2.1. Animals and ICH model
All experimental protocols and procedures conformed to the guidelines of the Chinese Council for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Harbin Medical University, China. Adequate measures were taken to ensure minimal pain or discomfort to rats. A total of 440 adult male Sprague-Dawley rats (200–250 g) were purchased from the Center for Experimental Animals, Harbin Medical University, China. Rats were allowed free access to food and water.
The rats were anesthetized with chloral hydrate (350 mg/kg, i.p.) and then placed in a stereotaxic frame (Bilaney Consultants, Germany). A midline scalp incision was made, and a hole was drilled in the left side of the skull (3 mm lateral to the midline, 0.2 mm anterior to the bregma). Autologous whole blood (50 μL) was collected from the arteria caudilis into a needleless sterile insulin syringe without any anticoagulant. After blood collection, pressure was applied to stop the bleeding. A 26-G needle attached to the syringe was inserted stereotaxically into the left caudate nucleus (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.0 mm lateral to the bregma). The blood was infused over 5 min, and the needle was left in place for another 5 min to minimize backflow. The syringe was then removed slowly. The hole in the skull was sealed with bone wax and the scalp was sutured. Body temperature was maintained at 37.0±0.5°C during surgery with the use of a feedback-controlled heating pad. Rats were sacrificed for analysis at 2 h, 3 h, 6 h, 10 h, 12 h, 24 h, 2 days, or 5 days after blood infusion. Control rats were infused with the same volume of saline. None of the rats died as a result of the anesthesia, surgery, or blood infusion.
2.2. Assessment of histopathology
Rats were sacrificed by an overdose of chloral hydrate and perfused transcardially with ice-cold saline. Their brains were then harvested, dehydrated, and embedded in paraffin. The brains were sliced coronally into 4-μm sections from the rostral to the caudal portion of the injection site. Three consecutive brain sections selected from the injection level and from 300 μm on each side were stained with hematoxylin and eosin for evaluation of brain damage.
2.3. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
At 2 h, 3 h, 6 h, 12 h, 2 days, and 5 days after ICH, rats were deeply anesthetized with chloral hydrate and perfused intracardially with ice-cold, RNase-free phosphate-buffered saline. The brains were rapidly removed and dissected. The ipsilateral hemispheric brain tissue was placed in liquid nitrogen prior to RNA extraction. Total RNA was prepared with Trizol Reagent (Invitrogen, Carlsbad, CA).
PCR reactions were carried out in 96-well plates with an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green to monitor in real-time the amplification of the target gene. The reaction mix contained 200 nM of each primer, 5 μL cDNA (corresponding to ~3 ng DNA), and 10 μL 2 × SYBR Green Master Mix Reagent (Applied Biosystems) in a total volume of 25 μL. Aliquots from the same cDNA sample were used with all primer sets in each experiment. Reactions were run according to the manufacturer's recommended cycling parameters: 93°C for 2 min followed by 40 cycles at 93°C for 1 min and 55°C for 1 min. Each PCR reaction was completed in triplicate. Standard curves were generated from serial dilutions of cDNA standards. The mRNA level was expressed in arbitrary units. Primers used were as follows: PAR-1, 5′-TGCCTTTAAGCCCAGTAGG TG-3′ (forward) and 5′-AAGCACAAGGTCCTGGGT TC-3′ (reverse); AQP-4, 5′-AGATCAGGGTGCTCCAGTCG-3′ (forward) and 5′-TGTGCCCCTCTATTCCTGGT-3′ (reverse). Five rats per group were assessed at each time point.
Brain sections were de-waxed and rehydrated, rinsed with distilled water and phosphate-buffered saline, quenched with 3% H2O2, blocked in 10% normal goat serum, and incubated overnight at 4°C with primary antibodies: mouse anti-PAR-1 monoclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-MMP-9 polyclonal antibody (1:100; Santa Cruz). The sections were then incubated with biotinylated goat anti-mouse IgG or rabbit anti-goat IgG (1:1000; DAKO) for 1 h at room temperature, washed three times, and incubated with streptavidin-peroxidase for 30 min. Finally, the immunoreactions were visualized with diaminobenzidine-H2O2 solution. Sections were washed, successively dehydrated in ethanol, and defatted in xylenes. Cover slips were placed over the sections with Permount (Fisher Scientific, Pittsburgh, PA). Control sections were processed without the primary antibodies and showed no positive signals.
To quantify the number of immunoreactive cells labeled with PAR-1 or MMP-9, three sections (from the injection site and 300 μm on each side) per rat were selected, and positively stained cells were counted randomly in 10 comparable fields (magnification, × 400) adjacent to the hematoma. The numbers of immunoreactive cells from 30 locations per rat (10 fields per section × three sections per rat) were averaged and expressed as positive cells per high-power field, as previously reported (Wang and Doré, 2007a
; Wang et al., 2003
). Five brains per group were assessed at each time point.
2.5. Western blot analysis
Rats were deeply anesthetized and perfused transcardially with ice-cold saline before being sacrificed at different time points post-ICH. The ipsilateral hemispheric brain tissue was homogenized and the protein samples (80 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After being blocked with 5% nonfat milk in Tris-buffered saline–Tween 20, membranes were probed with mouse monoclonal anti-PAR-1 (1:500, Santa Cruz), mouse monoclonal anti-AQP-4 (1:500, Santa Cruz), goat polyclonal anti-thrombin (1:100, Santa Cruz), or goat polyclonal anti-MMP-9 (1: 100, Santa Cruz) overnight at 4°C. Then membranes were incubated with a 1:7000 dilution of secondary antibody (IRDye 700DX conjugated rabbit anti-goat antibody or IRDye 800CW conjugated rabbit anti-mouse antibody, Rockland Immunochemicals, Gilbertsville, PA). The antigen-antibody complexes were visualized with the Odyssey Infrared Imaging System (LI-COR, Lincoln, Nebraska). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a protein loading standard. The relative intensities of bands were analyzed with Image-Pro Plus 6.0 (MediaCybernetics, Bethesda, MD) and values were normalized to the value of a saline-infused control animal on each gel. Each experiment was performed in triplicate. Five rats per group were assessed at each time point.
2.6. Brain water content
Brain water content was determined by the wet/dry weight ratio method. Rats were euthanized by an overdose of chloral hydrate. After the olfactory bulbs, brain stem, and cerebellum were removed, the rest of the brain was immediately weighed on an electronic analytical balance (Changzhou Instruments, Inc., Changzhou, China) to obtain the wet weight and then dried at 100°C for 24 h in an Electric Blast Drying Oven (Chongqing Sida Apparatus, Inc, Chongqing ,China) to obtain the dry weight. Brain water content was expressed as (wet weight – dry weight)/wet weight of brain tissue ×100. Five rats per group were assessed at each time point.
2.7. Statistical analysis
All data are reported as mean ± standard deviation (SD). The statistical significance of the data was determined by analysis of variance (ANOVA) and post hoc tests. The correlation between brain water content and target protein expression was analyzed by Spearman correlation test with SPSS 13.0 for Windows (SPSS Inc, Chicago, IL). P < 0.05 was regarded as statistically significant.