A neurotropic demyelinating strain of mouse hepatitis virus, MHV-A59, was used from our previous studies , for infecting mice and primary meningeal cultures to study the effect on gap junction protein Cx43.
Inoculation of Mice
Four-week old C57BL/6 mice were intracranially inoculated with 50% LD50 dose of a neurotrophic demyelinating strain of mouse hepatitis virus, MHV-A59 (2000 pfu) as described previously . Mice were monitored daily for symptoms of disease and mortality. Mock-infected controls were inoculated similarly with the vehicle containing phosphate-buffered saline (PBS) containing 0.75% bovine serum albumin (BSA) and maintained in parallel. All animals were euthanized at day 5 post-infection (p.i.) and brain tissues were harvested as described below. All experimental procedures were approved by the Institutional Animal Ethical Committee and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA, India).
Mice were perfused transcardially with PBS followed by PBS containing 4% paraformaldehyde (PFA) at day 5 p.i. Brain tissue was collected, post-fixed in 4% PFA overnight, and embedded in paraffin . Brains were sectioned at 5 μm and stained with hematoxylin and eosin (H&E) to evaluate inflammation or meningitis. The paraffin-embedded slides were first deparaffinized on a hot plate followed by xylene treatment for 10 mins. Sections were then rehydrated gradually through different alcohol grades from 100 to 50%. Sections were then treated with hematoxylin for 1 min and excess stain was washed under running tap water. Subsequently, sections were dehydrated by passing through 50 to 95% ethanol. Sections were then stained with eosin stain for 30 s and washed with 95% ethanol twice. Further dehydration was carried out with 100% ethanol and xylene. Sections were mounted with Refrax mounting medium (Anatech Ltd., MI, USA) and observed under the upright light microscope (Nikon Eclipse 50i) and analyzed with Nikon imaging software (NIS, Nikon Corp. Tokyo, Japan).
Immunofluorescence and Confocal Microscopy of Brain Cryosections
Mock and MHV-infected mice at day 5 p.i. were transcardially perfused and fixed with 4% PFA. Brain tissues were further post-fixed in 4% PFA overnight and isotonically equilibrated in 30% PBS sucrose. Tissues were embedded in OCT cryomatrix (Tissue Tek, Hatfield, PA) and sagittal sections (10 μm) were done on cryostat (Thermo Scientific). Tissue sections were mounted on charged slides and processed as described earlier . Briefly, sections were incubated with 1 M glycine in PBS for 1 h at room temperature (RT) to reduce non-specific cross-linking and subsequently treated with 1 mg/ml NaBH4 in PBS for 10 min at RT to reduce autofluorescence. Sections were washed in PBS and incubated with blocking serum containing PBS with 0.5% Triton X-100 and 2% normal goat serum. Sections were incubated overnight at 4 °C with anti-Cx43 in combination with either anti-vimentin or anti-viral nucleocapsid (N) antisera prepared in blocking serum at dilutions listed in Table 1. The following day, the sections were washed and incubated in appropriate FITC or Texas Red conjugated fluorescent secondary antibody (1:200; Jackson ImmunoResearch Inc., PA, USA) for 1 h at RT. All incubations were done in humid chambers. Slides were washed and mounted in Vectashield® mounting media containing DAPI (Vector Laboratories, CA, USA). Sections were visualized and imaged using a Zeiss® confocal microscope (LSM710; Carl Zeiss AG, Germany).
Primary Meningeal Culture
Primary meningeal cultures from day 0–1 C57BL/6 mouse pup brains were prepared as described earlier, with minor modifications . Briefly, the meninges were carefully removed, homogenized in Hank’s Balanced Salt solution (HBSS), and passed through a 70-μm cell strainer. The resulting flow-through were collected and centrifuged at 1000 rpm for 10 mins to pellet the meningeal cells. The cell pellet was washed twice with HBSS and re-suspended in growth medium containing Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, and 1% penicillin–streptomycin. The cells were plated and allowed to grow at 37 °C in a humidified CO2 incubator. After 72 h, all non-adherent cells were removed and fresh medium was added. Adherent cells were maintained as meningeal fibroblast culture in growth medium till confluency with a media change every 2–3 days.
Mixed Glia Cultures
Primary cultures of mixed glia from newborn (day 0–1) C57BL/6 mouse pup brains were prepared as described earlier . Briefly, the meninges were removed and brain tissue was minced and incubated in a shaking water bath at 37 °C for 30 min in HBSS containing 300 mg/ml DNase I (Sigma®) and 0.25% trypsin (Sigma®). Enzyme dissociated cells were triturated with 0.25% of FBS, washed and pelleted at 300×g for 10 min. The pellet was resuspended in HBSS and passed through a 70-μm strainer and centrifuged at 300×g for 10 min. The pellet was resuspended in astrocyte-specific medium (DMEM containing 1% l-glutamine, 10% FBS, and 1% penicillin–streptomycin), plated in T25 flasks, and allowed to grow at 37 °C in a humidified CO2 incubator. After 24 h, all non-adherent cells were removed and fresh astrocyte-specific medium was added. Adherent cells were allowed to grow to confluence with a media change every 3 to 4 days.
