Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

注：このプロトコルを見直し、ピッツバーグ大学の施設内動物管理使用委員会によって承認された。 修正クレブス溶液と試験薬品アリコートの調製<ol><li> 117.9のNaCl、4.7のKCl、25のNaHCO 3、1.3のNaH 2 PO 4、1.2をMgSO 4、2.5のCaCl 2、11.1 D-グルコース、2酪酸ナトリウム、20酢酸ナトリウム（単位はmM）を含有する改変クレブス液を6 Lを作る0.004ニフェジピン（自発的な筋収縮をブロックする）、及び0.003インドメタシン（内因性プロスタグランジンの合成をブロックする）。それぞれ、組織切開とシングルファイバー記録に氷のように冷たいと暖かいクレブスのソリューションを使用してください。 </li><li>任意の化学溶液を調製する（ 例えば、IS、カプサイシン、ATP）は、所望の濃度の分量で。 </li></ol>結腸直腸-神経組織の2解剖<ol><li>中（30グラム - - 8週齢、20 6）雄マウスを麻酔し、安楽死させるマウスは、胸部の動きの欠如によって示されるように、完全に呼吸が止まるまで、毎分チャンバ容積の30％ - 10を変位させる流量でCO 2室。 </li><li>直ちに、胸部室切開右心房を穿孔し、氷冷したクレブスの十分な体積（〜500ml）中でのマウスカーカスを浸漬することにより安楽死、放血させると、以下の（4°C）溶液カルボゲン（95％O 2でバブリング、5％CO 2）。 </li><li>慎重にすべての内臓が、コロンと骨盤臓器を取り除く。少し横隔膜より上T12脊髄分節全体の半分にマウスを横断すると氷の寒さを含む解剖室に尾側半分を転送し、クレブス溶液を泡立て。 </li><li>実体顕微鏡下では、尿道への接合部で離断によって膀胱と生殖器官を削除し、それが総腸骨動脈に分岐するまで下行/腹部大動脈を削除します。 PNまたはそれらのsurroundinからLSNを解放鈍的切開によるグラム組織および（PN用）L6とS1脊柱または（LSN用）T13とL1脊柱へのその腹側のエントリポイントまでの腸骨稜の外から神経に従ってください。 </li><li>恥骨結合と左右の寛骨臼関節を切断し、腸骨を取り除く。神経が脊柱に入る場所まで、大腸に近いから添付の筋肉や結合組織からのPNまたはLSNのどちらかを慎重無料。 </li><li>慎重に遠位大腸を露出するように腸骨を切除。連続で添付PNまたはLSNと遠位大腸を解剖。 </li><li>組織室の浴コンパートメントに装着神経と直腸を転送します。さらに切開により過度の結合組織を外し、抗腸間膜の国境に沿って縦方向に大腸を開きます。 </li><li>上に向け粘膜側では、chambeのシリコーンベースに記録コンパートメントに隣接した結腸直腸腸間膜エッジをピンrおよび力アクチュエータに接続されたフックのレーキに大腸のantimesenteric長さを取り付け（ 図1に示され、 図2Aで撮影）。 </li><li>マウスの穴とゲートで風呂コンパートメントに接続されている記録をコンパートメントの中にPNまたはLSNを拡張します。静かに神経接着させるための親水性表面を提供記録コンパートメント、小さなガラスミラーに神経幹を置く。暖かいと一緒にお風呂コンパートメントSuperfuse（30から32°C）を、Krebs溶液を酸素化し、鉱物油との記録コンパートメントを埋める。 </li></ol> 3.シングルファイバーの記録と受容野の局在<ol><li>慎重に高倍率（ - 60X 50）での実体顕微鏡下でPNまたはLSNから神経上膜（神経鞘）をバックはがします。 〜100μmの厚さの8神経束 - 細かい鉗子を使用して、5に神経幹をいじめる。 </li><li>白金 - イリジウムリファレンスを置き組織チャンバ内のクレブス溶液と接触する電極。順次同一の材料からなる記録電極上に個々の神経束を置く。 </li><li>穏やかになでることで大腸表面下大腸求心性神経からのAPを呼び起こすために柔らかいペイントブラシを使用してください。検出可能なAP（活動電位）の録音を通じてコロンを支配する神経束（s）を見つけます。 注：PNとLSNも膀胱や他の骨盤内の臓器を支配する。 </li><li>さらに〜10μmの厚さの細い束のフ​​ィラメントに神経束を分割し、記録電極上に個々のフィラメントを配置するために30 G針先端のペアを使用してください。 </li><li>電気的に現在のスプレッドの〜2mmの半径を生成閾値上の刺激強度（10ミリアンペアの大きさ、0.3 Hzの@ 0.5ミリ秒の持続時間）、での求心性終末を励起するために粘膜表面に垂直なラウンドが先端に付いた同心円状の電極を配置します。体系的に電極を移動（〜1.5ミリメートルステップ）受容語尾をローカライズする扁平な大腸の長さと幅に沿って。 </li><li>求心性エンディングが励起されると、最小刺激強度（刺激しきい値）を必要とする活性化（受容野、RF）のサイトを特定するための電極位置を調整します。 &gt; 3ミリアンペア19刺激閾値と語尾を破棄。 </li><li>刺激アーチファクトと活動電位の開始との間の受容野（RF）での刺激電極と記録部位および2）伝導遅延（ 例えば、 図2B）との間の距離）1から伝導速度（CV）を計算する。 CV（10m /秒）=距離（mm）/伝導遅延（ミリ秒）。 </li></ol>機械受容大腸求心性神経の4機能分類<ol><li>電気刺激によってRFを見つけた後、RFに、次の3つの機械的刺激を適用します。 <ol><li> Calibraでの先端を押して、プロービング刺激を実施テッド·フォン·フレイのようなナイロンモノフィラメント（0.4および1グラム力）垂直に平坦化された大腸上のRF向けた。 </li><li>穏やかRF小さな表面せん断応力を生成するために細かいナイロンフィラメントストランド（10 mgの力）で大腸粘膜をなでることでなでる刺激を行う。 </li><li> ステップ2.8に記載されてフックのレーキを介して大腸の抗腸間膜縁に沿って円周方向に（ -は5mN /秒で170 MN 0）傾斜伸縮力を提供し、コンピュータ制御の力アクチュエータを用いて円周ストレッチを行う。 </li></ol></li><li>漿膜として、粘膜を（プロービング平滑末端にのみ反応する）求心性神経を分類筋肉（周ストレッチに対応し、プロービング平滑）筋肉/粘膜を（周ストレッチ、粘膜なでるに対応し、プロービング平滑）、または、（なでる粘膜およびプロービング平滑末端に反応する） MIA（三機械的刺激のいずれかに応答しない）。 </li><li>だけで（腸間膜求心性神経のために、電気刺激によって選択的に活性化する腸間膜のプロービング/機械的なストロークによってそれらの受容語尾を見つけることが困難なLSNの神経支配）。 </li><li>ストレッチに敏感な求心性（筋肉と筋肉の粘膜）の場合は、傾斜のストレッチ中に最初のAPを呼び起こす力として定義されている応答閾値を決定する。 </li><li>漿膜求心性神経のために、コンピュータ制御力アクチュエータで駆動される受容野のプロービング点状のレベルを昇順に彼らの応答を記録。 </li></ol> 5.化学アプリケーション/受容性終末のモジュレーション<ol><li>機械的な刺激に対するベースライン応答を記録（ すなわち、傾斜ストレッチ、プロービング点状、または粘膜ストロークへの応答）。 </li><li>コートチューブの一片（真鍮やステンレス鋼、10ミリメートル、高と4×4ミリメートル2正方形または4〜5ミリメートルの直径）の下端ワセリンとし、大腸に受容野の上に置きます。 </li><li>削除するチューブ内部のクレブス溶液、および3の受容エンディングさらす - 試験される化学物質（単数または複数）を含む溶液の170μlに5分。 </li><li>化学アプリケーションの間の求心性の応答を監視します（一部の求心性神経は、化学感受性である）。 </li><li>化学物質の作用を終了させる薬液とチューブを外します。 4内 - 6分、ベースライン応答と同様に機械的刺激への求心性応答をテストします。 </li><li>ウォッシュアウト（&gt; 15分）の十分な期間の後に再び機械的刺激を再適用します。 </li></ol> 6.録音および識別APスパイク<ol><li> 20 kHzで軸索から記録された電気信号をデジタル化し、コンピュータにデータを保存します。オーディオモニターにより、オンラインの信号を監視します。 </li><li>オフラインAPスパイクを分析し、個々のスパイク波形25の主成分分析に基づいて、単一のユニットを区別する。 注：Oneレコードがこれ以上目を含める必要があります2簡単に識別可能なアクティブユニット。 </li></ol>

