Ruptured abdominal aortic aneurysm (RAAA) accounts for 1-2% of deaths in the population older than 65 years of age. However endovascular therapy (EVAR), which is increasingly being performed, has decreased the mortality rate to 20–25% from 50–70% encountered in the open repair (OR) performed for the treatment of RAAA. Today, open repair is the most frequently (80%) performed method because of anatomical inconvenience, such as short neck and poor iliac arteries, or inadequate team and equipment in the vascular surgery centers [
Whilst ischemia and reperfusion occur only in the lower torso with the clamping and declamping during surgical treatment of intact aneurysm, preclamping hemorrhagic shock and related impaired tissue perfusion accompany the ischemia and reperfusion of the lower torso during surgical treatment of ruptured aneurysm. This complex situation leads to systemic inflammatory response syndrome (SIRS) and multiorgan failure (MOF) by causing local or distant organ damage such as lungs, liver, and heart and enhances the mortality rate in the surgical treatment of RAAA. Lindsay et al. created an experimental model and demonstrated that supramesenteric aortic clamping and hemorrhagic shock, each of which is unable to cause lung damage individually, caused distant organ damage when they are together, as in the surgical treatment of RAAA, and they used this model in numerous experimental studies [
Ethyl pyruvate (EP) is a simple ester derivative of pyruvic acid. In many animal models, in which various critical diseases including endotoxemia, sepsis, hemorrhagic shock, burn injury, pancreatitis, ileus, and myocardial, mesenteric and hepatic ischemia/reperfusion injuries were modeled; EP was demonstrated to reduce organ damage and have favorable effect on survival. EP is an effective anti-inflammatory agent and shows its efficacy by decreasing the secretion of proinflammatory proteins such as TNF-
The study aimed at investigating the effect of ethyl pyruvate on inflammatory response and lung injury by creating a rat model of ruptured abdominal aortic aneurysm.
The present study was performed in 30 Sprague-Dawley male rats with a mean weight of
In order to administer in the treatment groups, ethyl pyruvate (Sigma-Aldrich, Cat no. E4, 780-8) was prepared in Ringer’s solution as per 1 mL would include 20 mg ethyl pyruvate. Ringer’s Lactate solution was used in the control groups approximately in the same amount with EP. Ringer’s solution includes 129.3 mEq/L Na+, 5.0 mEq/L K+, 112 mEq/L Cl−, and 4 mEq/L Ca2+ and its osmolality is 275.5 mOsm/L (Polypharma, Turkey).
Intraperitoneal ketamine (50 mg/kg) and xylazine (10 mg/kg) were used for the anesthesia of the rats. The anesthesia was maintained with intermittent ketamine administration allowing spontaneous respiration. Rats were randomized in two groups as sham (Sh) and shock/clamp (S/C); each group was redivided into two groups (Sh (
Schematic diagram of the interventions in each study group.
Groups |
|
Shock |
Treatment | Heparin |
|
Clamp |
|
Reperfusion |
Blood and |
---|---|---|---|---|---|---|---|---|---|
Sh | 6 | − | RL | + | − | − | − | − | + |
Sh + EP | 6 | − | 40 mg/kg |
+ | − | − | − | − | + |
S/C | 9 | + | RL | + | + | + | + | + | + |
S/C + EP | 9 | + | 40 mg/kg |
+ | + | + | + | + | + |
Sh: sham; EP: ethyl pyruvate; S/C: shock/clamp; RL: Ringer’s lactate solution.
Cutdown was performed and the right internal jugular vein was cannulated for the venous access, and the right carotid artery was cannulated using number 22 cannula (Novacath, Medipro, Co., Istanbul, Turkey) to monitor mean arterial pressure (MAP). Heart rate, MAP, rectal temperature, and respiratory rate were monitored (Nikon Kohden BSM-4113). In the course of experiment, 3 mL/kg/h of saline (0.9% NaCl) infusion (Perfusor Compact S, Brown) was done to meet the insensible losses. Rectal temperature was kept around 36.5°C using heat lamb. Midline laparotomy was performed in all groups and abdominal aorta was isolated at the level of proximal side of superior mesenteric artery and iliac bifurcation. Laparotomy was closed using 5/0 Prolene suture in the sham groups and the rats were kept under anesthesia for 4 hours. In the S/C groups, blood sample was drawn through the carotid artery cannula into the heparinized plastic injector (500 U Heparin) (Nevparin, 5000 U/mL, Mustafa Nevzat, Turkey) after the monitoring and stabilization periods, and shock was created keeping the MAP at 50 mmHg for 60 minutes and aneurysm rupture was simulated; the blood was stored at room temperature. The amount of blood obtained from the rats was calculated not to exceed 30% of the total blood volume. Equal amount of Ringer’s solution was given to the S/C and Sh groups at the end of 60-minute hemorrhagic shock and equivalent period, respectively, whereas Sh + EP and S/C + EP groups received intraperitoneal (ip) 40 mg/kg EP. Abdominal aorta was explored through the midline laparotomy. All the groups underwent systemic heparinization by intravenous heparin given at a dose of 250 U/kg. In the S/C and S/C + EP groups, lower torso ischemia was created by clamping abdominal aorta separately at the level of superior mesenteric artery and iliac bifurcation using microvascular clamps. Half of the blood sample taken at that time was reinfused via venous route, and surgical x-clamp and resuscitation were simulated. Following 60-minute ischemic period, all of the remaining blood was reinfused just before the clamp was removed. After the clamps were removed, the abdomen was closed and the rats were kept in reperfusion for 120 minutes. The MAP was kept at 100 mmHg during reperfusion period by administering additional saline solution when needed. The amount of fluid administered in the course of experiment was recorded.
