This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

注：倫理の声明：すべての実験は、研究における動物使用のためのマサチューセッツ総合病院のガイドラインに従って行いました。 1.事前準備<ol><li>どのように多くの大人のゼブラフィッシュの6-12ヶ月のを傷つけるために決定します。必要とされるよりも10〜20％より多くの魚を傷つけることを計画。それらはほぼ同じ大きさであり、以下の遺伝的多様性を有するように可変性を最小限にするために、使用年齢は同じタンクで一緒に飼育魚の兄弟に一致しました。女性の魚が原因腹部の大容量に、男性の魚に比べて注入する方がはるかに簡単です。しかし、男性および女性は同様の結果を与えます。 </li><li>初めての実験を行うときに、魚の特定の株について適切なゲンタマイシン投与量を決定するために、用量曲線をプロットします。ゲンタマイシンの純度がバッチ毎に変化するため、実験で使用する前にゲンタマイシンの各新しいバッチをテストします。適切な用量はexpressiを評価することによって決定されるべきですこのようなlhx1aとして傷害マーカーの上。 （ - D図1Aを参照してください）。 </li><li>ゲンタマイシン溶液を調製します。ゲンタマイシンストック溶液を-20℃に保存し、後の使用のために解凍することができます。例えば、0.5グラムの重さの魚で80ミリグラム/ kgの投与量（体重の80mgのゲンタマイシン/ kg）のために、便利な使用液としてリン酸緩衝生理食塩水でのゲンタマイシンの2 mg / mlのを準備します。これは魚に腹腔内注射のためのゲンタマイシンの20μLを提供しています。 注：一般的に、80〜120ミリグラム/ kgで適切であるかもしれないの40mg / kgという低い良好な結果が、投与量を達成します。ゲンタマイシンはまた、研究室の人員への危険な暴露を最小限にするために、溶液中に購入することができます。 注：高用量でのゲンタマイシンは、毒性であり得ます。粉末を計量する際、手袋とマスクを着用してください。 </li><li>魚を麻酔するための0.016パーセントのトリカインの100mlの水を準備します。使用するまで4℃でpHを7と店に調整し、滅菌ミリQ水に溶解しトリカイン（4グラム/ 1 L）の25倍の在庫を準備します。 4メートルを希釈0.016パーセントの作業濃度用の魚の水100mlでL。 注：トリカインが取り扱う際は手袋を着用し、麻酔薬や皮膚刺激性です。 </li><li>魚がスクープ形状に電球をカットし、水を排出するために底部に2つのスロットを切断することにより、プラスチック転送ピペットのかき出すことを確認します。準備されたゲンタマイシン溶液と負荷（10μlの階調の）1ミリリットルの注射器と30½G針を取り付けます。任意の気泡を除去。針の角度のついた先端が離れて直面していると、シリンジのマーキングが前進し、読み取り可能で直面していることを確認するために、注射器の針をねじります。コントロール注射のために、滅菌PBSで注射器と針を準備します。 </li><li>清浄な表面でスケールを準備したり、魚を乾燥させるために、注入のために魚を保持するためのボートだけでなく、紙タオルの重量を量ります。 </li><li>観測一夜ポスト噴射のために魚を保持するための蓋と各小0.5リットルの容器を準備します。小さな透明プラスチック交配ケージは、この目的のために有用です。これらの容器は白であってはならない - 魚に当てる白色上皮キャストが黒の作業台の上に観察できるように、理想的に、彼らは明確にする必要があります。快適に泳ぐ魚のための十分な魚の水で各容器を満たします。 </li></ol>ゲンタマイシンの2腹腔内注射<ol><li>魚の水中の0.016パーセントのトリカイン溶液に魚を麻酔。遅くする鰓換気率のために、もはや触れること応答しないために魚を待ちます。 </li><li>えらやフィンが負傷しないようにするために、ヘッドからテイルに向かって移動し、魚のスコップを使って魚をすくい上げます。その側で静かに魚を回して余分な水分を吸収し、スクープから余分な水分を振り払うためにペーパータオルの上に魚を置きます。 </li><li>再び魚をすくい上げると、ゼロ化された規模で秤量ボートに置き、魚の重量を量ります。最寄りの0.25グラムにラウンドし、注入するゲンタマイシンの適切な量を計算します。 注：0.5グラムの魚のために8で20μlの注入を使用します0ミリグラム/ kgの用量。 </li><li>再び魚をすくい上げるとドライ折り畳まれた紙タオルの上に置きます。右手を使用して注入する場合は、左の手にペーパータオルを保持し、簡単にアクセス腹で、左向きの魚の頭を置きます。 </li><li>排出腔への腹の皮膚、前方に45°の角度で針と注射器を持ちます。角度を減少させ、内臓を避け、皮膚の下に前方に針をスライドさせ、皮膚のすぐ下に針を押してください。その後、針を撤回、液体が針の周りに出ていないことを確認する一時停止、プランジャ適量を押します。安定した魚を保持することは問題である場合は、それらを安定させるために胴体に対して肘を引き締めます。 注：ゲンタマイシン溶液を取り扱う際は手袋を着用してください。 </li><li>個々の容器にそれぞれの魚をドロップします。