This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

提示手順は、学術医療センターアムステルダム、登録番号NL41985.018の治験審査委員会によって承認された研究プロトコルの下で行われます。書面によるインフォームドコンセントは、すべての参加者から要求されている。 1.システム<ol><li>この実験のために、1,280-1,350波長帯13で動作する、フーリエドメインOCTシステムを使用する。フーリエ領域低コヒーレンス干渉法は、第一世代の時間領域OCTシステムと比較した場合、データ収集速度を増加させる連続スキャンを可能にする。注：OCTシステムは、約90°の角度で螺旋状に走査し、光ファイバプローブとインターフェースされる。これは、2.7Fの外径（0.9 mm）の135センチ挿入可能な長さを有している。プローブは、54ミリメートルのプルバック範囲で駆動モータと光コントローラ（取付ドック）を通じて10月コンソールに接続します。取得したOCTデータセットは、541の断面像（Bスキャ​​ン）ワットから成り15μmの距離分解能i番目（ 図1,2）。 </li><li> Kodach ら 14,15により、前述のように正確で再現性の減衰測定を保証するために、脂肪乳剤、（ 例えば 、イントラリピッド）の重量百分率に基づいてμ10月増加する濃度について測定することによって較正する。 要するに： <ol><li> 0.125、0.250、0.5、1.0、2.0、4.0、10、15、20（ストック）パーセントの濃度を達成するために、脱イオンH 2 O、20％の脂肪乳剤の標準的なバッチを希釈する。 <ol><li>脂肪乳剤の混合物200mlにOCTプローブを配置し、OCT計測を取得する。 </li><li>相互参照は、文献中で既知の値とμ10月の値を抽出した。 </li></ol></li></ol></li></ol> 2.タイム·アウトと患者位置決め<ol><li> PR、手順を開始する前に、名前、生年月日、手順を確認し、「タイムアウト」を実行ocedural側、抗凝固剤の使用、およびアレルギー。 </li><li>腫瘍の位置に応じて、どちらを起こしやすいまたは側臥位で患者を置く。十分な支援を患者に提供し、彼/彼女は20から40分間にわたってこの位置に快適であることを期待しているかどうかを確認。 </li><li>超音波（US）16を使用して、腫瘍をローカライズし、永久的なインクで皮膚に針落ち点をマーク。 注：コンピュータ断層撮影（CT）を使用する場合は、アクセス針の好ましい位置をローカライズするために柔軟な針誘導テンプレートを使用します。 </li></ol> 3.消毒と滅菌ドレープ<ol><li>手術帽と口カバーに入れてください。 </li><li>以前に配置された針落ちマーク（ステップ2.3）を削除しないように注意しながら、クロルヘキシジン/アルコール溶液を用いて、穿刺部位の周りの皮膚を清掃してください。広域消毒すると、予期しないアクセス針の再配置の場合の追加の洗浄の必要性を防ぐことができます。 </li><li> Rはた10mlシリンジ、鈍吸引針、21 G注射針、メス、15 G同軸導入針、18 Gのトロカール針、及び16：無菌コンテンツのegardを含む経皮的穿刺セットを開きGコア生検銃。 </li><li>その後手の消毒剤を適用して、徹底的に手を洗う。手術衣及び滅菌手袋を着用してください。 </li><li>無菌ドレープで患者をカバーしています。 </li><li>超音波プローブの周りに無菌カバーを適用し、所定の位置にニードルガイドを修正。 </li></ol> 4. 