We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their excellent technical assistance.

全ての実験は、ビジョンと眼科の研究のための協会（ARVO）の眼科と視覚研究における動物の使用に関する声明に従って。 1。動物<ol><li> 26.5℃の温度と10分の14時間の明/暗サイクル15と水中での標準的な条件下で6〜12ヶ月の間熟成さAB（オレゴン州）株の野生型ゼブラフィッシュ（ ゼブラフィッシュ ） を維持する。 </li><li>カントン獣医庁の承認後に動物実験のため、関係機関の動物保護ガイドラインに従ってください。 </li></ol> 2 MNUトリートメント<ol><li> N -メチル- N個の -nitrosourea（MNU）の150 mg / Lの乾燥物質を含む水を準備します。注意！ MNUは有毒である。胎児にがん、遺伝性の遺伝子損傷または危害の原因となります。 </li><li>室温で60分間、MNUを含む水中でのゼブラフィッシュをインキュベートする。 </li><li>で迅速にゼブラフィッシュをすすぐ新鮮な水とMNUずに新しい水槽に魚を入れた。限りの実験のために、必要に応じて標準的な条件下で魚を維持してください。 </li></ol> 3視力測定<ol><li> 、視運動システムを起動し、メニューから「テスト」を選択し、を「シンプルな階段」、「視力（周波数）」と「ランダム/セパレート「16オプションを設定します。 </li><li>視運動システム上で1メートル、約500 mlの水で満たされた輸液ボトルを取り付けます。 </li><li>検査室で1ゼブラフィッシュを置き、注入ボトルに接続します。視運動系での検査室を配置します。 </li><li>測定を開始し、コンピュータの画面上でリアルタイムに眼球運動を観察します。否定応答は、（「いいえ」）の駅で観察されたものと同様のランダムな目の動きを表しているのに対し、それによって、肯定的な応答は（「はい」）、正しい方向に連続したサッカードとして定義されている進の格子。ゼブラフィッシュ出品物の目が3つ以上の後続の視運動性応答（OKR）の場合、を押して「はい」。そうでない場合、キーを押し「いいえ」。 </li><li>メニューの「結果」の下に、視運動性刺激の空間周波数の閾値を決定することによって、ソフトウェアによって計算された視力を抽出する。 </li></ol> 4。組織学<ol><li>エチル3 - アミノ安息香酸メタンスルホネート（200〜300 mg / Lの）の過剰投与によりゼブラフィッシュを安楽死させると目を脱核。 </li><li> 12時間、4℃でリン酸緩衝生理食塩水（PBS）中4％パラホルムアルデヒド（PFA）で全体の目を固定した後、段階的なアルコール系でサンプルを脱水する。 </li><li>パラフィンにサンプルを埋め込む、視神経乳頭（ONH）を介して5μmの切片を切断し、ガラススライド上にマウントします。 </li><li> 4分間hemalumとdeparaffizedパラフィン切片を染色し、0.2％の塩酸ACIが続く蒸留水にスライドを2回浸しdおよび0.8％アンモニア。少なくとも10分（H＆E）水道水でhemalum染色の現像後3分間エオシン切片を染色する。 </li><li>メディアをマウントするには、脱水のスライドを埋め込むし、光学顕微鏡下でスライドを観察します。 </li></ol> 5。免疫組織化学<ol><li>ターミナルデオキシヌクレオチジルトランスフェラーゼdUTPニック末端標識（TUNEL） <ol><li> 15分間室温で20μg/ mlのプロテ​​イナーゼKで脱パラフィン切片をインキュベート。トリスでセクションを3回洗浄を5分間ずつ生理食塩水（TBS）をバッファリング。 </li><li> 60分間37℃の加湿チャンバ内で50μlのTUNEL反応混合物（10％標識溶液および90％の酵素溶液）を用いて切片をインキュベートする。 5分ごとにTBSで3回洗浄する。 </li><li> DAPIを含む封入剤でスライドをマウントし、蛍光顕微鏡下でスライドを観察します。 </li></ol></li><li>増殖細胞核抗原（PCNA）染色<ol><li> 3分間の抗原回復緩衝液（トリス-EDTA + 0.05％ツイーン20、pHが9.0）中で脱パラフィン化切片を沸騰し、各5分間TBSで3回洗浄。 100μlを1時間室温で溶液（TBS + 10％ヤギ正常血清+ 1％ウシ血清アルブミン、pH7.6の）ブロッキング遮断する。 </li><li> 4℃で一晩、加湿チャンバー内で200倍希釈1中の抗PCNA一次抗体で染色する。 5分間ずつ、TBS + Tween 20で3回洗浄する。 </li><li> 1時間室温で500倍希釈1で適切な二次抗体で検出を終了します。 </li><li> DAPIを含む封入剤でスライドをマウントし、蛍光顕微鏡下でスライドを観察します。 </li></ol></li></ol>

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The authors have nothing to disclose.

