Isolation of rat cortical cells
Female timed-pregnant Sprague Dawley rats (200–250 g) were purchased from the Charles River Laboratories (USA). Use of animals was performed in strict accordance with the Institutional Animal Care and Use committee guidelines as approved by the IACUC committee at Creighton University (protocol #0793). Timed Sprague Dawley dams were housed for up to 3 days in Creighton’s Animal Resource Faculty that is AALAC accredited. Ad libitum food and water and normal 24h light dark schedules were followed. Rat cortical cultures were established as described by Meberg and Miller . Briefly, Sprague-Dawley dams were euthanized by CO2 asphyxiation. For, CO2 asphyxiation dam were placed in a clear chamber with CO2 delivery at 20% of chamber volume per minute. After 1 min of cessation of all respiratory movement, toe pinch tests were performed to determine a lack of reflexive responses and a thoracotomy was used to ensure death. E16-E18 rat embryos were removed from their placental sacks and immediately decapitated. The brains were removed and the cerebral cortices were dissected from day 15-16 embryonic Sprague-Dawley rats (Sasco, Wilmington, MA), mechanically dissociated in Ca2+/Mg2+-free Hank’s balanced salt solution, with 0.035% sodium bicarbonate and 1mM pyruvate (pH 7.4) following 15 min digestion with 2.5% trypsin. Trypsin was neutralized with Dulbecco’s Modified Eagles Media (DMEM: Hyclone, Thermofisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum and the cell suspension was washed three times and resuspended with neurobasal media supplemented with B-27® and penicillin/streptomycin (Thermofisher Scientific, Waltham, MA). Cells were then plated onto poly-D-lysine coated plates and coverslips (Sigma, St. Louis, MO) at density of 1.5 x 106 cells/well in 6-well plates and 5 x 105 cells/well in 24-well plates and were maintained at 37 ºC in 5% CO2 in neurobasal supplemented media. Each cortical culture from 1 pregnant dam was considered to be a biological replicate because embryonic brain tissue is used for co-culture, immunocytochemistry, Multiplex ELISA, RTPCR and western blot experiments all performed in triplicate. In total, 24 Sprague Dawley rats were used for these data.
Cultivation of microglia
EOC 2 microglia isolated from brain tissue of Mus musculus were purchased from American Type Culture Collection (ATCC CRL-2467; Manassas, VA) and were maintained in DMEM (Hyclone, Thermofisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 1% l-glutamine, 1% penicillin/streptomycin, and 20% LADMAC conditioned media. Cells were grown in 100-mm tissue culture dishes at 37 ºC in 5% CO2 and allowed to reach 80% confluency before the cells were passed. LADMAC conditioned media was collected from Mus musculus bone marrow derived LADMAC cells (ATCC CRL-2420; Manassas, VA) 5-7 days after initial plating of cells at 1 x 105 in Eagle’s Minimal Essential Media (MEM; Hyclone) supplemented with 10% FBS, 1% l-glutamine, 0.1mM nonessential amino acids, 1.0 mM sodium pyruvate, and 1% penicillin/streptomycin. LADMAC conditioned media was collected, filter sterilized, and frozen until needed as a media supplement for microglia. LADMAC conditioned media was used to provide colony-stimulating factor 1 (CSF-1) to the microglial cultures as outlined by the ATCC culture instructions for EOC microglial cells (ATCC CRL-2420; Manassas, VA).
Neuronal – Microglial Co-Cultures
Primary cortical cells were cultured for 48 hours then either injured by mechanical transection using a sterile stylet or left uninjured . Briefly, mechanical transection using a sterile stylet involves the application of the sterile stylet tip directly to the cortical cell culture. Pressure is placed on the stylet while dragging the stylet tip across the cortical cell culture to form parallel sites of injury in the cortical culture. Microglia were pre-seeded onto 6-well permeable Transwells® at 5 x 105 cells/well or onto 24-well Transwells® at 4 x 104 cells/well (Corning,Tewksbury, MA) and cultured for 24 hours before being suspended above cortical cells using Transwells® in the co-culture model system. Microglia seeded onto Transwells® and injured or uninjured (control) primary cortical cells were co-cultured for an additional 48 hours in unsupplemented neurobasal media prior to cellular assays.
