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Association Between Ischemic Stroke
and Increased Oxidative Stress

Antonio Cherubini MD, PhD; Cristina Polidori, MD;
Claudia Benedetti, MD; Sara Ercolani, MD;
Umberto Senin, MD; Patrizia Mecocci, MD, PhD

Institute of Gerontology and Geriatrics, Perugia University Medical School, Perugia, Italy

EPIDEMIOLOGY OF ISCHEMIC STROKE
   Stroke is a leading cause of morbidity and mortality, particularly in the elderly (AHA,2001). Its incidence and prevalence sharply increase with age: in the United States 72% of the subjects suffering a stroke are age 65 and older while up to 88% of stroke deaths occur in this population (AHA, 2001) (fig. 1). Moreover stroke is an important cause of long-term disability: up to 40% of survivors are not expected to recover independence with self-care and 25% become unable to walk independently. Ischemic stroke accounts for 70 to 80% of all strokes. Cerebral infarction may be due to primary thrombosis in an artery or to occlusion of the vessel by an embolus.

Fig. 1 : Estimated prevalence of stroke by age and sex
(United States 1988-1994)

PATHOGENESIS OF BRAIN DAMAGE IN ACUTE ISCHEMIC STROKE
   Independently from the mechanism responsible for the vessel occlusion, ischemia causes a cascade of events that eventually lead to neuronal damage and death (Fisher M., Schaebitz W.,2000) (fig 2). The reduction of blood flow decreases the production of high energy phosphates. The energy failure causes membrane depolarization and uncontrolled release of excitatory aminoacids, such as glutamate, in the extracellular space (excitotoxicity). Glutamate acts on various types of receptors, e.g. NMDA and AMPA, eventually causing calcium overload of neuronal cells. Calcium activates proteolytic enzymes, that begin to degrade both intracellular and extracellular structures, and other enzymes, i.e. phospholipase A2 and cyclooxigenase, which can produce free radicals. Neuronal nitric oxide synthase is also calcium dependent and produces nitric oxide, which is able to react with superoxide generating the highly reactive radical peroxynitrite (Lee J-M. et al.,1999). Secondary to ischemia proinflammatory genes are expressed and several inflammatory mediators are released, such as tumor necrosis factor, interleukin 1▀. Adhesion molecules are also expressed and therefore neutrophils, monocytes and macrophages start to bind the endothelium causing microvascular occlusion and cross the vascular wall penetrating in the brain. These inflammatory cells can also produce free radicals.

Fig. 2: Pathogenesis of brain damage in acute ischemic stroke. IP3 = inositol triphosphate; nNOS= neuronal nitric oxide synthase. (Fisher M., Schaebitz W., 2000)

   Although excitotoxicity typically leads to necrosis, the occurrence of apoptosis has also been reported to occur after cerebral ischemia (Linnik M.D. et al.,1993) and it has been proposed that both processes are triggered in parallel during ischemia and that the specific conditions determine which one will predominate (Lee J-M. et al.,1999).

FREE RADICALS AND OXIDATIVE STRESS
   Free radicals are atoms or molecules which have at least one unpaired electron in the outer orbital. In aerobic organisms free radical containing oxygen and/or nitrogen are continuously generated (Halliwell B., Gutteridge JM. 1999).

   Mitochondria are the main source of oxygen free radicals under normal conditions. Free radicals can react with any biological molecule (proteins, lipids, sugars, DNA) altering its structure and often also its function. Therefore living organisms are provided with a rich system of antioxidant defenses whose main purpose is to prevent the free radicals attack to other molecules.

   Oxidative stress has been defined as "an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage". Oxidative stress has been involved in aging as well as in the pathogenesis of several diseases (atherosclerosis, cancer, neurodegenerative diseases including Alzheimer's dementia, stroke).

IS ACUTE CEREBRAL ISCHEMIA ASSOCIATED WITH AN INCREASED GENERATION OF FREE RADICAL?
Evidence from animal studies
   Since free radicals are extremely reactive, they have a short half life. Therefore they are difficult to measure directly. Many studies have used an indirect approach to demonstrate free radical production during cerebral ischemia, measuring the products of free radical reaction with other molecules, such as DNA, lipids or proteins or the levels of antioxidant, according to the hypothesis that their changes are a consequence of the reaction with radicals.

   Lipid peroxidation products have been particularly studied since the brain is very rich in polyunsaturated fatty acids, which are highly susceptible to free radical attack. An increase of conjugated dienes has been demonstrated after reperfusion following 30 minutes of severe ischemia (Watson BD et al, 1984). Lipid peroxidation products are also increased in this condition ( Sakamoto A. et al.,1991).

   Markers of protein oxidation have been evaluated in other studies: the levels of 3 nitro- tyrosine are increased both in the ischemic core and in the surrounding penumbra area during ischemia with a further augment during reperfusion only in the latter ( Fukuyama N et al, 1998) (Fig. 3).

