TNF-a rat ELISA kit, protocol KRC3011, Invitrogen with sensitivity <4 pg/mL, and coefficient of variation 4.3%e6.9%; IL-6 rat ELISA kit, protocol KRC0061, Invitrogen with sensitivity <5 pg/mL, and coefficient of variation 2.8%e5.8%. Endothelin 1 (ET-1) and C-reactive protein (CRP) were also measured in liver tissue according to the

following protocols: ET-1 rat ELISA kit, protocol ABIN366462, antibodies-online.com with a sensitivity of 0.31pg/mL and CRP rat ELISA kit, protocol KA1035, Abnova with a sensitivity of 0.5 ng/mL, and coefficient of variation 5%.

2.3.

Histopathologic examination

Histopathologic evaluation was performed by two pathologists, who were blinded to the study groups. Liver, small intestine, and lung samples were preserved in 10% formaldehyde solution, enclosed in paraffin, and sectioned by a microtome set at 5 mm thickness. Finally, they were stained with hematoxylin and eosin (H and E stain). Tissue injury scoring was based on a five-scaled classification proposed by Hauet et al. [33]. Hauet’s scale was modified in a way that not only the extent but also the severity of lesions determined the tissue score (Table 1).

2.4.

Statistical methods

Mean and standard deviation were calculated, and a test of distribution (KolmogoroveSmirnov) was run for all biochemical data. Distribution was normal in all parameters measured and one-way analysis of variance test was used to compare means. Nonparametric ManneWhitney U test was performed to evaluate histopathologic data. The level of statistical significance was set at P < 0.05. Statistical power of our sample (n ¼ 10 for each subgroup) was calculated >0.80 for all parameters measured. It is, therefore, sufficient to support the final conclusions of the study.

3.

Results

3.1.

Biochemical results

As previously analyzed, tissue homogenates were prepared for biochemical analysis. ELISA kits were used to measure the values of the proinflammatory markers, whereas the already mentioned specific assays were performed for PAB and carbonyl protein evaluation. Results of these biochemical markers and the markers of oxidative stress are analyzed in detail in the following: Beginning with the presentation of biochemical results in liver, TNF-a values in aprotinin subgroups were significantly lower than control subgroups (C60 versus AP60 P ¼ 0.001, C60 versus AP180 P ¼ 0.002, C180 versus AP60 P ¼ 0.016, and C180 versus AP180 P ¼ 0.022). Almost similarly, IL-6 was significantly

Table 1 e Five-scaled histopathologic score. Score 1 2 3 4 5

Extent and severity of injury No injury Mild lesions affecting only 10% of tissue Moderate lesions affecting 25% of tissue Severe lesions affecting 50% of tissue Very severe lesions affecting more than 75% of tissue

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decreased in aprotinin subgroups (C60 versus AP60 P ¼ 0.003, C60 versus AP180 P ¼ 0.014, and C180 versus AP60 P ¼ 0.012). As for ET-1 results in liver, although following the same trend, statistically significant difference arose only between C60 and AP60 subgroups (P ¼ 0.005; AP60 having lower values). CRP values were also lower in aprotinin subgroups (C60 versus AP60 P ¼ 0.004, C60 versus AP180 P ¼ 0.017, and C180 versus AP60 P ¼ 0.017). PAB results revealed significant difference only between AP60 and C60, C180 (P ¼ 0.006 and 0.015,

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respectively). Finally, values of carbonyl proteins were substantially lower in aprotinin groups compared with both control groups (C60 versus AP60 P ¼ 0.007, C60 versus AP180 P ¼ 0.010, C180 versus AP60 P ¼ 0.008, and C180 versus AP180 P ¼ 0.012). The previously mentioned results are graphically demonstrated in Figure 1AeF. Focusing on the small intestine, TNF-a values of aprotinin subgroups (AP60 and AP180) were significantly lower than the control subgroup with short time of reperfusion (C60; P ¼ 0.015

Fig. 1 e (AeF) Graphic presentation of TNF-a, IL-6, ET-1, CRP, PAB, and carbonyl proteins values in liver homogenates (*P < 0.05).

