Name: coordinate mapping of hyolaryngeal mechanics in swallowing
Uploaded: 2014-05-06T15:30:00
Description: Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

著者はKendrea Focht、CSCD、CCC-SLP、および音声のためのイヴリントラメル研究所を確認し、この方法論を示すために使用されるフ​​ァイルを画像化するMBSを共有するため、サウスカロライナ医科大学で飲み込む。トランスレーショナル科学とグラント番号1K24DC12801で（PI：マーティン·ハリス）を進めるためのナショナルセンターからこれらのMBSデータは、付与数TL1TR000061（Focht PI）によって資金を供給学外支援を通じて収集された難聴の国民の協会やその他のコミュニケーション障害から、とマークとイヴリントラメル·トラストからの校内支援。難聴やその他のコミュニケーション障害の国民の協会からの助成金番号F31DC011705でサポートされている間、これらの方法は、もともと主任研究員によって開発されました。内容はもっぱら著者の責任であり、必ずしも難聴の国民の協会やその他のコミュニケーション障害や国立研究所の公式見解を示すものではありません。健康。

1。コンピュータの設定<ol><li> ImageJの、MacXのビデオコンバーター無料版（Mac用）、およびQuickTime（材料/機器の表を参照）：Macintoshの場合、次のオープンソースまたはフリーソフトウェアをダウンロードする。 </li><li> ImageJの、MPEG Streamclipの（PC）、およびQuickTime（材料/機器の表を参照）：PCの場合、次のオープンソースまたはフリーソフトウェアをダウンロードする。 </li></ol><p class="jove_titl...

<ol>
	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. 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=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., 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=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

この研究は、嚥下運動hyolaryngeal複数の運動学的測定値を算出するために、解剖学的ランドマークの座標データを用いた方法の有用性を実証する。座標計算の変数の2初心者評価者を含む6評価者の評価者間信頼性は（ICC&gt; 0.90）強かった。健康的な非嚥下障害の成人のランダムなサンプルからの代表的な結果は、2つのボーラスのタイプに応じて、いくつかの運動学的変数の違いを示した。我々?...

