The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOTA: Tutte le procedure sugli animali relative descritte in questo protocollo sono stati precedentemente approvati dal Institutional Animal Care and Use Committee Duke University (DUIACUC). 1. Cancer Cell Culture e iniezione <ol><li> Coltivare cellule tumorali metastatiche fluorescente (ad esempio le cellule del cancro al seno MDAMB231-GFP umani, dono di Dr. Patricia Steeg, NCI, e le cellule del mouse sarcoma YFP-etichettati, dono del Dr. David Kirsch, Duke University Medical Center, Dipartimento di Radioterapia Oncologica) in appropriato terreno di coltura (per es Dulbecco Modified Eagle Medium (DMEM) con il 10% di siero fetale bovino e 1% di penicillina / streptavidina) a 37 ° C fino a circa il 90% confluenti. </li><li> Trypsinize cellule, lavarli 2 volte con PBS, contare, e poi iniettarli nelle vene coda di isoflurano-anestetizzato 10 settimane ratti nudi femminili a 5 milioni di cellule per animale, usando una siringa con un ago 27 G. Anesthes livello chirurgiciia viene verificata dalla mancanza di reazione al pizzico punta. </li></ol> 2. Monitoraggio delle metastasi Uso microCT <ol><li> Esaminate ratti volta alla settimana usando un micro-CT / micro-irradiatore, per rilevare la presenza di metastasi sopra di circa 2 mm di diametro di diametro. Il micro-CT è incaricato come descritto in precedenza 12. <ol><li> Ratti soggetto a 3% isoflurano anestesia prima di imaging. Confermare profonda anestesia pizzico punta. </li><li> Dopo insorgenza dell&#39;anestesia, trasferire rapidamente i ratti alla culla di imaging nella camera di imaging e connettersi tramite un naso-cono per una miscela isoflurano-aria a 2,5-3%. Regolare la posizione del ratto in culla in modo che il suo torace è in posizione nel fascio di fotoni dello scanner microCT, utilizzando i comandi di posizione esterni e delimitatore laser sul supporto imaging. Assicurarsi che la porta della camera di imaging è chiusa, per proteggere l&#39;investigatore dai raggi gamma. </li><li> Controllare la posizione dell&#39;animale utilizzando nuovamentela telecamera a colori. Eseguire un test di imaging CT a bassa risoluzione, e utilizzare l&#39;immagine risultante per regolare il campo visivo alle dimensioni XYZ della cavità toracica. </li><li> Immagine torace topo usando un filtro Al 2 mm 40 kVp, 2,5 mA, e 0.008 voxel a 7 FPS e rispedire l&#39;animale nella sua gabbia. Imaging di un animale dovrebbe prendere non più di 15 o 20 minuti. Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente per mantenere decubito sternale. </li><li> Conferma metastasi dalla comparsa di oggetti relativamente radiopachi che non può essere spiegato da vasi sanguigni intratoracica (Figura 1A) </li></ol></li></ol> Chirurgia 3. Finestra Camera <ol><li> Anestesia, segni vitali e cateterizzazione coda vena <ol><li> Selezionare gli animali con la presenza di malattia metastatica. Iniettare animale con un d intraperitonealeose di 50 mg / kg pentobarbital. Conferma a livello chirurgico anestesia pizzico punta prima di procedere. Nota: i protocolli di anestesia dovrebbero essere abbinati al rispettivo apparato sperimentale. Pentobarbital stato scelto qui come anestetico ad azione prolungata, per indurre anestesia profonda per lunghe procedure, offrendo la possibilità di facile ri-somministrazione. Tuttavia, la perdita di animali da sovradosaggio è un problema comune con anestesia pentobarbital. Un&#39;altra opzione che conserva riflessi autonomi in misura maggiore di pentobarbital è ketamina in combinazione con sedativi come la xilazina o medetomidina, che però consente solo per un singolo ciclo di ri-dosaggio. </li><li> Rasatura animali sul lato del corpo che ha la malattia metastatica, e nella zona del collo, utilizzando un clipper. Rimuovere tutti i restanti capelli sciolti dalla pelle. Dopo capelli sciolti viene rimosso, applicare una pomata veterinari per gli occhi, per evitare che si secchi. NOTA: i topi nudi atimici possono avere capelli residuo requirimozione res prima di procedere con procedure chirurgiche. E &#39;molto importante rimuovere completamente tutti i capelli, come può interferire con le procedure chirurgiche e di imaging. </li><li> Fissare l&#39;animale in posizione supina su una piastra di metallo che è posto su una piastra elettrica dell&#39;acqua 37 ° C circolare. Le estremità anteriori e posteriori sono fissati sulla piastra con nastro. NOTA: E &#39;utile per controllare e registrare i segni vitali, come i tassi di