Experimental animals
A total of 60 healthy, male Wistar rats (2–3 months, 150–200 g) were obtained from experimental animal center (Tongji Medical College, Huazhong University of Science and Technology) and acclimatized in the laboratory for 2 weeks prior to the experimental manipulation. Rats were reared in a cage kept at 25±3°C in a normal atmosphere and a 12 h light/dark cycle. Rats had free access to dry pellets and water with intermittent feeding of green fodder. Rats were randomly divided into three groups: Control, (n=20), PTSD (n=20) and intervention (PTSD+COX-2 inhibitor treatment, n=20). A PTSD model was established by single prolonged stress (SPS) as previously described by Liberzon et al (20,21). SPS was induced in three stages: Rats were restrained for 24 h, forced to swim for 20 min and administered ether anesthesia. All protocols involving animals were approved by the Ethics Committee for Experimental Animals (Wuhan No. 1 Hospital, Wuhan, China).
Rats in the control and PTSD groups were administered 0.9% normal saline and in the intervention group rats were treated with 25 mg/(kgd) COX-2 inhibitor celecoxib (Pfizer, Inc., New York, NY, USA) through a nasogastric gavage. Samples were collected after treatment for 2 weeks.
A pre-experiment was performed to select the optimum celecoxib concentration for treatment. A total of 9 rats from the PTSD group were divided into three groups and received celecoxib in dose of 15, 25 or 35 mg/(kgd) respectively via intraperitoneal route for one week, as previously described (22). COX-2 mRNA expression levels in all groups were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Behavioral detection
A total of four incandescent lamps (60 W) were placed in each of the four corners and served as an indoor light source for a behavioral laboratory, at a constant illumination of 150–300 Lux. In order to ensure clear observation of rat activity, the lights did not directly irradiate the detection equipment. The outdoor light was blocked by shading curtains. The open-field (OF), elevated plus maze (EPM) and Morris water maze (MWM) tests used in the present study are widely used procedures for examining the behavioral effects of anxiety (23,24). Horizontal movement distance and residence time are important indices of the open field test, while the open arm entry times and open arm residence times are indices for the EPM test. The results of the Morris water maze test were evaluated based on the escape latency. The open field test was performed as previously described by Choleris et al (25), the EPMtest was performed as previously described by Walf and Frye (26) and the Morris water maze test was performed as previously described by Morris (27). The primary method for data collection was a video-tracking system, which automatically detected and recorded the horizontal movement distance and residence time.
Immunohistochemical staining
Glass slides were pre-heated in an oven at 65°C for 1 h. Paraffin-embedded sections were dewaxed, rehydrated and treated with 3% hydrogen peroxide for quenching of endogenous peroxidase activity at room temperature. Sections were incubated with a rabbit anti-rat polyclonal COX-2 antibody (1:1,000; cat. no. BA0738, Boster Biological Technology, Pleasanton, CA, USA) overnight at 4°C. The spatial localization of COX-2 was visualized by incubation with mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; cat. no. PV-9000; ZSGB-Bio; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. A 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) chromogenic reagent was added dropwise until the nucleus had stained brown. After wards, the sections were rinsed with PBS, counterstained with hematoxylin for 3 min at room temperature, dehydrated with graded ethanol (75, 85, 95% and anhydrous ethanol) and xylene and mounted with Entellan (cat. no. 1.07960.0500; Merck KGaA). Sections were observed under a light microscope, six visual fields were selected and the mean number of positive cells was counted.
RNA extraction and RT-qPCR
Total RNA from rat hippocampi was extracted using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) and detected with an ultraviolet spectrophotometer and agarose-gel electrophoresis. For each sample, 1 µg RNA was reverse-transcribed to obtain first-strand cDNA using the PrimeScript® RT Reagent kit with a gDNA Eraser (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. Expression levels of COX-2were analyzed using RT-qPCR. The primers were designed and synthesized by TsingKe Biological Company (Wuhan, China). The primer specific to COX-2 was: Forward, 5′-TCGCTGTGCCTGATGATTG-3′ and reverse, 5′-TCGCTTATGATCTGTCTTG-3′; and a primer specific to the internal control β-actin was: Forward, 5′-TGACGTGGACATCCGCAAAG-3′ and reverse, 5′-CTGGAAGGTGGACAGCGAGG-3′. Each reaction mixture (20 µl total volume) contained 10 µl of 2× SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), 0.4 µmol/l each forward and reverse primer and 0.2±0.02 µg cDNA template. The thermocycling conditions were: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 58°C for 20 sec and 72°C for 20 sec. The relative transcription levels of genes were calculated using the 2−∆∆Cq method (28). The quantitation cycle (Cq) was determined for each reaction, and the Cq values for each gene of interest were normalized to the endogenous control gene β-actin. The quantification of target and reference genes was evaluated using standard curves and the ratio between the target and reference gene represented the relative expression levels of target gene. For each group three technical replicates of each measurement were obtained.
Western blot analysis
Western blot analysis was performed to determine the expression of COX-2. Rat hippocampi were homogenized and the total protein was extracted. The protein concentration was determined using a Bicinchoninic Acid kit (Bio-Swamp, Wuhan, China). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 5% skimmed milk in Tris-buffered saline (20 mmol/l Tris, 500 mmol/l NaCl and 0.05% Tween 20). Subsequently, the membrane was incubated with anti-COX-2 antibody (1:100; cat. no. ab52237; Abcam, Cambridge, UK) overnight at 4°C. An anti-β-actin antibody (1:10,000, cat. no. ab227387, Abcam) was selected as an internal reference. The membranes were washed with Tris-buffered saline and incubated with a rabbit anti-goat secondary antibody (1:10,000, cat. no. E030130, EarthOx, LLC, Millbrae, CA, USA) for 2 h at room temperature. Immunoreactivity was visualized by colorimetric reaction using enhanced chemiluminescence substrate buffer (EMD Millipore, Billerica, MA, USA). Membranes were scanned with a Gel DocEZ imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and bands were quantified using Quantity One software version 5.0 (Bio-Rad Laboratories, Inc.).
Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining
Paraffin-embedded sections of rat hippocampi were obtained. TUNEL assay was performed using a TUNEL kit (Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer's protocol. The sections were sealed with natural gum. Apoptotic cells were observed under a light microscope (magnification, ×400) and counted in 6 fields of vision. The apoptosis index was expressed as the number of apoptotic cells within 1 mm2, and the apoptosis of rat hippocampal neurons was defined as the number of apoptotic neurons/total number of neurons ×100%.
ELISA
The levels of IL-6, TNFα and PGE2 in rat hippocampi were evaluated by an ELISA assay. The supernatant of 10% rat hippocampal homogenate was extracted. IL-6 (cat. no. RA20607), TNF-α (cat. no. RA20035) and PGE2ELISA kits (cat. no. RA20013) were obtained from Bio-Swamp (Wuhan, China) and the assay was carried out according to the manufacturer's protocol.
NO detection
The NO content in rat hippocampi was detected using the Griess test (29). The Nitric Oxide Assay kit (cat. no. S0021, Beyotime Institute of Biotechnology, Shanghai, China) was used according to the manufacturer's protocol. The hippocampal tissue was treated with S3090 lysate (Beyotime Institute of Biotechnology) prior to detection.
Statistics analysis
All data are expressed as the mean ± standard deviation. Statistical differences were analyzed by one-way using SPSS (version 18.0, SPSS, Inc., Chicago, IL, USA). Duncan's test was used as a post-hoc test. P<0.05 was considered to indicate a statistically significant difference.