Los autores desean agradecer a Mauna Kea Technologies para obtener asistencia técnica. Los autores también agradecen a Shelly Hsiao para la asistencia técnica y Kathleen E. Mach para su examen crítico. Este trabajo fue apoyado en parte por el NIH R01CA160986 a JCL

1. Preparación del paciente <ol><li> El consentimiento del paciente programado para la cistoscopia diagnóstica y otros procedimientos endourológicas como la resección transuretral de tumor vesical (RTU) para el CLE. Incluir en el consentimiento una descripción del uso de fluoresceína intravesical y / o intravenosa como el agente de contraste. Investigar la historia de reacción de hipersensibilidad a la fluoresceína. </li><li> El paciente es colocado para la cistoscopia (típicamente en posición de litotomía) y preparado de forma estéril. </li><li> Proceda con la norma cistoscopia con luz blanca (WLC) con un cistoscopio rígido o flexible a través de la uretra. </li><li> Supervisar todas las regiones de la vejiga con luz blanca (Figura 1) y observar las regiones de interés para la investigación CLE. </li><li> Además de WLC, la vejiga puede formar una imagen con otras modalidades de formación de imágenes macroscópicas ópticos, incluyendo fluorescencia cistoscopia (Figura 2A) o formación de imágenes de banda estrecha (Figura 2B) para Identificaciónentify áreas adicionales de sospecha seguido de caracterización con CLE. </li><li> Utilice un electrodo de cauterización o resección con asa para colocar una marca de cauterización pequeña adyacente a las regiones de interés que ambos serán fotografiados y biopsia para la confirmación patológica. La marca de cauterización facilita la re-localización para la posterior obtención de imágenes CLE y biopsia. </li></ol> 2. Agente de contraste <ol><li> Instilación intravesical del agente de contraste <ol><li> Preparar por dilución de grado clínico de sodio 10% de fluoresceína en solución salina normal estéril (0,9% NaCl) a la concentración deseada. Por ejemplo, preparar 400 ml de 0,1% de fluoresceína diluyendo 4 ml de 10% de fluoresceína en 396 ml de solución salina normal. </li><li> Insertar un catéter urinario en la vejiga estéril e infundir agente de contraste diluido por gravedad usando un catéter de 60 ml jeringa de punta. </li><li> Sujetar el catéter urinario para mantener permanente del agente de contraste durante 5 min. </li><li> Escurrir agente de contraste de vejiga. Utilice la 60 ml catheter-punta de la jeringa para vaciar este contraste residual de la vejiga con solución salina normal, alrededor de 240-300 ml. </li><li> Proceda a CLE imágenes. </li></ol></li><li> La inyección intravenosa de fluoresceína <ol><li> Dibujar 1,0 ml de fluoresceína en la jeringa. Coloque la jeringa para inyectar el acceso intravenoso y 0,5 a 1,0 ml de fluoresceína por vía intravenosa como un bolo. Enjuague línea con solución salina normal. Esto se hace típicamente por el equipo de anestesia en la sala de operaciones. </li><li> Proceda a CLE imágenes. </li></ol></li></ol> 3. CLE imágenes <ol><li> Retire la sonda esterilizada CLE imágenes (Cellvizio) de su embalaje y conéctelo a la unidad de escaneo láser (Mauna Kea Technologies, París, Francia) </li><li> Calentar y calibrar la sonda de imagen, siguiendo las instrucciones del fabricante. </li><li> Vuelva a insertar el cistoscopio o resectoscopio en la vejiga. Utilice una lente de 0 grados con el cistoscopio rígido. </li><li> Avance el CLE imágenes de la sonda a lo largo del working canal del cistoscopio con la punta un poco más allá del cistoscopio. </li><li> Bajo la luz blanca, localizar las regiones previamente marcados de interés. El uso adicional de irrigación de solución salina normal como sea necesario para la visualización de la mucosa y mantener distensión moderada de la vejiga. </li><li> CLE manipular imágenes de la sonda con la mano del operador para el contacto directo en la cara de las regiones de interés. Para el seccionamiento óptico de las regiones de interés, suavemente aumentar y disminuir la presión a la sonda de formación de imágenes mientras se mantiene el contacto directo. <ol><li> Para llegar a los tumores localizados en el anterior de la vejiga, un puente estándar Albarran puede ayudar a facilitar la deflexión de la sonda de imagen para el contacto directo con la región de interés (Figura 2C). </li></ol></li><li> Regiones comunes de interés incluyen tumor papilar, tumor no papilar, la transición entre mucosa de aspecto normal y tumoral, y placas eritematosas, todos los cuales se pueden visualizar utilizando intraveSical o administración intravenosa de fluoresceína. Intravenosa de fluoresceína se requiere para la formación de imágenes del lecho de resección, la uretra prostática y la uretra del pene. </li><li> A medida que la sonda de imagen escanea región de las imágenes de interés de discos, para su posterior análisis. Tenga en cuenta la morfología celular global, la cobertura, la organización y la presencia de neo-angiogénesis. </li><li> Al término de formación de imágenes, eliminar CLE formación de imágenes de la sonda de canal de trabajo. </li><li> Si lo desea, obtener biopsias taza fría de las regiones correspondientes de interés que fueron analizados por CLE. Utilice las marcas de cauterización como puntos de referencia para el co-registro de las áreas con imagen que se biopsia. </li><li> Completar TURBT por rutina. En los pacientes que recibieron fluoresceína por vía intravenosa, considerar el uso de resectoscopios de flujo continuo para mejorar la visualización durante la resección, como la fluoresceína, se excreta por el riñón a la vejiga aproximadamente 5 min después de la administración intravenosa. </li><li> Recuperar guardados grabaciones de imágenes confocal de procesador y CONDUct análisis de imagen. </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>