cuore e sangue arterioso ossigenazione utilizzando un pulsossimetro, durante le procedure chirurgiche e sperimentali. </li></ol></li><li> L&#39;intubazione tracheale <ol><li> Per posizionare un catetere per la ventilazione dell&#39;animale, prima una incisione cutanea cervicale trasversale, seguita dalla separazione mediana della muscolatura ventrale longitudinale alla trachea. </li><li> Utilizzare ripetuta azione di apertura a chiusura con una pinza taglienti per creare un passaggio per la sutura attraverso il lato dorsale della trachea. </li><li> Fai una piccola incisione nella trachea su tegli lato ventrale, non superiore semicircolare, circa tra il secondo e il terzo anello tracheale. Lasciare sufficientemente lunga porzione della trachea esposta sulla superficie dorsale, per consentire il fissaggio del catetere tracheale. </li><li> Inserire una 2,5-3,0 mm &quot;Y&quot; cannula tracheale nella trachea, e stringere con una sutura 4-0 monofilamento. Verificare la cannula è collegata ad un ventilatore ciclato a pressione con una bottiglia collegata al condotto di scadenza che viene riempito con 6 cm di acqua, per mantenere la pressione polmonare positiva. Flusso gas deve essere al 100% di ossigeno, a meno che sperimentalmente desiderato altrimenti. </li><li> Inserire un catetere con un ago G 25-27, e riempito di soluzione fisiologica eparinizzata in una delle vene coda di ratto, e fissare in posizione con nastro. NOTA: Assicurare la pervietà del catetere vena della coda per tutta la procedura iniettando ripetutamente una piccola quantità di soluzione fisiologica eparinizzata nella vena della coda. Inoltre, intubazione oro-tracheale, cioè la guida di un trachealetubo attraverso la bocca dell&#39;animale anestetizzato e passato laringe nella trachea, è una possibile alternativa alla procedura tracheotomia descritta qui. Tuttavia, questo metodo richiede formazione e dell&#39;esperienza specifica, per evitare danni alla trachea, e anche per impedire la cannulazione accidentali dell&#39;esofago. </li></ol></li><li> Applicazione della finestra polmonare <ol><li> Rimuovere la pelle dal lato del torace in cui si trova la malattia metastatica, creando una incisione, e poi staccare la pelle con dissezione smussata. Nota: La pelle può essere eliminata e rimossa successivamente al distacco </li><li> Procedere sezionando i due strati di muscolatura sovrapposizione (pettorale, dentato, e gran dorsale), ma lasciando intatti i muscoli intercostali. Creare una perforazione nella cavità toracica di circa 1,5 cm di diametro, rimuovendo porzioni tipicamente due nervature adiacenti. Idealmente, individuare la perforazione della regione della sesta e settima ribs. </li><li> Osteotomia: <ol><li> Per ridurre al minimo il sanguinamento e danni alla superficie del polmone, ermeticamente tenere la nervatura da tagliare con dentate pinza chirurgica durante il taglio. Usando forbici chirurgiche, tagliare il lato mediale della nervatura prima, con un angolo di circa 45 °, lasciando la parte appuntita della costola rimanente indica all&#39;esterno. </li><li> Successivamente, tagliare la parte laterale della costola in modo simile, lasciando ancora la parte appuntita della costola punta verso l&#39;esterno, per evitare danni alla superficie del polmone. </li><li> Ripetere la procedura per la nervatura adiacente, quindi tagliare i muscoli intercostali e togliere il pezzo asportato. Durante questa procedura, mantenere la pressione polmonare in modo che l&#39;interazione meccanica tra la superficie polmonare e la gabbia toracica è minimizzato. Per fare ciò, opportunamente regolare la pressione ispirazione sul ventilatore. </li></ol></li><li> Inserimento della finestra: <ol><li> Inserire una finestra polmone misura, costituito da un coprioggetti che èattaccato ad una presa plexiglas (Figura 1B). Fissare la finestra alla presa mediante incollaggio, oppure applicando una piccola quantità di grasso per vuoto. Inserire la finestra in modo che le metastasi superficiali sono situati vicino al centro della finestra. Se necessario, regolare il foro inserito per portare la micrometastasi al centro della finestra allargando leggermente il foro al rispettivo lato. NOTA: La malattia metastatica sulla superficie pleurica può essere identificato come puntini bianchi chiaramente riconoscibili o aree all&#39;interno delle parti altrimenti rosa-a-salmone colorati sani della superficie polmonare che appaiono prevalentemente lungo le fessure. Mentre micrometastasi possono