Lipid Peroxidation and Antioxidant Consumption as Early Markers of Neurosurgery-Related Brain Injury in Children.

BACKGROUND AND AIMS: Lipid peroxidation represents a marker of secondary brain injury both in traumatic and in non-traumatic conditions-as in major neurosurgical procedures-eventually leading to brain edema amplification and further brain damage. Malondialdehyde (MDA), a lipid peroxidation marker, and ascorbate, a marker of antioxidant status, can represent early indicators of this process within the cerebrospinal fluid (CSF). We hypothesized that changes in cerebral lipid peroxidation can be measured ex vivo following neurosurgery in children. METHODS: Thirty-six children (M:F = 19/17, median age 32.9 months; IQR 17.6-74.6) undergoing neurosurgery for brain tumor removal were admitted to the pediatric intensive care unit (PICU) in the postoperative period with an indwelling intraventricular catheter for intracranial pressure monitoring and CSF drainage. Plasma and CSF samples were obtained for serial measurement of MDA, ascorbate, and cytokines. RESULTS: An early brain-limited increase in lipid peroxidation was measured, with a significant increase from baseline of MDA in CSF (p = 0.007) but not in plasma. In parallel, ascorbate in CSF decreased (p = 0.05). Systemic inflammatory response following brain surgery was evidenced by plasma IL-6/IL-8 increase (p 0.0022 and 0.0106, respectively). No correlation was found between oxidative response and tumor site or histology (according to World Health Organization grading). Similarly, lipid peroxidation was unrelated to the length of surgery (mean 321 ± 73 min), or intraoperative blood loss (mean 20.9 ± 16.8% of preoperative volemia, 44% given hemotransfusions). Median PICU stay was 3.5 days (IQL range 2-5.5 d.), and postoperative ventilation need was 24 h (IQL range 20-61.5 h). The elevation in postoperative MDA in CSF compared with preoperative values correlated significantly with postoperative ventilation need (P = 0.05, r2 0168), while no difference in PICU stay was recorded. CONCLUSIONS: Our results indicate that lipid peroxidation increases consistently following brain surgery, and it is accompanied by a decrease in antioxidant defences; intraventricular catheterization offers a unique chance of oxidative process monitoring. Further studies are needed to evaluate whether monitoring post-neurosurgical oxidative stress in CSF is of prognostic utility.

Association of Nutrients with Biomarkers of Alzheimer's Disease.

Prospective cohort studies, cross-sectional surveys, autopsy studies and intervention clinical trials that investigated the association between nutrients and Alzheimer's disease (AD) have been reviewed. To estimate the relationship between specific nutrient intake and the risk of AD, Cochrane Library, PubMed, EMBASE, and the Fisher Center for Alzheimer's Research Foundation were searched for this purpose. Most published observational studies found an inverse relationship between vitamins, n-3 fatty acids and AD. The majority of intervention studies support the beneficial effect of combined vitamins and n-3 fatty acids providing them in the early stages of the disease. Only vitamin E and Zn supplementation failed to show any significant difference on the study population. On the other hand, high dietary intake of saturated fat and brain metal accumulation were positively associated with the incidence of AD.

Distribution of vitamin C is tissue specific with early saturation of the brain and adrenal glands following differential oral dose regimens in guinea pigs.

Vitamin C (VitC) deficiency is surprisingly common in humans even in developed parts of the world. The micronutrient has several established functions in the brain; however, the consequences of its deficiency are not well characterised. To elucidate the effects of VitC deficiency on the brain, increased knowledge about the distribution of VitC to the brain and within different brain regions after varying dietary concentrations is needed. In the present study, guinea pigs (like humans lacking the ability to synthesise VitC) were randomly divided into six groups (n 10) that received different concentrations of VitC ranging from 100 to 1500 mg/kg feed for 8 weeks, after which VitC concentrations in biological fluids and tissues were measured using HPLC. The distribution of VitC was found to be dynamic and dependent on dietary availability. Brain saturation was region specific, occurred at low dietary doses, and the dose-concentration relationship could be approximated with a three-parameter Hill equation. The correlation between plasma and brain concentrations of VitC was moderate compared with other organs, and during non-scorbutic VitC deficiency, the brain was able to maintain concentrations from about one-quarter to half of sufficient levels depending on the region, whereas concentrations in other tissues decreased to one-sixth or less. The adrenal glands have similar characteristics to the brain. The observed distribution kinetics with a low dietary dose needed for saturation and exceptional retention ability suggest that the brain and adrenal glands are high priority tissues with regard to the distribution of VitC.

