Journal: Nature Communications

Article Title: Targeting C1q prevents microglia-mediated synaptic removal in neuropathic pain

doi: 10.1038/s41467-025-59849-1

Figure Lengend Snippet: a Volcano plot showing gene expression changes in neuropathic mice at day 7 post SNI. Each dot is a gene. Positive fold change indicates higher expression in the spinal cord of SNI-injured mice than in naïve. Genes highlighted in blue are related to complement and CX3CR1 pruning pathways. Dashed horizontal orange line indicates P -value of 0.05. b Drawing illustrates microglia and signaling molecules that participate in complement and CX3CR1 pruning mechanisms. c1 - e1 Representative images of C1q, C3, and CR3 labeling in the dorsal horn of SNI mice at day 7 post-SNI. c2 - e2 Quantifications of fluorescence intensity of dorsal horn C1q, and C3, as well as number of CR3 + microglia in laminae I-III ( n = 8 mice per group; 4 mice per sex). f Temporal expression changes of ipsilateral C1q, C3, and CR3 levels normalized to the contralateral side ( n = 5 mice per group; 3 males and 2 females). g1-h1 Representative dorsal horn images of CX3CL1 and CX3CR1 immunolabelling in SNI mice at day 7 post-SNI. g2-h2 Quantifications of fluorescence intensity of dorsal horn CX3CL1, and the number of CX3CR1 + microglia in laminae I-III ( n = 8 mice per group; 4 mice per sex). i1 High-resolution image of CX3CR1 (red) expression in microglia (blue) sampled from ipsilateral and contralateral dorsal horn (quantified i2 ) ( n = 8 mice per group; 4 mice per sex). j Temporal expression changes of ipsilateral number of CX3CR1+ microglia and CX3CL1 fluorescence intensity levels normalized to the contralateral side ( n = 5 mice per group; 3 males and 2 females). For transcriptomics, false discovery rate (FDR) is used to correct for multiple two-sided t-tests ( a ). Means are plotted with individual data points ± SEM. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 analyzed with paired two-tailed t-test ( c2 - h2 ) and unpaired two-tailed t-test ( i2 ). ns, analyzed with two-tailed t-test comparing day 3 and month 5 time points ( f , j ). Source data are provided as a Source Data file.

Article Snippet: For the C1q neutralizing antibodies experiment, animals received i.p injections of either the ANX-M1.21 anti-C1q function blocking antibody (provided by Annexon Biosciences) or IgG isotype control (provided by Annexon Biosciences) at 100 mg/kg every 4 days starting 1 day prior to peripheral nerve injury until the experimental endpoint.

Techniques: Gene Expression, Expressing, Labeling, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting C1q prevents microglia-mediated synaptic removal in neuropathic pain

doi: 10.1038/s41467-025-59849-1

Figure Lengend Snippet: a1 RNA scope for C1qa ( a1 : green) in contralateral and ipsilateral dorsal horn cells (quantified in a3 ) ( n = 5 mice per group; 3 males and 2 females). Absence of C1qa ( a2 : red) expression in inhibitory and excitatory neurons (blue) (quantified in a4 ) ( n = 5 mice per group; 4 males and 2 females). b1 Representative high-magnification confocal image of C1q protein (green) in microglia (blue) (quantified in b2 ) ( n = 6 mice per group; 3 mice per sex). Representative SIM images ( c1-2 ) captured from ipsilateral dorsal horn at 7 days post-SNI showing the co-localization of C1q (green) with inhibitory (VGAT in red), and excitatory synapses (VGLUT2 in blue). Dotted circles show synapses that are colocalized with C1q. c3 Quantifications of C1q co-localization with inhibitory and excitatory synapses ( n = 10 mice per group; 5 mice per sex). d1-2 Depletion of C1q expression in the dorsal horn of neuropathic mice chronically treated with vehicle and PLX3397 (quantified in d3 ) ( n = 6 mice per group; 3 mice per sex). d4 Quantifications of C1q co-localization with inhibitory and excitatory synapses in vehicle and PLX3397 treated mice ( n = 3 male mice per group). Means are plotted with individual data points ± SEM. *** p < 0.001, and **** p < 0.0001 analyzed by two-way ANOVA with Bonferroni post hoc test ( a3, c3 , and d3-4 ) and unpaired two-tailed t-test ( a4 and b2 ). Source data are provided as a Source Data file.

Article Snippet: For the C1q neutralizing antibodies experiment, animals received i.p injections of either the ANX-M1.21 anti-C1q function blocking antibody (provided by Annexon Biosciences) or IgG isotype control (provided by Annexon Biosciences) at 100 mg/kg every 4 days starting 1 day prior to peripheral nerve injury until the experimental endpoint.

Techniques: RNAscope, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting C1q prevents microglia-mediated synaptic removal in neuropathic pain

doi: 10.1038/s41467-025-59849-1

Figure Lengend Snippet: a A diagram showing the timeline of drug treatments, surgery, and behavior testing. b Pain behaviour in neuropathic mice treated either with ANX-M1.21 or IgG control (n = 20 mice per group; 12 males and 8 females). c1 Representative images from ipsilateral dorsal horn show the co-localization of C1q (green) with inhibitory synapses (VGAT; red), and excitatory (VGLUT2; blue) in different experimental groups (Quantified in c2 ) ( n = 6 mice per group; 3 mice per sex). d1 and e1 Representative 3D surface rendering of microglia (white) from ipsilateral dorsal horn of IgG control and ANX-M1.21 treated mice. A single plane enlarged image selected from the confocal stack illustrating the CD68 (magenta) co-localization with VGAT or VGLUT2 ( d1 , e1 : green). Insets within 3D reconstructions are enlarged views of VGAT ( d1 ) or VGLUT2 ( e1 ) co-localization with CD68 (quantified in d2 and e2 ) ( n = 10 mice per group; 5 mice per sex). f1 Representative images of inhibitory presynaptic (VGAT; red) and postsynaptic (gephyrin; blue) elements from different conditions and quantification in f2 (n = 20 mice per group; 12 males and 8 females). g1 Representative images of excitatory pre-synaptic (VGLUT2; cyan) and post-synaptic (homer1; magenta) elements from different conditions and quantification in g2 ( n = 20 mice per group; 12 males and 8 females). Means are plotted with individual data points ± SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001 analyzed with repeated measures two-way ANOVA with Bonferroni post hoc test ( b ) two-way ANOVA with Bonferroni post hoc test ( d2, e2, f2 , and g2 ), and t-test ( c2 ). Source data are provided as a Source Data file.

Article Snippet: For the C1q neutralizing antibodies experiment, animals received i.p injections of either the ANX-M1.21 anti-C1q function blocking antibody (provided by Annexon Biosciences) or IgG isotype control (provided by Annexon Biosciences) at 100 mg/kg every 4 days starting 1 day prior to peripheral nerve injury until the experimental endpoint.

Techniques: Control