Name: in vivo tracking of edema development and microvascular pathology in a model of experimental cerebral malaria using magnetic resonance imaging
Uploaded: 2017-06-08T12:30:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging at JoVE.com

Miriam Reinigの専門家の技術支援は感謝しています。 AHは、ハイデルベルク大学医学部のポスドク奨学金から資金を受けた。 MPはElse-Kröner-Fresenius財団の記念奨励金によって支えられています。 AKMは、DZIFアカデミー（DZIF）の母子保健給付の受給者です。 JPは、ハイデルベルク分子医学研究センター（HRCMM）のキャリア開発フェローシップの受領者です。さらに我々は、スポロゾイト運動の模範的な映画を提供してくれたJulia M. SattlerとFriedrich Frischknechtに感謝する。

この記事で報告されたすべての動物実験は、実験動物科学連盟（FELASA）カテゴリBおよび実験動物科学協会（GV-SOLAS）標準ガイドラインに従って実施され、カールスルーエの地方ドイツ当局（RegierungspräsidiumKarlsruhe） 、ドイツ）。 biosavetyレベル2は、蚊とPlasmodium berghei ANKAスポロゾイトの仕事に適用されます。 1.感染<ol><li> Anophelesは、配偶子母細胞で15分間飼育することにより、 Plasmodium berghei ANKAを用いて蚊を繁殖させる。感染した蚊を湿度80％、21℃に保つ。 </li><li>血液摂取の17〜22日後にケージから女性の蚊を収集する。それらを氷上に置き、麻酔する。 </li><li>鉗子を使用して、一滴の冷RPMI培地で覆われたガラススライド上に3-4匹の蚊を置く。スライドを顕微鏡の下に置きます。 </li> &lt;鉗子を使用して、慎重に頭と体の間に蚊を伸ばします。シリンジと針を用いて唾液腺を分離する。残りの蚊でこの手順を繰り返します。 </li><li>ガラススライドから唾液腺を採取し、それらをガラスピペットで吸い取り、1.5mLの遠心チューブに集める。 注：感染率に応じて、唾液腺あたり通常8,000〜15,000の感染性スポロゾイトを得ることができます。 </li><li>約3分間、唾液腺組織からスポロゾイトを単離するために、小さなプラスチックスティックで遠心チューブ内の分離した唾液腺を粉砕します。 </li><li>残りの組織からスポロゾイトを精製するために、1,000×gおよび4℃で3分間遠心分離する。 </li><li>スポロゾイト（SPZ）を含む上清を新しい遠心管にピペットで移し、精製したスポロゾイトをNeubauer血球計で数えます。 </li><li> phを加えて精製スポロゾイトの濃度を10,000 / mLに調整するオスフェート緩衝生理食塩水。 </li><li>近交系のC57BL / 6マウスの尾静脈に合計1,000個のスポロゾイト（0.1 mL）を注入して感染を開始させます。注射を容易にするために、C57BL / 6マウスを拘束具に置き、尾を静脈（約37℃）の水に入れて尾静脈の視覚化を助ける。 注記：注入自体は、数秒以内に実行できる短い手順です。 </li><li> 1日1回、SPZ感染後3日目以降の血液塗抹標本において、血液段階の寄生虫血症をチェックする。 注：Parasitemiaのモニタリングは、Mueller らの JoVEの記事で以前に可視化されています。 8 </li><li>スポロゾイト注射後5日目から急速マウス昏睡および行動尺度（RMCBS）スコアでマウスを1日1回評価する。 注：ビデオデモンストレーションを含むこの手順の詳細な説明は、Caroll et al。 6 </li><li>評価RMCBSスコアおよび対処すべき研究課題に従って、MRI画像を有するマウス。 6 </li></ol> 2.磁気共鳴イメージングセットアップ<ol><li>高周波伝送用のボリューム共振器と4チャネルフェーズドアレイの表面受信コイルを使用して、9.4T小型動物スキャナーでMRIを実行します。マウスの体温を維持するために、温度制御された水浴を42℃にする。 </li><li> 2％イソフルランおよび圧縮空気を用いて、マウスがつま先にもはや反応しなくなるまで、チャンバー内の麻酔を誘導する。麻酔を1-1.5％に維持する。 </li><li>マウスの尾静脈に尾静脈カテーテルを置く。頭の動きを最小限に抑えるために、ヘッドロックと歯のバーが装備された動物のベッドに傷つきやすいように置き、MRIのためにマウスを配置します。マウスの頸椎をまっすぐにしないように注意してください。 </li><li>造影剤注入システムを尾静脈カテーテルに接続する。 Gd-DTPA（0.3 mmol / kg）シリンジを充填したカスタムメードの注入システムを使用するか、Gd-DTPA（0.3 mmol / kg）シリンジに接続したPEチューブを使用してください。 </li><li>デクスパンテノール眼軟膏を両眼に塗布する。 4チャンネルフェーズドアレイサーフェスレシーバヘッドコイルをマウスの頭に置きます。マウスの後ろに呼吸パッドを置き、呼吸監視装置に接続します。 </li></ol> 3.イメージングプロトコル注：対処すべき研究課題に従って、以下に列挙したプロトコールからイメージング配列を選択する。記載されているすべてのパラメータはMRIソフトウェアで有効ですが、他のソフトウェアプログラムが使用されている場合は調整する必要があります。 <ol><li>マウスの脳がマグネットのアイソセンタにあることを確認するために、ローカライズスキャンを開始します。 </li><li> 血管原性浮腫を定性的に評価するには、マルチスライスレアシーケンスを選択して3D T2強調画像を使用します。 <ol><li>次のPAを入力してください繰返し時間= 2.000ms、エコー時間= 22ms、等方性分解能= 0.1mm、視野= 20×10×12mm 3 。マトリックス= 200×100×120、フリップ角= 90~180°、 （スピンエコー）、および希少因子= 8.シーケンスを開始し、10分48秒待って生の画像を取得する。 </li></ol></li><li> 血管形成性浮腫を定量的に評価するために、マルチスライス、多重スピンエコーシーケンスを選択することによってT2緩和測定を実施する。 <ol><li>繰返し時間= 3.100ms、エコー時間= 8-136ms、8msの増分、スライス数= 17、スライス厚さ= 0.7mm、面解像度= 0.116mm×0.116mm、視野20 x20mm2、マトリックス= 172x172、フリップ角= 90-180°、 （スピンエコー）。シーケンスを開始し、8分53秒待って生の画像を取得する。 </li></ol></li><li> 血管原性浮腫および細胞傷害性浮腫の両方を定量的に評価するために、拡散強調画像/見掛け拡散係数スピンエコーEPI拡散シーケンスを選択することによって、効率的（ADC）マッピングを可能にする。 <ol><li>反復時間= 3.400ms、エコー時間= 20ms、スライス厚= 0.7mm、スライス数= 17、拡散増感方向の数= 30、b値= 1.500s / mm 2 、δ= 3msのパラメータを使用する。 、Δ= 9ms、部分フーリエ符号化加速係数= 1.51、視野= 12×15mm 2 、マトリックス= 96×128、面内分解能= 0.125mm×0.117mm、フリップ角= 90-180度、および彩度スライス（矢状）の数= 1。シーケンスを開始し、生画像が取得されるまで7分56秒待つ。 </li></ol></li><li> 微小出血を評価するには、3D T2 *加重イメージングを使用する。フロー補正FLASHシーケンスを選択します。 <ol><li>繰り返し時間= 2.000 ms、エコー時間= 22 ms、等方性解像度0.08 mm、視野= 32 x 15 x 8 mm 3 、マトリックスサイズ= 400 x 188 x 100、およびfリップアングル= 12度。シーケンスを開始し、生の画像を取得するために15分40秒待つ。 </li></ol></li><li> 動脈の開存性を評価するには、3D FLASHシーケンスを選択して、飛行時間血管造影を使用します。 <ol><li>繰返し時間= 16ms、エコー時間= 3.5ms、スライス厚さ= 0.07mm、面内分解能= 0.104×0.104mm、視野= 20×20×10mm 3 、MRIソフトウェアの以下のパラメータを使用する。 = 192×192×142、フリップ角= 15度である。シーケンスを開始し、画像が取得されるまで7分16秒待ちます。 </li></ol></li><li> 血液脳関門の崩壊を評価するには、0.3 mmol / kgの造影剤注入の前後に3D T1強調画像を使用する。グローバルラジオ周波数の励起を使用して、ラジオ周波数で損なわれたFLASHシーケンスを選択します。 <ol><li> MRIソフトウェアでは、繰り返し時間= 5ms、エコー時間= 1.9ms、等方性分解能= 0.156mm、視野20×18.7×18.7mm 3 、マトリックス128×120×120、フリップ角= 8.5°。シーケンスを開始し、画像が取得されるまで1分14秒待ちます。 </li></ol></li></ol> 4.画像処理と解析<ol><li>血液脳関門の障害を解析するには、ImageJの算術ツールまたは画像計算ツールを使用して、強化されたT1強調画像から3D非拡張T1強調画像を差し引きます。減数画像を評価し、血液脳関門の障害に対応する信号の増加を確認する。 9 </li><li>脳の容積を分析するには、ネイティブ3D T1または3D T2加重データセットを使用します。セグメンテーションエディタを使用して、脳を嗅球から小脳まで描写する。 10 </li><li> 血管形成性浮腫 <ol><li> MRIソフトウェアでT2緩和測定データを処理するか、非線形最小二乗適合手順を使用します。 11 MRI sofを用いたADCマップを得るための拡散強調データの処理twareまたはFDTツールボックス（ 材料表を参照）。 </li><li>関心領域を異なる解剖学的領域に手動で配置します。 注記：重要な脳の腫脹により、関心領域の自動配置が正しく登録されないことがあります。選択された解剖学的領域のT2時間およびADC値がこのようにして得られる。 </li></ol></li><li>微小出血量を分析するには、セグメンテーションエディタを使用して、T2 *加重データセット上に卵形の暗い焦点として現れる微小出血を描写する。 </li></ol>

