著者らは、テクニカルサポートにマウナケア·テクノロジーズに感謝したいと思います。また、作者は批評のための技術支援とKathleen E. Machのためシェリーシャオに感謝します。この作品は、JCLにNIHのR01CA160986によって部分的にサポートされていました

1。患者の準備<ol><li>同意の患者が診断膀胱鏡検査やCLE用経尿道的膀胱腫瘍切除術（TURBT）などの他のendourological手続きを予定しています。同意の造影剤として膀胱および/または静脈内フルオレセインの使用の記述が含まれています。フルオレセインへの過敏反応の既往を尋ねる。 </li><li>患者は、膀胱鏡検査（一般的に砕石位で）のために配置し、滅菌方法で調製される。 </li><li>尿道から膀胱鏡リジッド又はフレキシブルに標準白色光膀胱鏡検査（WLC）に進む。 </li><li>白色光（ 図1）の下で膀胱の全地域を調査し、CLEの調査のために関心のある領域の点に注意してください。 </li><li> WLCに加えて、膀胱がidに蛍光膀胱鏡検査（ 図2A）または狭帯域光観察（ 図2B）を含む他の巨視的な光学イメージングモダリティで撮像することができるCLEと特性評価に続いて疑惑の追加領域をentify。 </li><li>病理学的に確認を画像化し、生検を実施され、両方の関心領域に隣接して小さな焼灼マークを付け焼灼電極または切除ループを使用します。焼灼マークは、その後のCLEイメージングおよび生検のために再局在化を容易にします。 </li></ol> 2。造影剤<ol><li>造影剤の膀胱内注入<ol><li>所望の濃度に滅菌生理食塩水での臨床グレードの10％フルオレセインナトリウム（0.9％NaCl）を希釈して準備します。例えば、生理食塩水396ミリリットルに10％フルオレセイン4 mlを希釈することにより、0.1％フルオレセインの400ミリリットルを用意します。 </li><li>膀胱に無菌尿カテーテルを挿入し、60ミリリットルカテーテルチップシリンジを用いて、重力によって希釈造影剤を植え付ける。 </li><li> 5分間留置造影剤を保持するために、導尿カテーテルをクランプします。 </li><li>膀胱から造影剤を排出します。 60ミリリットルのCAを使用する240〜300ミリリットル程度、生理食塩水を用いた膀胱から残留コントラストを洗い流すtheterチップシリンジ。 </li><li> CLEイメージングに進みます。 </li></ol></li><li>フルオレセインの静脈注射<ol><li>シリンジにフルオレセイン1.0mlを描く。静脈アクセスにシリンジを取り付け、0.5を注入 - フルオレセイン1.0mlを静脈内ボーラスとして。生理食塩水でラインをフラッシュします。これは、通常、手術室で麻酔チームによって行われます。 </li><li> CLEイメージングに進みます。 </li></ol></li></ol> 3。 CLEイメージング<ol><li>パッケージから滅菌CLEイメージングプローブ（Cellvizio）を取り外し、レーザースキャニングユニット（マウナケア·テクノロジーズ、パリ、フランス）に接続し</li><li>ウォームアップし、製造元の指示に従ってイメージングプローブのキャリブレーションを行います。 </li><li>膀胱に膀胱鏡または切除鏡を再度挿入します。リジッド膀胱鏡で0度のレンズを使用しています。 </li><li> WO沿っCLEイメージングプローブを進めるちょうど膀胱鏡を超えて先端と膀胱鏡のチャンネルをrking。 </li><li>白色光の下では、興味のある以前にマークされた領域を見つけます。粘膜の可視化のために、必要に応じて追加の生理食塩水灌漑を使用して、膀胱の中程度の膨張を維持します。 </li><li>関心領域の直接専用面接触のためのオペレータの手でCLEイメージングプローブを操作します。直接の接触を維持しながら、関心のある領域の光学的切片は、優しくイメージングプローブへの圧力を増加させると減少。 <ol><li>前方に位置する膀胱腫瘍に到達するために、標準Albarranブリッジは、関心領域（ 図2C）と直接接触するためのイメージングプローブのたわみを促進するのに役立つかもしれません。 </li></ol></li><li>興味のある一般的な領域はすべてどちらintraveを使用して画像化することができる乳頭状腫瘍、非乳頭状腫瘍、正常に見える粘膜と腫瘍の間の遷移、および紅斑のパッチが含まれてsicalまたは静脈内フルオレセイン管理。静脈内フルオレセイン切除ベッドのイメージング、前立腺尿道、陰茎尿道のために必要です。 </li><li>イメージングプローブとして、将来の分析のために関心、記録画像の領域をスキャンします。全体的な細胞形態、境界、組織、および血管新生の存在に注意してください。 </li><li>イメージングの完了時に、現用チャネルからCLEイメージングプローブを削除します。 </li><li>必要に応じて、CLEによってスキャンされた関心の対応する領域からコールドカップ生検を得る。生検アール画像形成領域の共同登録のためのランドマークとして焼灼マーキングを使用します。 </li><li>ルーチン当たりTURBTを完了します。静脈内フルオレセインを受けた患者で、切除時の可視化を向上させるために連続フローresectoscopesの使用を検討して、フルオレセインは、約5分間、静脈内投与後に膀胱に腎臓から排泄される。 </li><li>共焦点プロセッサとconduから保存された画像の記録を取得CT画像解析。 </li></ol>