Primary cortical cultures were plated onto sterile poly-D-lysine coated coverslips. Microglia were plated onto sterile glass coverslips suspended above cortical cultures in Transwells®. Following co-culture experiments, cortical cells and microglia were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with 1X PBS. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, washed, and blocked for 1 hour in PBS, 0.2% BSA, and 0.2% Triton X-100. Primary antibodies were applied and incubated overnight at 4 ºC in PBS, 0.2% BSA, 0.2% Triton X-100. Primary antibodies were purchased from RMD Millipore Sigma (Darmstadt, Germany) and included: mouse anti-Nestin (1:200, Millipore Cat# AB5922, RRID:AB_91107), rabbit anti-GFAP (1:400, Millipore Cat# AB5541, RRID:AB_177521), mouse anti-α internexin (1:100, Millipore Cat# AB5354, RRID:AB_91800), mouse anti-TUJI/beta tubulin III (1:200, Millipore Cat# MAB1637, RRID:AB_2210524) and mouse anti-NeuN (1:50, Millipore cat#MAB377l, RRID:AB_2298772). Primary antibody directed against neurofilament was a mouse monoclonal neurofilament antibody, p-NF-H (7H11) (1:200, Santa Cruz Biotechnologies, Cat# sc-20015, RRID: AB_670161). To confirm microglial characteristics, microglial cells were immunostained with rabbit anti-mouse CD11b conjugated to Alexa 488 (2 µg/100 µl, Caltag Laboratories, Burlingame, CA). Secondary antibodies were applied for 1 hour at a concentration of 1:500 for goat anti-rabbit IgG (H+L) rhodamine conjugate and goat anti-mouse IgG (H+L) fluorescein conjugate (Pierce, Rockford, IL). Nuclei were visualized using a DAPI stain (300 mmol, MP Biomedicals, Santa Ana, CA). Qualitative and quantitative analysis of immunocytochemistry was performed by acquiring images with a Leica DMI4000B inverted microscope with a cooled CCD camera (Q Imaging, Surrey, BC) and fluorescent capabilities. Images were analyzed with ImageProPlus software (MediaCybernetics, Rockville, MD). For image data, 3 field views of at least 100 cells from 3 separate experiments were analyzed for each condition.
Measurement of Cell Viability
Viability of cortical cells and microglia were measured by metabolism of thiazolyl blue, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich). Injured and uninjured cortical cells cultured with and without microglia were incubated with 100 µl MTT in 1 ml of media for one hour. Media was removed and cells were dissolved in 300 µl dimethylsulfoxide (DMSO) and aliquoted to 100 µl/well in 96-well plates. Absorbance was read at 540-590nm on an ELISA plate reader. Three experiments were performed in triplicate.
Measurement of Cortical Cell Proliferation
Cell proliferation was measured using Click-iT® EdU Alexa Fluor 647 according to the manufacturer’s instructions (C10340, Thermo Fisher Scientific, Waltham, MA). Briefly, Click-iT® EdU Alexa Fluor 647 is a modified thymidine analogue EdU (5-ethynyl-2'-deoxyuridine, a nucleoside analog of thymidine) that is incorporated into newly synthesized DNA. The EdU is fluorescently labeled with a photostable Alexa Fluor® dye during the click reaction. Briefly, uninjured and injured cortical cells co-cultured with and without microglia for 2 DIV (days in vitro) were fixed in 3.7% formaldehyde in PBS for 15 min at RT. Fixed cells were washed twice with 1 ml of 3% BSA in PBS. Cells were permeabilized in 0.5% Triton®x-100 for 20 min at RT, washed and 1X Click-iT® EdU reaction cocktail was added for 30 min at RT. The reaction cocktail was removed, cells were washed in 3% BSA and PBS, counterstained with DAPI, mounted and imaged for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20x magnification. Volocity (PerkinElmer,USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation. Experiments were performed in triplicate with at least 300 cells counted per experiment for each condition.
