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

Dr. Alan O. Perantoni (CDBL/ncı/NIH) bu el yazması sunulması destek için teşekkür ederiz. Ben de Dr Michael Hall (CDBL/ncı/NIH) ve Nirmala Sharma (CDBL/ncı/NIH) bu el yazması düzenleme için teşekkür ederim. Ben onun mükemmel fare yetiştiriciliği için Lai Thang (SAIC) için minnettarım. Bu eser kanser araştırmaları için Ulusal Kanser Enstitüsü sağlık ve Merkezi Ulusal Enstitüleri tarafından desteklenmiştir.

fareler (Wnt5a flox / flox fareler (Wnt5a tm1.1Tpy) ve Dll1Cre satır, UVJO fare modeli) 7 yönetilen bakım ve laboratuvar hayvanlarının kullanımı için NIH esaslarına göre ve ncı-Frederick hayvan bakım ve kullanım Komitesi tarafından onaylanmış bir protokol altında okudu.
 1. hazırlık Metilen mavisi boya çözüm <ol><li>ölçmek 0.1 gr Metilen mavisi pudra.</li>
	<li>10 mL normal salin veya 1 fosfat tamponlu tuz (PBS) x Metilen mavisi vortexing tarafından tamamen erimesi.</li>
	<li>Enjeksiyon sırasında tıkanma ortadan kaldırmak için bir şırınga filtre (membran gözenek boyutu 0.45µm) ile 10 mg/mL Metilen mavisi çözüm filtre.</li>
	<li>Montajı steril bir kafa derisi damar seti (27GX3/4 &#8243;) 3 mL tek kullanımlık şırınga ve dolgu 3 mL şırınga ile Metilen mavisi çözüm filtre. 
	Not: hidrostatik basınç ve sonraki Boya akışı önlemek için Metilen mavisi çözüm ile dolu şırınga yukarıda iğne ucu yerleştirin.</li>
</ol> 2. Doğum öncesi embriyo diseksiyon <ol><li>temiz makas ve Forseps ile % 70 etanol dissekan.</li>
	<li>E18.5 veya E19.5, Perinatal embriyo toplamak için ötenazi hamile fareler ilk CO 2 inhalasyon kullanarak ve servikal çıkığı NIH esaslarına göre gerçekleştirin. 
	Not: Hamile kadın fare genellikle E18.5, başlangıç doğurur, ancak, daha büyük böbrekler işlemek daha kolay. Bu nedenle, doğum E19.5 daha yakın, böbrekler üzerinde bu Çözümlemeyi gerçekleştirmek daha kolay olur. Ancak, ikili üriner sistem tıkanıklıkları die ile doğumdan sonra sersemler. Deneysel fare özellikleri bağlı olarak uygun toplama gün/saat ampirik olarak belirlenen.</li>
	<li>% 70 etanol karın ventral yüzeyde sprey ve karın boşluğu ventrally diseksiyon makası ve forseps kullanarak açın.</li>
	<li>Tüm rahim Asansör ve vücuttan makas rahim boynuzları ucunda diseksiyon ile keserek ayırın.</li>
	<li>1 x PBS ile tüm rahim bir kabında durulayın.</li>
	<li>Segmentally makas diseksiyon ile rahim kesme ve forseps sarısı sacs embriyoların ortaya çıkarmak için anatomi ile plasental decidua kaldırmak.</li>
	<li>Sarısı sac ilk perinatal embriyo kurtarmak için forseps diseksiyon ile amniyotik membran sökün ve.</li>
