Mol Cell Biochem DOI 10.1007/s11010-015-2425-z

Redox status in acute ischemic stroke: correlation with clinical outcome Dalibor Paspalj1 • Petar Nikic2 • Milan Savic2 • Dragan Djuric3 • Igor Simanic4 Vladimir Zivkovic5 • Nevena Jeremic6 • Ivan Srejovic5 • Vladimir Jakovljevic5



Received: 12 February 2015 / Accepted: 22 April 2015  Springer Science+Business Media New York 2015

Abstract Connection between oxidative stress and clinical outcome in acute ischemic stroke (AIS) has been poorly investigated. This study was aimed to assess redox state (through measurement of oxidative stress markers) of patients with acute ischemic stroke during different stages of follow-up period, and to find association between values of mentioned markers and clinical outcome. The investigation was conducted on 60 patients (both sexes, aged 75.90 ± 7.37 years) who were recruited in intensive care units at the Special Hospital for Cerebrovascular Diseases ‘‘Sveti Sava,’’ Belgrade. After verification of AIS, patients were followed up in four interval of time: (1) at admission, (2) within 24 h after AIS, (3) within 72 h after AIS, and (4) 7 days after AIS. At these points of time, blood samples were taken for determination of oxidative stress parameters [index of lipid

& Vladimir Jakovljevic [email protected] 1

Institute of Gerontology and Palliative Care, Belgrade, Republic of Serbia

2

Special Hospital for Prevention and Treatment of Cerebrovascular Diseases ‘‘Sveti Sava’’, Belgrade, Republic of Serbia

3

Institute of Medical Physiology ‘‘Richard Burian’’, School of Medicine, University of Belgrade, Belgrade, Republic of Serbia

4

Specialized Hospital for Rehabilitation and Orthopedic Prosthetics, Belgrade, Serbia

5

Department of Physiology, Faculty of Medical Sciences, University of Kragujevac, 69 Svetozara Markovica, 34000 Kragujevac, Republic of Serbia

6

Department of Pharmacy, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Republic of Serbia

peroxidation (measured as TBARS), nitric oxide (NO) in the form of nitrite (NO2 ), superoxide anion radical (O2 ), hydrogen peroxide (H2O2)], and enzymes of antioxidant defense system [superoxide dismutase (SOD) and catalase (CAT)] using spectrophotometer. Present study provides new insights into redox homeostasis during ischemic stroke which may be of interest in elucidation of molecular mechanisms involved in this life-threatening condition. Particular contribution of obtained results could be examination of connection between redox disruption and clinical outcome in these patients. In that sense, our finding have pointed out that O2 and NO can serve as the most relevant adjuvant biomarkers to monitor disease progression and evaluate therapies. Keywords Antioxidative system  Clinical outcome  Ischemic stroke  Oxidative stress

Introduction Acute ischemic stroke (AIS), the most common type of stroke, is defined as episode of neurological dysfunction caused by focal cerebral, spinal, or retinal infarction [1]. It is a consequence of different conditions in which blood flow is not sufficient for adequate oxygen and glucose supply [1]. Epidemiological data have shown that stroke is leading cause of adult disability and the second or third leading cause of death in the most of developed countries [2]. The main reason of reduced focal cerebral perfusion includes thrombotic and/or embolic events that result in ischemic brain injury [2]. Therefore, understanding of complex molecular mechanisms involved in ischemic insults is certainly the key point in creation of the most effective therapeutic approach to diminish brain damage.

