We thank Ellen Ytebrouck for her valuable information regarding method application in rat. This work was supported by the Research Council of the KU Leuven (OT09/22) and the Fund for Scientific Research-Flanders (FWO Vlaanderen). Jeroen Aerts is supported by a Ph.D. fellowship of the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen) and Julie Nys by a Ph.D. grant from FWO Flanders.

全ての実験は、KUルーベンの倫理調査委員会により承認され、2010年9月22日の欧州共同体理事会指令（63分の2010 / EU）に厳密に従ってとベルギーの法律（2013年5月29日のKB）であった。あらゆる努力は、動物の苦痛を最小限にし、動物の数を減らすために行われた。 1動物の治療と麻酔<ol><li>塩酸ケタミン（75 mg / ml）を生理食塩水でメデトミジン塩酸塩（1mg / kg）の混合物の腹腔内注射でマウスを麻酔。 </li><li>マウスが完全に鎮静されている保証するために鉗子でつま先をつまんで反射神経をチェックしてください。 </li><li>まぶたや綿棒を使って、目の周囲の領域を消毒するために70％エタノールを適用します。さらに鎮静の程度を評価するためにまぶた反射を確認してください。 </li></ol> 2アイの削除<ol><li>乾いた、動物が平らに常駐していることを確認して、滑らかな表面。 </li><li>湾曲した、鋸歯状の先端部（：0.5×0.4ミリメートル優先チップサイズ）鉗子を滅菌する。 </li><li>静かに眼球をソケットから変位して視神経が到達可能であるされるまで、鉗子で眼角（目の角）に押してください。 </li><li>目の後ろの鉗子ガイド。押しては、好ましくは、曲線の始点と鉗子のではない、非常に先端で、しっかりと視神経を開催しています。これはソケットから地球を持ち上げるようにし、完全な視神経をクランプするのに役立ちます。 </li><li>マウスは、平らな面にしたまま手が最も抵抗のある方に鉗子を保持した円の動きを作る。マウスは、手の動きの方向に応じて表面に沿ってスイングします。 </li><li>視神経が二つに絞られるまで漸増速度でこのアクションを実行します（通常は7〜15円の動きとの間に、約半分毎秒1フルターンまで）。したがって、デタッチ目玉です削除しました。 </li></ol> 3。術後ケア<ol><li> （まれ）出血の場合には、粘性の凝固および止血剤で軌道を埋める。 </li><li>腹腔内に生理食塩水にatipamezol塩酸塩を1mg / kgで注入して麻酔を元に戻します。 </li><li>痛みを和らげるために腹腔内に24時間毎メロキシカムの1mg / kgのを管理します。 </li><li>角膜の脱水を防ぐために、残りの眼に眼軟膏を適用します。 </li><li>動物は、加熱プレート上回復または体温を制御するために個別のケージに絶縁材料で動物をラップしてみよう。 </li><li>最低2日間毎日マウスの体重を測定します。重量の損失は、苦痛を示し、動物が完全に回復するまで、この場合には、メロキシカムの治療を継続することができる。 </li></ol>

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The authors have nothing to disclose.

私たちの方法に従って成功した除核を行うためには、考慮すべき最も重要なステップは、1）適切なサイズの湾曲や鋸歯状の先端を有するピンセットを用いて; 2）滑らかで乾燥した表面上の摘出を実施する。 3）徐々少なくとも摩擦方向に円の動きをスピードアップ。 効果的な結果のためには、湾曲した鋸歯状の先端部（;：2.15のx 1.3ミリメートル：マウス、ラットのx 0.4ミリメートル0.5優先先端サイズ）を特徴とする、適切な鉗子を使用することが不可欠である。曲率は眼球変位後視神経への容易なアクセスを可能にし、円運動を行う際に、正しい手の配置が必要である。視神経を保持しているときに、必要なグリップを欠いているので、スムーズなヒントが推奨されていません。円の動きの間、適切に視神経を保持しないと、眼動脈、眼の貧しい離脱ひいては貧困再現性の破壊をもたらす。そのためには、 生体内での方法を適用した後、最大の動物福祉を保証するために、取り扱い鉗子の最適化のために安楽死させた動物に最初の練習にこの手法をお勧めします。成功した眼の摘出は、最近、手動で動物体を旋回し、鉗子を静止状態に保つことを除いて同じ手法を用いて研究室でラットにおいて行われた。 技術の一つの制限は、おそらく網膜を損傷する可能性がある。したがって、この方法は、組織学5を実行するために網膜を収集するにはあまり適していない。眼球がまぶたを削除するか、切断することなく、ソケットから変位される必要があるので、私たちの方法は、年齢を開いて目を投稿する制限されています。 げっ歯類を含む異なる種における眼の摘出は、日常的に、多くの場合、まぶたの除去を必要と視神経18,23-25 ​​を切断する別の方法を用いて行われる。これらのメトdsはより侵襲的である傾向があり、ここに記載の技術よりも高い学習曲線を持っている。取り外しやまぶたを縫合する必要性がなければ、手術後の回復時間は、より高い動物福祉と、より再現性のある結果をもたらし、最小化される。

