The authors acknowledge the Carefusion Foundation for support via a clinical excellence grant of this work titled &#8220;Prospective evaluation of procedural povidone iodine rectal preparation to reduce infectious complications following ultrasound guided prostate needle biopsy.&#8221;

Diese patientenbezogenen Studie wurde von der Ethikkommission (IRB) für den menschlichen Forschungs genehmigt. 1. Patientenvorbereitung und Positionierung <ol><li> Bringen Sie den Patienten in die Verfahren suite und legen Sie sie auf ihren rechten oder linken Seite mit gebeugten Knien auf der Brust. </li><li> Führen Sie eine OP-Timeout, wobei die Patienten, chirurgische Verfahren, und Angabe, werden überprüft und bestätigt. </li><li> Wenn angegeben, zu verwalten intravenöser Sedierung über die Anästhesie-Service. </li></ol> 2. Führen Sie eine digitale rektale Untersuchung (DRE) <ol><li> Platz Größe geeignete Handschuhe auf beiden Händen. </li><li> Legen behandschuhten Finger nach der Schmierung in rektalen Gewölbe und prüfen, Prostata, um mögliche verdächtige Bereiche, in denen konzentrierte Aufmerksamkeit während der transrektalen Sonographie fühlen. 10 </li></ol> 3. Erstellung von Povidon-Iod Rectal Vorbereitung (PIRP) <ol><li> Pour 15 ml einer 10%im Handel erhältlich Povidoniod Lösung in ein steriles Becken. </li><li> 5 ml eines handelsüblichen 1% Lidocain-Gel auf diesem Becken. </li><li> Mischen Sie den Inhalt für 15 Sekunden, um eine Povidoniod Brei zu erstellen. </li></ol> 4. Grundschulverwaltung der PIRP <ol><li> Verwenden Sie eine sterile rektale Tupfer zur Kultur der rektalen Gewölbe. </li><li> Eröffnen eine 4 cm x 4 cm steriler Gaze, genießen in der Povidoniod Brei, und dann in den rektalen Gewölbe einfügen. Nur ein kleiner Schwanz aus Gaze wird links vom Anus ausgeht. </li><li> Lassen die Gaze in statt für 2 min, wonach es entfernt und verworfen. </li></ol> 5. Sekundäre Administration of PIRP <ol><li> Verwenden Sie eine Einweg-gynäkologischen Abstrichtupfer, um den perianalen Bereich mit dem PIRP Lösung zu malen. </li><li> Fügen Sie diesen gynäkologischen Tupfer in der rektalen Gewölbe zu einem Abstand von 3 cm von dem Anus. </li><li> Um die Exposition der rektalen Schleimhaut PIRP weiter zu maximieren, Tupfer die rektale vauLT in ähnlicher Weise mindestens dreimal. </li><li> Lassen Sie das Povidon-Iod-Lösung für 2 trocknen - 3 Min. Vor der Anwendung mit Standard-transrektale Sonographie und anschließende Biopsie fortfahren 11 Dies ist wichtig, da die bakterizide Wirkung des PIRP erfordern die Lösung auf der exponierten Schleimhäute trocknen. </li><li> Verwenden Sie eine sterile rektale Tupfer zur Kultur der rektalen Gewölbe. </li></ol> 6. Transrektale Sonographie und Biopsie <ol><li> Legen Sie die Ultraschallsonde über den Enddarm. </li><li> Führen Sie Messungen der Prostata und anschließende Biopsie in der Standard-Weise, die mit allen urologischen Praxis. 11,12 </li></ol>

<ol>
	<li>Nam, R. 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=Increasing+hospital+admission+rates+for+complications+after+transrectal+ultrasound+guided+prostate+biopsy.">Increasing hospital admission rates for complications after transrectal ultrasound guided prostate biopsy.</a> J Urol. 189, S12-S17 (2013).</li><li>Feliciano, J., 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+incidence+of+fluoroquinolone+resistant+infections+after+prostate+biopsy+are+fluoroquinolones+still+effective+prophylaxis.">The incidence of fluoroquinolone resistant infections after prostate biopsy are fluoroquinolones still effective prophylaxis.</a> J Urol. 179 (3), 952-955 (2008).</li><li>Kehinde, O., Al-Maghrebi, M., Sheikh, M., Anim, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+ciprofloxacin+and+amikacin+prophylaxis+in+the+prevention+of+septicemia+after+transrectal+ultrasound+guided+biopsy+of+the+prostate.">Combined ciprofloxacin and amikacin prophylaxis in the prevention of septicemia after transrectal ultrasound guided biopsy of the prostate.</a> J Urol. 189 (3), 911-915 (2013).</li><li>Taylor, A. 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=Targeted+antimicrobial+prophylaxis+using+rectal+swab+cultures+in+men+undergoing+transrectal+ultrasound+guided+prostate+biopsy+is+associated+with+reduced+incidence+of+postoperative+infectious+complications+and+cost+of+care.">Targeted antimicrobial prophylaxis using rectal swab cultures in men undergoing transrectal ultrasound guided prostate biopsy is associated with reduced incidence of postoperative infectious complications and cost of care.</a> J Urol. 187 (4), 1275-1279 (2012).</li><li>Gyorfi, J. R., 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=Peri+procedural+povidone+iodine+rectal+preparation+reduces+microorganism+counts+and+infectious+complications+following+ultrasound-guided+needle+biopsy+of+the+prostate.">Peri procedural povidone iodine rectal preparation reduces microorganism counts and infectious complications following ultrasound-guided needle biopsy of the prostate.</a> World J Urol. 32 (4), 905-909 (2014).</li><li>Park, D. S., 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=Control+of+infective+complications+of+transrectal+prostate+biopsy.">Control of infective complications of transrectal prostate biopsy.</a> Surg Infect Larchmt. 15 (4), 431-436 (2014).</li><li>Park, D. S., Oh, J. J., Lee, J. H., Jang, W. K., Hong, Y. K., Hong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+use+of+the+suppository+type+povidone+iodine+can+prevent+infectious+complications+in+transrectal+ultrasound+guided+prostate+biopsy.">Simple use of the suppository type povidone iodine can prevent infectious complications in transrectal ultrasound guided prostate biopsy.</a> Adv Urol. , (2009).</li><li>AbuGhosh, Z., 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=A+prospective+randomized+trial+of+povidone+iodine+prophylactic+cleansing+of+the+rectum+before+transrectal+ultrasound+guided+prostate+biopsy.">A prospective randomized trial of povidone iodine prophylactic cleansing of the rectum before transrectal ultrasound guided prostate biopsy.</a> J Urol. 189 (4), 1326-1331 (2013).</li><li>Gerber, G. S., Brendler, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Urological+Patient:+History,+Physical+Exam,+and+Urinalysis.">Evaluation of the Urological Patient: History, Physical Exam, and Urinalysis.</a> Campbell Walsh Urology. , (2015).</li><li>Hodge, K. K., McNeal, J. E., Terris, M. K., Stamey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+systematic+versus+directed+ultrasound+guided+transrectal+core+biopsies+of+the+prostate.">Random systematic versus directed ultrasound guided transrectal core biopsies of the prostate.</a> J Urol. 142 (1), 71-74 (1989).</li><li>Donoghue, P. M., McSweeney, S. E., Jhaveri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genitourinary+imaging:+current+and+emerging+applications.">Genitourinary imaging: current and emerging applications.</a> J Postgrad Med. 56 (2), 131-139 (2010).</li></ol>

The authors have nothing to disclose.

Infektionen nach TRUS PNB sind sekundär zu Translokation von rektalen Gewölbe Bakterien in die gefäß Prostata. Auf diesem Prinzip basierende, haben wir das einfache Verfahren der Verwendung eines Thema Antiseptikum, wie Povidon-Iod Mikroorganismenkoloniezählungen vor Prostatabiopsie reduzieren erforscht. Unsere Erfahrung hebt eine Reduzierung von 4,3% auf 0,6% (p = 0,02) mit der Nutzung dieser PIRP Regime zum Zeitpunkt der Biopsie. Darüber hinaus stellen wir die Hypothese, dass die 97% ige Reduktion der rektalen G...

Prostatakrebs ist die häufigste Krebserkrankung bei Männern und die zweithäufigste Ursache der durch Krebs verursachten Sterblichkeit. Im Jahr 2014 werden rund 233.000 Fälle in den Vereinigten Staaten mit fast 30.000 Mann zu erliegen dieser Malignität diagnostiziert werden. 1. Während die Prostata-spezifischen Antigens (PSA) Blutuntersuchung und digitale rektale Untersuchung (DRE) spielen eine wesentliche Rolle bei der Früherkennung von Prostatakrebs, transrektale Ultraschall-geführte Prostatabiopsie (TRUS PNB) ist die häufigste Mittel, um eine histologische Gewebediagnose zu erhalten. TRUS PNB beinhaltet das Einfügen einer Ultraschallsonde in das Rektum des Patienten und dann allgemein üblichen 12-14 Biopsien der Umfangszone der Prostata befindet sich nur nach vorne. Jedes Nadelbiopsie erfordert Durchgang einer 18 G Nadel durch die Mastdarmwand in die gefäß Prostata. Daher wird die theoretische Gefahr von Blutungen und Infektionen durch bakterielle Translokation vorliegt Befolgen des Verfahrens. KeineDestotrotz hat sich die versicherungsmathematischen Komplikationsrate nach dem Eingriff historisch niedrig blieben. Antibiotika-Prophylaxe vor der TRUS PNB wird routinemäßig mit Chinolon-basierte Antibiotika als die am häufigsten verwendeten Mittel verschrieben. Trotz adäquater Prophylaxe, impliziert zeitgenössische Forschung einen Anstieg der infektiösen Komplikationen nach der Biopsie. 2 Darüber hinaus Studien zurückzuführen diesen Anstieg der infektiösen Komplikationen folgenden TRUS PNB zur Entstehung von Chinolon resistenten Mikroorganismen besonders E. coli. 3 Diese steigende Rate von Chinolonresistenz legt nahe, die Notwendigkeit für alternative Prophylaxe-Schemata für TRUS PNB Verfahren. Zu diesem Zweck sind mehrere verschiedene Wege untersucht. Ein Ansatz umfasst die Verabreichung einer intravenösen oder intramuskulären Antibiotikum zum Zeitpunkt der Biopsie in Verbindung mit einer oralen Chinolon. 4 Diese Strategie ist Sepsis Ereignissen nach TRUS PNB reduzieren,ein Haupt Einschränkung dieses Ansatzes ist das Potenzial für die weitere Entwicklung resistenter Organismen gegen dieses Antibiotikum Familie. Weiteres suchten kürzlich Methodik beinhaltet die Verwendung eines vor der Biopsie rektaler Abstrich, um Patienten zu screenen mit Chinolon-resistenten rektale Flora besiedelt. 5 Falls solche Organismen erkannt werden, könnte ein &quot;targeted&quot; Antibiotika-Therapie auf der Basis des Empfindlichkeitsprofils des identifizierten resistenten Bakterien eingesetzt werden Belastung. Während die Methodik ist elegant, der eigentliche Prozess der Erlangung solcher Tupfer, selektiven Züchten auf einem Chinolon selektiven Medium, und Schneiderei Antibiotika erfordert danach eine klinische und Laborinfrastruktur, die in vielen klinischen Praxis fehlen könnten. Die Verabreichung einer antiseptischen Thema zu rektalen Gewölbe Mikrokoloniezahlen vor der Biopsie kann eine alternative Strategie, um TRUS PNB Infektionen begrenzen präsentieren zu reduzieren. Povidon iodone ist eine kostengünstige, leicht verfügbaren Mittel, das istreduziert Bakterienzahlen dokumentiert, wann Operationsstellen aufgetragen. Anwendungen in beiden kolorektalen und gynäkologischen Operationen sind gut bekannt. Daher wird der Einsatz Povidin Jod als rektale Zubereitung würde eine herstellbare, einfache und kostengünstige Methode reduziert TRUS PNB Infektionen ohne zusätzliche Vorbereitung vor der Biopsie zu präsentieren. In Bezug auf den urologischen Literatur, zuvor Arbeit hat eine Verringerung der klinischen Infektionen nach TRUS PNB festgestellt. 6-9 Dieses Video demonstriert die Technik der PIRP, um die Leichtigkeit und Einfachheit hervorheben, um in die klinische Praxis zu integrieren. Alle Patienten, die TRUS PNB wurden in einem Spezialklinik für Urologie Weisung oder erste Bewertung für Prostata-Krebs-Screening zu sehen. Diese Auswertung brachte eine Überprüfung des Patienten Serum Prostata-spezifischen Antigens (PSA) und die Leistung eines digitalen rektalen Untersuchung (DRU). Anomalie entweder der PSA oder DRE aufgefordert Empfehlungeine TRUS PNB. Die TRUS PNB Verfahren wurde bei allen Patienten und die damit verbundenen Risiken, einschließlich Infektionen, Blutungen diskutiert und Harnverhalt wurden geklärt. Alle Patienten erhielten drei Tage der oralen Antibiotika-Prophylaxe entweder mit einem Chinolon-basierte Medikamente (zB Ciprofloxacin) oder Trimethoprim-Sulfamethoxazol (Bactrim) vor der Biopsie.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Povidone Iodine Solution, 10% Topical Microbicide Antiseptic, 8 fl oz.</td>
			<td>Equate</td>
			<td>4067979</td>
			<td></td>
		</tr>
		<tr>
			<td>4x4 sterile gauze pads, white</td>
			<td>Dukal Corporation</td>
			<td>DKL1412</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable gynecologic swab&#160;</td>
			<td>numerous vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1% lidocaine gel&#160;</td>
			<td>numerous vendors</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

povidone iodine rectal preparation at time of prostate needle biopsy is a simple and reproducible means to reduce risk of procedural infection

Einzelinstitut und bevölkerungsbezogenen Studien zeigen, dass infektiöse Komplikationen nach transrektalen Ultraschall geführte Prostatabiopsie (TRUS PNB) steigen. Solche Infektionen sind vor allem auf resistente Keime, die die rektale Gewölbe besiedeln und in den Blutstrom während der Biopsie transloziert Chinolon. Eine Povidon-Iod-rektale Zubereitung (PIRP) zum Zeitpunkt der Biopsie ist ein einfaches, reproduzierbares Verfahren zur rektalen Mikrokoloniezahlen und damit resultierenden Infektionen nach TRUS PNB reduzieren. Alle Patienten werden vor der Biopsie verabreicht drei Tagen der oralen Antibiotika-Therapie. Die PIRP Technik beinhaltet zunächst Positionierung des Patienten in der üblichen Weise für eine TRUS PNB. Folgende digitale rektale Untersuchung werden 15 ml einer 10% igen Lösung von im Handel erhältlichen Povidoniod mit 5 ml 1% Lidocain Gelee gemischt, um eine Aufschlämmung zu schaffen. Ein 4 cm x 4 cm steriler Gaze ist soain dieser Aufschlämmung KED und dann in den rektalen Gewölbe für 2 min, wonach es entfernt eingesetzt wird. Danach wird ein Einweg-Baumwolle gynäkologischen Abstrichtupfer verwendet, um sowohl den perianalen Bereich und der rektalen Gewölbe zu einer Entfernung von 3 cm vom Anus zu malen. 3 min vor der Verwendung mit Standard transrektale Sonographie und nachfolgende Biopsie hend - das Povidon-Iod-Lösung wird dann für 2 trocknen. Diese PIRP Technik hat sich in der Praxis an unserer Hochschule seit März von 2012 mit einem zugeordneten Verringerung der post-Biopsie-Infektionen von 4,3% auf 0,6% (p = 0,02) war. Der Hauptvorteil dieser Prophylaxe Regime ist seine Einfachheit und Reproduzierbarkeit bei Verwendung eines leicht erhältlichen, kostengünstigen Mittels an Infektionen. Darüber hinaus vermeidet die Technik Aussetzen Patienten zusätzliche systemische Antibiotika mit potenziellen weiteren Ausbreitung der multiresistenten Organismen. Die Verwendung von PIRP bei TRUS PNB ist jedoch nicht für Patienten mit Jod oder Schalentiere einllergies.

Diese PIRP Technik hat sich in der Praxis seit Januar 2012 (bis heute) mit 165 Patienten, die in dieser Qualität Verbesserungsmaßnahme eingeschrieben gewesen. In dieser Zeit wurde nur bei einem Patienten (0,6%) eine infektiöse Komplikation in Form systemischer Sepsis erlebt. Dieser Patient wurde mit einer Multidrug-resistenten E. kolonisiert coli-Stamm. Rektalabstrichen nach der Verabreichung von PIRP bei dieser Patientin beachten, dass der rektalen Gewölbe war noch mit 2,1 x 10 5 KBE / ...

Watch this Scientific Journal Video about Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection at JoVE.com

Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection

Here, we present a protocol for use at the time of transrectal ultrasound guided prostate needle biopsy (TRUS PNB) that is a simple and cost-effective means to reduce infections following the procedure.

Povidoniod Rectal Vorbereitung bei der Prostata-Nadelbiopsie ist eine einfache und reproduzierbare Mittel zur Gefahr der Verfahrens Infektion zu verringern

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy ...

povidone-iodine-rectal-preparation-at-time-prostate-needle-biopsy-is

Research

JoVE Journal

Medicine

31.8K Aufrufe.  Penn State Milton S. Hershey Medical Center. Here, we present a protocol for use at the time of transrectal ultrasound guided prostate needle biopsy (TRUS PNB) that is a simple and cost-effective means to reduce infections following the procedure.

Serie: Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection

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.

Evaluation von Nanopartikel-Aufnahme in Tumoren in Echtzeit über 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 ...

Forschung

Medizin

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.

MicroRNA-Detektion in Prostatatumoren durch 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 ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombiniert<em&gt; In-vivo-</em&gt; Optische und μCT Imaging-Infektion, Entzündung und Knochenanatomie in einem Orthopädie Implantat-Infektion in Mäuse Monitor

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

Substernalen Schilddrüse Biopsie Mit Endobronchial Ultraschall-gesteuerte Transbronchiale Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Vorbereitung und Respirometrische Bewertung der Mitochondrien isoliert aus dem Skelettmuskelgewebe durch perkutane Nadelbiopsie Erhalten

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Eine einfache Kritische große Femoral Defect Modell an der Maus

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Eine einfache Vorrichtung, um schnell vorbereiten Whole Mounts des Mausdarm

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generation von Prostatakrebs-Patienten gewonnen Xenograft-Modelle von zirkulierenden Tumorzellen

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Eine einfache mechanische Verfahren Limbusstammzellinsuffizienzen in der Maus zum Erstellen

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional orientation and localization of structures that are not directly visible to the surgeon. This is especially critical for structures at risk like nerves or blood vessels. Optimization of the handling of cadaveric material and the combination of multiple techniques compensate for the limited availability of adequate specimens. The described protocol combines anatomical plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to help localize relevant structures, such as nerves, arteries, veins and to correctly position the portals during MIS procedures. Depiction of these structures in anatomy textbooks can differ from what is encountered in the surgical field; and for this reason, new anatomical studies with a surgical orientation are needed. However, this is a complex, time-consuming technique requiring specific training. The anatomical references described with the so-called 'clock method' provide the surgeon with an easy and reproducible system to locate the path of the nerves at risk in Hallux Valgus MIS procedures. This model can be extrapolated to many other minimally invasive surgical procedures.

Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures.

Eine anatomische Untersuchung der Nerven gefährdet während minimalinvasiver Hallux Valgus Operation

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional ...