Les auteurs tiennent à remercier Mauna Kea Technologies pour le support technique. Les auteurs remercient également Shelly Hsiao pour l&#39;assistance technique et Kathleen E. Mach pour un examen critique. Ce travail a été soutenu en partie par le NIH R01CA160986 de JCL

1. Préparation du patient <ol><li> Le consentement du patient prévu pour la cystoscopie de diagnostic et d&#39;autres procédures endo-urologiques telles que la résection transurétrale de la tumeur vésicale (TURBT) pour CLE. Inclure dans l&#39;autorisation d&#39;une description de l&#39;utilisation de la fluorescéine intravésicale et / ou intraveineuse comme l&#39;agent de contraste. Informez-vous des antécédents de réaction d&#39;hypersensibilité à la fluorescéine. </li><li> Patient est positionné pour la cystoscopie (généralement en position gynécologique) et préparé de façon stérile. </li><li> Procéder à la norme cystoscopie en lumière blanche (WLC) avec un cystoscope rigide ou flexible à travers l&#39;urètre. </li><li> Enquête toutes les régions de la vessie sous une lumière blanche (figure 1) et notez régions d&#39;intérêt pour l&#39;enquête CLE. </li><li> En plus de WLC, la vessie peut être visualisée à l&#39;aide d&#39;autres modalités d&#39;imagerie optiques macroscopiques dont la fluorescence cystoscopie (figure 2A) ou l&#39;imagerie à bande étroite (Figure 2B) Numéro d&#39;entify d&#39;autres domaines de suspicion suivie par la caractérisation des CLE. </li><li> Utiliser une électrode de cautérisation ou anse de résection pour placer une marque cautère petite adjacente aux régions d&#39;intérêt qui seront tous deux imagés et une biopsie de confirmation pathologique. La marque cautérisation facilite re-localisation pour la suite CLE imagerie et la biopsie. </li></ol> 2. Agent de contraste <ol><li> Instillation intravésicale d&#39;agent de contraste <ol><li> Préparer par dilution clinique de sodium de qualité fluorescéine à 10% dans une solution saline normale stérile (NaCl 0,9%) à la concentration souhaitée. Par exemple, préparer 400 ml de fluorescéine 0,1% en diluant 4 ml de fluorescéine à 10% dans 396 ml de solution saline normale. </li><li> Insérer un cathéter stérile dans la vessie urinaire et insuffler un agent de contraste dilué par gravité à l&#39;aide d&#39;un cathéter 60 ml-embout de seringue. </li><li> Fixer la sonde urinaire à demeure à tenir agent de contraste pendant 5 min. </li><li> Égoutter agent de contraste de la vessie. Utilisez le ca 60 mltheter-embout de la seringue pour rincer le contraste résiduelle de la vessie utilisant une solution saline normale, environ 240-300 ml. </li><li> Passez à l&#39;imagerie CLE. </li></ol></li><li> L&#39;injection intraveineuse de fluorescéine <ol><li> Aspirer 1,0 ml de fluorescéine dans la seringue. Fixez la seringue pour l&#39;accès intraveineux et injecter 0,5 - 1,0 ml de fluorescéine intraveineuse en bolus. Rincer ligne avec une solution saline normale. Cela se fait habituellement par l&#39;équipe d&#39;anesthésie en salle d&#39;opération. </li><li> Passez à l&#39;imagerie CLE. </li></ol></li></ol> 3. CLE Imaging <ol><li> Retirer la sonde d&#39;imagerie stérilisés CLE (Cellvizio) de son emballage et branchez l&#39;appareil à balayage laser (Mauna Kea Technologies, Paris, France) </li><li> Echauffez-vous et calibrer la sonde d&#39;imagerie en suivant les instructions du fabricant. </li><li> Réinsérez le cystoscope ou résectoscope dans la vessie. Utilisez un objectif de 0 degré avec le cystoscope rigide. </li><li> Faire progresser la CLE sonde d&#39;imagerie le long de la working canal du cystoscope avec la pointe juste au-delà du cystoscope. </li><li> En lumière blanche, localiser les régions précédemment marqués d&#39;intérêt. Utiliser une solution saline normale irrigation supplémentaire si nécessaire pour la visualisation de la muqueuse et de maintenir une distension de la vessie modérée. </li><li> Manipuler CLE sonde d&#39;imagerie avec la main de l&#39;opérateur pour un contact directe en face à des régions d&#39;intérêt. Sectionnement optique pour des régions d&#39;intérêt, doucement augmenter et diminuer la pression de la sonde d&#39;imagerie tout en maintenant le contact direct. <ol><li> Pour atteindre les tumeurs situées à la vessie antérieure, une norme Albarran pont peut aider à faciliter déviation de la sonde d&#39;imagerie pour un contact direct avec la région d&#39;intérêt (figure 2C). </li></ol></li><li> Régions d&#39;intérêt communs comprennent tumeur papillaire, non papillaire tumeur, la transition entre la muqueuse d&#39;apparence normale et tumorale, et des plaques érythémateuses, qui peuvent tous être visualisés en utilisant soit intraveineusessique ou l&#39;administration par voie intraveineuse de fluorescéine. Intraveineuse de fluorescéine est nécessaire pour l&#39;imagerie du lit de résection, de l&#39;urètre prostatique, et l&#39;urètre du pénis. </li><li> Comme la sonde d&#39;imagerie analyse d&#39;images région d&#39;intérêt record, pour une analyse ultérieure. Notez globale morphologie cellulaire, limite, l&#39;organisation et la présence de la néo-angiogenèse. </li><li> À la fin de l&#39;imagerie, retirez CLE sonde d&#39;imagerie de canal de travail. </li><li> Si vous le souhaitez, obtenir froid tasse de biopsies provenant de régions correspondantes d&#39;intérêt qui ont été analysés par CLE. Utiliser les marques de cautérisation des points de repère pour la co-registration des zones imagées qui sont biopsiés. </li><li> Remplissez TURBT par routine. Chez les patients qui ont reçu par voie intraveineuse de fluorescéine, pensez à utiliser résectoscopes à flux continu pour améliorer la visualisation lors de la résection, la fluorescéine est excrétée par le rein dans la vessie environ 5 minutes après l&#39;administration par voie intraveineuse. </li><li> Récupérer les enregistrements d&#39;images enregistrées à partir confocale processeur et propianalyse d&#39;image CT. </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>

