Dr. Eisenhardt is funded by the German Research Foundation (DFG) # EI 866/1-1 and #EI 866/2-1.

<ol>
	<li>Mavrikakis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+nerve+palsy:+anatomy,+etiology,+evaluation,+and+management.">Facial nerve palsy: anatomy, etiology, evaluation, and management.</a> Orbit. 27 (6), 466-474 (2008).</li><li>Pitanguy, I., Ramos, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frontal+branch+of+the+facial+nerve:+the+importance+of+its+variations+in+face+lifting.">The frontal branch of the facial nerve: the importance of its variations in face lifting.</a> Plast Reconstr Surg. 38 (4), 352-356 (1966).</li><li>McAllister, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+interventions+for+the+early+management+of+Bell's+palsy.">Surgical interventions for the early management of Bell's palsy.</a> Cochrane Database Syst Rev. 10 (2), (2013).</li><li>Terzis, J. K., Anesti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+facial+paralysis:+a+review.">Developmental facial paralysis: a review.</a> J Plast Reconstr Aesthet Surg. 64 (10), 1318-1333 (2011).</li><li>Momeni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stage+microsurgical+reconstruction+for+facial+palsy+utilising+the+motor+nerve+to+the+masseter.">Single-stage microsurgical reconstruction for facial palsy utilising the motor nerve to the masseter.</a> Handchir Mikrochir Plast Chir. 42 (2), 95-101 (2010).</li><li>Volk, G. F., Pantel, M., Guntinas-Lichius, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+concepts+in+facial+nerve+reconstruction.">Modern concepts in facial nerve reconstruction.</a> Head Face Med. 6, 25 (2010).</li><li>Mathes, S. J., Nahai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+the+vascular+anatomy+of+muscles:+experimental+and+clinical+correlation.">Classification of the vascular anatomy of muscles: experimental and clinical correlation.</a> Plast Reconstr Surg. 67 (2), 177-187 (1981).</li><li>Harii, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneurovascular+free+muscle+transplantation+for+reanimation+of+facial+paralysis.">Microneurovascular free muscle transplantation for reanimation of facial paralysis.</a> Clin Plast Surg. 6 (3), 361-375 (1979).</li><li>Borschel, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+motor+nerve+to+the+masseter+muscle:+an+anatomic+and+histomorphometric+study+to+facilitate+its+use+in+facial+reanimation.">The motor nerve to the masseter muscle: an anatomic and histomorphometric study to facilitate its use in facial reanimation.</a> J Plast Reconstr Aesthet Surg. 65 (3), 363-366 (2012).</li><li>Zuker, R. M., Goldberg, C. S., Manktelow, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+animation+in+children+with+Mobius+syndrome+after+segmental+gracilis+muscle+transplant.">Facial animation in children with Mobius syndrome after segmental gracilis muscle transplant.</a> Plast Reconstr Surg. 106 (1), 1-8 (2000).</li><li>Bannasch, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+evaluation+of+the+concomitant+use+of+the+implantable+Doppler+probe+and+the+Vacuum+Assisted+Closure+system+in+free+tissue+transfer.">A critical evaluation of the concomitant use of the implantable Doppler probe and the Vacuum Assisted Closure system in free tissue transfer.</a> Microsurgery. 28 (6), 412-416 (2008).</li><li>Sierra, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+sealant+adhesive+systems:+a+review+of+their+chemistry,+material+properties+and+clinical+applications.">Fibrin sealant adhesive systems: a review of their chemistry, material properties and clinical applications.</a> J Biomater Appl. 7 (4), 309-352 (1993).</li><li>Brien, B. M., Lawlor, D. L., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneurovascular+free+muscle+reconstruction+for+long+established+facial+paralysis.">Microneurovascular free muscle reconstruction for long established facial paralysis.