以前は、私たちのグループは、ゼブラフィッシュのシステム9にげっ歯類から光受容体変性のMNUモデルを転送した。その後のイベントは、説明し、30日まで追跡した。この期間では、完全な網膜の形態学的変性と再生は、初期治療後に発生した。まず、組織学はそれに対応して8日目に最低で一日3から減少ロッド細胞数を明らかにし、TUNEL染色は、3日MNU処理後の桿体のアポトーシスを識...

ビジョンは、人間のための最も本質的な意味であり、その障害は、高社会経済的影響力を持っています。先進国では、網膜変性疾患は、高齢者1の間で失明や失明の主な原因である。ほとんどの退行性網膜疾患の原因は部分的にしか理解されており、治療のソリューションは、失われた視力を回復することは非常に限られています。網膜色素変性症は、一次光受容体の損失2-3で網膜変性疾患の代表例である。N -メチル- Nの -nitrosourea（MNU）は網膜変性を誘導するので、広く一次光受容体細胞死4で疾患をモデル化するために、齧歯類において使用される。これは、アルキル化剤であり、通常、露光5-7後数ヶ月現れる良性および悪性腫瘍をもたらす。また、短期間の観察期間内の特定の光受容体細胞死を引き起こす。これにより、網膜層の損失structu再度、重要な網膜の菲薄化は、濃度依存的に観察された。網膜グリア細胞が活性化されたが、網膜色素上皮（RPE）には変化は見られなかった。小胞体（ER）ストレス関連アポトーシス、網膜8にMNU作用の主要経路であると思われる。 私たちは最近、ゼブラフィッシュ9の光受容体の変性を誘導するために、化学モデルとしてMNUを導入しています。他の理由の中で、ゼブラフィッシュ（ ゼブラフィッシュ ） は、他の脊椎動物10のそれに、その視覚系の類似性の視覚の研究に重要になってきた。外側の網膜は、紫外線、短、中にピーク感度、および可視スペクトルの長波長と1桿体タイプで四つの異なるコーンタイプに分類することができ、光受容体が含まれています。内顆粒層（INL）は、バイポーラ、水平、およびアマクリン介在ニューロンの細胞体はWELとして、発見されたミュラーグリア細胞の細胞体としてlである。外網状層（OPL）は光受容体と網膜内層の間のシナプス接合レンズに最も近い細胞層に対し、視神経および視神経管を備えた長い軸索を形成する成分神経節細胞層（GC）であり、形成されている。神経節細胞、および内顆粒層における細胞間のシナプス結合は内網状層（IPL）11が形成されている。 RPEは神経感覚網膜の外側にあると長い頂端微絨毛12で光受容体外側セグメントを取り囲んでいる。さらに、ゼブラフィッシュは、高度に再生し、傷害、脳、網膜、脊髄、心臓、および他の組織13を再成長させることができる。網膜損傷が発生すると、ミュラー細胞であると考えられているINL中の細胞は、活性化され、各種の網膜細胞型に分化する可能性を有している。さらに、それらはまた、ONLに位置しているロッド前駆体を生成する。もうそう新しい細胞と大人のゼブラフィッシュの網膜を供給するス間は、毛様体辺縁帯です。このソースは連続的に成長しているゼブラフィッシュの眼14に桿体光受容体の一定の密度を達成するために必要とされる。 MNUモデルは、網膜組織のための単純かつ再現性の変性/再生のアプローチとして使用することができる。によるゼブラフィッシュにおける生物学的プロセスの特定の類似性およびヒトにおいてこれは、関連細胞死経路を識別するためにドアを開ける可能性があり、潜在的な神経保護薬をスクリーニングする。私たちのグループからの以前の研究に基づいて、私たちは今、実験室のビデオ9に従って機能的な変更を含む、網膜変性およびその結果としての再生のこのMNU誘発のゼブラフィッシュモデルの方法を示している。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonia</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>294993</td>
			<td>0.80%</td>
		</tr>
		<tr >
			<td >Bovine serum albumine (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>05470</td>
			<td></td>
		</tr>
		<tr >
			<td >Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>45260</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td>100%, 96%, 70%</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>E10521</td>
			<td>Tricaine</td>
		</tr>
		<tr >
			<td >Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat anti-rabbit Alexa 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrochloric acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>320331</td>
			<td>0.20%</td>
		</tr>
		<tr >
			<td >In situ Cell Death Detection Kit</td>
			<td>Roche Applied Sciences, Rotkreuz, Switzerland</td>
			<td>11684795910</td>
			<td>TUNEL Kit</td>
		</tr>
		<tr >
			<td >Mayer&#39;s hemalum solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>109249</td>
			<td></td>
		</tr>
		<tr >
			<td >Methylnitrosourea (MNU)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>N4766</td>
			<td>Toxic</td>
		</tr>
		<tr >
			<td >OptoMotry</td>
			<td>CerebralMechanics, Lethbridge, AB, Canada</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr >
			<td >Proteinase K</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S3004</td>
			<td></td>
		</tr>
		<tr >
			<td >Rabbit anti-PCNA</td>
			<td>Santa Cruz Biotechnology, Santa Cruz, USA</td>
			<td>sc-33756</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr >
			<td >Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizma&#210; base</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr >
			<td >Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr >
			<td >Zebrafish (Danio rerio) AB (Oregon) strain</td>
			<td>University of Fribourg, Dept. of Biology</td>
			<td>n.a.</td>
			<td>Own fish facility</td>
		</tr>
	</tbody>
</table>

