We thank the highly collaborative and stimulating environment of the NeSt Lab members for useful discussion and comments on this work. Work at the NeSt Lab is currently funded by FONDECYT 1221213. The scheme in Figure 1A was created with BioRender.com.

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 muscle electroporation
<ol>
	<li>Preparation
	<ol>
		<li>Gently sedate the mouse in an acrylic induction chamber under inhalation of 2.5% isofluorane with 0.8 L/min oxygen (following institutionally approved protocols). After that, maintain mouse sedation using a nose tubing mask connected to an inhalation anesthesia machine under a dissecting stereomicroscope. 
		&#8203;NOTE: Ensure that the mouse is asleep before starting the procedure; test the touch reflex of the mouse by gently squeezing one foot with a forceps (do this step every time after inducing anesthesia).</li>
		<li>Disinfect the cervical area at the level of the sagittal suture of the skull with the antiseptic chlorhexidine gluconate 2% and inject subcutaneously 20 &#181;L of 2 mg/mL hyaluronidase in PBS using a Hamilton syringe to facilitate the DNA plasmid access to the muscle surface.</li>
		<li>Turn off the isofluorane flow and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Weigh&#160;the mouse to calculate the appropriate Meloxicam&#160;dose (see step 1.3.2.).</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the animal to&#160;normal housing for 30 min16.</li>
	</ol>
	</li>
	<li>Surgery procedure
	<ol>
		<li>Re-anesthetize the mouse by the inhalation of 2.5% isofluorane with a 0.8 L/min oxygen mixture (following institutionally approved protocols) and keep it sedated throughout the surgical procedure using an inhalation anesthesia machine, under a dissecting stereomicroscope.</li>
		<li>Shave the cervical area using a double-edged razor blade and disinfect with chlorhexidine and 70% ethanol 3 times.</li>
		<li>Make an 8 mm incision over the sagittal suture of the skull and expose the LAL muscle by gently removing the fat and connective tissue located just under the skin using scissors/forceps. 
		NOTE: The LAL muscle is the most superficial muscle in the cranial region after skin incision. It is easily recognized by the orientation of muscle fibers that run diagonally from the middle skull towards each pinna8,16.</li>
		<li>Using a Hamilton syringe, inject 10 &#181;L of the commercially obtained DNA (0.8-1 &#181;g/&#181;L) in PBS into the fascia that is distributed throughout the muscle, forming a bubble.</li>
		<li>Place two gold needle electrodes 5 mm apart, parallel, and over muscle fibers, occupying the entire length of the LAL muscle (Figure 1A).</li>
		<li>Using an electroporator, generate five pulses of 100 V/cm of 20 ms duration each, at 1 Hz frequency.</li>
		<li>Repeat the procedure in the contralateral LAL muscle (repeating steps 1.2.4-1.2.6).</li>
	</ol>
	</li>
	<li>Suture and recovery
	<ol>
		<li>Suture (5/0 strain size, HR15 needle) the skin with absorbable polyglycolic acid thread.</li>
		<li>Inject subcutaneously the analgesic Meloxicam in doses of 1-2 mg/kg of weight to reduce post-surgery pain.</li>
		<li>Turn off the isofluorane flow and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the animal to its regular housing and monitor it for 2 days after surgery or until its recovery. Monitor pain or stress signs, allocating scores for each of the different parameters considering the humane endpoint criteria previously described19. 
		NOTE: Position the mouse in a heated pad covered with a surgical pad to prevent its body temperature from dropping during surgery. Try not to take more than 30 min for the surgical procedure, thus preventing the mouse from dying due to excess anesthesia. If the expressed protein yields low fluorescence levels, it is recommended to co-electroporate with a tracer (e.g., tdTomato or GFP) in a 1:5 ratio (tracer: DNA of interest).</li>
	</ol>
	</li>
</ol>
2. Crush nerve injury-induced denervation
NOTE: This protocol is a modification of a previously described protocol17. Perform this procedure with a difference of at least 3 days from the electroporation. Depending on the research questions, this procedure can be performed before or after electroporation.
<ol>
	<li>Anesthesia and animal positioning
	<ol>
		<li>Perform the animal sedation in an acrylic induction chamber under inhalation of 2.5% isofluorane with 0.8 L/min oxygen (following institutionally approved protocols).</li>
		<li>Weight the mouse to calculate the Meloxicam&#160;dose (see step 2.3.2.).</li>
		<li>Maintain mouse sedation with a nose tubing mask connected to an inhalation anesthesia machine under a dissecting stereomicroscope.</li>
		<li>Position the mouse on its left side (to denervate the right LAL muscle). Tape the right ear towards the nose to expose the posterior area of the pinna.</li>
	</ol>
	</li>
	<li>Surgery procedure
	<ol>
		<li>Shave the posterior area of the pinna using a double-edged razor blade and disinfect with chlorhexidine and 70% ethanol 3 times.</li>
		<li>Locate the posterior auricular vein and make an incision in the skin, 2 mm away caudally from it, of approximately 5 mm with spring scissors. Avoid cutting blood vessels and/or muscle tissue.</li>
		<li>Cut the fat and connective tissues with forceps and scissors. Find the spinal accessory nerve; continue until finding the belly of the digastric muscle and the cartilaginous auditory canal (semi-transparent whitish tissue) (Figure 2A).</li>
		<li>Locate the beginning of the facial nerve branching just below the digastric muscle.</li>
		<li>Follow the posterior auricular branch (the most dorsal branch of the facial nerve), and gently remove the connective tissue surrounding it, carefully avoiding direct nerve manipulation.</li>
		<li>Crush the posterior auricular branch of the facial nerve for 30 s, applying constant pressure on the nerve with a forceps with a 45&#176; angled tip.</li>
	</ol>
	</li>
	<li>Suture and recovery
	<ol>
		<li>Suture (5/0 strain size, HR15 needle) the skin with absorbable polyglycolic acid thread.</li>
		<li>Inject subcutaneously the analgesic Meloxicam in doses of 1-2 mg/kg of weight to reduce post-surgery pain.</li>
		<li>Remove the tape from the ear, turn off the isofluorane flow, and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the mouse to its regular housing and monitor it for 2 days after surgery or until its recovery.</li>
		<li>After recovery, examine the mouse for failures in the movement of the affected ear as a sign of efficient denervation. 
		NOTE: In Sham control animals, make an incision in the skin, expose the facial nerve without touching it directly, and subsequently suture.</li>
	</ol>
	</li>
</ol>

