Gli studi sono stati supportati da NIH HL75503 a KP.

Tutti gli esperimenti condotti su animali erano come approvato dal Comitato Istituzionale cura degli animali e uso della University of Tennessee Health Science Center. 1. Tubing preparati perfusione Rat Lung <ol><li> Preparare un sistema di tubi con tubi Tygon (# 18) per la perfusione di sangue come mostrato in Figura 1. Collegare trasduttori di pressione (P23XL) e posizionare il tubo sul palco microscopio. </li><li> Regolare la temperatura del bagno d&#39;acqua a 37 ° C. </li><li> Collegare l&#39;ingresso del polmone e di uscita temporanea con tubo Tygon. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> Figura 1. Tubo per perfusione di sangue. Lo schema mostra la configurazione tubo utilizzato per far circolare il sangue attraverso la preparazione polmone isolato. Inoltre, sono indicati i componenti associati attraverso il quale l&#39;i tubis instradato. Uno schema di cuore e polmone è incluso per mostrare siti di collegamento tra il tubo e l&#39;arteria polmonare (PA), e atriale (LA) cannula (linee blu tratteggiate) sinistra. 2. Preparazione dei polmoni Rat isolati <ol><li> Anestetizzare ratti (ratti maschi Sprague-Dawley, 250-300 g) con ketamina (80-100 mg / kg) e con xilazina (5-10 mg / kg). Dopo garantendo un piano chirurgico di anestesia, posizionare l&#39;animale in posizione supina. </li><li> Eseguire tracheostomia, inserire una cannula tracheale (PE-90) e fissare con sutura chirurgica. </li><li> Infondere eparina (100-200 U) nel cuore mediante puntura cardiaca (21 G ago a farfalla), attendere 60 secondi e il sangue exsanguinate (~ 12-15 ml di sangue). </li><li> Preparare due cannule (tubi Tygon, 3 mm di diametro, lunghi 4 centimetri; svasati a una estremità) e riempire con soluzione fisiologica. </li><li> Eseguire una toracotomia. Fare un&#39;incisione (3 mm) sul ventricolo destro e far scorrere l&#39;estremità svasata di una cannula nell&#39;incisione verso la polmonarearteria. Fissare cannula dell&#39;arteria polmonare con suture chirurgiche. </li><li> Incidere (3 mm) all&#39;apice del ventricolo sinistro e far scorrere l&#39;estremità svasata della seconda cannula nell&#39;incisione. Guida la cannula in atrio sinistro. Fissare cannula al ventricolo sinistro con un nastro ombelicale (2 mm di larghezza). </li><li> Sezionare qualsiasi tessuto connettivo, e rimuovere il polmone e cuore insieme alle cannule allegate. Posizionare polmone e cuore su un piatto di Petri. Riposizionare il polmone modo che la superficie diaframmatica è in cima. I tre cannule devono affrontare la stessa direzione. </li><li> Posizionare la preparazione polmone in una fase che può essere manipolata nelle direzioni XY. Ogni fase che viene con un microscopio può essere modificato per tenere il tubo o una fase fatta su misura, se necessario. </li></ol> 3. Perfusione sanguigna dei polmoni Rat isolati <ol><li> Mescolare il sangue dissanguato con albumina uguale volume (5%) soluzione e aggiungere al serbatoio. Avviare la pompa peristaltica e impostare il flusso rmangiato a 14 ml / min. Lasciare che il sangue per riempire il tubo. </li><li> Con lo shunt aperto, rimuovere il connettore di ingresso-uscita del polmone temporaneo. Attaccare l&#39;arteria polmonare e sinistra cannula atriale all&#39;ingresso polmone e di uscita, rispettivamente. Assicurarsi che non ci siano bolle d&#39;aria nel tubo. </li><li> Collegare la cannula tracheale di un terzo trasduttore di pressione (P23XL). Gonfiare polmoni con il 30% di ossigeno attraverso la