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A., 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=plasticity+and+modulation+of+visceral+afferents.">plasticity and modulation of visceral afferents.</a> Brain Research Reviews. 60, 171-186 (2009).</li><li>Kiyatkin, M. E., Feng, B., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization.">Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization.</a> Am J Physiol Gastrointest Liver Physiol. 305, G638-G648 (2013).</li><li>Brumovsky, P. R., Feng, B., Xu, L., McCarthy, C. J., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystitis+increases+colorectal+afferent+sensitivity+in+the+mouse.">Cystitis increases colorectal afferent sensitivity in the mouse.</a> Am J Physiol Gastrointest Liver Physiol. 297, G1250-G1258 (2009).</li><li>Shinoda, M., Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+and+central+P2X+receptor+contributions+to+colon+mechanosensitivity+and+hypersensitivity+in+the+mouse.">Peripheral and central P2X receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse.</a> Gastroenterology. 137, 2096-2104 (2009).</li><li>Tanaka, T., Shinoda, M., Feng, B., Albers, K. M., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+&#945;-3+in+colorectal+afferents.">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor &#945;-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li></ol>

The authors have nothing to disclose.

ここに記載のインビトロ結腸直腸神経準備がうまく内臓感覚ニューロン上の他の非機能的アプローチ（ 例えば 、細胞、分子、および組織学的研究）を補完する個々の結腸直腸求心性の神経エンコーディング機能（研究するための強力なアプローチであることが証明され）詳細についてはレビュー27を参照してください。痛覚および長期直腸過敏症に寄与神経メカニズム?...

疼痛および過敏症が明らかに病理学的原因または組織損傷の非存在下で存在する過敏性腸症候群（IBS）を含む機能性胃腸障害に罹患している患者の主な不満である。例えば、IBS患者は、体の紹介（腹部の触診に、すなわち、圧痛）1の直腸バルーン膨満への応答を高め、正常な腸機能の間に感受性の増加、ならびに過敏を含む過敏症を呈する。大腸求心性神経を標的にすること（局所麻酔薬2,3の例、直腸内点滴注入、グアニル酸シクラーゼ-Cアゴニストリナクロチド4-6の経口摂取）をIBS患者の痛みや過敏症を緩和するのに有効であることが証明されているため、理解の向上大腸の求心性神経支配が重要です。 大腸求心性神経を含む内臓求心性神経、化学/ nutrient-および熱モダリティ（ 例えば、7-9）に応答することができる。機械的な刺激（ 例えば、管腔膨満、ストレッチ）は、一般的に不快感や痛み10-16含め意識的な感覚、を生じさせるものであるので、機械的刺激（ すなわち、機械受容求心性神経）への対応内臓求心性神経は、最も徹底的に研究されている。また、内臓はまた、一般的に、機械的に区別しない求心性（MIAs）によって神経支配サイレントまたは眠っ侵害受容器17と呼ばれている。正常な生理的条件下では、MIAsは機械的な刺激に応答したり、持っていない非常に高い応答が18をしきい値が、アクティブになり、病態生理学的状態において機械的感受性を取得し、過敏症に寄与することができる。 ここに記載のインビトロ調製およびプロトコルを使用して、我々は、海に電気刺激戦略を開発し、採用<html>大腸19の両方の機械受容とMIA終末の公平な識別を可能に受容語尾のためRCH。結腸直腸の神経支配は腰部内臓（LSN）および骨盤神経（PN）経路に由来し、5機械受容クラス（漿膜、粘膜、筋肉、筋肉、粘膜、腸間膜）と一つMIAクラス20に分類することができる直腸求心性線維を含む。このインビトロ準備を使用して、我々は大腸MIAs獲得した機械的感受性がLSN経路においてPN経路におけるMIAsの71％とMIAsの23％増感炎症スープ（IS）、への受容野の簡単な露出以下（感作）ことがわかった19。また、（ザイモサン21または2,4,6-トリニトロベンゼンスルホン酸（TNBS）22で結腸内処置を受けたマウス、すなわち、）長期間持続する行動内臓過敏の文脈でMIAsの（28日まで）の長期感作を文書化。 機械受容求心性神経のうちjove_content &quot;&gt;、筋肉と筋肉の粘膜求心性は持続的に大腸の周ストレッチをエンコード（ すなわち、ストレッチと小文字が区別されます）と有害な結腸直腸拡張23,24のエンコーディングを補助する唯一のクラスである。コンピュータ制御を使用して、フォースアクチュエータは、均質で、基準を適用し、かつ再現性が平坦化された大腸組織とさらに分類ストレッチセンシティブ求心低閾値および高閾値23の周方向に伸縮性を上げ、また、伸縮性の感作の時間経過を結腸内ザイモサン21またはTNBS 22治療後の感受性求心性内臓痛および過敏でストレッチに敏感な大腸求心性神経の役割を示唆し、行動内臓過敏の発症、持続性、および/ ​​または回復に対応している。

