デラウェア州谷のライオンズ·アイバンクのサポートは、この仕事のための非常に貴重でした。 ICP8に対する一次マウスモノクローナル抗体は、デビッドKnipe（ハーバード大学医学部）から親切な贈り物だった。我々はまた、役立つ議論や専門知識のために博士スティーブンジェニングス（医学ドレクセル大学カレッジ）とピーターLaibson（ウィルズ眼研究所）に感謝。

全ての試薬および機器は滅菌購入したか、前の手順に滅菌した。この記事は、事前除核目で始まるex vivoでのモデルを設定する手順について説明します。私たちの実験では、若い（8-12週）アルビノウサギ（PEL-FREEZバイオ、ロジャーズ、アーカンソー州）からの新鮮な全体の眼球を使用しています。加えて、我々は地元のアイバンクから得られたヒト角膜を使用しています。摘出を自分で行う場合は、手順中に角膜または角膜輪部に損傷を与えないように注意してください。ウサギ摘出プロトコルは別の場所で2見つけることができます。 パート1：角膜強膜ボタンの切除<ol><li> PBSでガーゼの狭いストリップを濡らし、手順の間にそれを保持促進するために眼球のまわりでそれを包む。 </li><li>約0.5〜30cm離れ縁から、強膜に切開接線を作るために鋭いメスを使用してください。 </li><li>切開を延長する鋭いはさみを使用してCを完全に切除するorneoscleralボタン。 </li><li>角膜強膜ボタンが切り離されると、鉗子で強膜の縁を押しながら、絞りを離れていじめる。内皮の損傷や角膜に過度のストレスをかけることは避けてください。 </li></ol>パート2：器官型培養角膜への導入<ol><li>植した角膜は非必須アミノ酸（1X）、L-グルタミン（2mM）、​​ペニシリン（200単位/ ml）およびストレプトマイシン（200μg/ ml）のを補足した最小必須培地（MEM）中で培養される。必要に応じて、例えばアンホテリシンなどの抗真菌剤は、添加することもできる。 </li><li> 55℃で培養液中の1％アガロース溶液を調製し、それを準備完了状態に保つ</li><li>徹底的にペニシリン（200 U / ml）およびストレプトマイシン（200μg/ ml）を含む滅菌PBSで角膜をすすいでください。 </li><li>無菌スポットプレートのウェルに角膜上皮の側を下にして置きます。 </li><li>ちょうど十分に角膜内皮凹部にアガロース含有培地を追加完全にそれを埋める。アガロースのために〜1分が凝固することができます。 </li><li> 35mmの組織培養皿に支えるゲル足場上皮側で角膜を置き、上皮の表面を覆うように培養液を追加します。 </li><li>角膜は、媒体ごとに変更が48時間で、一週間以上もこのような方法で培養してもよい。 </li></ol>パート3：HSV-1の感染と実験薬による治療<ol><li> 35mmの組織培養皿にウイルスの所望の量を含有する培地を1 mlを加える。我々は、通常歪みKOS角膜あたりHSV-1の1×10 4 PFUで感染します。当社の株式価は、CV-1単層上でプラークアッセイを行うことにより決定されます。 </li><li>ウイルス含有培地中にアガロース足場上皮側と一緒に角膜を下に置きます。 </li><li>断続的な揺れ15分間隔で1時間感染させるために組織培養インキュベーター中に角膜を置きます。 </li><li> 1時間後、ウイル​​スcontaiを吸引媒体をNingと残留ウイルス粒子を除去するためにPBSで2-3回すすいでください。 </li><li>その前の位置に戻して角膜上皮を置き、表面を覆うように新鮮な培地を追加します。 </li><li>実験的な治療法は、実験の性質に応じて、感染前または後に起動されることがあります。 </li><li>培地は48時間毎に変更されています。 </li></ol>パート4：サンプルコレクション<ol class="lalpha"><li> プラークアッセイ分析のための媒体が。