49 patients were from the Department of Neurosurgery, West China Hospital. Pre-surgery evaluation of each case included video electronencephalography (VEEG) monitoring, brain magnetic resonance imaging (MRI), positron emission tomography(PET), and/or Depth electrodes. All surgical samples were sliced into small pieces and preserved in nitrogen and 4% formalin respectively.MTLE was diagnosed according to 2011 International League Against Epilepsy (ILAE) Classification. Lumbar puncture was done before surgery in all patients.
Non-epilepsy control samples were obtained from 19 patients who had serious brain trauma or a vascular event and underwent a neurosurgical procedure. All samples (controls and cases) comprised neocortical tissue and/or hippocampal tissue.CSF of controls was obtained from patients with chronic headache whose diagnosis ruled out CNS infection and tumor. The study was approved by the Ethics Committee of the West China Hospital of Sichuan University. Informed consents were obtained from patients or their direct relatives for the use of brain tissue and CSF. Control brain samples were only included if histological examination was normal.
Nested PCR and RT-qPCR for virus DNA in brain tissue
DNA was extracted from every sample using a DNA purification kit (Qiagen, Germany). DNA amplification was performed using nested primers specific for highly conserved sequences in viral genome. The total reaction volume of external amplification was comprised with 4 µL of 5×PCR buffer, 2ul of 20% Glycerol, 2ul of 10×dNTP（2mM）, 0.5ul of forward primer (10uM) , 0.5ul of reverse primer (10uM), 1 ul of DNA sample, 0.5ul of HS-Taq (5 U/μL), 9.5ul of ddH2O. DNA was amplified followed by 20 cycles of PCR under the following conditions: 94 °C for 5 min, 95 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s , and 72 °C for 2 min. Then a total of 2 μL of PCR products was amplified using theinternal primers according above process. A total of 10 µL of the final PCR product was subjected to 1.5% agarose gel electrophoresis to determine the molecular weight for distinguishing HHV-6B positive samples from negative samples.
Then viral DNA in HHV-6B-positive samples werequantified by TaqMan PCR with internal primer. The total reaction volume of internal amplification comprised 3 µL of 10× buffer (Mg2+free), 3 µL of MgCl2 (25 mM), 0.36 μL of dNTP (25 mM), 1 µL of forward primer (10 μM), 1 µL of reverse primer (10 μM), 0.6 μL of Taqman probe (10 μM), 0.3 µL of Taq enzyme (5 U/μL), 17.74 μL of ddH2O, and 3 µL of DNA sample. It was amplified followed by 45 cycles of PCR under the following conditions: 94 °C for 2 min, 94 °C for 15 s, 60 °C for 30 s.Vascular endothelial growth factor (VEGF) was used as a control marker (two copies of VEGF is considered equal 1 cell). The viral DNA was expressed as viral copies/1 × 106 cells after the cycle threshold (Ct) of the virus was adjusted. All primers were showed in Supplemental Table 1.
Immunohistochemistry for HHV-6B in brain tissue
Samples from the cases were investigated for HHV-6B infection using Immunohistochemistry(IHC) described in the previous study. Specimens were deparaffinized and dehydrated in graded alcohol series, washed with PBS, and incubated for 1 h with blocking buffer (3% FCS, 1% goat serum, 0.1%Triton X-100 in TBS), overnight with primary antibodies, and then incubated with secondary antibodies for 1 h at room temperature. The primary antibodies include HHV-6A/B specific Gp116/64/54 and P41 and HHV-6B specific U94 (providedby the HHV-6 Foundation, dilute concentration was 1:400, 1:500, 1:500, respectively). Anti-NeuN (Rabbit antibody, 1:500, Abcam), anti-glial fibrillary acidic protein (GFAP, Rabbit antibody, 1:500, Abcam),Positive control was SUP-T1 T-cell line containing HHV-6B viral genome (providedby the HHV-6 Foundation). Immunoreactivity was observed under a PM20 microscope (Olympus, Japan).
Real-time quantitative PCR for Inflammatory cytokines in brain tissue
RNA in patient samples was purified with RNA purification kit (QIAGEN) and spectroscopy confirmed that the RNA 260/280 ration was >1.8. RNA was then reverse transcribed using a Bio-Rad iScript Kit. The cDNA was quantified by qPCR using SYBR Green (Bio-Rad).Each reaction was run in triplicate and analyzed following the ΔΔCt method as previously described using actin as a normalization control.
Nested PCR for virus DNA in CSF
CSF samples were completely thawed and centrifuged at 3000r/min for 10min before testing. DNA extraction and amplification were performed according method described above.
Suspension bead array for cytokines in csf
CSF cytokine protein level was detected by using MILLIPLEX MAP (EMD Millipore Corp, Missouri, USA). Twelve cytokines including TGF-α, IL-10, IL-1RA, IL-9, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, TNF-α and VEGF were detected. The procedure was performed according to the manufacture’s protocol for Cytokine kit, assayed in triplicate, and read on a Molecular microplate reader at 450nm (Menlo Park, CA). Standard curves and individual well concentrations were determined using the Softmax 2.34 software (Molecular Devices, Menlo Park, CA). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). A five-parameter regression formula was used to calculate the sample concentrations from the standard curves. All 96 samples were analyzed with the LINCOplex kit (Linco Research Inc).
SPSS 19.0 software for Windows was used (Chicago, USA). The Independent-sample t-test was used to compare differences among continuous variables of MTLE patients and control individuals. The chi-square (χ2) test with Fisher’s exact was used to compare categorical parameters among groups. MLTE cases were categorized into subgroups according to IHC results for HHV-6B. Univariate analysis was used for measuring the association of HHV-6 viral phase and clinical manifestations. Two-tailed tests were used and a P < 0.05 was considered statistically significant