At the end of experimental period, all rats were sacrificed by drawing blood through the carotid artery. Hilar regions of both lungs were clamped and removed; a part of the left lung tissue was stored at −80°C as frozen for biochemical analyses, whereas the other part was fixed with formaldehyde for histopathological examinations. Right lungs were reserved for the calculation of the wet/dry weight ratio.
The serum levels of malondialdehyde (MDA), myeloperoxydase (MPO), tumor necrosis factor-alpha (TNF-
MDA and MPO were measured in the lung tissue and histopathological examination was performed.
The red color that resulted from the reaction between MDA, a lipid peroxidation product, and thiobarbituric acid (TBA) was measured spectrophotometrically at 532 nm light. Plasma level was calculated as nanomol/mL (nanomoles per milliliter) [
Serum MPO levels were assessed using enzyme-linked immunosorbent assay (ELISA) kit (Hycult biotech, Catalog number HK105, The Netherlands). Absorbance of the samples was measured by VERSA brand (designed by Molecular Devices in California, USA) microplate reader at 450 nm. Results were given as nanogram/mL [
Serum TNF-
Serum IL-6 levels were measured by a method similar to that of TNF-
Reduced Cobalt-to-albumin binding capacity was evaluated by rapid and calorimetric assessment method developed by Bar-Or et al. Once blood samples were taken, serum and plasma specimens were prepared by centrifuging at 1.800 ×g for 15 minutes. The specimens were put into Eppendorf tubes and stored at −80°C until analysis. Reduced cobalt-to-albumin- binding capacity (IMA level) was analyzed using rapid colorimetric method. The results were given as absorbance units (ABSU) [
At the end of experimental period, 0.5 mL of blood was drawn from the carotid artery to remove the probable serum residues and was disposed. Subsequently, approximately 1.5 mL of arterial blood sample taken into heparinized injector was transferred into the cartridge and blood gasses were measured (IRMA TRU point Blood Analysis System).
A piece of lung tissue was used to measure MDA levels. The sample was minced and homogenized by an Ultra-TurraxT25 homogenizer (Janke and Kunkel IKA) in an ice-cold 1.15% KCl solution containing 0.05% Triton X-100. Tissue MDA levels were determined using the method described by Uchiyama and Mihara. Tetramethoxypropane was used as a standard. The MDA levels were calculated in nanomoles per gram of wet tissue [
Tissue MPO levels were assessed using enzyme-linked immunosorbent assay (ELISA) kit (Hycult biotech, Catalog No. HK105, The Netherlands). Absorbance of the samples was measured by VERSA brand (designed by Molecular Devices in California, USA) microplate reader at 450 nm. Results were given as nanogram/mL per gram of tissue.
As the experiment was finalized, sternotomy was performed and the right lung was removed by clamping at the hilus, separated from the surrounding tissues and weighed using microbalances. It was reweighed after being stored at 70°C for 48 hours. Wet/dry weight ratio was calculated, and an increase was interpreted in favor of lung edema.
