著者らは、香港江博士に感謝したいと思いますとディーパParathasarthy、MPHは、技術支援のためのBDS。

1。全血採取<ol><li>ヒトからまたはNEM / EDTAを含むチューブで実験動物からの静脈血を採取する。 </li><li>直ちに血漿および赤血球細胞ペレットを準備する7分間14300 RCF（相対遠心力）でベンチトップ遠心分離機で血液をスピンダウンする。 </li><li>高速液体クロマトグラフィー（HPLC）および化学発光検出（CLD）分析のために血漿サンプルを準備します。 HPLC：沈殿物の血漿タンパク質への10分間プラズマ、13200 rpmでボルテックスと遠心に冷メタノールの1:1のボリュームを追加します。 HPLC分析のために上清を収集。 CLD：アリコート特にアッセイニトロソチオールにスルファニルアミドと塩化第二水銀を使用してサンプルとpreinc​​ubate。 </li><li> HPLCおよびCLD分析のために赤色細胞ペレットを準備します。 HPLC：10mMのNEM、2.5 mM EDTAおよび10mMのフェリシアン化を含む低張性溶解液に追加午前1時04分赤い細胞ペレット。 Vortexは徹底的にと、1:1メタノール、渦とcentrifを追加する沈殿タンパク質に10分間13200 rpmでuge。 HPLC分析のために上清を収集。 CLD。 10mMのEDTA、2.5 mM EDTAおよび10mMのフェリシアン化を含む低張性溶解液に1時04分赤い細胞ペレットを追加します。ニトロソチオールの特異的検出のためのスルファニルアミドと塩化第二水銀を含有するチューブに分注しサンプル。 </li></ol> 2。組織の抽出と準備<ol><li> NO代謝産物の組織レベルを決定するために、それは、上述したように試料調製のために血中遊離組織を採取することがまず必要です。完全な血液の交換は、左心室の頂部を介して生理的な緩衝液を注入することによって行われます。すべての血液が取り除かれた後、興味のある組織は、その後収穫することができます。 </li><li>組織サンプルをホモジナイズし、HPLCとCLD分析のために上記のようにサンプルを準備します。 </li></ol> 3。血管内皮機能のために大動脈リングの分離ティッシュオーガンバス<ol><li>マウスはanestになりますもはや応答ピンチをつま先と、手術前に頸椎脱臼を受けるようになるまで、ジエチルエーテルでhetized。開胸術は胸と腹部大動脈を露出するために実行されます。 25ゲージのシリンジは、左心室の頂部に挿入し、酸素クレブスHenseleit緩衝液、血液の自由な灌流されています。 </li><li>右心房は血液の出口を提供するためにカットされています。腹部大動脈は、外膜の除去と洗浄されます。 </li><li>リングは2ミリメートル長さのセグメントに切断し、生理的な緩衝液に浸し、4チャンネルの組織器官浴（DMT 720MO、AD·インスツルメンツ）にマウントされます。 </li><li>容器リングは37℃で酸素クレブス緩衝液（95％O 2および5％CO 2）で10 mlの器官浴に保持されます一グラム主張は、それぞれの大動脈リング（以前の実験で決定される最適な血管機能のための適切な開始張力）に配置されます。 8チャンネル進ブリッジ（Powerlab）及びデータ収集ソフトウェア（チャートバージョン5.2.2）すべての力の測定値を記録するために使用再。 </li><li>リングは、各臓器浴中の緩衝液で80分間平衡化を許可されているすべての20分に変更しました。 80分間平衡化した後、1μMのフェニレフリンは、最大下収縮のために、各リングに追加されます。 </li><li>安定した後など、アセチルコリンなどの内皮アゴニストは、血管弛緩のNO産生と程度を決定しないように追加されます。アセチルコリン用量反応した後、お風呂は一酸化窒素、平滑筋の応答性を決定するために、100％の弛緩のための値を取得するためにニトロプルシドナトリウムの外因性の源​​ですすぎ、recontractedして、処理されます。 </li><li> NOのスカベンジャーは、明らかに血管弛緩のNOおよび抑制のリリースを示すために、浴に添加することはできません。 </li></ol> 4。代表的な結果 ENO-20専用のHPLCを使用するには、中の亜硝酸および硝酸の特異的かつ高感度に検出するためのハイスループットメソッドを使用して簡単に提供しています生体マトリックス。検出し、元のクロマトグラムの原理を図1に示されています。このメソッドは、亜硝酸及び硝酸を決定するための任意の生物学的サンプルに使用することができます。 NO代謝産物のCLDベースの検出では、NO分子のソースを決定するために、化学誘導体化ステップを必要とします。化学発光検出器の同時酸化脱ニトロソ化および還元的脱ニトロソ化の実験セットアップを図2に示されています。右側の反応容器にPBS pH7.4中800mmのフェリシアン化で満たされて、左側の反応容器を還元脱ニトロソ化のために酢酸（ 図2A）のヨウ化カリウム/ヨウ素混合物が充填されています。全体のセットアップは、 図2Bに示されています。 図3は、グループ固有の脱ニトロソ化亜硝酸を検出し、定量化できるアッセイ、ニトロソ、ニトロソアミンなどニトロシルヘムの製品を示しています。このメソッドは、以前にdescriされているベッドと4,5検証されています。これらの重要な生化学的解析が容易に実験動物の内皮NO産生を決定しないように隔離された大動脈リングの機能研究と相関することができます。この古典的な薬理実験では、簡単かつ正確に血管内皮機能と生産NOを評価することはできません。そのようなアセチルコリンなどの内皮アゴニスト血管反応性を測定する直接して実験動物の血液や組織で検出された生化学的なマーカーと相関させることができる内皮NO産生を決定することはできません。アセチルコリンの用量反応の典型的な表現は、 図4に示します。正常な内皮機能と健康な対照マウスは、リラックスしてアセチルコリンに応答します。内皮機能不全（高コレステロール血症のマウス）のマウスは、同じ刺激​​に対してNO産生を減少させないことが原因で減少し緩和を示しています。 <img alt="図1" src="/files/ftp_upload/3722/3722fig1.jpg" /> イチジクURE 1。ENO-20およびサンプルのクロマトグラムによる亜硝酸及び硝酸の検出の原理。亜硝酸塩と硝酸塩の検出ENO-20方式の（上）模式図。 （下）10 pmolの亜硝酸及び硝酸の標準chromotogramは、ENO-20（亜硝酸及び硝酸の100 nM溶液の100μl）を注入した。 100μlの注入量と各陰イオンは1 nMの感度。タンパク質または着色された種との干渉はありません。 <img alt="図2" src="/files/ftp_upload/3722/3722fig2.jpg" /> パージ一酸化窒素ガスのガス相の検出にヨウ化/ヨウ素アッセイを使用してフェリシアン化及び還元脱ニトロソ化により、両方の酸化的脱ニトロソ化を使用して図2。CLD実験。 <img alt="図3" src="/files/ftp_upload/3722/3722fig3.jpg" /> 図3（パネル）グループ固有のchemicを含むサンプル前処理によって還元脱ニトロソ化アッセイにおける亜硝酸塩の化学発光検出、RSNO、RNNOらの試薬。ピーク面積の減算は、亜硝酸塩とRSNOsの検出を可能にする。フェリシアの酸化脱ニトロソ化ソリューションを使用してニトロシルヘム種（パネルB）化学発光検出。このメソッドは、RSNOs（GSNOまたはSNO-アルブミン）、またはRNNO（NO-ピロリジンとN-ニトロソアルブミン）との交差反応性を有するNO-ヘム製品の固有のものです。 <img alt="図4" src="/files/ftp_upload/3722/3722fig4.jpg" /> 図4分離された大動脈リング上のEx vivoの器官浴は、HPLCとCLDで検出された生化学的なマーカーと相関させることができる内皮NO産生の直接の尺度を提供しています。この図は、NO産生を減少させないことが原因で血管内皮機能障害を持つマウスの減少の緩和を示しています。