Prevention of symptomatic vasospasm by continuous cisternal irrigation with mock-CSF containing ascorbic acid and Mg(2+).

BACKGROUND: Symptomatic vasospasm (SVS) is still a major cause of poor outcome in cases undergoing early surgical intervention for ruptured intracranial aneurysm. Among the numbers of therapeutic trials to prevent and ameliorate neurological deterioration due to SVS, removal or quenching of oxy-hemoglobin (OxyHb) from subarachnoid colts and administration of Mg(2+) (Mg) have especially been expected to be effective. In this report the authors investigated the effect of continuous cisternal irrigation (CCI) with mock CSF containing ascorbic acid (ASA) and Mg, performed after early surgery for ruptured aneurysm. METHOD: Sixty-three cases which had received CCI were retrospectively compared with 40 control cases as to the incidence of SVS and outcome. FINDINGS: Incidence of SVS was significantly less frequent (P < 0.05) in the CCI group (11%) than in the control group (25%). Severe and definitive SVS requiring additional specific treatment occurred only in 3.2% of the CCI group, while 22.5% in the control (P < 0.01). Overall outcome at discharge was significantly better in the CCI group than in the control (P < 0.01). CONCLUSIONS: Postoperative CCI with ASA and Mg was definitively effective in preventing SVS and in lessening severity of SVS if it occurs.

Ascorbic acid and rates of cognitive decline in Alzheimer's disease.

The brain maintains high levels of ascorbic acid (AA) despite a concentration gradient favoring diffusion from brain to peripheral tissues. Dietary antioxidants, including AA, appear to modify the risk of Alzheimer's disease (AD). The objective of this study was to test the hypothesis that neurodegeneration in AD is modified by brain levels of AA. Thirty-two patients with mild to moderate AD participated in a biomarker study involving standardized clinical assessments over one year. Cerebrospinal fluid (CSF) and serum were collected at baseline for AA and albumin content. Cognitive measures were collected at baseline and one year. CSF and plasma AA failed to predict cognitive decline independently, however, CSF: plasma AA ratio did. After adding CSF Albumin Index (an established marker of blood-brain barrier integrity) to the regression models the effect of CSF: plasma AA ratio as a predictor of cognitive decline was weakened. CSF: plasma AA ratio predicts rate of decline in AD. This relationship may indicate that the CSF: plasma AA ratio is an index of AA availability to the brain or may be an artifact of a relationship between blood-brain barrier impairment and neurodegeneration.

Oxidative stress in cerebrospinal fluid of patients with aseptic and bacterial meningitis.

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.

[Serum and cerebrospinal fluid concentration of vitamins A, E and C in patients with tick-borne encephalitis]. / Stezenie witamin A, E oraz C w surowicy krwi i plynie mózgowo-rdzeniowym chorych z kleszczowym zapaleniem mózgu--doniesienie wstepne.

We included in our study 18 patients hospitalized because of tick borne encephalitis (tbc) at the Departament of Infectious Diseases and Neuroinfections of Medical University of Bialystok. In this group, concentration of vitamins A, E and C in serum and cerebrospinal fluid (CSF) was measured before and after treatment. The control group consisted of 11 patients with no inflammatory changes in CSF were observed. We did not observe significant differences in concentration of vitamins in serum and CSF before and after treatment comparing to controls. However, we showed significant increase in concentration of vitamin E before and after treatment in both serum and CSF in patients with tbc in comparison with control group.

Increased levels of ascorbic acid in the cerebrospinal fluid of cognitively intact elderly patients with major depression: a preliminary study.

