著者感謝したい Yuliya Naumchuk (Loyola 大学シカゴ)、アグネ Žiniauskaitė (Experimentica 株式会社) 優れた技術と再生産のサポート。カジャ博士の研究プログラムをサポートするには、博士ジョン p. とテレーズ E. マルケイ恵まれて教授ロヨラ大学シカゴ校で眼科で。

すべての動物は、動物のための使用の眼科とビジョンの研究や、EC 指令 86/609/EEC 動物実験で承認され、フィンランドの動物実験委員会によって監視されているプロトコルを使用して ARVO 声明に従って扱われました。
1. レーザー マウス CNV モデル5
<ol><li>肉眼的に異常動物の目を検査します。</li>
	<li>マウスの重量を量る。</li>
	<li>計算し、動物、例えばメデトミジン (1 mg/kg), ケタミン (75 mg/kg)、および 1:1.5:2.5、またはケタミン (40-75 mg/kg) の割合で蒸留水 (0.9 %nacl 溶液) の混合物の重量に基づいて、使用する麻酔薬の適切な量を準備します。キシラジン (5 mg/kg)、および 1:2.; 5:1 の割合で蒸留水 (0.9 %nacl 水溶液)20 g のマウスのための混合物の 0.1 mL を注入します。</li>
	<li>腹腔内麻酔薬を注入します。</li>
	<li>動物に麻酔がかかるまでは、ケージと待機にマウスを置きます。マウスは正しくペダル反射の欠如によって麻酔を確認します。</li>
	<li>レーザー安全保護具の使用を確認します。</li>
	<li>スリッ トランプと 532 nm ダイオード レーザーを入れます。</li>
	<li>ケージや加熱パッド上の場所から、マウスを削除します。</li>
	<li>瞳孔の拡張のトロピカミドの一滴を適用します。フル (3 mm) 瞳孔の拡張のための 3-5 分を待ちます。</li>
	<li>スリッ トランプのステージ上にマウスを置きます。</li>
	<li>Applanate 角膜に coverslip の眼科液体ゲルの一滴を配置します。</li>
	<li>中心部で視神経乳頭とマウスの目を向けます。</li>
	<li>レーザー パワーを 100 にセット mW、100 ms に期間および 50 μ m スポット サイズ。</li>
	<li>レーザー光線を網膜色素上皮 (RPE) に焦点します。</li>
	<li>1 つ目にそれぞれ理想的で、4、8、および 12 ポジション、視神経乳頭周囲の網膜の血管を避けることによって 3 つのレーザー ショットを確認します。すべてレーザー網膜出血の不在のためのショット後の眼底を検査します。反対側の眼は非レーザ書込みコントロールとして機能します。</li>
	<li>加熱パッド背面 coverslip と場所のマウスを破棄します。</li>
	<li>両方の目に釘ゲルの滴の一滴を適用します。</li>
</ol>2. SD 10 月6,7
<ol><li>齧歯動物のアライメント ステージにマウスを置き、頭を固定します。</li>
	<li>X ・ Y ステージ コント ローラーを用いた生体内イメージングの目に直面する (例えばBioptigen/ライカ Envisu R2200) SD 10 月システムのレンズを合わせます。</li>
	<li>SD 10 月ブルッフ膜の中断を確認するスキャンを実行: 書込みサイトで基準線を手動で移動 SD-10 月は、全体の目をスキャンしたら。ブルッフ膜の区切りは、書込み領域 (図 1参照) で明確に見えるはずです。</li>
</ol>3. フルオレセイン造影7,8,9
<ol><li>ホルダー、マウスを取り外して、FA システム (例えば、ハイデルベルク Spectralis HRA2) の上に置きます。</li>
	<li>レーザーで焦点は、表示ウィンドウの中央に視神経の頭部と赤外モードを使用しての眼底部を燃やします。</li>
	<li>皮下や腹腔内 5% フルオレセイン ナトリウム塩 20 g のマウスのための 0.1 mL を注入します。</li>
	<li>脈絡膜レベルに焦点を当てます。</li>
	<li>脈絡膜のフォーカス レベルからイメージを取る。</li>
	<li>網膜のレベルを合わせるし、イメージを取る。</li>
	<li>30 s と繰り返し手順 3.4 3.6 を待ちます。</li>
	<li>マウスをホルダーから外し、加熱パッドの上に置きます。</li>
	<li>メデトミジン、アチパメゾール (0.5 mg/kg, i. p.)、または麻酔から動物の回復を待つのため α 2 拮抗薬による麻酔を逆にします。</li>
	<li> SD-10 月と FA イメージング麻酔下の動物 5、10、および 14 フォロー アップ日を繰り返します。</li>
</ol>4. CNV のグレーディング
<ol><li>グレード 10 月画像、次として 0 日目にレーザー加工後すぐに撮影した脈絡膜の FA 画像からブルッフ膜の損傷: 0 - ブルッフ膜が破損していません。 1 にブルッフ膜の成功した被害</li>
	<li>等級一連の次のように網膜の FA イメージの蛍光信号のダイナミクスを比較することによって観測された漏れがあった書込みのスポットから CNV の存在: 0 - 網膜の正常な外観 漏れの 0.5 - かすかな染 1.0 - 漏れの CNV 領域 注: は、OCT 画像における網膜流体の存在が CNV グレーディングお勧めしたい疑問 FA、CNV の確認を OCT イメージングを使用します。</li>
</ol>5. 網膜厚測定
<ol><li>網膜の厚さ測定の自動領域分割ソフトウェアを使用します。その合計の網膜厚は RPE (健康測定サイト)、または損傷 (書込みサイト) のサイトの周り RPE を結ぶ架空の線神経線維層からのすべての層の厚さとして考慮されることを確認 (図 7を参照)。</li>
</ol>

