Questo lavoro è stato supportato dal programma di ricerca intramurale del National Cancer Institute, Center for Cancer Research.

Il seguente protocollo descrive un metodo non invasivo, semplice e veloce per fornire materiali di interesse per i polmoni del mouse. Tale procedura è stata approvata dalla cura degli animali e del Comitato Usa National Cancer Institute. 1. Anestesia <ol><li> Primo, anestetizzare il mouse usando una miscela di chetamina e xilazina (100 mg / kg di peso corporeo e 10 mg / kg di peso corporeo, rispettivamente). Questo è il ACUC raccomandato anestesia e dose. Con questo importo, i topi sono incosciente, almeno per ~ 20 min. </li><li> Applicare vet unguento agli occhi del mouse per evitare l&#39;essiccazione degli occhi durante l&#39;anestesia. </li><li> Dopo alcuni minuti, pizzicare un piede del mouse per controllare la coscienza. Una volta confermata inconscio, posizionare il mouse su un supporto intubazione angolato a ~ 60 ° e tenerlo in posizione agganciando i suoi incisivi superiori su una piccola banda di gomma situato nella parte superiore dello stand. </li></ol> 2. Intubazione endotracheale </p&gt; <ol><li> Rientrare delicatamente la lingua del mouse per un lato con un Q-tip. </li><li> Quando si utilizza il Biolite intubazione Illuminazione, inserire con attenzione il sistema intubazione fino alla laringe viene visualizzato con l&#39;aiuto della guida di luce a fibre ottiche. </li><li> Una volta che l&#39;epiglottide e le cartilagini aritenoidee vengono visualizzati, inserire la fibra sopra l&#39;epiglottide e tra le cartilagini aritenoidee, e avanzare finché è stata inserita la corretta lunghezza di catetere. </li></ol> Nota: Per avere la corretta lunghezza di catetere da inserire, inizialmente misurare la lunghezza tra la bocca e il punto di biforcazione bronchiale utilizzando un mouse pratica di dimensioni simili in anticipo (Figura 1). La lunghezza dipende in gran parte dalla dimensione del mouse. Inserimento del catetere deve smettere di sopra del punto di biforcazione (circa 1,5 cm per mouse con ~ 25 g di peso corporeo). Questo assicura che il materiale intubato andrà a tutti lobi. Almeno il 50 pratica mice può essere richiesto per una persona che esegue la intubazione per diventare esperti nella tecnica (avanzato significa che il tasso di successo di intubazione è superiore al 95%). <ol start="4"><li> Una volta inserito il catetere, rimuovere rapidamente la guida di luce a fibre ottiche dal catetere per permettere all&#39;animale di respirare normalmente. Quando il sistema intubazione illuminazione non viene utilizzata, inserire direttamente un catetere come descritto. </li><li> Aggiungere una soluzione contenente materiali di interesse ad un catetere. Assicurarsi che la soluzione viene aspirata nei polmoni subito dopo l&#39;aggiunta. Cinquanta microlitri per ~ 25 g di peso corporeo del mouse viene abitualmente utilizzati. </li></ol> 3. Il recupero degli animali <ol><li> Non appena la soluzione viene aspirata nei polmoni, prendere giù il mouse dal cavalletto, e rimetterlo nella gabbia originale. </li><li> Osservare il mouse fino a quando non inizia a muoversi. </li><li> Una volta confermato che il mouse è in buone condizioni, riportare la gabbia di un rack. </li></ol>

<ol>
	<li>Das, S., Macdonald, K., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> J. Vis. Exp. (73), e50318 (2013).</li><li>Miller, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+murine+intranasal+dosing+efficiency+using+luminescent+Francisella+tularensis:+effect+of+instillation+volume+and+form+of+anesthesia.">Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.</a> PLoS One. 7, e31359 (2012).</li><li>Rayamajhi, M., Redente, E. F., Condon, T. V., Gonzalez-Juarrero, M., Riches, D. W., Lenz, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-surgical+intratracheal+instillation+of+mice+with+analysis+of+lungs+and+lung+draining+lymph+nodes+by+flow+cytometry.">Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.</a> J. Vis. Exp. (51), e2702 (2011).</li><li>Riesenfeld, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+salmeterol+and/or+fluticasone+alters+structure/function+in+a+murine+model+of+allergic+airways+disease.">Inhaled salmeterol and/or fluticasone alters structure/function in a murine model of allergic airways disease.</a> Respir. Res. 11, 22 (2010).</li><li>Rivera, B., Miller, S., Brown, E., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+endotracheal+intubation+of+mice+and+rats+used+in+imaging+studies.">A novel method for endotracheal intubation of mice and rats used in imaging studies.</a> Contemp. Top. Lab Anim. Sci. 44, 52-55 (2005).</li><li>Bonniaud, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+transforming+growth+factor+beta1-induced+lung+fibrosis+is+blocked+by+an+orally+active+ALK5+kinase+inhibitor.">Progressive transforming growth factor beta1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor.</a> Am. J. Res. Crit. Care Med. 171, 889-898 (2005).</li><li>Bivas-Benita, M., Zwier, R., Junginger, H. E., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+pulmonary+aerosol+delivery+in+mice+by+the+endotracheal+route.">Non-invasive pulmonary aerosol delivery in mice by the endotracheal route.</a> Eur. J. Pharm. Biopharm. 61, 214-218 (2005).</li><li>Haegens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+deficiency+attenuates+lipopolysaccharide-induced+acute+lung+inflammation+and+subsequent+cytokine+and+chemokine+production.">Myeloperoxidase deficiency attenuates lipopolysaccharide-induced acute lung inflammation and subsequent cytokine and chemokine production.</a> J. Immunol. 182, 7990-7996 (2009).</li><li>Jackson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+pregnant+mice+to+carbon+black+by+intratracheal+instillation:+toxicogenomic+effects+in+dams+and+offspring.">Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic effects in dams and offspring.</a> Mutat. Res. 745, 73-83 (2012).</li><li>Bodewes, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Influenza+A/H5N1+virus+infection+in+ferrets+differs+between+intranasal+and+intratracheal+routes+of+inoculation.">Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation.</a> Am. J. Pathol. 179, 30-36 (2011).</li><li>Winslow, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+lung+adenocarcinoma+progression+by+Nkx2-1.">Suppression of lung adenocarcinoma progression by Nkx2-1.</a> Nature. 473, 101-104 (2011).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Intubazione endotracheale qui descritto è un metodo semplice, ma ottima non invasivo per fornire in modo uniforme i materiali di interesse per il mouse del polmone. Questo metodo permette studio dell&#39;effetto e / o il ruolo del materiale somministrato sulla fisiologia e / o patologia polmonare. I materiali possono essere somministrati molecole endogene come le citochine, o materiali esogeni, quali sostanze chimiche xenobiotiche / farmaci, sostanze cancerogene, inquinanti, allergeni, o virus che provocano varie malat...

Polmone è un organo in cui molte malattie devastanti sono comunemente diagnosticati. Tra questi, il cancro del polmone è la seconda più cancro diagnosticati in uomini e donne, e la causa più comune di morte per cancro. Malattia cronica polmonare ostruttiva, noto anche come enfisema e bronchite cronica, è una malattia molto grave e la terza causa di morte negli Stati Uniti. Nel 2011, è stato stimato che 25,9 milioni di americani avevano l&#39;asma, tra cui 7,1 milioni di bambini al di sotto dei 18 anni. L&#39;asma è la terza causa di ospedalizzazione tra i bambini al di sotto dei 15 anni (American Lung Association, <a href="http://www.lung.org" target="_blank">http://www.lung.org</a> ). Per studiare la fisiopatologia e / o eziologia di queste malattie devastanti e dei loro meccanismi sottostanti, l&#39;uso di modelli accurati è critica in congiunzione con somministrazione conveniente e non invasiva di vari materiali di interesse per polmone. I topi forniscono uno dei migliori modelli distudiare i meccanismi molecolari alla base della patologia e fisiologia delle malattie polmonari a causa della crescente disponibilità di eliminazione diretta e topi transgenici e una grande quantità di informazioni genetiche disponibili. Vari metodi sono stati utilizzati da un certo numero di ricercatori in contesti diversi per fornire materiali di interesse per i polmoni del mouse, tra cui inalazione, instillazione intranasale, instillazione, e l&#39;intubazione endotracheale 1-4. Quest&#39;ultima procedura non è stato utilizzato di routine perché è considerato piuttosto difficile da eseguire. Intubazione endotracheale qui descritto è un metodo non invasivo, semplice e pratica per fornire materiali di interesse ai polmoni di topo per studiare l&#39;effetto dei materiali consegnati sul pattern di espressione genica, patologia e / o la fisiologia del polmone 5. Questa tecnica garantisce la consegna di materiali esogeni per un intero polmone e non comporta alcun intervento chirurgico sopravvivenza e quindi sarà likely essere approvato da tutti i comitati per la cura degli animali e uso istituzionale.

