Ringraziamo di cuore F. Bierry per l&#39;assistenza nella produzione video e di editing.

Tutte le procedure che coinvolgono soggetti animali sono stati approvati dal Comitato Istituzionale Hospital Animal Care University. 1. L&#39;inoculazione batterica Strappo <ol><li> Prima dell&#39;iniezione, anestetizzare conigli mediante iniezione intramuscolare di ketamina (30 mg / kg di peso corporeo) mescolato con xilazina (4 mg / kg) entro erettori spinali muscolare. Garantire gli animali sono completamente anestetizzato controllando che non riescono a rispondere a zampa pizzico. Questo protocollo prevede anestesia sufficiente per l&#39;iniezione intra-articolare del ginocchio. Posizionare gli animali sotto la maschera di ossigeno durante l&#39;anestesia. </li><li> Shave ginocchio animali. Preparare il ginocchio per iniezione asettiche da 3 alternati salviette di povidone-iodio macchia e alcol seguita dalla applicazione della soluzione di iodio povidone. </li><li> Identificare manualmente il tendine rotuleo come una struttura elastica alla faccia anteriore del ginocchio usando un tappo ago sterile come illustrato oppure una sonda sterile. </li><li&gt; Con il controllo visivo e utilizzando un approccio anteriore, inserire un ago G 25 percutanea al ginocchio destro di ciascuno dei 12 conigli attraverso il tendine rotuleo. </li><li> Iniettare ciascun ginocchio con 1 ml di una sospensione batterica di Staphylococcus aureus (ceppo Neumann) ad una concentrazione di 10 6 UFC / ml. Una iniezione senza resistenza conferma la posizione intrarticolare della punta dell&#39;ago. </li><li> Dopo procedure di iniezione, tenere l&#39;animale sotto fonte convettivo per evitare dispersioni di calore e monitorare la frequenza respiratoria osservando movimento del torace. Una volta sveglio, tenere gli animali in gabbie con libero accesso a cibo e acqua. </li><li> Gestire il dolore con la somministrazione di un&#39;iniezione intramuscolare di 0,1 mg / kg di buprenorfina ogni 8 ore. </li><li> Clinicamente osservare conigli ogni giorno, il monitoraggio quantità consumate di cibo e acqua, perdita di peso, la temperatura, e il comportamento generale. </li><li> Per l&#39;immagine della fase acuta dell&#39;infezione eseguire prima sessione di imaging MR appenacome animale presenta notevole perdita di peso (10%); generalmente questo avviene dopo circa 2 giorni. </li><li> Se è necessaria la somministrazione del farmaco per via endovenosa prima di un esame MRI, un accesso venoso auricolare può essere installato (cfr. di seguito). </li></ol> 2. Esame MRI Quando l&#39;imaging di animali vivi, di un&#39;installazione animale attento è una caratteristica fondamentale per garantire il comfort degli animali e l&#39;anestesia ottimale. Questo permetterà di ottenere i migliori risultati di imaging e di garantire una acquisizione di immagini riproduttiva per studi longitudinali. A causa della dimensione dell&#39;animale, l&#39;uso di una unità MR clinico di dimensioni (1,5 T o T 3) è più adatto. Bobina ginocchio clinico 8-canale viene utilizzato in quanto fornisce adatto risoluzione e rapporto di contrasto-rumore per coniglio esplorazione ginocchio. <ol><li> Prima dell&#39;installazione del sistema di imaging, installare un accesso venoso periferico attraverso una vena auricolare. Questo accesso servirà per iniettare il mezzo di contrasto. <ol><li> Shave e disinfettare l&#39;orecchio. </li><li&gt; eseguire l&#39;anestesia locale per iniezione sottocutanea di lidocaina. </li><li> Inserire un catetere G 25 dentro l&#39;auricolare vena laterale. </li><li> Valutare la permeabilità mediante iniezione di 1 ml di soluzione fisiologica sterile. </li><li> Mantenere il catetere con intercettazioni adesivo. </li></ol></li><li> Anestetizzare animali tramite iniezione intramuscolare di ketamina (30 mg / kg di peso corporeo) mescolato con xilazina (4 mg / kg). Gli animali sono completamente anestetizzato una volta non riescono a rispondere a zampa pizzico. Posizionare una maschera facciale sul coniglio, dandogli ossigeno con una portata di 200 ml / min. </li><li> Una volta completamente anestetizzato, prendere gli animali per l&#39;unità imaging MR. Posto animali così che le ginocchia sono situati nel centro della bobina MR. Posizionare le zampe dell&#39;animale in estensione completa, in modo che l&#39;asse lungo della tibia sia parallelo al tavolo unità di imaging MR. Utilizzare velcro o sacchetti di sabbia per mantenere la posizione ottimale delle gambe. </li><li> Monitoraggio cardio-respiratorio degli animali non è richiesta laprotocollo di anestesia permette anestesia profonda respirazione spontanea, ma gli animali devono essere coperti per evitare dispersioni di calore anestesia indotta. </li><li> Protocollo MR è dettagliato in Tabella 1. Il tempo medio per l&#39;acquisizione totale compreso installazione ottimale è di circa 20 min. </li><li> Eseguire la RM in piani diversi. Nondimeno, il piano trasversale delle ginocchia (ortogonale alla tibia) è il piano di riferimento in quanto garantisce la riproducibilità anatomica della posizione dell&#39;immagine per esami longitudinali sequenziali e permette correlazione ottimale con fette istologiche. </li><li> Al completamento di sequenze non potenziate, iniettare l&#39;agente di contrasto UPSIO attraverso l&#39;accesso venoso auricolare. Somministrare lentamente le particelle di ossido di ferro in una singola dose di 150 micromol Fe / kg. Risciacquare il catetere mediante iniezione di 1 ml di soluzione fisiologica sterile. </li><li> Ripetere la sequenza rafforzata GRE pesata 24 ore dopo l&#39;iniezione. Questo ritardo consente alle particelle di essere fagocitati dai macrofagi. </li><li> Per gli studi longitudinali che valutano effetto farmaci (antibiotici, steroidi, anticorpi anti-macrofagi, ecc.), Ripetere esami RM sequenziali utilizzando il protocollo sopra descritto. </li></ol> 3. Image Analysis <ol><li> Per la valutazione longitudinale eseguire misure allo stesso livello anatomico, e di definire un punto di riferimento facilmente identificabile. Per esempio, attraverso l&#39;acquisizione piano assiale in cui si osserva il maggior diametro della rotula è un modo affidabile per ottenere la misurazione riproducibile (Figura 1). </li><li> Eseguire l&#39;analisi delle immagini utilizzando un software DICOM-based (come OsiriX) o un software di elaborazione delle immagini (ImageJ). </li><li> Utilizzando il software ImageJ, eseguire la segmentazione del territorio sinoviale che dimostra perdita di segnale sulle USPIO-enhanced GRE immagine pesata in T2 con la &quot;zona irregolare&quot; strumento di disegno. Una volta che la membrana sinoviale con segnale scuro è delineato, fai clic su &quot;Analizza&quot; per ottenere l&#39;area /numero di pixel (figura 2). </li></ol>

