この作品は、NIHの助成金EY 012031（ET -）、FM星のカービィ財団（ET -）、失明、株式会社（MAZ）、ニュージャージー州ライオンズアイ研究財団（MAZ）を、防止するための研究から、賞によって支えられてニュージャージー州（MAZ）、ジョセフJ.とマルグリットDiSepio網膜研究基金（MAZ）、そしてジャニスミッチェルヴァッサーとアシュビーミッチェルフェローシップ（AMK）の眼科研究所。

1。細胞培養インサートのためのカスタムメイドのスタンドを作る 5ミリリットルピペットチップ（ISC BioExpress、＃P - 3250 - 19）のベースは完全に10 mmの底部のサイズ（12.6ミリメートル、外径）（内径）NUNCセルに一致する13ミリメートル、内径を持っていますカルチャーインサート（8μmの、ポリカーボネート膜、熱、＃137443）。スタンドを作るために、ピペットを6 mm長中空シリンダーを生産する基地の近くで切断されました。その後、作品は約5 mmでインサートの高さを上げリング（高さ2mm）、で3足（高さ4 mm）を作成するために火炎加熱ブレードを使用してシリンダーの側面から削除されました。 2。ブタ網膜の添付ファイルと文化のための滅菌濾紙の調製ワットマン4Mフィルターペーパー（カタログ番号1004）（のような殺菌のためにまたは一度に網膜が添付されたガラス管にろ紙を移動するために使用されていた小さな、三角形のハンドルと円形（直径6.3 mm）に切断し、 ）ビデオに示す。 3。網膜外植片の調製 5〜アイズ - 9ヶ月歳まで、150〜230ポンド、アメリカのヨークシャー豚は核出の3時間以内に現地屠殺場（氷の上で輸送）から得た。到着時に、目は、余分な組織を洗浄した（10％ポビドンヨード、パデューフレデリック社、スタンフォード、コネチカット州）betadine溶液に浸漬し、ダルベッコ改変イーグル培地（1グラム/ Lグルコース含有DMEM、L -グルタミン、で2回洗浄しとピルビン酸ナトリウム、Cellgro - mediatech社、マナッセス、VAは）2.5μg/ mlのアムホテリシンB（ギブコ- Invitrogen社、カールスバッド、カリフォルニア州）を補充した。前のセグメントは、神経網膜を露出、削除されました。六ミリトレフィンブレード（ストルツ眼科 - ボシュロム、マンチェスター、MO）は赤道、全層後部セグメントの外植片を切断するために使用された。網膜は、その後、光受容体を上にして、神経網膜上に持ち上げ、そして文化のインサートに接続されている網膜にろ紙を置き、ゆっくりと神経節細胞層の上に、乾燥滅菌ろ紙片を適用することにより、剥離した。インサートは、12穴培養皿（フィッシャー）で、完全に網膜をカバーすることを確認しながら、培養液中に置かれた。複数の外植片は、日常的に得られ、単一の目から培養した。 4。組織の文化網膜組織は、0.2 mg / mlのグルタミン、10μg/ mlのブタのインシュリン、1m​​Mのピルビン酸、0.1を添加したイーグル最小必須培地（MEM、Invitrogen社、ギブコ-ライフテクノロジーズ社、ロックビル、MD）、pH7.4の、で培養したmMのL -アスコルビン酸、100 U / mlペニシリン、100μg/ mlのストレプトマイシン、および250 ng / mlのアンホテリシンB（抗真菌抗生物質：シグマ、セントルイス、MO）、および加湿5％CO 2、バランスの空気を通気、 37℃培地は2日毎に交換した。 組織は、使用する試薬またはプローブのさまざまな治療、またはその後の生化学的または形態学的解析のために未処理のままにすることができます。以下のセクションでは、一貫性のある全層網膜の断面を作成する手順を説明します。 5。セクショニング<ol><li>クライオスタットを使用する<ol><li> 4℃で一晩4％parafomaldehydeの網膜組織を修復° C; </li><li>リン酸緩衝生理食塩水（PBS）で簡単に組織をすすいでください。 </li><li>それは解剖顕微鏡を使って35mmのディッシュにPBSにあるときに慎重にろ紙から組織を取り除く。 </li><li> 30％ショ糖に組織を転送し、4℃で一晩おく° C; </li><li>離れて吸引組織の周り余分な溶液を、組織をカバーし、それが組織に浸透するために、室温で2時間浸漬させるのに十分な組織- Tek社の培地（OCTコンパウンド4583）を追加します。 </li><li>歯科用ワックスと正方形の枠（10ミリメートル× 10ミリメートル、5mmの高さ）を構築し、組織の周囲に配置します。冷凍庫のフレーム（組織サンプルが平らであることを確認してください）​​と場所を埋めるために、より組織- Tek社の培地を追加する（ - 20 ° C）少なくとも1時間。 </li><li>サンプルのプラットフォームに凍結サンプルのブロックを転送する（サンプルブロックがプラットフォームに垂直であることを確認してください）​​。 </li><li>あらかじめ冷却クライオスタット内のサンプルプラットフォームをマウントし（-20℃、ライカ、CM1900）とブロックがブレードに垂直であることを確認してください。 </li><li>所望の厚さの組織を切片前にワックスとフレームのブロックを削除します。 </li><li>ゼラチン処理スライド上のセクションを置きます。 </li></ol></li><li>ビブラトームの使い方<ol><li>クライオスタットのために上記のような組織を固定し、すすぎ; </li><li>小さな容器で予備加熱、溶融4％寒天にティッシュを置き、完全に寒天は固まるまで4℃で保つ; </li><li>内部の組織サンプルの小さなブロックに寒天をトリミングする鋭い刃を使用します。 </li><li>クレイジー接着剤でビブラトームサンプルホルダー（組織サンプルの部分が目のプラットフォームに対して垂直であることを確認する上で接着剤サンプルブロックeのサンプルホルダー、すなわち、垂直翼に）と完全に冷たい水に浸し、サンプルブロックとビブラトーム切片システム（ライカ、シリーズ1000）のホルダーをマウントします。 </li><li> 50μm以下のセクションにサンプルブロックをカットし、柔らかいブラシでゼラチン処理したスライド上に置きます。 </li></ol></li></ol> 6。免疫組織化学任意の標準プロトコルを使用してください。免疫標識厚い切片は共焦点顕微鏡で見ることができます。 7。代表的な結果： 形態は文化の7日間の期間中保持された。培養前後の網膜の画像は、ビデオでご利用いただけます。

