この研究は、ヴァンダービルト - イングラムがんセンターサポート助成金（P30 CA068485）、小児脳腫瘍財団と米国立衛生研究所（NS042205）からの補助金によって支えられている。

1。担癌小脳および腫瘍組織の解離の顕微解剖<ol><li> 腫瘍組織の検索 <ol><li>髄芽腫をもつ病気のマウスは、しばしばrunted、および水頭症と後部麻痺と覆さときに姿勢を取り戻すために、障害を含む典型的な神経症状を、表示されています。腫瘍組織を取得するには、二酸化炭素の吸入によりマウスを安楽死させる。それは頚椎脱臼、後頭骨に圧力を発生し、腫瘍組織の整合性が損なわれる可能性が手順を実行しないようimporantです。 </li><li>断頭は、頭蓋骨の良い可視化のために髪と筋肉組織を可能な限り除去し、はさみを使って死の直後に実行されます。 95％エタノールを浸したキムワイプで頭蓋骨の表面を清掃してください。 </li><li>頭蓋骨の正中線に沿って開口部を切断し、その時点で腫瘍のある小脳を含めた脳全体が露出している、細かいピンセットを使って頭蓋骨の組織を除去するために微細なハサミを使用してください。 </li><li>健康な成人の小脳は、明確に定義された半球と虫部を表示しながら、担癌マウスの小脳では、多​​くの場合、滑らかな表面と顕著な血管を持つアモルファス拡大です。無菌技術を用いて、緩衝生理食塩水氷冷リン酸塩でピンセットと場所を使用して、小脳の腫瘍を取り出す（PBS）のMg 2なし+及びCa 2 +。 </li></ol></li></ol>注：すべての測定器は使用前にオートクレーブ処理95％エタノールと水蒸気で消毒されています。 <ol start="2"><li> 腫瘍組織の解離 <ol><li> PBSから腫瘍組織の約4倍の体積が50％のAccutase（PBSで希釈）に腫瘍組織を転送、37℃でインキュベーションに続いて、室温で3分間細かいハサミで組織をミンチ℃で4分間、そのあと組織は、さらに3分間1 mLのPipetmanで反復的なペッティングを受ける。このメソッドは、単一細胞と小さな細胞の集合体の混合物を生成する必要があります。 </li><li> PBSと1000xgでペレットに5分間遠心し、細胞と細胞懸濁液3倍に希釈する。 （上記の手順は、Joveのはマスコミで、&quot;髄芽腫の幹細胞の分離、濃縮とメンテナンス&quot;に記載されている） </li></ol></li></ol>グルタミン、ペニシリン - ストレプトマイシン、N2、B27、ヒトEGF（25 ng / ml）および塩基性FGF（25 ng / mL）をしてNeurobasal媒質で構成される作りたての神経幹細胞の培地中で細胞ペレットを再懸濁します。ソリューションの細胞密度を計算すると1つの滅菌ベベル先端10μLシリンジに、解離の腫瘍細胞の適切な数、例えば5 × 5と溶液4μLをロードできるように追加の神経幹細胞の培地に適切な更なる希釈を行う。接種する細胞の正確な数は、特定の実験的な要件に応じて研究者によって決定されるべきである。氷の上にミニ200μLマイクロ遠心チューブ（PCRチューブ）の細胞溶液を保管してください。 2。レシピエントマウスのための麻酔の手順外科手術の場合は、齧歯類の手術専用の消毒領域はプロシージャの実行中に必要です。理想的には、動物の準備、営業分野と動物の回復のため、独立しているが、隣接地域で長いベンチを設定。手術用手袋ではなく、試験の手袋は、手術のために必要とされています。無菌操作は、腰の上に手袋をはめた手を保持することにより維持し、乾燥した無菌面から無菌オブジェクトを処理するためにのみ使用されます。 <ol><li>ケタミン（マウスの体重1kg当たり100mgのケタミン）とキシラジン（マウスの体重1kg当たり10mgのキシラジン）の混合物を麻酔に使用されます。麻酔薬の腹腔内に投与する。 </li><li>それは一般的に麻酔薬が完全に有効にするために、3〜5分かかります。マウスがつま先/テールピンチで完全に麻酔があるかどうか判別します。