The overall goal of this new experimental protocol is to efficiently conduct in vivo gene transfer to Schwann cells in rodent sciatic nerves using electroporation. This method can help answer key questions in the neuroscience field such as molecular mechanisms of myelination and the demyelination in the peripheral nervous system. The main advantage of this method is that the surgeons can perform in vivo transfection with Schwann cells with a simple procedure.
To begin the procedure, autoclave all the surgical instruments and 0.9%sodium chloride solution. Next, pull some glass pipette using a pipette puller, and cut the tip of each pipette to a diameter of 30 to 50 microns. After anesthetizing a rat pup with isoflurane in an induction box, place the rat on a pre-heated electric warmer under a binocular microscope.
Perform toe pinching to confirm proper anesthesia. Then, apply ophthalmic ointment to both eyes to prevent dryness during surgery. Maintain anesthesia by continuously administering isoflurane through a face mask.
Next, fix the animal's legs with surgical tape and clean the skin of the posterior thigh with povidone iodine. Make an incision with the scalpel. Expose the sciatic nerve by creating an opening between the quadriceps femoris muscle and biceps femoris muscle with two sewing needles.
Then, apply a 0.9%sodium chloride solution to the nerve, and absorb excess water with lint-free paper. After that, insert the base of a glass micro-pipette into flexible tubing. Gently fill the micro-pipette with an adequate amount of DNA solution by aspiration.
Now, gently pull the distal side of the nerve using a needle to lift the exposed nerve. Insert the glass micro-pipette into the distal site on the nerve, and inject the DNA solution by applying slight positive pressure until the nerve appears green. Next, place a tweezer-type platinum electrode at about one to two millimeters from the nerve.
Fill the gap between the electrode and the nerve with 0.9%sodium chloride solution. Apply electric pulses to the injection site using an electroporator. After the first pulse set, invert the electrode and apply another pulse set.
Afterward, clean the electroporation site with 0.9%sodium chloride solution and repeat the procedure on the contralateral sciatic nerve. After electroporation, close the incision with cyanoacrylate glue. Then, clean the wound with povidone iodine.
Remove the face mask from the animal, and keep it on the electric warmer for at least an hour to allow full recovery from anesthesia. Subsequently, return the pup to the mother rat. Administer carprofen, a non-steroidal anti-inflammatory drug, or buprenorphine, an opioid analgesic, if required.
Shown here is a representative image of a transfected sciatic nerve. The nerve was transfected with an RFP-expressing plasmid at P3, and fixed at P7.This is a representative image of an RFP-transfected cell at P7, showing colloquialization with S100, a Schwann cell marker. And this is a representative image of an RFP-transfected sciatic nerve at P14, showing colloquialization with MBP, a myelinating Schwann cell marker.
A representative image of a sciatic nerve co-transfected with GFP and RFP-expressing plasmids is shown here. Transfected Schwann cell simultaneously expressed GFP and RFP. This figure shows an image of myelinating Schwann cells at P31, which were transfected at P3, when myelination begins, and an image of myelinating Schwann cells at P31 which were transfected at P14, when most large axons become myelinated.
These images suggest that transfection of myelinating Schwann cells can be achieved not only in neonatal nerves, but also in more mature nerves. After watching this video, you should have a good understanding of how to conduct electroporation to the sciatic nerves.