</a> Ann Chir Gynaecol. 71 (1), 65-69 (1982).</li><li>Eisenhardt, S. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cross+face+nerve+graft+with+masseteric+nerve+as+donor+nerves+for+free+functional+muscle+transfers+in+facial+reanimation+surgery].">Comparison of cross face nerve graft with masseteric nerve as donor nerves for free functional muscle transfers in facial reanimation surgery].</a> Handchir Mikrochir Plast Chir. 45 (4), 223-228 (2013).</li><li>Harrison, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectoralis+minor+vascularized+muscle+graft+for+the+treatment+of+unilateral+facial+palsy.">The pectoralis minor vascularized muscle graft for the treatment of unilateral facial palsy.</a> Plast Reconstr Surg. 75 (2), 206-216 (1985).</li><li>Frey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphometric+studies+in+patients+with+facial+palsy+treated+by+functional+muscle+transplantation:+new+aspects+for+the+surgical+concept.">Histomorphometric studies in patients with facial palsy treated by functional muscle transplantation: new aspects for the surgical concept.</a> Ann Plast Surg. 26 (4), 370-379 (1991).</li><li>Eisenhardt, S. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salvage+Procedures+After+Failed+Facial+Reanimation+Surgery+Using+the+Masseteric+Nerve+as+the+Motor+Nerve+for+Free+Functional+Gracilis+Muscle+Transfer.">Salvage Procedures After Failed Facial Reanimation Surgery Using the Masseteric Nerve as the Motor Nerve for Free Functional Gracilis Muscle Transfer.</a> JAMA Facial Plast Surg. , (2014).</li><li>Bae, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+commissure+excursion+following+gracilis+muscle+transplantation+for+facial+paralysis+using+a+cross-face+nerve+graft+versus+the+motor+nerve+to+the+masseter+nerve.">A comparison of commissure excursion following gracilis muscle transplantation for facial paralysis using a cross-face nerve graft versus the motor nerve to the masseter nerve.</a> Plast Reconstr Surg. 117 (7), 2407-2413 (2006).</li><li>Schaverien, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+masseter+muscle+during+normal+smile+production+and+the+implications+for+dynamic+reanimation+surgery+for+facial+paralysis.">Activation of the masseter muscle during normal smile production and the implications for dynamic reanimation surgery for facial paralysis.</a> J Plast Reconstr Aesthet Surg. 64 (12), 1585-1588 (2011).</li><li>Manktelow, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smile+reconstruction+in+adults+with+free+muscle+transfer+innervated+by+the+masseter+motor+nerve:+effectiveness+and+cerebral+adaptation.">Smile reconstruction in adults with free muscle transfer innervated by the masseter motor nerve: effectiveness and cerebral adaptation.</a> Plast Reconstr Surg. 118 (4), 885-899 (2006).</li><li>Eppley, B. L., Zuker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salvage+of+facial+reanimation+with+vascularized+adductor+magnus+muscle+flap:+clinical+experience+and+anatomical+studies.">Salvage of facial reanimation with vascularized adductor magnus muscle flap: clinical experience and anatomical studies.</a> Plast Reconstr Surg. 110 (7), 1693-1696 (2002).</li><li>Labbe, D., Huault, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lengthening+temporalis+myoplasty+and+lip+reanimation.">Lengthening temporalis myoplasty and lip reanimation.</a> Plast Reconstr Surg. 105 (4), 1289-1297 (2000).</li><li>Iblher, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+evaluation+tool+for+monitoring+devices+and+its+application+to+evaluate+the+implantable+Doppler+probe.">A new evaluation tool for monitoring devices and its application to evaluate the implantable Doppler probe.</a> J Reconstr Microsurg. 26 (4), 265-270 (2002).</li></ol>

