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1。ラット膵島のアデノウイルス形質導入と培養<ol><li>必要な数にメディアの2ミリリットル（8 mMグルコース、10％ウシ胎児血清、50ユニット/ mlペニシリン、50μg/ mlのストレプトマイシンを含むRPMI 1640培地）を添加することにより、6ウェル非組織培養コーティングしたプレートを準備します。井戸。無ウイルスの制御のためのそれぞれ、ウイルス制御（例えば、GFPを発現するアデノウイルス）、および実験群 - 例えば、典型的な実験は、3つのウエルが必要な場合があります。 </li><li>少なくとも30分間組織培養インキュベーターにそれを置くことによって37℃にプレートを温める。 </li><li>すぐに6ウェル非組織培養コーティングしたプレートの各ウェルに、ラットの膵島分離18,19、場所100から200島を後に。シックス膵島インスリン分泌とチミジン取り込みアッセイに必要とされる。残りの島は、遺伝子発現の研究​​や免疫ブロット法のためのタンパク質分離のためのRNAの分離に使用することができます。 </li></ol> [注：この時点から、バイオハザード物質の取扱い、使用、廃棄のための制度のプロトコルに従ってください。] <ol start="4"><li>優しくスワールウェルの中央に島をもたらすプレート。 </li><li>皿の中央にある小島に直接アデノウイルスをピペット。感染症の100から500多重度（MOI、ウイルスのプラーク形成単位に標的細胞の比）を使用します。 </li><li> 5分間島の残りをしましょう​​。 </li><li>組織培養インキュベーター（37℃、5％CO 2）のプレートを置きます。 </li><li> 24時間後、優しくスワールプレートのウェルの中心に島を持ってきても新鮮な培地を含む新しいP200にマイクロピペットを用いて膵島を転送することができます。島がプレートに取り付けられなくなった場合、それらは静かにピペットチップで外れすることができます。 </li></ol> [注：適切な伝達すると効率を確認するには島はその後、膵島コアにアデノウイルスの侵入を確認するには、共焦点顕微鏡を介して撮像することができるようにCY、コントロールウイルスを発現するGFPの使用は、有益である。] <ol start="9"><li>最適化のパイロット研究から実験の所望のタイミングに応じて追加の24〜72時間の培養では、膵島、。たとえば、増殖反応の誘導は、目的の遺伝子の24〜72時間またはノックダウンに至るまで時間が48時間または72時間が必要になる場合がありますが必要な場合があります。毎日新鮮なメディアへの膵島を転送します。 </li><li>実験の最後の24時間、文化1μCiの[メチル- 3 H] -thymidine/mlメディア（一般的には1μlのチミジン/ mlのメディア）を含むメディアの小島。 </li></ol> [注：この時点から、放射性物質の取扱い、使用、廃棄のための制度のプロトコルに従ってください。] 2。インスリン分泌アッセイ<ol><li>準備する分泌アッセイバッファー（SAB）10Xストック溶液（1.14 MのNaCl、47 mMの塩化カリウム、12mMのKH 2 PO 4、11.6 mMのMgSO 4）し、CaCl 2の 100Xストック溶液（0.25MのCaCl 2）。これらのストック溶液は、前もって準備し、室温で保存することができます。 </li><li>新鮮で作業SAB（10X SAB、1 MのHEPES、100XのCaCl 2 0.5mlの35％BSA、0.11グラムのNaHCO 3 0.28ミリリットルの1ミリリットルと、滅菌水に50ミリリットルの5 mL）中の50ミリリットルを準備50 mlコニカルチューブと37℃の水浴中で配置することにより、37°C​​に暖かい。 </li><li>ピペットで15 mlのコニカルチューブに作業SABの10mlと高グルコース（16.7 mM）のSABを準備する2.5 M D-グルコースの66.8μlを追加します。 </li><li>低グルコース（2.8 mM）のSABを準備する作業SABの残りの40 mlの2.5 M D-グルコースの44.8μlを添加します。 </li><li>ラベル6ウェルプレートの各ウェルの3つの1.7 mlマイクロチューブおよびリン酸緩衝生理食塩水（PBS）1 mlを加える。 </li></oL&gt; [注：小島は放射性であるので、放射性物質の取扱い、使用、廃棄のための制度のプロトコルに従ってください。] <ol start="6"><li>それぞれのマイクロ遠心チューブに20の島を配置します。各遠心管に比較的大型の膵島を追加するには、あらゆる試みを行います。たとえば、各チューブは、5小、10中期、5大型の膵島（ 図1を参照）を含めることができます。 </li></ol> [注：ランゲルハンス島では、解剖立体または標準的な顕微鏡のいずれかを使用して可視化することができます。] <ol start="7"><li>膵島は重力（〜2分）でチューブの底に定住した後、マイクロピペットでPBSを吸引除去し、廃棄します。 </li></ol> [注：重力により沈降する代わりに、チューブは1分間300×gで遠心分離することができます。] <ol start="8"><li>プレインキュベーションのために、低GLU 400μlのCOSE SABは、組織培養インキュベーター（37℃、5％CO 2）にチューブを（それらのキャップを開いた状態）に置き、60分間プレインキュベートします。プレインキュベーション低グルコースSABと廃棄を吸引除去する。 </li><li>基礎インスリン分泌、低血糖SAB 400μlを加えるために、組織培養インキュベーター（37℃、5％CO 2）にチューブを（それらのキャップを開いた状態）に置き、60分間インキュベートします。低グルコースSABを収集し、インスリンラジオイムノアッセイのために保存します。 </li><li>刺激インスリン分泌については、高グルコースSAB 400μlを加え、組織培養インキュベーター（37℃、5％CO 2）にチューブを（それらのキャップを開いた状態）に置き、60分間インキュベートします。高グルコースSABを収集し、インスリンラジオイムノアッセイのために保存します。 </li></ol> 3。チミジン取り込みアッセイ<ol><li>小島は重力によってチューブの底に定住した後、破棄され、マイクロピペットでPBSを吸引除去し、これを繰り返し、1 mlのPBSを追加します。一度ステップ実行します。 </li><li> 500μlの氷冷トリクロロ酢酸（TCA、10％w / v）の追加し、氷上で30分間インキュベートします。 </li><li> 4℃で3分間16 000 XG℃でチューブを遠心</li><li> TCAを吸引し、0.3 N NaOHの80μlを加え、室温で30分間インキュベートする。この間、5〜10秒間激しくボルテックスサンプルごとに10分。 </li><li> 7ミリリットルの液体シンチレーション計数管にエコノセーフ計数カクテルの4ミリリットルを追加します。 </li><li>シンチレーション計数管に試料の50μlを添加し、チューブのキャップを簡単に振ると、液体シンチレーションカウンターでカウントします。 </li><li>製造元のプロトコールに従ってビシンコニン酸（BCA）アッセイとサンプル10μlを用いてタンパク質濃度を測定します。 </li></ol> 4。データ解析<ol><li>製造業者のプロトコルに従ってインスリンラジオイムノアッセイを実行します。 </li><li>タンパク質の濃塩酸でインスリン分泌とチミジンの取り込みデータを正規化するentration。 </li></ol> 5。代表的な結果ラット膵島 ​​では膵島複製およびβ細胞機能を評価する実験の例を図2に示されています。この例では、架空の &quot;ジーン＃6&quot;そのアデノウイルスの過剰発現を示しています。確実にβ細胞機能を変更せずに膵島複製を刺激する。トップパネルには、チミジン取り込みアッセイの結果から、 &quot;遺伝子第6位&quot;の発現を増加させるようにチミジンの取り込みによって測定しDNA合成を増加させることを示している。ラット膵島の細胞のほとんどがβ細胞であるので、チミジンの取り込みの増加は、β細胞複製の増加を示している可能性があります。しかし、確証実験はしっかりとこれを確立するために実行する必要があります。下部のパネルに、インスリン分泌アッセイからの結果は &quot;遺伝子＃6&quot;の過剰発現を示して私は、すなわち、主なβ細胞の機能のいずれかを変化させなかった低域と高グルコースでnsulin分泌。アデノウイルスによる治療後に膵島の膵島分離と健康の質が低く、高グルコース濃度でのインスリン分泌倍に増加によって示されます。 &quot;ジーン＃6&quot;障害β細胞機能の発現を増加させる場合、これは可能性が高く、刺激グルコース濃度（16.7 mm）で分泌されるインスリンの減少として反映されます。グルコース濃度を変化させるための用量反応曲線も行うことができます。 <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="図1"/> 図1遺伝子発現のアデノウイルス媒介性の変化を以下の単離ラット膵島 ​​膵島複製およびβ細胞機能を評価するためのプロトコルの概要。新たに単離したラット膵島を24時間アデノウイルスにさらされ、その後96時間まで培養されています。チミジン取り込みにおけるインスリン分泌の測定に続いて、最後の24時間で評価される低および高グルコース。 <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="図2"/> 図2コントロールアデノウイルスおよびアデノウイルスを過剰発現する&quot;遺伝子第6位&quot;としてラベル付けされ架空の遺伝子を用いた実験からの結果。上部パネルには、インスリン分泌をチミジンの取り込みとボトムパネルを示しています。