cannula tracheale e mantenere la pressione alle 5 cm H 2 O. </li><li> Chiudere lo shunt con un morsetto per iniziare perfusione polmonare. Mantenere arteria polmonare e di sinistra pressioni atriali a ~ 10 e il 3 cm H 2 O, rispettivamente. </li></ol> 4. Preparazione del polmone per microvascolare Infusione <ol><li> Sollevare la parte posteriore del tubo di estensione per il catetere di sopra del livello del polmone e sicuro. </li><li> Preparare un catetere di infusione con l&#39;inserimento di circa 30 cm di 40 cm di lunghezza tubo PE10 in un tubo PE90 ~ 30 cm di lunghezza. Collegare l&#39;estremità esposta del tubo PE10 di un ago (30 G) atached ad una siringa (1 cc). </li><li> Inserire la combinazione catetere nella parte posteriore sollevata del tubo di estensione. Guida la combinazione catetere attraverso l&#39;atrio sinistro e nei polmoni finché non incontra resistenza. Quindi, spingere il catetere PE10 solo ulteriormente nei polmoni finché non incontra resistenza. Si noti che l&#39;applicazione di una forza eccessiva durante l&#39;inserimento del catetere ferire il polmone. </li><li> Riempire la siringa da 1 cc con soluzione di Ringer e legarsi a una pompa a siringa. Impostare la velocità di infusione a 10 ml / min. </li><li> Al sito di infusione diventerà chiaro rispetto al resto del polmone. </li><li> Inumidire il polmone con soluzione salina e coprire il polmone con involucro di plastica con un foro abbastanza grande per lasciare il sito di infusione scoperto. </li><li> Tampone del grasso rubinetto sul fondo di un O-ring (misura) e posizionare un vetrino di vetro (# 1.5, 22 mm di diametro) sul fondo della O-ring. Posizionare delicatamente l&#39;O-ring con il vetrino sul sito di infusione. Fissare l&#39;O-ring con una Hol provetta normader. La combinazione coprioggetto / O-ring non distorce le strutture alveolari sotto di esso, come recentemente riportato 15. </li><li> Posizionare un obiettivo (20X) di un microscopio a fluorescenza sopra l&#39;O-ring e concentrarsi sulla superficie polmonare. </li></ol> 5. Imaging fluorescente Dextran transito attraverso microvasi <ol><li> Preparare il microscopio per l&#39;imaging FITC fioritura. </li><li> Selezionare una regione per essere ripreso e acquisire immagini alla velocità di 1/minuto utilizzando software di acquisizione immagini. </li><li> Iniziare infusione di FITC-destrano 20 kD (0,5 mg / ml) utilizzando la pompa a siringa. Fluorescenza nei vasi sanguigni aumenterà gradualmente e raggiungere un massimo. Continuare infusione per 60 min. </li><li> Quindi, passare a infusione di Ringer per lavare via la fluorescenza luminale. Mantenere infusione per oltre 10 min. Continua l&#39;acquisizione di immagini a 1/min durante il lavaggio-off. </li></ol> 6. Image Analysis <ol><li> Aprire il file di acquisizione delle immagini. </li><li> Regioni postodi interessi per microvasi all&#39;interno della cornice dell&#39;immagine. </li><li> Riprodurre il file immagine del fotogramma per fotogramma e registrare l&#39;intensità di fluorescenza a ciascuna regione di interesse per tutti i fotogrammi. </li><li> Tracciare la variazione di intensità di fluorescenza per ogni regione di interesse in funzione del tempo. </li><li> Quantificare la massima intensità di fluorescenza e l&#39;intensità di fluorescenza residua dopo 10 min di washoff. </li><li> Calcolare il rapporto di massima intensità di fluorescenza residua in ogni regione di interesse per ottenere l&#39;indice di permeabilità per microvasi quella regione di interesse. </li></ol>