よく培地を混合し、-80℃でプラークアッセイまたはストアにすぐにそれを使う°Cの異なる時点で試料を採取した場合、後で使用するため。 </li><li> DNA、RNA、タンパク質、または角膜上皮由来の細胞 <ol><li>アガロース足場を取り除き、PBSでよく角膜をすすいでください。 </li><li>角膜上皮のみ材料が収集されることを保証するため、角膜から強膜切除する組織のリングを。 </li><li> Uメスを歌う、孤立しようとする材料の種類に応じて、適切なバッファに角膜上皮細胞を掻き。 DNAとRNAの単離のために、私たちはそれぞれDNeasyブラッド＆ティッシュキットとRNeasyミニキット（キアゲン、ヒルデン、ドイツ）を使用します。タンパク質溶解物の収集のために、私たちはLaemmli緩衝液を使用しています。角膜上皮細胞のフローサイトメトリー分析のために、私たちは細胞を緩め、メスでトリプシンにそれらを取り除くために、トリプシン少量の角膜をプレインキュベートする。組織の同量を得られない角膜上皮をこするので、サンプルを分析する場合に適切なコントロールを含めることが重要です。私達はウェスタンブロット用ローディングコントロールとしてqPCRsで参照遺伝子として定量RT-PCRを、GAPDHで基準転写物としての18S rRNAを使用し、ヌクレオリン。 </li></ol></li><li> 免疫組織化学または免疫蛍光のために組織サンプル <ol><li>アガロース足場を取り外して、トウモロコシをすすぐPBSでよくEA。 </li><li>映像に示すように、実験目的に応じて、、パラフィン組織ブロックまたは凍結組織ブロックを作るために角膜を使用しています。角膜組織のセクションを準備するときに我々は標準的な手順に従ってください。 </li></ol></li></ol>代表的な結果組織培養中の細胞を研究するための使用可能なメソッドのほとんどは、簡単に、HSV-1 のex vivoに感染して角膜での使用に適合することができます。ここに示されては、HSV-1感染症の研究に適用される最も一般的なテクニックのいくつかのための代表的な結果です-定量PCR（ 図1D）、定量RT-PCR（ 図1B）、ウエスタンブロット（ 図1F）、プラークアッセイ（ 図1C）、および組織免疫蛍光（ 図1E）。 <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="図1" /> 図1。 HSV-1 EX Vに感染して角膜の解析イヴォ。急性角膜上皮HSV-1感染の ex vivo での（a）モデルの概略図。（BF）このビデオ記事で概説された方法を使用して感染した角膜の解析。植したウサギ角膜株KOS HSV-1の1×10 4 PFU /角膜に感染して存在（PAA）またはホスホノ酢酸（400μg/ ml）を、HSVの複製の既知の阻害剤の非存在下（モック）で培養した（B）総RNAを示した時点で収集され、ウイルスの転写は、HSV-1ポリメラーゼのプライマーを用いて、定量RT-PCRによって評価した。 18Sに対してプライマーはrRNAをリファレンスとして使用した。 n = 3とする。エラーバーは±SEMを示す。（C）の培養液を48 HPIで収集され、感染性粒子の産生は、CV-1単層上のプラークアッセイにより評価した。（D）の全DNAを48 HPIで採取し、ウイルスゲノムの複製が評価したHSV-1のゲノムのプライマーを用いた定量PCRアッセイ。 PRGAPDHに対するimersを基準として使用した。 n = 3とする。エラーバーは、区分された、（E）は偽処理角膜を48 HPIで急速冷凍しました。値±SEMを示すものであり、感染した上皮内のウイルス初期蛋白（ICP8）の存在を間接免疫蛍光法により分析した。核はヘーヒスト33258で対比されています（F）タンパク質溶解物を48 HPIで採取し、ウイルスタンパク質（ICP0）の発現をウェスタンブロットにより評価した。 BCAアッセイは、サンプルの等しい負荷を確実にするために使用されていました。 HPI =時間後に感染。