<table border="1">
	<tbody>
		<tr>
			<td>KetaVed</td>
			<td>Vedco</td>
			<td></td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>AnaSed Injection 20 mg</td>
			<td>LLOYD</td>
			<td></td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>BioLite Stand</td>
			<td>Braintree Scientific</td>
			<td>RIS100</td>
			<td>For mice</td>
		</tr>
		<tr>
			<td>BioLite Intubation Illumination System</td>
			<td>Braintree Scientific</td>
			<td>BIO MI-KIT</td>
			<td>For mice</td>
		</tr>
		<tr>
			<td>22 G, 1 inch i.v. catheter</td>
			<td>Terumo</td>
			<td>SR-OX2225CA</td>
			<td>For mice below 30 g.&#160;Catheter is used only for the case where the Intubation Illumination System is not used.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>or</td>
		</tr>
		<tr>
			<td>20 G, 1 inch i.v. catheter</td>
			<td>Terumo</td>
			<td>SR-OX2025CA</td>
			<td>For mice over 30 g.&#160;Catheter is used only for the case where the Intubation Illumination System is not used.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Catheter is used only for the case where the Intubation Illumination System is not used</td>
		</tr>
	</tbody>
</table>

noninvasive intratracheal intubation to study the pathology and physiology of mouse lung

L&#39;uso di un modello che simula la condizione di malattie polmonari nell&#39;uomo è fondamentale per lo studio della fisiopatologia e / o eziologia di una particolare malattia e per lo sviluppo di un intervento terapeutico. Con la crescente disponibilità di knockout e transgenici derivati, insieme ad una grande quantità di informazioni genetiche, i topi forniscono una delle migliori modelli per studiare i meccanismi molecolari alla base della patologia e fisiologia delle malattie polmonari. Inalazione, instillazione intranasale, instillazione, e l&#39;intubazione endotracheale sono le tecniche più utilizzate da un numero di investigatori per amministrare i materiali di interesse per i polmoni del mouse. Ci sono pro e contro per ogni tecnica a seconda degli obiettivi di uno studio. Ecco un metodo non invasivo intubazione endotracheale in grado di fornire direttamente i materiali esogeni ai polmoni del mouse è presentato. Questa tecnica è stata applicata ad amministrare bleomicina ai polmoni topo come modello per lo studio della fibrosi polmonare. &lt;/ P&gt;

Inizialmente, soluzione verde-tinto è stato usato per intubare topi per la pratica. Il polmone è stato asportato immediatamente dopo l&#39;intubazione per esaminare come uniformemente il colore è stato distribuito nel polmone (Figura 2). Questa tecnica è stata applicata per studiare la fibrosi polmonare indotta da bleomicina utilizzando C57BL / 6 topi. Quando i topi sono stati intratracheale intubati con 1,2 U / kg di bleomicina o soluzione fisiologica come controllo e necroscopia dopo 3 settimane, ...

Watch this Scientific Journal Video about Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung at JoVE.com

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

L&#39;uso di un modello che simula la condizione di malattie polmonari nell&#39;uomo è fondamentale per lo studio della fisiopatologia e / o eziologia di una particolare malattia e per lo sviluppo di un intervento terapeutico. Ecco un metodo non invasivo intubazione endotracheale in grado di fornire direttamente i materiali esogeni ai polmoni del mouse è presentato.

Non invasivo intratracheale intubazione per studiare la Patologia e Fisiologia del mouse polmone

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

noninvasive-intratracheal-intubation-to-study-pathology-physiology

Research

JoVE Journal

Medicine

24.4K Views.  National Institutes of Health.  L'uso di un modello che simula la condizione di malattie polmonari nell'uomo è fondamentale per lo studio della fisiopatologia e / o eziologia di una particolare malattia e per lo sviluppo di un intervento terapeutico. Ecco un metodo non invasivo intubazione endotracheale in grado di fornire direttamente i materiali esogeni ai polmoni del mouse è presentato. 

Video: Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

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  ...

Ricerca

Medicina

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Un modello murino di ischemia miocardica e riperfusione lesioni da legatura della arteria discendente anteriore sinistra

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

L&#39;impianto di fibrina gel sul mouse polmone per studiare angiogenesi Lung-specifica

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Intubazione-mediata intratracheale (IMIT) instillazione: A non invasiva, Lung-specifico sistema di rilascio

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterizzazione cardiaca nei topi per misurare il rapporto pressione del volume: Indagare l&#39;effetto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Modello apicale resezione mouse per primo studio Mammalian rigenerazione cardiaca

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Combinato di microscopia Intravital e l'ecografia contrapporre-aumentata del Hindlimb Mouse per studiare la perfusione del muscolo e la vasodilatazione indotta da insulina

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Misurazione ecocardiografica dei parametri diastolici ventricolari a destra nel mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Indurre lesioni polmonari acute nei topi mediante instillazione diretta del lipopolioaccaride intracheale

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Elettrocardiografia non invasiva nel topo perinatale

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...