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

Summary

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.

Abstract

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.

Introduction

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 terminal^2,3,4. Upon NMJ denervation due to traumatic peripheral nerve injury, disease, or pharmacologic intervention, contractile muscle activity is lost^5,6. As certai....

Protocol

All experimental procedures are approved by the Bioethics Committee at Universidad Austral de Chile, Chile (protocol Nr. 503/2023), and followed the norms imposed by the Bioethics Committee of the National Research and Development Agency, Chile (ANID), as well as the guidelines of the European Council Directive for the Care of Laboratory Animals. The present study used adult (3-6 months old) CF-1 mice of both sexes (30-45 g). Euthanasia was accomplished by inhalant anesthetic overdose followed by exsanguination. The reagents and equipment used in the study are listed in the Table of Materials.

1. In vivo LAL....

Results

The high fluorescence quantum yield of the tdTomato protein²⁰ makes it an appropriate tracer for electroporation efficiency. Therefore, the expression of tdTomato protein in LAL muscles by in vivo electroporation was evaluated (Figure 1). LAL muscles were dissected 21 days after electroporation. Whole-mount preparations show the thin caudal band (cLAL, cyan polygon) of the LAL muscle with its two innervation regions, and the thick rostral band (rLAL, yellow p.......

Discussion

The combination of in vivo muscle electroporation and denervation is a valuable experimental approach to investigating a regenerative niche at the NMJ⁴. Since the LAL muscle is superficially exposed, these combined procedures can be readily complemented with the delivery of probes or drugs that could affect protein expression or activity¹². In addition, electroporation of reporter genes into the LAL muscle could provide a valuable tool to follow the activation or i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#5/45 Dumont forceps	Fine Science Tools	11251-35	Sterilize before use
anti 2H3	DSHB		Dilute 1 : 300
anti S100 Ready to Use	Dako	IR504	Dilute 1 : 5
anti SV2	DSHB		Dilute 1 : 50
Chlorhexidine gluconate 2%	DifenPharma
Cold light dissecting stereomicroscope	Motic	Model SMZ-171
DAPI	Invitrogen	D1306	Dilute 1 : 100
Dumont #5SF Forceps	Fine Science Tools	11252-00	Sterilize before use
Dumont Mini Forceps –Style 5	Fine Science Tools	11200-14	Sterilize before use
ECM 830 electroporator	BTX Harvard Apparatus
Gold needle-type electrodes	Genetrodes, BTX	45-0114	10 mm straight
Hamilton syringe	Hamilton	80400
Hyaluronidase	Sigma-Aldrich	H3884
Inhalation anesthesia machine	SciVerma Scientific	M3000 Table Top
Isoflurane	Baxter	218-082
Mice	Instituto de Salud Publica - Chile		Adult (3-6 months old) CF-1 mice, of both sexes (30-45 g)
Razor blades	Schick
Suture Glicosorb 5/0	TAGUM	GS0812.J	Absorbable polyglycolic acid thread
tdTomato plasmid	Addgene	54642	tdTomato plasmid under the control of the CMV promoter
Tramadol hydrochloride 5% (Triamcol)	Drag Pharma
Vannas Spring Scissors	Fine Science Tools	91500-09	Sterilize before use
WGA Alexa Fluor 488	Invitrogen	W7024	Dilute
α-BTX Alexa Fluor 488	Invitrogen	B13422	Dilute 1 : 500