American Journal of Emergency Medicine (2012) 30, 476-480

Brief Report

Quantification of superoxide radical production in 4 vital organs of rats subjected to hemorrhagic shock^?

Eleftheria S. Panteli MD ^a, Fotini Fligou MD, PhD ^a, Chrisaugi Papamichail MD ^a, Ioannis Papapostolou PhD ^b, Georgios Zervoudakis PhD ^b, Christos D. Georgiou PhD ^b,

Kriton S. Filos MD, PhD ^a,?

^aDepartment of Anesthesiology and Critical Care Medicine, School of Medicine, 26500 Rion, Greece

^bSection of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, 26504 Rion, Patras, Greece

Received 19 October 2010; revised 14 December 2010; accepted 23 December 2010

Abstract

Objective: The aim of this study was to measure the production of superoxide radical (O^-), a direct indicator of oxidative stress, in 4 vital organs of rats subjected to hemorrhagic shock. For this purpose, and for the first time, a new quantitative assay for the ex vivo measurement of O^- via an established 1:1 molar relationship between O^- and 2-OH-ethidium was used. The production of lipid hydroperoxides (LOOHs), a standard method of evaluation of oxidative stress, was also used for reasons of comparison. Methods: Sixteen male Wistar rats were divided into 2 groups: sham and hemorrhagic shock, targeting to a mean arterial pressure of 30 to 40 mm Hg for 60 minutes. Three hours after resuscitation, tissues were collected for measurement of LOOHs and O^- production.

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Results: Hemorrhagic shock induced increased production of LOOHs in the gut, liver, and lungs (P b .001), whereas the production of O^- was also increased in the gut (P b .001), liver (P b .001), and, to a lesser extent, in the lungs (P b .05). The oxidative load of the kidneys, as estimated by both techniques, remained unaffected.

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Conclusion: The results of this new O^- assay were comparable with the results of the established LOOHs method, and this assay proved to be accurate and sensitive in the detection and quantification of O^- production in all organs tested. Thus, the proposed direct measurement of O^- in critically ill patients often facing in extremis situations could be used as a Prognostic tool and as a method to evaluate therapeutic interventions in the setting of emergency medicine.

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(C) 2012

Introduction

The role of oxidative stress in critical care and emergency medicine has been increasingly studied in recent years and

^? Support was provided solely from institutional sources.

* Corresponding author.

E-mail addresses: [email protected], [email protected] (K.S. Filos).

found to have considerable impact on the outcome of critically ill patients [1-3]. Oxidative stress defines an imbalance in the production of pro-oxidants and their effective removal. Pro-oxidants are reactive oxygen (ROS) and nitrogen species that cause injury to DNA, polyunsat- urated fatty acids, and proteins that finally lead to cell death [4]. Among ROS, the superoxide radical (O^-) has been found to be the key radical, because it functions as a messenger in

0735-6757/$ - see front matter (C) 2012 doi:10.1016/j.ajem.2010.12.031

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signaling pathways and as an effector of the oxidative stress attributable to many toxic ROS, such as H₂O₂, OH^-, and peroxynitrite [5,6].

Hemorrhagic shock (H/S), an entity commonly seen in the setting of emergency medicine, is one of the most common causes of death all over the world, the complications of which are related to oxidative stress. The development of adult respiratory distress syndrome (ARDS) and multiple- organ dysfunction syndrome (MODS) are considered severe and delayed complications of H/S, which result in significant morbidity and mortality [7]. During H/S, the sustained reduction in blood flow leads to diminished microcirculatory perfusion and regional hypoxia. oxygen supplementation upon resuscitation results in the formation of ROS and other radicals at the ischemic region, and the generated oxidative stress is proposed as a fundamental mechanism of organ damage in these conditions [8,9]. Therefore, it deems necessary to profoundly and thoroughly understand the physiologic and pathologic nature of free radicals to disclose a new phenomenon or mechanism from which future clinical tests or therapeutic interventions could benefit.

A number of studies have shown that H/S and ischemia/ reperfusion (I/R) are associated with oxidative stress in various organs [9-12]. However, in most of these studies, indirect methods of determination of the oxidative load have been used, such as the measurement of antioxidants and total antioxidant capacity, the detection of oxidized biologic markers, and measurements of DNA damage. On the other hand, to the best of our knowledge, the direct quantitative measurement of O^- has not been described so far in experimental H/S models. The aim of the present study was to measure O^- production in various organs of the rat after acute severe H/S and subsequent resuscitation by using a new quantitative assay [13]. The production of organic lipid hydroperoxides (LOOHs), a standard but indirect method of evaluation of oxidative load, was also used for reasons of comparison with the new assay.

