Fig. 3
Atf3 is a key upstream target of mitochondrial transfer in regulating the expression of RAGs. (A) Schematic of the experiment. The expression of Atf3 in DRG cells was knocked down by injection of Atf3 siRNA in to L3/4/5 DRGs and electrotransfection. The non-targeting siRNA were employed as a control to Atf3 siRNA. The SNI models were constructed 2 days later. To construct the SNI model, the right thigh of each mouse was surgically exposed at the mid-thigh level to access the sciatic nerve. The sciatic nerve was then crushed with moderate force using a forceps for a duration of 10 s. For the non-targeting siRNA with mitochrondria injection therapy (non-targeting siRNA + Mito) and Atf3 siRNA with mitochrondria injection therapy (Atf3 siRNA + Mito) groups, 2 µL of mitochondria derived from 106 MSCs were injected into the crush sites of the injured nerves. The injured sciatic nerves were removed 4 days later. After fixation and clearing, the nerves were whole-mount stained utilizing primary anti-Tuj1 antibodies and fluorescent secondary antibodies, imaging by a fluorescence microscope; (B) Representative immunofluorescence images of whole-mount sciatic nerves in non-targeting siRNA, non-targeting siRNA + Mito, Atf3 siRNA and Atf3 siRNA + Mito groups (left). Quantification of the average length of the top 5 axons with the longest regeneration distance in each groups (right). (C) qPCR of the selected RAGs in DRG tissue from the above 4 groups. The mRNA expression was normalized against GAPDH. Data represent means ± SD. Statistically significant differences are indicated; n = 6; *P < 0.05, vs. Control