10月の準備<ol><li> 10月コンソールを起動し、コンソールインターフェイスを使用して、患者ID、姓、名、およびDOB（生年月日）をラベルの分野で患者の詳細を入力してください。 </li><li>無菌のコンテンツのに関しては、OCTプローブ、無菌の取り付けドックカバー、および5ミリリットルルアーロックシリンジを含む10月のパッケージを展開。 </li><li> 10月コンソールマウントドックに無菌のカバーを適用します。非無菌取り付けドックを導くことが必要ですアシスタントの助けを。 </li><li> 0.9％NaClで5ミリリットル注射器を記入し、フラッシングポートに取り付けます。水がプローブカバーの先端部に表示されるまで、OCTプローブをフラッシュします。 </li><li>取り付けドックにOCTプローブをロードします。回転させて、適切な機能を確認した赤色の光を放出するプローブをロードした後。損傷のリスクを最小限に抑えるために紅潮し、ロード中にその保護カバーにプローブのままにしておきます。 </li><li>そのカバーからOCTプローブを削除します。硬い表面にプローブを配置し、チップを短縮するメスを使用しています。光ファイバとプリズムに圧力を最小限にするために、切削中にプローブの先端部を固定します。オリエンテーションのために放出される（赤色）光を用いて、プリズムから遠位5ミリメートルをカット。 </li></ol> 5.穿刺<ol><li> 2％リドカインた（20mg / ml）を用いて、皮膚および深層を麻酔。リドカインを有効にするためにできるように、数分待ちます。痛みがある場合に患者情報を確認して下さい。 </li><li>ニードルガイドを使用して、配置する撮影により位置を確認する15 G同軸導入針。配置が満足のいくものである場合には、オブチュレータ（鋭い針芯）を削除。 </li><li>腫瘍を貫通、導入針を通じて18のGトロカール針を配置します。再びイメージングと針の位置を確認します。配置が満足のいくものである場合には、閉塞具を取り外します。 </li><li>抵抗を感じまで、トロカール針までのOCTプローブフィード。 </li><li> OCTプローブを固定して、腫瘍組織をOCTプローブを露出する、トロカール針を後退させる。腫瘍内のトロカール針の先端を維持することは、呼吸サイクル中のOCTプローブのよじれを最小限に抑えます。これは、プローブの損傷のリスクを低下させる。 </li><li> 10月スキャン： <ol><li>データセットあたり541 Bスキャンに設定コンソールで、10月のスキャンを実行します。ここで使用されるOCTシステムは、特定のパラメータ調整を必要としない5.4センチメートルの長さにわたって、自動プルバックを実行します。 </li><li> （品質、工芸品のためのスキャンおよび固形組織の外観を確認してください図1A）。アーティファクトは最も一般的に円形のバンドが通常の10月パターン（ 図1B）から出て立って現れる。 </li><li>人工物が再スキャン後に続く場合はプローブを交換してください。 </li></ol></li><li> 10月3日のデータセットの最小までの手順を繰り返し5.6が取得される。 </li><li>インプレース導入針を残して、OCTプローブ及びトロカール針を外します。 </li><li>コア生検銃をARMおよびイメージング上の位置を確認し、導入針を介して配置します。 </li><li>位置決めが満足のいくものである場合には、生検銃を発射。 </li><li>病理部門プロトコルに従って容器に生検材料を配置します。ここでは、紙のインレイとペトリ皿、上の場所を検は十分に0.9％NaClで飽和。 </li><li>コア生検の品質を確認し、十分な材料が得られるまでステップ5.9と5.10を繰り返します。 </li></ol>