	<li>Makas diseksiyon ile bir embriyo başını kesmek. Steril gazlı bez yastıkları ile aşırı kan yok etmek. Embriyo ventral yüzeyi ile yukarı üzerinde görüntüleme için bir dijital fotoğraf makinesi ile donatılmış diseksiyon mikroskop pin. Kuyruğunu Genotipleme için toplamak. 
	Not: bir kerede enjeksiyonları bir embriyo gerçekleştirmek.</li>
	<li>Dikkatli bir embriyo karın boşluğuna Forseps ile cilt yırtılma tarafından açın. O zaman, dikkatli bir şekilde fazla organ ve dokulara karaciğer, mide ve bağırsak gibi Forseps ile onları kesme veya böbrekler, Üreter ve dorsally yer alan mesane ortaya çıkarmak için dışarı çekerek çıkarın ( şekil 1A). Steril gazlı bez yastıkları ile disseke embriyo aşırı kan absorbe. 
	Not: Aşırı kan boya enjeksiyon renal pelvis tanımlaması etkilemektedir.</li>
</ol> 3. Metilen mavisi boya enjeksiyon Renal Pelvis ve izleme Boya akışı içine <ol><li>iğne ve iğne ucu çözümden Metilen mavisi tarafından hidrostatik basınç kovma tarafından tüp Kaldır kabarcıkları. Akışını başlatmak için iğne ucu seviyesinin üzerinde boya solüsyon içeren şırınga kaldırın ve sonra akışını durdurmak için şırınga düşürebilir.</li>
	<li>Bu kez yerleştirilen bozmamaya dikkat çekici proksimal üreter yakınındaki renal pelvis iğne takın. Böbrek, idrar yolları tıkanıklığı belirlemek için içine boya enjeksiyon gerçekleştirmek. 
	Not: Gerçekleştirmek boya herhangi bir anormal böbrek 7 içine enjeksiyon ilk onun idrar yolları tıkanıklığı belirlemek için aynı yolu izleyerek.</li>
	<li>Şırınga hidrostatik basınç sağlamak ve 15 µL - 60 µL boya çözüm akış izin yaklaşık 20 cm yukarı kaldır. Şırınga embriyo üzerindeki bu yükseklik için harekete geçirilen akış hızı yaklaşık 3 µL/s olacak.</li>
	<li>Renal pelvis sonra üreter uzunluğu ilk başlangıç boya mavi rengini izlemek ve son olarak mesane Lümen içinde. 
	Not: Bu yaklaşık 5 alır s üreter mesane düzgün eklenirse mesane Lümen içinde ortaya bir zayıf boya renk görmek için. Boya bloke sitesinde yaklaşık 15 birikmesine izin yok boya renk mesane Lümen görüntülenirse s.</li>
	<li>Dur Boya akışı aşağı şırınga yerleştirin ve böbrek iğne kaldırma.</li>
	<li>Böbrek, Üreter ve boya çözüm ile takip mesane almak görüntüleri kamera kullanarak ve program görüntüleme bağlı bir stereomicroscope.</li>
	<li>Hydronephrosis böbrek, hydroureter ve boya çözüm son konumunu kaydını yapmak bir laboratuvar defter.</li>
	<li>Gerçekleştir enjeksiyon kontralateral böbrek açıklandığı gibi bölümleri 3.1) 3.5) ve boya akışı yaklaşık 20 izin s toplam. 
	Not: Mesane Lümen üreter düzgün mesane Lümen eklediyseniz güçlü bir mavi renk tarafından belirtilen boya Çözümle değiştirebilirsiniz. Sonra değerlendirme idrar yolu Kavşağı tıkanıklığının, Üreter ve mesanenin kesit için sabit olabilir.</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>

