Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Representative photos showing hind paw of rat receiving CPIP model establishment. ( B ) Time course showing the thickness of ipsilateral hind paw of sham and CPIP model rats. n = 5 rats per group. ( C ) Time course showing 50% paw withdrawal threshold (PWT) from sham and CPIP model rats. n = 5 to 6 rats per group. ( D ) Flow chart of scRNA-seq. Created in BioRender. Pan, Y. (2025) https://BioRender.com/wd2ghrz . ( E ) UMAP plot of cell clusters identified by scRNA-seq. vSMC, vascular smooth muscle cell. ( F ) Cell populations identified from immune cell cluster by marker genes. ( G ) Distribution of specific immune cell population (including macrophages, neutrophils, and T or B cells) in DRG of sham versus CPIP model rats. ( H ) Summary of increase in the number of cells in DRG after CPIP modeling. ( I ) CD68 (red, marker for macrophage) and NeuN (green, marker for neuron) immunostaining in ipsilateral L4-L6 DRG from sham and CPIP groups. Scale bar, 50 μm. ( J ) Flow cytometry of macrophages expressing CD68 in cell suspensions from DRG of sham and CPIP groups on day 7. A total of 10,000 cells were analyzed for each. ( K ) Summary of the percentage of CD68 + cells in DRG as in (J). n = 3 to 4 rats per group. ( L ) Representative pictures showing CD68 immunostaining in ipsilateral L4-L6 DRG 3, 7, and 14 days after model establishment. 4′,6-Diamidino-2-phenylindole (DAPI) was used as counterstain. Scale bar, 50 μm. ( M ) Summary of the number of CD68 + cells/mm 2 as in (L). n = 5 to 6 rats per group. 7D, 7 days. ( N ) Representative pictures showing CD68 immunostaining in ipsilateral sciatic nerve. Scale bar, 100 μm. ( O ) Summary of the number of CD68 + cells/mm 2 as calculated in (N). n = 6 rats per group. * P < 0.05 and ** P < 0.01 versus sham group. NS, no significance. Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for [(B) and (C)]. One-way ANOVA followed by Tukey’s post hoc test for (M). Student’s unpaired t test for [(K) and (O)].

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Marker, Immunostaining, Flow Cytometry, Expressing

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Time schedule. ( B ) CD68 immunostaining in ipsilateral L4-L6 DRG of sham + IgG, CPIP + IgG, and CPIP + Reg3β-neutralizing Ab groups. Scale bar, 50 μm. ( C ) Summary of the number of CD68 + cells/mm 2 from three groups as in (B). n = 7 rats per group. ( D ) 50% PWT changes in three groups. n = 8 to 10 rats per group. * P < 0.05 and ** P < 0.01 versus sham + IgG group. ## P < 0.01 versus CPIP + IgG group. ( E ) Strategy for Reg3b -specific knockdown in DRG neurons using AAV-PHP.S with neuronal promoter hSyn. ( F ) EGFP detected in DRG of control (without viral injection), scramble-transfected, and Reg3b shRNA-transfected groups. Scale bar, 100 μm. ( G ) Summary of the percentage of EGFP + NeuN + cells in DRG of three groups as in (F). n = 6 rats per group. ( H ) Western blotting showing Reg3β expression in DRG of CPIP + scramble versus CPIP + Reg3b shRNA group. n = 5 rats per group. ( I ) CD68 + immunostaining in DRG of sham + scramble, CPIP + scramble, and CPIP + Reg3b shRNA groups. Scale bar, 50 μm. ( J ) Summary of the number of CD68 + cells in DRG of three groups of rats as in (I). n = 5 rats per group. ( K ) 50% PWT changes in sham + scramble, CPIP + scramble, and CPIP + Reg3b shRNA groups. n = 5 to 6 rats per group. ** P < 0.01 versus sham + scramble group. # P < 0.05 versus CPIP + scramble group. ( L ) Area under the curve (AUC) analysis of the curves shown in (K). n = 5 to 6 rats per group. ( M ) C-Fos immunostaining in ipsilateral SCDH of three groups. C-Fos was magnified and shown in the upper right, and its outline was illustrated in lower right. Scale bar, 50 μm. ( N ) Summary of the number of c-Fos + cells in ipsilateral SCDH per observation field as shown in (M). n = 5 to 6 rats per group. * P < 0.05 and ** P < 0.01. One-way ANOVA followed by Tukey’s post hoc test for [(C), (G), (J), (L), and (N)]. Student’s unpaired t test for (H). Two-way ANOVA followed by Tukey’s post hoc test for [(D) and (K)]. w, weeks; d, days.

