Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Comparison of L5-VRT and SNI-induced expression of CX3CL1 in bilateral ACC. Representative protein microarray results ( A ) and the quantification analysis ( B ) showing that SNI triggered upregulations of chemokines CX3CL1 and CCL11 in contralateral ACC.* p < 0.05, ** p < 0.01 versus sham group ( n = 3 rats/group, one-way ANOVA). C , D Representative western blotting of CX3CL1 protein levels in bilateral ACC following SNI ( C ) and L5-VRT ( D ) are shown on the top and the quantification results are shown below. Significant differences are observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group. * p < 0.05 versus sham group ( n = 3 rats/group, one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Comparison, Expressing, Microarray, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Nerve injury-induced CX3CL1 is expressed in ACC neurons. Double-immunofluorescence staining showing the co-localization of CX3CL1-IR (red) with NeuN (green, top), but not Iba1 (green, medial) and GFAP (green, below) 7d post-SNI. Scale bar = 50 μm. The fluorescence intensity curves for red and green from boxed areas are shown on the right side of each group

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Double Immunofluorescence Staining, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Effects of DREADD-hM3Dq and hM4Di on ACC CX3CL1 protein levels and the mechanical paw withdrawal thresholds. A Behavioral test paradigms are shown on the top and representative western blotting for bilateral ACC CX3CL1 in the mice injected with DREADD-Gq or DREADD-Gi are shown below. B The injection site of virus carrying hM3Dq-, hM4Di-mCherry or mCherry in contralateral ACC. C Quantification of bilateral CX3CL1 in the mice injected with DREADD-Gq and DREADD-Gi, respectively. * p < 0.05, *** p < 0.001 versus mCherry control groups ( n = 3 mice/group, two-way ANOVA). D Compared to the mCherry control mice, DREADD-hM3Dq induced significant decrease of threshold in ipsilateral hind paws of normal mice (left); DREADD-hM4Di reversed the SNI-induced decrease of ipsilateral threshold (right). * p < 0.05 versus mCherry control groups ( n = 6 mice/group, multiple t tests)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Western Blot, Injection, Virus, Control

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Microinjection of anti-CX3CL1 antibody into ACC attenuates SNI-induced mechanical allodynia and inhibits ACC TNF-α and Nav1.6 protein upregulations. A , B Behavioral test paradigms are shown on the first left, and the ipsilateral and contralateral test results are shown on the second and first right, respectively. Statistical significance was determined by Dunn's multiple comparisons test (* p < 0.05; ** p < 0.01 versus PO -1d) or multiple t tests (# p < 0.05 versus IgG control). C The mark left by the cannula implantation showing the drug injection site in Cg1 region. D Representative western blotting of CX3CL1, TNF-α and Nav1.6 in bilateral ACC following contralateral administration of anti-CX3CL1 antibody (1 μg/μl, 3 μl) 7d post-SNI. E The quantification results showing the inhibitory effects of anti-CX3CL1 antibody on SNI-induced CX3CL1, TNF-α and Nav1.6 expressions. * p < 0.05, ** p < 0.01, *** p < 0.001 versus IgG control (one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Microinjection, Control, Injection, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Pretreatment with anti-CX3CL1 antibody in contralateral ACC modulates the expression levels of spinal c-Fos, Iba1, TNF-α, IL-6 after SNI surgery. A Pretreatment with anti-CX3CL1 antibody in contralateral ACC (1 μg/μl, 3 μl) 30 min before nerve injury downregulates Iba1-IR in ipsilateral SDH 7d post-SNI. The injection site in ACC and the detection area by grey rectangular box in spinal cord are shown on the left. Representative images obtained from sham and SNI rats pretreated with IgG or anti-CX3CL1 antibody (upper right). Scale bar = 500 μm. Insets from ipsilateral SDH (lower right). Scale bar = 50 μm. B Quantification for mean fluorescence intensity of Iba1 from bilateral SDH (*** p < 0.001, two-way ANOVA). C – G The expression levels of Iba1, CD11b, c-Fos, TNF-α and IL-6 in ipsilateral SDH following anti-CX3CL1 treatment in contralateral ACC and quantification for western blot data. * p < 0.05, ** p < 0.01, *** p < 0.001 (paired t test or one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Expressing, Injection, Fluorescence, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Graphical abstract: anterior cingulate cortex (ACC), a first-order cortical region that responds to painful stimuli, may play important roles in the occurrence of mirror-image pain. In this study, we provide evidence that in ACC, stronger immune response to L5-VRT surgery contributes to mirror pain, and CX3CL1 mediates the descending facilitation and aggravates the spinal neuroinflammation. Strategies targeting chemokine-mediated ACC hyperexcitability may develop novel therapeutics for neuropathic pain

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques:

Journal: Veterinary immunology and immunopathology

Article Title: Porcine Treg depletion with a novel diphtheria toxin-based anti-human CCR4 immunotoxin

doi: 10.1016/j.vetimm.2016.10.014

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: The monovalent, bivalent and single-chain foldback diabody anti-human CCR4 immunotoxins as well as DT390 alone were produced in our lab using unique diphtheria toxin resistant yeast Pichia Pastoris expression system ( Wang et al., 2015 ) and biotinylated as previously described ( Wang et al., 2015 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Name Clone# Source Cat# FITC- mouse anti-pig CD1 76-7-4 In house FITC-mouse anti-pig CD3 898H-2-6-15 In house FITC-mouse anti-pig CD4a 74-12-4 In house FITC-mouse anti-pig CD45RA Fg2F9 In house Biotin-mouse anti-pig CD45RA Fg2F9 In house FITC-mouse anti-pig CD16 G7 In house PerCp-Cy5.5-mouse anti-pig CD4a 74-12-4 BD Bioscience 561474 PE-mouse anti-pig CD8α 76-2-11 BD Bioscience 559584 Alexa Fluor® 647-mouse anti pig CD8α 76-2-11 BD Bioscience 561475 PE-mouse anti-human CD21 B-ly4 BD Bioscience 555422 PE-rat anti-pig γδ T lymphocytes MAC320 BD Bioscience 561486 PE-mouse anti-pig CD172a 74-22-15A BD Bioscience 561499 PE-mouse IgG 2b κ MPC-11 BD Bioscience 559529 Fluorescein-mouse anti-human/rat CCR4 205410 R&D Systems FAB1567F PE-mouse anti-human/rat CCR4 205410 R&D Systems FAB1567P Mouse IgG2B fluorescein isotype control 133303 R&D Systems IC0041F PE-mouse anti-human CD194 (CCR4) L291H4 BioLegend 359412 PE-mouse IgG1, κ isotype control MOPC-21 BioLegend 400139 PE-streptavidin BioLegend 405204 APC-streptavidin Biolegend 405207 FITC-rat anti-mouse Foxp3 FJK-16s eBioscience 11-5773-82 FITC-rat IgG2a, κ isotype control eBR2a eBioscience 11-4321-42 Propidium Iodide Sigma 81845 7-Aminoactinomycin (7-AAD) Sigma A9400 Open in a separate window Antibodies used in this study

Techniques: Control