Confluent cultures were trypsinized and washed twice in flow buffer (PBS containing 2% FBS). Following centrifugation (300×g, 5 min), the cell pellet was resuspended in flow buffer and approximately 1 × 106 cells were distributed per polystyrene round-bottom 5-ml flow tubes. Cells were fixed in 100 μl Cyto-Fix buffer (BD Biosciences, San Jose, CA) for 15 min at RT. Following a wash in flow buffer, cells were incubated with anti-vimentin and anti-glial fibrillary acidic protein (GFAP) antibody diluted (1:40) in 1× BD Perm/Wash Buffer for 30 mins at RT. Cells were washed, centrifuged, and labeled with goat anti-mouse TRITC and goat anti-rabbit FITC diluted (1:40) in BD Perm/Wash buffer and incubated for 30 mins at RT. After a final wash, all tubes were resuspended in 500 mL of flow buffer and subjected to flow cytometry in a BD FACS Verse™ flow cytometer and analyzed using BD FACSuite™ software .
Infection of Primary Meningeal Culture
Primary meningeal fibroblasts were infected with MHV-A59 for investigating the effect of virus infection on meningeal gap junction protein Cx43, following previously published protocols . In brief, meningeal cultures were infected with MHV-A59 diluted in inoculation medium (DMEM containing 2% FBS and 1% penicillin–streptomycin) at a multiplicity of infection of 2 and incubated for 1 h at 37 °C in a humidified CO2 chamber. After initial viral adsorption for 1 h, medium was changed and infected cells were maintained in normal growth medium for 24 h. For mock infection, parallel cultures were initially incubated in the inoculation medium for 1 h followed by normal growth medium. All cells were collected 24 h p.i. and used for immunolabeling and biochemical assays.
Immunofluorescence and Confocal Microscopy
Immunofluorescence staining of primary meningeal fibroblasts was performed as described previously , with minor modifications. In brief, cells fixed with 4% PFA for 15 mins at RT were permeabilized in PBS containing 0.5%Triton X-100 and incubated in blocking serum (PBS containing 0.2%TritonX-100 and 2.5% goat serum) for 1 h. Cells were incubated for 1 h at RT with a combination of anti-Cx43, anti-vimentin, anti-N anti-GFAP, and anti-calnexin antisera at dilutions listed in Table 1. Subsequently, the cells were washed and incubated for 1 h at RT with appropriate combination of FITC or Texas Red conjugated secondary antibody (1:200) diluted in blocking serum. Cells were washed and mounted in Vectashield mounting media containing DAPI. Cells were visualized in Zeiss® Confocal Microscope (LSM710) or Axio observer microscope with apoptome module (Carl Zeiss, Germany) and images were acquired and processed with Zen 2012 (Carl Zeiss AG, Germany) software.
Mock and virus-infected cells (24 h p.i.) were washed with PBS and homogenized in ice-cold protein extraction buffer (25 mM Tris (pH 7.6), 1 mM MgCl2, 1% Triton X-100, 0.5% SDS with 1X EDTA-free complete Protease Inhibitor (Roche, Mannheim, Germany) and phosphatase inhibitors (1 mM NaVO4 and 10 mM NaF)) by mild vortexing at regular time interval for 1 h. Lysed cell suspension was centrifuged at 4 °C for 10 min at 13,200 rpm using an Eppendorf® 5415 R centrifuge. The resulting supernatant was quantified for total protein concentration using a Pierce® BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amount (5 μg) of total protein was resolved by 12% SDS-PAGE . Proteins were transferred to PVDF membrane in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% v/v Tween-20 (TBST) for 1 h at RT and incubated overnight at 4 °C with anti-Cx43 and anti-vimentin antibodies at dilutions listed in Table 1. Membranes were then washed with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2000; Jackson ImmunoResearch Inc., PA, USA) for 2 h at RT. Membranes were developed using Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) and images were captured using Syngene G: Box™ ChemiDoc system and GENEsys software. Membranes were subsequently re-probed with γ-actin antibody and densitometric analysis of immunoreactive bands was carried out using ImageJ software.
Lucifer Yellow Scrape-Loading and Dye Transfer Assay
To evaluate gap junction coupling, meningeal fibroblasts were grown on 35-mm culture dishes to 100% confluency. Permeability mediated by gap junctions under control and MHV-A59-infected conditions (24 h p.i.) was determined by gap junction permeable Lucifer yellow (LY) dye transfer experiment, using a protocol described previously , with minor modifications. LY dye (Sigma, Saint Louis, MO, USA) dissolved in PBS (4 mg/ml) was scrape loaded onto confluent monolayer of meningeal fibroblasts. After 1 min of incubation, LY was thoroughly washed with PBS, fresh 10% DMEM was added, and dye spreading from scrape line was imaged using an Olympus IX-81 microscope system appended with a Hamamatsu Orca-1 CCD camera. Images were processed and analyzed using ImagePro (Media Cybernetics) and ImageJ software and the distance of dye spread from the scrape-loading line was measured.
In Situ TritonX-100 Solubilization
Cells plated on coverslips were washed with PBS and treated with 1%Triton X-100 at 4 °C for 30 mins to solubilize the Cx43 molecules not involved in gap junction plaques while the Cx43 molecules involved in cell surface gap junction plaques remain insoluble. Upon thorough washing and extraction of solubilized Cx43 molecules, cells were fixed and immunofluorosence labelling was performed as described previously . Cells were visualized in Axio observer microscope with apoptome module (Carl Zeiss, Germany) and images acquired and processed with Zen 2012 (Carl Zeiss AG, Germany) software.
Data are presented as mean ± SEM. Significance of the difference between two experiment groups for the LY dye transfer assay and western blotting was determined by unpaired two-tailed Student’s t test. The data from cell enrichment experiment were analyzed by one-way analysis of variance, and pairwise comparisons were made using the post hoc Tukey method for multiple testing. Statistical significance was set at p < 0.05. All statistical analysis were carried out using GraphPad prism (ver. 7) software (GraphPad Software, Inc).