Direct Vagus Nerve Injection Protocol For Rats

We conduct preclinical studies in gene replacement therapy. We use lab animals to establish the optimum method of drug delivery to address issues of autonomic, and peripheral nervous systems. Vector-based gene replacement therapy is one of the emerging technologies in gene therapy. The application of adino associated viral vector is one of the potential options available now. AAV9 has been widely used in gene therapy research to deliver the gene of interest into the central and peripheral nervous system. Researchers do not have enough options available to deliver drug agents directly into the peripheral nervous system. There are situations which involve neurological disorders caused by peripheral or the autonomic nervous system. However, there is a lack of an effective method of drug delivery to address those situations.

This method demonstrates a way to deliver the drug agents directly into the Vagus nerves to address the problem of autonomic nervous system in preclinical studies using rats. Additionally, this method can be exported to deliver drugs through other peripheral nerves with necessary modifications in other species. The gray lab will continue AV mediated preclinical gene therapy research using both rodents and large animals. Additionally, we were focused on developing new methods applicable to pave the strategy of gene replacement therapy.

[Narrator] To begin, connect a 100 microliter glass syringe with the plunger removed to a 27 gauge needle. Connect one end of approximately 45 centimeters of sterilized polyethylene tubing to the 27 gauge needle, then fit a three milliliter plastic syringe with a 27 gauge needle, and load the syringe with approximately 0.5 milliliters of 0.9% normal saline. Use the 0.9% normal saline to fill the polyethylene tubing from the open end, ensuring the full length of the tubing, and the barrel of the glass syringe are filled until the saline overflows. Once done, remove and discard the three milliliter syringe and its needle. Now insert the plunger into the barrel of the glass syringe, and push it slightly forward to the middle of the barrel, and place the glass syringe in the syringe pump. Use the syringe pump to pull the plunger slightly back, creating an airspace of approximately 1.5 centimeters at the open end of the tubing. Pipette at least five microliters of dye, and candidate drug mixture using a 10 microliter micro pipette onto a sterilized piece of para film. Withdraw the candidate drug mixture into the tubing with the help of the syringe pump, and mark the level of the injection mixture with a marker. Fit a 35 gauge needle to the tubing, and secure it to a clean surface on the surgical stage with tape, ensuring it remains fixed, sterilized, and does not touch anything else. Start the syringe pump for 10 to 15 seconds until the injection solution begins to emerge from the needle tip. Transfer the anesthetized rat to a table equipped for hair removal and site preparation. Apply artificial tearing ointment in both eyes of the rat to prevent excessive dryness. Administer analgesics according to the approved institutional animal protocol to control pain when the rat regains consciousness. Shaved the ventral side of the neck at the cervical region, and extend it up to approximately two centimeters perpendicularly from the midline on both sides from the chin to the sternum. Sterilized the shaved area three times with 70% ethyl alcohol and 10% Betadine, wiping outward in a circular pattern. Transfer the rat to the surgical stage in a supine position under the dissecting microscope. Ensure the heating pad beneath the rat is tempered appropriately, and adjust the microscopes focused to visualize the cervical region for surgery. Hook the rats up or jaw teeth with a non-absorbable suture. Fix the suture's open end inside the nose cone to keep the nostrils within the nose cone throughout anesthesia. Position the rat so its head lies on the left side of the right-handed surgeon. Place a sterilized gauze cushion under the neck to align, and straighten the cervical region. Cover the rat's body with a sterilized drape leaving the surgical area exposed. Fix four retractor pins with magnetic fixators at the four corners of the surgical stage. Now, inject local anesthetics epidermal at the midline of the cervical region where the incision will be made. Use a scalpel blade to make a two centimeter longitudinal skin incision at the midline between the chin and sternum in the ventral cervical region. Then with sterilized cotton tips, blunt separates the cut skin edges. Using the retractor tips, retract the separated skin edges in opposite directions. Separate the fascia using cotton tips to go deeper, and push the salivary glands laterally. Now, separate the sternum mastoid muscles with cotton tips and retract them laterally with retractor pins. Continue separating tissue on the left side of the trachea until the carotid sheath is visible. Use fine forceps to create a small hole in the carotid sheath without damaging the carotid artery, and carefully separate the left vagus nerve from the common carotid artery. Once the nerve is separated from the artery using forceps, use a 25 gauge curved needle fitted to a one milliliter syringe to hook, and hold the nerve with the non-dominant hand. Hold the needle loaded with the candidate drug dye mixture with the dominant hand. To facilitate a smooth prick, keep the nerve straight by gently pulling it using the 25 gauge hook. insert the needle into the nerve, and advance it forward by more than 0.5 centimeters while keeping the needle parallel to the Vagus nerve. Pull slightly back so that approximately 0.4 to 0.5 centimeters of the needle remains inside the nerve. Limit the number of nerve pricks with a single needle to no more than three times. After confirming the needle insertion, retain its position without movement, and turn on the syringe pump. Monitor the infusion to ensure it remains inside the nerve, and flows smoothly. Use the marked point on the infusion tubing to track the movement of the drug dye mixture. Observe as the nerve starts to develop the color of the dye. Remove the 25 gauge curved needle, and using sterile cotton tips, gently move tissues back to their original position. Then remove the retractor tips. Close the wound with an absorbable or non-absorbable suture, and apply a tiny amount of tissue glue between the stitches if necessary to ensure the wound closes perfectly.