Fig. 3: Formation of 3-Nitrotyrosine, a marker of protein oxidation, in noninfarct (light blue), perinfarct (yellow), and core-of-infarct regions (red) of rat brain after 2-hour ischemia, 2-hour ischemia followed by 3-hours reperfusion, and 2-hour ischemia followed by 3-hour reperfusion in animal treated with NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor, at the dose of 50 mg/Kg).
*P<0,05 versus noninfarct; **P<0,05 versus periinfarct in 2-hour ischemia. (Fukujama N. et al., 1998)

   Recent researches have shown that also DNA is a target of oxidative attack during reperfusion following cerebral ischemia. It is currently believed that peroxynitrite, superoxide and particularly hydroxyl radical can produce different types of oxidative DNA damage (Liu PK et al., 1996; Cui J et al., 1999; 2000).

   It has been proposed that antioxidant changes reflect an altered redox balance in several pathological states (Halliwell B., Gutteridge JM. 1999). In other words antioxidants would be consumed in the reaction with free radicals. Therefore the measurement of antioxidant concentration or activity has been performed to estimate the amount of oxidative stress.

   Brain levels of vitamin C have been found to be reduced both due to ischemia (Flamm..1978; Kinuta Y et al., 1989; Ranjan A. et al., 1993) or ischemia followed by reperfusion (Oriot D. et al., 1995; Katz LM et al., 1998). Analogously alpha tocopherol and ubiquinon 9 content in the rat brain have been shown to decrease both during ischemia and reperfusion, together with a rise in their oxidized forms (Yoshida et al.,1982; Kinuta Y et al., 1989). However other studies in rats failed to confirm a reduction of ascorbate, glutathione or alpha-tocopherol during cerebral ischemia (Cooper A.J.L. et al., 1980, Rehncrona S et al. 1980; Yue T-L et al.,1993).

   Reports concerning endogenous SOD level or activity in cerebrovascular ischemia are not consistent: SOD activity and concentration in brain tissue after ischemia-reperfusion have been found either decreased (Chan P.H. et al.,1988; Tokuda Y et al.,1993) or increased (Sutherland G. et al.,1991; Matsuyama T. et al.,1993; Ohtsuki T. et al.,1993)

   The occurrence of a higher generation of free radicals has been confirmed using compounds that trap free radicals generating stable products detectable using electron spin spectroscopy or high performance liquid chromatography. However also these techniques have some methodological weaknesses. Transient occlusion of MCA induces an augmented production of hydroxyl radical which starts during the ischemic phase in the rat penumbra and further increases during reperfusion (Morimoto T. et al.,1996). The method used is based on the use of a trapping technique: salicylate reacts with radicals generating stable products (2-3, 2-5 DHBA). Experimental studies in animals, using mycrodialisis technique, have also shown that hydroxyl radicals are produced following ischemia followed by recirculation (Zini I. et al.,1992, Piantadosi C.A., Zhang J. ;1996). Also electron paramagnetic resonance provided evidence that free radicals are generated after ischemia (Kirsch J.R. et al.,1987).

   Consistent with these results are those of Peters O. et al., who directly showed an increase in superoxide in the penumbral region by means of in vivo chemiluminescence not only during the initial ischemic phase but also after recirculation (Peters O. et al.,1996) (Fig. 4).

Fig. 4: Lucigenin-enhanced Chemiluminescense for monitoring reactive oxygen species during control condition (open circle), permanent middle cerebral artery occlusion (MCAO) (solid square), and reperfusion for 2 hours after 1 hour MCAO (solid diamond). N=6 for each experimental group. (Peters O. et al., 1996)

IS ACUTE ISCHEMIC STROKE ASSOCIATED WITH AN INCREASED GENERATION OF FREE RADICAL?
Evidence from human studies
   While there are several studies in animal models of ischemic stroke showing that this disease is associated with an increased generation of free radicals, data in humans have been particularly scanty. Methodological problems in the measurement of free radicals have certainly contributed to the paucity of studies addressing this topic.

   There are studies that measured blood and urine levels of antioxidants and oxidatively modified molecules as possible markers of oxidative stress taking place in the brain in patients with ischemic stroke. Lower plasma levels of vitamin A, C , E and carotenoids have been reported in patients with stroke compared to healthy controls (Lin,1975; Hume R.,1982; Chang C-Y,1998; El Kossi MMH, Zakhary MM, 2000)

   Vitamin C decreased and thiobarbituric reactive substances (TBARS), an index of lipid perodixation, increased two days after the onset of cerebral ischemia with respect to the levels measured immediately after it (Sharpe P.C. et al.,1994). However not in all the studies the levels of antioxidants were reduced in stroke patients: for instance similar vitamin A levels were found in acute stroke patients and age-matched controls (Barer,1989; De Keyser J et al.,1992;Chang C-Y. et al.,1998). The antioxidant enzyme SOD concentration was unchanged after stroke in serum in one study (Adachi T. et al.,1994), increased in cerebrospinal fluid (Strand T. et al.,1992) and in both cerebrospinal fluid and plasma in other studies (Gruener N. et al.,1994). Red blood cells SOD activity has been found unchanged in stroke patients (Imre SG. Et al.,1994). More recently, the extracellular SOD activity was found to be lower in acute stroke patients but increased to the level of controls within 5 days from the cerebrovascular accident (Spranger M. et al.,1997).

