Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Exogenous BMP4-induced allodynia, microglial activation and polarization. ( A ) PWT in rats after intrathecal BMP4 application showed a significant effect on time: F (4.143, 66.29) = 16.09, P<0.01, left; F (3.960, 63.35) = 12.67, P<0.01, right, intrathecal application: F (1, 16) = 161.3, P<0.01, left; F (1, 16) = 160.5, P<0.01, right and interaction: F (7, 112) = 11.76, P<0.01, left; F (7, 112) = 5.653, P<0.01, right. Compared with the Sham group, rats in the BMP4 group developed a significant decrease in bilateral PWT for the whole first week, P<0.01, n=9 at each time point for both groups. ( B ) Representative Western blotting showed a sustained increase of CD11b (P<0.01) and CD16 (P<0.01) expressions for the whole 1st week in the BMP4 group compared with the Sham group. Meanwhile, ARG-1 levels increased at day 1 (P<0.01) and day 4 (P<0.01), then fell below the levels of the Sham group (P<0.01). n=3 for each column. ( C and D ) Double-immunofluorescence further detected that compared with the Sham group (P7), CD11b expression was elevated in the dorsal horn of spinal cord after BMP4 treatment. Moreover, expressions of CD16 and ARG-1 showed similar pattern with Western blotting results, which were both mainly accumulated with CD11b + cells. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test ( A ) and one-way ANOVA, followed by Sidak’s multiple comparisons test ( B – D ) were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Exogenous BMP4 drove the imbalance of the M1/M2 ratio. ( A ) The steps and gating strategies of CD11b + microglia. ( B and C ) Representative flow cytometry images showed that exogenous BMP4 induces a sustained elevation of CD11b + CD86 + M1 microglia from day 1 to day 7. In the meantime, CD172 + CD11b + M2 microglia increased at day 1 and day 4, then fell to the Sham level at day 7. Moreover, the M1/M2 ratio persistently increased from P1 to P7 throughout the whole 1st week, indicating BMP4 induced a lasting imbalance of M1/M2 ratio. n=3 for each time point for both groups. One-way ANOVA, followed by Sidak’s multiple comparisons test were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Noggin relieved allodynia, attenuated microglial activation, and changed the polarized pattern after SNL. ( A ) PWT in rats receiving intrathecal Noggin application after SNL showed a significant effect on time: F (3.463, 55.41) = 162.3, P<0.01, intrathecal application: F (1, 16) = 44.85, P<0.01 and interaction: F (7, 112) = 3.540, P<0.01. Compared with the SNL group, Noggin application provided marked pain relief at day 3, day 4, day 5 and day 7 after SNL. n=9 at each time point for both groups. ( B and C ) Representative Western blotting showed SNL induced CD11b and CD16 upregulation at the first week, while ARG-1 expression remained unchanged compared with the Sham group; Moreover, Noggin effectively decreased the CD11b expressions at P1 and P4 and the CD16 expressions consecutively for the whole week. Simultaneously, the ARG-1 levels significantly elevated from P1 to P7 after Noggin treatment. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test (A) and one-way ANOVA, followed by Sidak’s multiple comparisons test (B and C) were performed to analyze the statistical differences. **Represented P<0.01 compared with the Sham group, # Represented P<0.05 and ## Represented P<0.01 compared with the SNL group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Inflammation Research

Article Title: The Role of Bone Morphogenetic Protein 4 in Microglial Polarization in the Process of Neuropathic Pain

doi: 10.2147/JIR.S356531

Figure Lengend Snippet: Noggin reversed the imbalance of the M1-M2 ratio in the process of NP. ( A and B ) Representative flow cytometry images showed that SNL induced a significant elevation of CD11b + CD86 + M1 microglia from day 1 to day 7, while CD172 + CD11b + M2 microglia decreased at P4 in the SNL group compared with the Sham group. Then, in the SNL+NOG group, Noggin treatment markedly decreased the proportion of CD11b+CD86+ M1 microglia from P1 to P7, while increased the proportion of CD172 + CD11b + M2 microglia at P7. Moreover, M1/M2 ratio markedly decreased from P4 to P7 after Noggin treatment. n=3 for each time point. One-way ANOVA, followed by Sidak’s multiple comparisons test were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group; # Represented P<0.05 and ## Represented P<0.01 compared with the SNL group.

Article Snippet: All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity).

Techniques: Flow Cytometry