<ol>
	<li>Madri, J., Kissane, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+healing.">Inflammation and healing.</a> Anderson's Pathology. , 67-110 (1990).</li><li>Sephel, G., Woodward, S., Rubin, R., Strayer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair,+regeneration,+and+fibrosis.">Repair, regeneration, and fibrosis.</a> Rubin's Pathology. , 71-98 (2008).</li><li>Verdrengh, M., Tarkowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+macrophages+in+Staphylococcus+aureus-induced+arthritis+and+sepsis.">Role of macrophages in Staphylococcus aureus-induced arthritis and sepsis.</a> Arthritis Rheum. 43 (10), 2276-2282 (2000).</li><li>Heale, J., Speert, D., Burke, B., Lewis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+bacterial+infection.">Macrophages in bacterial infection.</a> The Macrophage. , 210-252 (2008).</li><li>Bierry, G., 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=Macrophage+activity+in+infected+areas+of+an+experimental+vertebral+osteomyelitis+model:+USPIO-enhanced+MR+imaging--feasibility+study.">Macrophage activity in infected areas of an experimental vertebral osteomyelitis model: USPIO-enhanced MR imaging--feasibility study.</a> Radiology. 248 (1), 114-123 (2008).</li><li>Bierry, G., 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=MRI+of+macrophages+in+infectious+knee+synovitis.">MRI of macrophages in infectious knee synovitis.</a> AJR Am. J. Roentgenol. 194 (6), W521-W526 (2010).</li><li>Lutz, 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=Detection+of+synovial+macrophages+in+an+experimental+rabbit+model+of+antigen-induced+arthritis:+ultrasmall+superparamagnetic+iron+oxide-enhanced+MR+imaging.">Detection of synovial macrophages in an experimental rabbit model of antigen-induced arthritis: ultrasmall superparamagnetic iron oxide-enhanced MR imaging.</a> Radiology. 233 (1), 149-1457 (2004).</li><li>Weissleder, 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=Ultrasmall+superparamagnetic+iron+oxide:+an+intravenous+contrast+agent+for+assessing+lymph+nodes+with+MR+imaging.">Ultrasmall superparamagnetic iron oxide: an intravenous contrast agent for assessing lymph nodes with MR imaging.</a> Radiology. 175 (2), 494-498 (1990).</li><li>Lefevre, 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=Septic+arthritis:+monitoring+with+USPIO-enhanced+macrophage+MR+imaging.">Septic arthritis: monitoring with USPIO-enhanced macrophage MR imaging.</a> Radiology. 258 (3), 722-728 (2011).</li><li>Sigovan, 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=Rapid-clearance+iron+nanoparticles+for+inflammation+imaging+of+atherosclerotic+plaque:+initial+experience+in+animal+model.">Rapid-clearance iron nanoparticles for inflammation imaging of atherosclerotic plaque: initial experience in animal model.</a> Radiology. 252 (2), 401-409 (2009).</li></ol>