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Although there are ample of both pre- and clinical stroke researches designed to investigate these issues, we are still far from elucidation underlying molecular mechanisms. The previous and the newest studies have proposed an intimate association between marked oxidative stress and development of neuronal death in cerebral ishemia [3, 4]. It is well known that oxidative stress represents an imbalance between oxidants-reactive oxygen species (ROS) and antioxidant defense system (ADS) in favor of oxidants, potentially leading to cell injury [5]. Several factors have been suggested to stimulate excessive ROS generation in ischemic brain tissues. Among them, the most common are impairment of mitochondrial function [6], activation of neuronal nitric oxide synthase (nNOS) [7], and cytotoxic activity of neutrophils [8]. The importance of linking oxidative stress and stroke lies not only in unquestionable role of redox status in the pathogenesis of cerebral ischemia but also in its contribution to clinical outcome [3, 9]. In last two decades, increasing number of studies has proposed that, besides intensive ROS accumulation, activity of antioxidative defense system can also be changed in the ischemic cerebral conditions. Both enzymatic (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) [10], and nonenzymatic antioxidants (retinol, ascorbic acid, tocopherol, carotenoids) [11] have been investigated in stroke patients. However, most of these studies are cross-sectional or have short follow-up periods after stroke. On the other hand, there are lack of researches trying to find connection between oxidative stress and course of disease, with controversial and inconsistent data. Therewith this study was aimed to assess redox state (through measurement of oxidative stress markers) of patients with acute ischemic stroke during different stages of acute phase of stroke, and to find connection between values of mentioned markers and clinical outcome.

Excluding criteria are reflected in the following: (1) signs of intracranial hemorrhage or hemorrhagic transformation of ischemic stroke, (2) ischemic stroke in the posterior circulation areas (under irrigation of vertebral, basilar, or posterior cerebral arteries), (3) seizure at the beginning of the disease, (4) previous usage of any form of antiplatelet and/or corticosteroid drugs, (5) systemic infections, (6) clinically unstable medical condition based on clinical presentation, medical history, and performed diagnostic procedures, such as acute myocardial infarction, pulmonary embolism, severe chronic renal insufficiency, and cancer. The study was approved by competent Ethical committee. Protocol After verification of AIS, patients were followed up: (1) at admission, (2) within 24 h (1 day) after AIS, (3) within 72 h (3 days) after AIS, and (4) 7 days after AIS. Blood samples were taken for determination of oxidative stress parameters (index of lipid peroxidation measured as TBARS), nitric oxide (NO) in the form of nitrite (NO2 ), superoxide anion radical (O2 ), hydrogen peroxide (H2O2)), and enzymes of antioxidant defense system (superoxide dismutase (SOD) and catalase (CAT)) using spectrophotometer. Biochemical Assays Blood samples (up to 3.5 ml) were taken from the antecubital veins into Vacutainer test tube containing sodium citrate anticoagulant. Blood was centrifuged to separate plasma and red blood cells (RBCs). Index of Lipid Peroxidation (Thiobarbituric Acid Reactive Substances, TBARS) determination

Materials and methods Study population The investigation was conducted on 60 patients (both sexes (50:50 %), aged 75.90 ± 7.37 years) who were recruited from intensive care units at the Special Hospital for Cerebrovascular Diseases ‘‘Sveti Sava,’’ Belgrade. Criteria for including in the study were following: (1) clinically manifested symptoms of AIS, (2) neuroimaging findings showing signs of acute ischemia in the area of a. cerebri media, consistent with clinical diagnosis, (3) functional deficits based on NIHSS (National Institutes of Health Stroke Scale) [12] score: 14–25, (4) absence of other neurological diseases, (5) information consent for inclusion in the study.

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The degree of lipid peroxidation in plasma was estimated by measuring of thiobarbituric acid reactive substances (TBARS) using 1 % TBA (thiobarbituric acid) in 0.05 NaOH, incubated with plasma at 100 C for 15 min, and read at 530 nm. Distilled water was used as a blank probe. TBA extract was obtained by combining 0.8 mL plasma and 0.4 mL TCA (trichloroacetic acid), then samples were put on ice for 10 min and centrifuged for 15 min at 6000 rpm. This method was described previously [13]. Nitrite (NO2 ) determination Nitric oxide (NO) decomposes rapidly to form stable metabolite nitrite/nitrate products. Nitrite (NO2 ) was determined as an index of nitric oxide production with Griess

Mol Cell Biochem

reagent [14]. 0.1 ml 3 N PCA (perchloric acid), 0.4 ml 20 mM EDTA (ethylenediaminetetraacetic acid), and 0.2 ml plasma were put on ice for 15 min, then centrifuged 15 min at 6000 rpm. After pouring off the supernatant, 220 ll K2CO3 was added. Nitrites were measured at 550 nm. Distilled water was used as a blank probe.

parameters that mostly change in AIS, univariate logistic regression was used. Parameters that were marked as significant in univariate logistic regression entered multivariate logistic regression. Multivariate logistic regression marked parameters that were under independent effect of AIS.