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

このビデオでは、詳細に、マウスの大腸-神経準備を用いたin vitroの単繊維電気生理学的記録プロトコルを示しています。アプローチは公平な識別と個々の大腸求心性の機能解析を可能にします。 1または少数の求心性から生じる伝播し活動電位（APS）の細胞外記録は（ すなわち、単一ファイバ）大腸内受容野（RFS）はからかわ神経線維線維束から作られています。大腸は、骨盤（PN）または腰椎内臓（LSN）神経取り付けられており、縦方向に開いたのいずれかで削除されます。組織は、記録チャンバーに入れ、平らに固定し、酸素化クレブス溶液で灌流される。フォーカル電気刺激は、機能的に5 mechanosensiに求心性神経を分類するために、さらに三つの異なる機械的刺激によってテストされている大腸求心性終末を、（プロービング、粘膜なでると周ストレッチを鈍ら）を見つけるために使用されます電性のクラス。これらの機械的刺激のいずれにも応答エンディングは、機械的に小文字を区別しない求心性（MIA​​s）に分類される。機械受容およびMIAs両方は、化学物質へのRFの局所露光部（ 例えば、炎症性スープ（IS）、カプサイシン、アデノシン三リン酸（ATP））によって（ すなわち、強化された応答を低減閾値、および/ ​​または機械的感受性の取得）感作のために評価することができる。私たちは、機器や結腸直腸-神経録音の準備、添付のPNまたはLSNと大腸の収穫、大腸内のRFの識別、神経線維束から単心記録、およびRFへの化学物質のローカライズされたアプリケーションを記述する。また、標準化された機械的な刺激の準備とアプリケーションの課題も議論されている。

セットアップは、 図1に示されている。これは、シリコーン裏打ち浴区画と隣接する鉱物油が充填されたコンパートメントで取り付け神経における大腸を収容するカスタムメイドの組織チャンバを含む。二区画室は、CNC機によるアクリルプラスチックの固体ブロックから機械加工された。両方の区画の底部は、その後の結腸直腸組織の下に簡単にピンを許可するようにしっかりと...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

マウス大腸求心性終末の体外機能解析では、

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K ビュー.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

ビデオ: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

医学

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

プライマリマウス大腸腫瘍のin vitroオルガノイド培養で

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

マウスモデルにおけるUTIとCAUTIの樹立とその性状

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

心臓形態学的および心エコーイメージングによって横大動脈狭窄のマウスモデルにおける機能の変更の評価

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

縦<em&gt;インビボ</em&gt;脳血管系のイメージング：中枢神経系疾患との関連

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

開発とマウス免疫寛容樹状細胞の機能解析

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内在的に発現したヒトRYR1変異体の機能特性評価

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).