This study outlines a method for enhancing sensory axon regeneration in mice by transfecting dorsal root ganglia (DRG) with RNA constructs. The approach involves microinjection and electroporation techniques to facilitate the delivery of constructs that modulate protein production in sensory neurons.
Take an anesthetized mouse with exposed L4 and L5 dorsal root ganglia containing sensory neuron cell bodies.
Load a microinjection pipette with fluorophore-tagged RNA constructs that regulate axon regeneration and a tracer dye.
Inject the mixture into the DRGs.
Using electroporation electrodes, apply electric pulses to the DRGs.
This disrupts the membranes of sensory neuron cell bodies, creating pores that allow the constructs to enter the cytoplasm, facilitating transfection.
Suture the incision and allow the mouse to recover.
After a few days, re-anesthetize and secure the mouse.
Make an incision lateral to the midline on the left. Dissect the muscles to expose the sciatic nerve.
Crush the nerve to injure sensory neuron axons. Mark the site with a suture knot.
Suture the incision and allow the mouse to recover.
The transfected constructs target mRNAs in DRG sensory neuron cell bodies, transiently modulating protein production to enhance sensory axon regeneration post-injury.
For DRG injection, load the DNA plasmids or RNA oligos into the glass capillary pipette. Then insert the tip of the capillary glass pipette carefully into the DRG. And gradually inject 1 microliter solution of DNA plasmids or RNA oligos using the intracellular microinjection dispense system.
For electroporation, pinch the target DRG with the electrodes gently, and apply 5-square electric pulses with the electroporation system. Subsequently, close the muscle, then the skin layers with nylon sutures. Two or three days after the DRG electroporation, anesthetize the mouse intraperitoneally, tape its limbs on the corkboard, and make a 1-centimeter incision 0.5 centimeters to the left side along the midline.
Next, cut the muscles, such as gluteus maximus muscle and piriformis muscle longitudinally. Then expose the segment of the sciatic nerve between the greater sciatic foramen and the sciatic notch. Crush the nerve with microsurgery forceps for 12 seconds, and mark the crush site with a nylon epineural suture. Afterward, close the muscle and skin layers with nylon suture.
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