Journal: Cellular & Molecular Biology Letters

Article Title: Trigeminal nerve root compression induced neuroinflammatory response promotes mechanical allodynia through the CGRP/SP-Piezo2 axis via Ca 2+ signaling

doi: 10.1186/s11658-025-00831-6

Figure Lengend Snippet: PKC signaling initiates Piezo2-dependent allodynia by activating the CGRP/SP intracellular pathway. a Representative images of triple immunostaining for CGRP (red), SP (green) and DAPI (blue) in the whisker pads of TN and sham animals after the CCT operation. b Representative images of triple immunostaining for CGRP (red), SP (green) and DAPI (blue) in the TG of TN and sham animals after the CCT operation. Scale bar = 50 μm. c–d The results of fluorescence intensity analysis showed that the expression of CGRP and SP both increased at 21 days after the CCT operation in the TG and whisker pads (mean ± SEM, n indicates the number of Merkel cells or TG neurons in the independent tissue section from at least three animals; ** P < 0.01, *** P < 0.001; unpaired t test). e – h Western blot analysis of Piezo2 and CGRP in the sham group, TN group and TN + Go6983 group TG and whisker pad tissues at 21 days after CCT operation. The relative expression levels of Piezo2 and CGRP in the different groups were normalized to those of GAPDH. Injection of Go6983 into TG significantly attenuated the expression of CGRP and Piezo2 in both the TG and whisker pads at 21 days after CCT operation (mean ± SEM, n = 4; n indicates the number of independent animals; * P < 0.05, ** P < 0.01, *** P < 0.001 , ns, no significant difference; one-way ANOVA). i Measurement of allodynia grade in the three groups after the operation. The allodynia grade was significantly greater in the TN group than in the sham group at PODs 14 and 21, and Go6983 attenuated the effect of the CCT operation (TN or TN + Go6983 vs. Sham; mean ± SEM, n = 4–5; n indicates the number of independent animals; ** P < 0.01 , ns, no significant difference; two-way ANOVA). j The severity of allodynia was increased after CCT. Subcutaneous injection of LV-scramble shRNA into the whisker pad had no significant effect on allodynia severity after the CCT operation (TN vs. TN + LV-scramble shRNA; mean ± SEM, n = 5, n indicates the number of independent animals; ** P < 0.01, *** P < 0.001, ns, no significant difference; two-way ANOVA). CCT-induced mechanical allodynia was markedly attenuated 14 d after CCT surgery when LV- Piezo2 shRNA was subcutaneously injected into whisker pads (TN + LV-scramble shRNA vs. TN + LV- Piezo2 shRNA-skin; mean ± SEM, n = 5, n indicates the number of independent animals; ns, no significant difference, ** P < 0.01, ** P < 0.001; two-way ANOVA). k Administration of SQ22536 into the whisker pads of TN model animals produced significant alleviation of mechanical allodynia at 24 h post-injection (mean ± SEM, n = 5, n indicates the number of independent animals; *** P < 0.001; unpaired t test). Intra-whisker pad injection of dbcAMP still significantly increased allodynia severity 24 h post-injection in Piezo2-knockdown animals (mean ± SEM, n = 5, n indicates the number of independent animals; *** P < 0.001; unpaired t test). l Coadministration of LV- Piezo2 shRNA into the TG and whisker pad had no significant effect on allodynia severity compared with that in the sham group (mean ± SEM, n = 6–9; n indicates the number of independent animals; ns, no significant difference; two-way ANOVA). m Intra-whisker pad injection of PBS (vehicle control) did not significantly affect mechanical allodynia severity when assessed 24 h post-injection (mean ± SEM, n = 4, n indicates the number of independent animals; ns, no significant difference; unpaired t test). Intra-whisker pad injection of db cAMP significantly induced mechanical allodynia at 24 h post-injection in sham-operated animals (mean ± SEM, n = 5, n indicates the number of independent animals; *** P < 0.001; unpaired t test). At 24 h after intra-whisker pad injection of db cAMP, sham-operated animals with concurrent LV-Piezo2 shRNA-mediated knockdown in both TG and whisker pad innervation zones exhibited no significant development of mechanical allodynia (mean ± SEM, n = 5; n indicates the number of independent animals; ns, no significant difference; unpaired t test)

Article Snippet: In parallel, SQ22536 (1 mM; MCE Cat# HY-100396, Monmouth Junction, NJ, USA) was injected into TN model animals.

Techniques: Triple Immunostaining, Whisker Assay, Fluorescence, Expressing, Western Blot, Injection, shRNA, Produced, Knockdown, Control