Measurement of Cortical Cell Apoptosis
Apoptosis of cortical cultures was measured following co-culture with microglia for 2 DIV (days in vitro) using a Click-iT® TUNEL Alexa Fluor 488 imaging assay (C10245, Thermo Fisher Scientific, Waltham, MA). Manufacturer’s instructions were followed. Briefly, injured and uninjured cortical cells cultured with or without microglia were fixed with 4% PFA in PBS for15 min then permeabilized with 0.25% Triton-X® 100 for 20 min. Each condition was incubated with 100 µl of TdT reaction buffer for 10 min at RT then removed. Cells were incubated in 100 µl TdT reaction cocktail for one hr at 37 oC. Cells were washed twice in 3% BSA in PBS for 2 min then incubated with Click-iT® reaction buffer with additive for 30 min at RT protected from light. Cells were rinsed and counterstained with DAPI, mounted, and cover slipped for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20x magnification. Volocity (PerkinElmer,USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation. Experiments were performed in triplicate with at least 300 cells counted per experiment for each condition.
Conditioned media was collected from uninjured and injured neuronal and microglia co-cultures from three separate experiments and cytokine expression was determined by Q-Plex™ mouse cytokine –Inflammation multiplex assay. Concentrations of mouse microglia-derived cytokines MCP-1, IFN-g, MIP-1a, TNFa, RANTES, IL-1a, IL-1b, IL-2, IL-4, IL-3, IL-6, IL-10, IL-12, 1L-17 and GM-CSF were evaluated by Quansys Biosciences (#110449MS, Logan, UT). Cytokine concentrations in media collected from uninjured cortical cells cultured with microglia were used as the reference and control for these experiments. In order to use cytokine concentrations from uninjured cortical and microglial co-cultures as our control condition, each cytokine concentration measured in the uninjured cortical cell and microglia co-culture condition was normalized and set equal to one. Cytokine concentrations in media collected from injured cortical cells co-cultured with microglia were measured, normalized, and expressed as the percent change in cytokine concentration as compared to uninjured control concentrations for that cytokine. Multiplex ELISA assays were run in triplicate in three biological replicate experiments. Significance of the percent change from control was determined using student T test with Bonferroni correction. The percent change in cytokine concentration was considered significant if p<0.05, error bars represent the standard error of the mean of the percent change. Cytokine levels that were not consistently detected in either condition or were not significantly different in control and experimental conditions are not shown.
For real-time PCR analysis of cytokines and mature miRNAs, total RNA was extracted using the mirVana miRNA Isolation kit (Ambion). An amount of 200 ng total RNA was reverse-transcribed using the Invitrogen™ NCode™ miRNA First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Comparative real-time PCR was performed using the Invitrogen™ SYBR GreenER™ qPCR SuperMix Universal (Thermo Fisher Scientific) on the Bio-Rad CFX96 Touch™ Real-Time PCR Detection System. Primers were purchased from QIAGEN (Ccl3, Ccl5, Ifng, Mcpt1, Tnfα, Gapdh). Normalization was performed using Gapdh. Relative expression was calculated using the comparative Ct (ΔΔCt) method.
Following co-culture, protein collected separately from cortical cells or microglia was assessed using western blot analysis. Cortical cells or microglia were lysed with 500 µl lysis buffer (10x lysis buffer, Cat#9803, Cell Signaling, Danvers, MA), supplemented with 0.1M PMSF (Cat # 36978, Thermo Fisher Scientific, Waltham, MA), and HALT™ protease and phosphatase inhibitor diluted to 1X (Cat#78446, Thermo Fisher Scientific, Waltham, MA) per 3 wells of the 6-well plates or per 3 Transwells®.Lysates were spun 10,000 RPM for 10 minutes at 4 °C. Lysate supernatant were collected and heated at 95 °C for 5 minutes with 4X sample buffer plus 10mM DTT. Denatured protein samples were separated by SDS-PAGE gel electrophoresis on 10% TGS gels. Proteins were transferred to PVDF membranes in Tris-glycine transfer buffer. After transfer, membranes were blocked using BSA Blocking Buffer™ in TBS(Cat#37520, Thermo Fisher Scientific, Waltham, MA) for 1 hour and then incubated with primary antibody diluted in BSA Blocking Buffer™ in TBS overnight at 4 °C. Primary antibodies include rabbit anti-phospho-p44/42 MAPK (1:1000, 1:2000, Cell Signaling Technology Cat# 4376, RRID:AB_331772), rabbit anti-pan AKT (1:1000, Cell Signaling Technology Cat# 4691, RRID:AB_915783) rabbit anti-phospho-AKT (1:100, Cell Signaling Technology Cat# 9270, RRID:AB_329824). Following washing in Tris Buffered Saline with 0.1% Tween® 20 and BSA blocking buffer™, appropriate secondary antibodies (anti-rat IgG, HRP-linked antibody,1:1000, Cell Signaling Technology, Danvers, MA) were applied for detection. Membranes were developed using chemiluminescence SuperSignal™ ELISA Pico Chemiluminescent Substrate (Cat#37069, Thermo Fisher Scientific, Waltham, MA) following manufacturer’s instructions. Statistical analyses involved semi-quantitative measurements of chemiluminescence using BioRad ChemiDoc QRS (Hercules, CA) imaging system and software. Total protein loading was assessed by detecting GAPDH in each sample. Three separate experiments were performed for measurements of protein expression by densitometry.
