Figure 4. Time course of DLK-dependent gene expression in DRG neurons and corresponding spinal cord microgliosis.

(A–C) Representative images and quantification of gene expression changes in ipsilateral L4 DRGs of WT mice 6, 18, 24, 48 and 72 hr post-SNT (sciatic nerve transection). (A) and (D) Representative images of Atf3 (green), Csf1 (orange), Gal (cyan) and Tubb3 (red) in situ hybridizations in iL4 DRGs after SNT. Scale bars 100 μm. Quantification of Atf3 (B), Csf1 (C), and Gal (E) expression changes in L4 DRGs ipsilateral (green, orange, cyan iL4) vs contralateral (black, cL4) to injury 6–72 hr post-SNT. n = 4 (2 males, 2 females) for 6, 18, 24, and 48 hr groups, n = 2 (1 male, 1 female) for 72 hr group. ns: not significant; ***p<0.001, ****p<0.0001 by 2-way ANOVA followed by Sidak’s multiple comparisons post test comparing values between ipsilateral and contralateral L4 DRG at each time point. Note in (D), in representative images at 48 and 72 hr, that Gal comes on in fewer cells than for Atf3 and Csf1 (A), and these include a mixture of large and small diameter neurons. (F–G) Iba1 immunolabeling in cleared lumbar spinal cord shows no region of microglial reactivity at 18 hr post-SNI. Microgliosis becomes evident by 3 dpi and robust by 5 dpi. Representative images of 3D views from the top and the ipsilateral side (F) and quantification (G). DLK cKO prevents microgliosis in lumbar spinal cord 3 and 5 days post-SNI, as observed previously at 7 dpi (Figure 3D–H). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Tukey’s multiple comparisons.