Name: assessing urinary tract junction obstruction defects by methylene blue dye injection
Uploaded: 2017-10-12T12:30:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

この原稿の提出を支援博士アラン o. Perantoni (CDBL、NCI、NIH) をありがちましょう。私もこの原稿を編集の博士マイケル ホール (CDBL、NCI、NIH) とニルマラ シャルマ (CDBL、NCI、NIH) に感謝します。私は彼の優れたマウス飼育のライ タン (SAIC) に感謝しています。この作品は、癌研究のための健康、国立癌研究所とセンターの国立衛生研究所によって支えられました。

マウス (Wnt5a flox/flox マウス (Wnt5a tm1.1Tpy) と Dll1Cre ライン、UVJO マウス モデル) 7 ケアと実験動物の使用のための NIH のガイドラインに従って管理していましたNCI フレデリック ・動物ケアおよび使用委員会によって承認されたプロトコルに師事。
 1 メチレン ブルー色素溶液の調製 <ol><li>0.1 g メチレン ブルー粉を測定。</li>。
	<li>10 mL 生理食塩水入りのリン酸緩衝生理食塩水 (PBS) x 1 メチレン ブルーをボルテックスによって完全に分解します</li>。
	<li>注入時に詰まりを除去するためにシリンジ フィルター (膜孔径 0.45 μ m) で 10 mg/mL メチレン ブルー溶液をフィルター処理します</li>。
	<li>アセンブル滅菌頭皮静脈セット (27GX3/4 &#8243;) フィルターのメチレン ブルー溶液を 3 mL シリンジ 3 ml シリンジの塗りつぶしと 。
	注: 静水圧と後続の染料の流れを防ぐためには、上記のメチレン ブルー溶液で満たされた注射器の針の先端を配置します</li>。
</ol> 2。出生前の胎児の解剖 <ol><li>解剖はさみおよび 70% エタノールでピンセットをクリーンします</li>。
	E18.5 または E19.5、周産期の胚を収集する <li>は、妊娠マウスを安楽死させる最初に CO 2 吸入を使用し、NIH のガイドラインに従って頚部転位を実行します 。
	注: 妊娠中の雌マウス通常出産 E18.5 で開始、しかし、大きい腎臓が操作しやすくなります。したがって、出産に近い E19.5 で腎臓はでこの分析を実行しやすくなります。しかし、出生後両側尿路障害物金型で子犬します。実験的マウスの特性に応じて適切なコレクションの日/時刻を経験的に決定されるべき</li>。
	<li>腹部腹側で 70% エタノールをスプレーし、腹解剖はさみや鉗子を使用して腹腔内を開きます</li>。
	<li>子宮全体を持ち上げ、解剖子宮角の先端にはさみで切断することによって、体からそれを分離します</li>。
	<li>シャーレの 1x PBS で子宮全体をすすぎします</li>。
	<li>はさみを解剖前に子宮を切り取って、卵黄嚢胚を公開する鉗子を解離性大胎盤脱落膜を削除します</li>。
	<li>卵黄嚢を最初に削除し、周産期の胚を解放するために鉗子を解剖と羊膜を削除します</li>。
	<li>解剖はさみを持つ胚の首をはねます。滅菌ガーゼパッドで余分な血を拭き取る。胚その腹側表面にピン イメージングのデジタル カメラを搭載した解剖顕微鏡。必要に応じて遺伝子型の尾を収集します 。
	注: 一度に注射 1 つの胚を実行します</li>。
	<li>は慎重にピンセットで皮膚を引き裂くことによって胎児の腹部キャビティを開きます。その後、慎重に削除過剰な臓器や肝臓、胃、腸などの組織鉗子でそれらを切断または背部にある膀胱、尿管、腎臓を公開するそれらを引き出すことによって ( 図 1 a)。必要に応じて滅菌ガーゼパッドで切り裂かれた胚から過剰の血液を吸収です 。
	注: 過剰血干渉色素注入の腎盂の識別と</li>。
</ol> 3。腎盂と色素の流れの監視にメチレン ブルー色素の注入 <ol><li>針とチューブ静水圧によって針の先端からメチレン ブルー溶液を排除することで泡を削除します。フローを開始する針の先端のレベルの上の色素溶液の入った注射を持ち上げ、それから降ろしての流れを停止する注射器です</li>。
	<li>は、いったんそれを邪魔しないように世話、近位尿管近く腎盂に針を挿入します。色素を注入、尿路通過障害を判断するための腎臓を実行します 。
	: 注以下の尿路通過障害を決定する同じ方法で任意の異常な腎臓 7 注入の最初を染める</li>。
	<li>は、静水圧を提供し、15 μ L - 染料水溶液流の 60 μ L 約 20 cm を注射器を持ち上げて取り外します。この高さ、胚の上に注射器が発生した場合、流量は約 3 μ L/s になります</li>。
	<li>を監視し、腎盂、尿管の長さでまず色素の青の色、最終的に膀胱内腔にします 。
	注: かかる約 5 s 場合は、尿管が膀胱内に正しく挿入されて膀胱腔内に出てくる弱い染料の色を表示します。約 15 のブロックのサイトで蓄積する色素を許可する s 膀胱内腔に染料の色が表示されない場合します</li>。
	<li>色素フローを止めるまで注射器を置き、針を腎臓から削除します</li>。
	<li>カメラを使用して、プログラムをイメージング顕微鏡に接続されている染料の解決とトレース膀胱、尿管、腎臓を取るイメージ</li>。
	<li>、実験室のノートで、腎臓、尿管、および染料の解決の最後の位置の水腎症のレコード</li>。
	対側腎の <li>実行注射の説明に従ってセクション 3.1) 3.5) ができ、染料の流れ約 20 合計で s 。
	注: 膀胱内腔を染料の解決、尿管が膀胱内腔に正しく挿入されている場合に強い青色で示された塗りつぶされます。尿路移行部狭窄を評価した後、尿管と膀胱を区分のため固定することができます</li>。
</ol>

