Supplementary Figure 4: Additional analyses on mechanical hypersensitivity and dorsal horn plasticity in VT3^Lbx1-ablated (VT3^Lbx1-Abl) mice, SOM^Lbx1-ablated (SOM^Lbx1-Abl) mice and their control littermates

(a) Loss of brush-evoked dynamic (brush assay) mechanical hypersensitivity, but not von Frey-evoked punctate hypersensitivity, following SNI in both male and female VT3^Lbx1-ablated mice (VT3^Lbx1 Abl). (Left, dynamic) Male: n = 8, Control; n = 6, VT3^Lbx1 Abl; two-way ANOVA with Bonferroni’s post hoc analysis; F_{1, 72} = 556.20, P < 0.001. Female: n = 5, Control; n = 5, VT3^Lbx1 Abl; two-way ANOVA with Bonferroni’s post hoc analysis; F_{1, 72} = 510.10, P < 0.001. (Right, punctate) Male: control versus VT3^Lbx1 Abl mice; before SNI, two-tailed student’s unpaired t test, t₁₂ = 3.392, P = 0.0053; after SNI, two-way ANOVA with Bonferroni’s post hoc analysis, F_{5, 72} = 8.35, P = 0.1469. Female: control versus VT3^Lbx1 Abl mice; before SNI, student’s unpaired t test, t₈ = 2.603, P = 0.0315; after SNI, two-way ANOVA with Bonferroni’s post hoc analysis, F_{5, 48} = 1.248, P = 0.5823. (b) Mechanical hypersensitivity following intrathecal injection of bicuculline and strychnine in SOM^Lbx1-ablated mice (SOM^Lbx1 Abl) and their control littermates. Loss of dynamic hypersensitivity (n = 5, Control; n = 5, SOM^Lbx1 Abl; two-way ANOVA with Bonferroni’s post hoc analysis, F_{(1, 56)} = 675.40, P < 0.0001;), and loss of punctate hypersensitivity (n = 5, Control; n = 5, SOM^Lbx1 Abl; before SNI, two-tailed student’s unpaired t test, P < 0.0001; after SNI, two-way ANOVA with Bonferroni’s post hoc analysis, P < 0.0001). (c) Microglia activation following SNI (day 7 after SNI) marked by increased expression of Iba1. An increase in Iba1 expression in the ipsilateral (“Ipsi”) dorsal horn in comparison with the contralateral (“Cont”) dorsal horn both in wild type control mice (student’s unpaired t test, t₍₁₆₎ = 2.831, P = 0.0120, t₍₁₆₎ = 2.831) and in SOM^Lbx1 Abl mice (two-tailed student’s unpaired t test, t₍₁₆₎ = 3.519, P = 0.0028). No significant (“N.S.”) difference in Iba1 expression in the ipsilateral dorsal horn (“Ipsi”, normalized to contralateral side) was detected between control mice and SOM^Lbx1 Abl (Student’s unpaired t test, t₍₁₆₎ = 0.6419, P = 0.5300; n = 9 sections, from 3 Control mice; n = 9 sections, from 3 SOM^Lbx1 Abl mice). *, P < 0.05, **, P < 0.01. Our studies do not rule out unknown molecular changes. Data are represented as mean ± SEM. The scale bar represents 100 μm.