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Materials and methods

Animals

The study was carried out on male Wistar rats (350-400 g). All animals were housed in stainless-steel cages, with a wood- chip bedding, and had free access to tap water and pellet chow. The animals were maintained on a 12-hour/12-hour light/dark cycle and a constant temperature at 22 +- 2?C. The study protocol was approved by the local ethics committee.

Experimental design

Sixteen animals were divided into 2 groups: sham (n = 8) and H/S (n = 8). Rats were fasted overnight before the

Fig. 1 Experimental model.

experiment. Anesthesia was induced with intraperitoneal injection of ketamine (60 mg/kg body weight [bw]) and midazolam (5 mg/kg bw) and was maintained by supple- mentary ketamine.

Sham rats were subjected to artery cannulation only. In the H/S group, the femoral artery was catheterized with a 26- gauge catheter (Abbott Laboratories, Abbott Park, Ill), which was connected to a TruWave disposable pressure transducer (Edwards Life Sciences LLC) for the measurement of mean arterial blood pressure (MAP), displayed on a monitor (DINAMAP PLUS, CRITIKON; GE Healthcare). After the catheterization, the cardiovascular parameters were allowed to stabilize for 10 minutes. Blood was then withdrawn until MAP was reduced to 30 to 40 mm Hg within 15 minutes. Thereafter, MAP was maintained at this level for 60 minutes, after which the shed blood was reinjected to the animals within 15 minutes (Fig. 1). Upon resuscitation, the catheter was removed, the artery was ligated, the incision was sutured, and the rats were placed back in their cages.

Tissue sampling

One hundred five minutes after resuscitation, the rats received 8.5 mg/kg bw dihydroethidine (DHE) subcutane- ously dissolved in 40% dimethyl sulfoxide to a final volume of 1 mL under light ether anesthesia. Three hours after resuscitation and under anesthesia with ketamine, the rats were subjected to midline laparotomy, and the left hepatic lobe, the left kidney, and part of the terminal ileum were

excised. The left hemidiaphragm was then opened, and the left lung was harvested. After tissue sampling, the rats were killed by exsanguination.

Reagents

Dihydroethidine, horseradish peroxidase, DNA type III (from salmon testes), bovine serum albumin (fraction V), butylated hydroxyanisole, xylenol orange, Coomassie Bril- liant Blue G250, Triton X-100, and Dowex 50X-8 (mesh 400) were obtained from Sigma (St Louis, Mo). Dimethyl sulfoxide, acetone, chloroform, acetonitrile, absolute meth- anol and ethanol, hydrogen peroxide, sodium cyanide, ammonium ferrous sulfate, sorbitol, and trifluoroAcetic acid were obtained from Merck (Darmstadt, Germany), whereas Hydrophobic Oasis HLB 1 cm³ (30 mg) extraction cartridges were from Waters Corp (Milford, Mass). All reagents and solvents used were of the highest purity.

Tissue treatment

Rat organ tissues were homogenized with a glass-glass Potter- Elvehjem homogenizer in 1:1 (for lung and intestine) or 3:1 (for kidney and liver) tissue wet wt/vol ice-cold phosphate buffer (50 mmol/L, pH 7.8), containing 10 mmol/L sodium cyanide.

Superoxide assay

The method is based on the reaction between O^- and DHE administered to the rats in vivo [13] and in excess so as to trap efficiently the O^- formed over experimental incubation periods up to 75 minutes. The administered proper DHE dose to meet this criterion was assessed by preliminary experiments, where control/tested tissue samples were incubated with various doses of DHE at several time intervals to establish the following criteria: (a) the minimum dose of DHE, above which the rate of formed 2-HO-ethidium remains constant (at the tested time intervals), and (b) the detection of unreacted DHE in the tested rat organs to ensure DHE excess during incubation.

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The assay is based on the reaction of O^- with DHE that results in the formation of the specific product 2-OH- ethidium, the formation rate of which is measured and converted to superoxide production rate. 2-OH-ethidium is estimated after being extracted from the tissue in alkaline acetone, isolated via cation and hydrophobic microcolumn chromatographies and quantified by the use of its fluores- cence properties and its reaction with hydrogen peroxide.

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Lipid peroxidation assay

Lipid hydroperoxides were determined by the FOX-1 assay. The assay is based on the reaction of Fe³⁺ (resulting from Fe²⁺ after its reaction with XOOH or XOO) with the

reagent dye xylenol orange and the formation of a chromogenic product absorbing at 560 nm [14].

Protein concentration assay

Protein in sample homogenates was determined by a modification of a CBB-based method [15].

Statistical analysis

All data were analyzed by using analysis of variance, and the results are expressed as the mean +- SD. Statistical significance was reached when P <= .05.