<ol>
	<li>Rasouly, H. M., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+development+and+disease.">Lower urinary tract development and disease.</a> Wiley Interdiscip Rev Syst Biol Med. 5, 307-342 (2013).</li><li>Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Box+Genes+in+the+Kidney+and+Urinary+Tract.">T-Box Genes in the Kidney and Urinary Tract.</a> Curr Top Dev Biol. 122, 245-278 (2017).</li><li>Bohnenpoll, T., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureter+growth+and+differentiation.">Ureter growth and differentiation.</a> Semin Cell Dev Biol. 36, 21-30 (2014).</li><li>Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H., Knoers, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic,+environmental,+and+epigenetic+factors+involved+in+CAKUT.">Genetic, environmental, and epigenetic factors involved in CAKUT.</a> Nat Rev Nephrol. 11, 720-731 (2015).</li><li>Tripathi, P., Wang, Y., Casey, A. M., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+canonical+Smad+signaling+in+ureteral+and+bladder+mesenchyme+causes+ureteropelvic+junction+obstruction.">Absence of canonical Smad signaling in ureteral and bladder mesenchyme causes ureteropelvic junction obstruction.</a> J Am Soc Nephrol. 23, 618-628 (2012).</li><li>Aoki, Y., 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=Id2+haploinsufficiency+in+mice+leads+to+congenital+hydronephrosis+resembling+that+in+humans.">Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans.</a> Genes Cells. 9, 1287-1296 (2004).</li><li>Yun, K., Perantoni, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydronephrosis+in+the+Wnt5a-ablated+kidney+is+caused+by+an+abnormal+ureter-bladder+connection.">Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.</a> Differentiation. 94, 1-7 (2017).</li><li>Uetani, N., Bouchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plumbing+in+the+embryo:+developmental+defects+of+the+urinary+tracts.">Plumbing in the embryo: developmental defects of the urinary tracts.</a> Clin Genet. 75, 307-317 (2009).</li><li>Murawski, I. J., Watt, C. L., Gupta, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+urinary+tract+defects+in+mice:+methods+to+detect+the+presence+of+vesicoureteric+reflux+and+urinary+tract+obstruction.">Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.</a> Methods Mol Biol. 886, 351-362 (2012).</li><li>Weiss, A. C., 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=Nephric+duct+insertion+requires+EphA4/EphA7+signaling+from+the+pericloacal+mesenchyme.">Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.</a> Development. 141, 3420-3430 (2014).</li></ol>

著者は、何を開示します。

マウス腎臓機能の先頭 E16.5 で、色素注入試験はこの時点から理論的に可能です。しかし、腎臓は色素溶液を注入するのには小さすぎる、これらの表現型は、尿異常尿搬送による構築の二次的効果によって水腎症や水尿管などの表現型が明瞭に観察されません。UPJO または UVJO などの障害物からのこれらの表現型は膨張した腎臓と尿管で E18.5 で明白であります。E19.5 または新生児子犬で胚の大...

