This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

注：すべての本実験は（クイーンズランド大学、解剖学的バイオサイエンス倫理委員会によって承認された）クイーンズランド大学のロー​​カル動物倫理手順に従って実施しました。 1.膵島単離<ol><li>試薬調製<ol><li>酵素の混合物<ol><li>膵臓消化のために、リベラーゼおよびコラゲナーゼIV型の混合物を使用しています。リベラーゼTL DMEMの26ミリリットルとの5mg（低サーモリシン）1バイアル（ダルベッコ改変イーグル培地）を希釈します。 </li><li> 5mMのHEPES、0.5mMのCaCl 2を、は0.1mg / mlのDNA分解酵素とは​​1mg / mlウシ血清アルブミンを補充した0.5mgの/ mlであり、濃度のハンクス緩衝液中のコラゲナーゼIV型を準備します。 </li><li> すなわち 、（体積で）1 4ミリリットルのLiberase TL + 1ミリリットルのコラゲナーゼ：4の割合でリベラーゼTLとコラゲナーゼ溶液を混合。 -20℃でそれぞれ店舗を2.5ミリリットルする酵素の混合物を分取。 注：組織のインキュベーション時間は、V酵素のバッチ間のビット（30秒〜1分差）ARY。したがって、バッチ試験が必要とされ得ます。 </li></ol></li><li> RPMIアイソレーションメディア<ol><li>室温で水1LにRPMI 1640粉末の1つのバイアルを溶解し、2グラムのNaHCO 3、4.02グラムのHEPESで補います。 </li><li> 、マグネチックスターラーで混ぜ、大きな三角フラスコにRPMI分離媒体を組み合わせてNaOHでpHを7.4に調整し、フィルターを滅菌し、4℃で保存。 </li></ol></li><li> RPMI培養培地</li><li> 10％ウシ胎児血清および1％抗生物質（ 例えば 、ペニシリン100 U / mlのストレプトマイシンを100μg/ ml）を、RPMI培地を使用します。 </li></ol></li><li>膵島単離および培養<ol><li>動物のいけにえ<ol><li>地元の機関の倫理手順に従って、頸椎脱臼またはCO 2窒息によりマウスを生け贄に捧げます。 70％エタノールで徹底的にマウス本体スプレー。 </li></ol></li><li>膵臓灌流<ol><li> UV字状のフラップを作るために腹部の皮膚と腹膜を通って横方向に2長いカットをSE。 、全体の腹腔を明らか腸を脇に置くと、総胆管（ 図1および図2）を明らかにする折り畳まれたティッシュペーパーによって所定の位置に肝臓を維持するために皮膚のフラップを引き上げます。その結果、外科医に向かって頭と尾離れ外科医から動物を置きます。 </li><li>十二指腸（ 図1）に入るから注入された酵素をブロックするために十二指腸壁に膨大部で血管クランプを適用します。 注：それは注入された酵素が膵管に入ると（クランプ以上）注射部位に膵臓や逆流を灌流または十二指腸に入るかどうかを決定し、これは（クランプの下）重要なステップです。 </li><li>共通の肝管と胆嚢管（ 図1）の接合部付近31 G針で総胆管にカニューレを挿入し、二回目の注射のための余地を残す番目の場合Eの膵臓は初めてを灌流することができません。ゆっくりと（〜10秒以上）マウス総胆管に冷たい酵素混合物の約2mlを注入。 注：クランプが正しい位置にある場合に膵臓が急速に灌流されます。 </li><li>針が総胆管の内部にあるが、膵臓を灌流することができない場合（ すなわち 。クランプを超える場合は肝臓から少し離れてクランプを移動したり、クランプの下であれば肝臓に少しそれを下に移動）クランプ位置を調整します。 注：針が時々周囲のカプセルではなく、ダクトの内腔に入ります。注入する際、針が総胆管の内側にあるかどうかを確認するのに便利な記号は、ダクトの拡張です。膵臓が灌流するために失敗した場合、代替は、酵素混合物が予想より低い収率で膵臓の複数の部位に注入することができたです。膵臓（脾臓ローブ）の左サイトの灌流は、膵島収量の最大化のために重要です。 </li><li>膵臓は、pした後erfused、すぐに、膵臓を除去バイアル（〜20ミリリットル、ガラス）に入れ、37℃の水浴中で〜19分間インキュベートします。 