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. 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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=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. 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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...

Der Gesichtsnerv wird in der Augenschutz, Artikulation, orale Kontinenz beteiligt und das ästhetische Erscheinungsbild des Gesichts stark beeinflusst. Wertminderung von diesem Nerv ist daher mit einer erheblichen Morbidität und sozialem Rückzug verbunden. Trotz bedeutender Fortschritte in der Therapie kann die Vielzahl von Problemen mit der gelähmten Gesicht assoziiert nur mit einem breiten Spektrum an zusätzlichen Verfahren ausgerichtet sein. Gemeinsam ist allen Operationstechniken ist die Notwendigkeit einer genauen anatomischen Kenntnisse. Gesichtsnerv Anatomie Der Gesichtsnerv besteht aus einem Kiemenmotorkomponente für Willkürmotorik der Gesichtsmuskulatur und einer viszeralen Motorkomponente für parasympathischen Kontrolle Tränen, submandibularis und sublingualis. Außerdem gibt es zwei sensorische Komponenten für die Innervation des äußeren Gehörgangs und dem Geschmack in den vorderen zwei Drittel der Zunge. Der Verlauf der Gesichtsnerven kann bintrakranielle, im Schädelbein und extratemporal: e in drei Segmente unterteilt. Im Hirn Segment erhält der oberen Gruppe von Neuronen, die die Stirnmuskels und der periauricular Region innervieren bilaterale kortikale Eingang. Neuronen, die die übrigen Gesichtsmuskeln innervieren erhalten kontralateralen kortikalen Eingang exklusiv. Als Folge davon wird frontalis Funktion ipsilateral supranukleärer Läsionen gehalten. Die Schädelbein Segment kann ferner in drei Abschnitte unterteilt werden. Im labyrinthischen Segment verlässt der N. petrosus major den Kofferraum, um die Tränendrüse mit parasympathischen Fasern 1 zu liefern. In dem Mastoid Segment läuft das dünne stapedius Nerv zum Muskel-Korrespondent. Parasympathischen Zweige innervieren die submandibularis, sublingual und vorderen Zungendrüsen, während die vorderen zwei Drittel der Zunge sind mit Geschmacksfasern (Chorda) geliefert. Der Hauptstamm des Gesichtsnervs verlässt den knöchernen Kanaldurch die Foramen stylomastoideum. Dies ist der Beginn des extratemporal Segment jedoch Verästelungen nicht vor Eintritt in die Parotis starten. Der Nerv wird zuerst in 3 bis 4 Motor Divisionen, die intraparotideale Plexus und schließlich führen die zeitliche, Jochbein, bukkale Unterkiefer und zervikale Verzweigungen 2 unterteilt. Differentialdiagnose von Gesichtslähmung Als Ursache der Gesichtslähmung ist breit und schwer zu unterteilen, sollte der Unternehmensbereich, der betroffen ist zunächst zu berücksichtigen. Intrakranielle Gesichtslähmung kann durch lakunäre Infarkte oder Tumoren der Schädelhöhle verursacht werden. Bakteriellen und viralen Infektionen, Cholesteatom und Bell-Lähmung kann Gründe für Schädelbein Nervenschäden sein. Neoplastischen bösartigen Erkrankungen und die damit verbundenen chirurgischen Therapie sind die dominierenden Ursachen für extratemporal Gesichtslähmung. Obwohl Bell-Lähmung ist die häufigste Diagnosebei Patienten mit Gesichtslähmung, erholen sich die meisten Patienten völlig ohne Folgeerscheinungen und nicht Chirurgie 3 benötigen. Die zweithäufigste Ursache von Gesichtslähmung ist Trauma. Hier Frakturen des Schläfenbeins sind die vorherrschende Traumamechanismus 4. Die Behandlung Arten von Gesichtslähmung Zahlreiche chirurgische Optionen gibt es für die Behandlung von Gesichtslähmung, und sie können in Reinnervation, statische und dynamische Rekonstruktion Rekonstruktion klassifiziert werden. Typischerweise werden 2 Jahre als die Zeit von der Verletzung in dem Funktion nach reinnervation kann zufriedenstellend reinnervating Verfahren 5 wiedergewonnen werden können. Später Denervierung Atrophie der Gesichtsmuskeln schließt ihre Nützlichkeit für die weitere Rekonstruktion. Reinnervation können durch primäre Nervenreparatur, interpositional Nerventransplantate, Quergesichtsnerven Pfropfen oder Hirnnervenübertragung erzielt werden. Statische Rekonstruktionstechniken sind darauf gerichtet, zu korrigierenfunktionellen Behinderungen, (Schutz der Hornhaut, die Verbesserung der Nasenatmung und Prävention von Speichelfluss) und der Symmetrie in Ruhe zu verbessern. Typische Verfahren sind für die Stirn Ptosis oder