This work has been supported by NIH HL-1034.

L&#39;Università di cura e utilizzo Comitato Animal Johns Hopkins ha approvato tutti i protocolli di animali. 1. apparecchiature Il sistema composito costituito, pronti per misurare la curva PV è mostrato in Figura 1. <ol><li> Misura Volume: <ol><li> Generare un tasso costante di gonfiaggio e sgonfiaggio utilizzando una pompa a siringa con un interruttore che consente all&#39;utente di invertire rapidamente la pompa dopo aver raggiunto i limiti di pressione. Per le curve del mouse FV, utilizzare un molto leggermente unta siringa di vetro 5 ml con il volume iniziale (prima di inflazione) fissato a 3 ml di aria. 3 ml è sufficientemente ampio per misurare i volumi in quasi tutte le curve del mouse fotovoltaici. </li><li> Misurare il volume erogato dalla pompa collegando un trasformatore differenziale lineare al corpo pompa, con una piccola asta sensore collegato alla siringa movimento stantuffo. Nota: Un mezzo empirici per correggere per la compressione del gas nel sistema è descritto sotto il cu PVsezione registrazione rve. </li></ol></li><li> Misurazione della pressione: <ol><li> Utilizzare un manometro economico standard con una gamma di 0-60 cm H 2 O (0-1 PSI). </li></ol></li><li> Misura di registrazione: <ol><li> Per registrare la curva PV utilizzare qualsiasi registratore digitale con funzionalità XY (ad esempio, Powerlab). Impostare un canale per registrare il segnale volume corretto e un altro canale per registrare la pressione transpolmonare (PTP), al fine di rappresentare graficamente la curva PV. Utilizzare un preamplificatore ponte che collega alla Powerlab principale per misurare la pressione. Calibrare il canale di pressione 0-40 cm H 2 O, e calibrare il canale del volume da 0-3 ml. </li></ol></li></ol> 2. Correzione per Compressione Gas Nota: Questa è una fase critica iniziale nel set up, poiché, come la pressione aumenta, diminuisce di volume di gas, e quindi il volume d&#39;aria erogata al mouse sarà sempre inferiore lo spostamento del syringe barile. <ol><li> Chiudere il rubinetto che collegherà l&#39;impianto fotovoltaico ai polmoni, in modo che nessun gas può lasciare il sistema. Avviare l&#39;infusione e osservare se il canale Volume corretto sul registratore mostra eventuali cambiamenti misurabili come la pressione aumenta a circa 40 cm H 2 O. Se è così, allora preciso nei passaggi successivi. <ol><li> Corretta per la compressione del gas empiricamente sottraendo dalla misura dello spostamento dello stantuffo (cioè, il volume non corretta) un termine proporzionale alla pressione di gonfiaggio. Effettuare questa operazione su un canale Powerlab (chiamato Vc) per mostrare il segnale volume di meno di un coefficiente volte la pressione. </li><li> Determinare il coefficiente nell&#39;equazione. In primo luogo, fare un tentativo iniziale, ruotare il grafico registrazione su, e avviare la pompa. Poiché il tubo di gonfiaggio è sigillato, regolare il moltiplicatore coefficiente