저자는 기술 지원 마우나 케아 기술을 진심으로 감사드립니다. 저자는 또한 중요한 검토 기술 지원 및 캐서린 E. 마하에 대한 셸리 샤오 감사드립니다. 이 작업은 작업 제어 언어에 NIH R01CA160986에 의해 부분적으로 지원되었다

1. 환자 준비 <ol><li> 동의 환자는 진단 cystoscopy과 같은 CLE을위한 방광 종양 transurethral 절제술 (TURBT)과 같은 다른 endourological 절차를 예정. 동의에 대비 에이전트로 intravesical 및 / 또는 정맥 주사 fluorescein의 사용에 대한 설명을 포함합니다. fluorescein에 대한 과민성 반응의 역사를 문의 해주십시오. </li><li> 환자는 cystoscopy (일반적으로 쇄석술 위치에)에 위치하고 살균 방식으로 준비가되어 있습니다. </li><li> 요도를 통해 엄격한 또는 유연 cystoscope과 표준 흰색 빛 cystoscopy (WLC)를 진행합니다. </li><li> 하얀 빛 (그림 1) 아래에있는 방광의 모든 영역을 조사하고 CLE 조사에 대한 관심 지역을 확인합니다. </li><li> WLC뿐만 아니라, 방광은 형광 cystoscopy (그림 2A) 또는 ID에 좁은 밴드 영상 (그림 2B) 등 다른 매크로 광학 이미징 modalities과 이미징 할 수 있습니다CLE와 특성에 이어 의혹의 추가 영역을 entify. </li><li> 병적 확인 이미징과 검사 했어 될 것 모두 관심 지역에 인접한 작은 소작 표시를 할 수있는 소작 전극 또는 절제 루프를 사용합니다. 소작 마크는 다음 CLE 이미징 및 생검을 위해 다시 현지화를 용이하게한다. </li></ol> 2. 대비 에이전트 <ol><li> 대비 에이전트 Intravesical 떨어 뜨림 <ol><li> 원하는 농도로 멸균 일반 생리의 임상 학년 10 % fluorescein 나트륨 (0.9 % NaCl)을 diluting하여 준비합니다. 예를 들어, 정상적인 생리의 396 ML에 10 % fluorescein 4 ML을 diluting하여 0.1 % fluorescein 400 ML를 준비합니다. </li><li> 방광에 오줌 카테 테르를 살균를 삽입하고 60 ML 카테 테르 - 팁 주사기를 사용하여 중력에 의해 희석 대비 에이전트를 심어. </li><li> 5 분에 대비 에이전트 내주하시는을 보유 할 수있는 오줌 카테 테르를 고정. </li><li> 방광에서 대비 에이전트를 배수한다. 60 ML CA를 사용하여theter - 팁 주사기 240-300 ML에 대해, 정상적인 생리를 사용하여 방광에서 잔류 대비를 씻어합니다. </li><li> CLE 이미지로 진행합니다. </li></ol></li><li> fluorescein의 정맥 주사 <ol><li> 주사기로 fluorescein의 1.0 ML을 그립니다. 정맥 접근 주사기를 연결하고 0.5를 삽입 - fluorescein의 1.0 ML를 intravenously bolus로. 정상적인 생리와 라인을 플러시. 이것은 일반적으로 수술실에서 마취 팀에 의해 이루어집니다. </li><li> CLE 이미지로 진행합니다. </li></ol></li></ol> 3. CLE 이미징 <ol><li> 그 포장에서 살균 CLE 이미징 프로브 (Cellvizio)을 제거하고 레이저 스캐닝 유닛 (마우나 케아 기술, 파리, 프랑스)에 연결 </li><li> 몸을 따뜻하게하고 제조업체의 지침에 따라 이미징 프로브를 보정. </li><li> 방광에 cystoscope 또는 resectoscope를 다시 삽입합니다. 강성 cystoscope으로 0도 렌즈를 사용합니다. </li><li> 앨리스를 따라 CLE 이미징 프로브를 사전단지 cystoscope 이외의 팁과 cystoscope의 채널을 rking. </li><li> 하얀 빛에서 관심 이전에 표시된 영역을 찾습니다. 점막의 시각화를 위해 필요에 따라 추가 정상적인 생리 관개를 사용하여 방광의 중간 팽만를 유지하고 있습니다. </li><li> 관심 지역의 직접 실내 얼굴 연락처에 대한 운영자의 손으로 CLE 이미징 프로브를 조작. 관심 영역의 광학 sectioning를 들어, 부드럽게 증가와 직접 접촉을 유지하면서 이미징 프로브에 압력을 낮출 수 있습니다. <ol><li> 앞 방광에있는 종양에 도달하려면, 표준 Albarran 다리가 관심 지역 (그림 2C)와 직접 접촉에 대한 이미징 프로브의 편향을 촉진하는 데 도움이 될 수 있습니다. </li></ol></li><li> 관심의 일반적인 지역은 중 intrave를 사용하여 이미징 할 수 유두 종양, 비 유두 종양, 정상 나타나는 점막과 종양 사이의 전환 및 erythematous 패치를 포함sical 또는 정맥 주사 fluorescein 관리. 정맥 fluorescein은 절제 침대의 이미징, 전립샘 요도 및 음경 요도가 필요합니다. </li><li> 이미징 프로브는 미래 분석이자, 기록 이미지의 지역을 검색. 전체 세포 형태, 경계, 조직 및 신 혈관 신생의 존재를합니다. </li><li> 이미지가 완료되면 채널을 작동 CLE 이미징 프로브를 제거합니다. </li><li> 원하는 경우, CLE에 의해 스캔 된 관심 대응하는 지역에서 차가운 컵 biopsies를 구하십시오. 검사 했어 된 이미징 분야의 공동 등록 랜드 마크로서 소작 표시를 사용합니다. </li><li> 일상적인 당 TURBT를 완료합니다. 정맥 fluorescein을받은 환자에서 fluorescein은 정맥 주사 관리 후 약 5 분 방광에 신장으로 배설되기 때문에, 절제하는 동안 시각화를 개선하기 위해 지속적인 흐름 resectoscopes를 사용하시기 바랍니다. </li><li> 공 촛점 프로세서와 condu에서 저장된 이미지 레코딩를 검색중부 표준시 이미지 분석. </li></ol>