注：インキュベーション時間は、マウス株と年齢に応じてビットを変化させることができます。 </li><li>インキュベーション時間の後、〜冷分離培地を20mlを添加することによって、消化プロセスを停止します。膵臓を破壊し、滅菌50mlチューブに、通常の茶ふるい（1mmの孔径）を介して、それを注ぐ穏やかにバイアルを振ります。ブレーキをオフにして、4℃で1分間、400×gで分離培地と遠心でチューブをトップ。 </li><li>再び分離培地でペレットを溶解した後、ブレーキをオフにして、4℃で1分間、400×gで遠心分離し、2 15ミリリットルチューブに移します。吸引上清後、ボルテックスまたはピペッティングによりチューブあたり密度勾配細胞分離媒体の6ミリリットル（ヒスト1077溶液）とよく2ペレットを混合します。そしてそっとの上にチューブの側面に分離培地の〜6ミリリットルを追加ブレーキをオフにして、4℃で15分間100×gでの密度勾配分離媒体および遠心分離機。 </li><li>上部（分離媒体）に上清を収集し、3つの別々の皿の中央と下部（密度勾配細胞分離媒体）の層。解剖顕微鏡下でピペットを用いて、これら3つの皿で膵島を自分に都合の良いように選びます。 注：中間層は、島のほとんどが含まれています。膵島のサイズは約50〜500μmのコンパクトな構造（ 図3）として認識されています。我々は、WTマウスにおける〜200の島とされたdb / dbマウスにおける300- &lt;100膵島を得ます。 </li></ol></li><li>膵島培養<ol><li>島はライブ前に2日間酵素消化、5％/ 95％CO 2 / O 2、37℃、で6ミリリットルRPMI培地（10mMのグルコース）と25センチメートルシャーレで40島の文化·グループからの回復を支援するために、 2光子イメージングを-cell。毎日の培地を変更します。 </li></ol></li></ol></li></ol> 2。ライブセル2光子イメージング<ol><li>試薬の調製<ol><li>外バッファ<ol><li> 140のNaCl、5mMのKCl、2.5mMのCaCl 2を、1mMのMgCl 2、5mMのNaHCO 3および10mMのHEPESと2光子アッセイのためのナトリウムの豊富な細胞外のバッファを準備します。 </li></ol></li><li>外色素<ol><li> 2光子アッセイのために、細胞外のバッファの株式SRB 8ミリメートル、-20℃での小さなチューブやストアにアリコートを準備します。 0.8mmの作業濃度を得るために、新鮮な外のバッファにストックSRB 1:10に希釈します。 </li></ol></li></ol></li><li>光子撮像<ol><li>まず、30分間3 mMのグルコースの細胞外緩衝液中で2日間培養した膵島を前インキュベートします。 注：私たちは、文化の中で2〜5日の間の島を使用しています。 </li><li>その後、顕微鏡ステージ上に搭載サーモ制御室にスライドガラスを配置します。液の漏れを防止するために、グリースを使用します。 </li><li> extracellの500μlのチャンバーをカバー異なるグルコース濃度0.8 mMのSRB（3ミリモル、6ミリモル、15ミリモル、20 mMグルコース）を含有する緩衝液ウラル。 </li><li>次に、チャンバーの中央にプレインキュベート膵島を配置し、げっ歯類の小島で、インスリンについて免疫染色すると、ほとんどの細胞は、β細胞であることを示し、島に3-4細胞層に焦点を当てています。 注：SRBはすぐに島のすべての細胞膜を概説し、膵島は、画像への準備ができています。 </li><li> （ 例えば 、11のscanimage）60X油浸対物レンズ（NA 1.42）およびイメージングソフトウェアで2光子顕微鏡を使用して4は、典型的な顕微鏡の構成を示す図 。蛍光を有する膜880 nmのフェムト秒レーザーパルスによって励起された非透過性蛍光細胞外マーカーとしてSRBを使用して画像エキソサイトーシスのイベントは、550〜650 nmで検出しました。刺激に対する膵島の応答を記録します。 </li><li>小さな蛍光スポットの突然の出現によって、単一の細胞外のイベントを識別（解像度10ピクセル/1; m）および関心領域（〜0.78μmの2）の上に、蛍光信号の高速立ち上がり段階で確定してください。 </li></ol></li></ol>