Superoxide Anion Radical (O2 ) determination

Results The level of superoxide anion radical (O2 ) was measured using NBT (nitro blue tetrazolium) reaction in TRIS-buffer combined with plasma samples and read at 530 nm [15]. Hydrogen peroxide (H2O2) determination The protocol for measurement of hydrogen peroxide (H2O2) is based on oxidation of phenol red in the presence of horseradish peroxidase [16]. Two hundred lL sample with 800 lL PRS (phenol red solution) and 10 lL POD (horse radish peroxidase) were combined (1:20). The level of H2O2 was measured at 610 nm. Determination of antioxidant enzymes: Catalase (CAT) and Superoxide Dismutase (SOD) Isolated RBCs were washed three times with 3 volumes of 0.9 mmol/l ice-cold NaCl and hemolysates containing about 50 g Hb/l (prepared according to McCord and Fridovich) [17] were used for the determination of catalase (CAT). CAT activity was determined according to Beutler [18]. Lysates were diluted in distilled water (1:7 v/v) and treated with chloroform-ethanol (0.6:1 v/v) in order to remove hemoglobin. Then 50 ll catalase buffer, 100 ll sample, and 1 ml 10 mM H2O2 were added. Measurement was done at 360 nm. Bidistilled water was used as a blank probe. Superoxide dismutase (SOD) activity was determined by the epinephrine method of Misra and Fridovich [19]. 100 ll lysate and 1 ml carbonate buffer were mixed, and then 100 ll epinephrine was added. Determination was done at 470 nm. Statistics The statistical analysis was performed with SPSS 10.0 for Windows. Results are expressed as the means ± standard deviation (median). Data on figures are presented as mean ? standard deviation. After checking data distribution, the appropriate parametric or nonparametric test was used. The differences between two groups were assessed using t-test or Mann–Whitney test, while the differences between more than three groups were assessed using oneway ANOVA or Kruskal–Wallis test. To define the

Table 1 represents summary data referring the differences in the levels of oxidative stress parameters between survivors and deceased patients during follow-up period (at the admission, 24 h (1 day) after, 72 h (3 days) after, and 7 days after AIS). At the end of the follow-up after AIS, 24 patients (41 %) had deceased, whereas 34 (58 %) patients were still alive. Primary cause of death was bronchopneumonia (4 patients), cardiovascular complications (6 patients), neurological deterioration (10 patients), while no information was available for 4 patients (Table 2). Deceased patients are divided into three subgroups: deceased within 3 days after AIS, deceased from 3–7 days after AIS, and deceased 7 days after AIS. TBARS values showed trend of increase during followup period in the group of patients who died 7 days after AIS, but without statistical significance (due to large standard deviations). In other subgroups of deceased, as well as within survived patients, there were no significant changes during whole range of follow-up period in the value of this marker. Also it was observed that no significant differences between any of deceased subgroups and survived patients in any of follow-up points. Levels of NO2 during follow-up period showed slight trend of increase in the groups of patients who died between third and seventh day and 7 days after AIS, but without statistical significance. When compared between the subgroups of diseased and survived patients with regards to points of follow-up, it was observed statistically significant difference at admission point between the patients who experienced a lethal outcome in less than 3 days and patients who died from third to seventh days after AIS (Mann–Whitney test: P = 0.005), between patients who died from third to seventh days and patients who died after more than 7 days after AIS (Mann–Whitney test: P = 0.008), and between patients who died from third to seventh days after AIS and survived patients (Mann– Whitney test P = 0.023). During follow-up period, values of O2 also showed slight trend of increase in the groups of patients who died between third and seventh day and 7 days after AIS, as well as decrease in survived patients, but this changes

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Mol Cell Biochem Table 1 Differences in the levels of oxidative stress parameters recorded at admission, 24 h (1 day) after, 72 h (3 day) after, and 7 days after AIS, among deceased and survivors (X ± SD) Outcome