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Fig. 2 e (AeD) Graphic presentation of TNF-a, IL-6, PAB, and carbonyl proteins values in small intestine homogenates (*P < 0.05).

and P ¼ 0.008, respectively). Aprotinin subgroups also showed lower IL-6 values; however, this finding had no statistical significance. The results of PAB assay were almost similar with those of IL-6, as statistically significant difference was present only between C60 and AP180 (P ¼ 0.018). Values of carbonyl proteins in small intestine were lower in the AP60 subgroup compared with C60 (P ¼ 0.005; Fig. 2AeD). Furthermore, results of oxidative stress measured in lung samples followed the same tendency. PAB values were significantly lower in AP180 subgroup when compared with both C60 and C180 (P ¼ 0.019 and 0.029, respectively). In addition, aprotinin suppressed carbonyl protein production in the lungs (C60 versus AP60 P ¼ 0.035, C60 versus AP180 P ¼ 0.001, and C180 versus AP180 P ¼ 0.011). Lung results are graphically demonstrated in Figure 3A and B. It is worth mentioning that in the majority of biochemical markers measured in tissues, sham group had significantly lower values than the control group, which confirms the effect of increased intra-abdominal pressure on splanchnic circulation. Concerning serum results, PAB and carbonyl protein values, although showing the tendency observed in tissue samples, revealed no statistically significant difference between control and aprotinin groups (Fig. 4A and B).

3.2.

Histopathologic results

Pathology, combined with biomarkers, is always useful to verify the conclusions. In our study, histopathologic examination revealed even clearer results in favor of aprotinin. Scores of aprotinin subgroups in all tissues examined were significantly lower than those of control groups. Hepatic specimens showed milder periportal infiltration by inflammatory cells, less hyperemia, milder hydropic involution of hepatocytes, and hepatic veins and sinusoids congestion in aprotinin than in control subgroups. These lesions are demonstrated in Figure 5A. Small intestine specimens revealed more extensive and severe lesions in control subgroups (structural deformities of the villi, mucosal ulceration, and lymphocyte infiltration), compared with aprotinin subgroups (Fig. 5B). Finally, in the lungs, higher amount of alveolar exudate, interstitial edema, and capillary congestion were observed in control subgroups than in aprotinin subgroups (Fig. 5C). It is worth stating that AP60 showed milder lesions in liver, intestine, and lungs than AP180, a finding, which was also statistically confirmed. Histopathologic scores of the three tissues are graphically demonstrated in Figure 6AeC. Tissue scoring and statistical data are summarized in Tables 2 and 3.

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Fig. 3 e (AeB) Graphic presentation of PAB and carbonyl proteins values in lung homogenates (*P < 0.05).

4.

Discussion

The present experimental model was designed to reproduce ischemiaereperfusion syndrome induced by pneumoperitoneum in rats and to investigate the protective role of aprotinin on this phenomenon. Several studies have also investigated the anti-inflammatory and antioxidant effects of aprotinin. Most of them agree that anti-inflammatory action of the drug is biochemically and histologically detectable, at least on animals [27e29,35,36,38]. Apart from the various experimental protocols, two clinical studies were published in 2013, trying to justify the anti-inflammatory properties of aprotinin in humans. Although they are not based on ischemiaereperfusion protocols, their findings are extremely important. The first study included 60 patients undergoing cardiopulmonary bypass; of those, 30 patients received aprotinin and 30 received tranexamic acid intravenously. In the aprotinin group, significantly lower levels of procalcitonin, IL6, TNF-a, and IL-8 were detected [39]. The second clinical study investigated the role of aprotinin and tranexamic acid on

postoperative gene expression of inflammatory mediators and cytokines by measuring their messenger RNA with polymerase chain reaction. According to the results, suppression of eight genes was observed in the aprotinin group, and in addition, suppression of three more genes was detected in both aprotinin and tranexamic acid groups [40]. Returning to the present study, some important comments about dosage, time, and route of aprotinin administration should be made. The dosage administered during cardiac surgery (as given in Trasylol DatasheeteBayer) was chosen for our experiment, after making the appropriate adjustments to the animals’ body weight. Our aim was to succeed nearly perfect match to the clinical practice. The only difference was the intermittent administration of maintenance dose instead of constant infusion applied in humans. Nevertheless, equivalent total dose was finally administered. Higher doses were not used, taking into consideration that aprotinin was temporarily disapproved because of greater incidence of thromboembolic events in humans. Although interesting to test it in an animal model, a higher dose would be dangerous if used in clinical practice. In addition, comparable dosage has

Fig. 4 e (AeB) Graphic presentation of PAB and carbonyl proteins values in plasma. No statistically significant difference was observed between control and aprotinin groups.