Assays for Intracellular Signal Transduction
Primary cortical and microglial co-cultures were established as described above. Stock solutions of kinase inhibitors in DMSO were prepared at stock concentrations recommended by the manufacturer. Stocks were stored at -20oC and diluted into cell culture media prior to use. Four hours prior to injury and co-culture with microglia, signaling pathway inhibitors were added at concentrations of 0 µM, 10 µM, or 40 µM to the cortical cultures. Inhibitors tested were the following: MAPK inhibitor PD98059 (Cat#9900S), PI3K/AKT inhibitor LY294002 (Cat#9901S), PKC and Glycogen synthase kinase-3 inhibitor GF109203X (Cat#984150), Janus kinase 2 inhibitor AG490 (Cat#14704S). All inhibitors were purchased from Cell Signaling Technologies (Danvers, MA). Uninjured and injured cortical cells that were not cultured with microglia were used as controls. For control experiments, DMSO vehicle diluted in culture media was used in the experiments. After 48 hours, cells were analyzed using MTT assays (see above) or fixed with 4% PFA in PBS to observe expression for neurogenic markers, Nestin, α-internexin, and GFAP using immunocytochemical methods as described above. To quantify imaging data, 3 field views of at least 100 cells from 3 separate experiments were analyzed for each condition.
Immunoprecipitation for AKT/pAKT Analysis
Immunoprecipitation for AKT and pAKT was used to increase specificity and detection of AKT protein in cellular lysates. Cellular cultures were lysed as described above and each condition was split into two aliquots (200 µl each). Primary antibodies AKT (pan) (C67E7) rabbit mAB (Cell Signaling Technology Cat# 4691, RRID:AB_915783) and Phospho- AKT (Thr308) rabbit mAB (Cell Signaling Technology Cat#9275, RRID:AB_329828) were added at 1:50 for each sample and rotated overnight at 4 °C. A 50% slurry of EZview Red Protein A Affinity Gel Beads (Cat#P6486, EDM Millipore Sigma, Darmstadt, Germany) were added at 1:10 for each sample and rotated for 1 hour at 4 °C. Cells were centrifuged at 8,200g for 1 minute and washed with lysis buffer 3 times. Samples were heated at 95 °C for 5 minutes with 25 µl 3X sample buffer. Samples were run on 4-20% gradient SDS-polyacrylamide gels (Cat#4561096, BioRad, Hercules, CA) using SDS-PAGE and then transferred to PVDF membrane. After transfer, membranes were blocked using BSA Blocking Buffer™ in TBS for 1 hour and then gently rocked with the primary AKT or pAKT antibody at 1:1000 in BSA Blocking Buffer™ in TBS overnight at 4°C. Blots were washed and incubated in secondary anti-rabbit HRP conjugated antibody for 1 hour at RT. Membranes were developed using chemiluminescence as described above. Three separate experiments were performed. Statistical analysis involved analysis of densitometric images acquired with BioRad ChemiDoc QRS imaging system and software (BioRad,Hercules CA) were performed as described above.
Data are expressed as mean values and error bars represent standard error of the mean (SEM). Student T test with Bonferroni’s correction or one- way ANOVA followed by Tukey-Kramer post hoc tests were performed where appropriate. Values of p<0.05 were considered to be significant. All statistical analyses were performed with Graphpad Prism 6 (La Jolla, CA).