Results

The production of LOOHs in the gut of rats subjected to H/S showed a 100% increase compared with sham (P b

.001), whereas in the liver, the increase reached the 70% of the baseline value (P b .001). The production of LOOHs increased to a lesser extend in the lungs (31.3% compared with sham, P b .001), whereas no changes were observed in the kidneys (Fig. 2).

The rate of O- production after H/S, as measured by the new quantitative method, increased in the gut and the liver of the rats by 55.9% and 65.9%, respectively (P b

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.001), whereas a less excessive but still statistically significant increase (17.1%, P b .05) was observed in the lungs. Finally and in concordance with the results of the LOOHs assay, O^- production remained unchanged in the kidneys (Fig. 3).

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Fig. 2 Production of organic hydroperoxides in the gut, liver, lungs, and kidneys. Sham (n = 8): rats were subjected to the

surgical artery cannulation only. H/S (n = 8): rats were subjected to H/S. ??P b .001 compared with Sham group. Values are expressed as mean +- SD.

Fig. 3 Production of superoxide radical in the gut, liver, lungs, and kidneys. Sham (n = 8): rats were subjected to the surgical artery cannulation only. H/S (n = 8): rats were subjected to H/S. ?P b .05 compared with sham group. ??P b .001 compared with sham group. Values are expressed as mean +- SD.

Discussion

In the present study, we describe for the first time, in an experimental rat model of H/S, a method of quantitative direct measurement of O^- production in 4 vital organs. The DHE-based assay described measures O^- via an established 1:1 molar relationship of this radical with 2-OH-ethidium and is characterized by high specificity and proven sensitivity [13]. The importance of this report is obvious, given the fact that O^- formation is the first step in a sequence of reactions leading to the oxidation of biologic structures [4]. Consequently, this assay directly estimates the oxidative burden, in contrast to other indirect methods used in various previous studies [10,11]. To test the reliability of the new described assay, we also measured the production of LOOHs, which is a well-established method of oxidative load assessment.

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Acute severe H/S produced a substantial increase in the production of O^-, as well as in the production of LOOHs in the gut and the liver of the rat, whereas a less pronounced increase was found in the lungs. Regarding the kidneys, the oxidative load remained unaffected, as estimated by both methods. It is obvious that the almost identical results obtained by the 2 methods give more reliability to our new assay, which might be of value in conditions like H/S and sepsis in the Emergency care setting, where fast and reliable results are of critical importance.

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The results of the present study suggest that the gut and the liver are the organs primarily affected by H/S, in terms of oxidative stress. Significant increase of the oxidative load was also documented in the lungs. Likewise, a number of previous studies have shown that H/S results in acute gut, liver, and lung injury related to oxidative stress[11,12,16,17]. Specifically, it has been shown that H/S-induced intestinal

injury is mediated by oxidants derived from xanthine oxidase [18]. Furthermore, there is evidence that O^- is implicated in the pathogenesis of liver injury following I/R and H/S [11,12]. More precisely, an experimental study indicates that the oxidative stress-induced liver injury after H/S is mediated through the NADPH-derived O - [19], whereas in a model of liver I/R, blocking the O - production by xanthine oxidase with allopurinol prevents mitochondrial oxidative stress and lipid peroxidation [19]. Nevertheless, these observations indirectly show that O - production is responsible for the posthemorrhagic oxidative injury in the liver, whereas in the present study and for the first time, the direct quantification of O - was achieved. Finally, the lung injury due to H/S seems to be mediated by proInflammatory mediators of the mesenteric lymph, which have been shown to injure the Endothelial cells through oxidant- mediated pathways, such as lipid peroxidation [20-22].

Regarding the kidneys, I/R injury has been associated

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with increased lipid peroxidation and depletion of the antioxidant enzymatic pool [23,24]. However, in the present study, the oxidative state of the kidneys remained unaffected after H/S. This may be due to the duration of H/S performed, or even the time interval between resuscitation and tissue sampling, because these factors differ between the various study protocols.

Oxidative stress plays a major role in the outcome of critically ill patients, making biomarkers of oxidative stress a major area of research to predict outcome [3,6]. Moreover, it is obvious that there is a need for the development of new, more accurate, and preferably direct methods for the assessment of oxidative load, which in the near future could affect the daily practice in the ED.

In conclusion, in the present preliminary study, we presented a novel quantitative assay to detect the production of O - in 4 vital organs of rats subjected to H/S. Further studies are warranted so that the application of this method contributes to the better understanding of the O -induced tissue damage after H/S or other critical conditions in the ED. Moreover, this assay can open new routes for the use of O - as a diagnostic tool in clinical applications where blood and other biologic fluids are used [25]. For instance, the measurement of O - in critically ill patients with H/S could be used as a prognostic tool or a method to evaluate the various therapeutic interventions in the setting of emergency and critical care medicine.

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