나는이 원고를 제출 하 여 지원 박사 앨런 O. Perantoni (CDBL/NCI/NIH) 감사 합니다. 나 또한이 원고 편집에 대 한 박사 마이클 홀 (CDBL/NCI/NIH) 및 Nirmala Sharma (CDBL/NCI/NIH)을 감사 합니다. 감사 해요 라 Thang (SAIC)에 그의 뛰어난 마우스 축산에 대 한. 이 작품은 암 연구에 대 한 건강, 국립 암 연구소 및 센터의 국가 학회에 의해 지원 되었다.

쥐 (Wnt5a flox / flox 마우스 (Wnt5a tm1.1Tpy) 및 Dll1Cre 선, UVJO 마우스 모델) 관리 및 실험 동물의 사용에 대 한 NIH 지침에 따라 관리 7 NCI 프레드릭 동물 관리 및 사용 위원회에 의해 승인 프로토콜 아래에서 공부 하는 고.
 1. 메 틸 렌 블루 염색 솔루션의 준비 <ol><li>측정 0.1 g 메 틸 렌 블루 가루.</li>
	<li>10 mL 정상적인 염 분 또는 버퍼링 하는 인산 염 (PBS) x 1에서 메 틸 렌 블루를 vortexing에 의해 완전히 분해.</li>
	<li>주입 동안 막힘 제거 주사기 필터 (막 기 공 크기 0.45µm) 10 mg/mL 메 틸 렌 블루 솔루션 필터.</li>
	<li>어셈블 메 마른 두 피 정 맥 세트 (27GX3/4 &#8243;) 3 mL 일회용 주사기와 채우기 3 mL을 주사기 메 틸 렌 블루 솔루션 필터링. 
	참고: 수압 및 후속 염료 흐름을 방지 하려면 배치 메 틸 렌 블루 솔루션으로 채워진 주사기 위에 바늘 팁.</li>
</ol> 2. 출생 전 태아의 해 부 <ol><li>깨끗 한가 위와 집게를 70% 에탄올과 해 부.</li>
	E18.5 또는 E19.5, perinatal 배아를 수집 <li>임신 마우스를 안락사 먼저 CO 2 흡입을 사용 하 고 다음 수행 NIH 지침에 따라 자 궁 경부 전위. 
	그러나 참고: 임신 여성 마우스 일반적으로 출산 E18.5에서 시작,, 더 큰 신장은 조작 하기 쉽습니다. 따라서, 신장 E19.5 가까이에 출생에는에이 분석을 수행 하기 쉽습니다. 그러나, 양측 요로 방해 된 출산 후 새끼. 실험 쥐의 특성에 따라 적절 한 컬렉션 요일/시간 수 경험적으로 결정 합니다.</li>
	<li>복 부 복 부 표면에 70% 에탄올을 스프레이 하 고 해가 위와 집게를 사용 하 여 ventrally 복 부 구멍.</li>
	<li>전체 자 궁 고 해 부가 위 자 궁 뿔의 끝을 절단 하 여 몸에서 분리.</li>
	<li>1 x PBS 가진 전체 자 궁 페 트리 접시에 린스.</li>
	<li>Segmentally가 위 해 부와 자 궁을 잘라내어 집게 노른자위 낭에서 태아를 폭로 해 부와 placental decidua 제거.</li>
	<li>노른자위 sac를 먼저 제거 하 고 해방 perinatal 배아를 집게 해 부와 양수 막 제거.</li>
	<li>배아가 위 해 부와 목을 벨. 멸 균 거 즈 패드와 함께 과잉 혈액을 닦아. 태아 그것의 복 부 표면 위로 핀 해 현미경 이미징에 대 한 디지털 카메라를 갖춘. 필요한 경우 유전형 꼬리를 수집. 
	참고: 한 번에 주사 한 배아를 수행.</li>
	<li>신중 하 게 피부를 찢 어 여 집게로 태아의 복 부 구멍을 엽니다. 다음, 조심 스럽게 제거 초과 장기와 조직 간, 위, 소장 등 집게와 그들을 절단 또는 신장, ureters, 방광 dorsally는 노출에 그들을 밖으로 당기 ( 그림 1A). 필요한 경우 멸 균 거 즈 패드와 함께 해 부 배아에서 과잉 혈액을 흡수. 
	참고: 초과 혈액 신장 골반 염료 주입에 대 한 식별과 방해.</li>
</ol> 3. 신장 골반 및 염료 흐름 모니터링 Methylene 청색 염료의 주입 <ol><li>바늘에 액체 정역학 압력에 의해 바늘 팁에서 메 틸 렌 블루 솔루션을 추방 하 여 튜브 제거 거품. 시작 흐름, 바늘 팁의 수준 이상의 염료 솔루션이 포함 된 주사기를 들어올린 다음 낮은 흐름을 막으려고 주사기.</li>
	<li>신장 골반 근 ureter, 한 번 배치 그것을 방해 하지 않도록 주의 복용 근처에 바늘을 삽입 합니다. 수행의 요로 방해를 결정 하기 위해 신장에 염료 주입. 
	참고: 수행의 요로 장애를 결정 하는 같은 방법에 따라 어떤 비정상적인 신장 7에 주입을 첫 번째 염색.</li>
	<li>15 µ L-60 µ L 염료 솔루션 흐름의 액체 정역학 압력을 제공 하는 약 20 cm를 주사기를 들어올립니다. 유량 주사기 태아 위에이 높이 발생 약 3 µ L/s 됩니다.</li>
	<li>모니터링 신장 골반에서 다음 ureter의 길이에 처음 시작 하는 염료의 파란색 그리고 마지막으로 방광 루멘에서. 
	참고: 그것은 걸립니다 약 5 s ureter을 방광에 올바르게 삽입 하는 경우 방광 루멘 내에서 등장 하는 약한 염료 색깔을 보고. 차단 된 사이트에 대해 15에서 축적 하는 염료를 허용 염료 색깔 방광 루멘에 표시 하는 경우 s.</li>
	<li>중지 염료 흐름으로 주사기를 놓고 바늘을 제거 하는 신장에서.</li>
	<li>신장, ureter, 방광 염료 솔루션 추적의 걸릴 이미지는 stereomicroscope에 연결 하는 카메라를 사용 하 여 및 프로그램 이미징.</li>
	<li>실험실 노트북에는 신장, hydroureter, 및 염료 솔루션의 최종 위치 hydronephrosis의 기록을 만들기.</li>
	<li>수행 주입 contralateral 신장에 설명 된 대로 섹션 3.1) 3.5) 염료 흐름 약 20 수 총에서 s. 
	참고: 방광 루멘 ureter을 방광 루멘에 올바르게 삽입 하는 경우 강한 파란 색깔에 의해 표시 된 염료 해결책으로 가득 합니다. 요로 분기점 방해의 평가, 후에 ureters, 방광 고쳐질 수 있다 단면에 대 한.</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>

