このプロトコルを使用した研究は、心臓脳卒中財団(G-18-0020952)、クレイグ・H・ニールセン財団(542675)、カナダ自然科学工学研究評議会(RGPIN-2014-05498)、オンタリオ州研究イノベーション科学省(ER16-12-013)によって資金提供されました。

この研究は、大学保健ネットワークの研究倫理委員会によって承認されました。署名されたインフォームド・コンセントは、調査に登録する前に各参加者から得られました。署名されたインフォームド・コンセントは、ビデオ録画に登場する介護者や世帯員からも得られました。
1. 個人に対するプロトコル適用性の検証
注:このプロトコルは、障害を持つが、完全に不在の手の機能を持つ個人に適用されることを意図しています(特定の基準は、人口および/または関心のある質問に適応することができます)。
<ol><li>参加者に、影響を受ける手がADLを実行する能力に影響を与えるかどうかを尋ねます。 注: 影響を受ける手で独立して実行できるタスクとできないタスクの例をいくつか参加者に示すことをお勧めします。</li>
	<li>モントリオール認知評価(MoCA)の合計スコアが21を超えているか確認し、プロトコル手順を理解し、次の手順を実行する潜在的な困難を避けます。</li>
</ol>2. 参加者の日常の決定
<ol><li>過去2週間の毎日の日課を思い出してもらいます。毎日のタスクがどの程度、どのくらいの期間、そしておよそ何時に実行されるのかを文書化します。</li>
	<li>参加者とのコラボレーションで、ビデオを録画する時間をそれぞれ1.5時間の3つのタイムスロットを選択します。異なる曜日に分散されるタイムスロットを選択し、通常は手を含むADLが順番に実行されるときに発生します。 注: 選択した ADL は、各参加者の典型的な活動を表すものであり、参加者が意味を持つものとして認識する必要があります。録画期間を異なる日にスケジュールすることは、録画されたADLの多様性を増やし、有用で意味のあるデータのキャプチャを促進することを目的としています。 メモ:録画タイムスロットは録画効率を上げるためにスケジュールされていますが、参加者は録画を開始するタイミングと停止するタイミングを完全に制御できる点を理解する必要があります。</li>
</ol>3. 参加者との録画スケジュールとターゲットビデオコンテンツに関する合意
<ol><li>記録スケジュールに関する各参加者の同意を得る, 彼らが持っている可能性のある懸念を議論した後.</li>
	<li>2週間にわたってビデオの3時間の目標を設定します。動画が足りない場合は、録画期間の延長につながる可能性があることを参加者に知らせます。</li>
</ol>4. ADLを自然に行うことの重要性を強調する
<ol><li>記録する特定のアクティビティを指定するのではなく、現実的なルーチンをキャプチャすることに集中するように参加者に指示します。指示の目的は、参加者が自分のために一般的であるよりも多くの量で特定の活動を人為的に記録するのを思いとどまらせることです。</li>
</ol>5. 自宅での録画中の潜在的なプライバシーの問題の通知
<ol><li>参加者は、プライバシーの問題を避けるために公共の場所ではなく、自宅内ですべての録音が行われるべきであることを理解してください。</li>
	<li>入浴、ドレッシング/脱衣、機密情報のチェックなど、プライバシーに関する懸念を引き起こす可能性のある例をいくつか挙げてみましょう。録音に顔を見せることがある鏡に気づくように参加者に思い出させます。</li>
	<li>ビデオでできるだけ家族や介護者などの他の人の存在を避けることを参加者に提案します。 注:研究の文脈では、他の人の存在が避けられない場合には、それらの個人からインフォームド・コンセントを得る必要があります。</li>
</ol>6. カメラとタブレットの指示
注:参加者が最初の接触の間に、彼らの毎日のニーズの多くのために介護者の援助を必要とすることを示した場合、介護者はまた、研究訪問に出席し、後で参加者を支援できるように、機器の使用に関する訓練を受けることを奨励されます。
<ol><li>参加者に対して自己中心カメラ (材料表) を使用する方法をデモンストレーションします。<ol>