<ol>
	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <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+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <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+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

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このビデオの記事では 、ex vivo モデルにおける急性角膜上皮HSV-1感染を研究するための方法を説明します。それは細胞培養結果の生理学的に正確な検証を行うことができ、それにより、行わなければならない動物実験の量を制限しているので、このモデルでは、in vivo モデルにおいて、in vitroおよびin間の有用な足がかりです。また、ex vivoのモデルは、無傷の<...

<table border="1"><tbody><tr><td> 試薬/消耗品 </td><td> 会社 </td><td> カタログ＃ </td></tr><tr><td>最小必須培地（MEM） </td><td>メディアテック</td><td> 10から010-CV </td></tr><tr><td>非必須アミノ酸（100倍液） </td><td>メディアテック</td><td> 25から025-CI </td></tr><tr><td> L-グルタミン</td><td>メディアテック</td><td> 25から005-CI </td></tr><tr><td>ペニシリンGナトリウム塩</td><td>シグマ</td><td> P3032 </td></tr><tr><td>ストレプトマイシン硫酸塩</td><td>シグマ</td><td> S9137 </td></tr><tr><td>アガロース</td><td>インビトロジェン</td><td> 15510-027 </td></tr><tr><td>リン酸緩衝生理食塩水（PBS） </td><td>ラボで準備</td><td> N / A </td></tr><tr><td>トリプシンEDTA（1X）&lt;/ TD&gt; <td>メディアテック</td><td> 25から053-CI </td></tr><tr><td> DNeasyブラッド＆ティッシュキット</td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Miniキット</td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Laemmli緩衝液</td><td>ラボで準備</td><td> N / A </td></tr><tr><td>最適切断温度（OCT）化合物</td><td>組織 - Tek社</td><td> 4583 </td></tr><tr><td>全体ウサギの眼（ヤング） </td><td> PEL-FREEZ </td><td> 41211から2 </td></tr><tr><td>ヒト角膜および全アイズ</td><td>地元のアイバンク</td><td> N / A </td></tr><tr><td> 12まあ磁器スポットプレート</td><td>アボガドロラボサプライ</td><td> 1877 </td></tr><tr><td> 35mmの組織培養皿</td><td>ファルコン</td><td> 353001 </td></tr><tr><td>まあ6細胞培養プレート</td><td>グライナーバイオワン</td><td> 657160 </td></tr><tr><td> Cryomold中級</td><td>組織 - Tek社</td><td> 4566 </td></tr></tbody></table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

ヘルペス性角膜炎は、単純ヘルペスウイルス1型（HSV-1）に関連付けられている最も深刻な病態の一つである。ヘルペス性角膜炎は、現在、先進国の両方の角膜由来と感染関連失明の主要な原因である。ヘルペス性角膜炎の典型的なプレゼンテーションでは、瘢痕のような恒久的な角膜の病理につながる間伐、不透明度1、角膜上皮の感染、時には深い角膜実質と内皮細胞が含まれています。 角膜のHSV-1感染は、伝統的に実験モデルの2つのタイプに研究されている。角膜上皮細胞の単層培養をペトリ皿に感染されているin vitroモデルで 、シンプルさ、再現性の高いレベル、高速実験、比較的低コストを提供しています。一方、このようなウサギやマウスなどの動物が角膜に直接接種されているin vivoモデルで 、高度に洗練された生理を提供していますシステムが、コストが高く、長い実験では、必要に応じて動物の世話、及び変動の大きい程度を持っています。 このビデオの記事では、我々 のin vitroおよび in vivoモデルの両方の長所を組み合わせた角膜上皮のHSV-1感染症の新しいex vivoでモデルの詳細なデモンストレーションを提供する。 ex vivoのモデルはorganotypically培養で維持とHSV-1に感染した無傷の角膜を利用しています。 ex vivoでのモデルの使用は非常に生理的に基づく結論を可能にし、まだそれはむしろ安価で、in vitroモデルに匹敵する時間のコミットメントを必要とします。

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

このビデオの記事では、我々は新しいの使用を記述<em&gt; ex vivoで</em急性ヘルペスの&gt;モデルは、ウイルスI型角膜上皮感染を単純。

急性上皮単純ヘルペスウイルスI型感染の ex vivo器官角膜モデル

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

ex-vivo-organotypic-corneal-model-acute-epithelial-herpes-simplex

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Neuroscience

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

12.6K ビュー.  Drexel University College of Medicine. このビデオの記事では、我々は新しいの使用を記述 ex vivoでモデルは、ウイルスI型角膜上皮感染を単純。

ビデオ: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

全体の開発、 ショウジョウバエの脳のex vivoで培養

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

神経科学

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

無傷の鋤鼻器官と副嗅球のex vivoでの準備

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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 ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脈絡叢上皮細胞の繊毛運動の観測<em&gt;エクスビボ</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶媒クリア脳組織における狂犬病ウイルス感染の高解像度3Dイメージング

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

その場で 急性 エクスビボ 胚性脳スライス培養における軸索成長および成長円錐ダイナミクスの可視化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

ヒト幹細胞移植および検証のための ex vivo モデルとしての成体ヒト皮質の有機型培養

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

AMD研究のためのヒトドナー眼のOCTベースマルチモーダルイメージング

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

著者スポットライト:加齢性黄斑変性症の研究のためのヒトドナー眼のEx Vivo OCTベースのマルチモーダルイメージング  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

エクスビボ 加齢黄斑変性症研究のためのヒトドナー眼のOCTを用いたマルチモーダルイメージング

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...