The authors have nothing to disclose.

Even though different surgical techniques have been described to regain a dynamic smile in patients with long standing facial paralysis, the two-stage repair with initial cross facial nerve grafting and consecutive free gracilis muscle transfer is seen as the &#8220;criterion standard&#8221;.
Although a two-stage procedure, the technique allows for spontaneity of the smile which is seen as a crucial factor to overcome the social handicap associated with facial palsy13.In cases of bi...

The facial nerve is involved in ocular protection, articulation, oral continence and greatly affects the aesthetic appearance of the face. Impairment of this nerve is therefore associated with significant morbidity and social withdrawal. Despite significant advances in therapy, the multitude of problems associated with the paralyzed face can only be targeted with a broad spectrum of additional procedures. Common to all surgical techniques is the need for an exact anatomical knowledge.
Facial nerve anatomy
The facial nerve consists of a branchial motor component for voluntary motor control of the facial musculature and a visceral motor component for parasympathetic control of lacrimal, submandibular, and sublingual glands. Besides, there are two sensory components for the innervation of the external auditory canal and for the taste in the anterior two third of the tongue. The course of the facial nerve can be subdivided into three segments: intracranial, intratemporal, and extratemporal. In the intracranial segment, the upper group of neurons that innervate the frontalis muscle and the periauricular region receives bilateral cortical input. Neurons that innervate the remaining facial muscles receive contralateral cortical input exclusively. As a consequence, frontalis function is maintained in ipsilateral supranuclear lesions. The intratemporal segment can be further divided into three segments. In the labyrinthine segment, the greater petrosal nerve leaves the trunk to supply the lacrimal gland with parasympathetic fibers1.
In the mastoid segment, the thin stapedius nerve runs to the correspondent muscle. Parasympathetic branches innervate the submandibular, sublingual and anterior lingual glands, whereas the anterior two thirds of the tongue are supplied with gustatory fibres (chorda tympani). The main stem of the facial nerve exits the bony canal through the stylomastoid foramen. This is the beginning of the extratemporal segment, however arborisation does not start before entering the parotid gland. The nerve is first divided into 3 to 4 motor divisions that form the intraparotid plexus and ultimately give rise to the temporal, zygomatic, buccal, mandibular and cervical branches2.
Differential diagnosis of facial paralysis
As etiology of facial paralysis is broad and hard to classify, the division that is affected should be considered first.
Intracranial facial paralysis can be caused by lacunar infarcts or tumors of the intracranial cavity. Bacterial and viral infections, cholesteatoma and Bell&#8217;s palsy can be reasons for intratemporal nerve damage. Neoplastic malignancies and associated surgical therapy are the dominant causes for extratemporal facial paralysis. Although Bell&#8217;s palsy represents the most common diagnosis in patients with facial paralysis, most patients recover completely without sequelae and do not need surgery3. The second most common cause of facial paralysis is trauma. Here, fractures to the temporal bone are the predominant trauma mechanism4.
Treatment types of facial paralysis
Numerous surgical options exist for the treatment of facial paralysis and they may be classified into reinnervation, static reconstruction and dynamic reconstruction. Typically, two years are considered to be the time from injury in which function after reinnervation can be regained satisfactory with reinnervating procedures5. Later on, denervation atrophy of facial muscles precludes their usefulness for further reconstruction. Reinnervation can be obtained by primary nerve repair, interpositional nerve grafts, cross-facial nerve grafting or cranial nerve transfer. Static reconstruction techniques are directed to correct functional disabilities, (protection of the cornea, improvement of nasal airflow, and prevention of drooling) and to improve symmetry at rest. Typical procedures are browlift for brow ptosis, or canthoplasty for lower lid ectropion. Static reconstruction is preferred in elderly patients with significant comorbidities or in massive facial defects secondary to trauma or cancer resection.
The surgical techniques of dynamic reconstruction can be subdivided into regional muscle transfer and free microneurovascular muscle transfer with either coaptation to the masseter motor branch or cross-facial nerve grafting. The latter represents the criterion standard for the reconstruction of the smile in facial paralysis, as no other treatment option reliably achieves a spontaneous smile that is crucial to improve social handicap. Coaptation to the masseter motor branch is the treatment modality of choice for patients with bilateral paralysis, however indications have extended, as elderly patients or patients with significant comorbidities usually prefer a single-stage procedure6.
Gracilis flap
Regional anatomy
The gracilis muscle is a rather superficial muscle of the medial thigh, representing the longest muscle of the adductor muscles. It originates from the lower symphysis and the inferior ramus of the pubis. Running distally, the muscle becomes narrow and inserts distal to the knee joint on the tibia that allows not only adduction of the thigh but also flexion of the knee. The gracilis muscle has a type II circulatory pattern after Mathes and Nahai with an arterial supply from a dominant and some minor vascular pedicles7. The dominant artery exits the muscle at the hilum to course laterally and usually terminates in the medial circumflex artery. Rarely, the dominant artery terminates directly in the deep femoral artery. Venous supply of the gracilis muscle is usually achieved through two venae comitantes, that course deep to the adductor longus muscle to follow the dominant artery. Innervation of the muscle is achieved through an anterior branch of the obturator nerve that enters the muscle 1-2 cm superiorly to the hilum.
Clinical use
The gracilis muscle is a valuable donor muscle for reconstructive microsurgery and has become the muscle of choice for many surgeons for functional free muscle transfer. This is owed to the fact that there is little donor site morbidity and the flap shows optimal proportions with regards to excursion of the muscle and vascular pedicle dimensions respectively. A long single innervated motor nerve alleviates functional flap harvest8.
We here demonstrate the case of a 49 year old female, who initially presented with a complete picture of left peripheral facial paralysis following resection of an acoustic neuroma (vestibular schwannoma) 2 years earlier. The patient was most afflicted by facial asymmetry, particularly when smiling. Other pre-existing comorbidities were not documented.
Upon clinical examination, the patient showed a complete paresis of the frontalis muscle, however a satisfactory forehead symmetry at rest. Lid closure was insufficient on the left with a lagophthalmos of 5 mm and Bell&#8217;s phenomenon. Signs of cornea irritation and ectropion were absent. At rest, the patient showed a moderate asymmetry of the corner of the mouth with a tragus-modiolus distance of 11 cm on the right and 11.5 cm on the left at rest. Upon smiling, tragus-modiolus distance scaled down to 9 cm on the right and elongated on the left to 12 cm. After extensive counseling, the patient wished for a single-staged dynamic reconstruction of the smile with a free functional gracilis transfer using the masseter as donor nerve. The patient was also informed about various techniques of lid closure reconstruction, however declined surgical treatment at this stage. The course of the treatment was uncomplicated. Signs of reinnervation were first noticed three month postoperatively. Four months after surgery, the patient presented with unremarkable scarring along the former modified facelift incision line. Symmetry at rest and deliberate smiling were excellent with a satisfactory definition of the nasolabial crease. The patient also showed a completely spontaneous smile. Pre- and 9-month postoperative range of excursion of the corner of the mouth was documented by videography.