<ol>
	<li>Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+metabolism+and+breakdown.">Nitric oxide metabolism and breakdown.</a> Biochimica et Biophysica Acta. 1411, 273-289 (1999).</li><li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetycholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetycholine.</a> Nature. 288, 373-376 (1980).</li><li>Ignarro, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+factor+produced+and+released+from+artery+and+vein+is+nitric+oxide.">Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.</a> Proc. Natl. Acad Sci. U.S.A. 84, 9265-9269 (1987).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+S-,+N-,+and+heme-nitrosylation+in+biological+tissues+and+fluids:+implications+for+the+fate+of+NO+in+vivo.">Concomitant S-, N-, and heme-nitrosylation in biological tissues and fluids: implications for the fate of NO in vivo.</a> FASEB J. 16, 1775-1785 (2002).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+nitric+oxide+levels+in+the+red+cell:+validation+of+tri-iodide-based+chemiluminescence+with+acid-sulfanilamide+pretreatment.">Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.</a> J. Biol. Chem. 281, 26994-27002 (2006).</li><li>Angelo, M., Singel, D. J., Stamler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+S-nitrosothiol+(SNO)+synthase+function+of+hemoglobin+that+utilizes+nitrite+as+a+substrate.">An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 8366-8371 (2006).</li><li>Bryan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+NO+in+human+red+blood+cells:+fact+or+artifact.">Bound NO in human red blood cells: fact or artifact.</a> Nitric Oxide. 10, 221-228 (2004).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Processing+of+Nitrite+in+Hypoxia:+An+Intricate+Interplay+of+Nitric+Oxide-Generating+and+-Scavenging+Systems.">Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.</a> J. Biol. Chem. 283, 33927-33934 (2008).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic. Biol. Med. 43, 645-657 (2007).</li><li>Lundberg, J. O., Weitzberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NO+generation+from+inorganic+nitrate+and+nitrite:+Role+in+physiology,+nutrition+and+therapeutics.">NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and therapeutics.</a> Arch. Pharm. Res. 32, 1119-1126 (2009).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nat. Rev. Drug. Discov. 7, 156-157 (2008).</li></ol>

