Gli autori desiderano ringraziare Mauna Kea Tecnologie per il supporto tecnico. Gli autori hanno anche ringraziare Shelly Hsiao per l&#39;assistenza tecnica e Kathleen E. Mach per la revisione critica. Questo lavoro è stato in parte finanziato dal NIH R01CA160986 a JCL

1. Preparazione del paziente <ol><li> Il consenso del paziente in programma per la cistoscopia diagnostica e altre procedure endourologiche come resezione transuretrale del tumore della vescica (TURBT) per CLE. Includere nella consenso una descrizione dell&#39;uso di fluoresceina intravescicale e / o endovenosa come agente di contrasto. Informarsi storia della reazione di ipersensibilità ad fluoresceina. </li><li> Paziente viene posizionato per cistoscopia (tipicamente in posizione litotomica) e preparato in modo sterile. </li><li> Procedere con lo standard cistoscopia a luce bianca (WLC) con un cistoscopio rigido o flessibile attraverso l&#39;uretra. </li><li> Indagine tutte le regioni della vescica in luce bianca (Figura 1) e annotare le regioni di interesse per le indagini CLE. </li><li> Oltre al WLC, la vescica può essere ripreso con altre modalità di imaging ottico comprendente macroscopici cistoscopia a fluorescenza (Figura 2A) o imaging banda stretta (Figura 2B) per identify ulteriori aree di sospetto seguita da caratterizzazione con CLE. </li><li> Utilizzare un elettrodo di cauterizzazione o loop resezione per inserire un segno piccolo cauterio adiacenti alle regioni di interesse, che saranno entrambi impressi e biopsia per la conferma patologica. Il marchio cauterizzazione facilita ri-localizzazione per la successiva CLE imaging e biopsia. </li></ol> 2. Di contrasto <ol><li> Instillazione intravescicale di mezzo di contrasto <ol><li> Preparare diluendo clinica sodio fluoresceina grade 10% in soluzione salina normale (0,9% NaCl) alla concentrazione desiderata. Per esempio, preparare 400 ml di 0,1% fluorescina diluendo 4 ml di 10% fluoresceina in 396 ml di soluzione salina normale. </li><li> Inserire un catetere sterile in vescica urinaria e infondere agente di contrasto diluito per gravità utilizzando un catetere da 60 ml-siringa. </li><li> Fissare il catetere urinario per contenere l&#39;agente di contrasto interiore per 5 min. </li><li> Scolare agente di contrasto di vescica. Usa la 60 catheter-siringa per lavare contrasto residua dalla vescica con soluzione fisiologica, circa 240-300 ml. </li><li> Procedere alla CLE imaging. </li></ol></li><li> L&#39;iniezione endovenosa di fluoresceina <ol><li> Disegna 1,0 ml di fluoresceina nella siringa. Collegare la siringa per iniettare l&#39;accesso per via endovenosa e 0,5-1,0 ml di fluorescina per via endovenosa in bolo. Lavare linea con soluzione fisiologica. Questo viene fatto tipicamente dal team di anestesia in sala operatoria. </li><li> Procedere alla CLE imaging. </li></ol></li></ol> 3. CLE Imaging <ol><li> Rimuovere il sterilizzato CLE immagini della sonda (Cellvizio) dalla confezione e collegarlo all&#39;unità di scansione laser (Mauna Kea Technologies, Parigi, Francia) </li><li> Riscaldare e calibrare la sonda immagini seguendo le istruzioni del produttore. </li><li> Reinserire il cistoscopio o resettoscopio nella vescica. Utilizzare uno 0 lente gradi con il cistoscopio rigido. </li><li> Far avanzare il CLE immagini della sonda lungo il working canale del cistoscopio con la punta appena oltre il cistoscopio. </li><li> In luce bianca, individuare le regioni precedentemente contrassegnati di interesse. Utilizzare ulteriori irrigazione normale soluzione salina, se necessario per la visualizzazione della mucosa e mantenere moderata distensione della vescica. </li><li> Manipolare CLE immagini della sonda con la mano dell&#39;operatore per contatto diretto en face delle regioni di interesse. Per sezionamento ottico delle regioni di interesse, delicatamente aumentare e diminuire la pressione alla sonda di imaging restando a contatto diretto. <ol><li> Per raggiungere i tumori situati nella vescica anteriore, uno standard Albarran ponte può facilitare deflessione della sonda di imaging per il contatto diretto con la regione di interesse (Figura 2C). </li></ol></li><li> Regioni comuni di interesse tumore papillare, non papillare del tumore, la transizione tra mucosa normale e tumore, e chiazze eritematose, che possono essere esposte utilizzando via endovesico o la somministrazione per via endovenosa di fluoresceina. Fluoresceina endovenosa è necessaria per l&#39;imaging del letto resezione, uretra prostatica e uretra peniena. </li><li> Mentre la sonda esegue la scansione di immagini regione di interesse, di registrare le immagini per le analisi future. Nota generale morfologia cellulare, confine, l&#39;organizzazione, e la presenza di neo-angiogenesi. </li><li> Al termine dell&#39;imaging, rimuovere CLE immagini sonda dal canale di lavoro. </li><li> Se lo si desidera, ottenere freddo tazza biopsie dalle regioni corrispondenti di interesse che sono stati analizzati da CLE. Utilizzare marcature cauterio come punti di riferimento per la co-registrazione delle aree sotto forma di immagini che vengono sottoposti a biopsia. </li><li> Completa TURBT per routine. Nei pazienti che hanno ricevuto per via endovenosa di fluoresceina, considerare l&#39;utilizzo di resettoscopi flusso continuo per migliorare la visualizzazione durante la resezione, come fluoresceina viene escreto dal rene in vescica di circa 5 minuti dopo la somministrazione per via endovenosa. </li><li> Recupera le registrazioni di immagine salvati da confocale processore e conduct analisi dell&#39;immagine. </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. 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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. 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Nessun conflitto di interessi dichiarati.

Raggiungere e mantenere solidi contatti en face tra la sonda di imaging e la mucosa della vescica è il passo più importante per acquisire una qualità ottimale dell&#39;immagine. C&#39;è approssimativamente 3-5 apprendimento paziente di sviluppare l&#39;abilità di manipolare la sonda imaging e tenere la sonda costante durante l&#39;acquisizione dell&#39;immagine. Inoltre, poiché questo procedimento è condotto in vivo, i movimenti del paziente (cioè respiratoria) e pulsazioni vascolari p...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Cellvizio Serie 100 </td><td> Mauna Kea Technologies </td><td> Serie 100 </td><td> Include processore confocale e LSU: F400-v2 a 488 nm </td></tr><tr><td> Cellvizio confocale Miniprobe </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

Probe-based laser confocale endomicroscopy (CLE) è una tecnologia emergente di imaging ottico che permette in tempo reale in microscopia in vivo di superfici mucose durante il normale endoscopia. Con applicazioni attualmente nelle vie respiratorie e gastrointestinali 1, 2-6 CLE è stato analizzato nel tratto urinario per la diagnosi del cancro della vescica. 7-10 morfologia cellulare e microarchitettura del tessuto può essere risolto con risoluzione scala micron in tempo reale, oltre a l&#39;imaging dinamico del sistema vascolare normale e patologico. 7 La sonda basata CLE sistema (Cellvizio, Mauna Kea Technologies, Francia) costituita da una sonda a fibre ottiche di imaging riutilizzabile accoppiato ad un&#39;unità di scansione laser 488 nm. La sonda di imaging viene inserito nei canali di lavoro standard, endoscopi flessibili e rigidi. Un endoscopio basato CLE sistema (Optiscan, Australia), in cui il functionalit endomicroscopy confocaley è integrato sulla endoscopio, è utilizzato anche nel tratto gastrointestinale. Data la portata diametro più grande, tuttavia, applicazione nel tratto urinario è attualmente limitato all&#39;uso ex vivo. 11 acquisizione immagine confocale viene fatto attraverso il contatto diretto della sonda imaging con il tessuto bersaglio e registrata come sequenze video. Come nel tratto gastrointestinale, endomicroscopy del tratto urinario richiede un exogenenous contrasto agente più comunemente fluoresceina, che può essere somministrato per via endovenosa o per via endovescicale. Somministrazione endovescicale è un metodo consolidato per introdurre agenti farmacologici localmente con minima tossicità sistemica che è unico per il tratto urinario. Fluoresceina rapidamente le macchie della matrice extracellulare e ha un profilo di sicurezza. Imaging 12 sonde di diversi diametri garantire la compatibilità con endoscopi di calibro diversi. Fino ad oggi, 1,4 e 2,6 millimetri sonde sono state valutate con il flessibile e ricistoscopia gid 10. recente disponibilità di un &lt;1 mm di imaging sonda 13 si apre la possibilità di CLE nel tratto urinario superiore durante ureteroscopia. Cistoscopia a fluorescenza (cioè diagnosi fotodinamica) e l&#39;imaging a banda stretta sono ulteriori endoscopio a base di modalità di imaging ottico 14 che possono essere combinati con CLE per raggiungere l&#39;imaging multimodale del tratto urinario. In futuro, CLE può essere accoppiato con mezzi di contrasto molecolari come peptidi contrassegnati in modo fluorescente 15 e anticorpi per l&#39;imaging endoscopico di processi di malattia con specificità molecolare.

