Fig 2: BDNF recombinant protein intervention reversed electroacupuncture relieving the occurrence of hyperalgesic priming. (A)Schedule of establishing hyperalgesic priming model and BDNF recombinant protein intervention. BDNF recombinant protein or PBS was administered through the intrathecal catheter to HP model rats for 3 consecutive days starting on day 7, 1 h before the detection of mechanical withdrawal threshold. (B)The mechanical withdrawal threshold (MWT) of rats that received 3 consecutive days BDNF recombinant protein intervention. (C) The quantification of the Western blots results and a representative Western blot showing KCC2 protein isolated from the ipsilateral SCDH 48 h after 2nd injection. (D)Representative immunofluorescence images of KCC2 (green) merged with Dapi (blue) in ipsilateral L4-6 SCDH of BDNF recombinant protein intervention rats. (E)The immunofluorescence quantification of KCC2 in ipsilateral L4-6 SCDH. (F)Evaluation of KCC2 gene expression in ipsilateral L4-6 SCDH using qPCR in the HP + PBS, HP + EA + PBS, HP + EA + BDNF groups 48 h after 2nd injection. Data are presented as mean ± SEM, n = 5; *p < 0.05, **p < 0.01 vs HP + PBS group, #p < 0.05, ##p < 0.01 vs HP + EA + PBS. Two-way ANOVA with Tukey's post-hoc test was applied in. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Poly(I:C) injection during pregnancy resulted in overexpression of NKCC1 in PFC of MIA offspring. (A) Representative image of NKCC1/KCC2 expression in PFC of offspring at P14. (B) Quantification of NKCC1/Tubulin in panel (A), *** p < 0.001, n = 6. (C) Quantification of KCC2/Tubulin in panel (A), n = 6. (D) Representative image of NKCC1/KCC2 expression in PFC of offspring at P28. (E) Quantification of NKCC1/Tubulin in panel (D), *** p < 0.001, n = 6. (F) Quantification of KCC2/Tubulin in panel (D), n = 6. (G) Representative image of NKCC1/KCC2 expression in PFC of offspring at P42. (H) Quantification of NKCC1/Tubulin in panel (G), n = 6. (I) Quantification of KCC2/Tubulin in panel (G), n = 6.

Fig 4: Protein expression levels in the amygdala and hippocampus. (A-B) Level of K+/Cl- cotransporter (KCC2) oligomers normalized to KCC2 monomer (C-D) Level of Na+-dependent K+/Cl- cotransporter (NKCC1) (E-F) Level of total calcium calmodulin kinase (tCaMKII) (G-H) Level of total extracellular regulated kinase (tERK). Box central marks indicate the median, bottom and top edges indicate the 25th and 75th percentiles, n = 3–6 / group, asterisks denote the effect of genotype, *p < .05

Fig 5: Phosphor-serine activation of m-calpain dominants KCC2 regulation in 0 Mg2+ seizure model. (A,B) Immunoblotting detection of both plasma membrane (A) and cytoplasm (B) KCC2 expression level change in hippocampal slice 0 Mg2+ model treated with either μ-calpain or m-calpain selective inhibitor CI I and CI IV, respectively. (C) Expression of either μ- or m-calpain from seizure slices induced by 0 Mg2+. (D) Serine phosphorylation level by IP method. Naïve or 0 Mg2+ treated slices were used as inputs to form immune complex with μ- or m-calpain antibodies and detected by phosphor-serine antibody. Phosphor-serine levels were normalized to corresponded calpain levels. (E) Phosphor-serine level of m-calpain from 0 Mg2+, 0 Mg2+ + PD98059 or K252a treated slices. (F) Immunoblotting showed KCC2 level in 0 Mg2+ + MDL-28170 or PD98059 treated slices, compared with none treated or 0 Mg2+ treated alone slices. (G) KCC2 level of in vivo rats pre-treated with either MDL-28170 or SL-327 before PTZ kindling, in compared with vehicle PTZ treated rats. CI I, Calpain Inhibitor I; CI IV, Calpain Inhibitor IV; Calp, calpain; p-Ser, Phosphor-serine. *p < 0.05 compared with Ctrl group; **p < 0.01 compared with Ctrl group; ***p < 0.001 compared with Ctrl group; #p < 0.05 compared with 0 Mg2+ group; ##p < 0.01 compared with either 0 Mg2+ or with PTZ group; ###p < 0.001 compared with PTZ group.