マウスの眼に眼軟膏の少量を置きます。常にマウスが正常に呼吸していることを確認する監視、動作領域に完全に麻酔マウスをもたらす。 </li></ol> 3。腫瘍細胞のグラフト頭蓋内<ol><li>中脳と小脳の上に後部頭蓋骨を明らかにするために、バリカンを使用して、マウスの背側後部頭部を剃る。 </li><li>アルコールパッドで拭いてその後に綿の先端のアプリケーターを、使用して公開頭皮上Betadineソリューションを適用します。これら二つの殺菌のステップを2回繰り返します。 </li><li>滅菌メスを用いて後方頭皮の4分の1インチの切開を加えます。右〜0.5mm穿頭孔2mmと滅菌歯科用ドリルを使用してラムダに後方2 mmのドリル。 </li><li>フレームのホールドにその歯をフックすることで定位のフレームの上にマウスを置きます。慎重に下るとバリ穴への腫瘍細胞の溶液4μLをロードベベル先端が10μLシリンジを置きます。 syriのベベル一度ゲイン誤差の針は、頭蓋骨の表面の下にある、別の3 mmの下降と、0.5 mmの登る。ゆっくりと受信者の小脳に、30秒のタイムフレームで安定した力で、腫瘍細胞の所望量を注入する。追加の2分間の注入が完了した後にその場所に針を残す。その後、注射針とシリンジを取り外します。 autoclipを使用して傷をクリップ。 </li><li>その体温を維持するために手術後の温暖化の毛布の上にマウスをおいてください。モビリティと呼吸パターンが連続して観察される。マウスは、麻酔から回復すれば、滅菌住宅のケージに戻して置きます。食料と水の摂取量、行動、毛づくろい、そして目の赤の染色のために毎日監視する。切開部位での感染の兆候を検査する。 7日後の手術でオートクリップを取り外します。 2-6ヶ月の平均のためのマウスを維持し、さらなる研究のために典型的な髄芽腫の症状を表示し、それらを生け贄に捧げる。 </li></ol> 4。代表的な結果レシピエントマウスで開発された二次髄芽腫は、非常に原発性腫瘍のそれと類似していた。全体のマウントとして見たとき、二次腫瘍を有する小脳は、しばしば明白な異所性血管によるアモルファス、拡大された。二次腫瘍組織の免疫組織化学的解析は、腫瘍細胞は、発現する強力なSmoM2 - YFPなどMath1のとPax6などの小脳顆粒神経前駆細胞のマーカー非常に増殖していることを明らかにした。解離と報告された方法を（&quot;髄芽腫の幹細胞の分離、濃縮とメンテナンス&quot;、印刷中のJove）を使用して培養、二次髄芽腫の腫瘍細胞が急速に拡大することができるときは、速達、複数の幹細胞マーカー、クローン原性であり、さらに、腫瘍形成を開始することができます。追加の免疫不全レシピエントに移植。 <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="図1" /> 図1。典型的なホールマウントおよび二次髄芽腫の組織の断面 二次髄芽腫の組織の典型的な例は、5 × 10 5プライマリ腫瘍細胞の注射後26日間に開発。ヘマトキシリン​​染色は、高い核/細胞質比および核の多型を含む、髄芽腫の典型的な細胞形態を、明らかに。 Ki67の発現によって示されるようにこれらの二次性腫瘍細胞は高度に増殖され、彼らはまた、確実にSmoM2 - YFP膜発現する。 <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="図2" /> 図2。二次髄芽腫の細胞を培養し、 試験管内で展開することができる 初代マウス髄芽腫細胞（&quot;分離、濃縮と髄芽腫の幹細胞の維持&quot;、印刷中のJove）のために記述されたプロトコルを使用して、これらの二次髄芽腫の細胞を培養し、複数の通路を拡張することができます。写真は4日と14日で培養同じ代表の腫瘍のコロニーを示しています。培養腫瘍細胞は、わずかな細胞質と細胞の集合体の高密度コアから頻繁に放射すると伸長バイポーラ、です。彼らは成長の接触阻害を示さない。小さな丸い細胞が赤血球です。