Major depressive disorder (MDD) in the elderly is a risk factor for dementia, but the precise biological basis remains unknown, hampering the search for novel biomarkers and treatments. In this study, we performed metabolomics analysis of cerebrospinal fluid (CSF) from cognitively intact elderly patients (N = 28) with MDD and age- and gender-matched healthy controls (N = 18). The CSF levels of 177 substances were measured, while 288 substances were below the detection limit. Only ascorbic acid was significantly different, with higher levels in the MDD group at baseline. There were no correlations between CSF ascorbic acid levels and clinical variables in MDD patients at baseline. At the 3-year follow-up, there was no difference of CSF ascorbic acid levels between the two groups. There was a negative correlation between CSF ascorbic acid and CSF amyloid-ß42 levels in all subjects. However, there were no correlations between ascorbic acid and other biomarkers (e.g., amyloid-ß40, total and phosphorylated tau protein). This preliminary study suggests that abnormalities in the transport and/or release of ascorbic acid might play a role in the pathogenesis of late-life depression.

Total antioxidant/oxidant status in meningism and meningitis.

The objective of this study was to investigate the antioxidant/oxidant status of serum and cerebrospinal fluid in children with meningismus and acute bacterial meningitis. Twenty-three children (age range, 0.75 to 9 years) with fever and meningeal signs that required analysis of the cerebrospinal fluid, but no cytologic or biochemical evidence of meningitis in their serum and cerebrospinal fluid, constituted the meningismus group. Thirty-one children (age range, 0.5 to 10 years) with acute bacterial meningitis constituted the meningitis group. Twenty-nine healthy children (age range, 0.5 to 11 years) were recruited as control subjects. Antioxidant status (ascorbic acid, albumin, thiol, uric acid, total bilirubin, total antioxidant capacity, catalase and ceruloplasmin concentrations) and oxidant status (lipid hydroperoxide and total oxidant status) were measured. The serum antioxidant status was lower, and oxidant status levels higher in both meningitis and meningismus subjects than in the control children (P < 0.001). Cerebrospinal fluid oxidant status was lower in the meningitis group than in the meningismus group (P < 0.05). These results indicate that serum antioxidant status was lower, and serum oxidant status was higher in children in the meningismus and meningitis groups, whereas cerebrospinal fluid oxidant status was higher in the meningismus group than in the meningitis group.

Age-dependent change in Vitamin C status: a phenomenon of maturation rather than of ageing.

Several reports have shown that Vitamin C is depleted in animals with age. Based mainly on comparisons between young animals that have not yet reached maturity and old animals, it appears to be the general assumption that the change in Vitamin C status occurs at a late stage in life and that this phenomenon may either contribute to or result from the ageing process. In the present study, young (3 months old, n = 8) and old (36 months old, n = 8) female guinea pigs were followed for 6 months with monthly blood samplings and monitored for Vitamin C status as measured by plasma ascorbate and erythrocyte ascorbate recycling capacity after which the animals were euthanized. While remaining unchanged in the old animals, plasma Vitamin C status of the young animals significantly declined to that of the old animals within 3 months. During the following 3 months, the Vitamin C status of the young animals remained unchanged. Furthermore, post mortem Vitamin C analyses of the animals now aged 9 and 42 months, respectively, showed no effect of age on Vitamin C in plasma, liver, kidney, heart and brain between the groups while concentrations were significantly increased in cerebrospinal fluid and lung with age (p < 0.05). Moreover, a significantly elevated ascorbic acid oxidation ratio was observed in young compared to old animals (p < 0.05). The present data suggest that the decline in Vitamin C status with age occur early in life and is a phenomenon of maturation rather than of ageing. Data from other species and humans are discussed.

Biochemical analysis of the cerebrospinal fluid: evidence for catastrophic energy failure and oxidative damage preceding brain death in severe head injury: a case report.

OBJECTIVES: To compare biochemical and clinical parameters in a case of fatal severe traumatic brain injury (TBI) with secondary insult. DESIGN AND METHODS: A TBI patient was catheterized for intracranial pressure (ICP) monitoring and cerebrospinal fluid (CSF) analysis of ascorbate, malondialdehyde, oxypurines, and nucleosides. RESULTS: Oxidative brain damage preceded ATP catabolite increment in the CSF even with ICP below 20 mm Hg. Sustained oxidative stress caused irreversible energy state derangement followed by a refractory ICP rise. Massive oxypurine and nucleoside release was recorded 36 h before brain death. CONCLUSIONS: Molecular events, detected by biochemical CSF analysis and preceding modification of clinical parameters in severe TBI with secondary insult, are discussed.

