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

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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. 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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...

Le nerf facial est impliqué dans la protection oculaire, l&#39;articulation, la continence orale et affecte considérablement l&#39;aspect esthétique du visage. Dépréciation de ce nerf est donc associée à une morbidité importante et le retrait social. Malgré des avancées significatives dans la thérapie, la multitude de problèmes liés à la paralysie du visage ne peut être ciblée avec un large éventail de procédures supplémentaires. Commune à toutes les techniques chirurgicales est la nécessité d&#39;une connaissance de l&#39;anatomie exacte. L&#39;anatomie du nerf facial Le nerf facial est constitué d&#39;un composant de moteur branchies pour le contrôle volontaire du moteur de la musculature faciale et d&#39;un composant de moteur viscérale pour le contrôle parasympathique du lacrymal, sous-maxillaires et les glandes sublinguales. En outre, il ya deux composantes sensorielles pour l&#39;innervation du conduit auditif externe et pour le goût dans les deux tiers antérieur de la langue. Le cours du nerf facial peut be subdivisé en trois segments: intracrânienne, intratemporelle et extratemporale. Dans le segment intracrânienne, le groupe supérieur de neurones qui innervent le muscle frontal et la région reçoit périauriculaire entrée corticale bilatérale. Les neurones qui innervent les muscles du visage restants reçoivent entrée corticale controlatéral exclusivement. En conséquence, la fonction frontal est maintenu dans les lésions supranucléaires ipsilatérales. Le segment intratemporelle peut être divisé en trois segments. Dans le segment labyrinthique, le nerf grand pétreux quitte le tronc de fournir la glande lacrymale avec des fibres parasympathiques 1. Dans le segment mastoïdien, l&#39;étrier mince nerf passe au muscle correspondant. Branches parasympathiques innervent le sous-maxillaire, sublinguale et antérieure glandes linguales, tandis que les deux tiers antérieurs de la langue sont fournis avec des fibres gustatives (corde de timbales). La tige principale du nerf facial quitte le canal osseuxà travers le trou stylomastoïdien. Ce est le début du segment extratemporale cependant arborisation ne démarre pas avant d&#39;entrer dans la glande parotide. Le nerf est d&#39;abord divisé en trois à quatre divisions automobiles qui forment le plexus intraparotidienne et donnent finalement lieu à la temporelle, zygomatique, buccale, mandibulaire et branches cervicales 2. Le diagnostic différentiel de la paralysie faciale Comme étiologie de la paralysie faciale est vaste et difficile à classer, la division qui est touchée devrait être considéré en premier. La paralysie faciale intracrânienne peut être provoqué par un infarctus ou des tumeurs de la cavité intracrânienne lacunaires. Infections bactériennes et virales, cholestéatome et la paralysie de Bell peuvent avoir des raisons de lésions nerveuses intratemporelle. Néoplasiques malignes et traitement chirurgical associé sont les causes dominantes de paralysie faciale extratemporale. Bien que la paralysie de Bell représente le diagnostic le plus fréquentchez les patients avec une paralysie faciale, la plupart des patients se rétablissent complètement sans séquelles et ne ont pas besoin de chirurgie 3. La deuxième cause la plus fréquente de la paralysie faciale est un traumatisme. Ici, les fractures à l&#39;os temporal sont le mécanisme de traumatisme prédominante 4. types de paralysie faciale de traitement De nombreuses options chirurgicales existent pour le traitement de la paralysie faciale et ils peuvent être classés en réinnervation, la reconstruction statique et dynamique reconstruction. Généralement, deux ans sont considérés comme le temps d&#39;une blessure dans laquelle la fonction après réinnervation peut être regagnée satisfaisant avec reinnervating procédures 5. Plus tard, dénervation l&#39;atrophie des muscles du visage se oppose à leur utilité pour favoriser la reconstruction. Réinnervation peut être obtenue en réparation primaire du nerf, les greffes nerveuses interposition, croix-du nerf facial greffe ou transfert de nerf crânien. Techniques de reconstruction statiques sont adressées à corrigerincapacités fonctionnelles, (protection de la cornée, l&#39;amélioration des flux d&#39;air nasale, et la prévention de bave) et pour améliorer la symétrie au repos. Procédures typiques sont browlift pour le front ptosis, ou canthoplastie pour ectropion inférieure du couvercle. Reconstruction statique est préférable chez les patients âgés atteints de comorbidités importantes ou des défauts massifs visage secondaires à un traumatisme ou d&#39;une résection du cancer. Les techniques chirurgicales de reconstruction dynamique peuvent être subdivisés en transfert musculaire régionale et le transfert musculaire microneurovascular gratuit avec soit coaptation à la branche automobile masséter ou contre-nerf facial greffage. Ce dernier représente le critère de référence pour la reconstruction du sourire dans la paralysie faciale, comme aucune autre option de traitement permet d&#39;obtenir de manière fiable un sourire spontané qui est crucial d&#39;améliorer handicap social. Coaptation à la branche automobile masséter est la modalité de traitement de choix pour les patients atteints de paralysie bilatérale, cependant indications ont extfini, que les patients âgés ou les patients présentant des comorbidités significatives préfèrent généralement une procédure en une seule étape 6. Gracilis rabat Anatomie régional Le muscle est un muscle gracilis assez superficielle de la cuisse médiale, qui représente le plus long muscle des muscles adducteurs. Il provient de la symphyse inférieure et la branche inférieure du pubis. Exécution de manière distale, le muscle devient étroite et insère en aval de l&#39;articulation du genou sur le tibia qui permet non seulement adduction de la cuisse, mais aussi de flexion du genou. Le muscle gracilis circulatoire a un motif de type II après Mathes et Nahai avec un approvisionnement artériel d&#39;un dominant et des pédicules vasculaires mineures 7. L&#39;artère dominante quitte le muscle au niveau du hile au cours latéralement et en général se termine dans l&#39;artère circonflexe médial. Rarement, l&#39;artère dominante termine directement dans l&#39;artère fémorale profonde. Approvisionnement veineux de la graCilis musculaire est habituellement réalisé par deux veines satellites, ce cours profonde au muscle long adducteur de suivre l&#39;artère dominante. Innervation du muscle est atteint par une branche antérieure du nerf obturateur qui pénètre dans le muscle 1-2 cm supérieurement au hile. L&#39;utilisation clinique Le muscle droit interne est un muscle de donateurs précieuse pour la microchirurgie reconstructive et est devenu le muscle de choix pour de nombreux chirurgiens de transfert musculaire libre fonctionnelle. Ce est dû au fait qu&#39;il ya peu de la morbidité du site donneur et le volet montre proportions optimales en ce qui concerne à l&#39;excursion du muscle et pédiculaires dimensions vasculaires respectivement. Une longue unique nerf moteur innervée soulage la récolte du volet fonctionnel 8. Nous démontrons ici le cas d&#39;une femme âgée de 49 ans, qui a d&#39;abord présenté avec une image complète de paralysie faciale périphérique gauche après résection d&#39;une acoustiqueneurinome (schwannome vestibulaire) deux années plus tôt. Le patient a été plus affligé par l&#39;asymétrie faciale, en particulier lors du sourire. Autres comorbidités préexistants ne étaient pas documentés. Après examen clinique, le patient a montré une paralysie complète du muscle frontal, mais une symétrie de front satisfaisante au repos. fermeture du couvercle était insuffisante sur la gauche avec une lagophtalmie de 5 mm et le phénomène de Bell. Les signes d&#39;irritation de la cornée et ectropion étaient absents. Au repos, le patient a montré une asymétrie modérée du coin de la bouche avec une distance tragus-modiolus de 11 cm sur la droite et 11,5 cm sur la gauche au repos. Sur sourire, la distance tragus-modiolus réduite à 9 cm sur la droite et allongée sur la gauche à 12 cm. Après une vaste consultation, le patient a souhaité une reconstruction dynamique unique de la mise en scène sourire avec un transfert de gracilis fonctionnel libre utilisant le masséter comme donneur nerf. Le patient a également été informé diverses techniques de la reconstruction de fermeture du couvercle, cependant diminué traitement chirurgical à ce stade. Le cours du traitement est simple. Les signes de réinnervation ont d&#39;abord été remarqué trois mois après l&#39;opération. Quatre mois après la chirurgie, le patient a présenté avec des cicatrices banale long de l&#39;ancienne ligne d&#39;incision lifting modifié. Symétrie au repos et délibérée sourire étaient excellents avec une définition satisfaisante du pli naso-génien. Le patient a également montré un sourire complètement spontanée. Plage avant et 9 mois post-opératoire de l&#39;excursion du coin de la bouche a été documenté par vidéographie.