   Malondialdehyde (MDA) and 4-hydroxynonenal (HNE), two breakdown products of lipid peroxidation, were increased in stroke patients with a cardioembolic source (Re,1997) and lipid peroxides were higher in subjects with ischemic stroke (Chang, 1998; El Kossi MMH, Zakhary MM, 2000). At variance, the urinary excretion of F2-isoprostane, a very specific marker of free radical damage to lipids, was not different in acute ischemic stroke patients compared to controls (van Kooten F. et al.,1997).

   The majority of these studies were cross-sectional or considered a maximum of two time points after stroke. We performed the first longitudinal study in which we measured plasma levels of cholesteryl ester hydroperoxides (CEOOH), a marker of free radical induced cholesterol ester oxidation, and of vitamin C, the most important water soluble antioxidant, in 32 patients with large cortical stroke and in 13 patients with lacunar infarcts (Polidori M.C. et al., 1998). All patients were included in the study within 24 hours from stroke onset and they were followed up for up to ten days. Blood samples were collected on admission and every other day afterwards. The results showed that plasma vitamin C concentration was reduced in patients with large ischemic stroke while CEOOH were significantly increased (Fig. 5). No CEOOH levels were detectable in young healthy controls. Since CEOOH are more specific markers of lipid peroxidation than TBARS or malondialdehyde (MDA), these data suggested that free radical production was increased in the brain during ischemia and that this process was detectable even in plasma. We therefore undertook another study to evaluate the usefulness of measuring CEOOH levels to predict prognosis and to monitor therapeutic interventions in ischemic stroke patients. Unexpectedly, we were unable to replicate our previous findings in this second cohort of patients with ischemic or hemorrhagic cerebrovascular disease. In particular, we could not detect any level of CEOOH in their plasma, even in those who were in the worst clinical conditions or died, as well as in that of controls. After several unsuccessful attempts, we decided to use a more sensitive method, that allowed us to measure CEOOH levels, which were much lower than in previous studies and were not different between patients and controls (Fig. 6). Moreover they did not show either a correlation with the severity of the disease or any consistent temporal pattern. We do not presently have an explanation for the conflicting results between this study and our previous one. The patients were recruited using the same criteria, in the same Intensive care unit and using the same tubes to collect the blood and the same method to measure CEOOH. They had similar risk factors to those in previous studies and we are not aware of any change in the management or in the pharmacological treatment occurred in the short interval between the studies which may account for this discrepancy. These conflicting results suggest that CEOOH are probably not a useful marker to monitor oxidative stress in ischemic stroke patients.

Fig. 5: Plasma cholesteryl ester hydroperoxides (CEOOH) concentrations (nM) in patients with cortical stroke as compared to patients with lacunar stroke. There was a statistically significant difference between the groups, p<0,00. (Polidori M.C. et al., 1998)


Fig. 6: Plasma levels of CEOOH in patients with different cerebrovascular diseases and healthy controls are not significantly different. (Cherubini A. et al., unpublished)

MECHANISMS OF FREE RADICAL GENERATION IN CEREBRAL ISCHEMIA
   The increased production of free radicals in the setting of cerebral ischemia, with or without reperfusion, can arise from several mechanisms: glutamate stimulation of NMDA receptors (Lafon-Cazal M. et al., 1993), mytochondrial dysfunction (Braughler J.M., Hall E.D., 1989; Piantadosi C.A., Zhang J.,1996) (fig. 7), activation of neuronal Nitric Oxide Synthase (Lipton S.A., Rosenberg P.A., 1994), induction of nitric oxide synthase (Iadecola C., 1996) or cyclooxigenase 2 (Iadecola C. et al.,1999), autooxidation of catecholamines (Braughler J.M., Hall E.D., 1989); metabolism of free fatty acids, particularly arachidonic acid, released during ischemia (Schmidley J.W., 1990; Traystman R.J. et al.,1991), migration of neutrophils and leukocytes able to generate superoxide anions (Matsuo Y. et al.,1995; Walder C.E: et al., 1997), the conversion of xanthine dehydrogenase to xanthine oxidase (Traystman R.J. et al.,1991; Phillis JW, 1994; Fisher 2000), although the occurrence of latter event is still controversial (Betz A.L. et al.,1991).