利害の衝突が宣言されません。

複数の生物学的区画内の関連するNO代謝産物の定量化のためにここで説明する方法は、内皮細胞によるNOの機能的な測定値と相関させることができ、健康と疾患におけるNOの生物学のフィンガープリントが可能になる。これらのメソッドは、高スループットのために適応させるための可能性のあるシンプルなサンプル調製を必要とします。これらの分子の相対量は、病気の実験モデルとヒト患?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> N-エチルマレイミド</td><td>サーモフィッシャーサイエンティフィック</td><td> 23030 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich社</td><td> E7889 </td></tr><tr><td>フェリシアン化カリウム</td><td>フルカ</td><td> 60299 </td></tr><tr><td> HPLC </td><td> Eicom株式会社</td><td> ENO-20 </td></tr><tr><td>オートサンプラ</td><td>オールコット</td><td></td></tr><tr><td> DMTミオグラフ</td><td> AD·インスツルメンツ</td><td></td></tr><tr><td> PowerLab </td><td> AD·インスツルメンツ</td><td></td></tr><tr><td>化学発光</td><td> EcoPhysics </td><td> CLD 88Y </td></tr><tr><td>遠心分離</td><td>エッペンドルフ</td><td> 5415D </td></tr><tr><td>アセチルコリン</td><td> Sigma-Aldrich社</td><td> A6625 </td></tr><tr><td> R-（ - ）フェニレフリン</td><td> Sigma-Aldrich社</td><td> P6126 </td></tr></tbody></table>