<table><tbody><tr><td>Nylon Suture, 9-0</td><td>Serag Weissner</td><td>Z0039490</td><td>&amp;deg;</td></tr><tr><td>Polypropylene Suture&amp;nbsp;</td><td>Ethicon</td><td>-</td><td>Multiple thread sizes</td></tr><tr><td>Suprarenin 1mg/ml</td><td>Sanofi</td><td>-</td><td>&amp;deg;</td></tr><tr><td>Cook-Swartz Doppler Probe</td><td>Cook Medical</td><td>G03014</td><td>&amp;deg;</td></tr><tr><td>DP-M350 Blood Flow Monitor</td><td>Cook Medical</td><td>-</td><td>&amp;deg;</td></tr><tr><td>Surgical Microscope OPMI Vario</td><td>Carl Zeiss</td><td>-</td><td>&amp;deg;</td></tr><tr><td>Microsurgical instruments lab set</td><td>S&amp;amp;T</td><td>767</td><td>&amp;deg;</td></tr><tr><td>Biemer vessel clip</td><td>Diener</td><td>64,562</td><td>&amp;deg;</td></tr><tr><td>Applying forceps</td><td>Diener</td><td>64,568</td><td>for Biemer vessel clip</td></tr><tr><td>Cefuroxim 1500mg</td><td>Fresenius</td><td>J01DC02</td><td>&amp;deg;</td></tr><tr><td>Braunoderm</td><td>Braun Melsungen</td><td>3881105</td><td>&amp;deg;</td></tr><tr><td>Octenisept</td><td>Schuelke &amp;amp; Mayr</td><td>5702764</td><td>&amp;deg;</td></tr><tr><td>ISIS Neuromonitoring System</td><td>Inomed</td><td>-</td><td>&amp;deg;</td></tr><tr><td>Tissucol</td><td>Baxter</td><td>1.33052E+12</td><td>Fibrin glue</td></tr><tr><td>Jackson-Pratt Wound Drainage</td><td>Medline</td><td>SU130-1060</td><td></td></tr><tr><td>Myacyne Ointment</td><td>Schur Pharma</td><td>-</td><td></td></tr></tbody></table>