CLE immagini sono salvate come file di sequenze di video in scala di grigi a 12 fotogrammi al secondo. L&#39;interpretazione dell&#39;immagine avviene in tempo reale e possono essere utilizzati per influenzare il processo decisionale clinico in un protocollo sperimentale. Analisi offline, che include la revisione della sequenza video, un&#39;ulteriore elaborazione delle immagini come mosaicatura, 7 e confronto con standard di patologia, è importante durante la fase di apprendimento associata con la tecnologi...

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

Sonda basata endomicroscopy laser confocale consente in tempo reale la microscopia del tratto urinario umano durante cistoscopia, fornendo dinamico, imaging intravitale di stati patologici come il cancro della vescica con risoluzione cellulare. Endomicroscopy può aumentare l&#39;accuratezza diagnostica di livello luce bianca endoscopia e fornire orientamenti immagine intraoperatoria per migliorare la resezione chirurgica.

Probe-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

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JoVE Journal

Medicine

20.7K Views.  Stanford University School of Medicine. Sonda basata endomicroscopy laser confocale consente in tempo reale la microscopia del tratto urinario umano durante cistoscopia, fornendo dinamico, imaging intravitale di stati patologici come il cancro della vescica con risoluzione cellulare. Endomicroscopy può aumentare l'accuratezza diagnostica di livello luce bianca endoscopia e fornire orientamenti immagine intraoperatoria per migliorare la resezione chirurgica.

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.

Analisi biofilm orale di espansori palatali da Fluorescence In Situ-ibridazione e microscopia confocale a scansione laser

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

Ricerca

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.

Vescica muscolo liscio Striscia contrattilità come un metodo per valutare Lower Urinary Tract Farmacologia

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 Bengala Photothrombosis da confocale Optical Imaging<em&gt; In Vivo</em&gt;: Un modello di singolo vaso 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.

Utilizzando una tecnica di laminazione per l&#39;esecuzione di Microscopia confocale della sclera umana


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.

Metalli di imaging in tessuto cerebrale di Laser Ablation - plasma accoppiato induttivamente - spettrometria di massa (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.

Spaziali quantificazione delle droghe nelle lesioni di tubercolosi polmonare di Laser Capture Microdissection liquido cromatografia spettrometria di massa (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).

Ultrasonografia del tratto urinario maschile adulto per test funzionali urinari

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.

Modifica tecnica del Terminal Ureter durante la Nefroroureterectomia laparoscopica transperitoneale per il carcinoma uroteliale superiore urinario

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.

Imaging perfusione Doppler laser nell'hindlimb del mouse

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.

Caratterizzazione di una nuova tecnica di coltura retinica organotipica umana

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