Influence of repetitive transcranial magnetic stimulation on disease severity and oxidative stress markers in the cerebrospinal fluid of patients with spinocerebellar degeneration.

Ataxia severity, cerebellar hemispheric blood flow (CHBF), ascorbate free radical (AFR), superoxide dismutase protein, superoxide scavenging activity, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cerebrospinal fluid (CSF) were compared before and after an 8-week course of repetitive transcranial magnetic stimulation (rTMS) in 20 patients with spinocerebellar degenerations (SCD). SCD patients showed higher AFR, 8-OHdG, and superoxide scavenging activity than 19 controls. In SCD patients, AFR and ataxia severity declined, and CHBF increased after rTMS. As the SCD patients showed negative correlations between ataxia severity and CHBF or superoxide scavenging activity, the therapeutic mechanism of rTMS may involve decreased oxidative stress and increased CHBF.

Concentration gradients for HVA, 5-HIAA, ascorbic acid, and uric acid in cerebrospinal fluid.

Concentrations of HVA, 5-HIAA, ascorbic acid, and uric acid in the lumbar and cisternal cerebrospinal fluid (CSF) were measured in psychiatric and neurologically impaired patients. The concentration of HVA is 6.1 times and of 5-HIAA 2.7 times higher in cisternal than in lumbar samples, the cisternal level of uric acid is half that of the lumbar region, but no significant differences were found in ascorbic acid concentrations. Correlation between lumbar and cisternal metabolite concentrations is high for 5-HIAA and ascorbic acid, and is less for HVA and uric acid. In cisternal CSF there is a significant correlation between levels of HVA-5-HIAA, 5-HIAA-ascorbic acid, and 5-HIAA-uric acid. These correlations disappear in lumbar CSF. These findings indicate that extrapolations to cisternal neurotransmitter metabolite concentration from lumbar measures are unwarranted for HVA, but not for 5-HIAA.

The total peroxyl radical-trapping ability of plasma and cerebrospinal fluid in normal and preeclamptic parturients.

Plasma and cerebrospinal fluid total peroxyl radical-trapping antioxidative parameter (TRAP) and the main antioxidant components of TRAP (vitamin E, ascorbic acid, uric acid, protein sulfhydryl groups, and the unidentified antioxidant proportion) were analyzed in 11 preeclamptic parturients, 9 healthy parturients with an uncomplicated pregnancy, and 10 healthy nonpregnant women. In addition, the possible effects of ongoing labor were studied in 10 healthy parturients. The samples of plasma and cerebrospinal fluid (CSF) were collected at cesarean section (pregnant women) or minor surgical procedure (nonpregnant women). Normal pregnancy or ongoing labor induced no significant changes in total TRAP, as compared with nonpregnant women, but significant changes in the percentage contributions of individual antioxidants were noted in plasma and CSF. In preeclampsia, a significant increase in TRAP was noted in both plasma and CSF. This increase was mainly due to an increased proportion of uric acid and unidentified antioxidants in plasma samples, and an increased proportion of unidentified antioxidants in CSF. The concentration of CSF ascorbic acid was decreased in preeclampsia, and a negative correlation between CSF ascorbic acid and blood pressure was observed.

The effect of ascorbate and ubiquinone supplementation on plasma and CSF total antioxidant capacity.