di pressione per rendere il canale Vc leggere zero la pressione sale 0-40 cm H 2 O. Se si va su o giù, semplicemente regolare il fattore di correzione fino arimane piatta su questo campo di pressione. Questo fattore di correzione sarà sempre la stessa, se le stesse 3 ml partire volume nella siringa non è cambiato. </li></ol></li></ol> 3. Le prove sperimentali in topi <ol><li> Procedura per la misurazione della curva PV nei topi. Tutti i protocolli di animali sono stati approvati dall&#39;Università Cura e uso Comitato Animal Johns Hopkins. <ol><li> Anestetizzare topi (C57BL / 6 topi a 6-12 settimane di età) con ketamina (90 mg / kg) e xilazina (15 mg / kg), e confermare l&#39;anestesia dall&#39;assenza di movimento riflesso. Nota: La curva PV può essere completato in topi anestetizzati in meno di 10 minuti ed è una procedura terminale. </li><li> Tracheostomize i topi con un 18 G stub ago cannula. A tale scopo, una piccola incisione nella pelle sovrastante la trachea, localizzare la trachea, poi facendo una piccola fessura nella trachea, in cui l&#39;ago stub può essere inserito. Fissare la cannula legando con filo. </li><li> Lasciare i topi a respirare 100% di ossigeno per almeno 4 min. Questo può avvenire tramite la respirazione spontanea da un sacchetto o con un ventilatore nominalmente impostato con un volume corrente di 0,2 ml a 150 respiri / min. </li><li> Chiudere la cannula tracheale e lasciare 3-4 min per il mouse di assorbire tutto l&#39;ossigeno. Questa procedura assorbimento di ossigeno risulta la morte degli animali e in un degasaggio quasi completa del polmone 11. Conferma morte del mouse misurando la cessazione del battito cardiaco con elettrodi ECG o osservazione diretta. </li><li> Una volta che il degasaggio del polmone è completa e il volume polmonare è zero, cominciare a gonfiare il polmone con aria ambiente nella pompa a siringa ad una velocità di 3 ml / min. Monitorare la traccia pressione sul registratore digitale, e quando raggiunge i 35 cm H 2 O, invertire la pompa. </li><li> Seguire la curva di deflazione fino pressione raggiunge negativo del 10 cm H 2 O, momento in cui le vie aeree sono crollati, intrappolando l&#39;aria in alveoli che impediscono un&#39;ulteriore riduzione del volume. Invertire immediatamente tegli pompa di nuovo, consentendo al polmone per rigonfiare le vie respiratorie crollati aprire. Questa apertura eterogenea è normalmente evidenti dalla rumorosa dell&#39;arto gonfiaggio guardando la parte iniziale di questo 2a gonfiaggio. </li><li> Quando la pressione raggiunge nuovamente 35 centimetri H 2 O, invertire la direzione della pompa, e continuare a sgonfiare il polmone fino a questo 2a sgonfiaggio arto raggiunge 0 cm H 2 O. Quindi fermare la pompa. </li><li> Visualizza il record grafico PowerLab di pressione e portata e la curva PV. Poi analizza la curva PV per rilevare i cambiamenti fenotipici di parenchima polmonare che si verificano con diverse patologie polmonari. </li></ol></li></ol>