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 for reinnervation of the muscle graft was assessed in 5 patients (Table 1). Among them, acoustic neuroma resection was the main cause for facial paralysis. 3 candidates preferred the single-stage procedure rather than the &#8220;gold standard&#8221; two-stage approach. In two patients, the procedure served as a rescue procedure after insufficient stimulation by the cross-facial nerve graft (CFNG).
Reinnervation of the gracilis flap after single-...

Watch this Scientific Journal Video about Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer at JoVE.com

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

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 ...

single-stage-dynamic-reanimation-smile-irreversible-facial-paralysis

Research

JoVE Journal

Medicine

105.8K Views.  University of Freiburg Medical Centre. 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.

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

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

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 ...

Neuroscience

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 ...

Structured Motor Rehabilitation After Selective Nerve Transfers

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on the successful re-innervation of the targets in the periphery and on the motor re-learning process entailing cortical plasticity. While there is an increasing number of methods to improve rehabilitation, their routine implementation in a clinical setting remains a challenge due to their complexity and long duration. Therefore, recommendations for rehabilitation strategies are presented with the aim of guiding medical doctors and therapists through the long-lasting rehabilitation process and providing step-by-step instructions for supporting motor re-learning.
Directly after nerve transfer surgery, no motor function is present, and therapy should focus on promoting activity in the sensory-motor cortex areas of the paralyzed body part. After about two to six months (depending on the severity and modality of injury, the distance of nerve regeneration and many other factors), the first motor activity can be detected via electromyography (EMG). Within this phase of rehabilitation, multimodal feedback is used to re-learn the motor function. This is especially critical after nerve transfers, as muscle activation patterns change due to the altered neural connection. Finally, muscle strength should be sufficient to overcome gravity/resistance of antagonistic muscles and joint stiffness, and more functional tasks can be implemented in rehabilitation.

Here, we present a protocol for the motor rehabilitation of patients with severe nerve injuries and selective nerve transfer surgery. It aims at restoring the motor function proposing several stages in patient education, early-stage therapy after surgery and interventions for rehabilitation after successful re-innervation of the nerve&#8217;s target.

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on ...

Evaluating Biomaterials in Craniofacial VML Injury Using a Rat Model

Rodent Model of Masseter Volumetric Muscle Loss for Studying Bioengineering Materials

Craniofacial volumetric muscle loss (VML) injuries can occur as a result of severe trauma, surgical excision, inflammation, and congenital or other acquired conditions. Treatment of craniofacial VML involves surgical, functional muscle transfer. However, these procedures are unable to restore normal function, sensation, or expression, and more commonly, these conditions go untreated. Very little research has been conducted on skeletal muscle regeneration in animal models of craniofacial VML. This manuscript describes a rat model for the study of craniofacial VML injury and a protocol for the histological evaluation of biomaterials in the treatment of these injuries. Liquid hydrogel and freeze-dried scaffolds are applied at the time of surgical VML creation, and masseters are excised at terminal time points up to 12 weeks with high retention rates and negligible complications. Hematoxylin and eosin (HE), Masson's Trichrome, and immunohistochemical analysis are used to evaluate parameters of skeletal muscle regeneration as well as biocompatibility and immunomodulation. While we demonstrate the study of a hyaluronic-acid-based hydrogel, this model provides a means for evaluating subsequent iterations of materials in VML injuries.

Here, we describe a protocol for the surgical creation of a volumetric muscle loss (VML) injury in the rat masseter, providing a reproducible and accessible model for the study of craniofacial muscle injuries and their treatment using biomaterials such as the novel hydrogel.

Craniofacial volumetric muscle loss (VML) injuries can occur as a result of severe trauma, surgical excision, inflammation, and congenital or other ...