目を削除し、それにより不可逆的感覚受容体表面（網膜）を破壊、視覚路に沿って感覚入力のかなりの損失を課す。若年及び成体視覚系における摘出モデルは、異なる視覚中心1-4の発達、可塑性および機能を理解する上で貴重であることが証明された。この感覚遮断の、分子細胞および生理学的結果は、皮質回路が対処し、経験ではそのような大規模な変更に対応して、その構造と機能をどのように変化するか正常な発達調節されているとどのように確立された洞察を提供することができます。 視覚的剥奪の異なる方法が存在し、それらはすべて、ビジョン関連の研究では、それらの具体的な利点があります。例えば、暗い飼育は、具体的にはまだそれが自発的な網膜活性に影響を与えない、視覚的に駆動アクティビティを排除します。同様に、蓋縫合糸またはアイパッチは、パターン化された映像設備を削除L入力自発活動を妨害することなく、彼らは、閉じた目を通して分散光の透過を可能にする。これらの方法は、可逆的であり、開発6-8中に皮質回路を彫刻における眼の入力のパターン化視覚および低レベルの相関の役割を理解する上で貴重であることが示されている。それは視神経9,10を構成網膜神経節細胞の入力の進行性の喪失を確立するため、緑内障の研究では、成体動物における視神経挫滅モデルが広く用いられている。目的は不可逆的に一度に自発的でパターン化された両方のビジョンを削除するときに、目とは、このように、網膜が完全に瞬時に取り除かれる一方、脱核、上、剥奪の適切な選択である。また、活動マッピング研究11,12における信号対雑音比を向上させることができる強固な眼内活動の不均衡を誘発する。目に摘出に応答して、機能的および構造的変化を比較する大証、例えば蓋縫合糸としてはあまり思い切った方法によって、剥奪した後、また、可塑性の恒常性とシナプス型の両方の自発網膜活動の役割に新たな洞察を公開する可能性があります。 除核は、直接網膜標的における栄養の影響の損失をトリガします。例えば、BDNFレベルが有意に外側膝状核（LGN）と成人の除核したラット13の上丘で下方制御されている。構造的リモデリングを媒介するメッセンジャー分子として機能する反応性酸素種もまた、成体ラットの視覚システム14の皮質下の構造で検出された。さらに、マウスの異なる皮質下の視覚ターゲット構造全体のミクログリアおよびアストログリアの活性化は、一週間15の具体的なポスト摘出時間枠で起こる。一緒に、グリア、構造的および分子レベルで異なる皮質下の応答における視神経求心路遮断をもたらす。これらの皮質下にもかかわらず効果は、必ずしも、皮質レベル16での効果を巻き込むません。奪わ視覚野への非視覚的な入力の強化の隣に他の感覚分野の修正を含む注目すべきは、クロスモーダル皮質可塑性は、単眼（ME）3,4,17,18および双眼（BE）1,17の両方の後に発生摘出。 別に視覚神経科学に貢献するから、求心路遮断のタイプとして除核は、中枢神経系の19の神経保護及び神経変性20-22特性のバランスを研究するために使用することができる。 摘出を実行するための異なる手順は、既に文献に記載されている。ラットとマウスにおけるin vivo私の ​​ためにいくつかの方法論が原因軌道筋肉や組織23〜25の不必要な切片にあまり簡単である。そのようなマハジャンらのような他の出版。（2011）5を用いて詳細なプロトコルを提供<html>遺伝子型 - 表現型の相関関係を研究するための、目の高スループット収集、おそらく検死のための鈍的切開。その目的のために、この方法は、便利で速い。一つは（生きた動物での）除核ではなく、目自体、次求心視覚路を勉強することを選択するときしかし、この方法は、in vivoでの摘出にはあまり適しています。このような設定では、後の摘出生存率は非常に重要である。また、 生体内の損傷や視神経の保存および眼窩組織における最小限のは有利で ​​ある。 ここで、私たちはファゲらにより記載されたものと類似の代替摘出方法を提示する（2008）26、すなわち、特定の有利な特性を提供する：それは、迅速な術後回復に関連しており、非常に低い学習しきい値によって特徴付けられる研究者のための。眼の形態学的または視覚的経路の研究：一般的に、異なる方法が、その後の研究の焦点に応じて相補的である。 ve_contentは&quot;&gt;要するに、摘出は恒常性とクロスモーダル脳の可塑性、グリア応答特性、および軸索の安定性の調査に向けてのビジョンの研究から適用することができます。この可視化の記事では、私たちがin vivoでの眼の摘出のために実行可能で信頼性の高い方法を実証マウス。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine hydrochloride (Anesketin)</td>
			<td>Dechra Veterinary Products (Eurovet)</td>
			<td>BE-V136516</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine hydrochloride (Domitor)</td>
			<td>Orion Corporation (Janssen Animal Health)</td>
			<td>BE-V151742</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol hydrochloride (Antisedan)</td>
			<td>Orion Corporation (Elanco Animal Health)</td>
			<td>BE-V153352</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim Vetmedica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (Fucithalmic)</td>
			<td>Leo Pharma nv-sa</td>
			<td>BE 144654</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC31 Forceps - Serrated Curved</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
			<td>For application in the mouse. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps - Curved</td>
			<td>Fine Science Tools</td>
			<td>11003-13</td>
			<td>For application in the rat. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Hemostatic cotton wool</td>
			<td>Qualiphar</td>
			<td>N/A</td>
			<td>Other hemostatic agents are equally suitable (e.g. Viscostat, #649, Ultradent Products)</td>
		</tr>
	</tbody>
</table>

