Surgery: Controlled cortical impact (CCI) surgery or sham surgery was performed on anesthetized animals under a protocol approved by the San Francisco VA Medical Center Animal Care Committee. Briefly, bupivacaine was administered subcutaneously above the skull, and an incision was made followed by a 2.5mm circular craniectomy. TBI was inflicted by a 2 mm circular, flat pneumatic piston traveling at 3 m/s, penetrating 1.5 mm, for 150 ms (Amscien Instruments, Richmond, VA with extensive modifications by H&R Machine, Capay, CA). Target brain coordinates for the center of injury were 1.5 mm lateral, 2.3 mm posterior to the bregma point. After minor bleeding had ceased, the skin was clipped together and animals were monitored for recovery. Sham animals received all surgical procedures without piston impact. As needed, animals were given rehydration therapy and liquid diet for the first three days. Brain and blood leukocyte isolation: Brain leukocytes were harvested according to previously published methods (Sedgwick et al., 1991). Briefly, peripheral blood was collected through the inferior vena cava, and then total body perfusion was performed through the heart. Brains were obtained and mechanically disassociated through a 100 μm cell strainer. Washed cells were treated with 400 U/ml DNase I (Sigma-Aldrich) and 0.5 mg/ml collagenase type I (Worthington) at 37°C for 30 min. Leukocytes were isolated by separation on a Percoll gradient (Amersham Biosciences). For PBL isolation, mononuclear cells were separated from peripheral blood using ficoll-hypaque (GE Healthcare). Flow cytometry and antibodies: Fc receptors were blocked with 10% rat serum (Sigma) and cells were stained with fluorescent antibodies. Leukocyte analysis used a combination of the following antibodies: anti-CD45 (clone Ly5) APC (eBioscience), anti-CD11b (clone M1/70) PE (Invitrogen), anti-Ly6G (clone 1A8) PE-Cy7 (BD Biosciences), F4/80 (clone BM8) PE-Cy5 (eBioscience). Sytox blue (Invitrogen) was used to gate out dead cells. Cells were sorted on a FACSAria (BD Biosciences) directly into cell lysis buffer (Ambion). Sorted samples were frozen at -80C°.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using an RNAqueous-Micro kit (Ambion) according to the manufacturer's instructions. Samples were eluted in water.
Label
Cy3
Label protocol
Further sample preparation, labeling, and array hybridizations were performed according to standard protocols from the UCSF SABRE Functional Genomics Facility. RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies), and RNA was amplified by use of a whole transcriptome amplification kit (Sigma-Aldrich). Subsequent Cy3-CTP labeling was performed by using a NimbleGen one-color labeling kit (Roche-NimbleGen Inc). The quality of the amplified products was assessed using an Agilent 2100 Bioanalyzer and Nanodrop ND-8000 (Nanodrop Technologies, Inc.).
Hybridization protocol
The products were hybridized to Agilent Whole Mouse Genome 4x44K microarrays according to the manufacturer's protocol.
Scan protocol
Arrays were scanned with an Agilent microarray scanner, and raw signal intensities were extracted with Feature Extraction v10.5 software.
Data were normalized by using the quantile normalization method (Bolstad et al., 2003). No background subtraction was performed, and the median feature pixel intensity was used as the raw signal before normalization. A one-way ANOVA linear model was fit to the comparison to estimate the mean M values and calculated moderated t-statistic, B statistic, false discovery rate and p-value for each gene for the comparison of interest. Adjusted p-values were produced by the method proposed by Holm (1979). All procedures were carried out using functions in the R package limma in Bioconductor [Smyth (2004), Gentleman (2004)].