<ol>
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None of the authors have any financial interests that would be in conflict with the material presented in this paper.

In questo documento un metodo semplice e riproducibile è stato descritto per misurare in topi un metodo classico di elasticità fenotipizzazione polmonare, la curva totale PV polmonare. Tali curve sono state fondamentali per la scoperta del surfattante polmonare e la sua importanza nel fornire stabilità al polmone. Qui è mostrato come la curva PV è anche utile nel fornire un mezzo per misurare diverse variabili collegati elasticità polmonare nei polmoni topo adulto. Ci sono stati cambiamenti molto significativi in ...

Il mouse è ora il modello animale primario di una varietà di malattie polmonari. Nei modelli di enfisema o fibrosi, i cambiamenti fenotipici essenziali sono meglio valutati misurando le variazioni di elasticità polmonare. Sebbene vi siano molti modi per misurare l&#39;elasticità, il metodo classico è quello della curva di pressione-volume totale (PV) misurata dal volume residuo (RV) alla capacità polmonare totale (TLC). Questa misura è stata fatta su polmoni adulti da quasi tutte le specie di mammiferi risalenti quasi 100 anni 1-3. Tali curve PV anche svolto un ruolo importante nella scoperta e la comprensione della funzione del surfattante polmonare nello sviluppo del polmone fetale 4-7. Nonostante l&#39;importanza della curva PV come misura del fenotipo del polmone, non vi è stato alcun modo standardizzato effettuare tale misurazione. E &#39;stato fatto semplicemente gonfiando e sgonfiando polmone con passi discreti (attesa per un tempo variabile equilibramento dopo ogni) o con pompepuò continuamente gonfiare e sgonfiare il polmone. La curva PV è spesso fatto in un intervallo di volume tra zero e alcune capacità polmonare dall&#39;utente definire, ma la durata di ogni ciclo pressione volume riportato da diversi laboratori è estremamente variabile, che varia da pochi secondi a 8 ore 2. Alcuni ricercatori si riferiscono a questa curva totale PV polmone come statico o quasi statica, ma questi sono termini qualitativi che offrono visione poco, e che non sono usati qui. Inoltre, la curva PV non è stato ampiamente riportato nel topo, nonostante il fatto che essa può fornire informazioni utili sugli effetti macroscopici di cambiamenti strutturali nel polmone. Diversi problemi hanno portato a variabilità acquisizione curva PV tra cui: 1) il tasso di inflazione e la deflazione; 2) le escursioni di pressione per l&#39;inflazione e la deflazione; e 3) mezzi per la determinazione di una misura assoluta volume polmonare. Nel metodo qui presenti, una velocità di 3 ml / min è stato scelto come compromise, non essendo troppo breve da riflettere l&#39;elasticità dinamica associata con ventilazione normale e non troppo lento da rendere impraticabile la misura, in particolare quando si studia grandi coorti. Poiché una capacità nominale polmonare totale in un C57BL / 6 sana mouse è dell&#39;ordine di 1,2 ml 9, questo tasso consente tipicamente due completo chiusi PV cicli essere fatto in circa 1,5 min. In letteratura estesa in cui sono state riportate le curve PV, la pressione di gonfiaggio di picco utilizzato è stato estremamente variabile, variando da un minimo di 20 ad oltre 40 cm H 2 O. Una parte di questa variabilità può essere correlato alla specie, ma un obiettivo primario di impostare il limite di pressione superiore per le curve PV è di gonfiare il polmone di capacità totale polmonare (TLC), o il volume polmonare massimo. La TLC nell&#39;uomo è definita dallo sforzo massimale volontaria un individuo può fare, ma purtroppo non può mai essere duplicato in qualsiasi modello animale. Così, il volume massimo in curve sperimentali FV è scoraggiareminato da una pressione massima fissato arbitrariamente dallo sperimentatore. L&#39;obiettivo è quello di impostare una pressione in cui la curva PV è piatta, ma purtroppo l&#39;arto inflazione di una curva PV polmone dei mammiferi non è mai piatta. Quindi molti ricercatori impostare una pressione in cui la curva di gonfiaggio inizia ad appiattirsi sostanzialmente, genere il 30 cm H 2 O. Nel topo, tuttavia, la curva PV è ancora più complessa con una doppia gobba sull&#39;arto gonfiaggio, e dove tale parte dell&#39;inflazione spesso ancora a picco al 30 cm H 2 O 10, quindi 30 non è un punto finale per il buon curva PV. Per questo motivo, usiamo 35 centimetri H 2 O come il limite di pressione per la curva del mouse PV, che è una pressione alla quale gli arti gonfiaggio di tutti i ceppi che abbiamo esaminato cominciano ad appiattirsi. Poiché la curva PV è molto lineare, l&#39;aspetto di un loop PV dipenderà dal volume da dove la curva inizia. Alcuni ventilatori commerciali consentono agli utenti di fare grandi anse fotovoltaici, partendo da FRC, ma se il volume FRC è sconosciuto, allora è impossibile interpretare variazioni di tale curva PV con qualsiasi patologia, poiché questi cambiamenti potrebbero semplicemente derivare da un cambiamento di volume di partenza, e non alterazioni strutturali nel polmone. Così, senza una misura del volume assoluto, curve PV sono quasi impossibili da interpretare e quindi hanno scarsa utilità. Anche se, ci sono diversi modi per misurare i volumi polmonari, questi sono spesso ingombranti e richiedono attrezzature speciali. Nella semplice metodo qui descritto, la curva PV inizia a volume zero dopo una procedura di degasaggio in vivo. In sintesi, questo lavoro dimostra un metodo semplice per standardizzare la misurazione della curva PV polmonare nel polmone del mouse, e definisce alcuni parametri che possono essere calcolati da questa curva che sono collegate alla struttura del polmone. La curva PV fornisce quindi un test di funzionalità polmonare, che ha applicazione diretta di essere in grado di rilevare i cambiamenti strutturali fenotipiche nei topi con commsulle patologie polmonari come l&#39;enfisema e fibrosi.

