我々は、健康補助R01NS057476、P50NS010828、P01NS055104、R01EB000790、K99NS0670​​50、R01HL081273とR01EB007279とアメリカ心臓協会の助成金0855772Dの米国国立研究所からの支援に感謝します。

1。ラットの皮質血管系におけるPO2のビューイメージングの幅広い分野<ol><li>ラット（250グラム-350 g）は、最初はイソフルランで麻酔し、大腿動脈と静脈は、心拍数、血圧、および血液ガスだけでなく、点滴静注用を監視するためにカテーテルをされています。体温は、± 0.1 37℃に維持され気管切開が行われ、ラットは、空気と酸素の混合物で換気されています。 </li><li>血液ガスを測定するための血液サンプルは、すべて30〜45分を取られて、換気や麻酔は通常の生理的範囲内で血液ガスを保つために調整されます。 </li><li>サイズの頭頂骨4ミリメートル× 4mmの上に閉じた頭蓋窓は、イメージングのために用意されています。骨と硬膜が除去され、頭蓋窓は1.5％アガロースを充填し、顕微鏡のカバーガラスで封止されている。前頭骨上に追加の1 MM 2バリ穴は、KClの皮質内マイクロインジェクション（〜10μlの1 M）でCSDを誘導するために使用されます。細心の注意が皮質血管系の損傷を避けるために注意すべきである。余分な熱、機械的な圧力、または低酸素症の短い期間では、血液脳関門を危うくし、間隙に血管から染料の漏れの原因となります。 </li><li>光学的に不透明な材料からマスクが頭蓋窓の周囲に配置されます。マスクの目的は、硬膜の端から組織への漏出色素から来ているりん光信号を吸収することです。間隙空間内の色素が測定を台無しにすることができます非常に明るい燐光の源です。間隙空間における色素の増加明るさは、低酸素圧と低消光速度と環境に色素の蓄積の結果です。実験では血管系の損傷は、したがって、容易に実験データが拒否される場合には明るい燐光スポット、の出現で認識されます。 </li><li>手術終了後、イソフルランは廃止され、麻酔がalphachloralose（40 mgの/量（kg h）の注入に続いて50 mg / kgの静脈内ボーラス）に切り替えられます。 </li><li>動物は人工呼吸器、血圧と温度モニター付きカートで転送されます。部屋の空気を呼吸しながら、動物はすぐに、イメージングセットアップに移動し、空気と酸素の適切な混合物で流量計に再接続されます。 </li><li>動物を保持する定位固定フレームは、客観的の下に配置されます。頭蓋窓は、客観的で視野の中央に配置し、焦点面に平行に配置されている。 </li><li>パルスレーザーの電源が入っていて、最小限のパルスエネルギーに設定されています。光ビームは、10以上のミリジュール/ cm 2ではなくなるように調整されたパルスのエネルギーメーターとサンプルに配信パルスのエネルギーに向けられている。ビームISSは、〜60度の斜入射角と頭蓋窓を中心とレーザーISSがオフになって。 </li><li>動物のすべての生理学的パラメータは正常な生理的範囲内になるまで血液ガスの測定値と換気と麻酔の調整が行われている。 </li><li>その血液量を仮定すると燐光プローブOxyphor R3の適切な量のプローブの4 × 10 -5 Mの血中濃度を達成するために生理食塩水1 mLに溶解させ、体重の約7％です。プローブ溶液は大腿静脈を介して注入される。 </li><li>塩化カリウムの1M溶液を調製し、約1 ULは、CSDを誘導する前頭骨上バリ穴から注射器で注入されます。 </li><li>レーザーがオンになっていると画像はすぐにKCl溶液の注入後に開始されます。リン光のイメージングは​​、〜10分中に実行されます。 </li><li>別の血液ガスの測定は、動物の生理学的パラメータが正常範囲内に残っていることを確認するために実行されます。 </li><li>リン光寿命は、Matlabの統計的重み付き非線形正方形のフィッティングを使用して単一の指数関数的減衰をフィッティングすることにより全ての画素に対して得られる。リン光寿命は経験的スターンボルマーのような関係を（下記参照）を使用してPO2値に変換されます。 </li></ol> 2。マウスの皮質微小血管系の高分解能二光子PO2イメージング<ol><li> isofluoreneと大腿動脈が心拍数、血圧、および血液ガスだけでなく、色素を投与するために監視するためにカテーテルを挿入されているとマウスを麻酔しています。