crush nerve injury&#45;induced denervation in mouse levator auris longus muscle to assess nmj reinnervation and maintenance

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

The neuromuscular junction (NMJ) is the peripheral synapse that controls muscle contraction and, indirectly, the coordinated movement of organisms1. It is formed by a presynaptic motor axon terminal, a muscle postsynaptic domain enriched in acetylcholine receptors (AChRs), and non-myelinic terminal Schwann cells covering the axon terminal2,3,4. Upon NMJ denervation due to traumatic peripheral nerve injury, disease, or pharmacologic intervention, contractile muscle activity is lost5,6. As certain permissive microenvironments allow timely and efficient functional recovery, the search for proteins that could help the regeneration process is a permanent necessity. Here, two procedures have been combined to specifically evaluate the potential role of manipulating muscle protein expression in the short-term regeneration of the NMJ after nerve injury.
The levator auris longus (LAL) muscle, located on the dorsal surface of the skull, is a thin and flat muscle that controls the movement of the pinna. The LAL muscle consists of rostral and caudal bands, each containing two or three layers of muscle fibers7,8. The posterior auricular branch of the facial nerve innervates the LAL muscle, generating a well-described innervation pattern consisting of five rostral (R1-R5) and two caudal (C1-C2) innervation regions8,9,10. The LAL is a superficially exposed and easily accessible muscle, so its use requires minimally invasive procedures. This allows visualization of the NMJ using real-time microscopy, drug delivery, as well as the intervention of complete muscle preparations both in vivo and ex vivo11,12. Altogether, these features make the LAL muscle an excellent model to study NMJ behavior and function10,13.
In vivo electroporation is a gene-transfer technique that allows the incorporation of exogenous DNA into the tissue through the transient permeabilization of cell membranes and the mobility of DNA inside the cells, both generated after inducing an electric field14,15. This procedure is local and can be performed at any stage of the animal&#39;s development. Considering the unique features of the LAL muscle, its in vivo electroporation represents a minimally invasive, highly efficient, and quick procedure16, making it possible to observe robust protein expression two or three days after electroporation.
The facial nerve crush procedure has been widely employed in research as it offers several advantages5,17,18. This protocol has been previously modified to specifically target the posterior auricular branch of the facial nerve that innervates the cranial muscles, including the LAL muscle, to avoid facial paralysis; consequently, mice do not require special care after surgery (e.g., lubricating eye ointment for the loss of blink reflex). As NMJ reinnervation at the LAL muscle begins between 7-9 days after injury6, it is a good short-term reinnervation model compared to the commonly used nerve crush injury model of the sciatic nerve to denervate hindlimb muscles, where reinnervation occurs around three weeks post nerve injury18.
The combination of in vivo electroporation and facial nerve injury at the LAL muscle will enable researchers to evaluate the behavior of synaptic components against muscle-derived protein modulation at different times of NMJ regeneration, including short-term and long-term morphological effects6,16, through a wide variety of techniques including immunohistochemical, histological, and functional assays.

Author Spotlight: Regenerative Roles of Muscle Proteins at Neuromuscular Junction Post-Nerve Injury

Combined In Vivo Electroporation and Short&#45;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 overexpressing 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 protein's generation, paving the way for more intricate gene-editing techniques. Moreover, these protocols are minimally invasive. This combination of experimental methods has been employed to investigate the role of Wnt signaling or NMJ regeneration.By overexpressing agonists or inhibitors of the Wnt pathway, we analyzed nerve damage and the subsequent regeneration processes. Our future research will examine the impact of muscle-derived proteins on the organization and functionality of the neuromuscular synapse in various contexts, including aging and certain pathologies, such as ALS.

Crush Nerve Injury&#45;Induced Denervation in Mouse Levator Auris Longus Muscle to Assess NMJ Reinnervation and Maintenance

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 facial nerve. Using forceps with a 45-degree angled tip, apply a 30-second crush to the nerve with constant pressure.Finally, using a 5/0 string size and an HR15 needle, close the incision with absorbable polyglycolic acid sutures. After recovery and meloxicam treatment, examine the mouse's LAL microscopically for denervation. Confocal microscopy imaging of LAL muscles showed complete neuromuscular junction denervation five days after nerve injury.

crush-nerve-injury-induced-denervation-mouse-levator-auris-longus

Research

JoVE Journal

Neuroscience

31 Views. 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 cartilagino...

Video: Crush Nerve Injury-Induced Denervation in Mouse Levator Auris Longus Muscle to Assess NMJ Reinnervation and Maintenance

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

In Vivo Levator Auris Longus Skeletal Muscle Electroporation to Study NMJ Regeneration in Mice

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 5 millimeters apart in parallel alignment over the muscle fibers, covering the full length of the LAL muscle. Now, use an electro operator to apply five electrical pulses at 100 volts per 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.