<ol>
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The authors of this article have nothing to disclose.

この公報では、腎臓の10​​月、ベースの経皮的、針の実現可能性について報告する。これは、「光学的生検」と呼ば腫瘍分化のための臨床的に適用可能な技術、に10月の発展に不可欠な最初のステップです。私たちの最初の25人の患者が簡単で安全手順であることが、経皮10月を示している。光学的生検は、従来のコア生検上の2つの利点がある。まず、OCTデータのリアルタイム取得および分?...

過去数十年、腎大衆1,2の発生率の着実な増加を示している。今まで、腎腫治療の決定は、主にMRIとCT撮像特性、年齢および併存疾患に基づいて行われてきた。しかしこれらの診断法と臨床パラメーターは本当に腎質量の悪性の可能性を検出するためのフィネスを欠いている。病理学的評価のための十分な組織（診断）とコア生検または細針吸引95〜100％3の範囲の感度と特異性の両方を持つ客観的な腫瘍の分化を提供します。そのため生検は不審な腎大衆4,5の評価で受け入れを集めています。しかし、十分な組織生検なしでは、遅延、診断、または腎実質（非診断）法線が全体の10〜20％の割合で発生し、小さな腎腫瘤では30％までで（&lt;4センチのSRM）を確立する追加のための頻繁な必要性に起因する診断プロセス生検手順3,5。 光コヒーレンストモグラフィ（OCT）は、腎腫分化における上記の障害を克服する可能性を有する新規なイメージングモダリティである。近赤外光の後方散乱に基づいて、10月2~3程度の効果的な組織浸透に15μmの距離分解能を有する画像を提供する（ 図1,2）。ファーバーらによって記載されるように6：組織浸透のミリメートル当たり信号強度の損失、組織特異的な光散乱の結果は、減衰係数（ミリメートル-1μOCT）として表される。組織学的特徴は、組織分化の定量的パラメータ（ 図3）を提供するのOCT値をμと相関することができる。 発癌中は、悪性細胞が増加した数、とのより大きく、より不規則な形の核を表示<html>高い屈折率およびより活性なミトコンドリア。による細胞成分のこの過剰発現、μ10月の変化が良性腫瘍または非罹患組織7に悪性腫瘍を比較する際に予想される。 最近では、良性と悪性の腎腫瘤8,9を区別する表面的な10月の能力を研究した。 16人の患者では、腫瘍組織の術中のOCT測定は、外部に配置されたOCTプローブを用いて得た。同じ患者の非罹患組織のOCT測定値からなる制御アーム。正常組織は、腫瘍の分化のために、OCTの可能性を確認し、悪性組織と比較して有意に低い中央値の減衰係数を示した。この定量分析は、尿路上皮癌10,11及び外陰上皮新形成分化12などの悪性組織のグレード他のタイプと同様の方法で適用されている。 と診断された患者でENT &quot;&gt;我々は、現場での腫瘍分化と組み合わせたリアルタイムイメージングを提供し、光学的生検に10月を開発することを目指しています。現在の研究の目的は、経皮、針ベースを記述することで、10月のアプローチ腎臓の質量を強化する固体。この方法の説明には、我々の知る限り、腎腫瘍の10月に基づく針の可能性を評価する最初のものです。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

最初の25の腫瘍（23例）のうち、24成功したOCTの手順の合計を行った。あるケースでは、プローブの故障は、OCTスキャンを取得することができないことにつながった。二つの有害事象（AE）は、議論の項で詳細に記載されており、発生した。一般的な患者の特徴を表1に記載されている。 10月コンソールは、取得したデータセットの即時定性分析のために、リア...

Watch this Scientific Journal Video about In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses at JoVE.com

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

生体内 、経皮、ニードルベース、腎大衆の光コヒーレンストモグラフィーで

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

in-vivo-percutaneous-needle-based-optical-coherence-tomography-renal

Research

JoVE Journal

Medicine

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K ビュー.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

ビデオ: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

網膜循環のドップラー光コヒーレンストモグラフィー

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

医学

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

糖尿病研究におけるβ細胞の質量分布のアセスメント用の赤外線光学投影断層近く

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

経皮針生検によって得られた骨格筋組織から単離されたミトコンドリアの調製と呼吸計測評価

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

コンシャスげっ歯類における腎機能の経皮評価

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高解像度マウス光コヒーレンストモグラフィー網膜遺伝子治療研究における眼内注入を支援するために

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1gfp/gfpレポーター マウスの画像

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

糸球体疾患のマウス ・ モデルにおける腎機能の評価

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

生体内 、経皮、ニードルベース、腎大衆の光コヒーレンストモグラフィーで

生体内、経皮、ニードルベース、腎大衆の光コヒーレンストモグラフィーで