Design
A randomized, controlled, animal study.
Time and setting
Experiments were conducted in the Institute of Nautical Medicine, Nantong University, China from January 2009 to December 2010.
Materials
Animals
A total of 40 healthy adult male Sprague-Dawley rats, aged 6–8 weeks, weighing 220–250 g, were provided by the Laboratory Animal Center of Nantong University [license No. SCXK (Su) 2008-0010]. They were housed at 25 ± 2°C with 40–60% humidity in natural day/night cycle and allowed free access to food and water. Animal procedures were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[35].
Drugs
Carbenoxolone, with the chemical formule of C34H48O7Na2, purity ≥ 98%, was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Methods
Posttraumatic epilepsy model establishment
A posttraumatic epilepsy model was established by injection of FeCl3 as previously described[36] after some modifications. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (350 mg/kg) and placed in a stereotaxic instrument (Stoelting, Wood Dale, IL, USA). Following routine skin disinfection, a 3.0 cm scalp incision was made along the vertex median line. According to the Rat Brain in Stereotaxic Coordinates[37], a hole of 2.0 mm diameter was drilled at 2.0 mm posterior to coronal suture, 2.0 mm lateral to sagittal suture of the left cranium (Figure 4), and a micro-injection pump (WPI, Sarasota, FL, USA) was used to slowly inject 10 μL FeCl3 solution (0.1 M; Shanghai Jinshan Medicine Chemical Industry Factory, Shanghai, China) to a depth of 2.5 mm, 1 μL/min. The needle was maintained for 10 minutes to prevent FeCl3 leakage. The skin was closed, and rats were removed from the instrument after routine disinfection. The sham-surgery group was injected with 10 μL normal saline into the sensorimotor area of the left cortical frontal lobe. Rat behaviors were observed during and 120 minutes after injection, and 9:00 a.m. to 11:00 a.m. every day thereafter. Behaviors of rats were evaluated according to Racine scale of epileptic seizure[38]: grade 0, no behaviors of epileptic seizure; grade 1, face clonus, chewing, yawning; grade 2, nodding, face clonus, neck muscle convulsion in addition to behaviors of grade 1; grade 3, forelimb or hindlimb convulsion in addition to behaviors of grade 2; grade 4, hindlimb standing, sudden standing in addition to behaviors of grade 3; grade 5, rhythmic convulsion of four limbs, hindlimb stiffness and dorsal flexure or convulsion in addition to behaviors of grade 4. According to the Racine scale, rats at grade 4 or higher were selected as models.
Carbenoxolone pretreatment and treatment
The carbenoxolone pretreatment group was intraperitoneally injected with 20 mg/kg carbenoxolone 30 minutes prior to model establishment. The carbenoxolone treatment group was injected intraperitoneally with 20 mg/kg carbenoxolone 30 minutes following model establishment, once a day for 14 consecutive days[39].
Immunohistochemistry for cortical glial fibrillary acidic protein and connexin 43 expression
At 14 days after model establishment, the rats were anesthetized, followed by thoracotomy to expose the heart. The rats were perfused with normal saline and 4% paraformaldehyde phosphate buffer through the left ventricle. The rats were sacrificed and the brain was harvested, postfixed with 4% paraformaldehyde PBS for 24 hours and dehydrated with phosphate buffer containing 20% and 30% sucrose. The cross-section of 3.20–3.80 mm posterior to Bregma was harvested for serial sections on ice, 6 μm thick. Immunohistochemistry was conducted using the streptavidin peroxidase conjugated method[40]. Briefly, the brain sections were rinsed with 0.01 M PBS, placed in peroxidase blocking solution at room temperature for 10 minutes, washed and incubated with mouse anti-rat glial fibrillary acidic protein monoclonal antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-connexin 43 polyclonal antibody (1:100; Bioss, Beijing, China) at 4°C for 1 day. The antibodies were removed, and the sections were washed, incubated with secondary antibodies, ready-to-use goat anti-mouse/rabbit IgG (1:50; Maixin, Fuzhou, China) at room temperature for 15 minutes. The secondary antibody was removed, and sections were washed, visualized using diaminobenzidine (Maixin), stained with hematoxylin (Sinopharm Chemical Reagent Co., Shanghai, China) for 30 seconds, washed with tap water, dehydrated, mounted, and observed using a light microscope (Nikon, Tokyo, Japan). The negative control was treated with immunohistochemical staining primary antibody dilution buffer (Beyotime, Nantong, China) rather than primary antibody. Six serial sections from each rat were selected, and the number of glial fibrillary acidic protein- and connexin 43-positive cells was quantified under a 400 × magnification light microscope. The mean value was calculated as the number of positive cells in each rat.
Statistical analysis
Data were analyzed using Stata version 10.0 (Stata, College Station, TX, USA) and expressed as mean ± SEM. Comparison among groups was conducted using one-way analysis of variance, and intergroup comparison using q-test and paired comparison using two-sample t-test. A value of P < 0.05 was considered statistically significant.
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