Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway

All animals were treated humanely and with regard for alleviation of suffering and pain. All surgical protocols involving animals were performed by sterile/aseptic techniques and were approved by the Institutional Animal Care and Use Committee at Morehouse School of Medicine prior to the initiation of experimentation. Adult male Sprague-Dawley rats (250–300 g; Charles River Laboratory International, Inc., USA) were housed in standard cages in a temperature-controlled room (22 ± 2 °C) on a 12-h reverse light-dark cycle. Food and water were provided ad libitum.

Animals were randomly allocated into three groups: sham (control), middle cerebral artery occlusion (MCAO) + vehicle treatment (MCAO) and MCAO + NRG-1 (MCAO + NRG1). Rats were anesthetized with a ketamine/xylazine solution (100/10 mg/kg, i.p.) prior to surgery. After anesthesia administration, a rectal probe monitored the core body temperature and a Homoeothermic Blanket Control Unit (Harvard Apparatus, Hollister, MA) was used to ensure the body temperature maintained at 37 °C. Cerebral blood flow was monitored throughout the length of the surgery by a continuous laser Doppler flowmeter (Perimed, Ardmore, PA), with a laser Doppler probe placed 7 mm lateral and 2 mm posterior to bregma in a thinned cranial skull window.

Rats in the treatment groups (MCAO and MCAO + NRG1) were subjected to a left transient MCAO. MCAO was induced by the intraluminal suture method as previously described [3]. Briefly, a 4-cm length 4-0 surgical monofilament nylon suture coated with silicon (Doccol Corp., Sharon, MA) was inserted from the external carotid artery (ECA) into the internal carotid artery (ICA) and then into the Circle of Willis, to occlude the origin of the left middle cerebral artery (MCA). After 1.5 h of ischemia, the nylon suture was removed and the ischemic brain was reperfused for 22.5 h before sacrifice. Rats in the sham control group underwent the same procedure as those in the treatment groups, but a filament was not inserted into the ICA. Animals were randomly assigned into treatment groups and either administered 50 μl of NRG-1β reconstituted with 1 % bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (MCAO + NRG1; 20 μg/kg; EGF-like domain, R&D Systems, Minneapolis, MN) or vehicle (MCAO; 1 % BSA in PBS) All treatments were administered by bolus injection into the ICA through ECA immediately before MCAO. Animals were sacrificed 24 h after MCAO. All NRG-1 and vehicle treatment studies were performed in a blinded manner.

Microarray analysis was performed as we previously described [3, 48, 49]. Animals were sacrificed, and their brains removed 24 h following MCAO. Removed brains were sliced into 2-mm coronal sections (approximately +3.0 to −5.0 from bregma) using a brain matrix. Tissue from the ipsilateral tissue from the two middle slices (+1 to −3 from bregma) of MCAO treated (n = 3) and MCAO + NRG1 treated (n = 3) and sham animals (n = 3) were used for RNA isolation, while the two outer slices of tissue were used for staining with 2,3,5-triphenyltetrazolium chloride (TTC), in order to confirm infarct formation. Total RNA extraction was performed using TRIzol Reagent (Life Technologies, Rockville, MD, USA), cleaned (RNAqueous Kit Ambion, Austin, TX, USA), and converted to double-stranded complementary DNA (cDNA) (Invitrogen, Superscript Choice System, Carlsbad, CA, USA) using T7-(dT)24 primer. Cleanup of double-stranded cDNA used Phase Lock Gels (Eppendorf, Westbury, NY, USA)-Phenol/Chloroform/Isoamyl Alcohol (Sigma, St. Louis, MO, USA). cRNA was synthesized using a RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY, USA). Biotin-labeled cRNA was cleaned up using a GeneChip Sample Cleanup Module (Affymetrix Inc., Santa Clara, CA, USA) and quantified using a spectrophotometer. Twenty micrograms of the in vitro transcription product was fragmented by placing at 94 °C for 35 min in fragmentation buffer. Following fragmentation, 15 μg of the biotinylated cRNA was hybridized to an Affymetrix Rat Genome U34A GeneChip. The chips were hybridized at 45 °C for 16 h, and then washed, stained with streptavidin-phycoerythrin, and scanned according to the manufacturing guidelines.

