Name: simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging
Uploaded: 2017-05-07T15:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

動物のケア、準備、および実験プロトコルは、東京農工大学の動物研究委員会によって承認されました。この方法のために、ラットは、食物および水を自由に摂取して、制御された環境（24℃、12時間の明/暗サイクル）に収容されています。 従来のマルチスペクトル拡散反射イメージングシステムの構築1。 <ol><li>マウントの中心波長を有する9つの狭帯域光干渉フィルタ500、520、540、560、570、580、600、730、及び電動フィルタホイールのフィルタ孔760 nmでした。 </li><li>広帯域白色光源、狭帯域干渉フィルタの上記セット、ライトガイド、集光レンズ、ビデオズームレンズ、及びモノクロCCDカメラで電動フィルターホイールを使用して、マルチスペクトル画像化システムを構築します。 図 2に示される光学部品のレイアウトは、目のために参照することができます構築手順です。 注：照明の角度は約45°で試料表面に対してです。 </li><li>干渉フィルター、光ガイド、及び集光レンズを介して試料の表面を照明するハロゲンランプ光源をオンにします。 </li><li> CCDカメラの動作するソフトウェアを開きます。 </li></ol> 2.動物の準備注：このプロトコルでは、ラットは、将来の実験のために使用されていなかった、それはマルチスペクトル画像の測定直後に殺しました。 <ol><li>管を麻酔機の出口ポートに誘導室の入口ポートを接続します。第二管を麻酔機の入口ポートに誘導チャンバーの出口ポートを接続します。 </li><li>誘導室にラットを置き、5.0％イソフルランで麻酔を誘導します。ラットは、つま先のピンチに応答しないような深さで麻酔を維持します。 L麻酔機にロータリーノブを使用して2.0％イソフルランにower。 </li><li>定位フレームにラットの頭を固定してください。定位フレームに麻酔のためのマウスピースを取り付けます。 </li><li>管を麻酔機の出口ポートにマウスピースの入口ポートを接続します。管を麻酔機の入口ポートにマウスピースの出口ポートを接続します。 </li><li>皮膚表面が表示されるまで、バリカンを使用して将来の切開部位を越えて頭部を剃ります。 </li><li>外科用メス使用頭部の正中線に沿って約20mm長い縦切開を行う（ 図1（a）参照 ）および皮下結合組織を露出させる（ 図1（b）参照 ）。 </li><li>鋭いキュレットまたはペンチを使用して、皮下結合組織を除去し（ 図1（c）参照 ）頭蓋骨を露出させるためにヘッドの両側にそれを引っ張ります。 </li><li>頭蓋内sutur頭蓋骨上の楕円溝を掘ります（冠状縫合、矢状縫合、およびラムドイド縫合）を、高速ドリルを用いて行った（ 図1（d） ）。 </li><li>高速ドリルを使用して溝の中の骨をゆっくりと均一に掘削します。 </li><li>脳血管が出現した後、骨の太さと強度を推定するために、狭窄した頭蓋骨の表面を軽く押す。間引かれた頭蓋骨領域が容易に押し下げられる場合、高速ドリルで頭骨の縮小を終了する。 </li><li>狭窄した頭蓋骨の楕円形の境界線を、かぎ針の先端または小さな外科用はさみを用いて短く切る。 </li><li>細い頭蓋骨を脳の表面からゆっくりと、やさしくピンサーを使って外す。 </li><li>生理食塩水で頭蓋の窓を静かに浸し、厚さ約0.1mmの透明なガラス板で覆う。 </li></ol> 3.インスピレーション酸素の割合を調節する注：呼吸条件ンは、吸入酸素（のFiO 2）の割合を調節することによって変更することができます。 <ol><li>チューブを使用して、別のY字管コネクタ（コネクタ2）の第1のポートにY字管コネクタ（コネクタ1）の第1ポートを接続します。 </li><li>チューブコネクタ1の第2のポートにマウスピースの入口ポートを接続します。 </li><li>チューブを用いて、酸素濃度監視装置にチューブコネクタ1の第3ポートに接続。 </li><li>管を、麻酔機の出口ポートにチューブコネクタ2の第2ポートを接続します。 </li><li>チューブを使用して、ガス混合装置の出口ポートにチューブコネクタ2の第3のポートを接続します。 </li><li>チューブを用いて5％CO 2ガスボンベ-高圧95％O 2へのガス混合装置の一つの入口ポートを接続します。 </li><li>チューブを用いて5％CO 2ガスボンベ-高圧95％のN 2へのガス混合装置の他の入口ポートを接続します。 </li><li> Oガスの流量を変更しますf O 2およびN 2をガス混合装置の回転ノブを使用して測定する。 </li><li>酸素濃度モニタ装置を使用してFiO 2を確認し、調整する。 </li></ol> 4.マルチスペクトル拡散反射像の取得<ol><li> 参照画像の取得 注：この実験で使用される光源、光ファイバー、検出器などの光学部品は、独自のスペクトル特性を持っています。したがって、これらの成分を通過する光の強度は、基準画像として記録されるべきである。基準画像は、光源からの光で照明された標準的な白色ディフューザで撮影された画像である。 <ol><li>ステージ上に標準の白色ディフューザーを水平に置きます。 </li><li>鏡筒のズームリングを回して、白いディフューザーの表面にカメラのレンズを合わせます。 </li><li>カメラからの適切な値を選択して、カメラの積分時間を調整します。ドロップダウン積分時間のリストをカメラのオペレーティングソフトウェアの光の最大量が最大カウントの約75％である信号を生成するようになっています。画素値のヒストグラムを見ながら、信号強度レベルが最大カウントの約75％になるまで、積分時間を調整します。 </li><li>ファイルに画像を保存するために、ファイルメニューから「保存」コマンドを選択します。 </li><li>フィルタホイールを回転させることにより、フィルタの位置を変更します。 </li><li>上述した方法に従って、他の波長の画像を保存します。ファイル名には、サンプルと使用波長（ 例えば、W500、W520、W540 ... W760）を特定すべきです。 </li></ol></li><li> サンプル画像の取得 注：9つの波長で露出したラット脳の拡散反射光強度の画像が捕捉され、同一の取得条件を用いて、パーソナルコンピュータのハードドライブに保存されています。 <ol><li>静かにRを配置ステージ上では、ゆっくりとカメラがラット脳の表面に集中できるように、ステージレベルを調整します。 </li><li>ファイルに画像を保存するために、ファイルメニューから「保存」コマンドを選択します。 </li><li>フィルタホイールを回転させることにより、フィルタの位置を変更します。 </li><li>上述した方法に従って、他の波長の画像を保存します。ファイル名には、サンプルと波長（ 例えば、R500、R520、R540 ... R760）を特定すべきです。 </li></ol></li><li> 暗い画像の取得 注：CCDカメラは、電気信号に応答して光強度を生成することができます。しかし、光が検出器に入らない場合であっても、原因の電気回路及び検出器のノイズに若干の出力があります。これは、暗電流ノイズと呼ばれています。正確に光の分光強度を測定するため、暗電流成分は、暗画像として記録し、次いで測定信号から減算されるべきです。暗い画像はイメージテイクですブロックされた光路を有するn。 <ol><li>ハロゲンランプ光源をオフにします。 </li><li>遮蔽板を用いたCCDカメラシステムの光路を遮断します。 </li><li>ファイルに画像を保存するために、ファイルメニューから「保存」コマンドを選択します。ファイル名は、試料（ 例えば、ダーク）を特定すべきです。 </li></ol></li></ol> 5.ヘモグロビン含有量および光散乱パラメータの可視化注：マルチスペクトル拡散反射画像のセットは、パソコンのハードドライブに保存し、オフラインで分析されます。 9つの波長（500、520、540、560、570、580、600、730、760 nm）におけるマルチスペクトル拡散反射画像のモンテカルロシミュレーション19によって補助重回帰分析は、2つの次元を視覚化するために行われます酸化ヘモグロビン濃度、脱酸素化ヘモグロビン濃度、総ヘモグロビン濃度、局所脳酸素飽和度、及びスキャットのマップ電源をtering。詳細なアルゴリズムは文献17、18に掲載されました。 <ol><li>基準画像と各波長でのサンプル画像の両方から暗画像を差し引きます。 </li><li>各波長λにおける参照画像がサンプル画像を正規化します。拡散反射画像Rとして正規化された画像を扱います。 </li><li>各波長λでの拡散反射画像Rの逆数の対数をとることによって、吸光度（または光学密度）画像Aを計算します。 <img alt="式（1）" src="/files/ftp_upload/55399/55399eq1.jpg" /> （1） </li><li> y平面脳表面について得られた構造情報を示し、zは γ軸スペクトル情報を示す- xは 、それらの波長のオーダーで吸光度画像を積層した三次元マトリックスを生成します。 </li><li> PE座標各XYでの吸光度スペクトルA（λ）は、重回帰分析をrform。 </li><li>ステップ5.5の独立変数と従属変数とHBO（λ）ε酸素化ヘモグロビンのモル吸光係数スペクトル及び脱酸素化ヘモグロビンεのHBR（λ）として吸光度スペクトルA（λ）を使用する（ のHbOεの値を公開（λ）そしてεHBR（λ））を 表1に示します。 </li><li> 3重回帰係数HBO、HBR、0の二次元マップ（画像）を確認。 </li><li>順HBO、HBR、及び0に重回帰係数の画像を積層した三次元マトリックスを生成します<eM&gt; X - y平面は、脳の表面およびz -軸について得られた構造情報を示しているが、複数の回帰係数を示しています。 </li><li>次の実験式を用いて座標酸素化ヘモグロビン濃度C HBO、脱酸素化ヘモグロビン濃度C HBRを計算し、多重回帰のセットからの散乱電力Bは、それぞれXYでHBO、HBR、及び0係数 （彼は、βの値HBO、I、βHBR、I、およびβ0、I（I = 0,1,2,3）は、表2に提供されます）。 <img alt="式（2）" src="/files/ftp_upload/55399/55399eq2.jpg" /> （2） <img alt="式3" src="/files/ftp_upload/55399/55399eq3.jpg" /> （3） <img alt="式4" src="/files/ftp_upload/55399/55399eq4.jpg" /> （4） </li><li>酸素化ヘモグロビン濃度C HBO、脱酸素化ヘモグロビン濃度C HBR、散乱電力Bの二次元マップ（画像）を確認。 </li><li>各xにおけるC のHbO及びC HBRを加算することにより総ヘモグロビン濃度C HBTの二次元マップを計算- y座標 。 </li><li>各xにおける総ヘモグロビン濃度C HBTによって酸素化ヘモグロビン濃度C のHbOを分割して局所脳酸素飽和度RSO 2の二次元マップを計算- y座標 。 </li></ol>