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Immunostaining, Knockdown, Control, Injection, Transfection, shRNA, Western Blot, Expressing

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Strategy for Reg3b -specific overexpression in naïve rat DRG neurons using AAV-PHP.S with neuronal promoter hSyn. i.pl, intraplantar. ( B ) Time points for experiments in this study. ( C ) EGFP detected in ipsilateral DRG of control (nontransfected) and AAV-PHP.S-hSyn-EGFP (AAV-EGFP)–transfected rats. Scale bar, 100 μm. ( D ) Summary of the percentage of EGFP + NeuN + cells in DRG as shown in (C). n = 5 rats per group. ( E ) Western blotting examining Reg3β expression in DRG of AAV-EGFP versus AAV-PHP.S-hSyn- Reg3b -EGFP (AAV- Reg3b -EGFP) groups of rats. n = 6 rats per group. ( F ) EGFP signals in ipsilateral/contralateral SCDH of control and AAV-EGFP–transfected naïve rats. Scale bar, 100 μm. ( G and H ) Summary of the normalized fluorescence intensity of EGFP in ipsilateral/contralateral SCDH of control and AAV-EGFP–transfected naïve rats as shown in (F). n = 5 to 6 rats per group. ( I ) 50% PWT changes in the injected hind paw (ipsilateral hind paw) of naïve rats transfected with AAV-EGFP or AAV- Reg3b -EGFP. n = 5 rats per group ** P < 0.01 versus AAV-EGFP group. ( J ) Immunostaining of CD68 + cells (in red) in ipsilateral DRG of rats receiving AAV-EGFP or AAV- Reg3b -EGFP transfection. DAPI is in purple. Scale bar, 50 μm. ( K ) Summary of the number of CD68 + cells/mm 2 in DRG of two groups of rats as shown in (J). n = 5 to 6 rats per group. ** P < 0.01. Student’s unpaired t test for [(D), (E), (G), (H), and (K)]. Two-way ANOVA followed by Tukey’s post hoc test for (I).

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Over Expression, Control, Transfection, Western Blot, Expressing, Fluorescence, Injection, Immunostaining

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) KEGG analysis of signaling pathways activated in macrophages in DRG of CPIP rats. ( B ) Tnfa expression by qPCR in DRG. n = 5 rats per group. ( C ) Tnfa , Il6 , Il1b , and Cxcl1 expression in DRG of sham, CPIP + Lipo, and CPIP + Clodro groups. n = 4 to 5 rats per group. ( D ) Tnfa (red) expression in ipsilateral DRG by RNAscope. DAPI was in white. Negative control probe against bacterial gene Dapb shows no staining. ( E ) Summary of Tnfa + signals in DRG as in (D). n = 5 rats per group. ( F ) Colocalization of Cd68 (green) with Tnfa (red) in DRG of CPIP rats by RNAscope. ( G ) Summary of Tnfa expression in certain types of cells in DRG of CPIP rats by scRNA-seq. NK, natural killer. ( H ) Schedule. ( I ) 50% PWT in ipsilateral hind paws of sham + Veh, CPIP + Veh, and CPIP + ETA groups. ** P < 0.01 versus sham group. ## P < 0.01 versus CPIP + Veh group. ( J ) Normalized AUC analysis of curves in (I). n = 6 rats per group. ( K ) APs in small-sized DRG neurons of three groups. Five hundred–picoampere depolarizing current (1000 ms) was used to stimulate APs firing. ( L ) Number of APs elicited by 500-pA depolarizing current as in (K). n = 23 to 25 cells per group. ( M ) Summary of the number of APs triggered by depolarizing current steps in DRG neurons. n = 23 to 25 cells per group. * P < 0.05 and ** P < 0.01 versus sham + Veh group. # P < 0.05 and ## P < 0.01 versus CPIP + Veh group. ( N ) APs elicited by ramp current. ( O ) Summary of the number of APs triggered by ramp current. n = 23 to 27 cells per group. ( P ) Summary of current threshold for eliciting APs by ramp current. n = 23 to 27 cells per group. One-way ANOVA followed by Tukey’s post hoc test for [(B), (C), (E), (J), (L), (O), and (P)]. Two-way ANOVA followed by Tukey’s post hoc test for [(I) and (M)].

