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 border="0" cellpadding="0" cellspacing="0" width="612">
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			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="20" style="height:20px;">Nylon Suture, 9-0</td>
			<td>Serag Weissner</td>
			<td>Z0039490</td>
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			<td>-</td>
			<td>Multiple thread sizes</td>
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			<td height="20" style="height:20px;">Suprarenin 1mg/ml</td>
			<td>Sanofi</td>
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			<td>&#176;</td>
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			<td>Cook Medical</td>
			<td>G03014</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">DP-M350 Blood Flow Monitor</td>
			<td>Cook Medical</td>
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			<td>&#176;</td>
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			<td height="20" style="height:20px;">Surgical Microscope OPMI Vario</td>
			<td>Carl Zeiss</td>
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			<td>&#176;</td>
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			<td height="20" style="height:20px;">Microsurgical instruments lab set</td>
			<td>S&#38;T</td>
			<td>767</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">Biemer vessel clip</td>
			<td>Diener</td>
			<td>64,562</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">Applying forceps</td>
			<td>Diener</td>
			<td>64,568</td>
			<td>for Biemer vessel clip</td>
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			<td height="20" style="height:20px;">Cefuroxim 1500mg</td>
			<td>Fresenius</td>
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			<td height="20" style="height:20px;">Braunoderm</td>
			<td>Braun Melsungen</td>
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			<td>&#176;</td>
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			<td height="20" style="height:20px;">Octenisept</td>
			<td>Schuelke &#38; Mayr</td>
			<td>5702764</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">ISIS Neuromonitoring System</td>
			<td>Inomed</td>
			<td>-</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">Tissucol</td>
			<td>Baxter</td>
			<td>1.33052E+12</td>
			<td>Fibrin glue</td>
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			<td height="20" style="height:20px;">Jackson-Pratt Wound Drainage</td>
			<td>Medline</td>
			<td>SU130-1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Myacyne Ointment</td>
			<td>Schur Pharma</td>
			<td>-</td>
			<td></td>
		</tr>
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</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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

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

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Chapters in this video

In Vivo Canine Muscle Function Assay

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

Patient Derived Cell Culture and Isolation of CD133⁺ Putative Cancer Stem Cells from Melanoma

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue