Siamo in debito con la signora Ina Wesselman per la formazione e la pianificazione del personale OR.

1. Intraoperatoria Camera fluorescenza multispettrale <ol><li> Una misura sistema di telecamere è stato sviluppato presso sviluppato presso l&#39;Istituto per il biologico e Medical Imaging (IBMI, Università Tecnica / Helmholtz Zentrum di Monaco di Baviera, Germania) in stretta collaborazione con SurgOptix (SurgOptix Inc, Redwood Shores, CA, USA). L&#39;installazione del sistema di fluorescenza multispettrale fotocamera intraoperatoria è illustrato nella figura 2. </li><li> Luce passa attraverso un sistema di ottiche ed è separata in luce visibile, luce alla (fluorescenza) della lunghezza d&#39;onda di emissione e luce alla lunghezza d&#39;onda di eccitazione banda. Questi bundle vengono rilevati da telecamere CCD (CCD) (Figura 2). Multi-spettrale segnali provenienti da tutte le telecamere sono trattati al fine di correggere gli artefatti e la resa quantitativa vero fluorocromo bio-distribuzione. Colori e segnali di fluorescenza possono essere visualizzati come immagini separate su monitor esterno o sovrapposti in una sola immagine. </li><li> I filtri di eccitazione ed emissione della fotocamera sono impostate nel documento di misura per la raccolta a 750 nm e 800 ± 20 nm rispettivamente. </li></ol> 2. Ottica di contrasto Preparazione agente <ol><li> L&#39;agente di contrasto fluorescente verde indocianina (ICG, Pulsion AG, Monaco di Baviera, Germania) è preparato in condizioni sterili. Una concentrazione di 0,5 mg / ml viene utilizzato. E &#39;importante utilizzare acqua distillata sterile e non cloruro di sodio (NaCl), in quanto quest&#39;ultimo causerà l&#39;ICG di aggregare. </li><li> Dopo la preparazione, la soluzione deve essere conservata in un luogo buio e fresco per evitare il deterioramento veloce della fluorescenza di sbiancamento. Nota: da questo punto in avanti mantenere l&#39;ICG riparo dalla luce. </li></ol> 3. Intraoperatoria Imaging - Configurazione e Iniezione di ICG <ol><li> La telecamera è posizionata e avviato in sala operatoria prima dell&#39;intervento chirurgico per ridurre al minimo le interferenze con la procedura chirurgica standard, e collegata alla schermi ad alta definizione (Figura 2B). </li><li> La fotocamera è coperto in standard teleria sterile (Carl Zeiss Vision BV, Sliedrecht, Paesi Bassi, REF Drape OPMI 306071) dallo sperimentatore in sterile o abbigliamento. </li><li> Nota: si ha cura di utilizzare solo agenti disinfettanti chiara, colorata come agenti disinfettanti sono spesso autofluorescenti e potrebbe interferire con la procedura di imaging. Inoltre, teleria sterile e marcatori chirurgiche possono essere autofluorescenti. Si consiglia di testare tutti i materiali utilizzati nel campo OR e il sistema operativo di autofluorescenza prima della chirurgia. </li><li> L&#39;agente di contrasto fluorescente verde indocianina (ICG) è preparato in condizioni sterili. Una concentrazione di 0,5 mg / ml viene utilizzato. E &#39;importante utilizzare acqua distillata sterile e non NaCl, in quanto quest&#39;ultimo causerà l&#39;ICG di aggregare. Dopo la preparazione, la soluzione deve essere conservata in un luogo buio e fresco per evitare il deterioramento veloce della intensità di fluorescenza. Nota: si consiglia di cambiare i guanti dopo la preparazione ICG, come anche una piccola quantità di fuoriuscita di ICG può influenzare la qualità dell&#39;immagine. </li><li> La procedura di imaging inizia dopo l&#39;apertura dell&#39;addome. Quando l&#39;area di interesse è esposta, la fotocamera è manovrato nel campo operativo. Lo zoom e messa a fuoco vengono regolati in condizioni sterili. Le luci in sala operatoria sono spenti per un migliore rilevamento del segnale di fluorescenza. </li><li> 1,0 mL di 0,5 mg / ml ICG è mescolato con 1,0 mL dello standard colorante blu (blu di brevetto / bleu patente, Guerbet, Francia) in una siringa. Il chirurgo inietta il mezzo di contrasto in quattro quadranti intorno al tumore primario, evitando di versare l&#39;agente. Nota: in caso di fuoriuscita di ICG i guanti chirurgici, è necessario cambiare i guanti. Immagini fisse o video in tempo reale vengono acquisiti del flusso linfatico e l&#39;aspetto del nodo fluorescenti linfonodi sentinella (Figura 3). Tutte le immagini ei video vengono salvati direttamente sul computer. </li><li> Oltre a fluorescenza, il linfonodo sentinella è anche rilevata in base al protocollo standard, utilizzando una sonda gamma o mediante ispezione visiva per una colorazione blu, o entrambi. Il tracciante radioattivo viene generalmente somministrato un giorno prima dell&#39;intervento, dopo che un lymphoscintigram viene eseguita per la rilevazione di un SLN radioattivo. Questa è parte della procedura standard e non ha posto nel protocollo di imaging di fluorescenza. </li><li> In tempo reale l&#39;asportazione del linfonodo sentinella è guidata da entrambe le colorazione a fluorescenza e blu dei linfonodi. Il tempo reale caratteristica della fotocamera consente di rilevare per ogni residuo fluorescenza o localizzazioni inaspettato di nodi fluorescenti. </li><li> Dopo l&#39;asportazione del linfonodo sentinella, ex vivo le immagini sono acquisite di tutti i linfonodi asportati per la presenza di un segnale fluorescente. Il tempo di esposizione può essere prolungata per una maggiore risoluzione durante la cattura delle immagini (Figura 4). </li><li> L&#39;esame istopatologico dei linfonodi rivela la presenza ol&#39;assenza di cellule tumorali nel linfonodo sentinella. </li></ol> 4. RRisultati epresentative Linfonodi fluorescenti possono essere rilevati con un elevato rapporto segnale-background rapporto. Inoltre, il flusso di ICG attraverso i vasi linfatici possono essere monitorati in tempo reale, consentendo la mappatura dei linfonodi. I risultati possono essere influenzati da aumento della profondità del nodo, possibilmente che necessitano di procedure di illuminazione differenti per raggiungere il rilevamento. Futura applicazione di specifiche tumore agenti mirati a fluorescenza possono fornire rilevamento intraoperatoria del linfonodo sentinella positivo con le cellule tumorali con questa tecnologia. <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="Figura 1" /> Figura 1. Il linfonodo sentinella (SLN) teoria. SLN è il primo nodo linfatico drenante (s), dal tumore. <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="Figura 2" /> Figura 2. Quadro schematico del sistema di telecamere multispettrali di fluorescenza (A). La configurazione di base del sistema di telecamere in sala operatoria (B). <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="Figura 3" /> Figura 3. Multispettrali di imaging di fluorescenza di un linfonodo nel cancro vulvare. Immagine a colori di un linfonodo in vivo (A). Immagine di fluorescenza del linfonodo stesso, in vivo (B). Pseudocolori fluorescenza immagine sovrapposta l&#39;immagine a colori (C). <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="Figura 4" /> Figura 4. Multispettrali di imaging di fluorescenza di un ex-vivo dei linfonodi nel tumore del collo dell&#39;utero. Immagine a colori di un linfonodo ex vivo (A). Immagine di fluorescenza del linfonodo stesso, ex vivo (B). Pseudocolori fluorescenza immagine sovrapposta l&#39;immagine a colori (C).

<ol>
	<li>Tinga, D. J., Bouma, J., Aalders, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+squamous+cell+versus+adeno(squamous)+carcinoma+of+the+cervix,+what+factors+determine+the+prognosis.">Patients with squamous cell versus adeno(squamous) carcinoma of the cervix, what factors determine the prognosis.</a> Int J Gynecol Cancer. 2 (2), 83-91 (1992).</li><li>McMahon, C. J., Rofsky, N. M., Pedrosa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+metastases+from+pelvic+tumors:+anatomic+classification,+characterization,+and+staging.">Lymphatic metastases from pelvic tumors: anatomic classification, characterization, and staging.</a> Radiology. 254 (1), 31-46 (2010).</li><li>Zivanovic, O., Khoury-Collado, F., Abu-Rustum, N. R., Gemignani, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+biopsy+in+the+management+of+vulvar+carcinoma,+cervical+cancer,+and+endometrial+cancer.">Sentinel lymph node biopsy in the management of vulvar carcinoma, cervical cancer, and endometrial cancer.</a> Oncologist. 14 (7), 695-705 (2009).</li><li>Fuller, J., Guderian, D., Kohler, C., Schneider, A., Wendt, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+edema+of+the+lower+extremities+after+lymphadenectomy+and+radiotherapy+for+cervical+cancer.">Lymph edema of the lower extremities after lymphadenectomy and radiotherapy for cervical cancer.</a> Strahlenther Onkol. 184 (4), 206-211 (2008).</li><li>Ayhan, A., Celik, H., Dursun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+mapping+and+sentinel+node+biopsy+in+gynecological+cancers:+a+critical+review+of+the+literature.">Lymphatic mapping and sentinel node biopsy in gynecological cancers: a critical review of the literature.</a> World J Surg Oncol. 6, 53-53 (2008).</li><li>Tanis, P. J., Nieweg, O. E., Valdes Olmos, R. A., Rutgers, E. J., Kroon, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+sentinel+node+and+validation+of+the+technique.">History of sentinel node and validation of the technique.</a> Breast Cancer Res. 3 (2), 109-112 (2001).</li><li>Ghobashy, A. E. E. l., Saidi, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+sampling+in+gynaecological+cancers:+techniques+and+clinical+applications.">Sentinel lymph node sampling in gynaecological cancers: techniques and clinical applications.</a> Eur J Surg Oncol. 35 (7), 675-685 (2009).</li><li>Alford, R., Simpson, H. M., Duberman, J., Hill, G. C., Ogawa, M., Regino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+organic+fluorophores+used+in+molecular+imaging:+literature+review.">Toxicity of organic fluorophores used in molecular imaging: literature review.</a> Mol Imaging. 8 (6), 341-354 (2009).</li><li>Themelis, G., Yoo, J. S., Soh, K. S., Schulz, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+intraoperative+fluorescence+imaging+system+using+light-absorption+correction.">Real-time intraoperative fluorescence imaging system using light-absorption correction.</a> J Biomed Opt. 14 (6), 064012-064012 (2009).</li></ol>

VN e GMvD sono membro del consiglio scientifico del SurgOptix Inc. Prima della procedura, il protocollo di ricerca è stato approvato dal Consiglio locale Review Investigational (IRB) e il consenso informato del paziente è acquisito.

Questo video mostra l&#39;applicazione di multispettrali intraoperatorio tecnologia di imaging di fluorescenza per la rilevazione intraoperatoria del linfonodo sentinella (SLN) in oncologia ginecologica. La metodologia presenta alcuni vantaggi rispetto alla procedura convenzionale SLN. L&#39;iniezione avviene durante l&#39;intervento chirurgico stesso con il paziente anestetizzato, piuttosto che un giorno prima dell&#39;intervento, che è più paziente-friendly, soprattutto nei tumori ginecologici. Inoltre, l&#39;imaging intraoperatorio fornisce al chirurgo un riscontro visivo immediato del flusso di linfa e il suo modello di drenaggio per il linfonodo sentinella, piuttosto che indirettamente tramite un contatore Geiger-cassiere. Nel cancro del collo dell&#39;utero, in cui i linfonodi si trovano in profondità nel bacino, questo può aiutare a migliorare il rilevamento. Tuttavia, il più grande importanza sta nel passaggio verso l&#39;alto in direzione di imaging mirato. Mirati mezzi di contrasto fluorescenti possono essere fusi con tumore anticorpi specifici o substrati specificamente alle cellule tumorali. In questo modo, l&#39;imaging intraoperatorio fluorescenza ha un grande potenziale di modificare radicalmente la prassi corrente di chirurgia oncologica.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=2225">Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons</a>. There was an error with an author's name. The author name was corrected to include a first initial as follows:
K. Tim Buddingh
instead of:
Tim Buddingh

A correction to an authors name has been made for the article Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology.

Erratum: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

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<td>Indocyanine Green (ICG)</td>
<td>&nbsp;</td>
<td>Pulsion Medical Systems AG, Munich, Germany</td>
<td>D-81829</td>
<td>A solution of ICG in distilled water is used and not in NaCl 0.9%.</td>
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<td>Patent blue / Bleu patent&eacute;</td>
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<td>Guerbet, Paris, France</td>
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<td>Sterile water for injection</td>
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<td>B. Braun</td>
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<td>Multispectral fluorescence camera system </td>
<td>&nbsp;</td>
<td>Institute for Biological and Medical Imaging (IBMI), Technical University, Munich, Germany and SurgOptix Inc, USA</td>
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multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology

La prognosi in quasi tutti i tumori solidi dipende dalla presenza o assenza di metastasi linfonodali. 1-3 Il trattamento chirurgico il più delle volte combina l&#39;asportazione radicale del tumore con una linfoadenectomia completa nel bacino idrografico del tumore. Tuttavia, l&#39;asportazione dei linfonodi è associata ad un aumento della morbilità a causa di infezioni, rottura delle ferite e linfedema. 4,5 In alternativa, la procedura del linfonodo sentinella (SLN) è stato sviluppato da diversi decenni fa per rilevare il primo nodo linfodrenante dal tumore . 6 In caso di diffusione lymphogenic, il SLN è il primo linfonodo che è interessato (Figura 1). Quindi, se il linfonodo sentinella non contiene metastasi, nei linfonodi a valle sarà inoltre libera da metastasi tumorali e non devono essere rimossi. La procedura di SLN è parte del trattamento per molti tipi di tumore, come il cancro della mammella e del melanoma, ma anche per il cancro della vulva e della cervice. 7 L&#39;attuale metodologia standard per SLN-rilevamento mediante iniezione peritumorale di radiocolloid un giorno prima dell&#39;intervento, e un colorante intraoperatoriamente. Svantaggi della procedura di cancro cervicale e vulvare sono iniezioni multiple nella zona genitale, con conseguente aumento disagio psicologico per il paziente, e l&#39;uso di colloidi radioattivi. Multispettrali di imaging di fluorescenza è una metodica di imaging emergente che può essere applicato durante l&#39;intervento senza la necessità di iniezione di radiocolloid. Per intraoperatorio fluorescenza imaging, due componenti sono necessari: un agente fluorescente e un sistema quantitativo ottico per l&#39;imaging intraoperatorio. Come un fluoroforo abbiamo usato indocianina verde (ICG). ICG è stato usato per molti decenni per valutare la funzione cardiaca, la perfusione cerebrale e perfusione epatica. 8 Si tratta di un farmaco inerte di una cassaforte farmaco-biologico profilo. Quando sono eccitati a circa 750 nm, emette luce nel vicino infrarosso dello spettro intorno a 800 nm. Una misura di fluorescenza multispettrale sistema termocamera è stata utilizzata. 9. Lo scopo di questo articolo il video è quello di dimostrare la rilevazione del linfonodo sentinella con intraoperatorio fluorescenza imaging in pazienti con cancro cervicale e vulvare. Fluorescenza imaging è utilizzato in combinazione con la procedura standard, composto da radiocolloid e un colorante blu. In futuro, l&#39;imaging intraoperatorio fluorescenza potrebbe sostituire l&#39;attuale metodo ed è anche facilmente trasferibile ad altre indicazioni come il cancro della mammella e melanoma.

Watch this Scientific Journal Video about Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology at JoVE.com

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

L&#39;imaging di fluorescenza è una modalità innovativa per l&#39;immagine promettente chirurgia guidata in oncologia chirurgica. In questo video possiamo descrivere la procedura tecnica per la rilevazione del linfonodo sentinella mediante imaging di fluorescenza come esposti in ginecologiche oncologicy. Un sistema di telecamere multispettrali di fluorescenza, insieme con l&#39;agente fluorescente verde indocianina, viene applicata.

Multispettrali in tempo reale immagini a fluorescenza per il rilevamento intraoperatoria del linfonodo sentinella in Oncologia Ginecologica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

multispectral-real-time-fluorescence-imaging-for-intraoperative

Research

JoVE Journal

Medicine

23.0K Views.  University Medical Center Groningen. L'imaging di fluorescenza è una modalità innovativa per l'immagine promettente chirurgia guidata in oncologia chirurgica. In questo video possiamo descrivere la procedura tecnica per la rilevazione del linfonodo sentinella mediante imaging di fluorescenza come esposti in ginecologiche oncologicy. Un sistema di telecamere multispettrali di fluorescenza, insieme con l'agente fluorescente verde indocianina, viene applicata.

Video: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Ricerca

Medicina

Stereotactic Radiosurgery for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3 Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4 patient mouthpiece active breathing coordination,5 respiration-correlated computed tomography,6 or image-guided tracking of fiducials implanted within and around a moving tumor.7-9 The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10 Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8 Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET).8 Use of 18F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.

Radiochirurgia stereotassica for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Rilevamento MicroRNA in tumori della prostata dal Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Rilevazione intraoperatoria di endometriosi sottile: un nuovo paradigma per la rilevazione e il trattamento del dolore pelvico associato con la perdita di integrità peritoneale

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Trapianto nella camera anteriore dell&#39;occhio per longitudinale, non invasiva<em&gt; In vivo</em&gt; Imaging con Single-cella di risoluzione in tempo reale

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modellazione Piazza precoci di cancro ovarico Diffusione in una cultura Organotipica della Cavità peritoneale umano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.