No hay conflictos de interés declarado.

Lograr y mantener un contacto sólido entre la cara en la sonda de imagen y la mucosa de la vejiga es el paso más crítico en la adquisición de una calidad de imagen óptima. Hay aproximadamente una curva paciente 3-5 aprendizaje para desarrollar la destreza para manipular la sonda de imagen y de mantener la sonda estable durante la adquisición de imagen. Además, ya que este procedimiento se lleva a cabo in vivo, los movimientos del paciente (es decir, respiratorio) y pulsaciones vasculare...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Cellvizio 100 Series </td><td> Mauna Kea Tecnologías </td><td> 100 Series </td><td> Incluye procesador confocal y LSU: F400-v2 a 488 nm </td></tr><tr><td> Cellvizio Confocal minisonda </td><td> Mauna Kea Tecnologías </td><td> Gastroflex UHD </td><td></td></tr><tr><td> AK-FLUOR 10% </td><td> Akorn, Inc. </td><td> NDC 17478-253-10 </td><td></td></tr></tbody></table>

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

Sonda basada en láser confocal endomicroscopía (CLE) es una tecnología emergente que permite formación de imágenes ópticas en tiempo real de microscopía in vivo de las superficies mucosas durante la endoscopia estándar. Con las aplicaciones actualmente en los tractos respiratorio y gastrointestinal 1, 2-6 CLE también se ha explorado en el tracto urinario para el diagnóstico de cáncer de vejiga. 7-10 morfología celular y la microarquitectura del tejido se puede resolver con la resolución escala del micrón en tiempo real, además de formación de imágenes dinámica de la vasculatura normal y patológica. 7 La sonda basada en CLE sistema (Cellvizio, Mauna Kea Technologies, Francia) consiste en una sonda de imagen reutilizable fibra óptica acoplada a una unidad de exploración de 488 nm láser. La sonda de imagen se inserta en los canales de trabajo de estándar endoscopios flexibles y rígidos. Un endoscopio basado CLE sistema (Optiscan, Australia), en la que el functionalit endomicroscopía confocaly está integrado en el endoscopio, se utiliza también en el tracto gastrointestinal. Dado el diámetro mayor alcance, sin embargo, la aplicación en el tracto urinario se limita actualmente al uso ex vivo. 11 de adquisición de imagen confocal se realiza a través del contacto directo de la sonda de imagen, con el tejido diana y se registró como secuencias de vídeo. Como en el tracto gastrointestinal, endomicroscopía del tracto urinario requiere un agente de contraste exogenenous-más comúnmente fluoresceína, que se puede administrar por vía intravenosa o por vía intravesical. Administración intravesical es un método bien establecido para introducir agentes farmacológicos localmente con toxicidad sistémica mínima que es única para el tracto urinario. Fluoresceína rápidamente las manchas de la matriz extracelular y tiene un perfil de seguridad establecido. 12 sondas de imagen de varios diámetros permitir la compatibilidad con endoscopios de diferentes calibres. Hasta la fecha, 1,4 y 2,6 mm sondas han sido evaluadas con flexible y ricistoscopia gid. disponibilidad reciente de 10 a &lt;1 mm Sonda de imágenes 13 abre la posibilidad de CLE en el tracto urinario superior durante la ureteroscopia. Fluorescencia cistoscopia (diagnóstico fotodinámico es decir) y las imágenes de banda estrecha son adicionales basados ​​endoscopio modalidades de imágenes ópticas 14 que se pueden combinar con CLE para lograr imágenes multimodal del tracto urinario. En el futuro, CLE se puede acoplar con agentes de contraste molecular tales como péptidos marcados con fluorescencia 15 y anticuerpos para la obtención de imágenes endoscópicas de los procesos de enfermedad con especificidad molecular.