a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening

摘出または眼の外科的除去は、一般的に、神経求心路遮断のためのモデルとして考えることができる。これは、哺乳動物の視覚系1-4に沿って視覚的に、クロスモーダル·発生の可塑性の異なる側面を研究するための貴重なツールを提供します。 ここでは、大人2名まで20日齢のマウスでは検証されたマウスの1の除去または両眼のためのエレガントで簡単な方法を実証する。簡単に言うと、消毒湾曲した鉗子は、目の後ろに視神経をクランプするために使用されます。その後、円の動きは、視神経を収縮し、眼球を除去するために行われる。この技術の利点は、高い再現性、無出血への最小限の、迅速な術後の回復や実験者のための非常に低い学習しきい値です。したがって、動物の大量の最小限の労力で操作し、処理することができる。技術の性質は、わずかに損傷を誘導する可能性が手順中の網膜。この副作用は、マハジャンらと比較して、この方法はあまり適しています。（2011）5ゴールは、網膜組織を収集し、分析することであれば。眼球がまぶたを削除せずにソケットから変位させる必要があるので - ：また、私たちの方法は、ポストアイ開口の年齢（13年以降P10マウス）に制限されています。この原稿に記載のin vivoでの除核技術は、最近成功したラットにおいて軽微な変更を加えて適用され、一般的にはげっ歯類の求心視経路を研究するために有用であると思われています。

図1に記載したプロトコルを用いて眼の正常な除去を示し、出血の非存在下または眼窩組織または眼窩（ 図1A、1B）に明らかな物理的損傷によって特徴付けられる。さらに、削除された眼は完全に無傷の地球儀（ 図1C）の指標と、滑らかな角膜、脈絡膜および光ディスクを持っています。私たちのプロトコルは、目と機械旋盤の後ろに視神経のクランプを包含しているため、削除された眼の視神経は、網膜（ 図1D）の基部にくびれている。周囲の脳領域（ 図1E）を損傷することなく、きれいなカットの視神経に記載されたプロシージャの結果を実行する。 単眼摘出は、活動マッピング（ 図2）との組み合わせで、一方が急激に反対映像設備で機能的または眼特定の入力領域を描写することができサル28のような高次の哺乳動物におけるマウス12,27、さらには眼優位コラムlの皮質。 マウスを用いた実験では、一方の除去（ME）または両眼（BE）は、ターゲットを絞った視覚刺激と組み合わせ、 をZif268のmRNAまたはc-Fos蛋白質発現レベルの検出は、視覚野12,27の地域ニューロンの活性化を明らかにするために適用した。視覚的に刺激された対照（ 図2A）とは対照的に、BEマウスは、視覚入力（ 図2B）の欠如を完了することにより、視覚野において基底活性を示した。このように、非視覚野（ すなわち 、より前方部分より後方のセクションの聴覚野における体性感覚皮質）との視覚との間に境界線が発見された。 1週間の生存時間で私のマウスからの結果は、反対側の視覚皮質に目特定の入力領域を可視化した。 2単眼駆動型の領域は、低活動だったとメディ位置アルと中央両眼ゾーン（ 図2C）の横。 <img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" /> 眼窩、取り出し、目と視神経の後の除核状態の図1。定性的評価。曲がったピンセット（A）は出血や損傷が眼窩（B）に観察されないと、眼を除去した後。角膜および脈絡膜（C、D）の通常の出現によって反映されるように除去された眼は、完全に無傷である。視神経は、目（D）を残す視神経乳頭に絞られている。脳の腹側の部分の検査は、きれいにカット視神経（アスタリスク）およびその他の構造（E）の明白な損傷を明らかにしている。 C、D中のスケールバー：1ミリメートル。 E中のスケールバー：5ミリメートル。 ：前方; L：左; P：後部; R：右。 図摘出によって明らかにされたマウス視覚野における2。機能が目を入力固有の下位区分 。目や皮質を接続黒と灰色の線は、網膜求心と目特定の入力領域の上の交差を表す。神経活動は、コントロールの冠状断面上で可視化（A）、（B）BE、そしてブレグマレベルの周りのZif268のためのin situハイブリダイゼーション （ISH） の放射性によるME（C）マウス（グレースケール）-3.40 mmである。コントロール動物（A）は、両半球での視覚野は、視覚的な刺激後に高活性を発現する。一方または両方の眼（群）を摘出した場合、活動信号の明らかな減少が奪わ皮質領域の対応に表示されます。単眼は（C）マウスは、高ACTIのゾーンを示して除核削除された眼の反対単眼ゾーンで減少した信号に囲まれた視覚野の両眼ゾーンでVITY。スケールバー：2ミリメートル。ヴァンブリュッセルら 12からの許可を得て転載。

Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

また、脱核と呼ばれる目の除去は、それが不可逆的な部分（単眼）または完全な（両眼）視力低下を誘導するため、視覚的なクロスモーダル、および哺乳動物の視覚系に沿って発達可塑性の側面を研究するための有用な戦略を提供しています。ここでは、 生体内で脱核に実行するには、非常に再現性と簡単な方法について説明します。

実行するには非常に再現性と簡単な方法<em&gt;インビボ</emアイ開口後のマウスにおける&gt;眼摘出

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

a-highly-reproducible-straightforward-method-to-perform-vivo-ocular

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Biology

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

23.1K ビュー.  KU Leuven - University of Leuven. また、脱核と呼ばれる目の除去は、それが不可逆的な部分（単眼）または完全な（両眼）視力低下を誘導するため、視覚的なクロスモーダル、および哺乳動物の視覚系に沿って発達可塑性の側面を研究するための有用な戦略を提供しています。ここでは、 生体内で脱核に実行するには、非常に再現性と簡単な方法について説明します。 

ビデオ: A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

マウスで大網に膵島移植法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

自己 カエノールハブディティス 線虫の生態学的調査を行うための高度にスケーラブルなアプローチ

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin that is secreted by conjunctival goblet cells (CGCs) onto the ocular surface contributes to multiple ocular surface diseases. The elimination of exogenous sex hormones is essential to obtain consistent results during in vitro study of sex-based differences in CGCs. This paper describes a method to minimize the presence of exogenous hormones in the study of sex-based differences in CGCs while maintaining their physiological function. CGCs from postmortem human donors of both sexes were cultured from pieces of the conjunctiva in RPMI medium with 10% fetal bovine serum (FBS) (referred to as the complete medium) until confluency. Nearly 48 h before the start of the experiments, CGCs were transferred to RPMI medium without phenol red or FBS but with 1% BSA (referred to as phenol-red-free medium). The normal cellular function was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) after carbachol (Cch, 1 x 10-4 M) stimulation using fura 2/acetoxymethyl (AM) microscopy. The result shows that CGCs maintained normal function in the phenol-red-free media after 48 h. No significant difference in [Ca2+]i response was observed between phenol red-free RPMI medium and complete medium upon Cch stimulation. Therefore, we recommend using the phenol-red free RPMI medium with 1% BSA to eliminate exogenous hormones without altering the normal function of CGCs in the study of sex-based differences.

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences.

イン・ビトロ 結膜杯細胞の性差を研究する方法

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

網膜変性症のモデリングと解析

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

著者スポットライト:加齢性黄斑変性の病因を解明し、潜在的な治療法を発見する 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.