<ol>
	<li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest Ophthalmol Vis Sci. 49, 3503-3512 (2008).</li><li>Kretz, A., Marticke, J. K., Happold, C. J., Schmeer, C., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+technique+of+adult+retina+for+regeneration+studies+on+adult+CNS+neurons.">A primary culture technique of adult retina for regeneration studies on adult CNS neurons.</a> Nat Protoc. 2, 131-140 (2007).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Koizumi, A., Zeck, G., Ben, Y., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+physiologically+functional+adult+mammalian+retinas.">Organotypic culture of physiologically functional adult mammalian retinas.</a> PLoS One. 2, e221-e221 (2007).</li><li>Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+adult+rabbit+retina.">Organotypic culture of adult rabbit retina.</a> J Vis Exp. , (2007).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> J Neurosci. 12, 840-853 (1992).</li><li>Kobuch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+adult+porcine+retina+and+retinal+pigment+epithelium+in+perfusion+culture:+characterisation+of+an+organotypic+in+vitro+model.">Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.</a> Exp Eye Res. 86, 661-668 (2008).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> J Neurosci Methods. 173, 47-58 (2008).</li><li>Guduric-Fuchs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+pig+retinal+development.">Immunohistochemical study of pig retinal development.</a> Mol Vis. 15, 1915-1928 (2009).</li><li>Chandler, M. J., Smith, P. J., Samuelson, D. A., MacKay, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+density+of+the+domestic+pig+retina.">Photoreceptor density of the domestic pig retina.</a> Vet Ophthalmol. 2, 179-184 (1999).</li></ol>