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複製を刺激し、β細胞の機能を強化するために調節することができる確立する経路は、糖尿病の両方の主要な形態に関連しています。機能β細胞塊は、これらの決定を評価し、インスリン分泌細胞の存在と機能に依存しているため、同時にその利点を持っています。このプロトコルは、タンパク質の過剰発現または抑制し、in vivoでの有効性をテストすることができ、in vitroで機能...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> RPMI 1640培地</td><td>ギブコ</td><td> 11879 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>ギブコ</td><td> 15140 </td><td></td></tr><tr><td> 6ウェルプレート</td><td> BD-ファルコン</td><td> 35から1146 </td><td>非TC扱わ</td></tr><tr><td> [メチル- 3 H] -チミジン</td><td>パーキンエルマー</td><td> NET027Z001MC </td><td> 1 mCiの/ mlの</td></tr><tr><td>マイクロ遠心チューブ</td><td>デンヴィル</td><td> C2170 </td><td> 1.7ミリリットル</td></tr><tr><td> NaClを</td><td>シグマ</td><td> 59888 </td><td></td></tr><tr><td>塩化カリウム</td><td>アクロス</td><td> 42409 </td><td></td></tr><tr><td> KH 2 &lt;/ SUB&gt; PO 4 </td><td>アクロス</td><td> 20592 </td><td></td></tr><tr><td> MgSO 4で </td><td>アクロス</td><td> 41348 </td><td></td></tr><tr><td> CaCl 2を </td><td>アクロス</td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td>シグマ</td><td> H0887 </td><td> 1 M溶液を</td></tr><tr><td> 35パーセントBSA </td><td>シグマ</td><td> A7979 </td><td></td></tr><tr><td> NaHCO 3を </td><td>アクロス</td><td> 42427 </td><td></td></tr><tr><td> D-グルコース</td><td>シグマ</td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td>フィッシャー·サイエンティフィック</td><td> SA9410-1 </td><td>の10％w / vの</td></tr><tr><td> NaOHで</td><td>アクロス</td><td> 12426 </td><td></td></tr><tr><td>シンチレーション計数管</td><td>ザルスタット</td><td> 58.536 </td><td> 7ミリリットル、PP </td&gt; </tr><tr><td>シンチレーション計数管のキャップ</td><td>ザルスタット</td><td> 65.816 </td><td></td></tr><tr><td>エコノセーフカウントカクテル</td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td>インスリンRIA </td><td>ジーメンス</td><td> TKIN2 </td><td></td></tr><tr><td> BCAアッセイキット</td><td>サーモフィッシャーサイエンティフィック</td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>遠心分離</td><td>エッペンドルフ</td><td> 5415R </td><td></td></tr><tr><td>シンチレーション計数管ラック</td><td>ザルスタット</td><td> 93.1431.001 </td><td></td></tr><tr><td>液体シンチレーションカウンター</td><td>パーキンエルマー</td><td>トライ炭水化物2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

