Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: Bradykinin and its B2 receptor are persistently upregulated following pIONT (a) Behavioral testing (facial nocifensive scoring) revealed that pIONT model mice exhibited significant mechanical nocifensive responses on postoperative days 3, 7, and 21 (compared to sham-operated (Sham), *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by the Bonferroni test). (b) Immunofluorescence staining showed that the nerve injury marker ATF3 was specifically expressed in TG neurons on postoperative day 7 in the pIONT group. Scale bar: 200 μm. (c) Quantitative analysis indicates a significant increase in the proportion of ATF3-positive cells (***P<0.001, Student’s t-test). (d–f) qPCR analysis of mRNA levels in the ipsilateral TG for BK (d), B1 receptor (e), and B2 receptor (f) (compared to sham-operated groups: *P < 0.05, **P < 0.01,***P < 0.001, one-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Immunofluorescence, Staining, Marker

Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: B2 Receptor was predominantly expressed in TG neurons following pIONT (a) Western blot analysis shows that the protein levels of the B2 receptor in the TG were significantly upregulated after pIONT compared to the sham-operated group (Sham). (b) Quantitative analysis of BDKRB2 (normalized to GAPDH) further confirmed the significant increase in B2 receptor protein levels in the pIONT group (***P<0.001, Student’s t-test). (c) Immunofluorescence staining shows that the expression of the B2 receptor (red) in the TG was significantly increased on postoperative day 7 in the pIONT group compared to the sham-operated group (Sham 7d). Scale bar:100 μm. (d) Quantitative analysis indicates a significant increase in the proportion of B2 receptor-positive cells (**P<0.01, Student’s t-test, scale bar: 100 μm). (e–g) Immunofluorescence co-localization analysis demonstrates that the B2 receptor (red) is primarily expressed on myelinated neurons (NF200+, green, e), peptidergic neurons (CGRP+, green, f), and non-peptidergic neurons (IB4+, green, g). The bar charts on the right show the quantitative co-localization ratios of the B2 receptor with each cell marker. Scale bar:100 μm.

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Marker

Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: Blocking the B2 receptor effectively alleviates pIONT-induced neuropathic pain and reverses neuronal hyperexcitability. (a–b) Local injection of Icatibant significantly alleviated mechanical allodynia on postoperative day 1 (pIONT 1d) in pIONT mice (*P < 0.05, pIONT 1d + Vehicle vs. pIONT 1d + Icatibant; Two-way ANOVA followed by the Bonferroni test). (c–d) On postoperative day 7 (pIONT 7d), mechanical allodynia was similarly attenuated by Icatibant treatment (**P < 0.01, pIONT 7d + Vehicle vs. pIONT 7d + Icatibant; two-way ANOVA followed by Bonferroni test). (e-f) Representative traces of action potentials evoked by step-current injection in TG neurons. (e) Rheobase current responses in Sham (0 pA), pIONT + Vehicle (20 pA), and pIONT + Icatibant (30 pA) neurons (Δ = 10 pA, 1000 ms). (f) Firing patterns at 100 pA in Sham, pIONT + Vehicle, and pIONT + Icatibant neurons. Scale bar: 0.2s, 20 mV. (g) pIONT reduced the rheobase of the first action potential in Vehicle-treated mice, whereas Icatibant administration preserved rheobase values comparable to sham controls (*P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test). (h) pIONT increased the firing frequency elicited by step-current injection in Vehicle-treated mice, an effect abolished by Icatibant(**P < 0.01, pIONT + Vehicle vs. pIONT + Icatibant). (i) pIONT enhanced the firing frequency in response to ramp-current stimulation in vehicle-treated mice, which was prevented by Icatibant co-treatment (*P < 0.05, **P < 0.01, ***P < 0.001, pIONT + vehicle vs. pIONT + Icatibant; two-way ANOVA followed by Bonferroni test). (j) The discharge RMP of action potentials was not altered by pIONT or Icatibant treatment. (P > 0.05, pIONT + vehicle vs. pIONT + Icatibant; one-way ANOVA followed by Tukey’s post-hoc test). (K) pIONT decreased action potential half-width in vehicle-treated mice. (l-m) Neither the peak amplitude (l) nor the discharge threshold (m) of action potentials was altered by pIONT or Icatibant treatment (P > 0.05, pIONT + vehicle vs. pIONT + Icatibant; one-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Blocking Assay, Injection