<ol>
	<li>Dobi, E. T., Puliafito, C. A., Destro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+experimental+choroidal+neovascularization+in+the+rat.">A new model of experimental choroidal neovascularization in the rat.</a> Arch. Ophthalmol. Chic. Ill 1960. 107, 264-269 (1989).</li><li>Tobe, T., 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=Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors.">Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.</a> Invest. Ophthalmol. Vis. Sci. 39, 180-188 (1998).</li><li>Seo, M. 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=Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor.">Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.</a> Am. J. Pathol. 154, 1743-1753 (1999).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Prog. Retin. Eye Res. 29, 500-519 (2010).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> J Vis Exp. (106), e53502 (2015).</li><li>Giani, 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=In+vivo+evaluation+of+laser-induced+choroidal+neovascularization+using+spectral-domain+optical+coherence+tomography.">In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.</a> Invest. Ophthalmol. Vis. Sci. 52, 3880-3887 (2011).</li><li>Gong, Y., 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=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PloS One. 10, e0132643 (2015).</li><li>Sheets, K. G., 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=Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse.">Neuroprotectin D1 attenuates laser-induced choroidal neovascularization in mouse.</a> Mol. Vis. 16, 320-329 (2010).</li><li>Hoerster, R., 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=In-vivo+and+ex-vivo+characterization+of+laser-induced+choroidal+neovascularization+variability+in+mice.">In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.</a> Graefes Arch. Clin. Exp. Ophthalmol. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 250, 1579-1586 (2012).</li><li>Sulaiman, R. 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=A+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization.">A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.</a> J. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. 31, 447-454 (2015).</li></ol>

著者 Symantas Ragauskas、博士は従業員 (研究員) と提供契約のこの記事で使用されている臨床の CNV モデルを用いた研究サービス Experimentica 会社の株主です。
エヴァ Kielczewski の著者は、この記事で使用される SD OCT システムを生み出すライカマイクロシス テムズの社員 (研究アプリケーション エンジニア、10 月) です。
著者ジョセフ ・ ヴァンスは従業員 (NA 10 月セールス ・ ディレクター) のこの記事で使用される SD OCT システムを生み出すライカマイクロシス テムズ。ジョセフ ・ ヴァンスは、また社長と目的と、LLC のマネージング ディレクターです。
著者サイモン カジャ、博士はコンサルタント最高科学責任者および前臨床契約研究機関提供受託研究サービス、この記事で使用される臨床 CNV モデル税込 Experimentica 株式会社の株主です。サイモン カジャ、博士も CEO の K & P 科学的な LLC は、コンサルティング会社、生命科学、博士ジョン p. とテレーズ E. マルケイ恵まれて教授ロヨラ大学シカゴ、Stritch 医学部眼科として提供しています。この整理の言葉が検討されており、その利益相反ポリシーに従ってロヨラ大学シカゴ校によって承認されました。
著者 Giedrius Kalesnykas 博士は従業員 (CEO) と提供契約のこの記事で使用されている臨床の CNV モデルを用いた研究サービス Experimentica 会社の株主です。

マルチ モーダル イメージングでは、CNV の病理学的評価のための貴重なツールを提供しています。FA から成るイメージング プロトコルの紹介、SD OCT と CNV 病理、再現可能な迅速かつ信頼性の高い評価のための自動セグメンテーション。ブルッフ膜レーザー管理が確認された後の休憩。また、この段階で SD 10 月の使用はまたの結果の解釈を混同するかもしれない可能な網膜と網膜出血の即時?...