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D., Leitner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyper-spectral+video+endoscope+for+intra-surgery+tissue+classification+using+auto-fluorescence+and+reflectance+spectroscopy.">Hyper-spectral video endoscope for intra-surgery tissue classification using auto-fluorescence and reflectance spectroscopy.</a> Proc. SPIE. 8087, 808711 (2011).</li><li>Basiri, 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=Use+of+a+multi-spectral+camera+in+the+characterization+of+skin+wounds.">Use of a multi-spectral camera in the characterization of skin wounds.</a> Opt. Express. 18 (4), 3244-3257 (2010).</li><li>Nishidate, I., Maeda, T., Niizeki, K., Aizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+melanin+and+hemoglobin+using+spectral+reflectance+images+reconstructed+from+a+digital+RGB+image+by+the+Wiener+estimation+method.">Estimation of melanin and hemoglobin using spectral reflectance images reconstructed from a digital RGB image by the Wiener estimation method.</a> Sensors. 13 (6), 7902-7915 (2013).</li></ol>

The authors have nothing to disclose.

このプロトコルの中で最も重要なステップは、頭蓋窓を作るために薄くなった頭蓋骨領域の除去です。これは予期せぬ出血を避けるために、慎重に行われるべきです。この工程は、高い精度で反射率画像を拡散多重スペクトルの高品質を得るために重要です。可能な場合は実体顕微鏡の使用は、外科的処置のために推奨されます。ゼラチンスポンジの小片は、止血のために便利です。 <p cl...

マルチスペクトル拡散反射率イメージングは​​、皮質組織に固有の光信号（IOSs）の空間的マップを取得するための最も一般的な技術です。形態学的変化によって誘導される光散乱性の光吸収の変化とによる皮質血行動態の散乱特性、低減又はミトコンドリアにおけるシトクロムの酸化に依存して吸収の変化、および変形：IOSsは、主に3つの現象に起因するインビボ脳で観察され1。 近赤外（NIR）スペクトル範囲（VIS）可視光の光を効果的に吸収され、生体組織によって散乱されます。 インビボで脳の拡散反射スペクトルは、吸収および散乱スペクトルによって特徴づけられます。換算散乱係数は単調散乱スペクトルの展示にVIS-に-NIR波長範囲の結果で脳組織のs &#39; を μより長い波長で小さい大きさをINGの。等価散乱係数μスペクトルS「（λ）は、μSなどのべき乗則関数2、3の形態であると近似することができる」（λ）×λ-bを =。散乱電力Bは、組織2,3生体内生物学的散乱体の大きさに関連しています。組織及び皮質生体組織の生存率の減少の形態学的変化は、生物学的散乱体4、5、6、7、8、9の大きさに影響を与えることができます。 マルチスペクトル拡散反射結像光学系は、容易白熱リチウムから構築することができますGHTソース、簡単な光学部品、及びモノクロ電荷結合素子（CCD）。したがって、様々なアルゴリズムおよびマルチスペクトル拡散反射撮像のための光学系が皮質血行動態および/または組織形態10、11、12、13、14、15、16、17、18を評価するために使用されてきました。 この資料に記載された方法は、血行動態及び従来のマルチスペクトル拡散反射イメージングシステムを用いて、in vivoでのラット脳組織の光散乱特性の両方を可視化するために使用されます。代替技術に比べて、この方法の利点は、脳の血行動態および皮質組織の両方における時空間変化を評価する能力です形態学だけでなく、様々な脳機能障害の動物モデルへの適用。したがって、この方法は、外傷性脳損傷、てんかん発作、脳卒中、および虚血の調査のために適切であろう。

<table><tbody><tr><td>150-W halogen-lamp light source</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LA-150SAE</td><td></td></tr><tr><td>Light guide</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LGC1-5L1000</td><td></td></tr><tr><td>Collecting lens</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>SH-F16</td><td></td></tr><tr><td>Interference filters l@ 500 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65088</td><td></td></tr><tr><td>Interference filters l@ 520 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65093</td><td></td></tr><tr><td>Interference filters l@ 540 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65096</td><td></td></tr><tr><td>Interference filters l@ 560 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67766</td><td></td></tr><tr><td>Interference filters l@ 570 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67767</td><td></td></tr><tr><td>Interference filters l@ 580 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65646</td><td></td></tr><tr><td>Interference filters l@ 600 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65102</td><td></td></tr><tr><td>Interference filters l@ 730 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65115</td><td></td></tr><tr><td>Interference filters l@ 760 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67777</td><td></td></tr><tr><td>Motorized filter wheel&amp;nbsp;</td><td>Andover Corporation, NH, USA</td><td>FW-MOT-12.5</td><td></td></tr><tr><td>8-bit monochromatic CCD camera</td><td>THE IMAGINGSOURCE, Germany</td><td>DMK21BU618.H</td><td></td></tr><tr><td>Video zoom lens</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>VZM&lt;sup&gt;TM&lt;/sup&gt;300i</td><td></td></tr><tr><td>Spectralon white standard with 99% diffuse reflectance</td><td>Labsphere Incorporated, North Sutton, NH, USA</td><td>SRS-99-020</td><td></td></tr></tbody></table>

simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

インビボラットの脳から得られた拡散反射の代表的なスペクトル画像を図3に示す.500,520,540,560,570,580nmの画像は、大脳皮質の血管の緻密なネットワークを明瞭に視覚化する。 600,730,760nmの画像で観察される血管と周囲組織との間のコントラストの低下は、より長い波長およびNIR波長でのヘモグロビンによる光のより低い吸収を反映する。 <p class="jo...

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

脳血行動態およびin vivoでのラット脳組織の光散乱特性の同時評価は、従来のマルチスペクトル拡散反射イメージングシステムを用いて実証されています。

脳血行動態との光散乱特性の同時評価<em&gt;インビボ</emマルチスペクトル拡散反射率イメージングの使用&gt;ラット脳

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

Research

JoVE Journal

Neuroscience

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

7.6K ビュー.  Tokyo University of Agriculture & Technology. 脳血行動態およびin vivoでのラット脳組織の光散乱特性の同時評価は、従来のマルチスペクトル拡散反射イメージングシステムを用いて実証されています。 

ビデオ: Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

どのようにレーザーを構築するためには、血流を監視するためにコントラストイメージング（LSCI）システムのスペック

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

神経科学

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

乳幼児の脳代謝と血行動態の非侵襲的光計測

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

医学

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

マウス脳低酸素性虚血の間に同時PET / MRIイメージング

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

脳血流ベースの安静状態機能性コネクティビティ(光学拡散相関分光法)

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

生物工学

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

組織診断のためのマウス脳のリアルタイム、2色刺激ラマン散乱イメージング

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

軽度の外傷性脳損傷を有するラットの海馬における高度な拡散イメージング

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

外傷性脳損傷に対する神経炎症および血行動態応答のシステム解析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

Two-Dimensional Super-Resolution Visualization of Rat Brain Microvasculature Using Ultrasound Localization Microscopy

The cerebral microvasculature forms a complex network of vessels essential for maintaining brain function. Diseases such as stroke, Alzheimer's disease, gliomas, and vascular dementia can profoundly disrupt the microvascular system. Unfortunately, current medical imaging modalities offer only indirect observations at this scale. Inspired by optical microscopy, ultrasound localization microscopy (ULM) overcomes the classical trade-off between penetration depth and spatial resolution. By localizing and tracking individual injected microbubbles (MBs) with sub-wavelength precision, vascular and velocity maps can be generated at the micrometer scale. Here, we present a robust protocol for super-resolution imaging of the brain microvasculature in vivo in rats using a commercial ultrasound platform. This method achieves 12.5 &#181;m spatial resolution, reconstructing the microvascular architecture and providing detailed information on blood flow direction and velocity, greatly enhancing our understanding of cerebral microcirculation. The protocol can be extended to rat disease models, offering a powerful tool for the early diagnosis and treatment of neurovascular diseases.

Here, we describe a protocol for ultrasound localization microscopy (ULM), which achieves 12.5 &#181;m spatial resolution to image the brain microvasculature in rats. It enables detailed visualization of blood flow direction and velocity, offering a powerful tool for advancing studies of cerebral circulation and vascular disorders.

超音波ローカリゼーション顕微鏡を用いたラット脳微小血管系の2次元超解像可視化

The cerebral microvasculature forms a complex network of vessels essential for maintaining brain function. Diseases such as stroke, Alzheimer's disease, ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

びまん性光学分光法による神経クリティカル患者のリアルタイムモニタリング

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

脳血行動態との光散乱特性の同時評価

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