verificarsi in altri settori dell&#39;esterno pleurico, le linee cellulari esaminati mostrano quasi sempre affioramento micrometastasi nell&#39;area perforata, una volta metastasi può essere rilevato radiologicamente. </li><li> Dopo aver inserito la presa nella perforazione, e creando il contatto diretto con la pleura viscerale lung, suturare i bordi della cornice della finestra al muscolo intercostali circostante, utilizzando 4,0 sutura monofilamento (Figura 1C). Utilizzare un leggero aumento della pressione di inspirazione del ventilatore per aiutare aria residua di fuggire e per creare una tenuta. Nota: Il tasso di respirazione di ratti può variare notevolmente, a seconda dello stato di anestesia, lo stato di eccitazione o ansia, concentrazione di ossigeno dell&#39;aria inspirata (FiO2), ecc E &#39;consigliabile regolare la frequenza respiratoria tra 70 e 90 bpm. La pressione ispirazione deve essere regolata con cautela e non può essere impostato a più di ca. 8 centimetri H 2 O (0,6 mmHg), per evitare danni alla superficie del polmone. </li></ol></li><li> Posizionare l&#39;animale in un dispositivo di immobilizzazione progettato su misura che è progettato per eliminare Z-direzionale movimento (Figura 1D) su una piastra di acciaio che è posizionato su un termostatica (elettrica) termocoperta, sotto un microscopio a fluorescenza. Controllare il corpo dell&#39;animaletemperatura con un termistore rettale. Regolare le viti del dispositivo di immobilizzazione degli animali, e la pressione di inspirazione del ventilatore per ottenere il controllo ottimale di movimento laterale. Nota: la respirazione naturale in mammiferi coinvolge tutte e tre le direzioni di movimento del polmone e l&#39;estensione del torace: bilaterali, dorso-ventrale, e cranio-caudale. Per preservare il movimento respirazione naturale per quanto possibile, è importante minimizzare la compressione Z-direzionale nella misura necessaria. Poiché il sistema di ritenuta Z-dimensionale ha il potenziale per introdurre artefatti che possono influenzare il flusso di sangue e altri parametri, si consiglia di mantenere le condizioni di costante durante serie di misure ripetute dello stesso animale. </li></ol></li></ol> 4. Imaging e misurazione del flusso sanguigno Microcircolatoria <ol><li> Raccogliere i globuli rossi tramite puntura cardiaca ed etichettarli con DII (1,1 = -dioctadecyl-3,3,3 perclorato =, 3 = tetrametil-indocarbocyanine), come descritto in precedenza &lt;sup&gt; 10. </li><li> Iniettare 300 ml di globuli rossi marcati nella vena della coda del topo prima dell&#39;intervento camera di finestra è fatta, per evitare effetti primo passaggio adesione alla finestra di vetro. Eliminare eventuali bolle d&#39;aria nella siringa o del catetere, come introduzione di aria nella vena inibirà ulteriori iniezioni. </li><li> Immagine flusso di sangue con una telecamera CCD microscopio a -40 ° C di temperatura di raffreddamento di chip, e circa 100X risoluzione complessiva (cioè con un obiettivo microscopio 10X, 10X e pre-camera oculare). Utilizzare set di filtri Rhodamine / TRITC normali (eccitazione 450-490 nm, emissione&gt; 515 nm). Registrare il frame rate e pixel la risoluzione effettiva delle sequenze di immagini risultanti. Record di almeno 200 (idealmente circa 300) le immagini per stack, per garantire l&#39;analisi di successo della velocità del flusso. </li><li> Rifornire perdita di liquidi negli animali iniettando circa 1 ml di soluzione fisiologica ip ogni hr. NOTA: Le impostazioni sperimentali che coinvolgono un intervento, ad esempio,un farmaco che modifica la velocità del flusso sanguigno in una metastasi del cancro polmonare, richiede misurazioni ripetute di velocità del flusso sanguigno nel tumore metastatico polmonare. Per questi esperimenti estesi, è importante ricostituire l&#39;animale con sufficiente liquido. </li><li> Euthanize animali per infusione di 3 ml di 3N KCl nella vena della coda </li><li> Valutare le pile di immagini utilizzando una pubblicazione, pubblicamente disponibile algoritmo computer basato su Matlab che creerà il flusso di velocità in scala di grigi ea colori codificati mappe per tutto il flusso di sangue tracce 10,11. Successivamente valutare le risultanti immagini in scala di grigi utilizzando software di analisi dell&#39;immagine commerciale o pubblicamente disponibili, come ad esempio Immagine J, dopo thresholding off valori che indicano nessun movimento di cellule del sangue, cioè non vascolare attiva. </li></ol>

