Formamide (molecular formula: HCONH2, Chongqing Chuanjiang Chemical Reagent Factory, Chongqing,China), urethane (molecular formula: C3H7NO2, Wuxi Yangshan Biochemical), Evans blue (Beijing Hengye Zhongyuan Chemical), 4% paraformaldehyde (Wuhan Boster Biological Technology), urokinase (Guangdong Livzon Pharmaceutical), glutamate (Sigma), derivatization reagent borate buffer (Agilent Technologies, USA), FMOC reagent Agilent PN5061-3337 (Agilent Technologies, USA), OPA reagent Agilent PN5061-3335 (Agilent Technologies, USA, 2,4-DNFB (Japan) and HPLC-grade acetonitrile and methanol (Germany) were used in this study.
We used the following instruments: a ZH-Lanxing B-Type rabbit stereotaxic Apparatus (Huaibei Zhenghua Biological Instrument & Equipment), electronic scales (Satourious, Germany), a Rainbow Type-722 grating spectrophotometer (Shandong Gaomi Rainbow Analytical Instrument), a 5415R high-speed centrifuge (Frozen, Heraeus Company), micropipettes (Eppendorf), a 202–2 constant temperature oven (Shanghai Luda Laboratory Apparatus), a digital display thermostat water bath (HH-2; Guohua Electric Appliance), a tabletop general centrifuge (TGL-16B; Shanghai Anting Scientific Instrument Factory), a −80°C freezer (Forman Scientific Company), a refrigerator (Qingdao), a CT provided by the Guiyang Medical College, a high-performance liquid chromatograph (HP-1100; Agilent Technologies, USA), a G1315 A diode-array detector (DAD, Agilent Technologies, USA), a pH meter (410 A, ORION, USA), an Agilent 1313A Automatic Sampler (Agilent Technologies, USA), a column oven (Agilent Technologies, USA) and scales (Beijing Gangdong Hengye Instrument).
The present study was approved by the Animal Care and Use Committee of Guiyang Medical College.
Seventy-two male rabbits (2.8-3.4 kg) were provided by the Animal Center of Guiyang Medical College. The animals were divided randomly into an ICH group and a NC group (n = 36 each), and they were equally divided into 6 subgroups (n = 6 each) that were sacrificed at 6, 12, 18, 24, 48 or 72 h after ICH induction. ICH was induced in all animals in the model group.
ICH model preparation
Rabbits were fasted for 12 hours and water restricted for 4 hours prior to the experiment. The rabbits were then anesthetized by injecting 20% urethane (5 ml/kg) into the ear vein. Slow breathing, a slow corneal reflex and no pain reaction were used as indicators of complete anesthetization. The head of the rabbit was then shaved to expose the skin for surgery.
The anesthetized rabbit was fastened to the stereotaxic apparatus, and the skin in the operation field was disinfected with 75% alcohol. A 3-cm incision was made along the mid-line between the two post-orbital margins, and the subcutaneous fascia was stripped to expose the skull. A 3% H2O2 solution was used to open the periosteum and expose the bregma and lambdoid sutures. The head was then adjusted to make the bregma 1.5 mm higher than the lambdoid suture. The position of the internal capsule was located according to the rabbit stereotaxic atlas. The coronal plane crossing the center of bregma was used as the coronal zero plane (AP0); A1 represented the coronal plane 1 mm rostral to the AP0, and the internal capsule was estimated to be between A5 and P2. The present experiment used the A1 level and the bregma as base points, with the puncture point 6 mm left of the coronal suture and 1 mm parallel to the sagittal suture. A hole was drilled in the skull, and a #12 needle and a 1-ml syringe were used to deliver 0.8 ml autologous arterial blood taken from the central ear artery. The syringe was then connected to a #7 needle in without a tip. Air was completely removed from the syringe, leaving 0.3 ml of blood. The #7 needle was quickly inserted vertically 12 mm into the skull, and the blood was slowly injected into the basal ganglia. The injection lasted for approximately 3 minutes. The needle was left in place for 8 min after the injection to prevent blood backflow, and then the needle was slowly removed. Local hemostasis was induced by compression for 2 min.
The drill hole was then covered using gutta-percha. A CT scan was performed 3 hours later. A hyperdensity shadow in the basal ganglia region without a shadow in the lateral ventricle was considered as a successful model of ICH.
After successful ICH induction confirmed by CT scan, the animals were returned to the housing facility. All the animals recovered from anesthesia within 5 hours after intravenous injection of 20% urethane. Exclusion criteria included visualization of back flow along the needle track, blood in the ventricle, and death.
NC group treatment
Procedures performed in the ICH group were identical in the NC group, but autologous arterial blood was not injected into the basal ganglia to induce intracerebral hematoma.
Medical treatment of the animals
Animals received an intramuscular injection of penicillin (400,000 U) to prevent infection. They were housed as usual until they were sacrificed. No other medical treatment was administered.
Intracerebral hematoma volume and neurological deficit score
To demonstrate that ICH was successfully modeled, hematoma volume was measured with a CT scan, and a neurological deficit scale was used to assess neurological functions (Purdy et al. 1989). The scale included tests of motor function (Parker et al. 2010; Miller et al. 2007; Shi et al. 2011; Lampl et al. 2005), consciousness (Parker et al. 2010; Miller et al. 2007; Shi et al. 2011; Lampl et al. 2005), head turning (0–1), circling (0–1) and hemianopsia (0–1). A total score of 11 indicates maximum impairment (comatose or dead rabbit), whereas 2 denotes complete normality. Tests were conducted by an observer blinded to the animals. The tests were conducted by an observer blinded to the animal’s group.