<ol>
	<li>Guariguata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li><li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li><li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia,+independent+of+plasma+lipid+levels,+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db/db+mouse+model+of+diabetes.">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li><li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li><li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13 (2003).</li></ol>

The authors declare they have no competing financial interests.

膵島単離の中で最も重要な要因は、膵臓の初期灌流です。かなり低い膵島収量の灌流膵臓結果の下に。他の要因も、このような消化時間と部分的に灌流のレベルを補うことができる振とうのレベルとして分離の質に影響を与えます。例えば、完全に灌流膵臓を静かに振盪しながら下には、膵臓が困難に振盪しながら〜20分の消化を必要とする可能性が消化対照的に、18分30秒〜37℃でインキュ?...

疾患におけるβ細胞機能不全の役割が広く3,4認識されています。このようなMIN6及びINS-1などの細胞株は、β細胞の挙動の生物学を理解するための便利なツールです。しかし、インスリン分泌の生理学的制御は、ランゲルハンス島内で起こります。これらの島は密に詰まったβ細胞の数千、ならびに血管および他の内分泌細胞型を含みます。膵島内のこの環境は、インスリン分泌に影響を与え、糖尿病において重要である可能性があります。したがって、インスリン分泌の生理学的制御、および疾患の病態生理を理解するためには、無傷島を研究することが必須です。 膵島単離ヒト膵島ライブ及び、特に、2型糖尿病患者からのヒト膵島を得ることが困難です。また、ヒト膵島は、実験分子操作のための限られた可能性があります。研究者はそのためである使用してきました動物や2型糖尿病の動物モデルからすることができます。そのような疾患モデルは、DB / dbマウスです。これは、モデルが密接にヒトの疾患5,6と平行した表現型の進行と2型糖尿病自然突然変異です。糖尿病のdb / dbマウスの膵島単離のためにここで紹介するプロトコルは、より良好な収率、精製および強化された膵島の生存のためのいくつかの改良と、他のグループと共通の多くのステップがあります。 2光子イメージング生細胞2光子アッセイは、ここで説明した数を定量化し、糖尿病7の多くの細胞および野生型の島8,9から単一のインスリン含有顆粒の特性を評価するために研究者を可能にします。

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase TL 5mg</td>
			<td style="width:173px;">Roche</td>
			<td style="width:119px;">-5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type IV-1g</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histopaque 1077 500ml</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium (10X) 1L</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">R1383</td>
			<td>to prepare isolation media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 (1X) 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin 100ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>to prepare cultured media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">10099-141</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">11966-025</td>
			<td>to dilute the liberase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metamorph program</td>
			<td style="width:173px;">Molecular Devices, USA</td>
			<td style="width:119px;"></td>
			<td>to analyze the 2-photon images</td>
		</tr>
	</tbody>
</table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

2型糖尿病は、過去20年の間に、2型糖尿病におけるβ細胞機能不全の役割が明確に2を確立されている。2013年382万人に影響を与える慢性疾患であり、2035年15.92億に上昇すると予想されます。研究の進歩は、膵島の単離のための方法が必要でした。野生型および糖尿病LEPR デシベル （DB / DB）マウスの両方から分離された膵島の収量と品質を向上させるためにいくつかの変更を加えて、ここでは他のグループからのプロトコルと共有多くの共通の手順を提示膵島単離のプロトコル。生細胞2光子イメージング方法は、次いで、膵島内のインスリン分泌の制御を研究するために使用することができるが提示されます。

膵島収量および精製正常な野生型マウスの場合、約200膵島が期待されます。健康な島は、明るく見える丸い形と滑らかな境界線を持っています。アンダー消化バッチが少ない膵島と腺房細胞取り付け膵島（ 図3）を有し、一方の上に消化分離バッチは通常小さく、ファジー島を持っています。より良い血糖マウスは100の下半透明の外観（と小さい島を持って?...

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

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, ...

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

9.4K ビュー.  The University of Queensland. 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.

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

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 ...

医学

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 ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

ミュンヘンWistar系ラットにおける2光子顕微鏡を用いた蛍光高分子の糸球体透過性を定量化

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

ヒト胎児膵臓細胞クラスターの単離、培養、およびイメージング

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

同所膵臓癌マウスモデルのダイナミックコントラスト強化磁気共鳴画像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

内分泌と外分泌リプログラミングおよび増殖のためのモデルとしての膵管の連結を介して、マウスの膵臓への外科的損傷

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

マウス モデルにおける膵島の門脈内移植

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

前眼室におけるヒト膵島移植および寄与宿主細胞のイメージングと定量

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

2光子顕微鏡を使用して、糸球体プロセスに対する慢性片側尿管閉塞の影響を定量化

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...