我々は、ビデオ撮影の準備を整理するために彼女の優秀な技術支援とマヘシュクマーテリーためブリッタKaltwasserに感謝したいと思います。

1。動物<ol><li>実験は、実験動物の管理と使用のためのNIHのガイドラインに従って実施され、地元当局によって承認された。すべての実験は、C57BL6 / J野生型マウスでは、ApolipoproteinE - / - （アポKO）とSV129マウス（12-16週齢、26〜30グラムの体重、実験群当たり5から6匹）を使用した。 </li></ol> 2。着色ラテックスと脳血管の染色<ol><li> EP管で1:20の割合でラテックス化合物の0.5ミリリットル（Pebeo、フランス）と25μlのカーボンブラックインク（Herlitz、ドイツ）の混合物を準備し、水浴中で37℃で混合物を温める。動物をanaesthetizing前に28-30Gの針で2 ml注射器内の混合物を収集します。 </li><li>滅菌生理食塩水1mlにpapavarine塩酸塩の粉末50mgを溶解します。インスリン注射器で液を収集する。別の無菌のインスリン注射器から針を折ります。 20 cの終わりにこの針を置きメートル長いPE10チューブ。今すぐ血管拡張と血管の適切な充填を確実にするために大腿静脈を介して注入するpapavarine塩酸溶液を含むインスリン注射器の針でこのPE10チューブを取り付けます。 </li><li>別の2つのインスリン注射器に生理食塩水をそれぞれ含む1ミリリットルを用意します。インスリン注射器は、正確な位置での注入された流体のスムーズな配信を可能にします。しかし、ラテックスの高粘度のために、取り外し可能な広い針で代替1ミリリットル注射器を使用する必要があります。 </li><li>オペレーティングステージに近く、すべての注射器や滅菌解剖器具を取る。運転段階の周りに手術ドレイパーシートを広げた。運転段階が十分に照らされるべきであり、必要に応じて手術用顕微鏡を用いてもよい。 </li><li>今すぐC57BL6 / Jマウスの体重を測定し、次いで、5％、70％、N 2 O、30％O 2中イソフルラン気化麻酔を誘導する。気化イソフルランの1％で麻酔を続行します。適切positionin後に手足のその背中と固定上でマウスのグラム、麻酔の深さを評価する。次に、左大腿静脈上の皮膚を切り取って、静脈の位置を確認します。 PE10チューブ上に配置され、針の尖った先端を挿入し、ゆっくり分当たり100μl（50 mg / kg体重）の速度でpapavarine塩酸塩溶液を注入します。 </li><li>注射後、腹腔とピアスダイアフラムを切った。それを傷つけたり、あざができずに心臓を露出するためにリブをカットします。動脈鉗子で胸部下行大動脈をクリップ。静脈血を流出可能にするために、右心房オーバー鋭いカットを行います。 18から20秒以上、わずかな圧力を使用して手動で着色ラテックスを注入し、続いて左心室に生理食塩水2mlを注入します。プレフィルドシリンジ別の後にいずれかを使用します。任意の気泡を注入することは避けてください。 </li><li>ラテックスの注入後、5分間動物を残す。動物の首を、慎重に脳全体を削除します。容器のアーキテクチャとコルテを傷つけない良い世話をするX頭蓋骨、髄膜を除去しながら。写真を撮るために4％PFAの6-8 mlを含む60mmの細胞培養皿の中で脳を置きます。透明な皿の下に測定定規を置きます。手術用顕微鏡に取り付けられたカメラで背と脳の腹側表面の25倍の倍率で写真を撮る。写真は元の距離を定量化するために、ペトリ皿と顕微鏡対物間に90°​​の角度で取られるべきである。 </li><li>別の4月5日、動物における手順を繰り返します。 </li></ol> 3。カーボンブラックインクの混合物を用いて脳血管の染色<ol><li>我々のプロトコルを持つラテックスベースの脳血管染色の結果を比較するために、1.5ミリリットルEP管でペリカンScribtolシュワルツインク（CB2）900μlでHerlitz Stempelfarbeインク（CB1）100μlの混合物を調製する。 1:9の比率でCB1とCB2 2mlの混合物の総容積を作るために2、EPチューブを準備します。 2つの別々のインスリン注射器（1の混合物を集めるミリリットルずつ）。 