Combined In Vivo Electroporation and Short-Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

Our research aims to understand the mechanisms underlying the reiteration of the neuromuscular synapse and to evaluate the impact of inhibiting or over expressing specific muscle derived proteins on this regenerative process. Through muscle in vivo electroporation, we have observed the impact of muscle derived proteins on the organization of postsynaptic acetylcholine receptors. Additionally, our denervation protocol has enabled us to identify various morphologies of postsynaptic domains and correlate them with their stability.

This technique serves as an initial experimental approach to evaluate the impact of a specific muscle proteins on NMD regeneration, paving the way for more intricate gene editing techniques. Moreover, the protocols are minimally invasive. This combination of experimental methods has been employed to investigate the role of wind signaling on NMD regeneration.

By or expressing agonist or antagonist of the wind pathway, we have analyzed nerve damage and the subsequent regeneration processes. Our future research will examine the impact of muscle derived proteins on the organizational functionality of the neuromuscular synapse in various contexts, including aging and certain pathologies such as ALS. To begin, make sure all dissection tools and the working space have been autoclaved and are clean.

Place an anesthetized CF-1 mouse on the surgical table. Use a Hamilton syringe to subcutaneously inject 20 microliters of 2 milligrams per milliliter Hyaluronidase in PBS. Weigh the mouse to determine the approximate meloxicam dose and place the animal in an empty cage for a 30 minute recovery.

After re-anesthetizing the mouse, make an 8 millimeter incision over the sagittal suture of the skull. Using scissors and forceps, gently remove the fat and connective tissue beneath the skin to expose the levator auris longus or LAL muscle. With a Hamilton syringe, inject 10 microliters of commercially sourced DNA into the fascia of the muscle.

Position two gold needle electrodes five millimeters apart in parallel alignment over the muscle fibers covering the full length of the LAL muscle. Now, use an electroporator to apply five electrical pulses at 100 volts centimeter for 20 milliseconds each with a frequency of 1 hertz. Microscopic examination showed that a high proportion of muscle fibers from the rostral LAL portion were positive for the expression of tandem dimer Tomato, confirming efficient in vivo electroporation mediated gene transfer of LAL muscles.

To begin, position an anesthetized mouse on its left side to denervate the right levator auris longus or LAL muscle. Secure the right ear towards the nose to reveal the posterior area of the pinna. After shaving the skin and locating the posterior auricular vein, make a five millimeter incision in the skin, two millimeters caudally away from it.

With forceps and scissors, remove fat and connective tissues to expose the spinal accessory nerve, the digastric muscle, and the cartilaginous auditory canal. Gently remove the surrounding connective tissue to expose the posterior auricular branch of the fascial nerve. Using forceps with a 45 degree angled tip, apply a 32nd crush to the nerve with constant pressure.

Finally, using a 5-0 strain size and an HR-15 needle, close the incision with absorbable polyglycolic acid sutures. After recovery and meloxicam treatment, examine the mouse LAL microscopically for denervation. Confocal microscopy imaging of LAL muscles showed complete neuromuscular junction denervation five days after nerve injury.