Fig. 7: Recovery of hydroxylated salicylate, a product of the reaction between hydroxyl radical and the spin trapping agent salicylic acid, from brain interstitium after transient cerebral ischemia in the rat. Hydroxylated salicylic acid (2,3-DHBA) in brain dialysate was quantified by high-performance liquid chromatography and electrochemical detection. In control animals (n=5), the recovery of 2,3-DHBA was stable or declined during the experiments. In rats with brain ischemia (n=5), 2,3-DHBA concentration increased progressively, but the treatment with the mitochondrial complex 1 inhibitor rotenone almost completely inhibited the 2,3-DHBA increase. (Piantadosi C.A., Zhang J., 1996)

   Both the cerebral parenchyma and the vascular endothelium have the potential to produce free radicals.
A considerable degree of attention has been devoted to the relationship between oxygen concentration and free radical generation. In the complete absence of oxygen it is clear that oxygen free radicals cannot be generated. However, even in the ischemic core, where permanent ischemia occurs, the cessation of blood flow is not achieved instantaneously but small amount of oxygen might remain for some time. Moreover there is an area surrounding the ischemic core, the so called penumbra, of reduced blood flow where there is still oxygen available. Moreover the blood flow can change over time, leading to reperfusion or to more severe ischemia. If blood flow is restored in both partial and complete ischemia the so called reperfusion takes place, that is followed by a dramatic increase of free radical generation (Braughler J.M., Hall E.D., 1989).

   Although many studies showed that the production of free radicals is particularly increased after transient ischemia (Nelson C.W. et al.,1992), i.e. ischemia followed by reperfusion, others demonstrated that free radicals are also produced in permanent ischemia, particularly in the ischemic penumbra (Peters O. et al.,1996).

OXIDATIVE STRESS IN ACUTE ISCHEMIC STROKE: DOES IT REALLY MATTER?
   When free radicals overcome the cellular antioxidant defenses oxidative stress takes place potentially leading to damage of all the principal cellular molecules. The brain is especially prone to free radical damage for several reasons. First it is very rich in polyunsaturated fatty acids, which are particularly vulnerable to free radical induced peroxidation. Then it has a low content of antioxidant enzymes, such as catalase and glutathione peroxidase, while it contains a significant amount of iron despite its iron binding capacity is not very high. Iron ions are known to stimulate free radical generation (Halliwell B., 1992).

   As previously discussed several lines of evidence support the occurrence of oxidative stress in acute ischemic stroke. However, the important question is if oxidative stress contributes to
the pathogenesis of brain damage in this setting or it is just a consequence of other, more relevant, biological processes.

OXIDATIVE STRESS IN ACUTE ISCHEMIC STROKE: DOES IT REALLY MATTER?
Evidence from pharmacological trials in animals
   If oxidative stress is responsible, at least in part, of ischemic cerebral damage, then antioxidant treatment should be able to reduce the consequences of ischemic stroke.

   Several studies have demonstrated a protective effect of both natural and synthetic antioxidant compounds in animal models of stroke.

   Dietary and supplemental natural antioxidants, such as vitamin C (Ranjan A et al.,1993; Henry PT, Chandy MJ, 1998) and vitamin E (van der Worp H.B. et al., 1998), reduce infarct size and neurological impairment after permanent ischemia or ischemia followed by reperfusion in rats and primates. Vitamin E prevents apoptosis due to transient ischemia in stroke-prone spontaneously hypertensive rats (Tagami M. et al., 1999) (Fig. 8). Conversely vitamin E depleted rats are more susceptible to lipid peroxidation (Yoshida S. et al.,1985).

Fig. 8: Vitamin E prevents apoptosis of hippocampal neurons in cerebral ischemia and oxygen reperfusion.
CI= cerebral ischemia; 6R= 6 days of reperfusion; 9R= 9 days of reperfusion; VE= vitamin E.
WKY= Winster Kyoto rats; SHRSP= spontaneously hypertensive stroke-prone rats.
(Tagami M. et al., 1999)

   Several studies have also demonstrated that ischemic brain injury is reduced by treatment with antioxidant enzymes that have been modified in order to become able to cross the blood brain barrier - such as polyethylene glycol-conjugated superoxide dismutase (SOD) and catalase (Liu T.H. et al.,1989) or liposome-entrapped SOD (Imazuimi S. et al., 1990).

   Several other drugs with antioxidant activity [(tirilazad mesylate, a 21-aminosteroid and a lipid peroxidation inhibitor (Hall E.D. et al., 1994; Mori E. et al., 1995)]; spin trapping agents, such as a phenyl N tert butyl nitrone (PBN) (Nakashima M. et al.,1999) and NXY-059 (Jonathan W.B.et al.,2001); Ebselen, a seleno-organic compound having a glutathione peroxidase like action (Dawson D.A. et al.,1995); salen-manganese compounds, that are synthetic SOD/catalase mimetics (Baker K. et al., 1998); BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation (Chabrier P-E. et al.,1999) (Fig. 9); ginko biloba and a lipoic acid (Clark W.M. et al.,2001) have been shown to be able to reduce infarct volume and, in some models, neurological impairment after brain ischemia. Some agents have been shown to be protective even when administered up to some hours after the onset of ischemia, e.g. NXY-059. The latter compound reduces functional disability, including motor and cognitive deficits, in monkeys following permanent occlusion of right middle cerebral artery (Jonathan W.B.et al.,2001).

