Activation of the growth program of RGCs and inactivation of RhoA have synergistic effects in vivo. a-d, GAP-43-positive axons visualized in longitudinal sections through the adult rat optic nerve 2 weeks after axotomy with (b, d) or without (a, c) lens injury (asterisk indicates crush site). RGCs were transfected with AAV expressing GFP alone (a, b) or C3 plus GFP (c, d). a, Absence of regeneration after axotomy alone. b, Lens injury increases the number of GAP-43+ axons proximal to the injury site (to the left of the asterisk) and the number that regenerate beyond this point. c, C3 expression stimulates some regeneration even in the absence of lens injury. The inset shows regenerating axons at increased magnification and illumination. d, C3 expression greatly enhances axon regeneration when axotomized RGCs are exposed to the effects of lens injury. e, Quantitation of outgrowth (number of axons growing ≥500 μm beyond the injury site per optic nerve). f, RGC survival (TUJ1⁺ RGCs per retinal cross section). Axot, Axotomy; LI, lens injury. ^***Effect of C3 expression significant at p < 0.001; †††effect of intravitreal macrophage activation significant at p < 0.001. Scale bar, 200 μm.