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La preparazione isolato polmone sangue perfuso accoppiato con immagini in tempo reale fornisce un semplice strumento per la determinazione delle variazioni di permeabilità in microvasi polmonari singoli. Abbiamo applicato questo metodo per definire cambiamenti di permeabilità in risposta alle infusioni di LPS. I nostri dati indicano chiaramente che LPS infusione provocato un aumento della permeabilità microvascolare. Inoltre, i dati indicano anche che i cambiamenti di permeabilità indotte da LPS erano simili in entr...

Aumento della permeabilità microvascolare nei polmoni porta allo sviluppo di edema alveolare e gli scambi gassosi compromessa ed è un importante caratteristica del danno polmonare acuto (ALI) 1-3. Pertanto, le stime di permeabilità vascolare sono importanti nel definire l&#39;entità del danno polmonare e l&#39;efficacia degli interventi terapeutici proposti. Analisi gravimetrica come rapporto polmone gratuito sangue wet-to-dry e coefficiente di filtrazione microvascolare sono ampiamente utilizzati metodi per stimare la permeabilità 4,5. Altri metodi includono quantificare la ritenzione di sonde radioattive o fluorescenti nel tessuto polmonare 6-8. Tuttavia, i metodi di cui sopra richiedono un&#39;elaborazione postexperiment di campioni di tessuto polmonare verso chiarire i dati di permeabilità. Inoltre, poiché un animale può essere utilizzato solo per un protocollo singolo trattamento, possono essere necessari grandi numeri di animali per uno studio completo. Una caratteristica comune dei metodi sopra descritti è che essi determinano la permeabilità vascolare medio pertutti i vasi sanguigni all&#39;interno del campione di tessuto. Tuttavia, è noto che polmonari micro-e macro-vasi sono fenotipicamente diversi 9. Quindi, risposte permeabilità possono essere eterogenei tra i vari segmenti di flotta e 9,10. Così, quantificando la permeabilità media di tutti i vasi polmonari in un campione di tessuto può non riflettere adeguatamente tale eterogeneità. Nella preparazione isolato polmone sangue perfuso, vasi sanguigni sulla superficie polmonare possono essere visualizzati tramite un microscopio verticale 4,11,12. In questo modo le risposte che caratterizzano in singoli vasi e, quindi, affrontare qualsiasi eterogeneità nelle risposte 13. Inoltre, utilizzando imaging di fluorescenza di microvasi, saggi di fluorescenza base possono essere incorporati. Inoltre, un microcatetere atriale sinistro può essere utilizzato per fornire agenti e sonde di fluorescenza nei vasi sanguigni 11,14. Microcatetere limita la consegna di una piccola regione polmonare, così exche propone solo i vasi sanguigni all&#39;interno della regione agli agenti infusi e fluorofori. Questo consente a più piccole regioni dello stesso polmone da utilizzare per esperimenti separati, portando ad una riduzione complessiva animali necessari per uno studio. L&#39;imaging in tempo reale consente l&#39;acquisizione dei cambiamenti dinamici nella vascolare e fluorescenza extravascolare singoli microvasi della preparazione polmone isolato. Così, per ogni microvasi all&#39;interno di un campo immagine, cambiamenti nella fluorescenza durante l&#39;infusione di fluorofori e washoff possono essere registrati, e quantificati in linea 14. Utilizzando i valori di fluorescenza massima e residua vascolare, un indice di permeabilità per ogni microvasi nel campo dell&#39;imaging può essere determinato. Per determinare le variazioni di permeabilità in risposta ad agenti infiammatori o pregiudizievoli, l&#39;agente desiderato può essere somministrato prima e poi l&#39;indice di permeabilità determinato. Inoltre, il campo dell&#39;immagine può essere regolato dovunque nella regione polmonare infusa dallomicrocatetere, consentendo così un elevato grado di flessibilità nella scelta della rete vascolare desiderato. Così, l&#39;isolato di sangue perfuso preparazione del polmone in tandem con le immagini in tempo reale fornisce un modello sperimentale interessante per quantificare permeabilità dei microvasi polmonari singoli.

<table><tbody><tr><td>Tygon Tubing</td><td>Fisher Scientific</td><td></td><td>#18</td></tr><tr><td>Pressure Transducer</td><td>Data Sciences International</td><td>P23XL</td><td>Need quantity 3</td></tr><tr><td>Butterfly Needle</td><td>Greiner Bio-One</td><td>450081</td><td>21 G</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer</td><td>Masterflex L/S</td><td></td></tr><tr><td>PE-90 tubing</td><td>Becton Dickinson</td><td>427421</td><td>30 cm needed</td></tr><tr><td>PE-10 tubing</td><td>Becton Dickinson</td><td>427401</td><td>40 cm needed</td></tr><tr><td>Syringe Pump</td><td>Braintree Scientific</td><td>BS8000</td><td></td></tr><tr><td>O-ring</td><td></td><td></td><td>Custom made with a 20 mm diamter hole and a handle to secure O-ring to holder</td></tr><tr><td>Upright fluorescence microscope</td><td>Olympus America</td><td>BX61WI</td><td></td></tr><tr><td>Image Acquisition Software</td><td>Molecular Devices</td><td>Metamorph</td><td></td></tr><tr><td>FITC Dextran 20KD</td><td>Sigma Aldrich</td><td></td><td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&amp;nbsp;</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Aldrich</td><td></td><td>Serotype 0111:B4</td></tr></tbody></table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