泌尿器系は、腎臓、尿管および一般的な膀胱と尿道のペアで構成されます。泌尿器系の主な機能は、血液の水分・電解質のバランスを管理することによって体の恒常性を維持することです。腎臓はコントロール電解質濃度、酸塩基平衡、血液をろ過し、余分な水分と溶質と代謝産物などを含む廃棄物を排泄する尿を生成します。Bladderin 一方向方法で保存および尿道1で最終的には排除する腎臓の腎盂から尿管を尿後転送します。
尿管がストレート管 nephric 管から発生しました。マウスの萌芽期の日 10.5 (E10.5) で nephric のダクトから出芽後、尿管茎を延長し、E12.5 と E16.5 マウス間透水性である尿路上皮と呼ばれる多層構造に区別します。尿管茎を取り巻く間葉系細胞はまた間質細胞の内側、中間厚い平滑筋細胞と外側の外繊維芽細胞から成る 3 つの層に区別されます。尿2,3マウス1E16.5 でから作り出されるを輸送するため膀胱に尿管壁の平滑筋層を通って腎盂で開始する尿管の蠕動波が反映されます。
腎臓や尿路 (先天) の先天異常は最も頻繁に遺伝的疾患、胎児1,4の約 1% に存在の中で、さまざまな水腎症や水尿管などの表現型から成る。腎臓と尿管の尿の異常な蓄積, 水腎・水尿管の形成で起因します。水腎症や水尿管形成の原因の 1 つは、尿路の閉塞です。腎盂尿管移行部狭窄 (UPJO) は近位尿管と腎盂、腎と湾曲または永続的な折り畳み式5、近位尿管狭窄の結果との間の閉塞が原因の異常尿の流れによって引き起こされる 6。また、膀胱壁や生殖器官に遠位尿管異所挿入尿管移行部狭窄 (UVJO) と呼びます。UVJO することができますも水腎症および hydromegaureter の形成7、8を誘発します。その他尿管ジャンクション (UVJ) 欠陥が度逆流症 (VUR です)。VUR は UVJ で腎臓への膀胱からの逆行性尿の流れによって特徴付けられます。UVJO と比較して、vur が認められた周産期胚はっきりと示さない膨張, 水腎または尿管の深刻な表現型9。
実験室マウスの腎孟9へのメチレン ブルーなどの色素の注入によって尿の流れを調べることができます。青 injectedmethylene ソリューションは、腎盂、尿管、膀胱からの尿軌道がトレースされます。水腎症は、腎臓で色素の展開によって認識できます。UPJO 色素近位尿管腎盂が膨張した5流の閉塞として検出できます。尿管の拡大径によって示される任意の拡張は、尿管の例を示します。最後に、色素蓄積膀胱壁、または生殖管のサイトでは膨張した, 水腎と UVJO を示し、hydromegaureter7,10を拡張します。膀胱に VUR を検出する色素溶液を注入し、腎臓9以降の逆行フローを監視します。
ここでは、周産期胎児の腎孟にメチレン ブルー色素注入のプロトコルが表示されます。このプロトコルにより、腎盂尿管、膀胱からの尿の流れのトレースと UPJO や UVJO など潜在的な尿路接合の障害物を確認します。

<table><tbody><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>Quality Biological Inc. NORMAL SALINE - 500ML</td><td>Fisherscientific</td><td>50-983-204</td><td></td></tr><tr><td>3 mL disposable syringe with BD Luer-Lok tip</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Acrodisc Syringe Filters with Supor Membrane</td><td>Pall corporation</td><td>4614</td><td></td></tr><tr><td>Exel Scalp Vein (Butterfly) Sets; 27G x 3/4&quot; 12&quot;&amp;nbsp;</td><td>EXELINT</td><td>26709</td><td></td></tr><tr><td>Delicate Operating Scissors</td><td>Roboz Surgical Instrument Co.</td><td>RS-6702</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Roboz Surgical Instrument Co.</td><td>RS-5135</td><td></td></tr><tr><td>Dumont Tweezers; Pattern #5</td><td>Roboz Surgical Instrument Co.</td><td>RS-5045</td><td></td></tr><tr><td>BD PrecisionGlide Needles 30 G x 1/2&quot;</td><td>BD</td><td>305106</td><td></td></tr></tbody></table>

assessing urinary tract junction obstruction defects by methylene blue dye injection