37℃まで温め注射器にフタをし、水浴中に置いてください別の2つのインスリン注射器に生理食塩水2mlを収集し、同様に水浴中に置いてください。 </li><li>動物を麻酔し、papavarine塩酸塩注射用以外は上記と同じ手順を繰り返します。左心室に2ミリリットルsalaineの注入後、カーボンブラックインクの代わりに着色ラテックス2mlの混合物を注入します。 mlあたり18から20秒以上、わずかな圧力で手で注射を行う。注射の前に胸部下行大動脈をクリップ。 </li><li>動物を犠牲にすることは、脳を取り外して、写真を撮る。 </li><li>フローティング脳は全くsubmergesまで、脳の長期保存の場合は、（5％〜15％が続いた後、30％）、徐々に増加する濃度のショ糖とのインキュベーションによってPFAは一晩続いた4％に脳を入れた。着色ラテックスで灌流動物の脳も同じように保存することができる。 </li><li>実行する別の4月5日、動物における手順。異なる遺伝的背景や歪みに起因する脳血管解剖の違いを比較するには、それぞれアポKOおよびSV129マウスの同数にプロトコルを繰り返す。 </li><li>深麻酔（手術の詳細な説明は、この記事の範囲を超えています下にMCAの腔内閉塞の標準プロトコルによると、45分間または90分間一過性局所脳虚血を誘発し、虚血条件下で血管解剖を勉強すること。読者はDoeppnerら、2010）と呼ばれています。梗塞体積を識別するために、再灌流期間（1日〜5日）は計画の終了時に、CB1 + CB2のインクのみで血管染色を行い、5〜10分間37℃で2％TTC溶液を用いて脳全体を染色。 TTCはミトコンドリアの酵素（特にコハク酸デヒドロゲナーゼ）によって赤色のホルマザンに還元される。 TTC染色した後、深い赤色の代謝活性組織の汚れ梗塞組織がunstaiままネッドとによる機能不全や変性したミトコンドリアの酵素に白で表示されます。上記と同様の方法で画像を収集しています。 </li></ol> 4。脳血管領の研究<ol><li>どちら脳血管、脳の背側表面の画像着色ラテックスで染色するか、又はCB1の肉眼解剖学を分析するために、+ CB2インクはImageJのソフトウェア（画像処理と解析に使用されるJavaベースのパブリック·ドメイン·ソフトウェア）を用いて分析することができます。 CB1 + CB2灌流が腹側と背側の両面にすべての血管を染色する一方着色ラテックスで灌流脳は、背側表面に位置する血管染色の可変度が表示される場合があります。 </li><li> ImageJのソフトウェアで画像ファイルを開きます。 ACAとMCAとの間のすべての吻合のポイントを識別します。吻合ポイントは血管径はそれぞれ2、狭いまたはACAとMCAの枝の最寄りの分岐点間の距離の半分であるサイトとして定義されています。 <sアップ&gt;折れ線ツールとプラグイン&quot;ドットライン&quot;を描くの助けを借りて、吻合のポイントをマークすることで、仮想線（吻合線）を描画します。 </li><li> mm単位でスケールを設定します。吻合線と直線描画ツールと &quot;測定&quot;ツールを使用して脳の前頭極に尾4ミリメートルで正中線間の距離を測定します。尾前頭極から6 mmで、同じ操作を行います。動物あたり3から4の画像を解析し、平均値を計算します。脳血管の解剖学的構造の変化を識別するために動物の異なるグループ間の値を比較します。 C57BL6 / Jの動物では、ACAとMCAの間に吻合線は尾側前頭極から6 mmで4mm以下に近い正中線にまで遡ることができる。アポEノックアウトマウスでは、この点で有意差を提示しません。それどころか、SV129マウスで吻合線は、前頭極から尾側に両方4ミリメートルと6ミリメートルで正中線にさらにと平行に位置します。 </li><li>血管解剖を分析するためにInの虚血状態は、上記と同様の計算を実行します。それぞれ、代謝活性組織および壊死組織染色を表す赤と白の色のついたティッシュマージンとして梗塞の境界を識別します。 4ミリメートルで、脳の前頭極から尾6ミリメートルで正中線から梗塞の境界線の距離を測定します。動物あたり3から4の画像から平均値を収集します。別の虚血再灌流期間の間の結果を比較します。 </li></ol>