Yazar ifşa etmek hiçbir şey vardır.

Fare böbreklerde fonksiyonel E16.5 başında ve bir boya enjeksiyon testi bu zaman noktasından teorik olarak mümkün. Ancak, böbrek boya çözüm ile enjekte edilir çok küçük ve bu fenotipleri anormal idrar taşıma nedeniyle inşa idrar ikincil bir etkisi olduğundan fenotipleri hydronephrosis ve hydroureter gibi açıkça gözlenir değil. UPJO veya UVJO, gibi engellerin üzerinden bu fenotipleri şişmiş böbrek ve üreter tarafından E18.5 açık durumdadır. E19.5 veya neonatal pups embriyolar daha büyük ...

Üriner sistemin bir böbrek ve Üreter ve ortak bir mesane ve üretra ile oluşur. Üriner sistemin ana kan su ve elektrolit dengesi yöneterek vücut homeostazı korumak için fonksiyonudur. Böbrekler denetim elektrolit konsantrasyonları ve asit-baz dengesi kan filtre ve aşırı su ve solutes ve metabolitleri dahil olmak üzere atık salgılamak idrar üretmek. İdrar sonra böbrek renal pelvis üzerinden üreter nerede depolandığı ve sonuçta ortadan üretra1yolu ile tek yönlü bir şekilde bladderin için aracılığıyla taşınır.
Üreter düz borular nephric kanalı kaynaklanan vardır. Sonra fare embriyonik günde 10.5 (E10.5), tomurcuklanma nephric kanalı üzerinden üreter SAP hisli ve çok katmanlı bir yapıda fare arasında geçirimsiz olan urothelium adı verilen E12.5 ve E16.5 ayırır. Üreter sapı çevreleyen Mezenkimal hücreler de iç stromal hücreler, orta kalın düz kas hücreleri ve dış adventitial fibroblastlar içeren üç katmanlara farklı. Renal pelvis başlatılıyor üreteral Peristaltik dalgalar E16.5 içinde fare1başlayarak üretilen idrar2,3, taşımak için mesane üreter duvara düz kas tabakası yoluyla yayılır.
Konjenital anomalileri, böbrek ve idrar yolu (CAKUT) en sık görülen genetik hastalıklar, insan fetusa1,4, yaklaşık % 1'mevcut arasındadır ve fenotipleri hydronephrosis ve hydroureter de dahil olmak üzere çeşitli oluşur. İdrar hydronephrotic böbrek ve hydroureter oluşumu böbrek ve üreter sonucu anormal birikimi. Bir hydronephrosis ve/veya hydroureter oluşumu idrar yollarının bir obstrüksiyon nedenidir. Ureteropelvic junction obstrüksiyonu (UPJO) anormal idrar akışı proksimal Üreter ve renal pelvis, hydronephrosis ve proksimal hydroureter bükülme veya kalıcı katlanır5daralma sonuçlanan arasında bir tıkanma nedeniyle neden olur, 6. Buna ek olarak, mesane duvar veya üreme sistemi içine distal üreter Ektopik ekleme ureterovesical junction obstrüksiyonu (UVJO) denir. UVJO hydronephrosis ve hydromegaureter oluşumu7,8de tetikleyebilir. Vesicoureteric reflü (VUR) bir ek ureterovesical Kavşağı (UVJ) kusurdur. VUR doğru UVJ, böbrek mesane retrograd idrar akışı ile karakterizedir. UVJO için karşılaştırıldığında, VUR ile perinatal embriyo belirgin şişmiş hydronephrotic böbrek veya şiddetli hydroureter fenotip9gösterme.
Laboratuvar fare idrar akışı Metilen mavisi gibi bir boya enjeksiyon renal pelvis9içine tarafından incelenebilir. İnjectedmethylene mavi çözüm idrar yörünge renal pelvis Üreter ve mesanenin içine üzerinden iz olur. Hydronephrosis boya böbrek yönelik kabul edilebilir. UPJO Boya akışı şişmiş renal pelvis5ile proksimal üreter, tıkanması olarak tespit edilebilir. Genişletilmiş bir çapı belirtilen üreter herhangi bir genişleme hydroureter örneği gösterir. Son olarak, boya birikimi mesane duvar veya sitenin üreme sistemi ile şişmiş hydronephrotic böbrek UVJO gösterir ve hydromegaureter7,10santim açılmış. VUR algılamak için boya çözüm mesane enjekte edilir ve sonraki retrograd akışı böbrek9' izlenir.
Burada, perinatal embriyo içine renal pelvis Metilen mavisi boya enjeksiyon için bir protokol sunulmuştur. Bu iletişim kuralı renal pelvis Üreter ve mesanenin idrar akışı izleme sağlar ve UPJO veya UVJO gibi potansiyel idrar yolu Kavşağı engel olunur.