<ol>
	<li>Thiberville, L., 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=Human+in+vivo+fluorescence+microimaging+of+the+alveolar+ducts+and+sacs+during+bronchoscopy.">Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.</a> Eur. Respir. J. 33, 974-985 (2009).</li><li>Dunbar, K. B., Okolo, P., Montgomery, E., Canto, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+endomicroscopy+in+Barrett's+esophagus+and+endoscopically+inapparent+Barrett's+neoplasia:+a+prospective,+randomized,+double-blind,+controlled,+crossover+trial.">Confocal laser endomicroscopy in Barrett's esophagus and endoscopically inapparent Barrett's neoplasia: a prospective, randomized, double-blind, controlled, crossover trial.</a> Gastrointest. Endosc. 70, 645-654 (2009).</li><li>Buchner, A. M., 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=Comparison+of+probe-based+confocal+laser+endomicroscopy+with+virtual+chromoendoscopy+for+classification+of+colon+polyps.">Comparison of probe-based confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps.</a> Gastroenterology. 138, 834-842 (2010).</li><li>Pech, O., 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=Confocal+laser+endomicroscopy+for+in+vivo+diagnosis+of+early+squamous+cell+carcinoma+in+the+esophagus.">Confocal laser endomicroscopy for in vivo diagnosis of early squamous cell carcinoma in the esophagus.</a> Clin. Gastroenterol. Hepatol. 6, 89-94 (2008).</li><li>Goetz, M., 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=In+vivo+confocal+laser+endo+microscopy+of+the+human+liver:+a+novel+method+for+assessing+liver+microarchitecture+in+real+time.">In vivo confocal laser endo microscopy of the human liver: a novel method for assessing liver microarchitecture in real time.</a> Endoscopy. 40, 554-562 (2008).</li><li>Meining, A., 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=Detection+of+cholangiocarcinoma+in+vivo+using+miniprobe-based+confocal+fluorescence+microscopy.">Detection of cholangiocarcinoma in vivo using miniprobe-based confocal fluorescence microscopy.</a> Clin. Gastroenterol. Hepatol. 6, 1057-1060 (2008).</li><li>Wu, K., 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=Dynamic+real-time+microscopy+of+the+urinary+tract+using+confocal+laser+endomicroscopy.">Dynamic real-time microscopy of the urinary tract using confocal laser endomicroscopy.</a> Urology. 78, 225-231 (2011).</li><li>Sonn, G. A., 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=Optical+biopsy+of+human+bladder+neoplasia+with+in+vivo+confocal+laser+endomicroscopy.">Optical biopsy of human bladder neoplasia with in vivo confocal laser endomicroscopy.</a> J. Urol. 182, 1299-1305 (2009).</li><li>Sonn, G. A., 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=Fibered+confocal+microscopy+of+bladder+tumors:+an+ex+vivo+study.">Fibered confocal microscopy of bladder tumors: an ex vivo study.</a> J. Endourol. 23, 197-201 (2009).</li><li>Adams, W., 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=Comparison+of+2.6-+and+1.4-mm+imaging+probes+for+confocal+laser+endomicroscopy+of+the+urinary+tract.">Comparison of 2.6- and 1.4-mm imaging probes for confocal laser endomicroscopy of the urinary tract.</a> J. Endourol. 25, 917-921 (2011).</li><li>Wiesner, 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=Confocal+laser+endomicroscopy+for+the+diagnosis+of+urothelial+bladder+neoplasia:+a+technology+of+the+future?.">Confocal laser endomicroscopy for the diagnosis of urothelial bladder neoplasia: a technology of the future?.</a> BJU Int. 107, 399-403 (2011).</li><li>Wallace, M. B., 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=The+safety+of+intravenous+fluorescein+for+confocal+laser+endomicroscopy+in+the+gastrointestinal+tract.">The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract.</a> Aliment. Pharmacol. Ther. 31, 548-552 (2010).</li><li>Konda, V. J. A., 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=First+assessment+of+needle-based+confocal+laser+endomicroscopy+during+EUS-FNA+procedures+of+the+pancreas+(with+videos.">First assessment of needle-based confocal laser endomicroscopy during EUS-FNA procedures of the pancreas (with videos.</a> Gastrointest. Endosc. 74, 1049-1060 (2011).</li><li>Liu, J. -. J., Droller, M., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Optical+Imaging+Technologies+in+Bladder+Cancer:+Considerations+and+Perspectives.">New Optical Imaging Technologies in Bladder Cancer: Considerations and Perspectives.</a> J. Urol. 188, 361-368 (2012).</li><li>Hsiung, P. -. L., 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=Detection+of+colonic+dysplasia+in+vivo+using+a+targeted+heptapeptide+and+confocal+microendoscopy.">Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy.</a> Nat. Med. 14, 454-458 (2008).</li></ol>

