RIG-I contributes to the innate immune response after cerebral ischemia

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Miami (protocol number 12–200). To induce transient middle cerebral artery occlusion (MCAO), male Sprague–Dawley rats were anesthetized with 3 % isoflurane in a mixture of 70 % N₂O/30 % O₂ for 5 min. Rats were intubated and immobilized with pancuronium bromide (0.35 mg/kg, IV) and mechanically ventilated. Brain and body temperature were monitored and the animals were maintained at a normothermic (37 °C) temperature. A catheter was implanted into the tail artery for blood-gas monitoring during surgery. The right common carotid artery was exposed with a midline neck incision and dissected from the surrounding nerves. The external carotid artery and superior thyroid artery were then ligated. A 4-cm length of 3–0 suture that was heat-blunted with a 20 mm section coated with poly-l-lysine solution (0.1 % wt/vol) was used for the occlusion. This suture was inserted at the proximal external carotid artery into the internal carotid artery to the MCA. The MCA was occluded for 90 min, then released for reperfusion. The neck incision was closed with staples and the animals were allowed to wake-up from the anesthesia. The animal was then taken to a cage supplied with food and water until termination of the study. Animals that presented with difficulty eating, presented blood pressure abnormalities, respiratory difficulties or brains that were not well perfused were euthanized and excluded from the study.

For detection of RLR proteins after MCAO, protein lysates ipsilateral to the ischemic side, of hippocampal brain areas were obtained and resolved by immunoblotting. We did not detect any effects of MCAO on RIG-I signaling on the contralateral side (data not shown). Sham animals were used as controls. Animals were sacrificed at different time points after ischemia (1 h, 4 h, 1d and 3d after MCAO) and then lysates were prepared and resolved by immunoblotting as described in de Rivero Vaccari et al. [16] using antibodies against RIG-I (1:1000, Anaspec), IFN-α (1:1000, Abcam) and β-actin (1:5000, Sigma). Data was normalized to β-actin.

For immunohistochemical analysis rats were anesthetized and perfusion-fixed with 4 % paraformaldehyde (PFA) as described in de Rivero Vaccari et al. [16].

Immunostained brain sections of sham and 4 h MCAO animals were examined with an Olympus FV-1000 Laser Scanning Confocal Microscope (Olympus). Rats were perfused with 4 % paraformaldehyde as described [16] and processed for cryostat sectioning (Leica SM 2000R Sliding Microtome). Sections (50 μm) were prepared for immunohistochemical analysis as described in de Rivero Vaccari et al. [16] with a primary antibody to RIG-I (1:500, Abcam) and with the astrocytic marker anti-glial fibrillary acidid protein (GFAP) (Millipore Bioscience Research Reagents). Alexa-Fluor secondary antibody conjugates (1:500, Invitrogen) were used. Secondary antibodies alone were used as control for antibody specificity.

Primary rat astrocyte cells (Lonza) were plated and grown in culture for 7 days prior to experimentation at a density of 4×10⁴ cells/9.5 cm² as described in de Rivero Vaccari et al. [14]. Cells were grown in Astrocyte Basic Medium (Lonza).

Astrocyte cultures were grown as described above. For OGD, cultures were exposed to 95 % nitrogen and 5 % CO₂ atmosphere maintained by constant gas flow at 37 °C in all experiments. Oxygen tension was kept at 5 % during the duration of the experiment. The Nitrogen/CO2 content was controlled with the ProOx P110 Oxygen Controller with an E702 Oxygen Sensor (model #P-110-E70) with single set point controller (#P110) (Biospherix). The media used in these OGD experiments consisted of hypoglycemic medium containing normal salts, 1 mg/mL bovine serum albumin (BSA) and 55 μM glucose (1 % of the normal glucose concentration). Control groups were grown under normal culture conditions (5 % CO₂ and 95 % Oxygen).

To test for NF-κB activation, protein lysates (Control, 2, 3 and 4 h after OGD) were obtained from rat astrocytes and assayed with the PathScan Phospho-NF-κB p65 (Ser536) Sandwich Elisa kit (Cell Signaling) according to manufacturer’s instructions. Briefly, 100 μl of cell lysate were added and sealed to the appropriate wells for a 2 h incubation period at 37 °C. Each well was washed 4 times with 1X Wash Buffer. A detection antibody was added to each well and incubated at 37 °C for 1 h. Then 100 μl of reconstituted HRP-Linked secondary antibody were added to each well and incubated for 30 min followed by adding 100 μl of TMB Substrate to each well for 10 min and a STOP solution. The plate was read using a spectrophotometer (Victor³ 1420 multilabel counter, Perkin Elmer) at 450 nm.

Astrocytes were grown in culture as described above in a 96-well plate. RLR signaling was stimulated in cells with poly(I:C)LMW (low molecular weight) at 3 different concentrations (1, 3 and 6 μg/ml, dissolved in sterile water) for 24 h. On day 2, 150 μl per well of B16-BLUE IFN-α/β cells (Invivogen) in suspension (50,000 cells) were added and incubated overnight at 37 °C in 5 % CO₂. On day 3, 50 μl per well of the supernatant were added to 150 μl of QUANTI-Blue (Invivogen). The plate was incubated for 2 h at 37 °C and read at 600 nm in a spectrophotometer (Victor³ 1420 multilabel counter, Perkin Elmer). A similar procedure was carried to test n-propyl-gallate (n-PG, dissolved in dimethyl sulfoxide (DMSO), MP Biomedicals) as a blocker of type I IFN signaling at different concentrations (5, 10 and 50 μM).

Statistical comparisons between the different groups were done using a two-tailed Student’s t-test or a one-way ANOVA followed by Dunnett’s multiple comparison tests. P-values of significance used were P < 0.05.