Subjects
A total of 52 human cerebral cortex specimens were collected from the Department of Neurosurgery at Xinqiao Hospital (Army Medical University, Chongqing, China) and were divided into the FCDIIb (n = 20), TSC (n = 20) and control groups (n = 12). All procedures for this study were conducted according to the guidelines approved by the Army Military Medical University Ethics Committee. All specimens were collected and used in accordance with the Declaration of Helsinki. Prior to surgery, patient underwent a complete preoperative evaluation and signed an informed consent form for research using the removed brain tissue. All cases were diagnosed independently by 2 neuropathologists based on the International League Against Epilepsy classification. Control tissue was collected from the cadavers of 12 patients who had no history of epilepsy or any other neurological disease. According to our previous studies (Sun et al. 2016), all specimens were collected within 24 h of death, since most proteins are stable during this postmortem time (Hynd et al. 2003).
Tissue preparation
During surgery, epileptogenic foci samples were divided into representative tissue blocks. All specimens were immediately divided into two parts. One part was fixed in 10% buffered formalin for 72 h and then embedded in paraffin. Paraffin-embedded tissue was sectioned at a thickness of 5 µm and used for histopathologic diagnosis and pathologic classification, followed by immunohistochemical and immunofluorescence staining. The other part was immediately placed in a cryovial containing buffered diethylpyrocarbonate (1:1000) for 24 h and was then snap-frozen in liquid nitrogen. Frozen samples were stored at −80 °C until they were used for ELISA, quantitative real-time polymerase chain reaction (Real–time PCR) and western blot analysis.
Real–time PCR
According to the manufacturer’s instructions, total RNA was isolated from the FCDIIb, TSC, and control specimens by using a TRIzol reagent isolation kit (Invitrogen, La Jolla, CA). PCR primers were designed based on the complementary DNA sequence. The primers used in this study are shown in Supplementary materials 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control gene. Finally, the quantitative results were calculated by the 2−ΔΔCT method.
Western blots
Total protein was extracted from human specimens and cultured cortical neurons by using a total protein extraction kit (Beyotime, Jiangsu, China). Equal amounts of protein (20 µg) were used for immunoblotting and primary antibodies are as follows: FPR2 (rabbit polyclonal, 1:800, Abcam, UK), 5-LOX (rabbit polyclonal, 1:800, Abcam, UK), 15-LOX (rabbit polyclonal, 1:800, Abcam, UK), NF-κB (rabbit polyclonal, 1:500, Cell Signaling Technology, USA), and GAPDH (rabbit polyclonal, 1:1000; Cell Signaling Technology, USA). Specific protein bands on the membranes were visualized using enhanced chemiluminescence, and densitometry was performed with ImageJ software (National Institutes of Health, USA). GAPDH levels were evaluated as a loading control.
Immunohistochemistry and immunofluorescence analysis
Paraffin-embedded slices a thickness of 5 µm were used for immunohistochemistry and immunofluorescence. The sections were incubated with anti-FPR2 (1:500, Abcam, UK) primary antibody overnight at room temperature. Next, the sections were incubated with goat anti-rabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer (Envision + System-HRP; Boster, Wuhan, China) for 1 h at 37 °C. The sections were counterstained with hematoxylin, dehydrated, and coverslipped. For double-labeled immunofluorescence staining, sections were incubated at 4 °C overnight with following primary antibodies: anti-FPR2 (rabbit polyclonal, 1:500, Abcam, UK), anti-NeuN (mouse polyclonal, 1:500; Millipore, Billerica, USA), anti-GFAP (mouse polyclonal, 1:500; Sigma, USA), and anti-Iba1 (mouse polyclonal, 1:500; Abcam, UK). Next, they were incubated with the corresponding goat anti-mouse Cy3- and goat anti-rabbit 488-conjugated secondary antibodies (both at 1:500, Invitrogen, La Jolla, CA) for 2 h at 37 °C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China). The fluorescent sections were captured and photographed by a Zeiss Axio Vert microscope (Zeiss, Oberkochen, Germany).
Evaluation of immunostaining
The mean optical density (MOD) of FPR2 was determined according to a previous report (Wang et al. 2021). Briefly, the stained sections were assessed using a light microscope (BX51, Olympus, Japan). The photographs were analyzed by using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA), and the MOD was measured.
ELISA
Frozen cortical lesion samples (50 µg) were collected and cleaned in precooled PBS to remove the blood. The RvD1 ELISA kit was purchased from Cayman Chemicals (USA). After incubation overnight at 4 °C and development by Ellman's reagent and the optical density was measured at 420 nm with a plate reader (Bio–Rad). The concentrations of the samples were calculated on the basis of a standard curve.
In vitro epilepsy model
Male neonatal Sprague-Dawley (SD) rats (less than 24 h old) were obtained from the Laboratory Animal Center of the Army Medical University (Chongqing, China). All animal procedures were approved by the Animal Studies Committee of the Army Medical University and were conducted according to the Guidelines for the Care and Use of Laboratory Animals. Primary cortical neurons were isolated from 1-day-old SD rats. To determine the signaling pathways associated with RvD1-FPR2, the cells were divided into four groups: the control group was treated with PBS; the KA group was treated with kainic acid (KA) (20 μM/ml); the RvD1 + KA group was treated with 150 nmol/L RvD1 (Cayman Chemical Company, USA) 30 min before KA administration; and the WRW4 + KA group was treated with 10 µg/mL WRW4 (Cayman Chemical Company, USA) 30 min before KA administration. Eight hours later, the cells were collected for further analysis. After the treatments, the cultures were harvested with a cell scraper, and centrifuged at 12,000 rpm for 10 min. For protein extraction, a total protein extraction kit (Beyotime, Jiangsu, China) was used. For RNA extraction, the EZ-10 Total RNA Mini-Prep Kit (Sangon Biotech, Shanghai, China) was used.
Statistical analysis
Differences in seizure frequency and epilepsy duration were evaluated using t tests. Spearman's rank correlation test was used to perform bivariate correlation analyses. Overall statistical significance was carried out using one-way analysis of variance (ANOVA), followed by Tukey's test for between-group comparisons. A p-value < 0.05 indicated a statistical difference. The data are shown as the mean ± SEM, and analysis was performed using SPSS 13.0 (SPSS, Chicago, IL, USA).