CLE imágenes se guardan como archivos de vídeo en escala de grises en la secuencia de 12 cuadros por segundo. Interpretación de las imágenes se realiza en tiempo real y puede ser utilizado para impactar toma de decisiones clínicas en virtud de un protocolo de investigación. Análisis fuera de línea, que incluye la revisión de la secuencia de vídeo, el procesamiento de imágenes adicionales, tales como mosaicos, 7 y la comparación con el estándar de patología, son importantes durante la fase de cur...

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

Basada en sondeos endomicroscopía láser confocal permite en tiempo real de la microscopía del tracto urinario humano durante la cistoscopia, proporcionando imágenes dinámicas, intravital de estados patológicos como el cáncer de vejiga con resolución celular. Endomicroscopía puede aumentar la precisión diagnóstica de la endoscopia estándar de luz blanca y proporcionar orientación intraoperatoria en la imagen para mejorar la resección quirúrgica.

Basada en sondeos endomicroscopía Láser Confocal del tracto urinario: la técnica

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 vistas.  Stanford University School of Medicine. Basada en sondeos endomicroscopía láser confocal permite en tiempo real de la microscopía del tracto urinario humano durante la cistoscopia, proporcionando imágenes dinámicas, intravital de estados patológicos como el cáncer de vejiga con resolución celular. Endomicroscopía puede aumentar la precisión diagnóstica de la endoscopia estándar de luz blanca y proporcionar orientación intraoperatoria en la imagen para mejorar la resección quirúrgica.

Video: 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.

Análisis de biofilm oral del paladar Expansores por fluorescencia de hibridación in situ y microscopía de escaneo láser confocal

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

Investigación

Medicina

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.

Vejiga contractilidad del músculo liso de Gaza como un método para evaluar tracto urinario inferior Farmacología

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.

Rosa de Bengala Photothrombosis por imagen óptica confocal<em&gt; En Vivo</em&gt;: Un modelo de solo recipiente Stroke

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.

Usando una técnica de laminación para realizar la microscopía confocal de la esclerótica humana


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.

Metales de imagen en el tejido cerebral por ablación con láser - plasma de acoplamiento inductivo - espectrometría de masas (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.

Cuantificación espacial de fármacos en lesiones de Tuberculosis pulmonar por Laser Capture Microdissection líquido cromatografía espectrometría de masas (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).

Ultrasonografía del tracto urinario masculino adulto para pruebas funcionales urinarias

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.

Modificación técnica del ureter terminal durante la nefroterectomía laparoscópica transperitoneal total para el carcinoma urotélial del tracto urinario superior

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.

Imágenes de perfusión doppler láser en el hindlimb del ratón

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.

Caracterización de una nueva técnica de cultivo de retina organotípica humana

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