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特別な利害関係は宣言されません。

イメージングプローブと膀胱粘膜の間に固体専用面接触を達成し、維持することは、最適な画質を取得する上で最も重要なステップです。イメージングプローブを操作すると、画像取得時にプローブが安定保持するために器用さを開発するために3月5日、患者の学習曲線は、ほぼあります。この手順は、生体内で行われるようにさらに、患者の動き（ すなわち呼吸器）と?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Cellvizio 100シリーズ</td><td>マウナケア·テクノロジーズ</td><td> 100シリーズ</td><td> 488nmでF400-V2：共焦点プロセッサとLSUを含む</td></tr><tr><td> Cellvizio共焦点Miniprobe </td><td>マウナケア·テクノロジーズ</td><td> Gastroflex UHD </td><td></td></tr><tr><td> AK-FLUOR 10パーセント</td><td> Akorn株式会社</td><td> NDC 17478-253-10 </td><td></td></tr></tbody></table>

probe-based confocal laser endomicroscopy of the urinary tract: the technique

プローブベースの共焦点レーザーendomicroscopy（CLE）は、標準的な内視鏡検査中に粘膜表面の生体顕微鏡でリアルタイムを有効に新興の光学イメージング技術です。 1呼吸器や消化管で現在のアプリケーションと、2-6 CLEはまた、膀胱癌診断のための尿路で検討されています7月10日細胞形態および組織マイクロアーキテクチャは、に加えて、リアルタイムでミクロンスケールの空間分解能で解決することができます正常および病的血管系の動的イメージング。7 プローブベースのCLEシステム（Cellvizio、マウナケア·テクノロジーズ、フランス）は488 nmのレーザースキャニングユニットに結合された再利用可能な光ファイバーイメージングプローブで構成されています。イメージングプローブは、標準的な柔軟性と硬性内視鏡のワーキングチャンネル内に挿入されている。共焦点内視鏡endomicroscopyのfunctionalitベースCLEシステム（Optiscan、オーストラリア）、yは、消化管にも使用され、内視鏡の上に統合されています。大きい範囲の直径を考えると、しかし、尿路内のアプリケーションは、現在、ex vivoでの使用に限定されています11共焦点画像取得が標的組織とイメージングプローブの直接接触を介して行われ、ビデオシーケンスとして記録されます。消化管のように、尿路のendomicroscopyはexogenenousコントラスト静脈内または膀胱内に投与することができるエージェント最も一般的にフルオレセインを必要とします。膀胱内投与では、尿路に固有のもので、最小限の全身毒性を使用してローカルに薬理学的薬剤を導入するための十分に確立された方法である。フルオレセインは急速に汚れ細胞外マトリックスを、確立された安全性プロファイルを持っています様々な直径の12イメージングプローブは、異なる口径の内視鏡との互換性を有効にしてください。現在までに、1.4と2.6ミリメートルのプローブは、柔軟かつ里と評価されてきたgidの膀胱鏡検査では、&lt;1ミリメートルイメージングプローブ13の10最近の可用性は、尿管の間に上部尿路内のCLEの可能性を開きます。蛍光膀胱鏡検査（ すなわち、光線力学的診断）と狭帯域光観察は、尿路のマルチモーダルイメージングを実現するために、CLEと組み合わせることができます追加の内視鏡をベース光学イメージングモダリティ14アール。将来的には、CLEは、分子特異性疾患プロセスの内視鏡イメージングのための蛍光標識ペプチド15および抗体などの分子造影剤と結合させることができる。

