Skip to main content
. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Pain. 2021 Jun 1;162(6):1758–1770. doi: 10.1097/j.pain.0000000000002171

Figure 7. DRG neurons from HomNav1.7I228M mutant mice display normal neurite length in vitro.

Figure 7.

(A-B) Representative large-field montage images of 7-day DRG cultures from (A) WT mice and (B) HomNav1.7I228M mutant mice (6-8 weeks). Each large-field image consists of 7×7 field views. Dotted lines distinguish individual field-of-view. Scale bar, 1000 µm. (C-D) Representative images of individual (C) WT and (D) HomNav1.7I228M DRG neuron with neurite growth. Scale bar, 50 µm. (E) Quantification of total length per cell of WT and HomNav1.7I228M neurons. Average normalised neurite length per cell of WT (n = 19 culture dishes from 5 WT mice) and HomNav1.7I228M (n = 19 culture dishes from 5 Nav1.7 I228M mutant mice) are 1.000 ± 0.1989 and 1.039 ± 0.4898, respectively. There is no significant difference in neurite length (p = 0.8338, nested t test) between WT and HomNav1.7I228M groups. (F-G) Representative images of (F) WT and (G) HetNav1.7I228M dorsal root ganglion neurons (from HetNav1.7I228M mutant mice by CRISPR gene editing) cultured in a monolayer, then fixed and stained with Tuj1 after 24 hours. (H) Neurites of cultured HetNav1.7I228M neurons (n=156 wells in 96 well plate, from 4 HetNav1.7I228M mutant mice by CRISPR gene editing) do not show evidence of degeneration, and in fact display longer neurite lengths per neuron than WT (n=167 wells in a 96 well plate, 4 mice) (p = 0.0215, two-tailed t test). (I) Example image of dorsal root ganglion neurons cultured in a spot culture, where neurons are plated in a dense spot, allowing neurites to grow radially outward. After 6 days of growth, neurons were fixed and stained with Tuj1. (J) Neurite length calculated by Sholl analysis, whereby the intersections of neurites with concentric rings drawn around the spot are quantified as a proxy for neurite length. Intersections at higher diameters indicate longer neurite lengths (WT, n=2; HetNav1.7I228M mutant by CRISPR gene editing, n=2). (K) Area under the curve from (J). The area under the curve, representing total neurite length per spot is not significantly different between WT and HetNav1.7I228M (p = 0.195050, two-tailed t-test).