Figure 3: PI3K signalling is required for activating proregenerative responses and subsequent axon regeneration in vivo.

(a) Schematics of in vivo electroporation and investigation of axon regeneration. (b,c) L4/ L5 DRGs of an adult mouse were electroporated in vivo with EGFP alone (control) or together with dominant-negative PI3K (dnPI3K) at a ratio of 1:3, followed by the sciatic nerve crush as depicted in a. The lengths of all identifiable regenerating axons in the whole-mount nerves were measured from the crush site (arrowheads) to the distal axon tips. Lengths of total of 120 axons from six control mice and 124 axons from six dnPI3K-transfected mice were measured. Quantification of axon length is shown in b and representative images are shown in c. Scale bar, 500 μm. Error bars represent s.e.m. ***P<0.00001, unpaired two-tailed Student’s t-test. (d) L4/L5 DRGs in adult mice were electroporated in vivo with EGFP (control) or dnPI3K (myc-tagged), immediately followed by sciatic nerve transection. At 7 days after sciatic nerve transection, neurons from L4/L5 DRGs were cultured overnight and stained for neuronal tubulin, TuJ1 (red) or the myc tag. Transfected neurons are shown in green (EGFP or the myc positive). Experimental scheme is depicted in the upper left. Representative images (right) and quantification of axon length (lower left) from three independent experiments are shown. Scale bar, 100 μm. Error bars represent s.e.m. ***P<0.001, unpaired two-tailed Student’s t-test. (e) Adult mice were first subjected to sciatic nerve transection. At 7 days after the nerve injury, L4/L5 DRGs were electroporated in vivo with EGFP (control) or dnPI3K (myc tagged). One day after the in vivo electroporation, neurons from L4/L5 DRGs were cultured overnight and stained for neuronal tubulin, TuJ1 (red) or the myc tag. Transfected neurons are shown in green (EGFP or the myc positive). Experimental scheme is depicted in the upper left. Representative images (right) and quantification of axon length (lower left) from three independent experiments are shown. Scale bar, 100 μm. Error bars represents s.e.m. ***P<0.001, unpaired two-tailed Student’s t-test.