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

利害の衝突は宣言されません。

その収量二次腫瘍の形成をallografting成功した髄芽腫の細胞を確保するためにいくつかの重要な要因は以下の通りである：まず、組織解離のためのわずか50％のAccutaseを使用し、長時間の酵素処理は細胞の生存率の大幅な削減につながるとして、組織を過剰に消化しないでください。小さなヒントでも解離細胞への物理的な損傷を生成することができるとしても、わずか1 mLサイズのPipetmanを使用...

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<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
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<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
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<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
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<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
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<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
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<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50% </td>
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<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
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<td>Foredom flexible shaft drill, hang-up style</td>
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<td>Stoelting </td>
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<td>10 &mu;L syringe, 26 ga., bevel tip</td>
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<td>Stoelting </td>
<td>51105</td>
<td>&nbsp;</td>
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<td>Drill bit .75mm</td>
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<td>Stoelting</td>
<td>51455-3</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

髄芽腫は神経系の最も一般的な小児腫瘍です。動物実験の大きな体は、髄芽腫1-4の細胞の起源としての小脳顆粒神経前駆細胞（CGNPs）に焦点を当てている。しかし、ヒトの患者（、結節性線維形成性、古典的および大細胞/未分化）の髄芽腫のサブタイプの多様な臨床症状、および髄芽腫はCGNPマーカー5のない異所性発現と人間の患者のサブセットで発見されているという事実は、ことを示唆している髄芽腫の細胞および分子の起源は、より複雑で、はるかに完全に解読されているからです。したがって、それはどの細胞型に特異的な治療法を開発することができますに基づいて、別の髄芽腫の腫瘍細胞の起源があるかどうかを判断するために不可欠です。この目的のために、受信者の二次腫瘍の開発のその後の分析に続いて遺伝的にマークされた腫瘍細胞のタイプのallografting頭蓋内同所では、腫瘍開始細胞の細胞起源を決定することができるようになります。ここでは、頭蓋内原発腫瘍組織に由来する髄芽腫細胞のallografting同所性、および、この手順のための実験プロトコルを記述するも、確立された細胞株からの細胞を移植するために使用することができます。

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

このプロトコルは、二次髄芽腫を開始するために、マウスの髄芽腫の組織の分離と解離を説明し、免疫不全レシピエントマウスに腫瘍細胞のallograftingそれに続く。

免疫不全マウスにおける髄芽腫細胞の頭蓋内同所Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

Research

JoVE Journal

Neuroscience

12.1K ビュー.  Vanderbilt University. このプロトコルは、二次髄芽腫を開始するために、マウスの髄芽腫の組織の分離と解離を説明し、免疫不全レシピエントマウスに腫瘍細胞のallograftingそれに続く。

ビデオ: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

髄芽腫の幹細胞の単離、濃縮、およびメンテナンス

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

神経科学

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

アデノ随伴ウイルスベクターの頭蓋内注射

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

頭蓋内薬物療法と痛みの試金の齧歯動物

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

マウスの神経幹細胞と脳における5-ヒドロキシメチルシトシンの検出

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

マウスにおけるインテコール内合成タンパク質の定量測定

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

マウスにおけるコフィリンを標的とするアデノ関連ウイルスを用いて操作した大脳皮質領域の電気コルチコチコ

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.