At admission

1 day after AIS

3 days after AIS

7 days after AIS

P

TBARS (lmol/ml) Decease \3 days

3.61 ± 3.40

2.95 ± 2.89

/

/

0.655

Decease 3–7 days

2.50 ± 2.71

4.66 ± 7.65

0.81 ± 0.11

/

0.264

Decease [7 days

5.45 ± 5.51

7.48 ± 9.28

6.85 ± 3.70

20.71 ± 25.16

0.615

Survived

6.62 ± 5.69

8.37 ± 7.66

10.37 ± 9.82

9.71 ± 10.33

0.392

P

0.141

0.317

0.080

0.380

NO2 (nmol/ml) Decease \3 days

5.93 ± 2.95

3.52 ± 1.09

/

/

0.180

Decease 3–7 days

1.58 ± 1.01

5.42 ± 7.39

6.59 ± 10.12

/

0.441

Decease [7 days Survived

7.73 ± 1.07 6.00 ± 6.41

7.92 ± 6.77 6.68 ± 8.63

8.34 ± 3.90 10.76 ± 11.25

8.64 ± 1.12 6.84 ± 6.86

0.145 0.958

P

0.034

0.651

0.611

0.242

O2 (nmol/ml) Decease \3 days

1.83 ± 1.66

6.66 ± 4.55

/

/

0.180

Decease 3–7 days

3.38 ± 1.65

5.10 ± 2.85

6.48 ± 3.81

/

0.264

Decease [7 days

4.90 ± 5.18

8.53 ± 10.53

4.61 ± 1.69

11.53 ± 6.52

0.145 0.185

Survived

7.91 ± 7.61

5.46 ± 5.53

6.59 ± 5.44

4.61 ± 2.24

P

0.042

0.453

0.855

0.076

H2O2 (nmol/ml) Decease \3 days

4.49 ± 2.50

5.39 ± 0.38

/

/

0.180

Decease 3–7 days

4.18 ± 1.96

4.37 ± 2.70

4.59 ± 3.34

/

0.264

Decease [7 days

5.83 ± 6.26

5.73 ± 6.50

1.56 ± 1.33

3.59 ± 0.87

0.615

Survived

3.43 ± 3.44

3.57 ± 2.88

3.20 ± 2.92

2.79 ± 1.31

0.319

P

0.272

0.600

0.057

0.380

SOD (J/g Hb 9 103) Decease \3 days

1059.85 ± 677.38

4855.00 ± 5796.86

/

/

0.180

Decease 3–7 days

1505.98 ± 1405.64

4136.47 ± 6195.86

4159.54 ± 2020.24

/

0.050

Decease[7 days Survived

2215.70 ± 2350.26 2891.73 ± 2139.93

3756.15 ± 2267.23 4057.47 ± 3371.69

1824.98 ± 1442.27 2754.28 ± 2697.78

3488.39 ± 2744.97 3238.45 ± 3311.39

0.615 0.134

P

0.135

0.360

0.334

0.770

37.10 ± 41.95

76.50 ± 30.40

/

/

Decease 3–7 days

89.10 ± 41.50

94.58 ± 66.03

136.25 ± 58.08

/

0.717

Decease [7 days

83.80 ± 39.47

93.88 ± 50.85

96.10 ± 46.00

75.50 ± 5.30

0.896 0.159

CAT (J/g Hb 9 103) Decease \3 days

Survived

97.55 ± 51.55

103.41 ± 61.85

99.42 ± 64.03

92.17 ± 80.06

P

0.081

0.633

0.337

0.770

Table 2 Primary cause of death in deceased patients Cause of death

Number of patients

Bronchopneumonia

4

Cardiovascular complications

6

Neurological deterioration No information

10 4

remained insignificant. When compared between the subgroups of diseased and survived patients with regards to points of follow-up, the only significant difference was

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0.655

observed at admission point between the patients who experienced a lethal outcome in less than 3 days after AIS and survived patients (Mann–Whitney test: P = 0.009). Levels of H2O2 during follow-up period showed slight increase in the group of patients who died within 3 days after AIS, but without statistical significance. When compared between the subgroups of diseased and survived patients with regards to points of follow-up, it was not observed any statistically significance. On the other hand, during follow-up, levels of SOD showed trend of increase in the group of patients died