体温が37℃に維持される± 0.1℃と動物が自発的に空気と酸素の混合物を呼吸している。 </li><li>頭蓋窓は当初、David K​​leinfeldとヴィンフリートデンクによって開発された技術に従って調製される。良いケアは、間隙に色素の漏れを防ぐために血管系の損傷を避けるために注意すべきである。 </li><li>血液ガスを測定するためのいくつかの血液サンプルは、製剤全体の手順の間に取られており、換気や麻酔が血液ガスを保つように調整されています正常な生理的範囲内にES。 </li><li>動物は、イメージングセットアップに移動されます。動物を保持している変更定位フレームが目的の下に配置されます。頭蓋窓はオリンパス4X目的の下、視野の中央に配置し、焦点面に平行に配置されている。 </li><li>頭蓋窓の画像は、接眼レンズを介してデジタルカメラで撮影され、そして4X目的はオリンパス20倍対物レンズ（NA = 0.95）に置き換えられます。 </li><li>動物のすべての生理学的パラメータは正常な生理的範囲内になるまで血液ガスの測定値と換気と麻酔の調整が行われている。 </li><li>その血液量を仮定すると、体重の約7％、燐光プローブPTP - C343の適切な量のプローブの〜15μMの血中濃度を達成するために生理食塩水0.2 mLに溶解されています。プローブ溶液は大腿動脈を介して注入される。 </li><li>各ピクセルでの色素励起と発光の収集のために必要な時間間隔を設定することにより、実験を始める。 </li><li>現在の深さで血管構造を表示するリン光強度の減衰の遅い2次元ラスタースキャンである調査のスキャンを取得する。目的は、頭蓋窓に垂直に移動されます（Z軸）とリン光強度の遅い2D調査のスキャンは、結像面での微小血管を見つけるために840 nmで最小のレーザパワーを用いて撮影されています。 </li><li>その深さで燐光の調査のスキャンに基づいて各画像の深さ、で、血管系内の点の集合が選択され、各点でのリン光減衰の記録は、平均の定義済みの数のために繰り返されます。 </li><li>希望する平均の量、測定間隔、およびテスト期間を設定し、測定を開始します。 PO 2の測定は、実験期間中に指定された測定間隔で、選択した場所に取得されます。 </li><li>測定中に、ソフトウェアは、ガルバノスキャンミラーの位置を変更することにより、選択した場所に励起レーザーを再指示する。ガルバノメータスキャナ、電気光学変調器、およびその他すべての機器の制御は、LabVIEWでカスタムメイドのソフトウェアによって実行されます。 </li><li>リン光寿命は、Matlabの非線形正方形のフィッティングを使用して単一の指数関数的減衰をフィッティングすることにより、選択したすべてのポイントで得られる。リン光寿命は経験的スターンボルマーのような関係を（下記参照）を使用してPO2値に変換されます。 </li><li>様々な皮質の深さでデータを収集した後、イメージングのための血管に微小血管構造のデキストランコンジュゲートフルオレセイン色素を注入する。 </li><li> 4チャンネルの検出器の緑のチャネルを使用してFITC蛍光の二光子励起イメージングを行うことにより、血管系の構造的な画像のスタックを取得する。 </li><li>動物実験は研究のアニマルケアのマサチューセッツ総合病院の小委員会によって定められたガイドラインおよび規制に準拠して行った。 </li></ol> 3。代表的な結果： <img src="/files/ftp_upload/1694/1694fig1.jpg" alt="図1" /> この図の左側の図1。パネル（）CSDの波の到着前に酸素の圧力のビューの画像の幅広いフィールドが表示されます。 （b）の右側のパネルは、パネル（）にマーク関心領域内のCSDの伝播中の平均酸素圧の時間変化を示しています。 図2（AVI動画）：この映画は、CSDの波の伝播中に、全体頭蓋窓の酸素圧の時間変化を示しています。スケールバーは水銀のミリメートル単位での酸素圧力を示します。 <img src="/files/ftp_upload/1694/1694fig3.jpg" alt="図3" /> 図3。撮像された血管系のスタックの3次元投影。グレーの影は、容積血管のマスクを表す構造的な画像に基づいて作成。測定されたPO 2値は色分けされています。スケールバーは200マイクロメートルである。ネイチャーパブリッシンググループから許可を得て転載。7