魚を観察し、麻酔からの回復を確実。 28.5℃で魚を一晩おいてください。 注：魚はすぐに復活しない場合は、プラスチック製のトランスを使用それを復活させるために、その鰓を横切って水を灌漑するFERピペット。 </li><li>ゲンタマイシンの同じ用量で複数の魚を注入する際、同じ注射器と針を使用してください。適切なバイオハザードシャープコンテナに使用される注射器と針を処分。 </li></ol>傷害の3ポスト噴射観測<ol><li>次の日（1日後の傷害）、暗い表面に、その容器内に注入された魚を置きます。負傷した魚によって排泄死んだ上皮組織のホワイトキャストが表示されるはずです。 （ 図1Eを参照してください- H）。何のキャストがない場合は、どちらかの魚がゲンタマイシン注射によりけがはなかった（投与量のいずれかが低すぎた、または射出プロセス中に漏れたゲンタマイシンの一部）や魚がひどく尿管および総排泄腔の完全な遮断をもたらす負傷しました酔っぱらった組織と。 注：魚は、その本体からキャストをクリアすることができない場合は、通常2〜3日以内に死亡し、長く、したがって使用できないでしょうアッセイ。何のキャストが表示されていない場合は、鰓の動きが停止した後、少なくとも10分間浸漬することによりトリカイン水中で魚を安楽死させます。 </li><li>白色組織キャストが表示されている場合は、別に魚を設定し、他の魚のチェックを続け。ゲンタマイシンの適切な用量は、魚が使用可能であることの80〜90％になります。負傷した魚をプールした後、必要に応じて、異なる処理群に分割して。 </li></ol>魚の回収4.ケア<ol><li>きれいな水と混雑環境で魚を保管してください。汚れた水と混雑は感染症や意図しない死につながります。これ以上6以下の魚/魚の水500mlを維持し、可能な場合は、毎日水を変更してみてください。 注：これは、空間の保全のための最小量であるか、または研究者は、高価な薬物治療を用いた実験を行いたい場合。 </li><li> 3日は、損傷後まで魚を餌を与えないでください。魚を回収すること、最初は食べないし、タンク内のすべての食品は、細菌グラムを分解し、推進していきますowth。 3日目に開始すると、損傷後、一日一回、少量を養います。 </li></ol>負傷腎臓の5分析<ol><li> lhx1a mRNA発現のためのin situハイブリダイゼーションのために、腎臓は、所望の時点で魚から採取することができます。鰓の動きが停止するまで、次にかみそりの刃でヘッドを取り外して、体腔を開き、鉗子で内臓を除去し、氷、少なくとも10分にトリカイン水に魚を安楽死させます。 </li><li>代わりに腎臓を残す（背側体壁に取り付けられた着色された臓器）、一晩PBS中の4％パラホルムアルデヒド中で魚を修正。腎臓15を解剖し、in situハイブリダイゼーション標準に進みます。 <ol><li>簡単に説明すると、室温で1時間、PBST中でプロテイナーゼK（10μg/ ml）で透過処理PBST（リン酸緩衝生理食塩水、0.5％のツイーン20）で腎臓を洗浄します。 4％パラホルムアルデヒドで後固定し、PBSTで再度洗浄しました。 </li><li>その後、68で一晩腎臓を事前ハイブリダイズ、ハイブリダイゼーション緩衝液（50％ホルムアミド、5×SSC、50μg/ mlのヘパリン、500 / mlのtRNAを、0.1％のTween20、pH6.0）で中°C。 </li><li>ハイブリダイゼーション緩衝液中でジゴキシゲニン標識プローブとサンプルをインキュベートし、その後、100％、75％、50％、68℃、25％ハイブリダイゼーション緩衝液で各々5〜10分間洗浄し、次いで、室温に移動しながら30分間、二回洗浄0.1％Tween20でSSCを2倍、0.1％Tween20で0.2×のSSCで30分間、二回洗浄します。 </li><li> MABのサンプル3×5分（0.1 Mマレイン酸、0.15 M塩化ナトリウム、pHは7.5）を平衡化し、10％ヤギ血清とM​​ABに4℃で一晩ブロッキングしました。その後、抗ジゴキシゲニンAPのFab 1で一晩試料をインキュベート：MABで5,000。 </li><li> MABで1時間サンプル5回洗浄し、NTMTで15分（0.1MトリスpH9.5、0.05 MのMgCl 2、0.01 MのNaCl、50μLのトゥイーン20）のために3回平衡化。 </li><li>その後、信号を検出するためにNBT / BCIPで腎臓を治療します。 4％パラホルムアルデヒドで腎臓を修正し、レモするジメチルホルムアミド中でインキュベート脱イオン水に一晩漂白、過剰NBT / BCIP VEの。腎臓を洗浄した後、カバーガラス下で50％グリセロールでPBSTで撮影。 </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

ゼブラフィッシュは、大人の中腎腎臓6を含む成人の臓器の再生を研究するための理想的です。最近の研究では、より良いネフロンの再生中に発生するステップを特徴づけるために分子マーカーと新しいトランスジェニックレポーターラインを利用しているとどのような細胞が7,12責任を負うことがあります。尿円柱の観察は、ヒト16における腎疾患を診断するために一...