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2014).</li><li>Seydel, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+swelling+and+death+in+children+with+cerebral+malaria.">Brain swelling and death in children with cerebral malaria.</a> N Engl J Med. 372 (12), 1126-1137 (2015).</li><li>Penet, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+experimental+cerebral+malaria+in+vivo:+significant+role+of+ischemic+brain+edema.">Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.</a> J Neurosci. 25 (32), 7352-7358 (2005).</li><li>de Souza, J. B., Riley, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+malaria:+the+contribution+of+studies+in+animal+models+to+our+understanding+of+immunopathogenesis.">Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.</a> Microbes Infect. 4 (3), 291-300 (2002).</li><li>Curfs, J. H., van der Meide, P. H., Billiau, A., Meuwissen, J. H., Eling, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei:+recombinant+interferon-gamma+and+the+development+of+parasitemia+and+cerebral+lesions+in+malaria-infected+mice.">Plasmodium berghei: recombinant interferon-gamma and the development of parasitemia and cerebral lesions in malaria-infected mice.</a> Exp Parasitol. 77 (2), 212-223 (1993).</li><li>Carroll, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+murine+coma+and+behavior+scale+for+quantitative+assessment+of+murine+cerebral+malaria.">A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.</a> PLoS One. 5 (10), (2010).</li><li>Hoffmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Cerebral+Malaria+Spreads+along+the+Rostral+Migratory+Stream.">Experimental Cerebral Malaria Spreads along the Rostral Migratory Stream.</a> PLoS Pathog. 12 (3), e1005470 (2016).</li><li>Mueller, A. K., Behrends, J., Blank, J., Schaible, U. E., Schneider, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+to+study+tuberculosis-malaria+coinfection+upon+natural+transmission+of+Mycobacterium+tuberculosis+and+Plasmodium+berghei.">An experimental model to study tuberculosis-malaria coinfection upon natural transmission of Mycobacterium tuberculosis and Plasmodium berghei.</a> J Vis Exp. (84), e50829 (2014).</li><li>Hynynen, K., McDannold, N., Sheikov, N. A., Jolesz, F. A., Vykhodtseva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+reversible+blood-brain+barrier+disruption+by+noninvasive+focused+ultrasound+at+frequencies+suitable+for+trans-skull+sonications.">Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications.</a> Neuroimage. 24 (1), 12-20 (2005).</li><li>Nag, N., Mellott, T. J., Berger-Sweeney, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+postnatal+dietary+choline+supplementation+on+motor+regional+brain+volume+and+growth+factor+expression+in+a+mouse+model+of+Rett+syndrome.">Effects of postnatal dietary choline supplementation on motor regional brain volume and growth factor expression in a mouse model of Rett syndrome.</a> Brain Res. 1237, 101-109 (2008).</li><li>Giri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T2+quantification+for+improved+detection+of+myocardial+edema.">T2 quantification for improved detection of myocardial edema.</a> J Cardiovasc Magn Reson. 11, 56 (2009).</li><li>Engwerda, C., Belnoue, E., Gruner, A. C., Renia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+cerebral+malaria.">Experimental models of cerebral malaria.</a> Curr Top Microbiol Immunol. 297, 103-143 (2005).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+plays+a+key+role+in+spatiotemporal+pathogenesis+of+cerebral+malaria.">Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria.</a> Cell Host Microbe. 15 (5), 551-563 (2014).</li><li>Nacer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cerebral+malaria+pathogenesis--hemodynamics+at+the+blood+brain+barrier.">Experimental cerebral malaria pathogenesis--hemodynamics at the blood brain barrier.</a> PLoS Pathog. 10 (12), e1004528 (2014).</li><li>Nacer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimmunological+blood+brain+barrier+opening+in+experimental+cerebral+malaria.">Neuroimmunological blood brain barrier opening in experimental cerebral malaria.</a> PLoS Pathog. 8 (10), e1002982 (2012).</li><li>Pai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+reveals+the+dynamics+of+leukocyte+behaviour+during+experimental+cerebral+malaria+pathogenesis.">Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.</a> PLoS Pathog. 10 (7), e1004236 (2014).</li><li>Shaw, T. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+Arrest+of+CD8++T+Cells+Is+a+Signature+of+Experimental+Cerebral+Malaria.">Perivascular Arrest of CD8+ T Cells Is a Signature of Experimental Cerebral Malaria.</a> PLoS Pathog. 11 (11), e1005210 (2015).</li><li>Potchen, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+brain+MRI+findings+in+120+Malawian+children+with+cerebral+malaria:+new+insights+into+an+ancient+disease.">Acute brain MRI findings in 120 Malawian children with cerebral malaria: new insights into an ancient disease.</a> AJNR Am J Neuroradiol. 33 (9), 1740-1746 (2012).</li></ol>