Mol Cell Biochem

within 3 days after AIS, and slight increase in the group died between 3 and 7 day, but without significance (due to large standard deviations). Similar to this, values of CAT showed trend of increase in the groups of patients died within 3 days after AIS and died between 3 and 7 day, while values decreased in group of patients died 7 days after AIS, but this changes remained insignificant (due to large standard deviations). When compared between the subgroups of diseased and survived patients with regards to points of follow-up, both SOD and CAT did not achieve significant changes.

Discussion Taken into consideration complexity of diagnosis, treatment and rehabilitation of patients with stroke, growing attention has been paid in identification of molecules that participate in neuronal death and ischemic brain injury. Phenomenon of ischemia–reperfusion which inevitably occurs during and after blockage of cerebral perfusion, leads to enlarged generation of free radicals, pointing out important role of oxidative stress development of neuronal damage [20]. This study was designed to evaluate the potential changes in dynamics of oxidative stress markers during course of disease and their connection with clinical outcome in patients with AIS. We decided to follow-up values of oxidative parameters in four periods of time, based on experience of the majority of relevant studies [21], in order to create most complete picture of redox state. It has been previously suggested that brain tissues are especially prone to the deleterious effects of free radicals [22]. The brain cellular membrane lipids are very rich in polyunsaturated fatty acid side chains, which are especially sensitive to free radical attack, making suitable foundation for lipid peroxidation. The degree of this process can be evaluated through measurement of index of lipid peroxidation, in this study expresses as TBARS. Recent studies have shown that concentrations of lipid peroxidation markers were higher in patients with AIS than those in control subjects [23]. However, most of these studies does not provide continuous monitoring of mentioned markers, which would be of interest to follow regarding their potential prognostic value. In our study, except group of patients who died 7 days after AIS, TBARS values were no significantly changed during whole range of follow-up period (Table 1). This could mean that TBARS may not be so sensitive marker for prediction of outcome, or monitoring of clinical course. On the other hand, L arginine/NO system has been proposed as another key-player in pathophysiology of cerebral ischemia–reperfusion injury [24]. NO is dominantly generated by endothelial nitric oxide synthase (eNOS) located in

the vascular endothelium and the choroid plexus [25]. In physiological conditions, NO is critical for the regulation of cerebrovascular hemodynamics and expresses anti-inflammatory, antioxidative, and anticoagulant activity [26]. In period of ischemia, NO concentration drop down due to oxygen deficiency [27], while immediately after reperfusion, biosynthesis of this molecule is triggered mainly by overactivation of neuronal nitric oxide synthase (nNOS) [27]. Literature data have showed that concentration of NO returns to physiological levels approximately 1 h after reperfusion [28] and increases again due to iNOS expression between 12 h and up to 8 days later [29], which is important from aspects of comparison with present study. In that sense, we observed slight trend of increase from first to last point of following (in the groups of patients who died between third and seventh day and 7 days after AIS), which correlates with mentioned findings [29]. However, it was interesting that values at admission were the highest in the group of patients who died seven days after AIS and survived patients, which indicate that in our study population NO may potentially have protective role and thus be associated with better outcome, or even surviving (Table 1). This result pointed out that NO may eventually have better prognostic significance compare to TBARS. The exact mechanism through which NO achieve its effect is still not clear. Until now it is assumed that type of nitric oxide synthesis (NOSs) may be responsible for action of NO. Unfortunately, one can not predict which form of NOSs can act predominantly, although all forms are present in brain tissue. Therefore, it is proposed that if nNOS predominates early neuronal injury could occur, while iNOS contributes to late neuronal injury, whereas eNOS activity might be protective [30]. To summarize, whether the effects of this molecule are beneficial or harmful depends on the cellular compartment in which NO is generated, on its concentration, on the environment’s redox state, and on the evolutive stage of ischemic brain injury [30]. Another very important pro-oxidative parameter which is unavoidable produced during ischemic injury is O2 . Animal investigations have suggested that hypoxic and ischemic conditions in brain create optimal surrounding for generation of O2 which represent one of most toxic ROS and consequently leads to cellular damages [31]. It is interesting that there are almost no reliable clinical investigations regarding potential role of this molecule in pathogenesis of ischemic stroke. In present study, we have shown that this marker may be very sensitive in terms of connection with outcome of disease. Namely, during follow-up period values of O2 showed slight trend of increase in the groups of patients who died (between third and seventh day and 7 days after AIS), while it was decreased in survived patients. Nevertheless, these changes remained