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それは一定の圧力2,3を暗示する具体的な装置を必要としないよう手動注入によるCB1 + CB2の灌流は集中的な訓練なしで正常に行うことができる。我々のプロトコルにおける灌流成果の異質性もごくわずかです。 20虚血動物のうち、1つだけ16非虚血動物のうち、動物と3は不完全な灌流を示しています。これらのケースでは、血管の閉塞につながる生理食塩水灌流時の気泡の混入はほとん?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社名 </td><td> カタログナンバー </td></tr><tr><td> Scribtolシュワルツ（CB2） </td><td>ペリカン、ドイツ</td><td> 221 135 </td></tr><tr><td> Stempelfarbe（CB1） </td><td> Herlitz PBS AG（ドイツ） </td><td> 10417202 </td></tr><tr><td> Gedeoラテックス</td><td> Pebeo、フランス</td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

脳血管の解剖学的構造は、脳の血行動態を決定する主要因だけでなく、虚血性侮辱後に損傷の重症度である。脳血管系は、動的にさまざまな病態生理学的な状態に応答し、それが系統間および遺伝子操作の条件下でかなりの相違を示す。本質的には、頭蓋内血管染色のための信頼性の高い技術は、虚血性脳卒中の病因を研究するために不可欠です。最近まで、さまざまなテクニックの集合は低粘度の樹脂の射出を含む脳血管系を可視化するために用いられてきた、アラルダイト、F、ゼラチン2または黒3個の炭素ことなく、様々な染料1（ すなわちカーミン赤、墨）やラテックスと混合。上行大動脈を通して白いラテックス化合物の灌流は、まずコイルとJokelainen 3によって報告されている。前田ら 2炭を追加することにより、プロトコルを変更した脳の生理食塩水を灌流後の血管の改善されたコントラストの可視化のためのラテックス化合物からn黒インク。しかしながら、非効率的な血流と血管の不十分な充填が頻繁にラテックス化合物4の高粘度のために経験している。そこで我々は、再現可能な方法で5脳血管系を可視化するために2つの市販のカーボンブラックインク（CB1およびCB2）の混合物を用いて、シンプルで費用対効果の高い手法を説明してきました。我々は、ラテックス灌流5と比較して高い密度で有意に小さい脳血管を染色したマウスの結果におけるCB1 + CB2でその血を示している。ここでは、前方（ACA）および異なる遺伝的背景を有するマウスの血管の変化を研究するために、中大脳動脈（MCA）の間に吻合ポイントを識別するために、我々のプロトコルについて説明します。最後に、我々は、CB1を組み合わせることにより、マウスにおける一過性局所脳虚血モデルにおいて、本手法の実現可能性を実証+虚血性損傷の様々な程度のTTC染色によるCB2媒介血管染色。

ここで説明されたプロトコルは齧歯類脳血管系の従来のラテックスベースの視覚化の技術的な限界を克服しています。 図1Aは、着色ラテックスのそれ以下の灌流を示し、腹面にのみ大型船は全体背側表面は汚れのないままに、染色されている。結果はまた、非常に可変です。 6番組のうちの1頭のみACAおよび脳の背側表面（データは示していない）上に表示され、MCAの部分的な染?...

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Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

げっ歯類の脳の解剖学的構造の分析は、実験的な脳卒中の研究に重要な役割を果たしている。この文脈において、着色ラテックスと血管内血流は数年のための標準ツールとして考えられてきた。しかし、この技術は、その再現性を弱体化させる明確な技術的な限界を意味しています。ここでは、再現可能な方法で脳の血管を可視化するための簡単​​な方法を説明します。高コントラストの可視化と脳血管の適切な充填における左心室の心筋の結果を介して2つの市販のカーボンブラックインクの混合物の注入。我々は、正常に異なる遺伝的背景をもつマウスの脳血管地域間吻合のポイントを識別するためにこの手法を適用している。マウスでの梗塞体積を観察し、分析するための広く使用されているツール - 我々は最終的に血管染色にこの小説と単純な方法は、塩化トリフェニルテトラゾリウム（TTC）染色と組み合わせることができるという証拠を与える。

カーボンブラックインクの血管内灌流は脳血管の信頼性の高い可視化を可能に

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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15.3K ビュー.  University of Duisburg-Essen Medical School. げっ歯類の脳の解剖学的構造の分析は、実験的な脳卒中の研究に重要な役割を果たしている。この文脈において、着色ラテックスと血管内血流は数年のための標準ツールとして考えられてきた。しかし、この技術は、その再現性を弱体化させる明確な技術的な限界を意味しています。ここでは、再現可能な方法で脳の血管を可視化するための簡単​​な?...

ビデオ: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

ラット中大脳動脈の血管縫合閉塞による局所脳虚血モデル

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

神経科学

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

アニマルコミュニケーションの研究のための自動化されたインタラクティブなビデオ再生

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

新生児げっ歯類から単離された脳幹・脊髄調製物の電気生理学は、ニューラルネットワーク呼吸器の出力の記録を可能にします

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

マウス脳血管の精製

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

単離および脳実質細動脈のカニューレ挿入

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

新生児の脳へのウイルス粒子と蛍光マイクロビーズの脳室内および血管内注入

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

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 ...