Design
A case-control study.
Time and setting
Experiments were performed at the Laboratory of Neurology, the First Affiliated Hospital, Sun Yat-sen University, China from April 2010 to July 2011.
Subjects
All of the cases were inpatients of the First Affiliated Hospital of Sun Yat-sen University and Guangdong 999 Brain Hospital. All procedures were conducted according to the Administrative Regulations on Medical Institutions, formulated by the State Council of China[28], and institutional approval was obtained from the hospital ethics committees. The patients were informed of the experimental protocol and risks, and informed consent was obtained from each patient.
Hippocampal sclerosis group
The subjects in this group had mesial temporal lobe epilepsy with hippocampal sclerosis. The inclusion criteria are as follows: (1) clinical diagnosis of mesial temporal lobe epilepsy according to the criteria proposed by the International League Against Epilepsy[29]; (2) the patients underwent resective surgery because of drug-resistance; and (3) the post-operative pathological examination confirmed sclerosis of the resected hippocampus.
Control group
The inclusion criteria for the controls are as follows: (1) The patient was diagnosed with symptomatic epilepsy with a focus near the hippocampus demonstrated by MRI and the focus was identified as the epileptogenic zone by depth electrode recording; (2) no aura before seizures was described in the clinical presentation; (3) the hippocampus could not be confirmed as normal by pre-operative MRI and was surgically removed with approval of the patient, but was identified as normal on pathological examination by two independent neuropathologists.
Methods
Specimen preparation
After excision, western blot analysis was performed on fresh hippocampal tissue that was frozen in liquid nitrogen. Immunohistochemistry was performed on fresh hippocampal tissue that was fixed with 4% paraformaldehyde.
Western blot analysis
Membrane proteins were extracted from each hippocampus according to the protocol supplied with the Membrane Protein Extraction Kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein concentrations were determined using the Micro BCATM Protein Assay Reagent (Thermo Scientific Pierce). For individual western blot experiments, 30 or 60 μg of protein per lane was electrophoretically separated and transferred to an immobilon polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 5% fresh non-fat milk for 60 minutes at room temperature and incubated with primary antibodies (Na+-K+-Cl– cotransporter 1, goat-anti-human, polyclonal IgG, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA; K+-Cl– cotransporter 2, goat-anti-human, polyclonal IgG, 1:100; Santa Cruz Biotechnology) overnight at 4°C, followed by washes in Tris-buffered saline with Tween. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (donkey-anti-goat IgG, 1:1 000; Santa Cruz Biotechnology) for 2 hours at room temperature. Immunodetection of proteins by chemiluminescence was followed by exposure to X-ray film. Rabbit-anti- human β-actin monoclonal antibody (1:2 000; Cell Signaling Technology, Boston, MA, USA) and horseradish peroxidase-conjugated secondary antibody (polyclonal IgG, donkey-anti-rabbit, 1:400; Thermo Scientific Pierce) were used to detect β-actin expression.
Immunohistochemistry
Frozen coronal sections (10 μm thickness) were cut on a cryostat and thaw-mounted onto polylysine-coated slides. They were thoroughly washed with 0.01 M PBS and then incubated in 1% H2O2 to block endogenous peroxidase activity. After being washed with PBS, the sections were incubated with a blocking solution (2% bovine serum albumin, 0.3% Triton X-100, and 5% normal pig serum in Tris-buffered saline) for 1 hour at room temperature and directly transferred into the primary antibody (Na+-K+-Cl– cotransporter 1, goat-anti-human, polyclonal IgG, 1:40, Santa Cruz Biotechnology; K+-Cl– cotransporter 2, goat-anti-human, polyclonal IgG, 1:50, Santa Cruz Biotechnology) overnight at 4°C. After being rinsed with PBS, the sections were placed in peroxidase-labeled secondary antibody (donkey anti-goat IgG, 1:50; Bioworld, Minneapolis, MN, USA) for 1 hour at room temperature. They were then washed with PBS again and incubated in 3,3’-diaminobenzidine (DAKO, Glostrup, Denmark) for 3 minutes[30]. Finally, sections were washed, treated with dehydrated alcohol and dimethyl benzene, and cover-slipped for analysis.
Image analysis and quantification
Images were captured under a biological microscope (BX51, Olympus, Tokyo, Japan) and analyzed using image analysis software (NIH Image J 1.42, Bethesda, MD, USA). For western blot analysis, densitometry analysis for the quantification of bands was performed as described on the NIH Image J analysis website (http://rsb.info.nih.gov/ij/)[31]. Band intensities were measured in duplicate for each homogenate. Reactive optical densities of specific reactive bands corrected for background were averaged per sample and the means were used for statistical analysis.
For immunohistochemistry, the absorbance of immunostained cells was chosen as the concentration of Na+-K+-Cl– cotransporter 1 or K+-Cl– cotransporter 2. The analysis was performed as described previously[32]. Six sections per sample and six cells per region were analyzed. Cells were chosen randomly for analysis from each region of each section. The mean absorbance value was obtained by averaging absorbance values of analyzed cells in each region. The background absorbance of each section was measured and subtracted from the mean absorbance. A single mean absorbance value was then obtained for each region of each section. As the analysis was carried out on six sections per sample, six mean absorbance values were obtained for each region in each sample. Last, the six mean absorbance values were averaged per sample and the means were used for statistical analysis.
Statistical analysis
All numerical values were expressed as mean ± SD. All statistical analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). The data in this study were normally distributed. Comparisons of two groups were carried out using independent sample t-tests. P < 0.05 was considered statistically significant.
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