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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>
			<td>-</td>
			<td>&#176;</td>
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			<td height="20" style="height:20px;">Surgical Microscope OPMI Vario</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<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>
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			<td>for Biemer vessel clip</td>
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			<td height="20" style="height:20px;">Braunoderm</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.

<html> L&#39;utilisation du nerf massétérine pour la réinnervation du muscle de la greffe a été évaluée chez 5 patients (Tableau 1). Parmi eux, le neurinome acoustique résection était la principale cause de la paralysie faciale. Trois candidats préférés de la procédure en une seule étape plutôt que l&#39;approche en deux étapes &quot;étalon-or&quot;. Chez deux patients, la procédure a servi en tant que procédure de secours après une stimulation insuffisante de la greffe de nerf facial...

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.

Une seule étape de la dynamique de réanimation Smile Irréversible paralysie faciale par Free fonctionnelle transfert musculaire

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 vues.  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.

Vidéo: 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.

In Vivo Assay Muscle Fonction Canine

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

Recherche

Médecine

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

Visualisation quantitative et la détection de cancer de la peau Utilisation dynamique imagerie thermique

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

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.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

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.

L&#39;utilisation de fibroblastes humains primaires pour la surveillance des phénotypes mitochondriales dans le domaine de la maladie de Parkinson

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).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

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.

Haut-débit cytométrie en flux test cellulaire pour détecter les anticorps de la N-méthyl-D-aspartate Receptor ou récepteur de la dopamine-2 dans le sérum humain

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.

Évaluation de ventriculaire droite Structure et fonction dans le modèle de souris de l&#39;artère pulmonaire constriction par échocardiographie transthoracique

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.

Évaluation Basé échographie de l&#39;artère coronaire de débit et de Flow Reserve coronaire Utilisation de la surcharge de pression modèle chez la souris

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).

Foie conservation et de récupération fonctionnelle humaine par des moyens de Subnormothermic machine à perfusion

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.

Longitudinal<em&gt; In Vivo</em&gt; Imagerie du Cerebrovasculature: Pertinence pour les maladies du système nerveux central

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.

Un modèle de transfert de tissu libre: Le Rat épigastrique Flap gratuit

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