저자는 공개 상관이 있다.

마우스 신장 E16.5 기능 시작 되며 염료 주입 테스트가 시간 포인트에서 이론적으로 가능 하다. 그러나, 신장이 너무 작아 염료 솔루션 주입 및 이후이 고기 때문에 비정상적인 소변 전송 쌓아 소변의 보조 효과 hydronephrosis 및 hydroureter 같은 고기 명확 하 게 관찰 하지는. UPJO 또는 UVJO, 같은 장애물에서이 고기 발포 신장, ureters, E18.5에서 분명 하다. E19.5 또는 신생아 강아지에 태아의 큰 신장은 E18.5에?...

비뇨 기 시스템은 신장, ureters 및 일반적인 방광과 요도의 한 쌍의 구성 됩니다. 비뇨 기 시스템의 주요 기능은 혈액의 물과 전해질 균형을 관리 하 여 신체의 항상성 유지 하는 것입니다. 신장 혈액 제어 전해질 농도 산 성-기본 균형을 필터링 하 고 초과 물과 용액 및 대사 산물 등 폐기물을 배설 하는 소변 생산. 소변은 신장 신장 골반에서 ureter 그것 저장 되 고 궁극적으로 요도1통해 제거 단방향 방식으로 bladderin 통해 다음 수송 된다.
ureters는 직선 튜브 nephric 덕트에서 발생. 마우스에서 배아 일 10.5 (E10.5)에서 nephric 덕트에서 신진, 후 ureter 스토킹 elongates 및 E12.5 및 E16.5 라는 마우스 사이 침투성은 urothelium 다층 구조로 차별화. Ureter 줄기 주변 중간 엽 세포 또한 내부 stromal 세포, 중간 두께 평활 근 세포, 그리고 외부 adventitial 섬유 아 세포로 구성 된 3 개의 층으로 구분 됩니다. 신장 골반에서 시작 ureteral 연동 파도 ureter 소변2,3, E16.5 마우스1부터 생산 되는 수송 방광 벽의 평활 근 층을 통해 전파 됩니다.
신장 및 요로 (CAKUT)의 선천적인 이상이 가장 빈번한 유전 질병, 인간 태아1,4의 약 1%에 중 고 고기 hydronephrosis 및 hydroureter 등의 다양 한 구성. 고 신장 ureter 소변의 비정상적인 축적 hydronephrotic 신장 및 hydroureter 형성에 결과. Hydronephrosis 또는 hydroureter 형성의 한 원인은 요로 방해 이다. Ureteropelvic 접속점 방해 (UPJO) 인접 ureter hydronephrosis 및 인접 hydroureter angulation 또는 영구 접는5, 축소의 결과로 신장 골반 사이 방해 때문에 탈 선 소변 흐름에 의해 발생 6. 또한, 방광 벽 또는 생식 관의 원심 ureter 소성 삽입 ureterovesical 접속점 방해 (UVJO) 이라고 합니다. UVJO 또한 hydronephrosis와 hydromegaureter 형성7,8을 유도할 수 있다. 추가 ureterovesical 접합 (UVJ) 결함은 vesicoureteric 환류 (VUR). VUR는 UVJ에서 신장으로 방광에서 역행 소변 흐름에 의해 특징입니다. UVJO에 비해 perinatal 배아 VUR로 표시 되지 않습니다 분명히 팽창된 hydronephrotic 신장 이나 심한 hydroureter 표현 형9.
실험실 마우스에서 소변 신장 골반9에 메 틸 렌 블루 등 염료의 주입에 의해 시험 될 수 있다. Injectedmethylene 블루 솔루션 통해 ureter, 방광으로 신장 골반에서 소변 궤적을 추적 한다. Hydronephrosis 염료는 신장에서의 확장에 의해 인식 될 수 있다. UPJO 발포 신장 골반5근 위 ureter에서 염료 흐름의 막힘으로 감지할 수 있습니다. 어떤 팽창 확대 직경으로 표시 ureter의 hydroureter의 예를 보여 줍니다. 마지막으로, 염료 축적 방광 벽에 또는 생식 관의 사이트에서 UVJO 발포 hydronephrotic 신장으로 나타내고 크기 hydromegaureter7,10. VUR를 검출 하기 위하여 염료 솔루션은 방광에 주입 하 고 후속 역행 흐름 신장9에서 모니터링 됩니다.
여기, 주 태아의 신장 골반에 methylene 청색 염료 주입에 대 한 프로토콜 제공 됩니다. 이 프로토콜 통해 ureter, 방광으로 신장 골반에서 소변 흐름의 추적을 허용 하 고 UPJO 또는 UVJO와 같은 잠재적인 요로 분기점 장애물을 확인 합니다.