Affymetrix Expression Console software (Affymetrix, Santa Clara, CA) was used for initial data processing. Affymetrix microarrays contain the hybridization, labeling, and housekeeping controls that help determine the success of the hybridizations. The Affymetrix Expression Analysis algorithm uses the Tukey’s biweight estimator to provide a robust mean signal value and the Wilcoxon’s rank test to calculate a significance or p value and detection call for each probe set. The detection p value is calculated using a discrimination score (R) for all probes. The discrimination score is a basic property of a probe pair that describes its ability to detect its intended target. It measures the target-specific intensity differences of the probe pair (perfect match (PM)–mismatch (MM)) relative to its overall hybridization intensity (PM + MM). Background estimation is provided by a weighted average of the lowest 2 % of the feature intensities. Mismatch probes are utilized to adjust the PM intensity. Linear scaling of the feature level intensity values, using the trimmed mean, is the default to make the means equal for all arrays being analyzed. False-negative and false-positive rates are minimized by subtracting nonspecific signal from the PM probe intensities and performing an intensity-dependent normalization at the probe set level. Calculation of fold change and the presence of genes in the tissue were analyzed in Microsoft Excel. Changes in gene expression were compared between the control (sham) and following MCAO and MCAO + NRG-1 treatment. Gene expression values that increased or decreased by twofold or more were statistically significant (p < 0.05) using two-way ANOVA. The list of genes upregulated twofold or more by ischemia and decreased 50 % or more by NRG-1 was analyzed using Ingenuity Pathway Analysis (IPA) software (Qiagen, Redwood City, CA; www.​ingenuity.​com) overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. Fischer’ s exact test was used to calculate a p value determining the probability that each biological function and/or disease assigned to that network is due to chance alone. The functions, canonical pathways, and gene networks that were most significant to the dataset were identified. IPA identified 64 inflammatory genes that were upregulated twofold or more by MCAO and reduced 50 % or more by NRG-1.

Using CONFAC software, we further examined the IPA-identified inflammatory genes [47, 48]. A tab-delimited text file containing the gene name and RefSEQ ID of the clusters of interest (genes) was uploaded to the CONFAC web browser interface (http://​confac.​emory.​edu/​). CONFAC software identified mouse orthologs from the uploaded gene list (from UCSC and ENSEMBL genomes). CONFAC software next identified significantly conserved sequences (e value <0.001), which were analyzed for transcription factor-binding sites (TFBS) using MATCH™ software. The final output table consisted of the cohort list of genes (column) and the position weight matrix (collected in TRANSFAC® database) identified potential TFBS and the number of TFBS determined for each gene of interest. The CONFAC program then allowed for additional analysis using the Mann-Whitney U test, which facilitated the statistical analysis of TFBS over-represented in the sample dataset compared to the seven control random control datasets provided by CONFAC.

The murine N9 microglial cell line was kindly provided by Dr. Celine Beamer (Department of Biomedical and Pharmaceutical Sciences, University Montana). N9 cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) with 4 mM l-glutamine, supplemented with 10 % fetal bovine serum (FBS), streptomycin (100 U/ml), and penicillin (100 U/ml). The cells were maintained in 95 % air and 5 % CO₂-humified atmosphere at 37 °C and were passaged every 3 days. For culturing with various stimulants, N9 cells were plated into 12-well plates in prepared culture medium and NRG-1 and lipopolysaccharide (LPS) were applied at the following final concentrations: LPS (10 μg/ml) and NRG-1 (100 ng/ml in 1 % BSA/PBS; recombinant human neuregulin1-beta1 EGF-like domain, R&D Systems). N9 cells were either pre-treated with NRG-1 (100 ng/ml) for 24 h followed by stimulation with LPS (10 μg/ml) or treated with LPS or NRG-1 alone for the indicated times (1–24 h).

Cell lysates were taken at 1, 3, 6, and 24 h time points following LPS treatment. The cytokine release was measured in cell culture supernatants by Luminex technology using the manufacturer’s protocol (Bio-Rad, Hercules, CA). Data analysis was performed using Bio-Plex Manager software on the Bio-Plex 200 system. Experiments were performed four times in duplicate wells.

Proteins were isolated using whole cell extraction reagent M-PER (Pierce Biotechnology, Rockford, IL) or NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific) according to the manufacturer’s instructions. Protein concentrations were determined by Bradford protein assay (Bio-Rad). Fifty micrograms of protein lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF) (Bio-Rad, CA). The membranes were blocked with 3 % nonfat dry milk in 0.1 % Tween-20/Tris-buffered saline for 1 h at room temperature and subsequently incubated with the primary antibodies overnight, followed by incubation in corresponding secondary horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were developed using immuno-western star ECL detection kits (Bio-Rad). The optical densities of the antibody-specific bands were analyzed by a Luminescent Image Analyzer, LAS-4000 (Fuji, Japan). The following antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used: NF-kB p65 (sc-372; 1:200), IkB-α (sc-371; 1:200), YY1 (sc-281; 1:200), and β-actin (sc-130656; 1:200).

The phosphorylation of IkB-α was determined using an enzyme-linked immunosorbent assay (ELISA)-based assay kit according to the manufacturer’s protocol (Affymetrix eBioscience, San Diego, CA). To analyze the results, we calculated the average values for each treatment group. This assay was run four times in duplicate.

Nuclear extracts were isolated using NE-PER Nuclear and Cytoplasmic Extraction kit according to the manufacturer’s protocol (Thermo Scientific, Waltham, MA). ELISA was performed using the manufacturer’s protocol. To analyze the results, the optical density was read using 450 nm with an optional reference wavelength of 655 nm. This assay was run three times in duplicate.