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Un modello che viene presentato è fattibile immagine cambia in microcircolazione del flusso sanguigno e altri processi dinamici in metastasi polmonari di ratti, utilizzando la microscopia intravitale e analisi del flusso di sangue computazionale. Mentre altri metodi esistono per effettuare microscopia a polmoni esposte in ribcages aperti di roditori, questo modello è anche il primo ad immagine metastasi polmonari attraverso una perforazione della parete toracica in un ambiente chiuso del torace. Usando questo metodo, ...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

La vascolarizzazione nei tumori solidi è noto a differire significativamente da normale apporto di sangue, mostrando maggiori gradi di tortuosità, e superiori distanze InterVascular 13. Di conseguenza, le tracce del flusso sanguigno nel cancro al seno e sarcoma metastasi polmonari sperimentali hanno forme irregolari e grandi lacune InterVascular (figura 2A, inferiori di due pannelli) rispetto alla microcircolazione polmonare normale (Figura 2A, pannello superiore). In uno s...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Automated Misura di microcircolatorio Portata in polmonare Metastasi di Ratti

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K Views.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Video: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Nella misura in vivo della superficie polmonare strato endoteliale mouse

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

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Medicina

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Microscopio video intravitale misurazioni della retina flusso sanguigno nei topi

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Misurazione automatica di enfisema polmonare e piccole vie aeree ritocca in sigaretta Mice-fumo esposti

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Aumentare polmonare Arteria flusso pulsatile Migliora ipossica Ipertensione polmonare nei suinetti

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Transcutanea microcircolatoria Imaging in pretermine neonati

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Un modello di rimodellamento cardiaco Attraverso Costrizione del Aorta addominale in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Elettrocardiografia simultanea registrazione e invasiva sangue pressione misura in ratti

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Valutazione della riserva del flusso coronario dopo la miaocardia Ischemia Reperfusione nei ratti

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Test di emocompatibilità degli impianti che si contattono con il sangue in un modello di flusso a ciclo continuo che imita il flusso sanguigno umano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.