齧歯動物で人間の CNV の病理学を模倣する最初の成功した試みはロング ・ ラット1クリプトン レーザーでほぼ三十年前を示した。その後、クリプトン レーザーは、最も人気のあるマウス緊張、c57bl/6 j2,3,4でブルッフ膜を破るに使用されました。FA と組織学的汚れ CNV 誘導の成功率を検証しました。OCT などの非侵襲的画像診断装置の急速な発展は、齧歯動物の前臨床モデルのフィールドの成長を促しています。大幅目と同じで複数の時点で網膜の形態変化を監視する機能は動物の使用の削減に寄与する、実験的研究の効率が向上します。CNV 病変の組織学的評価ではなく簡単で、レーザー管理、画像取得と画像解析ソフトを用いた面積/体積推定のサイトの周りの異常血管の増殖のラベリングを必要とします。対照的に、生体内のイメージ投射様相は、CNV の病理とその解釈のより複雑な分析をご紹介します。
ここで提示グレード誘導、進行、CNV の回帰してシンプルで比較的高速なメソッド FA、SD、OCT を使用して、マウスの自動抽出法レーザー誘起 CNV モデル。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">Medetomidine (commercial name Domitor)</td>
			<td style="width:123px;">Orion</td>
			<td style="width:344px;">Vnr 01 56 02</td>
			<td style="width:177px;">Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine</td>
			<td>Intervet</td>
			<td>Vnr 51 14 85</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0,9% NaCl</td>
			<td>B Braun</td>
			<td>357 0340</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylazine (commercial name Rompun vet)</td>
			<td>Bayer</td>
			<td>vnr 14 89 99</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tropicamide</td>
			<td>Santen</td>
			<td>Vnr 04 12 36</td>
			<td>Mydriatic agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Viscotears</td>
			<td>Alcon</td>
			<td>Vnr 44 54 81</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Systane</td>
			<td>Alcon</td>
			<td>&#160;-</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5% Fluorescein sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>F6377-100G</td>
			<td>Fluoresent agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Atipamezole (commercial name Antisedane)</td>
			<td>Orion</td>
			<td>Vnr 47 19 53</td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
</table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

レーザー誘発脈絡膜新生血管 (CNV) は、加齢に伴う黄斑変性症 (AMD) のウェット フォームを模倣する確立されたモデルです。このプロトコルでは血管新生のプロセス、マルチ モーダル縦から得ることができる強力な情報焦点を当てるをトリガーするレーザー誘起病変を生成の技術的な考察を通して単に読者を目指します生体内のフォロー アップ期間中イメージングします。
レーザー マウス CNV モデルは、ダイオード レーザーの管理によって生成されました。体内のマルチ モーダル イメージング技術は、CNV 誘導、進行と回帰を監視する使用されました。最初、ブルッフ膜の中断を確認するレーザー加工直後後スペクトル領域光干渉断層法 (SD 10 月) を行った。血管造影 (FA) を使用してその後体内イメージング脈絡膜レベルでシリアル画像からブルッフ膜の正常な損傷を確認しました。SD 10 月と FA の両方を使用して CNV の増殖と、レーザー加工後 5、10、および 14 日回帰の縦方向のフォロー アップを行った。シンプルで信頼性の高い FA 画像から漏れの CNV leasions の等級が表示されます。CNV のサイトで網膜の厚さの測定のための手動口径アプリケーションと組み合わせて、合計の網膜厚測定のための自動セグメンテーションは、浮腫の存在の公平な評価を許可します。最後に、脈絡膜の flatmounts に isolectin GS IB4 染色を使って CNV の組織学的検証を行います。どちらられる、染色は、ImageJ で isolectin 正面積が計算されます。
このプロトコルは、CNV の病理学のような高スループット スクリーニングを必要とすることができます高速、マルチ モーダル、治療研究と CNV 病理学および網膜浮腫の信頼性の高い分類に便利です。さらに、高解像度 SD OCT は網膜や網膜の流体の蓄積など、他の病理学的特徴の記録をできます。ただし、このメソッドは SD OCT 画像から手動で行う必要のあるボリュームの CNV 解析を自動化する可能性を行いません。

バブルやレーザー加工後すぐに出血は常に表示されません。したがって、SD 10 月は特にブルッフ膜の損傷を確認することが重要です。図 1は、異なる時点でレーザー投与後 OCT イメージングの例を示します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> 図 1<stro...

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In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

マウスのレーザー誘発脈絡膜新生血管の形態学的変化のフォロー アップの縦生体内イメージングの有用性を紹介します。

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

9.0K ビュー.  Experimentica Ltd.. マウスのレーザー誘発脈絡膜新生血管の形態学的変化のフォロー アップの縦生体内イメージングの有用性を紹介します。

ビデオ: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神経科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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

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

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

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

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

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

ex vivoでマウス神経上皮における単一細胞分裂のライブイメージング

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

感染ニューロンの3-Dイメージングと分析<em&gt;インビボ</em&gt;と<em&gt;トキソプラズマ</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

ラットの運動皮質におけるレーザー誘発脳損傷

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除されたマウス脛骨神経におけるニューロフィラメント輸送のイメージングと解析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

経瞳孔二光子マウス網膜のin vivoイメージング

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

生体内でマルチモーダルイメージングとマウスレーザー誘発脈絡膜新生血管モデルの解析