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. 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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. 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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. 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...

顔面神経は、眼の保護、関節、経口失禁に関与しており、非常に顔の美的外観に影響を与えている。この神経の障害は、そのための重要な罹患率と社会的引きこもりと関連している。治療における著しい進歩にもかかわらず、麻痺した顔に関連する問題の多数は追加の手順を広範囲に標的化することができる。すべての外科的技術に共通することは、正確な解剖学的知識の必要性である。 顔面神経解剖学顔面神経は、顔の筋肉組織の自主的なモータ制御のための鰓モーター部品および内臓モーター涙腺の副交感神経制御のためのコンポーネント、顎下腺、舌下腺から構成されています。また、外耳道の神経支配のためと舌の前3分の2で味のための2つの感覚のコンポーネントがあります。顔面神経のコースは、bにすることができます頭蓋内、intratemporal、およびextratemporal：eは3セグメントに分割。頭蓋内のセグメントでは、前頭筋とperiauricular地域を支配するニューロンの上位グループは、二国間の皮質入力を受け付ける。残りの顔の筋肉を支配するニューロンは、排他的に対側皮質の入力を受け取る。結果として、前頭機能は同側性核上性病巣内に維持される。 intratemporalセグメントはさらに3つのセグメントに分割することができる。迷路のような分野では、より大きな錐体神経が副交感神経繊維1と涙腺を供給するためにトランクを残します。 乳様突起セグメントでは、薄い鐙骨神経は、コレス筋肉に実行されます。副交感神経枝は顎、舌下を支配し、舌の前3分の2味覚繊維（鼓索）が供給されているのに対し、舌腺を前方。顔面神経の主茎は骨の運河を抜け茎乳突孔を通して。しかし、これはarborisationは耳下腺に入る前に起動しない、extratemporalセグメントの始まりである。神経は最初intraparotid叢を形成し、最終的に時間的、頬骨、頬側、下顎頸部枝2を生じさせる3~4モータ部門に分けられる。 顔面神経麻痺の鑑別診断顔面神経麻痺の病因は分類する広範かつ困難であるため、影響を受けている部門が最初に考慮されるべきである。 頭蓋顔面神経麻痺は頭蓋腔のラクナ梗塞や腫瘍によって引き起こされる場合があります。細菌やウイルス感染、真珠腫とベル麻痺はintratemporal神経損傷の理由であることができます。新生物の悪性腫瘍と関連する外科的治療がextratemporal顔面神経麻痺のための主要な原因となっている。ベル麻痺は、最も一般的な診断を表していますが顔面神経麻痺の患者では、ほとんどの患者は後遺症なしで完全に回復し、手術3は必要ありません。顔面神経麻痺の第二の最も一般的な原因は、外傷です。ここでは、側頭骨に骨折が優勢な外傷機構4である。 顔面神経麻痺の治療の種類多数の外科的選択肢は、顔面神経麻痺の治療のために存在し、それらが神経再生、静的および動的な再構成の再構成に分類することができる。一般的に、二年後には、神経再生機能は手順5 reinnervatingで良好取り戻し可能な損傷からの時間であると考えられる。その後、顔の筋肉の除神経萎縮はさらに復興のためにその有用性を排除する。再神経支配は、一次神経修復、中間物挿入神経移植片、クロス顔面神経移植または脳神経伝達することにより得ることができる。静的再構成技術は、修正するために指示されている安静時の対称性を改善するための機能的な障害者、（角膜の保護、鼻の気流の改善、及びよだれの防止）と。典型的な手順は、下蓋外反のための眉下垂、またはcanthoplastyのためbrowliftされている。静的再建は重要な併存疾患を持つ高齢の患者や外傷または癌切除に続発する大規模な顔の欠陥が好ましい。 動的再構成の外科技術は、地域の筋肉の転送と咬筋モーター枝やクロス顔面神経移植への接合のいずれかとの自由なmicroneurovascular筋肉の転送に分割することができます。他の治療法の選択肢は確実に社会的なハンディキャップを向上させるために非常に重要である自発的な笑顔を達成しないように、後者は、顔面神経麻痺で笑顔の復興のための基準の標準を表します。咬筋モーターブランチへの接合は、兆候がEXTを持っているが、二国間の麻痺の患者のための選択の治療法です。重要な併存疾患を持つ高齢の患者または患者は通常、単一の段階の手順6を好むように、終わった。 薄筋フラップ地域解剖薄筋は、転筋の最長筋を表す、大腿内側のむしろ表面的な筋肉です。これは、下恥骨と恥骨の下枝に由来する。遠位に実行すると、筋肉が狭くなり、だけでなく、太ももの内転だけでなく、膝の屈曲を可能にする脛骨上の膝関節の遠位に挿入されます。薄筋が支配的といくつかのマイナーな血管茎7からの動脈供給とMathesのとNahai後にII型循環パターンを有している。支配的な動脈は内側回旋動脈で終了する通常横方向にコースへの門で筋肉を出て、。まれに、支配的な動脈は深い大腿動脈に直接終端していない。 GRAの静脈供給cilis筋は通常、主要な動脈に従うことが、そのコースの深い長内転筋に、2大静脈のcomitantesを通じて達成される。筋肉の神経支配は、門に1〜2センチメートル上方筋肉に入る閉鎖神経の前枝を介して達成される。 臨床用途薄筋は、マイクロサージャリのための貴重なドナーの筋肉で、機能的な自由な筋肉の転送のための多くの外科医のための選択の筋肉となっています。これは、そこに小さなドナー部位の罹患率であり、フラップがそれぞれ筋肉と血管茎の寸法の遠足に関して最適な割合を示しているという事実に負っている。長い一神経支配運動神経は機能的なフラップの収穫8を軽減。 ここでは音響の切除後の最初に左末梢性顔面神経麻痺の全体像を提示49歳の女性の場合を実証2年前に神経腫（前庭神経鞘腫）。患者はほとんど笑顔特に顔面非対称、に悩まされました。その他の既存の併存疾患が記載されていなかった。 臨床検査の際に、患者は、前頭筋の完全な麻痺、安静時が良好額対称性を示した。蓋の閉鎖は5ミリメートルとベル現象の兎眼と左には不十分であった。角膜の炎症や外反の徴候は存在しなかった。安静時、患者は右に11センチメートルと安静時の左の11.5センチメートルの耳珠-蝸牛軸距離と口角の適度な非対称性を示した。笑顔の際、耳珠-蝸牛軸の距離は、右の9センチメートルにスケールダウン12 cmの左側に細長い。豊富なカウンセリングの後、患者がドナー神経などの咬筋を使用して遊離官能薄転送と笑顔のシングル段の動的再構成のために望んだ。患者はまた、様々なTEについて知らされました蓋閉鎖再建のchniquesは、しかし、この段階で外科的治療の減少となりました。治療のコースは、合併症のないだった。神経再生の徴候は最初の手術後3ヶ月に気づいた。四ヶ月手術後、患者はかつての修正されたフェイスリフトの切開線に沿って目立たない瘢痕化を提示。安静時対称性と笑顔意図的にほうれい線しわの満足な定義に優れていた。患者はまた、完全に自発的な笑みを示した。口角の遠足の前および9ヶ月の術後の範囲は、ビデオ撮影によって文書化された。