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Protein-Protein interactions, Expressing, RNAscope, Negative Control, Staining

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Experiments schedule. ( B ) p-STAT3 and STAT3 expression in ipsilateral L4-L6 DRG of sham and CPIP model rats. n = 5 rats per group. ( C ) p-STAT3 immunostaining (green) in DRG of sham + Veh, CPIP + Veh, and CPIP + ETA groups. Neurons were stained with Nissl (red). ( D ) Normalized fluorescence intensity of p-STAT3 immunostaining in DRG of three groups as in (C). n = 5 rats per group. ( E ) p-STAT3 and STAT3 expression in ipsilateral L4-L6 DRG of three groups. n = 6 rats per group. ( F ) Overlapping of p-STAT3 + cells (green) with p-Syk + cells (red) and neurons (by Nissl, purple) in DRG of CPIP model rats. White arrows indicate overlapped cells. ( G ) Reg3β immunostaining (in red) in DRG of sham + Veh, CPIP + Veh, and CPIP + S3I-201 groups. ( H ) Normalized fluorescence intensity of Reg3β in DRG of three groups as in (G). n = 6 to 7 rats per group. ( I ) Immunostaining showing overlapping of p-STAT3 + cells (green) with Reg3β + cells (red) in DRG of CPIP model rats. Purple: DAPI. White arrows indicate overlapped cells. ( J ) CD68 immunostaining (red) in DRG of sham + Veh, CPIP + Veh, and CPIP + S3I-201 groups. DAPI in purple. ( K ) The number of CD68 + cells in DRG of three groups as in (J). n = 6 rats per group. * P < 0.05 and ** P < 0.01. ( L ) 50% PWT changes in three groups. n = 6 rats per group.** P < 0.01 versus sham + Veh group. ## P < 0.01 versus CPIP + Veh group. Scale bar is as indicated. One-way ANOVA followed by Tukey’s post hoc test for [(D), (E), (H), and (K)]. Two-way ANOVA followed by Tukey’s post hoc test for (L). Student’s unpaired t test for (B).

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Expressing, Immunostaining, Staining, Fluorescence

Journal: Frontiers in Behavioral Neuroscience

Article Title: Characterization of pain-related behaviors and gene expression profiling of peripheral sensory ganglia in a mouse model of acute ankle sprain

doi: 10.3389/fnbeh.2023.1189489

Figure Lengend Snippet: LAS model mice showed increased c-Fos and p-ERK immunoreactivity as well as glial cell overactivation in the ipsilateral spinal cord dorsal horn. (A) Left panel: representative pictures showing c-Fos staining of spinal cord dorsal horn (SCDH) of sham and LAS model groups. Summary is shown on the right. (B) Left panel: representative pictures showing GFAP staining of SCDH of sham and LAS model groups. Summary is shown on the right. (C) Left panel: representative pictures showing CD68 staining of spinal cord dorsal horn (SCDH) of sham and LAS model groups. Summary is shown on the right. (D) Left panel: representative pictures showing p-ERK staining of SCDH of sham and LAS model groups. Summary is shown on the right. ** p < 0.01, * p < 0.05. n = 5–6 mice/group. Student's t -test was used for statistics.

Article Snippet: The primary antibodies used were mouse anti GFAP (1:500, #c9205, Sigma), rabbit anti c-Fos (1:500, #2250, CST), rabbit anti p-ERK (1:500, #4370, CST), and rabbit anti CD68 (1:500, #NB100-683PE, Novus).

Techniques: Staining