The Conditioning Lesion Response in Dorsal Root Ganglion Neurons Is Inhibited in Oncomodulin Knock-Out Mice

L5 DRG explants from both WT and Ocm KO mice exhibited a CL response at 24 h (a) and 48 h (b), although ganglia from Ocm KO mice showed a significantly smaller response than those from WT ganglia after 48 h in culture (b). Micrographs represent WT (c) and Ocm KO (d) ganglia following a CL and 48 h in culture. N = 5/group. Scale bars: 250 μm. *p < 0.05, **p < 0.01, ****p < 0.0001.

DRG from Ocm KO mice do not exhibit a CL response in vivo. Nine days after a CL and 2 d after a crush injury, axons from WT mice exhibit enhanced growth response in vivo, whereas axons from Ocm KO mice did not (a). The nerves were immunostained for SCG10. The regeneration index is the distance from the crush site to the site where the staining for SCG10 is half that seen at the crush site (a, c–f). The regeneration ratio is the ratio of staining at a site to that at the crush site. This ratio was determined for distances from 0.5 to 3.0 mm distal to the crush site (b). Images represent WT crush only (c) and conditioned plus crush (d) and Ocm KO crush only (e) and conditioned plus crush (f). Asterisks in the images indicate the crush site for each nerve. Dashed rectangles indicate where the immunostaining for SCG10 is reduced by 50% compared with the staining at the crush site. N = 8/group. Scale bars: 500 μm. In the line graph, *p < 0.05, **p < 0.01 comparing WT conditioned versus WT crush only. #p < 0.01 comparing WT conditioned versus Ocm KO conditioned.

Macrophage accumulation in the distal sciatic nerve and DRG 2 d after a nerve crush with and without a prior CL in WT and Ocm KO mice. Macrophage accumulation observed by CD68 immunostaining in the distal sciatic nerve was similar after a CL in both genotypes (a). Macrophage accumulation was also increased in WT and Ocm KO DRG after a CL (f). Three adjacent fields from each sample were counted and summed. Images of WT crush only (b, g) and conditioned plus crush (d, i) and Ocm KO crush only (c, h) and conditioned plus crush (e, j). N = 5–6/group. Scale bars: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Flow cytometry studies on macrophage and neutrophil accumulation in the distal sciatic nerve and DRG in WT and Ocm KO mice after a nerve crush with and without a prior CL. Macrophages were determined by double staining with antibodies against CD11b and F4/80. Neutrophils were determined by double staining with antibodies against CD11b and Ly6G. For animals receiving a CL, the sciatic nerve was transected unilaterally 7 d before the nerve was crushed (samples labeled CL). Forty-eight hours later, the ipsilateral distal sciatic nerve and the ipsilateral DRG were taken for flow cytometry. The contralateral sciatic nerve received a crush only (samples labeled Crush). Representative dot plots for macrophages (a, d) and neutrophils (b, e) in the sciatic nerve (a, b) and DRG (d, e). Numbers in plots correspond to the percentage of total events in each quadrant. Events in quadrant outlined with a red box correspond to cells that are CD11b⁺F4/80⁺ (a, d, macrophages) or CD11b⁺Ly6G⁺ (b, e, neutrophils). Bar graphs indicate mean percent CD11b⁺F4/80⁺ and CD11b⁺Ly6G⁺ events in the sciatic nerve (c) and DRG (f). n = 4 animals per condition per genotype. *p < 0.05, **p < 0.01.

Myelin clearance from the distal sciatic nerve from WT and Ocm KO mice. Seven days after unilateral sciatic nerve transection changes in myelin clearance were determined by staining with luxol fast blue. Transected distal nerve segments from WT and Ocm KO mice showed significantly less staining than the sham-operated contralateral nerves, and no difference was seen between genotypes (a). The micrographs represent sections of WT sham-operated nerves (b) and transected distal nerves (d) and sections of Ocm KO sham-operated nerves (c) and transected distal segments (e). N = 5/group. Scale bar: 100 μm. ****p < 0.0001.