methylnitrosourea (mnu)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics

産業界における失明の大部分を光受容体の損傷アカウントを生じたとの網膜変性疾患、 例えば網膜色素変性症、。動物モデルは、そのような疾患を研究するために極めて重要重要である。この点において、感光体特異的毒素N -メチル- Nの -nitrosourea（MNU）が広く薬理学的に網膜変性を誘導するために、齧歯類において使用されてきた。以前は、私たちはゼブラフィッシュにおけるMNU誘発性網膜変性モデル、視覚的な研究の別の人気モデルシステムを構築しています。 哺乳動物への魅力的な違いは、大人のゼブラフィッシュの網膜の損傷後の再生に永続的な神経発生である。この観察を定量化するために、私たちは、大人のゼブラフィッシュで視力測定を採用している。これにより、視運動反射は、非麻酔下の魚における機能変化を追跡するために使用された。これは、組織学ならびに免疫組織化学的stainiを補充した開発形態変化を相関させるためにアポトーシス（TUNEL）および増殖（PCNA）のためにngの。 要約すると、光受容体のアポトーシスが三日外顆粒層（ONL）における細胞の顕著な減少が続いているMNU処理、後に発生します。その後、内顆粒層（INL）及びONLにおける細胞の増殖が観察される。ここで、私たちは完全な組織学的だけでなく、機能的な再生だけでなく、30日の時間経過にわたって生じることを明らかにした。今、私たちは、ビデオ形式でMNUを使用して、ゼブラフィッシュの網膜変性および再生を定量化し、フォローアップする方法を示している。

視力： 実験の設定[空間周波数：0.042サークル/度（c / d）前記コントラスト：100％;ドリフト速度：20度/秒（D / src）の;バックライト輝度：152 CD / m 2]と本研究の成人ゼブラフィッシュのOKR評価を可能にした。よく手順を容認それぞれゼブラフィッシュ、10分 - VA測定の平均時間は約5であった。 MNUの暴露前視力は0.577±0.014サイクル/度（C / D）であった。 図1に、...

Watch this Scientific Journal Video about Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics at JoVE.com

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

ここで私たちは、網膜変性および再生及び成人のゼブラフィッシュにおけるN -メチル- N個の -nitrosoureaを使用して視覚機能への影響の定量化を実証する。視力の喪失や減少感光番号が内顆粒層での増殖が行われました。完全な形態学的および機能的な再生は、30日、最初の治療後に発生した。

組織学的および機能的特性：メチルニトロソ尿素（MNU）は、網膜変性·再生ゼブラフィッシュでの誘発

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

methylnitrosourea-mnu-induced-retinal-degeneration-regeneration

Research

JoVE Journal

Biology

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

11.4K ビュー.  University of Bern. ここで私たちは、網膜変性および再生及び成人のゼブラフィッシュにおけるN -メチル- N個の -nitrosoureaを使用して視覚機能への影響の定量化を実証する。視力の喪失や減少感光番号が内顆粒層での増殖が行われました。完全な形態学的および機能的な再生は、30日、最初の治療後に発生した。 

ビデオ: Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

腎上皮再生を研究するためにゼブラフィッシュの前腎のレーザーアブレーション

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

生物学

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

プラナリアの再生を操作するために薬理学的および機能的遺伝学的アッセイ<emジャポニカ> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavagingアダルトゼブラフィッシュ

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

アダルトゼブラフィッシュ側線再生のためのアッセイ

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

筋生検における免疫標識筋線維変性

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

ニトロレダクターゼ/メトロニダゾール媒介アブレーションとゼブラフィッシュ網膜色素上皮の再生を研究するためのMATLABプラットフォーム(RpEGEN)

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

組織学的分析のためのヒトの眼からの網膜色素上皮/脈絡膜フラットマウントの効率的かつ一貫した生成

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...