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Fig. 5 e (A) Liver parenchyma (C180/AP180)dHematoxylineeosin stain 3200 showing milder lesions in AP180 specimen. (B) Small intestine (C180/AP180)dHematoxylineeosin stain 3100 showing less severe damage of tissue architecture and less extensive leukocyte infiltration in AP180 specimen. (C) Lung (C180/AP180)dHematoxylineeosin stain 3200 revealing remarkable histopathologic improvement in the AP180 specimen, especially regarding alveolar infiltration by lymphocytes. (Color version of figure is available online.)

been selected in other ischemiaereperfusion protocols in rats, which seems to be an adequate reason for not experimenting with lower doses of the drug [28,38,41,42]. Intraperitoneal injection of aprotinin has been used in several experiments. Because of its large surface and absorptive capacity, peritoneum is an ideal route of drug administration to animals. Aprotinin has been administered i.p. to mice [43], rats [38,44,45], and pigs [46], succeeding satisfactory bioavailability. Finally, intraperitoneal administration of the drug has been used in clinical protocols of acute pancreatitis [47] and peritoneal adhesions [48]. Regarding the exact timing of intraperitoneal injection, it is set at the beginning of ischemia in the majority of relevant studies, our protocol being no exception. Another parameter, which is worth commenting, is the amount of intra-abdominal pressure used in our protocol. Pressure of 12 mm Hg has been proved to be clinically safe and also appropriate for adequate working space. Furthermore, older studies have evaluated the effects of various intraabdominal pressure on splanchnic circulation, both in

animals and humans and concluded that the extent of ischemiaereperfusion injury was proportional to the amount of pressure [3e5]. Based on these protocols and their clear results, we avoided experimenting with different amounts of pressure. Moreover, our previous laboratory experience showed that higher pressures could result in unacceptable rates of animal mortality in both rats and rabbits [6,49]. Our final scope was to evaluate anti-inflammatory properties of aprotinin in a safe and clinically applicable environment. Concerning group selection, the plan decided on sham group involved only administration of anesthesia, avoiding any other intervention. The use of another sham group, with aprotinin administration as the only intervention, seems also reasonable to add more evidence. Indeed, the second sham group would be absolutely necessary if this was the first trial evaluating aprotinin or if the study aimed at determining the appropriate dose. However, aprotinin has been used in many experimental protocols as well as in the clinical field and the previously mentioned parameters have already been determined. Moreover, in several animal studies an “aprotinin

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Fig. 6 e (AeC) Liver, small intestine, and lung histopathologic scoredgraphs. Aprotinin attenuated inflammatory response in all tissues examined (*P < 0.05). sham” group was not used [28,29,34,35,38,41,42]. As a conclusion, although an aprotinin sham group should be a part of a flawless protocol, the absence of such a group does not affect the validity of our results concerning the protective role of aprotinin during severe oxidative stress. The selection of biomarkers measured was determined by the relatively short time of reperfusion period chosen in our protocol. Proinflammatory factors (TNF-a, IL-6, ET-1, and CRP) were selected, because of their rapid production and release following the harmful stimulus, unlike to other markers, which are characterized by more delayed elevation. TNF-a is produced in high amounts during the first steps of inflammatory response. IL-6 is also detectable within 60 min after the injurious event. ET-1 plays an important role in ischemiaereperfusion syndrome because of its vasoconstrictive properties. In a recent experimental study, suppression of

ET-1eattenuated ischemiaereperfusion injury in rat kidneys [50]. Moreover, the fact that endothelin receptor A (ETA) is expressed in hepatocytes, makes ET-1 a reliable marker for hepatic ischemiaereperfusion injury. CRP, which is produced in liver, has proved to be a useful marker for evaluating inflammatory response, specifically after ischemia induced by pneumoperitoneum [51]. Facing our results and trying to interpret numbers into words, the first conclusion drawn is that sham group had significantly lower values than control group in most of biochemical markers. Moreover, in sham group, histopathologic changes were very mild or absent compared with the severe lesions of control group. These results testify that oxidative stress and ischemiaereperfusion injury were present in the control group due to prolonged pneumoperitoneum. Biochemical markers in the aprotinin group were

Table 2 e Tissue score summary (mean and standard deviation).