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

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The authors have nothing to disclose.

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...

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.

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

combined in vivo electroporation and short&#45;term reinnervation of the cranial levator auris longus skeletal muscle

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.

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...

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

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.

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

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

combined-vivo-electroporation-short-term-reinnervation-cranial

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Research

JoVE Journal

Neuroscience

361 Views.  Universidad Austral de Chile. Ons onderzoek heeft tot doel de mechanismen te begrijpen die ten grondslag liggen aan de herhaling van de neuromusculaire synaps en de impact te evalueren van het remmen of overexpressie van specifieke spierafgeleide eiwitten op dit regeneratieve proces. Door middel van spier in vivo elektroporatie hebben we de impact van spier-afgeleide eiwitten op de organisatie van postsynaptische acetylcholinereceptoren waargenomen. Bovendien heeft ons denervatieprotocol ...

Video: Auteur in de schijnwerpers: Regeneratieve rollen van spiereiwitten bij neuromusculaire junctie na zenuwbeschadiging

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bell&#8217;s palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis.
The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity.
Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

The use of the masseteric nerve as donor nerve represents a single-stage alternative to the criterion standard two-stage procedure of cross-facial nerve grafting and free muscle transfer in facial paralysis. We provide a detailed description to safely perform this technique with a gracilis muscle transfer and discuss indications and limitations.

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic ...

Medicine

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Biology

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

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

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