관심 없음 충돌이 선언 없습니다.

취득 및 이미징 프로브 및 방광 점막 사이의 견고한 전용 얼굴 연락처를 유지하는 최적의 이미지 품질을 확보에서 가장 중요한 단계입니다. 이미징 프로브를 조작하고 이미지 수집하는 동안 탐사선이 안정적으로 보유 할 민첩성을 개발하는 3-5 환자 학습 곡선은 약이 있습니다. 이 절차는 생체로 진행되기 때문에 또한, 환자의 움직임 (즉, 호흡기)와 혈관 pulsations은 방광과 이?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> Cellvizio 100 시리즈 </td><td> 마우나 케아 기술 </td><td> 100 시리즈 </td><td> 488 nm의에서 F400-V2 : 공 촛점 프로세서와 LSU을 포함 </td></tr><tr><td> Cellvizio 공 촛점 Miniprobe </td><td> 마우나 케아 기술 </td><td> Gastroflex UHD </td><td></td></tr><tr><td> AK-형석 10% </td><td> Akorn 주식회사 </td><td> NDC 17478-253-10 </td><td></td></tr></tbody></table>

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

프로브 기반 공 촛점 레이저 endomicroscopy (CLE)는 표준 내시경 중에 점막 표면의 생체 현미경에 실시간있게 신흥 광학 이미징 기술입니다. 현재 한 호흡기와 위장 책자에서 응용 프로그램을, 2-6 CLE는 방광 암 진단 병명은 요로에 탐구되었습니다. 7-10 세포 형태와 조직 마이크로 아키텍처는뿐만 아니라, 실시간으로 마이크론 규모의 해상도로 해결할 수 있습니다 정상과 병적 vasculature의 동적 이미지. 7 프로브 기반 CLE 시스템 (Cellvizio, 마우나 케아 기술, 프랑스)는 488 nm의 레이저 스캐닝 유닛에 연결된 재사용 fiberoptic 이미징 프로브로 구성되어 있습니다. 이미징 프로브는 표준 유연하고 단단한 내시경의 워킹 채널에 삽입됩니다. 내시경 기반 CLE 시스템 (Optiscan, 호주) 된 공 촛점 endomicroscopy의 functionalitY는 내시경에 통합되어, 또한 위장관에서 사용됩니다. 더 큰 범위의 직경을 감안할 때, 그러나, 요로의 응용 프로그램은 현재 전 생체 사용으로 제한됩니다. 11 공 촛점 이미지 수집이 대상 조직과 이미징 프로브의 직접 접촉을 통해 수행 및 비디오 시퀀스로 기록됩니다. 위장관에서와 같이 요로의 endomicroscopy는 intravenously 또는 intravesically 관리 할 수​​있는 exogenenous 대비 에이전트 - 가장 일반적으로 fluorescein을 필요로합니다. Intravesical 관리는 요로에 고유 최소한의 전신 독성과 로컬 약리 에이전트를 소개하는 잘 알려진 방법입니다. Fluorescein는 빠른 속도로 얼룩 세포 외 기질을 다양한 직경의 12. 이미징 프로브가 다른 구경 내시경와의 호환성을 가능하게 설립 안전 프로파일이 있습니다. 현재까지, 1.4 및 2.6 mm 프로브는 유연하고 리와 평가 된GID의 cystoscopy이 있습니다. &lt;1mm 이미징 프로브 (13)의 최근 10 여부는 ureteroscopy 동안 상위 비뇨기 기관에 CLE의 가능성을 열어 준다. 형광 cystoscopy (예 : photodynamic 진단)와 좁은 밴드 영상은 요로의 multimodal 영상을 달성하기 위해 CLE와 결합 될 수있는 추가 내시경 기반의 광학 이미징 modalities 14아르. 앞으로 CLE는 이러한 분자 특이성과 질병 프로세스의 내시경 영상을위한 휘황 표시 펩티드 15 항체 등의 분자 대비 에이전트와 커플 링 될 수있다.

초당 12 프레임의 비디오 시퀀스 파일을 그레이 스케일로 CLE 이미지는 저장됩니다. 이미지 해석은 실시간으로 수행되고 investigational 프로토콜에서하는 임상 의사 결정에 영향을 미칠하는 데 사용할 수 있습니다. 비디오 시퀀스의 예 mosaicing, 7 및 표준 병리와 비교하여 같은 추가 이미지 처리를 검토 포함 오프라인 분석, 기술과 관련된 학습 곡선 단계에서 중요합니다. 인물 1B와 1C는 각각 in...

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

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

프로브 기반 공 촛점 레이저 endomicroscopy은 휴대 해상도 방광 암과 같은 병적 인 상태의 동적, intravital 이미지를 제공 cystoscopy 동안 인간의 비뇨기 기관의 실시간 현미경 할 수 있습니다. Endomicroscopy 표준 하얀 빛 내시경의 진단 정확도를 증가, 외과 절제술을 향상시키기 위해 intraoperative 이미지지도를 제공 할 수 있습니다.

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 ...

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

Research

JoVE Journal

Medicine

20.7K 조회수.  Stanford University School of Medicine. 프로브 기반 공 촛점 레이저 endomicroscopy은 휴대 해상도 방광 암과 같은 병적 인 상태의 동적, intravital 이미지를 제공 cystoscopy 동안 인간의 비뇨기 기관의 실시간 현미경 할 수 있습니다. Endomicroscopy 표준 하얀 빛 내시경의 진단 정확도를 증가, 외과 절제술을 향상시키기 위해 intraoperative 이미지지도를 제공 할 수 있습니다.