analytical techniques for assaying nitric oxide bioactivity

一酸化窒素（NO）は1（血液の循環で1秒未満）、生物学的システムに住んでいた非常に短い二原子フリーラジカルです。 NOは含めて本質的な機能を調節する、私たちの体内で作ら最も重要なシグナル伝達分子の一つと考えられませんが、血圧、免疫応答と神経通信の規制に限定されない可能性があります。したがって、生体マトリックスでの正確な検出および定量は、健康と疾患におけるNOの役割を理解することが重要です。 NOのような短い生理的半減期で、NO生化学の反応生成物を検出するための代替戦略が開発されている。複数の生物学的区画内の関連するNO代謝産物の定量化は、 生体内で生産、生物学的利用能および代謝NOに関して貴重な情報を提供します。単にそのような血液または血漿のような単一コンパートメントをサンプリングすると、常に全体のBOの正確な評価を提供することはできません特に組織中のNOのステータスを、DYはありません。実験動物の選択組織と血液を比較する能力は、はるかに健康と病気におけるNOのバイオマーカーの診断および予後診断ユーティリティなどの基礎科学と臨床医学の間のギャップを埋めるのに役立ちます。したがって、興味のある特定の組織へ血漿又は血液の外挿なしの状態は、もはや有効なアプローチではありません。結果として、メソッドを開発し、in vivoでの実験動物の複数の区画内のNOとNO関連製品/代謝物の検出および定量化が許可されて検証され続けています。と硝酸塩（NO 3 - ） -亜硝酸（NO 2）にNO合成酵素によって生産からの可溶性グアニル酸シクラーゼ（sGC）の活性化への最終的な酸化にNO生化学の確立されたパラダイムは、in vivoでの NOの影響の一部を表すことができる。 S-nitrosothiを形成するタンパク質のチオール基、二級アミンと金属とのNOとNO由来の代謝物の相互作用OLS（RSNOs）、N-ニトロソアミン（RNNOs）、およびニトロシルヘムは、それぞれのNOのcGMP依存しない効果を表しており、NOによるSGCの活性化として、生理学的に可能性が同様に重要である。生理中のNOの真の理解は、同時に複数のコンパートメントをサンプリングin vivo実験から導出されます。一酸化窒素（NO）の手法は複雑で、しばしば混乱の科学と多くの議論とNO生化学に関するません議論の焦点となっています。我々の能力特異的に上のNOのヒンジを伴わない新しい機構やシグナル伝達経路の解明は、選択的かつ高感度検出とNOと関連するすべての複雑な生体マトリックス中のNOの製品や代謝産物を定量化する。ここでは、高速液体クロマトグラフィーによる亜硝酸及び硝酸の迅速かつ高感度分析のための方法と同様のNO分子のソースなどのex vivoで決定するために、化学derivitazationとin vitroでのオゾンベースの化学発光で使用して生体試料中の遊離NOの検出を発表臓器浴筋運動記録法。

Watch this Scientific Journal Video about Analytical Techniques for Assaying Nitric Oxide Bioactivity at JoVE.com

Analytical Techniques for Assaying Nitric Oxide Bioactivity

一酸化窒素（NO）の内因性産生は、生物多様な機能を調節する。それは、NOベースのシグナリングの中断または調節不全は、多くのヒト疾患に関与していることがますます明らかになりつつあります。関連するNOの代謝産物を定量化しないための方法は、ヒトの疾患の新しい診断または予後バイオマーカーを提供することがあります。

一酸化窒素の生物活性をアッセイするための分析手法

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

analytical-techniques-for-assaying-nitric-oxide-bioactivity

Research

JoVE Journal

Medicine

17.9K ビュー.  University of Texas Health Science Center at Houston. 一酸化窒素（NO）の内因性産生は、生物多様な機能を調節する。それは、NOベースのシグナリングの中断または調節不全は、多くのヒト疾患に関与していることがますます明らかになりつつあります。関連するNOの代謝産物を定量化しないための方法は、ヒトの疾患の新しい診断または予後バイオマーカーを提供することがあります。

ビデオ: Analytical Techniques for Assaying Nitric Oxide Bioactivity

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

高塩ダイエットとルイスラットにおける慢性腎臓病を誘発するために一酸化窒素合成酵素阻害と組み合わせた5/6thの腎摘出

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

医学

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

ローカライズされた病理組織学的分析のための人工網膜を移植処理アイズのための技術

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

正常および疾患米国でフィブリンクロット構造を調べるための実験とイメージング技術

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

網膜タックと2部網膜上インプラント：ローカライズされた病理組織学的分析のための人工網膜を移植した処理の目のための技術

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超常磁性鉄酸化物ナノプローブの合成、特性解析、応用による肺外結核検出

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP のリン酸化を測定することによって血液中の一酸化窒素の血小板ベースの検出

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

小さい干渉の RNA のマクロファージへの配信のための車両としてポリエチレンイミン被覆された酸化鉄ナノ粒子の in VitroとIn Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

外傷性脳損傷マウスモデルにおける鼻腔内送達後のMRIを用いた超常磁性酸化鉄標識間葉系幹細胞の追跡

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

胸膜普及のマウスモデルに対する近赤外光免疫療法

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...