Free radicals are thought to be involved in the onset of neuronal disturbances such as Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. It is also assumed that they play a role in cerebral injury caused by ischemia or trauma. Plasma and cerebrospinal fluid (CSF), Total (peroxyl) Radical-trapping Antioxidant Parameter (TRAP), and the known antioxidant components of TRAP, for instance, ascorbic acid, uric acid, protein sulfhydryl groups, tocopherol, and ubiquinol were analyzed and the remaining unidentified fragment was calculated in five healthy volunteers before and after 4 weeks of ascorbate and ubiquinone (Q-10) supplementation. In CSF, TRAP was significantly lower than in plasma. The major contributor to plasma's antioxidant capacity was uric acid (UA), whereas in CSF it was ascorbic acid (AA). In CSF, AA concentrations were four times higher than in plasma. Oral supplementation of AA (500 mg/d first 2 weeks, 1,000 mg/d following 2 weeks) and Q-10 (100 mg/d first 2 weeks, 300 mg/d following 2 weeks) induced a significant increase in plasma AA and Q-10. Surprisingly, in spite of the high lipophilicity of Q-10, its concentration did not change in CSF. The supplementation of AA increased its concentration in CSF by 28% (p < .05). However, the increase in AA did not result in an increase in CSF TRAP. This indicates that AA had lost one-third of its radical trapping capacity as compared to that in plasma. The facts that AA is the highest contributor to CSF TRAP and its effect on TRAP is concentration dependent could indicate that the peroxyl radical-trapping capacity of CSF is buffered by AA.

Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer's disease.

Because increased oxidation is an important feature of Alzheimer's disease (AD) and low concentrations of antioxidant vitamins C and E have been observed in cerebrospinal fluid (CSF) of AD patients, supplementation with these antioxidants might delay the development of AD. Major targets for oxidation in brain are lipids and lipoproteins. We studied whether supplementation with antioxidative vitamins E and C can increase their concentrations not only in plasma but also in CSF, and as a consequence decrease the susceptibility of lipoproteins to in vitro oxidation. Two groups, each consisting of 10 patients with AD, were for 1 month supplemented daily with either a combination of 400 IU vitamin E and 1000 mg vitamin C, or 400 IU vitamin E alone. We found that supplementation with vitamin E and C significantly increased the concentrations of both vitamins in plasma and CSF. Importantly, the abnormally low concentrations of vitamin C were returned to normal level following treatment. As a consequence, susceptibility of CSF and plasma lipoproteins to in vitro oxidation was significantly decreased. In contrast, the supplementation with vitamin E alone significantly increased its CSF and plasma concentrations, but was unable to decrease the lipoprotein oxidizability. These findings document a superiority of a combined vitamin E + C supplementation over a vitamin E supplementation alone in AD and provide a biochemical basis for its use.

Concentrations of the water-soluble vitamins thiamin, ascorbic acid, and folic acid in serum and cerebrospinal fluid of healthy individuals.

Thiamin, thiamin monophosphate, ascorbic acid, and folic acid were determined in serum and cerebrospinal fluid (CSF) in 31 outpatients who underwent a myelography because of back-pain. All subjects were otherwise healthy. The CSF concentration (mean +/- SD) was 8.6 +/- 3.9 nmol thiamin/L, 16.9 +/- 8.3 nmol thiamin monophosphate/L, 133 +/- 58.8 mumol ascorbic acid/L, and 44.9 +/- 13.2 nmol folic acid/L. The CSF-serum ratio was 2.1 +/- 0.8 for thiamin, 8.3 +/- 4.3 for thiamin monophosphate, 3.0 +/- 1.4 for ascorbic acid, and 3.3 +/- 0.8 for folic acid; the amount in CSF was significantly higher than in serum for each compound. These results support the existence of a saturated transport mechanism of water-soluble vitamins from serum into CSF for thiamin monophosphate, ascorbic acid, and folic acid. However, low CSF concentrations are correlated with low serum concentrations for the three vitamins. High serum concentrations should therefore be advocated to ensure high CSF concentrations.

Oxidative stress in bacterial meningitis in humans.