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nylon Suture, 9-0</td>
			<td>Serag Weissner</td>
			<td>Z0039490</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene Suture&#160;</td>
			<td>Ethicon</td>
			<td>-</td>
			<td>Multiple thread sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suprarenin 1mg/ml</td>
			<td>Sanofi</td>
			<td>-</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cook-Swartz Doppler Probe</td>
			<td>Cook Medical</td>
			<td>G03014</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DP-M350 Blood Flow Monitor</td>
			<td>Cook Medical</td>
			<td>-</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Microscope OPMI Vario</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgical instruments lab set</td>
			<td>S&#38;T</td>
			<td>767</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biemer vessel clip</td>
			<td>Diener</td>
			<td>64,562</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applying forceps</td>
			<td>Diener</td>
			<td>64,568</td>
			<td>for Biemer vessel clip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cefuroxim 1500mg</td>
			<td>Fresenius</td>
			<td>J01DC02</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braunoderm</td>
			<td>Braun Melsungen</td>
			<td>3881105</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Octenisept</td>
			<td>Schuelke &#38; Mayr</td>
			<td>5702764</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ISIS Neuromonitoring System</td>
			<td>Inomed</td>
			<td>-</td>
			<td>&#176;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissucol</td>
			<td>Baxter</td>
			<td>1.33052E+12</td>
			<td>Fibrin glue</td>
		</tr>
		<tr height="20">
			<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>
	</tbody>
</table>

single-stage dynamic reanimation of the smile in irreversible facial paralysis by free functional muscle transfer

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

<html>筋肉移植片の再神経支配のための咬筋神経の使用は、5人の患者（ 表1）において評価した。その中でも、聴神経腫の切除は顔面神経麻痺の主な原因だった。 3候補は、単一段階手順ではなく、「ゴールドスタンダード」二段階アプローチが好ましい。 2人の患者では、手順がクロス顔面神経移植片（CFNG）で不十分な刺激後レスキュー手順を務めた。 シン?...

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.

フリー機能性筋肉転送により不可逆顔面神経麻痺が笑うの単段ダイナミック蘇生

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 ビュー.  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.

ビデオ: 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.

定量的な可視化と動的な熱画像を用いた皮膚癌の発見

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 ...

医学

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.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

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).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

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.

ハイスループットフローサイトメトリー細胞ベースのアッセイN-メチル-D-アスパラギン酸受容体またはヒト血清中のドーパ​​ミン-2受容体に対する抗体を検出する

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.

経胸壁心エコー検査による肺動脈狭窄のマウスモデルにおける右室の構造と機能の評価

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.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

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).

Subnormothermicマシン灌流によって機能的なヒト肝臓保全と回復

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.

縦<em&gt;インビボ</em&gt;脳血管系のイメージング：中枢神経系疾患との関連

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.

無料組織転送のモデル：ラット心窩部無料フラップ

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.