<li>カメラのオンとオフを切り上げる方法をデモンストレーションします。</li>
		<li>カメラを使用して録画を制御する方法 (開始、一時停止、停止) をデモンストレーションします。</li>
	</ol>
</li>
	<li>プリインストールされたカメラ アプリでタブレット (表) を使用して、該当する場合は録画を制御する方法をデモンストレーションします。 注: デモには、カメラ アプリからの録画の制御、および録画したビデオの再生と編集 (トリミングまたは削除など) が含まれます。カメラリモコンは当初検討されていましたが(補足ファイル)、実際には、参加者がカメラやタブレットを使って録画を開始したり停止したりすることに慣れていたため、実際には使用されませんでした。<ol>
<li>タブレットのオン/オフを切り上げる方法をデモンストレーションします。</li>
		<li>カメラ アプリを使用してタブレットをカメラに接続する方法をデモンストレーションします。</li>
		<li>カメラ アプリから録画を制御する方法をデモンストレーションします。</li>
		<li>カメラ アプリから録画したビデオを確認する方法をデモンストレーションします。</li>
		<li>カメラ アプリでビデオをトリミングまたは削除する方法をデモンストレーションします。</li>
	</ol>
</li>
	<li>参加者の頭に合わせて調整可能な弾性ヘッドバンドを使用して、カメラをドン アンド オフする方法をデモンストレーションします。 注: 図 1を参照してください。 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61898/61898fig01.jpg"> 図 1.ウェアラブルカメラのセットアップ。 (A)自己中心カメラの位置決め。(B)カメラからの視野角。<a href="https://www.jove.com/files/ftp_upload/61898/61898fig01large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
	<ol>
<li>カメラを参加者の額に置きます。ヘッドバンドを調整して、カメラを快適かつ着実に装着します。</li>
		<li>額に対してカメラの最適な角度を確保します。</li>
		<li>参加者に、手を前に動かしながら、オブジェクト(タブレットなど)を操作しながら、ビデオの短いセグメントを記録するように依頼します。</li>
		<li>録画したビデオを確認し、操作作業を行っている間に、シーンの中央領域に両手がはっきりと見えるようにします。</li>
		<li>彼らは能力を実証するまで、参加者とその介護者とカメラとタブレットの使用を練習します。</li>
	</ol>
</li>
</ol>7. 機器を与える
<ol><li>すべての機器を備えたキットを参加者に提供し、自宅でADLを記録します。カメラとタブレットに加えて、キットには、カメラのバッテリー、カメラとタブレットの両方のバッテリー充電器、充電ケーブル、カメラ用のヘッドバンド、およびカメラを使用するためのガイドラインの印刷されたセットが含まれています( 補足資料を参照)。</li>
</ol>8. 実験的なトラブルシューティングとフォローアップ
<ol><li>自宅で実際の記録中に障害物を解決するために研究者の連絡先情報を提供します。1週間後、研究者は参加者に電話して記録の進捗状況を文書化し、潜在的な技術的な問題を解決します。</li>
</ol>9. 機器・映像の検索
<ol><li>直接またはプリペイドメール小包を通じて参加者からのすべての機器やビデオを取り消します。</li>
	<li>参加者が返されたすべての動画を共有することに同意していることを確認します。参加者は、収集したすべてのビデオを研究チームに戻す前に確認し、共有したくない部分を削除することをお勧めします。</li>
	<li>調査では、返されたビデオを確認し、同意を得ずにビデオに表示される人がいるかどうかを確認します。その場合は、同意書を送信するか、動画に表示される個人に電話して、動画の使用に同意を得てください。個人が到達できない場合は、それらが表示されるビデオの一部が研究者によって削除されます。</li>
</ol>