<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

요로 분기점 방해 결함은 선천적인 변형을 유도 hydronephrosis 및 hydroureter. Murine 요로 분기점 방해 결함 비뇨 기 시스템 내에서 메 틸 렌 블루 염색 흐름을 추적 하 여 평가 될 수 있다. Methylene 청색 염료 perinatal 배아 신장의 신장 골반에 주입 하 고 염료 흐름은 액체 정역학 압력을 적용 한 후 신장 ureter 통해 및 방광 루멘에의 신장 골반에서 모니터링 합니다. 염료 축적 정상적인 perinatal 요로, 방광 루멘에 분명 있을 것입니다 하지만 요로 문제 발생 하는 경우 신장 골반 사이 비정상적인 ureter의 강요 될 것 이다. 이 방법은 요로 분기점 장애물 확인 및 hydronephrosis 및 hydroureter의 시각화를 촉진 한다. 이 원고는 요로 분기점 장애물을 확인 하 신 골반에 methylene 청색 염료 주입에 대 한 프로토콜을 설명 합니다.

신장과 더 낮은 오 줌 시스템 등 간, 대 장 등 대부분의 다른 내부 장기에 있습니다. 제거한 후에 이러한 다른 내부 장기, 신장, ureters과 단일 방광의 쌍 표시 (그림 1A)는. 성공 해 부에 신장 골반에 주입 하는 염료 ureter 통해 방광으로 신장에서 흐름 것입니다. 신장 골반 ureter는 신장에서의 깔때기 모양의 동 공이 확장 되어 근 위 부분 이다. 따라서, 염?...

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

Methylene 청색 염료 주입 신장 골반으로 마우스 배아 요로 개발 하는 동안 요로 분기점 방해 결함의 평가 촉진 한다. 여기, 신장 골반에 methylene 청색 염료 주입에 대 한 프로토콜을 설명 합니다.

Methylene 청색 색소를 주입 하 여 요로 분기점 방해 결함 평가

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 조회수.  National Institutes of Health. Methylene 청색 염료 주입 신장 골반으로 마우스 배아 요로 개발 하는 동안 요로 분기점 방해 결함의 평가 촉진 한다. 여기, 신장 골반에 methylene 청색 염료 주입에 대 한 프로토콜을 설명 합니다.

비디오: 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.

마우스 블레더 월 주입

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

연구

의학

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).

유방암 센티넬 노드 생검에 있는 단 하나 기술로 1% 메틸렌 청색염의 응용

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 ...

암 연구학

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.

각종 설치류 모델에 Cystometric 분석을 사용하여 방광 증원을위한 Biomaterials 평가

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

생체공학

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.

광 음향 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 ...

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.

미묘한 Endometriosis의 Intraoperative 감지 : 복막 무결성의 손실과 관련된 골반 통증의 감지 및 치료를위한 소설 패러다임

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.

요로의 프로브 기반 공 촛점 레이저 Endomicroscopy : 기술

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.

성인 남성 마우스 transurethral 주입 절차

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

면역학 및 감염병학

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).

요로 기능 검사를 위한 성인 남성 요로의 초음파 검사

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.

2광자 현미경을 사용하여 만성 일방적 요관 폐쇄가 사구체 과정에 미치는 영향을 정량화

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.

가역적 일방적 요관 폐색의 미성숙한 쥐 모델(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 ...