Brain tissues preparation
A 2% Evans blue (2 ml/kg) solution was injected into the ear vein 2 hours before euthanization. The animals were then anesthetized using 20% urethane, and the chest was quickly opened to expose the heart. A tube was inserted from the left ventricle into the aortic root, with a small hole cut in the right ventricle to allow the tube to exit. Rabbits were transcardially perfused with 400 ml 0.9% sodium chloride solution until the fluid flowed clear, at which time they were perfused with 100 ml 4% paraformaldehyde. The brain was then extracted and placed on ice. The needle track was used as the center to prepare coronal and sagittal sections, then the brain on the hematoma side was cut and divided into four parts: front-inner, front-outside, back-inner and back-outside. A total of 5 mm of brain tissue surrounding the hematoma was collected from each area. The front-inner and front-outside parts were used for amino acid testing, the back-inner part was used for assessing Evans blue content, and the back-outside part was used for testing BWC.
Perihematomal glutamate level measurement
Perihematomal glutamate content was determined using high-performance liquid chromatography.
A ZORBAX Eclipse-AAA (4.6 × 150 mm, 5 μm) chromatographic column was used. Mobile phase A was 40 mM Na2HPO4, pH 7.8 (5.5 g Na2HPO4 · H2O + 1 l water, NaOH was added to make the pH 7.8) and mobile phase B was 45:45:10 (V/V/V) ACN: MeOH:water. The column was run with a flow rate of 2 ml/min. Phase B increased from 0 to 57% between 0 and 18 min and from 57 to 100% between 18.1 and 18.6 min. It remained at 100% between 18.6 and 22.3 min and decreased from 100% to 0% between 22.3 min and 23.2 min. Between 23.2 and 26 min, Phase B remained at 0%. The column temperature was 40°C, and the sampling volume was 10 μl. The diode array wavelength was 262 nm, and the reference wavelength was 324 nm.
Derivative solution preparation
A total of 25 mg OPA was dissolved in 1 ml methanol. Sodium borohydride buffer (4 mol/l) was then added (pH 10.4), and the solution was stirred. The final solution was stored at 4°C.
Standard solution preparation
Glutamate and 0.2 mol/l NaHCO3 (pH 9.8) was used to make a 1-g/l standard stock solution.
The brain tissue was defrosted, weighed and placed in a dry glass homogenizer. Dilute hydrochloric acid (1:5 w/v, 0.1 mmol/l) was added, and the homogenized brains were placed into an ultrasonicator (Temp: 4°C; pulse for 2 s, rest for 2 s; intensity: 20%; 15 times in total). Samples were then centrifuged at 1,200 rpm for 20 min at 4°C. Borate saline buffer (2.5 μl) was then added to the supernatant solution and mixed for 20 min, followed by the addition of 0.5 μl OPA. The sample was then mixed for 30 s before FMOC (0.5 μl) was added and the solution was mixed for another 30 s. Finally, water (32 μl) was added, and the final sample was mixed for 30 s, and 10 μl of the sample was used.
Calculation of glutamate concentration
The peak area of glutamate from the HPLC was integrated and used as an external standard for the samples. The glutamate concentration for 1 g of brain tissue was then calculated according to the sample quality.
Determination of BBB permeability
Evans blue was used as a tracer to measure BBB permeability. Two hours before each experiment, 2% Evans blue (2 ml/kg) was injected into the ear vein. After 2 h, the brain tissue was quickly removed. The tissue surrounding the hematoma was weighed (within 0.1 mg) and then placed into a test tube with 4 ml formamide. The tube was then capped and placed in a 54°C water bath for 24 h to allow the Evans blue to spread throughout the brain tissue.
The samples were then centrifuged at 2,400 rpm for 5 min. The supernatant was placed in a quartz cuvette, and a spectrophotometer was used (λ = 632 nm) to measure the absorbance of the supernatant. Formamide was used as a blank control.
Setting up the standard curve
Evans blue (4 mg) was placed into a volumetric flask and weighed (within 0.1 mg). A total of 100 ml NS was added, and the solution was stirred. From this solution, 0.3 ml was removed and placed in 5.7 ml of formamide to make the standard buffer solution. A total of 3 ml of this solution was serially diluted in seven tubes each containing 3 ml formamide. The amount of Evans blue in each of the seven tubes was 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/ml. The tubes were capped and placed into a 54°C water bath for 24 hours. The aforementioned method was used to measure absorbance. Linear regressions were calculated for the absorbencies and Evans blue content. The final equation was y = 0.0053 × + 0.0608 (R2 = 0.9833).
Evans blue content computation
We used the formamide method to measure Evans blue content in brain tissue to assess BBB damage severity. The formula used was as follows: Evans blue content in brain tissue (μg/g wet brain) = B × formamide (ml)/wet weight (g), where B refers to the Evans blue content of the sample (μg/ml) given by the linear regression equation according to standard curve.
Measurement of BWC
The dry and wet weight method was used to measure BWC. The brains were quickly removed, and brain tissue from the back-outside portion of the hematomas was used. First, the weight of the wet tissue was obtained. The samples were then placed in an oven at 100°C for 48 h, and the dried samples were then weighed. BWC was then calculated as (wet weight–dry weight)/wet weight × 100%.
All data were analyzed using SPSS 11.5. Basic data are expressed as the mean ± standard deviation (X ± SD). A Kruskal-Wallis test was used to make comparisons across the whole time series among the groups. If the Kruskal-Wallis test detected significant differences, Bonferroni corrections were used to make comparisons between groups. A correlative analysis was also performed to assess the relationship between the glutamate level and the BBB permeability. A p value less than 0.05 was considered statistically significant. Statistical analysis was performed with the help of the Department of Biostatistics of Guiyang Medical College.