2. Methods

2.1. Animals
Ninety Wister rats (male, body weight: 250 g to 300 g) were provided by Experimental Animal Center, China Medical University. The rats were assigned randomly to three groups: normal group (N, n = 10, without any treatment), normal saline control (S, n = 40, intra-cerebral injection of normal saline as control) and intra-cerebral hemorrhage group (ICH, n = 40). The experimental protocol used for animals was approved by the Animal Care and Use Committee, China Medical University. The rats were subjected to intra-cerebral injection of normal saline or autologous whole blood and were sacrificed on 6 hours, 1 day, 3 days and 7 days after the injection, respectively. Five rats in each group at each time point were sacrificed by transcardially perfusion of normal saline followed by 4% buffered Para formaldehyde (pH = 7.4). The brains were removed, postfixed, embedded in paraffin, and cut into sections (7 μm) as described previously [8]. Five rats in each group at each time point were sacrificed by over dose injection of Ketamine. The brains were removed and stored at minus 80°C for RT-PCR and Western Blots.

2.2. Induction of Intracerebral Hemorrhage
Since most intracerebral hemorrhages occur in the basal ganglia clinically, in the present study, rat model of ICH was induced in left basal ganglia. Rats were anesthetized with intra-peritoneal sodium pentobarbital (60 mg/kg). The body (rectal) temperature was maintained at 37°C during the surgery using a feedback-controlled heating system. The rats were positioned in a stereotactic frame, and the scalp was incised along the midline. Using a sterile technique, a 1 mm burr hole was opened in the skull on the left coronal suture 3 mm lateral to the midline. A blunt 26-gauge needle was inserted into the left basal ganglia under stereotactic guidance (coordinates: 0.2 mm anterior, 6.0 mm ventral, and 3.0 mm lateral to the midline). Then, a 75 μL of autologous whole blood was infused at a rate of 20 μL/min with the use of a microinfusion pump. After the completion of the infusion, the needle was withdrawn quickly, cyanoacrylate glue was placed around the burr hole, and the skin incision was closed with sutures. For the normal saline control rats, only 75 μL normal saline was injected.

2.3. Detection of TLR4 mRNA by the Method of RT-PCR
Briefly, total RNA samples were isolated from peri-hemorrhage regions in left basal ganglia using Simply P Total RNA isolation Kit (BioFlux, HZ, China) according to the manufacturer's instructions. The quantity of total RNA was determined on a Nanodrop (ND-1000 Spectrophotometer) by measuring optical density at A260 and A280 nm wavelength. Then, the concentrations of mRNA were adjusted to 1.0 g/mL. RNA was reverse-transcribed into cDNA in 20 μL reaction system using Superscript First-Strand Synthesis Kit for RT-PCR (promega Inc) under conditions described by the supplier. One microliter of cDNA was used to amplify the TLR4 gene fragment with 1× PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 200 μM of primers, and 2u Taq DNA polymerase. The house keep gene (β-actin) was used as an internal control. The PCR primer sequences were designed according to the TLR4 and β-actin gene sequences reported in GenBank. TLR4 (145 bp): forward: 5′-AGCTTTGGTCAGTTGGCTCT-3′; reverse: 5′-CAGGATGACACCATTGAAGC-3′; β-actin (701 bp): forward: 5′-GCCAACCGTGAAAAAGATG-3′; reverse: 5′-CCAGGATAGAGCCACCAAT-3′. Reaction cycles were performed at the following condition: 94°C 2 min, 94°C 40 s, 55°C 40 s, and 72°C 60 s. The reaction was stopped after 31 cycles. PCR products were electrophoresed through agarose gel. Image Acquisition and Analysis software were used to determine band densities. The abundance of TLR4 mRNA was expressed as the ratio of TLR4 mRNA to β-actin mRNA.

2.4. Immunohistochemistry (IHC)
The IHC staining for TLR4 was performed as described previously [8]. The primary antibody employed was rabbit anti-TLR4 (WuHan, BA1717). The biotinylated secondary antibody and antibody-biotin-avidin-peroxidase complexes (ABC reagent, SC-2018) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Slides processed without primary antibodies served as negative controls.

2.5. Western Blots
Cellular proteins were prepared from ICH cerebral hemispheres, electrophoresed with SDS-polyacrylamide gel and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ). The ECL membranes were incubated with the appropriate primary antibody (WuHan, BA1717) followed by incubation with peroxidase-conjugated secondary antibodies (Golden Bridge, PV-6001). The signals were detected with the ECL system (Amersham Pharmacia). The same membranes were probed with anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Biodesign, Saco, Maine) after being washed with stripping buffer. The signals were quantified by scanning densitometry and computer-assisted image analysis.

2.6. Electrophoretic Mobility Shift Assay (EMSA)
Nuclear proteins were isolated from brain tissue. Briefly, after removal of supernatant (cytoplasmic extract), the pellets were resuspended in nuclear extraction reagent. The tubes containing pellets were put on vortex for 15 seconds every 10 minutes, for a total of 40 minutes. The tubes were centrifuged at maximum speed (~16,000 × g) in a micro centrifuge for 10 minutes. The supernatants (nuclear extract) were transferred to a clean prechilled tube and stored at −80°C until use. EMSA was performed as described previously [8]. NFκB binding activity was examined in a 15 μL binding reaction mixture containing 15 μg of nuclear proteins and 35 fmol [γ-32P] labeled double-stranded NFκB consensus oligonucleotide. A super shift assay using antibodies to P65 and P50 was performed to confirm NFκB binding specificity as previously described.

2.7. Statistical Analysis
All data were presented as mean ± SEM. The measurements were analyzed by one-way analysis of variance (ANOVA) or Student's t-test. The P < .05 level of probability was used as the criteria of significance.