Fig. 8
Effect of driving CL-macrophages toward an M1 phenotype on CL-enhanced regeneration. A. Diagram showing the CL paradigm performed on WT animals with daily CL injections to pharmacologically inhibit an M2 phenotype and promote an M1 phenotype at the CL. The arginase inhibitor, nor-NOHA (left, B-K), or an M1 stimulation cocktail, containing LPS and a STAT6 inhibitor (right, L-U), was injected daily into the CL site to polarize the macrophages from M2 to M1. A-B, L-M. Representative images for vehicle and arginase inhibitor (A-B), or M1 stimulation cocktail (L-M) treated mice showing CD68, Arg1, and iNOS, immunostaining. CD68 is shown in blue so that macrophages co-labeled with Arg1 will appear Cyan and those co-labeled with iNOS will appear magenta. Triple labeled cells will appear white. Note the increase in magenta cells and decrease in cyan cells in the nor-NOHA treated CL (C), compared to the increase in white cells with few magenta cells in the M1 stim CL (M). Scale bar = 100 μm in A, B, L, M. D, N. Macrophage number, displayed as cells per 0.01 mm2, was not altered by the treatment. E, O. The percentage of CD68+ macrophages expressing Arg1 after the arginase inhibitor (E) or M1 stimulation cocktail (O) treatment compared to vehicle controls. F, P. The percentage of CD68+ macrophages expressing iNOS after arginase inhibitor (F) or M1 stimulation cocktail (P) treatment compared to vehicle. G, Q. Axon regeneration quantified at 100 μm intervals as the fraction of regenerating axons relative to the crush site for arginase inhibitor (G) or M1 stimulation (Q) treated nerves. H, R. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating axons for arginase inhibitor (H) or M1 stimulation (R) treated nerves. I-K, S-U. Representative images of regenerating nerves treated with arginase inhibitor (I-K) or M1 stimulation cocktail (S-U) immunostained for regenerating axons with SCG10 in 40 μm sections. Unconditioned regeneration (I, S) was the same for both treatment groups. Conditioned regeneration (J-K, T-U) was also the same between treatments and significantly increased compared to contralateral uninjected nerves. The dotted line indicates the center of the crush site which was considered to be 500 μm wide, and the solid line is 3000 μm from the crush. Scale bar = 500 μm in I-K, S-U. * p < 0.05. *** p < 0.001. # p < 0.05 between injury groups within the same treatment group. N = 4–11 per group