Fig. 9 : Effect of BN 80933 (a dual inhibitor of neuronal nitric oxide syntase and lipid peroxidation) treatment on the infarct volume (up) and on the total neurological score (down) after 2 h of MCAO in rats. Vehicle or 0,3 mg/kg BN 80933 was administered i.v. at 4 and 24 h onset of ischemia. The infarct volume was assessed 7 days after MCAO. P< 0,005 for comparison between vehicle-treated rats (n=9) and BN 80933-treated rats (n=11). The neurologic score was evaluated on 15-point scale such that the higher the score, the greater was the neurological deficit. * p < 0,055; **p < 0,01 for comparison between vehicle-treated rats (n=13) and BN 80933-treated rats (n=12). (Chabier L. et al., 1999)

   However, negative trials have also been published (Takeshima R. et al.,1994; Clemens J.A., Panetta J.A.,1994).
Antioxidant drugs able to cross the blood brain barrier seem to have a higher efficacy in neuroprotection against cerebral ischemia than those purely acting on the vascular endothelium (Schmid Elsaesser R. et al.,1999).

EVIDENCE FROM STUDIES IN GENETICALLY ENGINEERED ANIMALS
   Genetically engineered mice represent a useful tool to evaluate molecular mechanisms of brain damage during both permanent and transient cerebral ischemia. The occurrence of oxidative stress in acute cerebral ischemia and its relevance to brain damage has been clearly shown in these animal models.

   The first studies showed that transgenic mice overexpressing the antioxidant enzyme SOD had reduced infarct size compared to the wild type when ischemia is followed by reperfusion (Kinouchi H et al.,1991; Yang G. et al, 1994;Sheng H. et al.,1999) (Fig. 10). Moreover the levels of antioxidants, such as ascorbic acid and glutathione, were higher in the penumbra area of the transgenic mice than in controls (Kinouchi H et al.,1991). Interestingly the protection was not present in the setting of permanent ischemia in one study (Chan P.H. et al.,1993). Conversely mice that are deficient of copper-zinc or manganese SOD, since the respective genes have been knocked-out, had an increased mortality (100% in those with no residual SOD activity), infarct size and brain edema, due to blood brain barrier breakdown, compared to controls (Kondo T. et al., 1997; Murakami K. et al.,1998 ). Mice lacking neuronal NOS gene were also more susceptible to ischemic damage (Huang Z. et al.,1994). Recently it has been shown in double mutant mice, i.e. lacking nNOS and over expressing SOD, that superoxide is probably more dangerous than NO since these animals were not more protected against ischemic injury than simple SOD transgenic mice (Sampei K. et al.,2000).

Fig. 10 : Cortical, subcortical and total cerebral infarct volumes in individual (open circle) wild-type and transgenic EC-SOD mice. Values from individual animals measured 24h after 90 min of MCAO are depicted. In all three regions, infarct size was significantly less in transgenic EC-SOD mice. Horizontal bars denote mean values for each group. (Sheng H. et al., 1999)

OXIDATIVE STRESS IN ACUTE ISCHEMIC STROKE: DOES IT REALLY MATTER?
Evidence from observational studies in stroke patients
   The relevance of oxidative stress in the pathogenesis of brain injury is indirectly supported by the results of some studies performed in stroke patients. Vitamin A but not vitamin E levels have been found to predict early prognosis after ischemic stroke (De Keyser J. et al.;1992). Moreover, low plasma antioxidant activity has been shown to inversely correlate with the volume of ischemic stroke and the degree of neurological impairment after an acute cerebrovascular event (Leinonen J.S. et al., 2000) (Fig. 11).

Fig. 11: The correlation between the volume of the cerebral infarction measured by MRI and plasma total peroxyl radical-trapping potential (TRAP) (r=-0,53; P=0,001). (Leinonen S.S. et al., 2000)

   We evaluated the longitudinal changes of plasma antioxidants after ischemic stroke to determine their utility in predicting the short-term outcome, in terms of survival and functional status. Patients over 65 years of age with acute ischemic stroke, admitted within 24 hours from the onset of symptoms to the Acute Geriatric Ward of the Perugia University Hospital, were consecutively enrolled. A CT scan of the brain was performed.

   Subjects with hemorrhagic stroke, other neurological diseases, or taking iron or antioxidant vitamins during the six months preceding the enrollment were excluded. Control subjects were age- and sex-matched healthy relatives of hospital employees (Cherubini A. et al.,2000).