La preparazione isolato polmone sangue perfuso è ampiamente usato per visualizzare e definire segnalazione in singoli microvasi. Accoppiando questa preparazione con la rappresentazione in tempo reale, diventa fattibile per determinare le variazioni di permeabilità delle singole microvasi polmonari. Qui descriviamo passi per isolare i polmoni di ratto e profumato con sangue autologo. Poi, abbiamo delineare misure per infondere fluorofori o agenti attraverso un microcatetere in una piccola regione del polmone. Utilizzando queste procedure descritte, abbiamo determinato un aumento della permeabilità nel ratto microvasi polmonari in risposta alle infusioni di lipopolisaccaride batterico. I dati hanno rivelato che lipopolisaccaride aumentata perdita di liquido in entrambi i segmenti venulare e capillare dei microvasi. Così, questo metodo permette di confrontare le risposte permeabilità tra segmenti vascolari e quindi, definisce alcun eterogeneità nella risposta. Mentre i metodi comunemente usati per definire la permeabilità polmonare richiedono post-elaborazione di campioni di tessuto polmonare, iluso di imaging in tempo reale evita questo requisito come evidente dal presente metodo. Così, la preparazione polmone isolato combinato con l&#39;imaging in tempo reale offre diversi vantaggi rispetto ai metodi tradizionali per determinare la permeabilità microvascolare polmonare, ma è un metodo semplice per sviluppare e implementare.

Un isolato preparazione polmone sangue perfuso collegato al tubo per perfusione e relative apparecchiature è mostrato in Figura 2. A scopo dimostrativo, abbiamo usato un ratto Sprague Dawley, se le procedure qui descritte possono essere utilizzati con qualsiasi specie di ratto. Infusioni attraverso un microcatetere atriale sinistra raggiungono solo una piccola regione del polmone. La regione infuso può essere identificato dal scolorimento infusione indotta (Figura 3). La preparazione ...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

La preparazione isolato polmone sangue perfuso rende possibile visualizzare le reti microvasali sulla superficie polmonare. Qui si descrive un metodo per quantificare permeabilità delle singole microvasi nei polmoni isolati utilizzando l&#39;imaging di fluorescenza in tempo reale.

Quantificare singolo microvasi Permeabilità in Isolata Blood-perfusione Rat Lung Preparazione

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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9.4K Views.  The University of Tennessee Health Science Center.  La preparazione isolato polmone sangue perfuso rende possibile visualizzare le reti microvasali sulla superficie polmonare. Qui si descrive un metodo per quantificare permeabilità delle singole microvasi nei polmoni isolati utilizzando l'imaging di fluorescenza in tempo reale. 

Video: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Un<em&gt; In vivo</em&gt; Test per la verifica della nave Permeabilità Sangue

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Ricerca

Medicina

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

L&#39;angiogenesi nel polmone del ratto ischemica

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Analisi videomorfometrica della vasocostrizione polmonare ipossica delle arterie intrapolmonari utilizzando fette polmonari tagliate con precisione murina

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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.

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. ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caratterizzazione del isolata, ventilata e polmone mouse Instrumented perfusi con flusso pulsatile

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Biologia

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

Chirurgia Shunt, Destra cuore cateterizzazione, e Vascolare Morfometria in un modello di ratto per Flow-indotta ipertensione arteriosa polmonare

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Valutazione dei meccanismi di controllo vascolare utilizzando Video microscopia del arterie di resistenza isolata dei ratti

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Quantificare singolo microvasi Permeabilità in Isolata Blood-perfusione Rat Lung Preparazione

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