Name: measuring the stiffness of ex vivo mouse aortas using atomic force microscopy
Uploaded: 2016-10-19T14:00:00
Description: Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

Tier Arbeit in dieser Studie wurde von den Institutional Animal Care und Verwenden Ausschüssen der University of Pennsylvania genehmigt. Die Verfahren wurden in Übereinstimmung mit den genehmigten Richtlinien durchgeführt. 1. Vorbereiten der Maus und Isolierung der Aorta <ol><li> Anästhesieren eine Maus mit Ketamin (80 - 100 mg / kg), Xylazin (8 - 10 mg / kg) und Acepromazin (1 - 2 mg / kg) intraperitoneal. Bestätigen Anästhesie mit einem Schwanz Kneiftest. Sobald die Maus vollständ...

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AFM Einbuchtung kann verwendet werden, um die Steifigkeit (Elastizitätsmodul) von Zellen und Geweben zu charakterisieren. In diesem Beitrag stellen wir ausführliche Schritt- für -Schritt - Protokolle , um die absteigende Aorta und Aortenbogen in der Maus und bestimmen die Elastizitätsmoduli dieser arteriellen Regionen ex vivo zu isolieren. Wir haben jetzt zusammenfassen und die technischen Probleme und Einschränkungen des Verfahrens in diesem Papier beschrieben diskutieren. Ver...

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">BioScope Catalyst AFM system</td>
			<td style="width:191px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE 200 inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Silicon nitride AFM probe</td>
			<td>Novascan Technologies</td>
			<td>PT.SI02.SN.1</td>
			<td>0.06 N/m cantilever; 1 &#181;m SiO2 particle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>See section 1.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5SF forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>See section 1.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fine Scissors-ToughCut</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td>See section 1.4 (medium sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas-T&#252;bingen spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td>See section 1.6 (small sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mmTC-treated cell culture dish</td>
			<td>Corning</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Krazy Glue instant all purpose liquid</td>
			<td>Krazy Glue</td>
			<td>KG58548R</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel-loading tips, 1-200 &#181;L</td>
			<td>Fisher</td>
			<td>02-707-139</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip Tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78092-CP</td>
			<td>See section 3.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mm, clear wall glass bottom dishes</td>
			<td>TED PELLA</td>
			<td>14027-20</td>
			<td>See section 4.4</td>
		</tr>
	</tbody>
</table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

Arterielle Versteifung ist ein signifikanter Risikofaktor und Biomarker für Herz-Kreislauf-Erkrankungen und ein Markenzeichen des Alterns. Rasterkraftmikroskopie (AFM) ist ein vielseitiges Analysewerkzeug für viskoelastische mechanischen Eigenschaften für eine Vielzahl von Materialien zu charakterisieren die von harten (Kunststoff, Glas, Metall, etc.) Oberflächen auf Zellen , die auf jedem Untergrund. Es wurde in großem Umfang verwendet, um die Steifigkeit der Zellen zu messen, aber weniger häufig verwendet, um die Steifigkeit des Aorten messen. In diesem Beitrag werden wir die Verfahren für die Verwendung von AFM im Kontaktmodus beschreiben die ex vivo - Elastizitätsmodul von unbelastetem Maus Arterien zu messen. Wir beschreiben unsere Verfahren zur Isolierung von Maus Aorten und dann detaillierte Informationen für die AFM-Analyse liefern. Dazu gehören die Schritt-für-Schritt-Anleitungen für die Ausrichtung des Laserstrahls eine Kalibrierung der Federkonstante und Ablenkung Empfindlichkeit der AFM-Sonde, und den Erwerb von Kraftkurven. Wir bieten auch ein detailliertes Protokoll für die Daten Analysis der Kraftkurven.

<html> 5A zeigt ein Phasenkontrastbild des absteigenden (Thorax) Aorta aus einem 6-Monate alten, männlichen C57BL / 6 - Maus. Die AFM - Cantilever ist an Ort und Stelle direkt über dem Gewebe und bereit für den Einzug. 5B und 5C zeigen repräsentative Kraftkurven durch AFM Vertiefung im Kontaktmodus erhalten. Grüne Linien in den 5B und 5C gezeigt stellen die besten Fitkurven für eine Kugel mit dem Hertzian Modell erhalten. In <stron...

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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JoVE Journal

Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

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