Kanthoplastik für untere Deckel Ektropium browlift. Statische Rekonstruktion bei älteren Patienten mit signifikanten Begleiterkrankungen oder in massiven Gesichtsdefekten nach Trauma oder Krebs Resektion bevorzugt. Die chirurgischen Techniken der dynamischen Rekonstruktion kann in regionale Muskeltransfer und kostenlose microneurovascular Muskeltransfer entweder Koaptation auf die masseter Motorabzweig oder Quergesichtsnerven Transplantation unterteilen. Letzteres stellt das Kriterium Standard für die Rekonstruktion des Lächelns in Gesichtslähmung, wie keine andere Behandlungsoption zuverlässig ein spontanes Lächeln, das entscheidend für die soziale Behinderung zu verbessern, ist erreicht. Koaptation auf die masseter Motorabzweig ist die Behandlungsmethode der Wahl für Patienten mit beidseitiger Lähmung jedoch Anzeichen haben extendete, wie älteren Patienten oder Patienten mit signifikanten Begleiterkrankungen der Regel lieber ein einstufiges Verfahren 6. Gracilis Klappe Regional Anatomie Der M. gracilis ist eine eher oberflächliche Muskel des medialen Oberschenkel, die die längste Muskel der Adduktoren. Es stammt aus der unteren Symphyse und dem unteren Ast des Schambeins. Lauf distal, wird die Muskel schmalen und fügt distal zu dem Kniegelenk des Schienbeins, die nicht nur die Adduktion des Oberschenkels sondern auch Beugung des Knies ermöglicht. Der M. gracilis hat eine Typ-II-Kreislauf-Muster nach Mathes und Nahai mit einem arterielle Versorgung aus einer dominanten und einige kleinere Gefäßstiele 7. Die dominierende Arterie verlässt den Muskel am Hilus Kurs seitlich und endet in der medialen Kranzarterie in der Regel. In seltenen Fällen endet die dominierende Arterie direkt in der tiefen Oberschenkelarterie. Venöse Versorgung des gracilis Muskel ist in der Regel durch zwei Venae comitantes erreicht, dass natürlich tief in die M. adductor longus, um die dominante Arterie zu folgen. Innervation des Muskels wird durch einen vorderen Ast des Nervus obturatorius, die den Muskel an den Hilus tritt 1-2 cm kranial erreicht. Klinische Anwendung Der M. gracilis ist eine wertvolle Muskel Spender für die rekonstruktive Mikrochirurgie und hat die Muskeln der Wahl für viele Chirurgen für funktionelle freier Muskeltransfer zu werden. Dies wird auf die Tatsache, dass es wenig Spenderstellenmorbidität und die Klappe zeigt optimale Verhältnisse in Bezug auf die Auslenkung des Muskels und Gefäßstiel Abmessungen jeweils verdanken. Eine lange einzigen innervierten motorischen Nerven lindert Funktionsklappe Ernte 8. Wir zeigen hier den Fall einer 49 Jahre alte Frau, die zunächst ein vollständiges Bild von links peripheren Gesichtslähmung präsentiert nach Resektion eines akustischenNeurom (Vestibularisschwannom) 2 Jahre zuvor. Der Patient wurde am stärksten von Gesichtsasymmetrie, besonders beim Lächeln heimgesucht. Andere bereits bestehenden Begleiterkrankungen wurden nicht dokumentiert. Bei der klinischen Untersuchung zeigte der Patient eine komplette Parese des Stirnmuskels jedoch eine zufriedenstellende Stirn Symmetrie in Ruhe. Deckelverschluss nicht ausreichend war auf der linken Seite mit einem Lagophthalmus von 5 mm und Bell-Phänomen. Zeichen der Hornhaut Reizungen und Ektropium fehlten. Im Ruhezustand, zeigte der Patient eine moderate Asymmetrie der Ecke der Mund mit einem Tragus-modiolus Abstand von 11 cm auf der rechten und 11,5 cm auf der linken Seite in Ruhe. Nach Lächeln, Tragus-modiolus Entfernung von 9 cm auf der rechten skaliert und auf der linken Seite von 12 cm gestreckt. Nach umfangreicher Beratung, wünschte der Patient für eine einstufige dynamische Rekonstruktion des Lächelns mit einer freien funktionellen gracilis Übertragung mit dem masseter als Spendernerv. Der Patient wurde auch über verschiedene te informiertchniques der Deckelverschluss Rekonstruktion jedoch sank die chirurgische Behandlung in diesem Stadium. Der Verlauf der Behandlung war unkompliziert. Zeichen der Reinnervation wurden zum ersten Mal bemerkt 3 Monate postoperativ. Vier Monate nach der Operation, der Patient mit unauffälligen Narben entlang der ehemaligen geändert Facelift Schnittführung vorgestellt. Symmetrie in Ruhe und bewusste lächelnd waren mit einem zufriedenstellenden Definition der Nasenlippenfalte ausgezeichnet. Der Patient zeigte auch eine völlig spontane Lächeln. Vor- und 9-Monatspostoperativen Bereich der Auslenkung des Mundwinkel wurde von Videografie dokumentiert.