   On admission, all patients underwent full physical and neurological examinations. Body temperature was measured on admission and daily until discharge. Vascular risk factors including hypertension, diabetes, and smoking habits were recorded. Caloric intake in the week before admission and during hospitalization was assessed by a food frequency questionnaire. The functional status of patients on admission based on Barthel Index (BI) score was compared to that preceding (two weeks) and following (one week) the admission. Proxy respondents were questioned when the patient was unable to provide all the requested information. Based on clinical and neuroradiological criteria, it was possible to distinguish patients as having lacunar or non-lacunar syndromes (including total anterior, partial anterior and posterior syndromes). The severity of the neurological deficit was measured by means of the Canadian Neurological Scale (CNS) administered on admission, and after three, five and seven days. Patients were divided into two groups according to the outcome after one week from the acute cerebrovascular event: those who died or experienced a decline in functional status measured on the basis of a decrease in the BI score (group W-worse), and those who did not undergo functional decline, as indicated by a stable BI score (group S-stable).

   In the patient group, an initial sample of blood was collected in a sodium heparin tube on admission (T1), then after 6 hours (T2), 24 hours (T3), three (T4), five (T5) and seven (T6) days. In the control group, blood was obtained in the morning after an overnight fast. Vitamin C and uric acid were detected by HPLC with electrochemical detection. Vitamin A and vitamin E were measured by HPLC with UV detection at 280 nm.

   The levels of the vitamins and of uric acid are expressed ad mmol/L. Since total-, HDL- and LDL-cholesterol and triglyceride levels were similar in stroke patients and controls, the concentration of vitamins A and E were not adjusted for lipids. Plasma and red blood cell SOD (U/ml) and plasma GPX (ÁM NADPH/min/ml) activities were measured using a spectrophotometer. Plasma concentrations of non enzymatic antioxidants and plasma and RBC activities of antioxidant enzymes over time in groups W and S were compared by two-way analysis of variance with Tukey used for post-hoc analyses. The study population consisted of 75 subjects: 38 patients with acute ischemic stroke admitted within 24 hours from stroke onset (mean time 8.0 ± 5.7 hours) (25 M, 13 F, 77.2 ± 7.9 y/o) and 37 controls (23 M, 14 F, 77.8 ± 8.1 y/o). According to the evaluation of stroke outcome, 25 patients were included in group W-worse (10 having a lacunar syndrome) and 13 in group S-stable (12 having a lacunar syndrome). Four patients died. On admission, mean antioxidant levels were lower in patients compared to controls. Significance was reached for vitamin C, vitamin A and uric acid concentrations and plasma SOD activity (p < 0.001) (Fig. 12). During the days following the acute event, all antioxidants tended to increase. After 1 week, antioxidant levels were similar in patients and controls, with the exception of vitamin C and plasma SOD activity, which remained lower, and of vitamin E and RBC-SOD activity, which became higher. Patients of group W had a higher degree of neurological impairment than group S patients on admission (CNS score 4.4 vs. 7.9, p < 0.001) and at every time point thereafter (data not shown). Age, sex and vascular risk factors did not differ between W and S groups. With respect to antioxidants, subjects in group W had significantly higher levels of vitamin A (Fig. 13) and of uric acid (Fig. 14) and a lower concentration of vitamin C (Fig. 14) with lower RBC SOD activity (Fig. 12).

Fig. 12: Red blood cell (RBC) superoxide dismutase (SOD) (circles) and plasma SOD (triangles) activities (mean ± S.E.M.) in patients who worsened (group W, closed symbols) or remained stable (group S, open symbols) in the first week after stroke. Plasma RBC-SOD activity was significantly lower (p < 0.01) in group W as compared to group S patients. (Cherubini A. et al. 2000)


Fig. 13: Vitamin E (circles) and vitamin A (triangles) plasma levels (mean ± S.E.M.) in patients who worsened (group W, closed symbols) or remained stable (group S, open symbols) in the first week after stroke. Plasma vitamin A levels were significantly higher (p < 0.001) in group W as compared to group S patients. (Cherubini A. et al., 2000)


Fig. 14: Uric acid (circles) and ascorbic acid (triangles) plasma levels (mean ± S.E.M.) in patients who worsened (group W, closed symbols) or remained stable (group S, open symbols) in the first week after stroke. Plasma vitamin C levels were significantly lower (p < 0.001) and plasma uric acid levels were significantly higher
(p = 0.001) in group W as compared to group S patients. (Cherubini A. et al., 2000)

   No differences were observed in vitamin E levels, plasma SOD and GPX activities. To our knowledge, this is the first study evaluating the time course of levels and activities of several enzymatic and non-enzymatic antioxidants in stroke patients during the first week following the occurrence of a cerebral infarct. Mean plasma levels of non enzymatic antioxidants and antioxidant enzyme activities were lower in patients as compared to healthy controls on admission. These levels gradually increased in the following days, although vitamin C and plasma SOD activity remained lower in patients as compared to controls while vitamin E and erythrocyte SOD activity became higher. Moreover, patients with the worst outcome (death or increasing disability after stroke) were characterized by a peculiar antioxidant profile, showing consistently lower RBC SOD activity and vitamin C levels and higher vitamin A and uric acid levels as compared to patients who survived the stroke without experiencing any functional decline. The relationship between antioxidant profile and early outcome of the cerebral infarct might provide new insights into the pathogenesis of ischemic stroke as well as open new therapeutic possibilities.