<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

İdrar yolu junction obstrüksiyonu kusurları Konjenital anomalileri hydronephrosis ve hydroureter inducing vardır. Fare idrar yolu junction obstrüksiyonu kusurları Üriner sistemin içinde Metilen mavisi Boya akışı izleme tarafından tespit edilebilir. Metilen mavisi boya perinatal embriyonik böbrek renal pelvis enjekte edilir ve boya akışı hidrostatik basınç uygulandıktan sonra Üreter ve mesane Lümen içine böbrek renal pelvis üzerinden izlenir. Boya birikimi normal perinatal idrar kesesi Lümen belirgin olacak ama idrar yolu engellerden oluşursa renal pelvis ve anormal bir üreter, bitiş noktası arasında kısıtlı olacak. Bu yöntem idrar yolu Kavşağı engelleri onayı ve görselleştirme hydronephrosis ve hydroureter kolaylaştırır. Bu el yazması için idrar yolu Kavşağı engelleri onaylamak için renal pelvis içine Metilen mavisi boya enjeksiyon protokolünü açıklar.

Böbrekler ve alt üriner sistem sırt en diğer iç organlara karaciğer ve bağırsak gibi yer alır. Bu diğer iç organlara kaldırdıktan sonra bir çift böbrek ve Üreter ve tek bir mesane görülebilir (şekil 1A) vardır. Başarılı diseksiyon renal pelvis enjekte boya böbrek mesane yoluyla üreter içine akar. Renal pelvis huni benzeri dilate proksimal üreter böbrek parçasıdır. Bu nedenle, boya enjeksiyon noktası görünür proksimal üreter...

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

Metilen mavisi boya enjeksiyon renal pelvis içine idrar yolu junction obstrüksiyonu kusurları değerlendirme sırasında fare embriyonik üriner sistem geliştirme kolaylaştırır. Burada, renal pelvis içine Metilen mavisi boya enjeksiyon için bir protokol açıklanmıştır.

İdrar yolu Junction obstrüksiyonu kusurları Metilen mavisi boya enjeksiyonla değerlendirilmesi

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

Research

JoVE Journal

Biology

15.6K Görüntüleme.  National Institutes of Health. Metilen mavisi boya enjeksiyon renal pelvis içine idrar yolu junction obstrüksiyonu kusurları değerlendirme sırasında fare embriyonik üriner sistem geliştirme kolaylaştırır. Burada, renal pelvis içine Metilen mavisi boya enjeksiyon için bir protokol açıklanmıştır.

Video: 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.

Fare mesane Duvar Enjeksiyon

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

Tıp

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).

Meme kanseri Sentinel düğüm biyopsi tek bir tekniği olarak 1% metilen mavi boya uygulaması

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 ...

Kanser Araştırmaları

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.

Çeşitli Kemirgen Modeller sistometrik Analizleri kullanarak Mesane Büyütme için Biyomalzeme Değerlendirilmesi

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

Biyomühendislik

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.

Foto akustik Sistografi

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.

Hafif Endometriozis İntraoperatif Algılama: Periton Bütünlüğü Kaybı ile İlişkili Pelvik Ağrı Tespiti ve Tedavisi İçin Yeni Bir Paradigma

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.

İdrar Yolu ve Probe tabanlı Konfokal Lazer Endomikroskopi: Tekniği

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.

Yetişkin erkek fare yordamda transüretral korumak

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

İmmünoloji ve Enfeksiyon

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).

İdrar Fonksiyonel Testi için Erişkin Erkek İdrar Yolu Ultrasonografisi

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.

Kronik Tek Taraflı Üreter Tıkanıklığının Glomerüler Prosesler Üzerindeki Etkilerini Ölçmek için 2-Foton Mikroskobu Kullanılması

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.

Geri Dönüşümlü Tek Taraflı Üreter Tıkanıklığının Olgunlaşmamış Bir Murin Modeli

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