Liver scores Small intestine scores Lung scores

Sham

AP180

AP60

C60

C180

1.33  0.52 1.17  0.41 1.00  0.0

1.88  0.64 2.00  0.53 2.37  0.52

2.75  0.46 3.00  0.53 3.25  0.46

3.71  0.76 4.14  0.69 4.86  0.38

3.86  0.69 4.43  0.79 4.71  0.49

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Table 3 e Tissue score mean comparison. Liver scores Groups Sham versus C60 Sham versus C180 Sham versus AP60 Sham versus AP180 C60 versus AP60 C60 versus AP180 C180 versus AP60 C180 versus AP180 C60 versus C180 AP60 versus AP180

Small intestine scores

Lung scores

P

Groups

P

Groups

P

0.002 0.002 0.002 0.112 0.013 0.002 0.005 0.001 0.674 0.011

Sham versus C60 Sham versus C180 Sham versus AP60 Sham versus AP180 C60 versus AP60 C60 versus AP180 C180 versus AP60 C180 versus AP180 C60 versus C180 AP60 versus AP180

0.002 0.002 0.001 0.012 0.006 0.001 0.004 0.001 0.404 0.004

Sham versus C60 Sham versus C180 Sham versus AP60 Sham versus AP180 C60 versus AP60 C60 versus AP180 C180 versus AP60 C180 versus AP180 C60 versus C180 AP60 versus AP180

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.530 0.007

lower in liver tissue (especially TNF-a, IL-6, CRP, and carbonyl proteins) with great statistical significance. Histopathologic results confirmed this tendency, providing the additional finding that AP180 subgroup had significantly milder lesions than AP60. Lung measurements and observations came to the same results: aprotinin ameliorated biochemical and histological findings in lung tissue as well. However, in the small intestine, there was a discrepancy between biochemical and histopathologic results. While pathologists observed a great improvement in rats under aprotinin, biochemical values were ambiguous, with some of the markers having no statistically significant difference between groups (even though aprotinin group had still lower values). To resolve this discrepancy and find out whether there is a real tendency in favor of aprotinin or not, total proteins were measured in intestinal homogenates using ELISA. After calculating the fraction of TNF-a and IL-6/total proteins, statistical process was repeated, providing results closer to the pathology observations (Fig. 7AeB). Regarding the different time of reperfusion (60 or 180 min), it appears that in the control group it did not make any difference. However, between the two aprotinin subgroups (AP60

and AP180), histopathologic results showed better performance in the AP180 subgroup, which was obvious in all tissues examined. AP180 had the lowest histologic score among all groups except sham. This result appears rather convincing, considering the pharmacokinetics of aprotinin. According to a scintigraphic study using 99mTc aprotinin, the drug diffuses rapidly from the plasma to the interstitial space and its concentration remains high in many organs (such as liver) for 24 h or more after administration [52]. It is, therefore, reasonable to argue that the prolonged action of aprotinin (180 min) had a beneficial effect compared with the shorter time of 60 min. As for the biomarker results in plasma, there are several reasons to explain why the values of PAB and carbonyl proteins were not comparable with tissue biomarkers and histopathology. First of all, free radical production and protein carbonylation are procedures that primarily take place in tissues suffering from ischemiaereperfusion. It is, therefore, rational to expect that the difference between control and aprotinin groups would be more evident in tissues than in plasma. Another possible explanation originates from aprotinin pharmacokinetics. In the scintigraphic study mentioned previously, the radiotracer was rapidly cleared from the

Fig. 7 e (A) TNF-a/total proteins graphdsmall intestine: both aprotinin subgroups take significant lower values compared with C180 (*P < 0.05). (B) IL-6/total proteins graphdsmall intestine: both aprotinin subgroups take significant lower values than both control subgroups (*P < 0.05).

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plasma as a biexponential function [52], which means that during the reperfusion phase of our experiment a low plasma concentration of aprotinin should be expected. Taking the previously mentioned data into consideration, we focused on examining the influence of the drug on tissue samples rather than on plasma. Finally, should one try to respond accurately to the assumptions made at the very beginning of this study, he will conclude that: (a) the administration of aprotinin resulted in reduction of cytokine values and oxidative stress in liver and lungs, whereas the trend was statistically less strong in the small intestine, though existent; (b) the animals that received aprotinin had less extensive and milder histopathologic changes not only in liver but also in the small intestine, which showed equivocal biochemical behavior; (c) the pulmonary lesions that reflect systemic inflammatory response were also milder in aprotinin subgroups, which was confirmed by both biochemical and histological examinations. Administration of aprotinin results in reduction of tissue damage caused by splanchnic ischemiaereperfusion, which is induced by pneumoperitoneum. This conclusion, combined with the findings of other relevant experimental protocols, could be a forerunner for the design of clinical trials, to investigate the anti-inflammatory effect of the compound on humans.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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