Intraspinal and Intrathecal Injection

The intraspinal injection was performed as described previously (Jiang et al., 2016). In short, after anesthetized with Nembutal, mice underwent hemilaminectomy at the L1-L2 vertebral segments. The intraspinal injection was carried out ipsilaterally on the left side. By using a glass micropipette, each animal received two injections (5 × 10⁵ TU per injection, 0.8 mm from the midline, 0.5 mm apart in rostrocaudal axis, 0.5 mm deep) of lentivirus following the L3-L4 dorsal root entry zone after exposure of spinal cord. The tip of glass micropipette should reach a depth of lamina II-IV of the spinal cord. Finally, the dorsal muscle and skin were sutured layer by layer.

Intrathecal injection of siRNA (20 μM, 10 μL) was performed daily for two to three consecutive days in sham or SNL mice. Mice were held firmly in place over the pelvic girdle and between L5 and L6 vertebrae inserted a 30-gauge needle attached to a 25 μL microsyringe. A slight flick of the tail after a sudden advancement of the needle confirmed the proper insertion of the needle into the subarachnoid space (Jiang et al., 2016). TurboFect in vivo transfection reagent (Thermo Fisher, R0541) was used to improve delivery and prevent siRNA degradation. Mmp24 siRNA1 (sense: 5′-GAG AUU CGU CUU CUU CAA ATT-3′, antisense: 5′-UUU GAA GAA GAC GAA UCU CTT-3′), Mmp24 siRNA2 (sense: 5′-GGA UAU UAC ACC UAC UUC UTT-3′, antisense: 5′-AGA AGU AGG UGU AAU AUC CTT-3′), Mmp24 siRNA3 (sense: 5′-CUA UCU UCC AAU UCA AGA ATT-3′, antisense: 5′-UUC UUG AAU UGG AAG AUA GTT-3′).