Aucun conflit d&#39;intérêt déclaré.

L&#39;atteinte et le maintien de solides face de contact entre la sonde en imagerie et de la muqueuse de la vessie est l&#39;étape la plus critique dans l&#39;acquisition de la qualité d&#39;image optimale. Il ya environ une courbe d&#39;apprentissage 3-5 patiente de développer la dextérité pour manipuler la sonde d&#39;imagerie et de maintenir la sonde stable pendant l&#39;acquisition des images. En outre, comme cette procédure est menée in vivo, les mouvements respiratoires du patient...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Cellvizio 100 Series </td><td> Mauna Kea Technologies </td><td> Série 100 </td><td> Comprend un processeur confocale et LSU: F400-v2 à 488 nm </td></tr><tr><td> Cellvizio confocale minisonde </td><td> Mauna Kea Technologies </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

Sonde laser confocal à base endomicroscopie (CLE) est une technique d&#39;imagerie optique émergeant qui permet en temps réel en microscopie in vivo de la surface des muqueuses pendant endoscopie standard. Avec les applications en cours dans les voies respiratoires 1 et gastro-intestinal, 2-6 CLE a également été explorée dans les voies urinaires pour le diagnostic du cancer de la vessie. 7-10 morphologie cellulaire et tissulaire microarchitecture peuvent être résolus avec la résolution échelle du micron en temps réel, en plus de imagerie dynamique de la vascularisation normale et pathologique 7. La sonde à base CLE système (Cellvizio, Mauna Kea Technologies, France) est constitué d&#39;une sonde à fibre optique d&#39;imagerie réutilisable couplée à une unité de balayage laser 488 nm. La sonde d&#39;imagerie est inséré dans les canaux de travail de types endoscopes flexibles et rigides. Un endoscope basé CLE système (Optiscan, Australie), dans lequel le functionalit endomicroscopie confocaley est intégré sur l&#39;endoscope, est aussi utilisé dans le tractus gastro-intestinal. Compte tenu de la portée plus large diamètre, cependant, l&#39;application dans les voies urinaires est actuellement limité à l&#39;utilisation ex vivo 11. Acquisition d&#39;image confocale se fait par contact direct de la sonde d&#39;imagerie avec le tissu cible et enregistré sous forme de séquences vidéo. Comme dans le tractus gastro-intestinal, endomicroscopie de l&#39;appareil urinaire nécessite un agent de contraste exogenenous-fluorescéine plus souvent, ce qui peut être administré par voie intraveineuse ou par voie intravésicale. Administration intravésicale est une méthode bien établie pour introduire des agents pharmacologiques au niveau local avec une toxicité systémique minimale qui est unique à l&#39;appareil urinaire. Fluorescéine rapidement les taches de la matrice extracellulaire et a un profil de sécurité établi. 12 sondes d&#39;imagerie de différents diamètres permettre la compatibilité avec les endoscopes de calibre différents. À ce jour, 1,4 et 2,6 mm sondes ont été évalués avec flexible et ricystoscopie gid 10. disponibilité récente d&#39;un &lt;1 mm Sonde d&#39;imagerie 13 ouvre la possibilité de CLE dans le haut appareil urinaire au cours de l&#39;urétéroscopie. Fluorescence cystoscopie (c.-à-diagnostic photodynamique) et l&#39;imagerie à bande étroite existe d&#39;autres endoscope à base de modalités d&#39;imagerie optiques 14 qui peuvent être combinés avec des CLE pour atteindre imagerie multimodale des voies urinaires. À l&#39;avenir, CLE peut être couplé avec des agents de contraste moléculaires tels que les peptides marqués par fluorescence 15 et des anticorps pour l&#39;imagerie endoscopique de processus pathologiques avec une spécificité moléculaire.

CLE images sont enregistrées en niveaux de gris des fichiers de séquences vidéo à 12 images par seconde. L&#39;interprétation des images se fait en temps réel et peuvent être utilisés pour influer prise de décision clinique en vertu d&#39;un protocole expérimental. Analyse hors ligne, ce qui comprend l&#39;examen de la séquence vidéo, traitement d&#39;image supplémentaires telles que mosaïquage, 7 et comparaison avec la norme pathologie, jouent un rôle important pendant la phase de la courbe d&...

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

Sonde à base endomicroscopie confocale permet la microscopie en temps réel de l&#39;appareil urinaire humain durant la cystoscopie, fournissant dynamique, imagerie intravitale d&#39;états pathologiques tels que le cancer de la vessie avec une résolution cellulaire. Endomicroscopie peut augmenter la précision diagnostique de l&#39;endoscopie standard de la lumière blanche et donner des orientations pour améliorer l&#39;image peropératoire résection chirurgicale.

Basée sur sonde endomicroscopie confocale laser de l&#39;appareil urinaire: La Technique

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 vues.  Stanford University School of Medicine. Sonde à base endomicroscopie confocale permet la microscopie en temps réel de l'appareil urinaire humain durant la cystoscopie, fournissant dynamique, imagerie intravitale d'états pathologiques tels que le cancer de la vessie avec une résolution cellulaire. Endomicroscopie peut augmenter la précision diagnostique de l'endoscopie standard de la lumière blanche et donner des orientations pour améliorer l'image peropératoire résection chirurgicale.

Vidéo: 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.

Analyse orale de biofilm palatine Expandeurs par fluorescence hybridation in situ et la microscopie confocale à balayage laser

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

Recherche

Médecine

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.

Vessie muscle lisse bande de contractilité comme une méthode pour évaluer urinaires inférieures Pharmacologie

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.

Rose Bengale Photothrombosis par imagerie optique confocal<em&gt; In Vivo</em&gt;: Un modèle de l&#39;unité des maladies du navire

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.

En utilisant une technique de plastification pour effectuer la microscopie confocale de la sclérotique humaine


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.

Métaux d&#39;imagerie dans le tissu cérébral par Laser Ablation - plasma à couplage inductif - spectrométrie de masse (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.

Quantification spatiale des drogues dans les lésions de la tuberculose pulmonaire par Laser capturer Microdissection liquide chromatographie en spectrométrie de masse (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).

Ultrasonographie de l'appareil urinaire masculin adulte pour l'essai fonctionnel urinaire

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.

Modification technique de l'uretère terminal au cours du nephroureterectomie laparopique transpéritonéen total pour le carcinome urothélial des voies urinaires supérieures

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.

Laser Doppler Perfusion Imaging dans l’hindlimb souris

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.

Caractérisation d’une nouvelle technique de culture rétinienne organotypique humaine

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