嚥下の咽頭相は、気道を保護しながら、食道に口腔からボーラスを転送するために20以上の筋肉や、複数の骨格要素が関与する複雑な過程である。咽頭収縮、hyolaryngeal複合体の要素（舌骨、喉頭、および上部食道括約筋を含め関連する構造）の前には、消化管に呼吸管を変換するために変位する。喉頭の前方に離れて対向ボーラスの軌道から、再配置され、上部食道括約筋を開放延伸される。ビデオ透視嚥下試験（また、MBSとして知られているバリウムツバメ調査を修正）から取られた運動学的測定がhyolaryngeal錯体1の複数の動きを定量化するための主要な研究方法である。 定量的なビデオ透視測定は、基準の異なる軸嚥下機能を測定するために有用であるが、及び運動学的測定値2の種々の方法のうちの互換性のない所見の測定結果のスカラ。臨床医は手動制御の下で、患者とX線透視装置の動きは、この複雑な生理的プロセスの測定精度を混乱させる。さらに重要なことは、運動学的な測定が必ずしも無秩序嚥下を評価するための重要な構造と機能の関係を反映していない。特に舌の運動学は、椎骨と整合解剖学的面を基準における前方または優れた方向への移動を追跡するために設計されています。しかし、この構成では、舌骨を一時停止する筋肉の作用線を表すものではありません。 嚥下の咽頭相におけるhyolaryngeal上昇の二スリング機構（ 図1）3,4同定されている。舌骨上筋は前部の筋肉スリングを含み、長い咽頭筋はPを含み、筋肉のスリングをosterior。これらの筋肉は、舌、喉頭、および上部食道括約筋を形成する構造を含むhyolaryngeal複合体の様々な要素を高める。 hyolaryngeal力学の座標マッピングは、前部と後部の筋肉のスリング（ 図2）のhyolaryngeal複雑表す接続点の3骨格のレバーや機能をマッピングするために9簡単に識別できる解剖学的ランドマークを利用しています。嚥下時、hyolaryngeal複合体の各骨格レバーと機能が動いている。座標を収集することによって、システムは、任意の時間枠内に捕捉することができる。三角座標の変換は、嚥下中にhyolaryngeal運動の複数の運動学的測定値を生成するために使用することができる。変数は、文献に報告所見との比較のために計算された、または対象の構造への関数関係を表す新しい測定値を生成するために使用することができる。 本稿の主な目的は、変更されたバリウムツバメ（MBS）の研究から収集された解剖学的ランドマーク座標の単一のセットから計算された複数の運動学的な測定値を生成する方法を示すことです。私たちは、ある専門家、経験を持つ3人の評価者、および2初心者を含む6種類の研究者の評価者間の信頼性を判断するために内相関係数を使用して、この方法の信頼性を文書化する。キネマの結果から、ボーラスの一貫性により力学を飲み込むの違いを評価する。最後に、舌の動きを測定する際に使用される基準の軸の重要性に関するMolefenterとスティールが提案し、問題が対処されている。この質問にアプローチするために、我々は両方のボーラスタイプの座標の同じセットから計算され、脊椎を参考にし、下顎を参考に舌の遠足の測定値を比較します。舌骨運動を測定するこれらの2つの方法は、関係機能する同一の構造を表す場合ヒップ、そして結果は、相関されなければならない。 この研究では、40側面のMBS研究は無作為ジョージアリージェンツ大学施設内倫理委員会によって承認された研究プロトコルの下や医科大学の音声と嚥下イヴリントラメル研究所との共同研究で139の通常の研究のコレクションから選ばれたサウスカロライナ州。この方法の有用性を実証するために、hyolaryngeal運動学を特徴付ける10の変数が座標データの同じセット（ 表1）から計算した。これらの計算された測定値の七は、以前含む文献で ​​使用されている：前方および脊椎5を参照するに優れた舌の距離測定;前部とも脊椎6を参照したC2〜4の長さの比、などの優れた舌の変位;脊椎7を参照すると喉頭の優れた運動; hyolaryngeal approximatイオン1;椎骨1を参照すると、最大舌遠足。また、3つの新規の測定が計算された：咽頭短縮が口蓋咽頭筋、茎突咽頭筋を表現する作用線以下の喉頭隆起し、舌骨上筋4,8の添付ファイルを近似舌遠足の添付ファイルを近似する。 専門家の頭頸部解剖（WP）、限られた経験服用測定（CJ、SR、TT）、二つの研究者、初心者（RS、JT）を有する3つの研究者らは、以下に記載のプロトコルを使用して、マッピング座標データを得た。専門家（WP）は、経験を持つ3人の評価者を訓練しており、これらは順番に2初心者評価者を訓練した。被験者が座標から算出された座標データ及び結果の評価者間信頼性は、内相関係数9により決定した。両側t検定をstatisticallを決定するために、各変数に対して行ったボーラス種類のY有意差。ピアソン相関係数と決定係数は5ミリリットル薄液体ツバメと5ミリリットルのための参照軸として下顎と舌遠足に対する参照軸として脊椎骨で計算舌の遠足の結果との間の合意を評価したプリンツバメ。

<table border="0" cellpadding="0" cellspacing="0" style="width:864px;" width="865">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:339px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;&#160;</td>
			<td style="width:71px;">NIH</td>
			<td style="width:339px;">http://rsbweb.nih.gov/ij/download.html</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MacX Video Converter Free Edition (Mac)</td>
			<td style="width:71px;">Digiarty</td>
			<td style="width:339px;">&#160;http://www.macxdvd.com/mac-video-converter-free/</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickTime&#160;</td>
			<td style="width:71px;">Apple</td>
			<td style="width:339px;">http://support.apple.com/downloads/#QuickTime</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;&#160;</td>
			<td style="width:71px;">NIH</td>
			<td style="width:339px;">http://rsbweb.nih.gov/ij/download.html</td>
			<td>For a PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPEG Streamclip (PC)&#160;</td>
			<td style="width:71px;">Squared 5</td>
			<td style="width:339px;">http://www.squared5.com</td>
			<td>For a PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickTime</td>
			<td style="width:71px;">Apple</td>
			<td style="width:339px;">http://support.apple.com/downloads/#QuickTime</td>
			<td>For a PC</td>
		</tr>
	</tbody>
</table>