腎臓は、身体からの体液の恒常性、血圧調節および廃棄物のろ過のために不可欠です。哺乳動物は怪我が分化した上皮細胞1-4を使用した後、既存のネフロンを修復することができますが、それらは予約済みの幹細胞5のプールを欠いているように見えるし、新しいネフロンデノボを形成することができません。哺乳類とは対照的に、成魚魚の成長および傷害6,7に応答をサポートするために、成人期を通じて新しいネフロンを形成することができます。ゼブラフィッシュ、 ゼブラフィッシュは 、臓器再生8-10の研究のための貴重なモデル生物であり、ヒトの腎臓の修復を操作するためのアプリケーションに強力な洞察を提供する可能性があります。 Tgは（Lhx1a：EGFP）導入遺伝子11は、しかし、機構が損傷rにlhx1a +細胞の応答を制御する、大人のゼブラフィッシュの腎臓12にネフロン前駆細胞のプールを標識することが示されています不明emain。 アミノグリコシドゲンタマイシンは、ヒト13における既知の腎毒性及び聴器毒性効果と広く使用されている抗生物質です。ゲンタマイシンの腹腔内注射は、魚6に急性腎損傷を誘導する確立された方法です。魚の模倣でこの傷害細管上皮や過剰摂取14ゲンタマイシンした後、ヒトに起こる糸球体の瘢痕化の損失。多くの新しいネフロンが生成され、同時に形成、増殖および分化の段階を経て進んでゲンタマイシン注射によってゼブラフィッシュにおける傷害を誘導することは、強力な、同期再生応答を誘導する便利な方法です。 このプロトコルは、外れ値を最小化するために非侵襲的な視覚的なスクリーニングプロセスを利用して大人のゼブラフィッシュの腎臓における堅牢性と再現性の損傷のための私達の方法を詳述します。私たちは、ゲンタマイシンとの損傷が、上皮腎臓組織と形式の死につながるという事実を利用しますその後、中腎管および排出腔に塊に蓄積し、尿細管円柱、のイオン。これらは、魚に渡され、水中で視覚的に観察することができます。これは、私たちはその後、さらなる実験のためにプールすることができる強力な非致死傷害、と魚をスクリーニングすることができます。これらは、実験のエンドポイントは、より均一で効率的なデータ収集と分析につながる達する前に死亡無傷魚または魚の数を最小限に抑えます。また、特別な装置や試薬は、この方法の費用対効果や学術や指導設定で使用するための適切なを作り、必要ありません。私たちの方法を使用して、ここでは、腎臓の再生にゲンタマイシン投与量の増大効果だけでなく、上昇した温度の影響を示します。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS for working solution</td>
		</tr>
	</tbody>
</table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

ゲンタマイシン損傷は水に腎上皮キャストの観察（ 図1）によって視覚的に確認することができます。ゲンタマイシンの異なる投与量は、再生腎臓（ - D図1A）にlhx1a +細胞凝集体の数を増加させることで、その結果、成体野生型TuABゼブラフィッシュを傷つけるために使用されました。ホワイトキャストが容易に損傷後の水1日（ - H図1E）で見ること?...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

ゲンタマイシン誘発性損傷によって大人ゼブラフィッシュにおける腎再生

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.8K ビュー.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

ビデオ: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

ゼブラフィッシュ胚における機械的血管損傷

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

発生生物学

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

胆道主導の肝臓の再生を研究するためにゼブラフィッシュにおける肝細胞特異的アブレーション

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

開発にターゲットマイクロインジェクションのテクニック<em&gt;アフリカツメガエル</em&gt;腎臓

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

前駆細胞が媒介する肝細胞の再生を研究するためにゼブラフィッシュにおけるエタノール誘発性線維症肝臓モデルの開発

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

多光子顕微鏡によるライブセルイメージングのための大人のトランスジェニックゼブラフィッシュ網膜外植片の文化

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

ゼブラフィッシュの発生における急性腎障害のモデル化のための正確な細胞除去法

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

ゼブラフィッシュにおける急性アルコール性肝障害の組織学的解析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

ナノ粒子を介した siRNA 遺伝子サイレンシング アダルト ゼブラフィッシュ心臓で

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...