利害の衝突は宣言されません。

ビジョンの研究者は、網膜幹細胞移植1、網膜再生2、および外来遺伝子発現3月5日などの問題、様々な勉強する器官系、を含む、網膜培養システムのさまざまなを確立している。しかし、成体哺乳類の網膜は、in vitroで光受容体6の高エネルギーの需要が主な要因を維持することは困難です。小泉らは、光受容体のための酸素供給が培養インサート?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> MEM </td><td>インビトロジェン-ギブコ</td><td> 10370-021 </td><td></td></tr><tr><td> 5 mLのピペットチップ</td><td> ISC BioExpress </td><td> P - 3250から19 </td><td></td></tr><tr><td> NUNCの細胞培養インサート</td><td>熱</td><td> 137443 </td><td> 8μm、ポリカーボネート</td></tr><tr><td>ワットマン4Mフィルターペーパー</td><td>ワットマン紙</td><td> 1004 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro - mediatech株式会社</td><td> 10から013 - CV </td><td></td></tr><tr><td>寒天</td><td>フルカ</td><td> 05040 </td><td></td></tr><tr><td> FungizoneアムホテリシンB </td><td>インビトロジェン-ギブコ</td><td> 15290018 </td><td></td></tr><tr><td>トレフィンブレード</td><td>ボシュロム</td><td> T3096 </td><td> 6.00ミリメートル</td></tr><tr><td> OCTコンパウンド</td><td>電子顕微鏡学</td><td> 62550〜01 </td><td> 4583 </td></tr><tr><td></td><td>科学</td><td></td><td></td></tr><tr><td>クライオスタット</td><td>ライカ</td><td> CM1900 </td><td></td></tr><tr><td>ビブラトーム</td><td>ライカ</td><td>シリーズ1000 </td><td></td></tr><tr><td>共焦点顕微鏡。 </td><td>カールツァイス</td><td> LSM510 </td><td></td></tr><tr><td>反シナプス小胞タンパク質2（SV2）マウスモノクローナル抗体</td><td> DSHB </td><td> N / </td><td></td></tr><tr><td>抗グリア線維性酸性タンパク質（GFAP）ポリクローナルウサギ抗体</td><td> DAKO </td><td> DAKO </td><td></td></tr><tr><td>ヨウ化プロピジウム（PI） </td><td>シグマ</td><td> P4170 </td><td></td></tr></tbody></table>

organotypic culture of full-thickness adult porcine retina

動物実験を取り替えるか、または減らすことができるin vitroモデルのための認識が求められています。ブタの網膜は、人間の網膜に似た神経細胞の構造を有し、したがって、人間の網膜損傷や変性疾患のメカニズムを研究するための貴重な種です。ここでは、食肉処理場から得られる目から分離された大人のブタの網膜の器官培養のための費用対効果の高い手法を説明します。前眼部を除去した後、トレパンのブレードは、大人のブタの眼の後部から複数の神経網膜-ブルッフの膜RPE -脈絡-強膜の外植片を作成するために使用されました。滅菌ろ紙片は、それぞれの外植片から神経網膜をリフトオフ使用されていました。ろ紙 - 網膜の複合体は、カスタムメイドのスタンドで培養皿の底部から離れて開催された挿入、上に（最大光受容体の側）培養した。スタンドは、網膜の両側に培地の良い循環が可能になります。全体として、この手順では再現性、シンプルであり、そして少なくとも7日間にネイティブ網膜構造の保存を許可し、それ哺乳類網膜上に、形態学的薬理学的、および生化学的研究の様々な有用なモデルとなっています。

Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

Organotypic Culture of Full-thickness Adult Porcine Retina

ここでは、七日のための大人のブタの網膜の器官培養のための費用対効果の高い手法を説明します。簡単に言えば、滅菌濾紙は、カスタムメイドのスタンドで発生したインサートにRPEと場所光受容体の側から神経網膜をリフトオフ使用されていました。

全層の大人のブタの網膜の器官培養

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

organotypic-culture-of-full-thickness-adult-porcine-retina

Research

JoVE Journal

Neuroscience

14.9K ビュー.  University of Medicine and Dentistry of New Jersey - UMDNJ. ここでは、七日のための大人のブタの網膜の器官培養のための費用対効果の高い手法を説明します。簡単に言えば、滅菌濾紙は、カスタムメイドのスタンドで発生したインサートにRPEと場所光受容体の側から神経網膜をリフトオフ使用されていました。

ビデオ: Organotypic Culture of Full-thickness Adult Porcine Retina

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神経科学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

末梢神経突起伸長のリアルタイムイメージングのためのGFP発現マウス胚の器官型スライス培養

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング<em&gt;ウテロの</em&gt;エレクトロポレーション

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

成人ラット尿膀胱からの一次ニューロンとグリアの分離と培養

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

成人大草原の神経起源ニッチ由来の神経球の培養

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...