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Mentre i mezzi di contrasto a base di gadolinio non specifici forniscono informazioni sul volume e perfusione dello spazio extracellulare, macrofagi imaging USPIO-enhanced MR permette una localizzazione anatomica precisa e una valutazione qualitativa di infiltrazione dei macrofagi all&#39;interno sinoviale infetto, senza la necessità di campionamento tessuto 9. Grazie alla loro alta sensibilità, USPIO può dimostrare anche sottile infiltrato cellulare presente nelle prime fasi di infezione. Sotto terapia an...

La risonanza magnetica (MRI) è considerato essere la modalità di imaging di scelta per la dimostrazione di sinovite infettiva dovuta alla sua elevata risoluzione spaziale e di contrasto dei tessuti molli. Cambiamenti del segnale osservati alla risonanza magnetica in artrite bassa T1 e T2 alta segnale che riflette la maggiore presenza di contenuto di acqua extracellulare, e un marcato enhancement dopo gadolinio basato contrasto amministrazione agente, coerenti con le istologicamente risultati di vascolarizzazione aumentata grazie alla vasodilatazione e l&#39;angiogenesi 1. Tuttavia, la RM è spesso in grado di dimostrare la risoluzione di infezione durante la terapia antibiotica come potenziamento persistente può essere osservato nelle articolazioni dei pazienti con clinicamente e biologicamente guarito artrite settica dovuta alla persistenza delle allargata spazio extracellulare (tessuto di granulazione, cicatrice fibrotica) 2. Oltre ai cambiamenti extracellulari, cellule infiammatorie, tra cui un intenso reclutamento di macrofagi massicciamente infiltrano l&#39;ininfetti sinovia. I macrofagi giocano un ruolo importante sia nella fase acuta e cronica di infezione batterica 3. Inducono la reazione infiammatoria necessaria per sradicare microrganismi patogeni, e coordinare risoluzione infiammazione, come macrofagi indotta infiammazione persiste fintanto tessuti necrotici non vengono rimossi, impedendo riparazione per iniziare 4. Così, la riduzione di infiltrazione di macrofagi all&#39;interno infetto synovia dopo terapia efficace può essere un indicatore affidabile della risoluzione dell&#39;infezione. Cellular imaging è in grado di dimostrare infiltrazione cellulare specifica all&#39;interno tessuto patologico. Macrofagi specifico RM utilizzando mirati di contrasto è stato ampiamente indagato e ha dimostrato la sua capacità di dimostrare l&#39;infiammazione delle articolazioni o infezione 5-7. Dopo iniezione endovenosa, questi agenti di contrasto mirati come USPIO particelle (ultrasmall ossidi di ferro superparamagnetiche) sono ripreso da fagociti come i macrofagi e,a causa delle loro proprietà magnetiche, indurre cambiamenti del segnale in tessuti che presentano infiltrazione di macrofagi 8. Il follow-up longitudinale di coloro segnale MR modifiche terapia consente una valutazione non invasiva in vivo di infiltrazione di macrofagi, e una riduzione della USPIO caricato-macrofagi relative variazioni di segnale dimostrerebbe successo terapia. Lo scopo di questa relazione è quello di presentare le modalità di esecuzione dei macrofagi RM per monitorare in maniera non invasiva artrite settica, dimostrando la riduzione di infiltrazione di macrofagi all&#39;interno della sinovia.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>P904</td>
			<td>Guerbet</td>
			<td></td>
			<td>Dose: 150 &#956;mol Fe/kg</td>
		</tr>
		<tr>
			<td>Ketamine (500 mg/ml ketamine)</td>
			<td>Virbac</td>
			<td></td>
			<td>Dose: 30 mg/kg</td>
		</tr>
		<tr>
			<td>Rompun (20 mg/ml Xylazine)</td>
			<td>Axience</td>
			<td></td>
			<td>Dose: 4 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Vetoquinol</td>
			<td></td>
			<td>Dose: 0.1 mg/kg/8 hr</td>
		</tr>
		<tr>
			<td>BD 22 G, 1 inch</td>
			<td>BD Biosciences</td>
			<td>381423</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 25 G, 5/8 inch</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo macrophage imaging using mr targeted contrast agent for longitudinal evaluation of septic arthritis

