Additionally, if the responses (nociception, protein expression, and mRNA expression) were measured only once after the stimulus injection, the differences between responses were evaluated by one-way ANOVA followed by Tukey’s test (for three or more groups) comparing all pairs of columns. and vidarabine shorten the duration of skin lesions and complications related to herpes zoster (Tyring, 2007). Although nonsteroidal anti-inflammatory drugs (NSAIDs), antidepressants, and sympathetic nerve blockers are used for the management of herpetic neuralgia, these treatments do not usually relieve severe pain (Gan et al., 2013). The underlying pathophysiological mechanisms of spontaneous and evoked pain in herpes zoster are not well comprehended. However, because of species specificity of contamination. After cutaneous contamination of GDC-0879 HSV-1, mice develop a zosteriform-like skin lesion, which is usually followed by herpetic pain-like behavior (Takasaki et al., 2000a; Takasaki et al., 2000b). This resembles some clinical and pathophysiological features of herpes zoster contamination and could be used to study the immune mechanisms involved with the development of herpetic neuralgia (Takasaki et al., 2000b). Using this model of HSV-1 cutaneous contamination, we describe here the neuroimmuneCglia mechanisms occurring at the sensory ganglia accounting for the development of herpetic neuralgia. Materials and Methods Animals. The experiments were performed using male C57BL/6 or BALB/C WT mice (20C25 g); LysM-eGFP mice, which GDC-0879 carry a knock-in mutation for enhanced green fluorescent protein (eGFP) in the lysozyme M-locus (Faust et al., 2000); mice deficient in TNFR1 (were as follows: PEBP2A2 forward: 5-TGTGCTCAGAGCTTTCAACAA-3; reverse: 5-CTTGATGGTGGTGCATGAGA-3; forward: 5-GTGGAAAAACCTCGTCCAGA-3; reverse: 5-GCTCGGCTTCCAGTATTGAG-3; forward: 5-AGGGCGAAGAAAACCGCATCACC-3; reverse: 5-CGGCCAAATCCGTTCAC-3; forward: 5-GTGACAGGCAAACTGCTTCA-3; reverse: 5-GGGCTATCAGAGGCTGTGTC-3; forward: 5-CATCTTCTTGTGCAGTGCCA-3; and reverse: 5-CGGCCAAATCCGTTCAC-3. Flow cytometry analysis. DRGs L3, L4, L5, and L6 were collected from infected mice and analyzed as a pool. DRGs were prepared as single-cell suspensions according to standard protocols. Briefly, DRGs were incubated in colagensase for 75 min at 37C. After this time, the tissues were digested through vortex and 1 ml of RPMI medium (10% SBF) was added to the cell suspension made up of collagenase. The cells were washed once with PBS and then suspended and incubated for 30 min at 4C in PBS made up of 2% BSA and FcgRI block mAb (CD16/CD32) to avoid nonspecific background staining. The following monoclonal antibodies (BD Biosciences) were used for staining: APC rat anti-mouse CD45, PE rat anti-mouse Ly6-G/Gr-1, FITC rat anti-mouse F4/80, FITC rat anti-mouse CD4, and PE rat anti-mouse CD8. Flow cytometry was performed in a FACSCanto instrument (BD Biosciences) and analyzed with FlowJo software (TreeStar). Western GDC-0879 blot analysis. DRGs L3, L4, L5, and L6 were collected, pooled, and homogenized in a lysis buffer made up of a mixture of protease inhibitors and phosphatase inhibitors (Sigma-Aldrich). The protein concentrations of the lysate were determined using a BCA Protein Assay kit (Pierce) and 30 GDC-0879 g of protein was loaded for each lane. Protein samples were separated on a SDS-PAGE gel (10% gradient gel; Bio-Rad) and transferred to nitrocellulose membranes (GE Healthcare). The filters were blocked with 5% dry milk and incubated overnight at 4C with a primary antibodies against COX-2 (1:500; Abcam), GFAP (1:1000; Cell Signaling Technology), and Kir4.1 (APC035A; Alomone). The membranes were washed and incubated for 2 h at room heat with an HRP-conjugated secondary antibody (1:10000; Jackson ImmunoResearch). Immunodetection was performed using an enhanced chemiluminescence light-detecting kit (GE Healthcare) for 1 min. A mouse monoclonal antibody against -actin (1:10000; Sigma-Aldrich) was used for loading controls. Images were used as representative blots. Densitometric data were measured after normalization to the control (housekeeping gene) using Scientific Imaging Systems Image labTM 3.0 software (BioRad). DRG immunohistochemistry. Mice were terminally anesthetized with ketamine/xylazine and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4 (4C). After the perfusion, DRGs L4 and L5 were removed and postfixed in the same fixative for 2 h, which was then replaced overnight with 20% sucrose. DRGs were embedded in OCT and sections (14 m) were cut in a cryostat and processed for immunofluorescence. All of the sections were blocked with 1% BSA in 0.1% Triton X-100 for 1 h at room temperature and incubated overnight at 4C with the following antibodies: mouse anti-TNFR1 (clone 55R-28, 1:400; Biolegend), mouse anti-TNFR1 (sc-8436; Santa Cruz Biotechnology), rabbit anti-glutamine synthase (GS, 1:400; AB73593; Abcam), rabbit anti-Kir4.1 (APC035A; Alomone) or goat anti-GFP (1:100, AB6662; Abcam). After washing, a mixture of Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen/Thermo Scientific) were added for 2 h at room heat. Finally, the slides were coverslipped with.