   In twenty-eight patients (19 men, 9 women, aged 76.9±8.7 years) of the original cohort we were also able to measure carotenoid levels, including lutein, zeaxanthin, b-cryptoxanthin, total lycopene, a- and b-carotene, by HPLC with UV/vis. detection and plasma MDA levels by HPLC with fluorescence detection. They were compared to 76 healthy, free-living, normolipidemic subjects (48 men, 28 women, aged 77.3±10.1 years). On admission, plasma levels of lutein, lycopene, a-carotene and b-carotene were significantly lower in patients as compared to controls. MDA plasma levels on admission were significantly higher (by almost 60%) in patients as compared to controls. All plasma carotenoid concentrations tended to decrease during the first 24 hours after admission, to rise afterwards up to the seventh day after stroke. When overtime changes of carotenoids and MDA were compared between groups W and S separately, significant differences were found for lutein and MDA, being plasma lutein levels significantly higher in S group as compared to W group patients (p < 0.01), and plasma MDA levels significantly lower in group S as compared to W group patients (p < 0.05). The main finding is that only lutein among carotenoids is significantly related to the outcome of stroke patients and to stroke severity. A significant inverse relation between fruit and vegetable intake and specifically lutein -but not other carotenoids- and risk for ischemic stroke was shown (Ascherio A. et al.,1999). Our results are in agreement with this observation, and suggest that low lutein plasma levels might influence not only the risk of stroke, but also its outcome. Along with the new observation of a relationship between plasma MDA levels and stroke outcome, these data are consistent with the occurrence of a condition of oxidative stress in patients with ischemic stroke. Due to the observational design of our study we cannot establish a causal relationship between carotenoids, and particularly lutein, and stroke incidence and outcome. however our findings supporting previous epidemiological evidence suggesting the need of experimental studies in the field of stroke and micronutrients contained in foods.

   Some limitations of these studies should be acknowledged. The lack of data regarding antioxidant levels before stroke onset hinders the possibility of ascertaining whether low antioxidants are a cause or a consequence of stroke. Several epidemiological studies have shown that low levels of vitamin C, vitamin E and carotenoids in the diet or in blood are associated with an increased risk of stroke. However, since almost all antioxidant levels were reduced immediately after the stroke and increased in the following days, it seems reasonable to assume that at least in part they declined after the cerebral infarct. We did not observe significant changes in dietary intake during hospitalization in patients, thereby excluding the possible influence of diet on differences in antioxidant levels between patients and controls. Moreover, after excluding from the analysis the patients who died, we obtained identical results.

   In conclusion our and previous studies suggest that antioxidant activity in plasma may be associated with a higher protection from neurological damage caused by stroke-associated oxidative stress. This might simply reflect the correlation between plasma and tissue antioxidant levels or it might imply that systemic stroke causes oxidative damage to biological molecules which extends outside the brain.

OXIDATIVE STRESS IN ACUTE ISCHEMIC STROKE: DOES IT REALLY MATTER?
Evidence from pharmacological trials in stroke patients
   In recent years a great deal of interest has been devoted to neuroprotective therapy, including antioxidant therapy, whose aims are to reduce the vulnerability of brain tissue to ischemia, to extend the therapeutic window for thrombolysis and to increase the efficacy of thrombolysis by reducing the reperfusion injury (Lee J-M. et al.,1999). Unfortunately, neuroprotective therapy has been shown to be effective in animal models of stroke but not conclusively so in humans (Fisher M., Schaebitz W.,2000).

   A randomized controlled trial with tirilazad mesylate in ischemic stroke patients was interrupted before the end due to the lack of efficacy of the drug (The RANTTAS investigators, 1996). Preliminary evidence from a pilot study shows that ebselen given within 24 hours from ischemic stroke onset can improve the functional outcome of patients. Ebselen is seleno-organic compound having a glutathione peroxidase-like antioxidant activity. Moreover it blocks the production of superoxide anions by activated leukocytes, inhibits inducible nitric oxide synthase and protects against peroxynitrite (Yamaguchi T. Et al., 1998).