グルコース恒常性は、主にそれぞれ、膵臓のβとα細胞から分泌され、内分泌ホルモンのインスリンとグルカゴンによって制御されます。機能β細胞塊を解剖ベータ細胞塊など栄養素の負荷に対応するβ細胞の能力によって決定されます。機能β細胞量の減少は、糖尿病1-3の両方の主要な形態の中心となります。 1型糖尿病の自己免疫攻撃から減少し、機能β細胞塊の結果に​​対し、2型糖尿病では、この減少は、適切なインスリンを分泌するβ細胞の無力とメカニズムの幹部からベータ細胞の破壊の両方から開発しています。従って、機能β細胞量を復元するための努力は、より良い治療と糖尿病の潜在的な治療に最も重要なことです。 努力は複製を刺激し、β細胞の機能を強化するために悪用される可能性が分子経路を特定するために進行中です。理想的には、治療の目標は、β細胞の増殖と機能の両方を向上させるだろう。しかしおそらくもっと重要なβ細胞の増殖を刺激する戦略は、β細胞機能（例えば、いくつかの遺伝子と同様に）、およびその逆を損なうことの代償かどうかを識別することである。 単離したラット膵島 ​​における標的遺伝子の発現を体系的に抑制または過剰発現することによって、人が増えて、機能のベータ細胞塊4-6の潜在的な治療標的を識別することができます。アデノウイルスベクターは、単離されたラット膵島​​4,7-15で効率的に過剰発現やノックダウンの蛋白質に用いることができる。ここでは、アデノウイルス形質導入を利用して遺伝子発現を操作し、膵島の複製と分離されたラット膵島 ​​のβ細胞機能（ 図1）を評価する手法を提案する。このメソッドは、β細胞の複製または関数5,6,8,9,16,17を調節する新たな標的を識別するために以前に使用されています。