<ol>
	<li>Kleinfeld, D., Friedman, B., Lyden, P. D., Shih, A. Y., Chen, J., Xu, Z., Xu, X., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+occlusion+to+surface+and+deep+vessels+in+neocortex+via+linear+and+nonlinear+optical+absorption,+Animal+Models+of+Acute+Neurological+Injuries.">Targeted occlusion to surface and deep vessels in neocortex via linear and nonlinear optical absorption, Animal Models of Acute Neurological Injuries.</a> Contemporary Neuroscience Series. , (2007).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Lebedev, A. Y., Cheprakov, A. V., Sakadzic, S., Boas, D. A., Wilson, D. F., Vinogradov, S. A., A, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Phosphorescent+Probes+for+Oxygen+Imaging+in+Biological+Systems.">Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems.</a> Applied Materials &#38; Interfaces. , (2009).</li><li>Finikova, O. S., Lebedev, A. Y., Aprelev, A., Troxler, T., Gao, F., Garnacho, C., Muro, S., Hochstrasser, R. M., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+microscopy+by+two-photon-excited+phosphorescence.">Oxygen microscopy by two-photon-excited phosphorescence.</a> Chemphyschem. 9, 1673-1679 (2008).</li><li>Sakad&#382;i&#263;, S., Yuan, S., Dilekoz, E., Ruvinskaya, S., Vinogradov, S. A., Ayata, C., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl Opt. 48, D169-D177 (2009).</li><li>Yaseen, M. A., Srinivasan, V. J., Sakadzic, S., Wu, W., Ruvinskaya, S., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+of+oxygen+tension+in+cortical+microvessels+with+confocal+microscopy.">Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy.</a> Opt Express. 17, 22341-22350 (2009).</li><li>Sakadzic, S., Roussakis, E., Yaseen, M. A., Mandeville, E. T., Srinivasan, V. J., Arai, K., Ruvinskaya, S., Devor, A., Lo, E. H., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7, 755-759 (2010).</li></ol>

我々は、燐光の酸素依存性消光に基づく皮質微小血管系におけるPO2測定の2つのアプリケーションを示した。 CCDイメージングに基づいて、最初の方法は、2光子顕微鏡に基づいて、皮質の微小血管系における酸素の分圧を測定​​し、PO2のビューの監視の幅広いフィールドを提供する一方で毛細血管の分解能を提供し、深さで撮影することができます。どちらの方法では、高速および高信号対?...

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<td>Glycopyrrolate</td>
<td>Reagent</td>
<td>American Regent Inc.</td>
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<td>Used to control pharyngeal, tracheal, and bronchial secretions.</td>
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<td>Lidocaine HCL</td>
<td>Reagent</td>
<td>Hospira Inc.</td>
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<td>Used as the local anesthesia during surgeries.</td>
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<td>Isoflurane</td>
<td>Reagent</td>
<td>Baxter Healthcare Corp.</td>
<td>NDC 10019-360-40</td>
<td>Used as a general inhalation anesthetic drug during surgeries and as a general anesthesia during experiments with mice.</td>
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<td>Alpha Chloralose</td>
<td>Reagent</td>
<td>Sigma</td>
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<td>Fluorescein isothio-cyanate&#x2013;dextran</td>
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cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence

脳血と組織の酸素化の時空間特性のモニタリングは、神経代謝、血管の関係をより良く理解するために重要です。高い空間および/または時間分解能を持つビューの大きなフィールドでPO2の同時モニタリングを持つ新しいPO2測定のモダリティの発展は、正常な脳の働きにより深い洞察を可能にし、次のような神経血管疾患の診断と治療に大きな影響を与えることになるも脳卒中、アルツハイマー病、および頭部外傷。 光学的画像診断法は、光スペクトルの可視および近赤外領域でのヘモグロビンの吸収に基づいて、高時空間分解能とPO2の定量的なイメージングを提供する大きな可能性を示している。しかし、脳の血液の酸素化のマルチスペクトル測定は、高度に散乱脳組織を介して光子の移行に依存しています。実験中の動的な変更を受けることができる組織の光学パラメータ、、の推定とモデリングは通常、血液の酸素化の正確な推定が必要です。一方、燐光の酸素依存性消光に基づく酸素の分圧（PO2）の推定は大幅に組織の光学的パラメータの変化の影響を受け、PO2の絶対的な尺度を提供すべきではない。酸素感受性色素を利用した実験系は、同様に生理的PO2範囲の正確な酸素の撮影が可能な強力な技術であること燐光消光を示し、組織培養中の酸素含有量を監視するためとして、潅流組織のin vivo試験で実証されている。 ここでは、どのように燐光寿命イメージングに基づいて、皮質血管系にPO2の測定を行うために二つの異なる画像診断法を示しています。最初のデモンストレーションでは、ラットの皮質表面でPO2のビューイメージングの幅広い分野を提示。この画像診断法は、CCDカメラおよびパルスグリーンレーザーに基づいて比較的単純な実験のセットアップを持っています。 Oxyphor R3染料の燐光寿命に基づいて、皮質拡散うつ病を監視の例が発表された。第二デモンストレーションでは、マウスの皮質微小血管系の高分解能二光子PO2のイメージングを示す。実験は、フェムト秒レーザー、電気光学変調器、およびフォトンカウンティング光電子増倍管を備えたカスタム構築された2光子顕微鏡を含みます。我々は、強化されたとPTP - C343色素小説2光子励起の断面積を用いて、イメージングの例毛細血管を含む皮質微小血管系におけるPO2異質性を提示する。 関連記事の表示するにはここをクリックして<a href="http://www.jove.com/Details.php?ID=1731">生体内の酸素イメージング用燐光ナノプローブの合成とキャリブレーションを。</a>

Watch this Scientific Journal Video about Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence at JoVE.com

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

我々は、燐光の酸素依存性消光に基づく脳血管系における酸素の分圧（PO2）を測定するための実験手順を示す。動物の準備と撮像法は、ラットとマウスにおけるPO2の2光子励起に基づくイメージングにおけるPO2のビューCCDベースのイメージングの大きなフィールドの両方に概説された。

燐光の酸素依存性の焼入に基づいて脳の血液の酸素化の測定

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
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cerebral-blood-oxygenation-measurement-based-on-oxygen-dependent

Research

JoVE Journal

Neuroscience

14.6K ビュー.  Massachusetts General Hospital and Harvard Medical School. 我々は、燐光の酸素依存性消光に基づく脳血管系における酸素の分圧（PO2）を測定するための実験手順を示す。動物の準備と撮像法は、ラットとマウスにおけるPO2の2光子励起に基づくイメージングにおけるPO2のビューCCDベースのイメージングの大きなフィールドの両方に概説された。

ビデオ: Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同時脳波、リアルタイム乳酸濃度の測定と齧歯類大脳皮質ニューロン活動のOptogeneticマニピュレーション

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

神経科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

栓塞狭窄軽度子宮内血流の低下のモデルラット

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鮮な人間の脳から脳の毛細血管の分離

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

生体内における脳タンパク質合成の局所速度の決定のための定量的自動放射線法

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

加圧齧歯動物に及ぼす影響の爆発による頭部外傷の中間の大脳動脈

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

痛みとかゆみに関連する行動の同時測定のための頬注射モデル

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

脳血流と電場のマッピング:マウスにおける電極配置

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

著者スポットライト:アルツハイマー病モデルにおける脳血流を改善するための刺激ベースのアプローチ 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

麻酔マウスにおける頭蓋外刺激電極の配置と脳血流および頭蓋内電界の測定

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...