To determine the involvement of oxidative stress in the pathogenesis of acute encephalopathy associated with human herpesvirus-6 (HHV-6) infection, we measured the levels of oxidative stress markers 8-hydroxy-2′-deoxyguanosine (8-OHdG) and hexanoyl-lysine adduct (HEL), tau protein, and cytokines in cerebrospinal fluid (CSF) obtained from patients with HHV-6-associated acute encephalopathy (HHV-6 encephalopathy)
Viral infection-associated acute encephalopathy/encephalitis is a serious complication with neurological sequelae. The main symptoms of the acute phase are impaired consciousness and convulsive status epilepticus with hyperpyrexia. Several subtypes of acute encephalopathy have been established based on clinical, radiologic, and laboratory findings. Acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) is a new subtype characterized by a prolonged febrile seizure (FS) on day 1, which usually lasts longer than 30 min, as the initial neurological symptom [
Oxidative stress originates from an imbalance between the production of reactive oxygen species (ROS) and, to a lesser extent, reactive nitrogen species (RNS), and the antioxidant capacities of cells and organs [
In the present study, we measured the levels of oxidative stress markers (8-hydroxy-2′-deoxyguanosine: 8-OHdG and hexanoyl-lysine adduct: HEL), tau protein, and cytokines in cerebrospinal fluid (CSF) obtained from patients with HHV-6-associated encephalopathy and complex FS associated with HHV-6 infection.
We analyzed CSF obtained in the acute phase of inpatients with HHV-6-associated encephalopathy (HHV-6 encephalopathy)
CSF samples were obtained from each patient at any point during the disease and immediately stored at −80°C until they were analyzed. The amount of DNA oxidative stress marker, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and the early stage lipid peroxidation marker, hexanoyl-lysine adduct (HEL), was examined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Japan Institute for the Aging, Shizuoka, Japan). Total tau protein was determined using sandwich ELISA (Invitrogen Corporation, Camarillo, CA). The levels of cytokines were evaluated by multiplex bead-based immunoassay (BioPlex 200 system) (Bio-Rad Laboratories, Inc., Hercules, CA). All assays were carried out according to the manufacturer’s protocols. The detection limit for each ELISA kit was 0.06 ng/mL (8-OHdG), 2.6 ng/mL (HEL), and 15 pg/mL (total tau protein).
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenging drug that is clinically used in Japan for treatment of acute ischemic stroke [
Data were analyzed by GraphPad Prism version 5.0. Differences in oxidative stress markers, tau protein, and cytokine levels among each group were analyzed by one-way analysis of variance (ANOVA) and Dunn’s multiple comparison test. Correlations between CSF-8OHdG and other biomarkers were evaluated using Spearman’s rank correlation coefficient. We used Fisher’s exact test to examine the relationship between increased levels of each biomarker and the presence or absence of neurological sequelae in HHV-6 encephalopathy. Comparisons of levels of CSF biomarkers before and after edaravone treatment were performed by paired
The characteristics of the patients included in the study are summarized in Table
Clinical characteristics of HHV-6 encephalopathy and febrile seizure patients.
HHV-6 encephalopathy | HHV-6 complex febrile seizures | Controls | |
---|---|---|---|
Number of patients | 16 | 10 | 16 |
Age (months) |
|
|
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Sex ratio (M : F) | 8 : 8 | 5 : 5 | 11 : 5 |
Sampling time (day of illness) | 1–8 | 1 | — |
MRI abnormality | 14/16 | ND | ND |
Outcome (without sequelae) | 5/16 | 10/10 | — |
HHV: human herpesvirus; No.: number; ND: not done; M: male; F: female; MRI: magnetic resonance imaging.
The CSF-8-OHdG levels in HHV-6 encephalopathy (
Descriptive statistics for the biomarkers examineda.
Biomarkers | Controls | HHV-6 complex FS | HHV-6 encephalopathy |
| |||
---|---|---|---|---|---|---|---|
Global | Controls versus HHV-6 FS | Controls versus |
HHV-6 FS versus HHV-6 encephalopathy | ||||
8-OHdG, ng/mL | 0.063 (0.01) | 0.116 (0.061) | 0.129 (0.07) | 0.0025 | <0.05 | <0.01 | ns |
HEL, nmol/L | 3.62 (1.08) | 5.24 (3.63) | 3.59 (1.87) | 0.1863 | ns | ns | ns |
Tau, pg/mL | 609.0 (342.0) | 654.7 (213.7) | 13,905.6 (14,201.1) | 0.0028 | ns | <0.05 | ns |
IL-6, pg/mL | 3.2 (3.0) | 5.8 (5.3) | 74.6 (116.9) | 0.0349 | ns | <0.01 | ns |
IL-10, pg/mL | 0.4 (0.3) | 0.6 (0.8) | 1.4 (2.1) | 0.1663 | ns | ns | ns |
TNF- |
0.1 (0.1) | 0.3 (0.5) | 3.4 (4.0) | 0.0036 | ns | <0.01 | <0.05 |
8-OHdG: 8-hydroxy-2′-deoxyguanosine; HEL: hexanoyl-lysine adduct; HHV-6: human herpesvirus-6; FS: febrile seizure; ns: not significant; IL: interleukin; TNF: tumor necrosis factor.
aValues are expressed as the mean (standard deviation).
Cerebrospinal fluid (CSF) levels of oxidative stress markers in HHV-6 encephalopathy, HHV-6 complex febrile seizures (FS), and controls. (a) 8-hydroxy-2′-deoxyguanosine: 8-OHdG, (b) hexanoyl-lysine adduct: HEL.
Total tau protein levels in HHV-6 encephalopathy patients
Cerebrospinal fluid (CSF) tau protein levels. The horizontal bar indicates the mean value of each group. CSF levels (mean ± SD) of tau protein in HHV-6 encephalopathy, HHV-6 complex febrile seizures (FS), and controls are
We next confirmed the elevation of CSF IL-6 and TNF-
Cerebrospinal fluid (CSF) cytokine levels. The horizontal bar indicates the mean value of each group. (a) Levels of CSF IL-6 in patients with HHV-6 encephalopathy, HHV-6 febrile seizures (FS), and controls are
We next examined correlations between CSF-8OHdG and other biomarkers in the HHV-6 encephalopathy group (Table
Correlation analysis of CSF biomarkers in HHV-6 encephalopathy.
Biomarker | 8-OHdG | HEL | Tau | IL-6 | IL-10 | TNF- | |
---|---|---|---|---|---|---|---|
8-OHdG | Spearman |
1.000 | −0.292 | −0.239 | −0.484 | −0.046 | −0.315 |
|
<0.0001 | 0.312 | 0.373 | 0.068 | 0.871 | 0.253 | |
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HEL | Spearman |
−0.292 | 1.000 | 0.277 | 0.476 | −0.286 | 0.497 |
|
0.312 | <0.0001 | 0.338 | 0.086 | 0.322 | 0.070 | |
|
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Tau | Spearman |
−0.239 | 0.277 | 1.000 | −0.036 | −0.224 | 0.091 |
|
0.373 | 0.338 | <0.0001 | 0.899 | 0.422 | 0.748 | |
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IL-6 | Spearman |
−0.484 | 0.476 | −0.036 | 1.000 | 0.226 | 0.783 |
|
0.068 | 0.086 | 0.899 | <0.0001 | 0.418 | 0.0006 | |
|
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Il-10 | Spearman |
−0.046 | −0.286 | −0.224 | 0.226 | 1.000 | 0.166 |
|
0.871 | 0.322 | 0.422 | 0.418 | <0.0001 | 0.555 | |
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TNF- |
Spearman |
−0.315 | 0.497 | 0.091 | 0.783 | 0.166 | 1.000 |
|
0.253 | 0.070 | 0.748 | 0.0006 | 0.555 | <0.0001 |
CSF: cerebrospinal fluid; HHV: human herpesvirus; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; HEL: hexanoyl-lysine adduct; IL: interleukin; TNF: tumor necrosis factor.