毎秒12フレームでビデオシーケンスファイルをグレースケールとしてのCLE画像が保存されます。画像解釈はリアルタイムで行われ、治験プロトコルの下で行う臨床判断に影響を与えるために使用されるかもしれません。このようなモザイク、7と標準病理との比較などのビデオシーケンスの見直し、追加の画像処理を含むオフライン分析では、技術に関連する学習曲線·フェーズで重要...

Watch this Scientific Journal Video about Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique at JoVE.com

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

プローブベースの共焦点レーザーendomicroscopyは、細胞レベルの解像度でこのような膀胱癌などの病理学的状態の動的な生体内イメージングを提供し、膀胱鏡検査中に人間の尿路のリアルタイム顕微鏡検査が可能になります。 Endomicroscopyは、標準的な白色光内視鏡検査の診断精度を補強し、外科的切除を改善するために術中画像ガイダンスを提供することがあります。

尿路のプローブベースの共焦点レーザーEndomicroscopy：テクニック

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

probe-based-confocal-laser-endomicroscopy-urinary-tract

Research

JoVE Journal

Medicine

20.7K ビュー.  Stanford University School of Medicine. プローブベースの共焦点レーザーendomicroscopyは、細胞レベルの解像度でこのような膀胱癌などの病理学的状態の動的な生体内イメージングを提供し、膀胱鏡検査中に人間の尿路のリアルタイム顕微鏡検査が可能になります。 Endomicroscopyは、標準的な白色光内視鏡検査の診断精度を補強し、外科的切除を改善するために術中画像ガイダンスを提供することが...

ビデオ: Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

蛍光in situハイブリダイゼーションおよび共焦点レーザー走査顕微鏡による口蓋パンダの口腔バイオフィルムの解析

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

下部尿路薬理学を評価するための方法として膀胱平滑筋収縮ストリップ

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

人間の強膜の共焦点顕微鏡法を実行するために積層技術を用いて、


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

誘導結合プラズマ -   - レーザーアブレーションによる脳組織におけるイメージング金属質量分析法（LA-ICP-MS）

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

レーザーによる肺の結核病変における薬物の空間の定量化をキャプチャ レーザーマイクロダイ セクション液体クロマトグラフィー質量分析法 (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

尿機能検査のための成人男性尿路の超音波検査

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment procedure. At present, different choices still exist for treating the ureteral end during laparoscopic ureteral bladder sleeve resection. Our center has adopted a new method for treating the ureteral end. This new method can increase the operating space and reduce the difficulty of the surgery compared with current methods.

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

上部尿路尿道尿道癌に対する全腹腹腔鏡下腎摘出術中の末端尿管の技術的改変

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

マウスの後肢におけるレーザードップラー灌流イメージング

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新しいヒトの言語性の高いレティナル文化技術の特徴付け

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...