Intraspinal Delivery of Recombinant Adeno-Associated Virus for Targeted Neuronal Transduction

Begin with a secured, anesthetized mouse in a stereotaxic frame, with the lumbar spinal cord exposed and three injection sites pre-punctured.

Align a micropipette filled with recombinant adeno-associated virus above one puncture site.

Lower the micropipette until it slightly depresses the spinal cord surface, confirming contact with the pia mater.

Advance the micropipette to the target depth in the dorsal horn, then retract slightly to relieve pressure and stabilize the tissue.

Slowly infuse the viral solution, allowing it to diffuse locally.

The virus enters neurons through endocytosis and delivers episomal DNA, enabling transgene expression.

Hold the micropipette in place to prevent backflow and allow pressure to equalize.

Retract the micropipette gradually and repeat the injection at two additional lumbar sites to cover spinal segments L3 to L5.

Remove the spinal clamps and cushion, then close the incision with sutures.

Apply an antiseptic and allow the mouse to recover.

The transduced mouse model, which expresses GFP in neurons, is ready for further analysis.

Use the stereotaxic arm to move the tip of the micropipette over one of the holes in the dura and then down until a slight depressi