Finally, we compared the CSF levels of oxidative stress markers in six patients with HHV-6 infection (5 patients with encephalopathy and 1 patient with febrile seizures) before and after edaravone treatment. Clinical profile of patients with edaravone treatment is shown in Table
Clinical profile of patients receiving edaravone treatment.
Patient | Clinical diagnosis | Age/sex | Initiation and dosage of edaravone treatment | Other treatments | Outcomes |
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1 | HHV-6 encephalopathy | 14 m/M | Day 5 |
Mannitol, Dexamethasone, |
Intellectual disability |
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2 | HHV-6 encephalopathy |
12 m/F | Day 5 |
Mannitol, Dexamethasone, |
Without sequelae |
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3 | HHV-6 encephalopathy | 14 m/M | Day 4 |
Mannitol, Dexamethasone, |
Hemophagocytic syndrome |
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4 | HHV-6 encephalopathy |
20 m/F | Day 7 |
Mannitol, Dexamethasone, |
Lt hemiparesis |
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5 | HHV-6 encephalopathy |
12 m/M | Day 3 |
DZP, MDL, |
Moderate psychomotor retardation |
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6 | HHV-6 febrile seizures | 10 m/F | Day 1 |
Mannitol, Dexamethasone, |
Without sequelae |
m: months; M: male; F: female; DZP: diazepam; MDL: midazolam; PHT: phenytoin; Lt: left; HHV: human herpesvirus; AESD: acute encephalopathy with biphasic seizures and late reduced diffusion.
Changes in cerebrospinal fluid (CSF)-8-OHdG (a) and CSF-HEL (b) levels before and after edaravone treatment in HHV-6-associated acute encephalopathy and complex febrile seizure (FS) patients. CSF-8-OHdG levels are decreased after edaravone treatment (
In the present study, we demonstrated that CSF-8-OHdG levels in HHV-6 encephalopathy and HHV-6 complex FS patients were significantly higher than in controls, suggesting increased oxidative stress is induced by HHV-6 infection. Recent studies revealed that oxidative damage is an emerging general mechanism of nervous system injury caused by viral infection. For example, oxidative injury is a component of acute encephalitis caused by herpes simplex virus type 1 (HSV-1) [
In contrast with the increased levels of 8-OHdG, there was no significant increase of CSF-HEL levels in HHV-6 encephalopathy compared with HHV-6 complex FS and controls. We previously demonstrated that oxidative stress of DNA contributes to early neuronal damage, whereas lipid peroxidation is related to subsequent neurodegeneration in subacute sclerosing panencephalitis [
Most patients with HHV-6 encephalopathy in this study (81.3%) were AESD, which has a high incidence of neurological sequelae. We previously indicated that levels of CSF tau protein were elevated in patients with AESD [
Edaravone is a free radical scavenger that interacts biochemically with a wide range of free radicals [
There were several limitations in this study. First, the initiation of edaravone treatment was delayed in HHV-6 encephalopathy because it is difficult to distinguish HHV-6 encephalopathy from HHV-6 complex FS during the initial seizures. Early diagnosis of HHV-6 encephalopathy, especially AESD, will be required to overcome this problem. Second, a clinical trial of edaravone for the treatment of acute encephalopathy might be difficult ethically, as placebo control cannot be used because of the severe nature of this disease. We confirmed that CSF-8-OHdG levels decreased after edaravone treatment, although there were no significant differences of mean values of other biomarkers between before and after the treatment. These results suggest edaravone treatment was partially effective for HHV-6 encephalopathy. Although these findings are encouraging, the therapeutic implications of ROS and RNS scavengers are complex, owing to their potential to exert toxic as well as protective effects [
In summary, we found oxidative DNA damage is involved in acute encephalopathy/febrile seizures associated with HHV-6 infection and may be independent of inflammatory reactions and subsequent axonal damage.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (22591147) and the Ministry of Health, Labour and Welfare (H23-Nanji-Ippan-107), Japan.