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著者らは開示するものは何もない。

cSCIや脳卒中などの上肢障害を持つ個人のウェアラブルカメラを使用して、自宅でADLのビデオを記録するためのプロトコルを発表しました。このプロトコルは柔軟性があり、特定のADLで手の機能性能をキャプチャしたり、自宅に住む人々のリハビリテーションの進行状況を遠隔で追跡したりするために目標指向にすることができます。自己中心的なビジョンパラダイムは、地域に住む個人の手...

手の機能は、上肢障害1、2を有する臨床集団全体の独立性と生活の質の決定要因である。在宅で手の障害を持つ人の手の機能を捉えて、リハビリ中とリハビリ後の日常生活(ADL)の活動を行う能力の進歩を評価するためには不可欠です。ほとんどの臨床手機能評価は、家庭3,4ではなく臨床環境または検査環境で行われます。自宅でADLへの影響を捉えようとする既存の臨床手機能評価は、アンケートであり、主観的自己申告評価5、6、7に依存しています。自宅での手機能に対するリハビリテーションの最終的な影響を評価するための客観的な評価はまだ不可能です。
近年、現実世界の環境で上肢機能を捉えるウェアラブル技術が数多く開発され、実装されています。加速度計や慣性測定ユニット(IUS)などのウェアラブルセンサーは、日常生活の上肢の動きを測定するために一般的に使用されてきました。しかし、これらのデバイスは、通常、検出されたエポックが機能的な上肢の動き8,9に属しているかどうかは区別しない。例えば、一部のウェアラブルセンサーは、上肢の機能的な動きではない歩行中の上肢のスイングの存在に敏感である。さらに、手首を装着した加速度計は上肢の動きを捉えますが、現実世界の環境では手の機能の詳細を捉えることはできません。感覚手袋は手操作10に関するより詳細な情報をキャプチャすることができますが、手の機能と感覚がすでに損なわれている人々にとっては面倒かもしれません。また、磁気計や指装着加速度計11、12、13を介して指の動きを捉えるためにウェアラブルアプローチも提案されているが、それらの動きの機能的解釈は依然として困難である14。従って、以前に提案されたウェアラブル機器は小型で使いやすいものの、手使いの詳細や機能的な文脈を説明するには不十分である。
ウェアラブルカメラは、神経リハビリテーションアプリケーション15、16、17、18、19のために、自宅でのADL中にこれらのギャップを埋め、手機能の詳細をキャプチャするために提案されています。コンピュータビジョンを用いた自己中心的な動画の自動分析は、手自体と実際のADL20で行われるタスクに関する情報を提供することで、文脈における手の機能を定量化する可能性が大きい。一方、連続録音の持続時間は、通常、バッテリ、ストレージ、および快適さの考慮事項によって約1〜1.5時間に制限されます。ここでは、これらの制約の中で、個人の日常生活を代表するデータを取得し、手関数評価のための有益なデータを得ることを目的とした自己中心的なビデオ収集プロトコルを提示する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Egocentric camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>GoPro Hero 4 and 5</td>
			<td>A camera that records from a first-person angle.</td>
		</tr>
		<tr>
			<td>Battery chager and batteries</td>
			<td>GoPro Inc., CA, USA</td>
			<td>MAX Dual Battery Charger + Battery</td>
			<td>Extra batteries for the camera and battery charger</td>
		</tr>
		<tr>
			<td>Camera charger</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Supercharger</td>
			<td>This charger is connected to the camera directly without disassembling the camera frame.</td>
		</tr>
		<tr>
			<td>Camera frame</td>
			<td>GoPro Inc., CA, USA</td>
			<td>The Frame</td>
			<td>The hinge of the camera frame can be used to adjust the angle of view of the camera.</td>
		</tr>
		<tr>
			<td>Headband for the camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Head Strap + QuickClip</td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk, CA, USA</td>
			<td>32GB microSD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet</td>
			<td>ASUSTeK Computer Inc., Taiwan</td>
			<td>ZenPad 8.0 Z380M</td>
			<td>The tablet is installed with the GoPro App in order to connect with the camera.</td>
		</tr>
	</tbody>
</table>

capturing representative hand use at home using egocentric video in individuals with upper limb impairment