尿路ジャンクション閉塞奇形は、水腎症や水尿管を誘導する先天異常です。マウス尿路ジャンクション閉塞奇形は、尿中のシステム内のメチレン ブルー色素の流れを追跡することによって評価できます。周産期の萌芽期の腎臓の腎盂にメチレン ブルー色素を注入し、静水圧を適用した後尿管と膀胱内腔に腎臓の腎盂から色素の流れを監視します。色素の蓄積は正常周産期尿路膀胱内腔で明らかになりますが、尿路通過障害が発生した場合に, 腎盂と異常な尿管の終了点の間拘束します。このメソッドは、尿路ジャンクション障害物の確認と水腎、水尿管の可視化を促進します。本稿では、メチレン ブルー色素を注入腎盂尿路接合の障害物を確認するためのプロトコルについて説明します。

腎臓と泌尿器系が肝臓や腸などの他のほとんどの臓器に背側に位置します。これら他の内臓を除去した後は、単一膀胱と尿管腎臓のペアは、表示されている (図 1 a) をします。成功術には、尿管から膀胱に腎臓から腎盂に注入された色素を流れます。腎盂は腎臓の尿管の漏斗のような拡張した近位部分です。したがって、色素注入ポイントは黒?...

Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

メチレン ブルー色素を注入、腎盂マウス胚性尿路開発中に尿路ジャンクション閉塞欠陥の評価が容易になります。ここでは、腎盂にメチレン ブルー色素液を注入するためのプロトコルを説明します。

メチレン ブルー色素注入による尿路ジャンクション閉塞欠陥の評価

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.6K ビュー.  National Institutes of Health. メチレン ブルー色素を注入、腎盂マウス胚性尿路開発中に尿路ジャンクション閉塞欠陥の評価が容易になります。ここでは、腎盂にメチレン ブルー色素液を注入するためのプロトコルを説明します。

ビデオ: Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

マウスの膀胱壁インジェクション

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

医学

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

乳癌センチネルノード生検における単一の技術としての1%メチレン色素の応用

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

がん研究

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

各種齧歯類モデルで内圧分析を用いた膀胱補強のためのバイオマテリアルの評価

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

生物工学

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光音響膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

微妙な子宮内膜症の術中検出：腹膜整合性の喪失に伴う骨盤痛の検出および治療のための新たなパラダイム

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

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

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

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 ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

大人の雄のマウスに経尿道的注入手順

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

免疫・感染学

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 ...

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

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

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

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

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

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

可逆的片側尿管閉塞の未熟マウスモデル

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

メチレンブルー色素注入による尿路ジャンクション閉塞欠陥の評価

要約

さらに動画を探す

この動画の章

マウスの膀胱壁インジェクション

乳癌センチネルノード生検における単一の技術としての1%メチレン色素の応用

各種齧歯類モデルで内圧分析を用いた膀胱補強のためのバイオマテリアルの評価

光音響膀胱造影

微妙な子宮内膜症の術中検出：腹膜整合性の喪失に伴う骨盤痛の検出および治療のための新たなパラダイム

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

大人の雄のマウスに経尿道的注入手順

尿機能検査のための成人男性尿路の超音波検査

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

可逆的片側尿管閉塞の未熟マウスモデル

メチレン ブルー色素注入による尿路ジャンクション閉塞欠陥の評価

要約

さらに動画を探す

この動画の章

マウスの膀胱壁インジェクション

乳癌センチネルノード生検における単一の技術としての1%メチレン色素の応用

各種齧歯類モデルで内圧分析を用いた膀胱補強のためのバイオマテリアルの評価

光音響膀胱造影

微妙な子宮内膜症の術中検出：腹膜整合性の喪失に伴う骨盤痛の検出および治療のための新たなパラダイム

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

大人の雄のマウスに経尿道的注入手順

尿機能検査のための成人男性尿路の超音波検査

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

可逆的片側尿管閉塞の未熟マウスモデル

メチレンブルー色素注入による尿路ジャンクション閉塞欠陥の評価