<table><tbody><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>55-2226</td><td>Infuse/withdraw syringe pump</td></tr><tr><td>Pump 22 reversing switch</td><td>Harvard Apparatus</td><td>552217</td><td>Included with pump</td></tr><tr><td>Linear displacement transformer</td><td>Trans-Tek, Inc.</td><td>0244-0000</td><td></td></tr><tr><td>5 ml glass syringe</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>Digital recorder</td><td>ADInstruments</td><td>PL3504</td><td>Several other possible vendors</td></tr><tr><td>Bridge amp signal conditioner</td><td>ADInstruments</td><td>FE221</td><td></td></tr><tr><td>Gas tank, 100% oxygen</td><td>Airgas, Inc</td><td></td><td>Any supplier or hospital source will work</td></tr><tr><td>Pressure transducer: 0 - 1 psi&amp;nbsp;mV output</td><td>Omega Engineering</td><td>PX-137</td><td>Range &amp;asymp; 0 - 60 cm H&lt;sub&gt;2&lt;/sub&gt;O</td></tr></tbody></table>

measurement of the pressure-volume curve in mouse lungs

Negli ultimi decenni topo è diventato il modello animale primario di una varietà di malattie polmonari. Nei modelli di enfisema o fibrosi, le variazioni fenotipiche essenziali sono meglio valutati mediante misurazione delle variazioni di elasticità polmonare. Per comprendere meglio i meccanismi specifici alla base di tali patologie nei topi, è indispensabile effettuare misurazioni funzionali che possono riflettere la patologia sviluppo. Sebbene vi siano molti modi per misurare l&#39;elasticità, il metodo classico è quello del polmone pressione volume totale (PV) Curva fatto sull&#39;intero intervallo di volumi polmonari. Questa misura è stata fatta su polmoni adulti da quasi tutte le specie di mammiferi risalenti quasi 100 anni, e tali curve fotovoltaici anche svolto un ruolo importante nella scoperta e la comprensione della funzione del surfattante polmonare in sviluppo polmonare fetale. Purtroppo, tali curve totale PV non sono stati ampiamente riportati nel topo, nonostante il fatto che essi possono fornire informazioni utili sul macroscEffetti OPIC di cambiamenti strutturali nel polmone. Sebbene curve parziali FV misura solo le variazioni di volume polmonare sono talvolta riportati, senza misura del volume assoluto, la natura non lineare della curva totale PV rende questi parziali molto difficile interpretazione. Nel presente studio, si descrive un metodo standardizzato per misurare la curva totale PV. Abbiamo poi testato la capacità di queste curve per rilevare i cambiamenti nella struttura del polmone del mouse in due patologie polmonari comuni, enfisema e fibrosi. I risultati hanno mostrato cambiamenti significativi in ​​più variabili coerenti con i cambiamenti strutturali attesi con queste patologie. Questa misurazione della curva PV polmone nei topi fornisce pertanto un mezzo semplice per monitorare la progressione dei cambiamenti fisiopatologici nel tempo e l&#39;effetto potenziale di procedure terapeutiche.

Anche se la procedura per le curve FV è dimostrato nel video solo per topi sani di controllo, abbiamo esaminato la capacità della curva PV per rilevare cambiamenti funzionali e patologici in topi con due diverse patologie comuni, enfisema e fibrosi. I dettagli di questi modelli tradizionali descritte altrove 12,13. Molto brevemente, dopo l&#39;anestesia con il 3% isoflurano l&#39;enfisema è stato causato da 3 o 6 U elastasi pancreatica porcina instillato nella trachea e ha studiato tre settimane più tardi...

Watch this Scientific Journal Video about Measurement of the Pressure-volume Curve in Mouse Lungs at JoVE.com

Measurement of the Pressure-volume Curve in Mouse Lungs

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Misura della curva di pressione-volume in mouse polmoni

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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18.5K Views.  Johns Hopkins University. Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung’s structural changes with developing pathology.

Video: Measurement of the Pressure-volume Curve in Mouse Lungs

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Pressione di ventilazione controllata per indurre lesioni polmonari acute nei topi

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Ricerca

Medicina

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Ventricolo destro di pressione sistolica Misure in combinazione con raccolta di campioni di tessuto polmonare e immunitaria nei topi

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Immunologia e infezioni

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Un metodo standardizzato per la misurazione della superficie interna polmonare tramite la polmonite e l&#39;impianto di protesi

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

Biologia dello sviluppo

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un metodo semplice di intubazione mouse polmone

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

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 ...

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

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

Valutazione della Meccanica Respiratoria sistema nei topi utilizzando la tecnica delle oscillazioni forzate

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Fissazione polmonare sotto pressione costante per la valutazione dell'enfisema nei topi

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Misurare funzione respiratoria nei topi mediante sfrenato Pletismografia corpo intero

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...