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insignificant. Moreover, values at admission were significantly lower in the group with the most negative outcome (lethal outcome in less than 3 days after AIS) compared with survived patients (Table 1). This means that although we could expect that in patients who first died O2 should be higher at beginning of ischemia, this was not a case in our study, and it started to significantly increase during follow-up period. Explanation for this result is difficult to find. We can assume that dominant generator of O2 , NADPH oxidase [31] becomes active not immediately after blockade of perfusion, nor hours and days later. Molecular interplay between ROSs and antioxidative enzymes is very complex and dynamic. Superoxide anion can be converted by SOD into H2O2, and the latter is then transformed into the toxic hydroxyl radical (•HO), through the Haber–Weiss reaction, converted to water by glutathione peroxidase (GPx) or diluted to water and oxygen through CAT [32]. Accumulation of H2O2 has been suggested to exert neurotoxic effects, although recent in vitro studies have demonstrated either physiological or protective roles of this molecule in the brain [32]. In our study, there were no significant changes during follow-up period except in group of patients who died within 3 days after AIS (slightly increased levels, but without statistical confirmation). On the other hand, values of this molecule were not statistically different between the subgroups of diseased or survived patients (Table 1). This result shows that H2O2 is not enough a sensitive marker in terms of outcome prediction. On contrary, Nanetti and his group have noted higher values of this molecule in the early stages of AIS with respect to their late evaluation [33]. However, they followed patients in just two intervals (at admission and after 1 month), and last interval was after quite longer period, leaving opportunity for CAT and GPx, for scavenging H2O2. Having in mind changes in production of oxidants during stroke [20], evaluation of antioxidative defense system seems to be logical part in assembling of redox puzzle in these patients. Pharmacological studies in animals showed that antioxidant molecules (SOD and CAT) are able to cross the blood–brain barrier, and thus reduce ischemic cerebral damage [34]. In addition, transgenic mice overexpressing SOD have reduced infarct size compared with wild-type mice [35]. Similar to previous cases, the number of relevant human investigations examining the role of antioxidative defense molecules in pathogenesis of stroke is very poor. Spranger and coworkers [36] have shown that levels of SOD are lower in stroke patients compare to control subjects. In the present study, although changes remained significant, in subgroups with most negative outcome (died within 3 days, and between 3 and 7 day after AIS), we have observed that both antioxidative

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markers (SOD and CAT) increased during follow-up period. Increased levels of this enzyme may be a consequence of antioxidative defense activation in response to raised production of O2 in the subgroups with worst outcome. Having in mind, absence of differences between the subgroups of diseased and survived patients, we can notice that antioxidative protection does not have important predictive significance in these patients. Our findings were in correlation with study mentioned before [21] where it was also noted increased antioxidative activity over time. Present study provides new insights into redox homeostasis during ischemic stroke which may be of interest in elucidation of molecular mechanisms involved in this lifethreatening condition. Particular contribution of obtained results could be the examination of connection between redox disruption and clinical outcome in these patients. In that sense, our findings have pointed out that O2 and NO can serve as the most relevant adjuvant biomarkers to monitor disease progression and evaluate therapies. Acknowledgments This work is supported by Grant no. 175043 from the Ministry of Science and Technical Development of the Republic of Serbia. Conflict of interest All authors of the present paper disclose no actual or potential conflicts of interest, including any financial, personal, or other relationships with people or organizations.

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Redox status in acute ischemic stroke: correlation with clinical outcome.

Connection between oxidative stress and clinical outcome in acute ischemic stroke (AIS) has been poorly investigated. This study was aimed to assess r...
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