coordinate mapping of hyolaryngeal mechanics in swallowing

hyolaryngeal動きを特徴づけることは嚥下障害の研究に重要です。従来の方法は、一方変性バリウムツバメ（MBS）を使用してhyolaryngeal力学のマッピングは、目的の複数の変数を計算するために1つの座標を使用して一つの座標運動学的測定値を得るために複数の測定を必要とする。デモのために、10動の測定は、2つの異なるタイプのボーラスを飲み込むの違いを決定するために、1つの座標から生成した。脊椎骨と下顎骨に対する舌骨の遠足の計算は、基準の軸の重要性を決定するために相関している。 マッピングの方法論を座標を示すために、40のMBSの研究は無作為には知られていない嚥下障害と健常被験者のデータセットから選択した。 5ミリリットル薄い液体ボーラスおよび5mlプリンツバメ各被験者から測定された。ナイン座標、頭蓋底、下顎、脊椎やヒョリの要素をマッピングする喉頭複合体は、最小および最大hyolaryngealエクスカーションのフレームで記録した。座標は、数学的にhyolaryngeal力学の10の変数に変換した。 評価者間信頼性は、内相関係数（ICC）により評価した。両側t検定は、ボーラス粘度による運動の差異を評価した。参照の異なる軸に対する舌エクスカーション測定は相関していた。 0.97 - 18座標の6人の評価者のうち、評価者間の信頼性は、ICC = 0.90の範囲であった。 10運動学的測定のスレートは、6の評価者間の対象者と比較した。一つ外れ値は拒否され、残りの信頼性スコアの平均は、ICC = 0.91、0.84だった - 0.96、95％信頼区間。 10運動学的変数（5ミリリットル薄液体対5ミリリットルプリンツバメ）を比較ボンフェローニ補正と両側t検定は、舌の遠足、喉頭の動き、および咽頭shorteninにおいて統計的に有意な差が認められた得た（p &lt;0.005）。参照の2の異なる軸から舌の遠足測定のピアソンの相関があった。R = 0.62、R 2 = 0.38、（薄液体）; R = 0.52、R 2 = 0.27、（プディング）。 ランドマークの座標を得ることは、嚥下障害の研究に有用なビデオX線透視画像から複数の運動学的変数を生成するための信頼できる方法である。

0.97 - 独立80ビデオ透視ファイルを分析した6研究者（40科目から2ボーラストライアル）によって収集された座標の内相関係数（ICC）は=、ICCから0.90の範囲であった。次のようにグループによるICCの座標のの内訳は次のとおりです＃1の座標 - 5（最小​​hyolaryngeal遠足の骨格要素は）平均= 0.93、0.91から0.95、95％信頼区間を、 9（最小hyolaryngeal遠足でhyolaryngeal複合体）= 0.94、092意味 - - ＃6座標0.9...

Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

座標マッピングは、嚥下の咽頭期にhyolaryngealバイオメカニクスの顕著な特徴を文書化する方法である。この方法は、解剖学的標識点の座標を記録するために画像解析ソフトウェアを使用している。これらの座標は、Excelマクロにインポートされ、嚥下障害の研究に有用関心の動変数に変換されます。

嚥下Hyolaryngeal力学のマッピングを調整する

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

coordinate-mapping-of-hyolaryngeal-mechanics-in-swallowing

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

単一絨毛膜性のツイン胎盤の血管のマッピングのためのガイドワイヤ補助カテーテルと有色色素注入

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

医学

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

ストレッチ誘発性心房細動の羊のモデルで高解像度の心内膜と心外膜光学マッピング

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

定量的免疫蛍光法を用いて腫瘍におけるタンパク質発現の不均一性のマッピング

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

非接触型誘電泳動によりラベルフリーの単離と細胞の濃縮

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

ガス吸入課題を経由して脳血管反応性のMRIのマッピング

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

マウスを用いた胃食道逆流の外科モデル

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高解像度光学マッピングとマウスの心房の電気生理学的評価

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

造影剤としてのプロトン磁気共鳴イメージングと酸素を用いたヒト肺における特定の換気の定量的マッピング

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

エレクトロマップを用いた光マッピングデータのハイスループット解析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

蛍光タグ付きサルコメアタンパク質を用いた多能性幹細胞由来心筋細胞のサルコメア短縮

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...