OBJECTIVE: To study reactive nitrogen species-mediated oxidative brain damage and antioxidant defenses in patients with acute bacterial meningitis. METHODS: Nitrotyrosine (a widely used marker for the formation of reactive nitrogen species, such as peroxynitrite) and the lipid peroxidation product 4-hydroxynonenal were detected by immunohistochemistry in brain specimens obtained at autopsy. CSF concentrations of nitrotyrosine were quantified by ELISA. CSF and serum concentrations of ascorbic acid, uric acid, and its oxidation product allantoin were determined by high-pressure liquid chromatography. RESULTS: Tyrosine nitration was strongly increased during meningitis. It was most evident in inflammatory cells and blood vessels in the subarachnoid space. The same cell types stained positive for the lipid peroxidation marker 4-hydroxynonenal, suggesting that reactive nitrogen species contribute to oxidative brain damage during meningitis. High CSF nitrotyrosine concentrations were associated with an unfavorable outcome according to the Glasgow Outcome Score. In the CSF, the increase of nitrotyrosine was accompanied by a depletion of the antioxidant ascorbic acid and an increased oxidation of the natural peroxynitrite scavenger uric acid to allantoin. CONCLUSION: These findings indicate that oxidative stress due to reactive nitrogen species and altered antioxidant defenses are involved in the pathophysiology of bacterial meningitis in humans.

A comparative study of ascorbic acid entry into aqueous and vitreous humors of the rat and guinea pig.

The transport rates of radiolabeled ascorbic acid and dehydroascorbic acid, as well as, labeled 3-O-methyl-D-glucose and L-glucose from a central plasma compartment into aqueous and vitreous humors and cerebrospinal fluid were studied in vivo. Normal, male albino Sprague-Dawley rats and English Short Haired guinea pigs were used to explore the mechanism of ascorbic acid entry into ocular humors in a species that can produce ascorbate (the rat) and one that cannot and, like humans, is dependent on dietary sources (the guinea pig). In vivo kinetic studies allowed for the calculation of entry rate constants, Ki (min-1), in double-labeled experiments using L-glucose as an internal passive control. Parallel TLC chromatographic studies were performed to monitor intraocular labeled molecules deriving from the plasma-introduced test molecule. In addition, resting levels of ascorbic acid and D-glucose were determined in order to obtain more reliable data than previously available. Resting levels of D-glucose revealed a consistent pattern of lower levels in aqueous and vitreous humors and CSF than found in plasma for both rat and guinea pig. However, ascorbate levels differed significantly, with the guinea pig demonstrating high ascorbate levels in the aforementioned humors: 58, 77 and 22, respectively, times the circulating plasma value of 0.2 +/- 0.2 mg/dl. In contrast, the rat, like the guinea pig, had low plasma ascorbate levels (3.3 +/- 0.8 mg/dl) compared to glucose (162 +/- 8 mg/dl), with even lower aqueous and vitreous values in a pattern similar to that of D-glucose. In vivo aqueous, vitreous and CSF transport results from the guinea pig indicate active transport mechanisms for ascorbic acid that prefer the ascorbate over the dehydroascorbate moiety and are probably different from the carrier-facilitated diffusion mechanisms for D-glucose, which do not move molecules against a concentration gradient. TLC studies, performed under nitrogen, revealed that only (14C)-ascorbic acid was present in aqueous or vitreous humors regardless of whether the radiolabeled pulse was of ascorbic or dehydroascorbic acid. The rat demonstrated little or no carrier involvement, with ascorbic acid crossing into ocular humors at rates very close to those of L-glucose, which is similar in size and is considered to cross the barriers studied via passive diffusion. Saturation studies with unlabeled glucose and glucose inhibitor drugs phloretin (10(-3) M) and phloridzin (10(-1)) had no apparent effect on ocular entry rates. Dehydroascorbic acid movement was also found to be passive.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of free radical scavengers in the spinal cord of rats with experimental autoimmune encephalomyelitis.

Antioxidants (ascorbic acid, glutathione, cysteine, alpha-tocopherol) and uric acid were measured using two high-pressure liquid chromatographic methods in 3 regions (cervical, thoracic, lumbar) of the spinal cord and in blood of Lewis rats during the attack and recovery of experimental autoimmune encephalomyelitis (EAE). Uric acid, which is thought to be a marker of free radical release, was greatly increased and glutathione correspondingly decreased in lumbar and thoracic regions. Cysteine and ascorbic acid were practically unchanged, whereas alpha-tocopherol was significantly increased during attack and recovery. Results, which could have therapeutic implications, generally support the hypothesis that free radicals are released during EAE.