Following the finalization of the experiment, tissue samples taken from approximately the same lung sections in all rats were separately kept in numbered storages containing 10% of neutral formaldehyde solution for histopathological examination. The bloody solution was changed after 30 minutes, and the tissues were fixed with 10% neutral formaldehyde solution for 48 hours. Tissues were dehydrated by passing through the graded series of alcohol and embedded into paraffin after being made pellucid. The paraffin blocks were cut in 5 micrometer (
Statistical analysis was performed using SPSS 15.0. Kruskal-Wallis variance analysis (the Mann-Whitney
The results of serum MPO, MDA, TNF-
Biochemical parameters and histopathological scores in all groups (mean
Parameters | Sh ( |
Sh + EP ( |
S/C ( |
S/C + EP ( |
---|---|---|---|---|
MPO (ng/mL) |
|
|
383.85 |
198.33 |
MDA (nmol/mL) |
|
|
2.95 |
1.96 |
TNF |
|
|
262.71 |
188.76 |
IL-6 (pg/mL) |
|
|
|
|
IMA (ABSU) |
|
|
0.84 |
0.74 |
Lung MDA (nmol/g) |
|
|
522.5 |
499.14 |
Lung MPO (ng/mL) |
|
|
6541.1 |
6019.12 |
W/D weight ratio |
|
|
5.55 |
3.77 |
Histopathological score |
|
|
2.66 |
1.44 |
Note: Sh: sham; EP: ethyl pyruvate; S/C: shock/clamp; MDA: malondialdehyde; MPO: myeloperoxidase; TNF
Whilst MPO level was
Lipid peroxidation is one of the reactions caused by free oxygen radicals, and MDA is one of the end products of lipid peroxidation. MDA levels were increased by two folds in the S/C group versus the sham groups and reached to
TNF-
IL-6 level was minimally increased in the S/C group versus the sham groups, but no statistically significant difference was found between the groups (whilst it was
This marker, which depends on the measurement of the amount of circulating albumin modified due to ischemic stress, was increased by two folds from
Lung tissue MDA level was significantly increased to
Likewise, lung MPO level increased to
There was no significant difference between the sham groups (
Blood gas analysis of the groups revealed increase in the acidosis and base gap in the S/C and S/C + EP groups versus the sham groups. pH decreased to
Blood gas values.
pH | PO2 | PCO2 | HCO3 | BE | |
---|---|---|---|---|---|
Sh |
|
|
|
|
|
Sh + EP |
|
|
|
|
|
S/C | 7.20 |
|
|
|
−9.38 ± 5.04a,b |
S/C + EP | 7.33 |
|
|
|
−5.64 ± 2.8a,b |
PaO2: arterial oxygen pressure; PaCO2: arterial carbon dioxide pressure; HCO3: bicarbonate; BE: base excess.
Histopathological examination of the lung tissues revealed normal lung tissue in the sham groups but diffused intra-alveolar edema, intra-alveolar hemorrhage, and leukocyte infiltration in the S/C group. Moderate intra-alveolar edema and alveolar epithelial thickening were observed in the S/C group that received EP (Figure
(Sh, Sh + EP) Normal histopathological examination of the lung tissues in Sh and Sh + EP groups. (S/C) Diffuse intra-alveolar edema (↑), intra-alveolar hemorrhage (▲), and leukocyte infiltration in the S/C group. (S/C + EP) Moderate intra-alveolar edema and alveolar epithelial thickening (↑) were observed in the S/C group that received EP. (Hematoxylin and Eosin, [H&E] ×200).
Whilst the mean arterial pressure (MAP) was stable in the sham groups over the course of experiment, the amount of saline given to achieve the baseline level of MAP was low in these groups.
After the baseline level was achieved, the MAP was kept around 50 mmHg in the S/C and S/C + EP groups in accordance with the experiment protocol. The amount of blood taken from the rats was
During reperfusion phase, MAP decreased rapidly and became lower than that in the sham groups; decrease in MAP in the S/C group was more remarkable through the end of 120-minute reperfusion period (Figure
Mean arterial blood pressure during the experiment. Values are mean ± SEM.
Total amount of extra fluid given in addition to the maintenance fluid was
In the present study, we investigated whether ethyl pyruvate reduces systemic inflammatory response and lung injury that result from shock, ischemia, and reperfusion in the surgical treatment of the rat model of ruptured abdominal aortic aneurysm. Systemic inflammatory response syndrome (SIRS) and related multiorgan failure are the most important causes of high mortality following surgical treatment of ruptured abdominal aortic aneurysm. Whilst the prevalence of multiorgan failure is 3.8% after surgical treatment of intact aneurysms, it is 64% after RAAA [
Unlike intact aneurysm, total body ischemia, which varies depending on the duration and deepness of hemorrhagic shock, occurs in ruptured aneurysms and lower torso ischemia and reperfusion, which result from aortic clamping, accompany this situation in the course of surgery. Although many systems including complement, coagulation, fibrinolytic, and kallixrein cascades are activated during inflammatory response triggered together by hemorrhagic shock, ischemia, and reperfusion, leukocytes play the most important role. Activation and interaction of leukocytes and endothelial surface initiate the cytokine release, endothelial microvascular permeability is increased, and transendothelial neutrophil migration occurs. Endothelial damage leads to endothelial cell swelling, capillary leak, edema, and organ dysfunction. This situation known as systemic inflammatory response is followed by multiple organ failure including the lungs and the kidneys. In many of the ischemia and reperfusion studies, some biochemical analyses are performed including cytokine levels such as TNF-
Pyruvic acid, which is a carboxylic acid with three carbons, is the end product of glucose metabolism as a member of aliphatic amino acids. The reaction between pyruvate and coenzyme A results in Acetyl CoA, which is a rate-limiting step in the oxidative metabolism of glucose by thiocarboxylic acid. Although pyruvate is a metabolic fuel, it also functions as an endogenous oxygen radical scavenger.
Ethyl pyruvate (EP) is a simple aliphatic ester derivative of pyruvic acid. In various animal experiments, it was demonstrated to be an effective anti-inflammatory agent by inhibiting proinflammatory signal pathways such as NF-
Lindsay et al. conducted an experimental study and created shock, ischemia, and reperfusion, and this method was called experimental model of ruptured abdominal aortic aneurysm [
Lipid peroxidation is one of the reactions caused by free oxygen radicals, and MDA is one of the end products of lipid peroxidation. Measurement of MDA level in the plasma and tissues gives information about free oxygen radicals. In a substantial proportion of experimental IR studies, information about lipid peroxidation in the serum and tissues was obtained by measuring serum MDA values using various methods. Jan et al. measured MDA values as a parameter to investigate whether ischemic preconditioning performed prior to shock reduces lung injury [
MPO, which is a peroxidase enzyme and mostly found in the neutrophils, is a lysosomal protein stored in the azurophilic granules of the neutrophils. Measurement of serum and tissue MPO values gives information about neutrophil activation. In the present study, increased MPO activity both in the systemic circulation and in the lungs due to S/C application was suppressed by EP. Wang et al. and Luan et al. demonstrated that MPO activity in the ileum was suppressed by EP in the extrahepatic cholestasis and in acute pancreatitis, respectively [
Tumor necrotizing factor-
İnterleukin-6 is responsible for the specific conditions in the inflammatory response of the host. It is produced as a response to the secretion of TNF-
Various studies demonstrated that ischemia-modified albumin (IMA) is increased in the event of elevated free radical levels such as hypoxia or ischemia-reperfusion that tissues are exposed to sepsis, acute infection, advanced cirrhosis, end-stage renal disease, and advanced cancer [
Blood gas analysis exhibits gas change at alveolar level and acid-base balance of the organism. Serum pH level was significantly decreased in the S/C group versus the sham group and acidosis occurred, base gap was developed, and a little improvement occurred in the group that received EP although being not significant. It is obvious that great changes in blood gas parameters could not be tolerated by the rats kept in spontaneous respiration in the room air and would result in death. One of the most important limitations of this experiment is leaving the rats in spontaneous respiration in the room air without intubating.
Histopathological examination revealed diffuse edema, leukocyte infiltration, and interalveolar and intra-alveolar hemorrhage in the lung tissue of the SIR group. Injury score of this group increased to 2.66 from 0.5 in the sham group. In the SIR + EP group, a decrease was observed in the intra-alveolar edema and leukocyte infiltration and injury score decreased to 1.44 from 2.66 in the SIR group. There was no difference in the sham + EP group as compared to the sham group; that is, EP alone has not cause any damage. There are many studies demonstrating that ethyl pyruvate acts as anti-inflammatory, antiedema, and antioxidant agent and treats organ injury in many experimental studies including hemorrhagic shock, ischemia- reperfusion, burn, sepsis, and acute necrotizing pancreatitis [
The present study is a unique experimental model, which provides the simulation of both hemorrhagic shock and ischemia-reperfusion in the same experimental model. In this respect, we think that it will provide an important contribution to the literature.
Over the course of experiment, the sham groups maintained stable blood pressure showing minimal need for fluid replacement; however, in the S/C group, a remarkable hypotension and need for fluid replacement were observed which particularly increased as the aortic clamp was removed and reperfusion was started. This resulted from the vasodilation caused by vasoactive mediators and certain metabolites that joined the systemic circulation from the ischemic tissues during reperfusion phase, as well as from the increased vascular permeability. In the group that received ethyl pyruvate, inflammatory cell activation was suppressed and microvascular permeability decreased, more stable blood pressure levels were obtained, and the need for replacement was reduced. Pyruvate is a compound that structurally resembles lactate and is shown to help survival in hemorrhagic conditions due to its antioxidant and anti-inflammatory effects [
In conclusion, ethyl pyruvate prevents the lung injury created by experimental rat model of RAAA and shows this efficacy reducing the systemic and local inflammatory response by means of suppressing neutrophil infiltration and production of free radicals.
None of the authors have any conflict of interests related to this paper.
This paper was supported by Karadeniz Technical University Scientific Research Projects (Project no. 114.02.15).