Design
A randomized controlled animal study.
Time and setting
Experiments were performed at the Institute of Neurosurgery, General Hospital of Shenyang Military Region, China from October 2011 to July 2012.
Materials
Healthy male Wistar rats (n = 112), weighing 240–330 g, were provided by the Animal Experimental Center, General Hospital of Shenyang Military Region (Experimental Center license No. SYXK (Jun) 2002-019) (Animal license No. SCXK (Jun) 2002-0174). The rats were allowed free access to food and water and were maintained at 24–26°C. The protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[37].
Methods
Establishment and 8-OH-DPAT intervention of the diffuse axonal injury rat model
With the exception of the normal group, rat models of diffuse axonal injury were established using the Marmarou method[18]. Rats were intraperitoneally anesthetized with 10% (w/v) chloral hydrate. The rats were fixed on a sponge bed in the prone position and hit with a free falling 450 g iron rod from a height of 1.3 m through an empty steel pipe. The iron rod hit the rat median calvaria (between the coronal suture and lambdoid suture) to induce diffuse axonal injury. The normal group did not receive any hit. The probe head of a thermometer (Taimeng Technology Co., Ltd., Chengdu, Sichuan Province, China) connected to an intellectual thermostatic controller (Taimeng Technology Co., Ltd.) was inserted into the rat anal tube to maintain body temperature at 37.0 ± 0.5°C (constant temperature was maintained using a blanket). At 15 minutes following diffuse axonal injury, 8-OH-DPAT was intraperitoneally administered (0.5 mg/kg) (5 mg/tablet; Sigma, St. Louis, MO, USA) in the constant temperature and 8-OH-DPAT. An equal volume of physiological saline was intraperitoneally injected in the model and normal groups. Excepting the constant temperature group, the blanket was removed in all other groups. Body temperature (37.0 ± 0.5°C) was maintained using a constant temperature blanket (Chengdu Kangyu Medical Equipment Engineering Co., Ltd., Chengdu, Sichuan Province, China) for 168 hours.
Determination of rat brain temperature
The head of the rat was fixed using a stereo-directional fixation system, which was supplied by the Laboratory of Neurosurgery, General Hospital of Shenyang Military Region, China. Approximately 1.5 cm skin from the median head was incised and cooled using physiological saline at room temperature under a light microscope (Nikon, Tokyo, Japan). Simultaneously, the bilateral frontal bone-parietal bone was drilled using a dental high speed drill, with a bone window of about 5 mm × 5 mm. The probe of the electrode (Testo 735 thermodetector; Testo, Shanghai, China) was placed 3 mm below the right frontal cortex of rats to monitor brain temperature.
Specimen collection
At 6, 12, 24, 72 and 168 hours following diffuse axonal injury, seven rats were collected from each group. Rats from the normal group were sacrificed at 24 hours following model induction. After measuring brain temperature, the rats were subjected to anesthesia overdose. Injured brain tissues were obtained at 24 hours following model establishment in the normal group. The heart was exposed by opening the chest. Intubation was performed in the left heart apex, and the right auricle was cut. The specimen was washed using 100 mL physiological saline, and perfused using 4% (w/v) paraformaldehyde buffer at 4°C, pH 7.4, for 0.5 hours. Subsequently, approximately 3 mm3 brain tissues at the edge of the wound area of the left frontal lobe was collected, fixed in 4% (w/v) paraformaldehyde buffer, and embedded in paraffin.
Hematoxylin-eosin staining for pathological changes in injured brain tissues following diffuse axonal injury
Paraffin-embedded specimens were sliced into 2 μm sections, and unfolded in warm water and hot water. The slide was baked in an oven (Yaguang Medical Electrical Technique Institute, Xiaogan, Hubei Province, China) for 24 hours. The specimen was rapidly dewaxed in xylene (I, II), immersed in dehydrated alcohol and gradient alcohol (95%, 90%, 80%), stained with hematoxylin and 2% (w/v) eosin for 2–3 minutes, washed with running water, 95% (v/v) and 80%(v/v) alcohol, and mounted in neutral gum.
Immunohistochemical staining for Bcl-2, Bax and caspase-3 expression in injured brain tissues
Sections were dewaxed and incubated with methanol containing 3% (v/v) hydrogen peroxide to inactivate endogenous peroxidase for 10 minutes at room temperature. Antigen was retrieved using a microwave oven (WD750S; Galanz, Zhongshan, Guangdong Province, China). Sections were blocked with confining liquid for 20 minutes, incubated with rabbit anti-Bcl-2, Bax, caspase-3 polyclonal antibodies (1: 100; Boster, Wuhan, Hubei Province, China) at 37°C for 1 hour, at 4°C overnight, treated with biotinylated goat anti-rabbit IgG (1:1 000) at 37°C for 20 minutes, developed with 3,3’- diaminobenzidine, and then mounted with neutral gum.
TUNEL for apoptosis in injured brain tissues
The in situ cell apoptosis detection kit was purchased from Boster. Sections were dewaxed with xylene, hydrated with gradient alcohol (95%, 90%, 80% (v/v)), digested with 20 μg/mL proteinase K for 20 minutes, and incubated with methanol containing 0.3% (v/v) hydrogen peroxide to inactivate endogenous peroxidase for 30 minutes. After being washed with sodium citrate buffer containing 0.1% (v/v) Triton X-100 for 5 minutes, sections were incubated with TUNEL 50 μL reaction mixture at 37°C for 1 hour, washed with PBS, incubated with 50 μL converter peroxidase at 37°C for 30 minutes, washed with PBS, and then developed with 3,3’-diaminobenzidine for 5–10 minutes.
Assessment of results
Three specimens were randomly collected from each rat at each time point in each group for measuring apoptosis, caspase-3, Bcl-2 and Bax expression. Five fields from each section were observed by two observers under a 400 × light microscope (Olympus, Tokyo, Japan), and the number of apoptotic cells was quantified. Absorbance values of caspase-3, Bcl-2 and Bax expression were obtained in each group using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA), and the average value was calculated and compared. Apoptosis is presented as the apoptotic index:
Apopototic index = number of apoptotic cells/(the number of apoptotic cells + the number of normal cells) × 100%.
|
Statistical analysis
Measurement data are expressed as mean ± SD, and were analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). The mean difference among groups was compared using two-way analysis of variance; Student-Newman-Keuls test was used for intergroup comparisons. A value of P < 0.05 was considered statistically significant.
.jpg)
.jpg)
.jpg)