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

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

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

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

형광에 원위치 하이브리드화 및 공촛점 레이저 스캐닝 현미경에 의해 맛이 좋은 확장기의 사정 Biofilm 분석

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

연구

의학

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

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

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

하부 요로 약리학을 평가하는 방법으로 방광 평활근 스트립 수축성

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

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

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

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

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

공 초점 광학 이미징에 의해 로즈 벵갈 Photothrombosis<em&gt; 생체</em&gt; : 단일 선박 스트로크의 모델

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

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

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

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

인간 공막의 공 초점 현미경을 수행하기 위해 코팅 기술을 사용하여


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

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

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

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

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

유도 결합 플라즈마 - - 레이저 박리에 의한 뇌 조직의 이미징 금속 질량 분석 (LA-ICP-MS)

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

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

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

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

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

레이저에 의해 폐 결핵 병 변에서 약의 공간 정량화 캡처 서 액체 착 색 인쇄기 질량 분석 (LCM-LC/MS)

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

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

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

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

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 ...

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

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

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

상부 요로 비뇨기과 암에 대한 총 복강경 복강경 신장 절제술 동안 터미널 요관의 기술적 수정

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

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

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

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

마우스 힌드림에 있는 레이저 도플러 관류 화상 진찰

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

Characterization of a Novel Human Organotypic Retinal Culture Technique

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

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

소설 인간 오르가노피 망막 문화 기술의 특성화

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