   Several reasons could explain why the results of trials with antioxidant drugs have not been as good in humans as they were in animals. Based on available evidence different strategies have been suggested to improve the design of clinical trials with neuroprotective agent in stroke: among them a more careful selection of patients, who in previous studies were probably too heterogeneous in terms of pathogenesis, severity, and the demonstration of the presence of salvageable cerebral ischemic tissue (Muir KW, Grosset DG, 1999). Combination therapy with different antioxidant might represent another strategy in the future.

MECHANISMS OF OXIDATIVE STRESS INDUCED DAMAGE
   Once generated free radicals can react with all the cellular macromolecules leading to lipid peroxidation, DNA and protein oxidation (Halliwell B., 1992) (Fig. 15). Lipid peroxidation can lead to membrane damage. Moreover the end products of lipid peroxidation, such as 4-hydoxynonenal, are toxic to neurons (Bruce-Keller AJ et al.,1998) and white matter, including axons and oligodendrocytes, being able to induce apoptosis (Montine T.I. et al., 1996; McCraken E. et al.,2000). Damage to proteins, particularly when they are enzymes, can lead to impairment of their function. Finally DNA oxidation can cause the activation of repair enzymes, such as poly(ADP-ribose) polymerase (PARP), that provokes a rapid depletion of intracellular energy leading to cell death (Szabo C., Dawson V.L., 1998). This mechanism is also suggested by studies that demonstrated a reduction in brain infarct volume in animals treated with PARP inhibitors (Takahashi K. et al.,1999).

Fig 15: Mechanisms of cell damage by oxidative stress. (Halliwell B., 1992)

   Nitric oxide (NO) is also a free radical and it has been shown to have a double role during ischemia in relationship to the isoenzyme of NO synthase that is activated: NO produced by the constitutive endothelium nitric oxide synthase has a vasodilatative effect and hence it is neuroprotective, while NO from neuronal nitric oxide and inducible nitric oxide synthases contained in microglia and endothelium has been shown to be neurotoxic, at least in part by reacting with superoxide leading to the highly reactive peroxynitrite (Wei G. et al., 1999; Chan P.H.,2001).

   Both neuronal necrosis (Patel M.et al., 1996) and apoptosis can be induced by oxidative stress. In addition to directly react with cellular molecules there is increasing evidence that free radicals can act also by redox sensitive signal transduction pathways (Chan P.K., 2001) (Fig. 16). Experimental studies suggests that free radicals can induce cytochrome c release from mitochondria, which is an important step in the induction of apoptosis (Fujimura M. et al., 1999; Kim G.W. et al.,2000). Moreover the contribution of oxidative stress to apoptotic cell death caused by cerebral ischemia is demonstrated by the efficacy of antioxidant treatment in attenuating caspase-3 activation, DNA fragmentation and lesion size in mice with cortical infarction (Kim G.W. et al., 2000). Another possible link between oxidative stress and apoptosis is the reduction in the activity of Apurinic apirimidinic endonuclease (APE/Ref-1) ,a nuclear protein which removes the oxygen radical induced AP site in oxidized DNA and regulates transcriptional factors, such as Ap-1, which are redox sensitive (Bennet R.A.O. et al.,1997). Finally oxidative stress has been shown to activate several transcription factors, particularly NF-kB (Dalton T.P. et al.,1999). NF-kb is activated after both transient (Gabriel C. et al., 1999) and permanent ischemia (Seegers H. et al.,2000). NF-kb can then induce the transcription of pro-apoptotic genes (Barkett M, Gilmore TD,1999).

Fig. 16 Reactive oxygen species signal transduction pathways involving DNA repair and transcription factors.
COX2: cyclooxygenase-2; iNOS: inducible nitric oxide; MMPs: metalloproteinases; ICAM-1: intracellular adhesion molecules; BBB: blood-brain barrier. * DNA repair enzyme; ** transcripton factor. (Chan P.H., 2001)

   Not only brain parenchyma but also cerebral vessels can be injured by oxidative stress. Free radicals produce arteriolar dilation, decrease vascular reactivity, induce lesions of the endothelium and muscle cells, impair blood brain barrier permeability (Chan P.H. et al., 1984; Traystman R.J. et al.,1991; Nelson C.W. et al.,1992)

CONCLUSIONS
- Several studies demonstrate that ischemic stroke, with or without reperfusion, is associated with an increased production of free radicals in animal models while studies performed in stroke patients indirectly support the occurrence of an increased oxidative stress.

- Studies with genetically engineered mice and pharmacological trials with antioxidant agents in animal stroke models demonstrate that oxidative stress contributes to the pathophysiology of brain damage during ischemia

- Pharmacological trials have not convincingly demonstrated the efficacy of antioxidant treatment in patients with acute stroke although preliminary evidence supports the efficacy of ebselen, a selenium compound with antioxidant activity similar to glutathione peroxidase. Additional studies, with an improved design and a more careful selection of patients, are needed to evaluate the efficacy of antioxidant drugs in improving the outcome of ischemic stroke.

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