Bioengineering

Force Assessment of Rat Masseter Muscle After VML Injury

In Vivo Functional Assessment of Rat Masseter Muscle Following Surgical Creation of a Volumetric Muscle Loss (VML) Injury

Volumetric Muscle Loss (VML) is prevalent in civilian and military populations and represents a debilitating skeletal muscle injury surpassing the body&#39;s natural regenerative capacity. These injuries disrupt not only muscle fibers but also nerves, blood vessels, and the extracellular matrix, overwhelming the regenerative capacity of skeletal muscle and leading to severe fibrosis and permanent deficiencies in muscle structure and function. Current clinical management has many limitations, and thus, research is ongoing to develop more effective therapeutic approaches. Notably, however, much of the preclinical emphasis on VML injuries has focused on limb and trunk muscles, with limited investigation into craniofacial muscles. Differences in developmental biology and regenerative capacity between craniofacial and limb/trunk muscles may provide crucial insights that drive more injury-specific VML treatment options. Moreover, evaluation of functional recovery is critical to establishing therapeutic efficacy. In this regard, in vivo testing of muscle contraction with percutaneous nerve stimulation is a minimally invasive method that allows for repeated functional assessment over the course of a study - in the same animal. In light of these considerations, this paper describes a method for the in vivo assessment of muscle function in the rat masseter muscle before and after a VML injury. This protocol is the first published instance to detail the creation and functional evaluation of a biologically relevant craniofacial VML injury in the rat.

Volumetric muscle loss (VML) injuries exceed endogenous regenerative ability, resulting in permanent functional deficits. Current VML research primarily focuses on limb and trunk muscles. To extend mechanistic studies of VML to craniofacial muscles, this article describes an in vivo method for longitudinal assessment of masseter muscle function pre- and post-VML injury.

Volumetric Muscle Loss (VML) is prevalent in civilian and military populations and represents a debilitating skeletal muscle injury surpassing the ...

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

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.

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.

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

Establishing an Orofacial Muscle Fibrosis Model in Mice via a Freezing Injury

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.

Author Spotlight: Exploring Orofacial Muscle Regeneration &#8211; Insights and Innovations

The goal of this protocol is to establish an orofacial muscle fibrosis model. Comparison of the histology between mice masseter and tibialis anterior muscle after freezing injury confirmed masseter muscle fibrosis. This model will facilitate further investigation into the mechanism underlying orofacial muscle fibrosis.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived ...

Biology

Establishment of Facial Nerve Injury Rat Model for Idiopathic Facial Paralysis Research

Idiopathic facial paralysis is the most common type of facial nerve injury, accounting for approximately 70% of peripheral facial paralysis cases. This disease can not only lead to a change in facial expression but also greatly impact the psychology of patients. In severe cases, it can affect the normal work and life of patients. Therefore, the research on facial nerve injury repair has important clinical significance. In order to study the mechanism of this disease, it is necessary to carry out relevant animal experiments, among which the most important task is to establish an animal model with the same pathogenesis as human disease. The compression of the facial nerve within the petrous bone, especially the nerve trunk at the junction of the distal end of the internal auditory canal and the labyrinthine segment, is the pathogenesis of idiopathic facial paralysis. In order to simulate this common disease, a compression injury model of the main extracranial segment of the facial nerve was established in this study. The neurological damage was evaluated by behavioral, neuroelectrophysiological, and histological examination. Finally, 50 g constant force and 90 s clamp injury were selected as the injury parameters to construct a stable idiopathic facial paralysis model.

The present protocol establishes a facial nerve injury rat model using microscopy to investigate the diagnostic and therapeutic mechanisms of idiopathic facial paralysis.

Idiopathic facial paralysis is the most common type of facial nerve injury, accounting for approximately 70% of peripheral facial paralysis cases. This ...

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

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Structured Motor Rehabilitation After Selective Nerve Transfers

Rodent Model of Masseter Volumetric Muscle Loss for Studying Bioengineering Materials

In Vivo Functional Assessment of Rat Masseter Muscle Following Surgical Creation of a Volumetric Muscle Loss (VML) Injury

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

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Establishment of Facial Nerve Injury Rat Model for Idiopathic Facial Paralysis Research