I macrofagi sono cellule-chiave nella iniziazione, lo sviluppo e la regolazione della risposta infiammatoria alle infezioni batteriche. I macrofagi sono intensamente e sempre reclutati nelle articolazioni settiche dalle prime fasi di infezione e l&#39;infiltrazione si suppone a regredire una volta efficace rimozione dei patogeni è ottenuto. La capacità di identificare in vivo l&#39;attività dei macrofagi in un giunto infetto può quindi fornire due applicazioni principali: rilevamento precoce di sinovite acuta e monitoraggio della terapia. In vivo rilevamento non invasivo di macrofagi può essere eseguita con la risonanza magnetica utilizzando nanoparticelle di ferro come ultrasmall superparamagnetico ossido di ferro (USPIO). Dopo somministrazione endovenosa o intra-articolare, USPIO sono specificamente fagocitate dai macrofagi attivati ​​e, a causa delle loro proprietà magnetiche, provocare variazioni del segnale in tessuti che presentano infiltrazione dei macrofagi. Un quantitativvalutazione e della infiltrarsi è fattibile, come la zona con perdita di segnale (numero di pixel scuri) osservata il gradiente echo immagini MR dopo iniezione di particelle è correlata con la quantità di ferro all&#39;interno del tessuto e quindi riflette il numero di cellule USPIO-caricati. Presentiamo qui un protocollo per eseguire l&#39;imaging macrofagi utilizzando USPIO-enhanced RM in un modello animale di artrite settica, consentendo un iniziale e longitudinale nella valutazione non invasiva in vivo di macrofagi infiltrazione e una valutazione delle iniziative terapia.

Sulle immagini non potenziate, sinovia delle ginocchia infetti presenta un gonfiore diffuso segnale intermedio che è non distinguibile dal tessuto circostante molli, mentre femore e rotula appaiono come strutture basse segnale (Figura 1). Sulle immagini USPIO avanzata, 24 ore dopo la somministrazione di contrasto, zona sinoviale contenente macrofagi USPIO-caricati dimostrerà perdita di segnale (figure 1 e 2). I...

Watch this Scientific Journal Video about In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis at JoVE.com

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Dimostriamo come eseguire macrofagi RM con mezzo di contrasto ultrasmall superparamagnetico (USPIO) in artrite settica, consentendo un iniziale e longitudinale<em&gt; In vivo</em&gt; Valutazione non invasiva di macrofagi infiltrazione e una valutazione della efficacia terapeutica.

In vivo Macrofago Imaging Utilizzando MR mirata di contrasto agente per la valutazione longitudinale di artrite settica

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

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Research

JoVE Journal

Medicine

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

9.2K Views.  University Hospital of Strasbourg. Dimostriamo come eseguire macrofagi RM con mezzo di contrasto ultrasmall superparamagnetico (USPIO) in artrite settica, consentendo un iniziale e longitudinale In vivo Valutazione non invasiva di macrofagi infiltrazione e una valutazione della efficacia terapeutica.

Video: In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Contrast Imaging nave migliorata utilizzando microCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Ricerca

Medicina

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

La valutazione longitudinale di perdita dell&#39;arto posteriore ossea del mouse dopo la lesione del midollo spinale tramite Novel, In vivo, Metodologia

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

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 ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imaging Molecolare del cancro alla prostata con un piccolo Molecular CLT1 Peptide mirato di contrasto agente

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinale<em&gt; In Vivo</em&gt; Imaging del Cerebrovasculature: Rilevanza per le malattie del sistema nervoso centrale

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Fase di contrasto risonanza magnetica nell'arteria carotica comune ratto

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Quantitativo [18F]-Naf-PET-MRI Analisi per la valutazione del fatturato osseo dinamico in un paziente con mal di schiena facetogenico

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mappatura quantitativa della ventilazione specifica nel polmone umano utilizzando l'imaging a risonanza magnetica protonica e l'ossigeno come agente di contrasto

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Visualizzazione longitudinale stabilizzata a livello cellulare in vivo del pancreas in un modello murino con una finestra di imaging intravitale pancreatico

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...