Fig. 5 |. CD14⁺Ly6G^low cells induce RGC axon outgrowth, in part, via secretion of growth factors.

a-d, Ly6G⁺ cells were purified from peritoneal lavage fluid that was collected 4 hours (4h NΦ) or 3 days (3d NΦ) following i.p. zymosan injection. a, 3d NΦ, 4h NΦ, the conditioned media of 3d NΦ (3d NCM), or heat shocked conditioned media of 3d NΦ (HS NCM), were added to primary RGC cultures, and neurite outgrowth was measured 24 hours later. RGCs were cultured with recombinant CNTF or particulate zymosan (Zym) as positive and negative controls, respectively. Each circle represents the mean neurite length of 200 RGCs countedin one experiment, n=3 independent experiments. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. Right panels, representative images. b, Upper panels, NGF and IGF-1 mRNA levels in 4h or 3d NΦ, quantified using qPCR, and normalized to Actβ. Lower panels, NGF and IGF-1 protein levels, measured in the CM of 4h or 3d NΦ by ELISA. c, NGF and IGF-1 protein levels, measured by ELISA, in vitreous fluid collected on day 5 following ONC injury and i.o. zymosan or PBS injection. Mice were injected i.p. with either NRS or αCXCR2 on days 0, 2 and 4 post-ONC injury. b,c Statistical significance determined using the two tailed unpaired Student’s t-test (n= 3 mice/group). One of two experiments shown. d, Primary RGC were cultured with conditioned media of 3d NΦ, in the absence or presence of neutralizing antibodies against NGF and/ or IGF-1, or isotype matched control antibodies. Neurite length was measured 24 hours later. Each symbol represents the mean neurite length of 100 RGCs counted in one independent experiment; n=10 experiments. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. e,f, Purified 3d NΦ were adoptively transferred, with or without neutralizing antibodies against NGF and/ or IGF-1 or isotype matched control antibodies, into the eyes of mice with ONC injury, as in fig. 4c. A negative control group was injected with PBS alone. Optic nerves and retinas were harvested 14 days later. e, Density of regenerating axons in optic nerve sections, at serial distances from the crush site. (n= 10 nerves per group). One experiment representative of 3 shown. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with PBS; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with αNGF+αIGF-1). f, Frequency of viable BRN3a⁺ RGC neurons in whole mounts, normalized to healthy retina (n=10 retina per group). One experiment representative of 2 shown. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. a, d, f, *^{, #}P < 0.05, **^{, ##}P < 0.01, ***^{, ###}P < 0.001, ****^{, ####}P < 0.0001, δ P=.058. a-f, Error bars depict mean +/− sem.