The authors declare that they have no conflicts of interest.

この記事では、実験的脳マラリアの変化を描写する全脳MRIプロトコルについて説明します。我々は、今日までマラリア研究においてMRIが十分に活用されておらず、我々のプロトコールが他の研究者を助けることを期待している。私たちは役に立つかもしれないいくつかの追加ポイントについて説明したいと思います。 重度の病気のマウスが画像化される場合、位置決め?...

マラリアは世界的に重要な健康問題です。重度のマラリアは部分的に脳の関与を特徴とし、しばしば不良な予後因子である。 5歳未満の小児では、マラリア伝播率の高い地域で脳関与が一般的であり、その年齢層でのマラリア関連死亡の主な原因となります。積極的な治療は命を救うことができますが、脳マラリアの検出は、特に初期段階では困難です。脳マラリアに関連する病理学的プロセスには、脳血管障害および脳浮腫が含まれ、重度の脳の腫脹を招く可能性がある。この記事では、実験的脳マラリア（ECM）の全脳インビボイメージングを可能にする磁気共鳴イメージング（MRI）プロトコルを紹介します。 ECMが中央でどのように開始するかについてはほとんど知られていないが、全脳高分解能イメージング法はこの疾患では十分に活用されていないあるいは特定の機序が原因となってこの疾患に至ることがあります。脳全体をカバーするインビボ MRIは、ECM病理のより良い理解を得るための重要な研究ツールである。 MRIは、最近、ECMだけでなくヒト大脳マラリアにおいても死の重要な予測因子であると最近認識されている世界的な脳の脳の腫脹を評価することができる。重度の脳の腫脹は、致命的な疾患で起こり、ECMモデルとヒトの疾患（炎症性および微小血管の両方の変化が特徴である疾患）との間のいくつかの病理学的特徴の1つを表す。 4 ECMは、致死的Plasmodium berghei ANKA感染によるCBAまたはC57BLマウスで誘導することができる。 ECMの発症は、感染後6日目から10日目までに典型的に起こり、フィッティング、運動失調、呼吸困難、昏睡をもたらし、これがラップにつながるイドの死。ラピッドネズミ昏睡および行動尺度（RMCBS）は、ECMの臨床症状を評価するのに役立つスコアです。これは、0から2のスコアを付けられた10のパラメータで構成され、最大20の可能なスコアを有する。6最近、我々はECMマウスにおけるRMCBSスコアの重症度とMRIによって示される病理学的変化との間に良好な一致を示した。 7このプロトコールでは、マウスにおけるECM誘導およびECMによるマウスのインビボ磁気共鳴イメージングについて記載する。

