This method provides an in-vivo gene transfection technique to manipulate gene expression in adult mouse sensory neurons for future studies of mammalian exo-regeneration. The main advantage of this technique is that it's less labor and time consuming, and can both overexpress and knock down the target genes simultaneously and separately. To begin this procedure, mark both sides of the iliac crest of an anesthetized mouse with a fine marker.
Draw a line connecting two points of the iliac crest to facilitate the identification of L5 DRGs positions. Then, make a three centimeter incision along the midline of the lower back with micro scissors. Afterward, detach the paraspinous muscles, such as the musculus multifidus and the musculus longissimus lumborum, from the L3 to S1 spinous processes, and expose the fascia joint of the L4-5 and L5-6.
Next, use a micro rongeur to remove the fascia joints of the L4-5 and L5-6. Subsequently, remove the left neural arch of L4 and L5, to expose the dorsal side of the DRGs. 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 one microliter solution of DNA plasmids or RNA oligos using the intracellular micro injection dispense system. For electroporation, pinch the target DRG with the electrodes gently, and apply five 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 cork board, and make a one 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. After profusion, separate the DRGs together with the nerve roots and sciatic nerve, carefully, with micro scissors and micro forceps under the dissection microscope.
Then, transfer the sciatic nerve directly in 4%PFA overnight at four degrees celsius. To image and measure the fluorescence labeled sensory axons under the dissection microscope, strip off the attached tissue and membrane on the fixed sciatic nerve with micro scissors and micro forceps, carefully. Then, wash the nerve with PVS three times.
Next, place the sciatic nerve on a slide and keep it straight. Add 80 microliters of anti-fade solution around the nerve, then lay a cover slip on it. Flatten the whole mounted tissue with pressure.
Afterward, place the flattened tissue under the inverted epifluorescence microscope, equipped with an accessory for mosaic acquisition and image processing. When measuring the length of regenerated axons, trace all identifiable fluorescently labeled axons in the sciatic nerve from the crush site to the distal axon ends. When applying the current, in-vivo electroporation method to study axonal regeneration, the sciatic nerve was flattened and imaged.
Every regenerated axon with distinctive trajectory and recognizable distal axon end, can be traced from the crush site indicated by the suture knot. The white arrowhead, arrow, and star, indicate three distinctive axon ends, all of which extend from the crush site. Additionally, the DRGs were electroporated with EGFP plasmids, and the spinal cord was harvested.
The EGFP labeled, dorsal column axons, in the spinal cord, were imaged and identified in both the longitudinal and sagittal projecting images. Here is a detailed view of axons in the dorsal column, and here is a detailed view of axons in the the dorsal root entry zone. This image shows the sagittal projection of the EGFP labeled axons within the dorsal column.
Alternatively, the confocal images can be processed with microscopy visualization software to reconstruct a 3D image. While attempting this procedure, it's important to remember to position the L4 and the L5 DRGs correctly, and to make a knot on the dural membrane without impaling the sciatic nerve, while labeling the crush site with a suture knot. After its development, this technique pave the way for researchers in the field of axon regeneration to explore genes that are required for PNS, natural axon regeneration, or genes that can further promote regeneration in adult mouse in-vivo.
