j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 2 3 8 e2 4 8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
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
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 2 3 8 e2 4 8
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
239
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
240
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 2 3 8 e2 4 8
(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
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
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
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 2 3 8 e2 4 8
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
239
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
240
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 2 3 8 e2 4 8
(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