<table><tbody><tr><td>Isoflurane</td><td>Baxter</td><td>1001747</td><td>for anesthesia</td></tr><tr><td>Dotarem</td><td>Guebert</td><td>1086923</td><td>Gd-DTPA contrast agent; 0.5 mmol/mL</td></tr><tr><td>Amira (Image Processing Program)</td><td>FEI Group</td><td></td><td>Version Amira 5.3.2</td></tr><tr><td>MATLAB&amp;nbsp;</td><td>The MathWorks, Inc.,</td><td></td><td>Release 2012b</td></tr><tr><td>FDT toolbox&amp;nbsp;</td><td>FMRIB&#039;s Software Library</td><td>&lt;a href=&quot;http://www.fmrib.ox.ac.uk/fsl/fdt/index.html&quot;&gt;http://www.fmrib.ox.ac.uk/fsl/fdt/index.html&lt;/a&gt;</td><td></td></tr></tbody></table>

in vivo tracking of edema development and microvascular pathology in a model of experimental cerebral malaria using magnetic resonance imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

C57BL / 6マウスでは、 P. berghei ANKAスポロゾイトによる感染後6日目〜10日目の間にECMの最初の臨床症状が観察される。 ECMは感染マウスの60〜80％で発症し、24〜48時間以内に急速に昏睡と死に進行する。対照的に、ECMを発症しないマウスは、過気管支喘息により重度の貧血から感染後第2週後に死亡する。 12 <p class="jove_content" fo:keep-together.within-pa...

Watch this Scientific Journal Video about In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging at JoVE.com

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

in-vivo-tracking-edema-development-microvascular-pathology-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

6.4K ビュー.  Heidelberg University Hospital. We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging. 

ビデオ: In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神経科学

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

免疫・感染学

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

頭蓋内注射と磁気共鳴画像による脳転移のモデル化

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

がん研究

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

医学

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

マウスでの磁気共鳴イメージング顕微鏡による実験的自己免疫性心筋炎における心臓の異常を非侵襲的に評価

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

高解像度MR画像に皮質脳Microinfarctsを評価

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

縦<em&gt;インビボ</em&gt;脳血管系のイメージング：中枢神経系疾患との関連

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

外傷性脳損傷マウスモデルにおける鼻腔内送達後のMRIを用いた超常磁性酸化鉄標識間葉系幹細胞の追跡

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites

Source: Hoffmann, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55334">In Vivo&#160;Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging.</a>&#160;J. Vis. Exp.&#160; (2017)
This video demonstrates the generation of an experimental mouse model of cerebral malaria. Sporozoites isolated from Plasmodium berghei-infected female mosquitoes are injected into a restrained mouse&#39;s tail veins, leading to blood-brain barrier disruption and brain inflammation, causing cerebral malaria. This infected mouse model offers a valuable tool to study in vivo microvascular pathology associated with cerebral malaria.

宿主蚊由来スポロゾイトを用いた実験的脳マラリアのマウスモデル作製

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

要約

さらに動画を探す

この動画の章

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流の定量的解析

使用して樹状細胞の移行の監視

頭蓋内注射と磁気共鳴画像による脳転移のモデル化

細胞追跡のためのin vivoでの¹⁹ F MRI における

マウスでの磁気共鳴イメージング顕微鏡による実験的自己免疫性心筋炎における心臓の異常を非侵襲的に評価

高解像度MR画像に皮質脳Microinfarctsを評価

縦

外傷性脳損傷マウスモデルにおける鼻腔内送達後のMRIを用いた超常磁性酸化鉄標識間葉系幹細胞の追跡

宿主蚊由来スポロゾイトを用いた実験的脳マラリアのマウスモデル作製

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

要約

さらに動画を探す

この動画の章

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

使用して樹状細胞の移行の監視

頭蓋内注射と磁気共鳴画像による脳転移のモデル化

細胞追跡のためのin vivoでの19 F MRI における

マウスでの磁気共鳴イメージング顕微鏡による実験的自己免疫性心筋炎における心臓の異常を非侵襲的に評価

高解像度MR画像に皮質脳Microinfarctsを評価

縦

外傷性脳損傷マウスモデルにおける鼻腔内送達後のMRIを用いた超常磁性酸化鉄標識間葉系幹細胞の追跡

宿主蚊由来スポロゾイトを用いた実験的脳マラリアのマウスモデル作製

脾臓の生体顕微鏡検査：寄生虫モビリティと血流の定量的解析

細胞追跡のためのin vivoでの¹⁹ F MRI における