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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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。

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		<tr height="36">
			<td height="36" style="height:36px;width:289px;">150-W halogen-lamp light source</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td style="width:119px;">LA-150SAE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Light guide</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LGC1-5L1000</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Collecting lens</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>SH-F16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@500nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@520nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@540nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@560nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67766</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@570nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@580nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@600nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@730nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@760nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized filter wheel&#160;</td>
			<td>Andover Corporation, NH, USA</td>
			<td>FW-MOT-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16-bit cooled CCD camera</td>
			<td>Bitran, Japan</td>
			<td>BS-40</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Video zoom lens</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>VZMTM300i</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:289px;">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>
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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

Universidad de Cartagena UDEC

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

齧歯類の脳における同時fMRIと電気生理学

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

神経科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

ハイフィールドのfMRIを使って大人の猫における聴覚皮質の機能イメージング

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

げっ歯類での同時のfMRIと脳深部刺激

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同時2光子<em&gt;インビボ</emマウス脳梁膨大後部皮質におけるシナプス入力とシナプス後ターゲットの&gt;イメージング

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

脳血管内皮細胞での生体エネルギー機能の評価

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィル テスト

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

回転加速度に基づくラットにおけるびまん軸索脳損傷の誘導

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

脳血行動態との光散乱特性の同時評価

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齧歯類の脳における同時fMRIと電気生理学

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

ハイフィールドのfMRIを使って大人の猫における聴覚皮質の機能イメージング

げっ歯類での同時のfMRIと脳深部刺激

同時

同時2光子

脳血管内皮細胞での生体エネルギー機能の評価

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィルテスト

回転加速度に基づくラットにおけるびまん軸索脳損傷の誘導

脳血行動態との光散乱特性の同時評価

要約

さらに動画を探す

この動画の章

齧歯類の脳における同時fMRIと電気生理学

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

ハイフィールドのfMRIを使って大人の猫における聴覚皮質の機能イメージング

げっ歯類での同時のfMRIと脳深部刺激

同時

同時2光子

脳血管内皮細胞での生体エネルギー機能の評価

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィル テスト

回転加速度に基づくラットにおけるびまん軸索脳損傷の誘導

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィルテスト