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Fig. 2 | Molecular Brain

Fig. 2

From: Histone modification of pain-related gene expression in spinal cord neurons under a persistent postsurgical pain-like state by electrocautery

Fig. 2

Analysis of chronic postsurgical pain-activated neurons. A Targeting strategy for the Fos-2A-iCreERT2 KI allele. PAM, protospacer adjacent motif. HDR, homology directed repair. Star; Two synonymous mutations (377L (CTG to TTA) and 378L (CTG to TTA)) were introduced to prevent binding and re-cutting of the sequence by gRNA after HDR. B Schematic diagram of EGFP labeling of electrocautery-activated neurons. iCreERT2 expression is driven by the activity-dependent cFos promoter to mediate 4-OHT-dependent recombination that permanently labels the active neurons with EGFP. (cFos-negative neurons: EGFP, cFos-positive neurons: EGFP+) C Protocol for EGFP-labeling of electrocautery-activated neurons. Workflow for isolation of electrocautery-activated neurons from the ipsilateral side of the spinal cord by MACS and FACS. D-ii Representative FACS plot. Fluorescence from cells double-labeled for Thy1 (APC on Y axis) and cFos-EGFP (X axis) logarithmic plots (a log10 scale). In Thy1-labeled neurons, cFos-negative (EGFP) and cFos-positive (EGFP+) neurons (left and right gates, respectively) on the X axis were represented by blue (left gate, cFos-negative (EGFP)) and red (right gate, cFos-positive (EGFP+)) dots. E mRNA expression levels of markers of Slc17a6+ excitatory neurons in the spinal dorsal horn in electrocautery-activated neurons (cFos-positive neurons) compared to those in cFos-negative neurons. (n = 4–5 animals per sample, unpaired t-test, *p < 0.05, **p < 0.01 vs. cFos-negative neurons) F Heat map analysis of transcriptional profiles related to the subpopulation (“Glut 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14- or 15-type”) of Slc17a6+ excitatory neurons in electrocautery-activated neurons. G, H Quantitative analysis of mRNA levels of Sprr1a G and Anxa10 H in cFos-positive neurons as compared to cFos-negative neurons. Each column represents the mean ± S.E.M. of 3 samples (n = 4–5 animals per sample, unpaired t-test, *p < 0.05 vs. cFos-negative neurons)

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