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			<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>Schuelke &#38; Mayr</td>
			<td>5702764</td>
			<td>&#176;</td>
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			<td>Inomed</td>
			<td>-</td>
			<td>&#176;</td>
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			<td>Baxter</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>
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			<td height="21" style="height:21px;">Myacyne Ointment</td>
			<td>Schur Pharma</td>
			<td>-</td>
			<td></td>
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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> Die Verwendung des massetericus Nerv reinnervation des Muskels Transplantat wurde bei 5 Patienten (Tabelle 1) bewertet. Unter ihnen Akustikusneurinom Resektion war die Hauptursache für die Gesichtslähmung. 3 Kandidaten bevorzugt, die einstufiges Verfahren anstatt der &quot;Goldstandard&quot; zweistufigen Ansatz. Bei zwei Patienten war die Prozedur als Rettungsverfahren nach unzureichende Stimulation durch den Quergesichtsnerventransplantat (CFNG). Reinnervation des g...

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.

Einstufige Dynamische Reanimation des Lächelns in Irreversible Fazialisparese von Free Funktionsmuskelüber

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

105.7K Aufrufe.  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.

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

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.

Quantitative Visualisierung und Erkennung von Hautkrebs mit 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 ...

Forschung

Medizin

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.

Die Verwendung von primären humanen Fibroblasten zur Überwachung Mitochondriale Phänotypen auf dem Gebiet der Parkinson-Krankheit

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).

Patienten abgeleitet Zellkultur und Isolierung von CD133<sup&gt; +</sup&gt; Mutmaßliche Cancer Stem Cells von Melanoma

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.

Hochdurchsatz-Durchflusszytometrie zellbasierten Assay auf Antikörper gegen N-Methyl-D-Aspartat-Rezeptor oder Dopamin-2-Rezeptor im menschlichen Serum Detect

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.

Beurteilung der rechtsventrikulären Struktur und Funktion in Maus-Modell der Lungenarterie Verengung durch transthorakale Echokardiographie

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.

Ultraschall Basierend Beurteilung der koronaren Herzfluss und koronare Flussreserve Mit der Drucküberlastung Modell an der Maus

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).

Funktionale Menschliche Leber Erhaltung und Rückgewinnung mittels Subnormothermic Maschine 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.

in Längsrichtung<em&gt; In Vivo</em&gt; Imaging des Cerebrovasculature: Bedeutung für ZNS-Erkrankungen

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.

Ein Modell der freien Gewebetransfer: Die Ratte Epigastrische Freie Flap

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.