神経学的傷害後の手機能障害は、独立性と生活の質に大きな影響を与える可能性があります。ほとんどの既存の上肢評価は直接行われ、必ずしも地域社会での手の使用を示しているわけではありません。リハビリテーション介入の真の影響を測定するために、日常生活の中で手の機能をキャプチャするための新しいアプローチが必要です。自己中心的なビデオとコンピュータビジョンを組み合わせて自動分析を行い、家庭での手の使用を評価することが提案されている。ただし、連続録画の継続時間には制限があります。私たちは、取得したビデオが参加者のプライバシーを尊重しながら、毎日のルーチンを代表するように設計されたプロトコルを提示します。 
代表的な録画スケジュールは、研究者と参加者の共同作業で選択され、ビデオが自然なタスクとパフォーマンスをキャプチャし、手の評価に役立つようにします。機器と手順の使用は参加者に示されます。合計 3 時間のビデオ録画が 2 週間にわたってスケジュールされます。プライバシーに関する懸念を軽減するために、参加者は録画の開始と停止を完全に制御し、ビデオを編集してから研究チームに戻す機会を与えます。リマインダーは、必要に応じて、ヘルプコールや家庭訪問だけでなく、提供されています。 
このプロトコルは、9人の脳卒中生存者と子宮頸部脊髄損傷を有する14人で試験された。得られたビデオには、食事の準備、食器洗い、編み物などの様々な活動が含まれていました。平均3.11±0.98時間のビデオが得られました。記録期間は、病気や予期せぬ出来事によって12-69 dから変化した。23名の参加者のうち22名からデータを取得し、6名の参加者が自宅記録期間中に調査官の支援を必要としました。このプロトコルは、神経学的傷害後に自宅で手機能に関する貴重な情報を含むビデオを収集するのに有効でした。

参加者の人口統計と包含基準 これらの研究のために23人の参加者のサンプルが募集されました:9脳卒中生存者(6人の男性、3人の女性)とcSCIを持つ14人(12人の男性、2人の女性)。募集したサンプルの人口統計情報と臨床情報の概要を 表 1に示します。
<table border="1" fo:keep-together.within-page="always"><tbody>
<tr>
<td rowspan="2"></td>
			<td r...

Watch this Scientific Journal Video about Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment at JoVE.com

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

自己中心的なカメラを使用して、毎日のルーチン中に手の障害を持つ個人の自然な手の機能をキャプチャするためのプロトコルが提案されています。プロトコルの目的は、記録が自宅での日常生活の活動中に個人の典型的な手の使用の代表であることを確認することです。

上肢障害を持つ個人の自己中心的なビデオを使用して自宅で代表的な手の使用をキャプチャ

Impaired hand function after neurological injuries can have a major impact on independence and quality of life. Most existing upper limb assessments are ...

capturing-representative-hand-use-at-home-using-egocentric-video

Research

JoVE Journal

Bioengineering

2.5K ビュー.  University of Toronto. 自己中心的なカメラを使用して、毎日のルーチン中に手の障害を持つ個人の自然な手の機能をキャプチャするためのプロトコルが提案されています。プロトコルの目的は、記録が自宅での日常生活の活動中に個人の典型的な手の使用の代表であることを確認することです。

ビデオ: Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

誘電泳動と層流を用いたマルチチャネルマイクロ流体デバイスの分離ビーズと細胞

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

生物工学

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

ビデオレートでスキャニング共焦点顕微鏡とMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同時にデュアルカメラエミッション分割システムを使用して、2つの発光チャンネルにリアルタイム画像のキャプチャ：アプリケーションは、接着性を細胞する

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

血管新生および血管組織工学を研究するための小さな動物モデルにおける動静脈（AV）ループ

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水質浄化のための膜のような防汚特性を持つハイドロゲルの合成

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

バイオミメティック化学ニューロモデュレーション神経伝達物質グルタミン酸の in Vitroラット網膜のための方法論

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

化学のエピジェネティックな修飾剤の細胞機械装置をリダイレクトすることによって遺伝子の転写を抑制

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

極薄ポリジメチルシロキサン マイクロ流体チップを用いた保水ライブ線虫個人の固定化

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

歩行とアクティビティ分析のためのホームベースのモニター

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...