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

このプロトコルは1つが糖尿病の治療のための潜在的な治療標的を見つけるために機能的なβ細胞量を調節する因子を同定することができます。プロトコルは、アデノウイルスによる遺伝子発現の操作後に膵島の複製と分離されたラット膵島のβ細胞機能を評価するための効率的な方法で構成されています。

Adenovirally形質分離されたげっ歯類膵島におけるレプリケーションおよびβ細胞機能を評価する

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

assessing-replication-beta-cell-function-adenovirally-transduced

Research

JoVE Journal

Medicine

13.9K ビュー.  Indiana University School of Medicine. このプロトコルは1つが糖尿病の治療のための潜在的な治療標的を見つけるために機能的なβ細胞量を調節する因子を同定することができます。プロトコルは、アデノウイルスによる遺伝子発現の操作後に膵島の複製と分離されたラット膵島のβ細胞機能を評価するための効率的な方法で構成されています。

ビデオ: Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

使用する絶縁マウスハーツの心機能およびエネルギー論の評価 31 P NMR分光法

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

医学

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

遠藤- PAT 2000で内皮血管拡張機能を評価する

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

単離骨格筋のミオ - 機械的分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

部分的に膵臓を摘出された患者からのヒト膵島の単離

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

ヒト膵島の染色プロトコル

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; DB</sup&gt; 2型糖尿病のマウスモデル：無傷の膵島の膵島単離およびライブセル2光子イメージング

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

ハイコンテンツ<em&gt;インビトロ</em&gt;膵島β細胞の複製ディスカバリー・プラットフォーム

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量的3D<em&gt;インシリコ</emアルツハイマー病の齧歯類モデルにおける脳アミロイドベータ食作用の&gt;モデリング（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

女性の小型げっ歯類における神経温存ミッド尿道閉塞（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...