Name: quantitative micro-ct analysis of aortopathy in a mouse model of &#946;-aminopropionitrile-induced aortic aneurysm and dissection
Uploaded: 2018-07-16T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ÃŽÂ²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

Vi vil gerne takke Mark Smith for hans hjælp med Radiografisk billeddannelse. Dette arbejde er støttet af NIH T32 Grant for tværfaglig forskning i hjerte-kar-sygdom (BOA), American Heart Association (SMC) og NIH R35 Grant (DKS).

Protokoller for dyr håndtering blev godkendt af institutionelle Animal Care og brug Udvalget af University of Maryland, Baltimore (animalske protokolnummer 0116024) og gennemføres i henhold til AAALAC internationale standarder.
1. forberedelse af reagenser
<ol><li>
Heparin
	<ol>
<li>Fortynd 250 µL af 1000 U/mL heparin sulfat i 50 mL af phosphat bufferet saltvand til at foretage en endelig koncentration på 5 U/mL.</li>
		<li>Varm heparinized (5 U/mL) fosfatbufferet saltop...

<ol>
	<li>Meszaros, I., 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=Epidemiology+and+clinicopathology+of+aortic+dissection.">Epidemiology and clinicopathology of aortic dissection.</a> CHEST. 117 (5), 1271-1278 (2000).</li><li>Kochanek, K. D., 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=Deaths:+final+data+for+2009.">Deaths: final data for 2009.</a> National Vital Statistics Reports. 60 (3), 1-116 (2011).</li><li>Li, J. S., Li, H. Y., Wang, L., Zhang, L., Jing, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+beta-aminopropionitrile-induced+aortic+dissection+model+in+rats+by+different+administration+and+dosage.">Comparison of beta-aminopropionitrile-induced aortic dissection model in rats by different administration and dosage.</a> Vascular. 21 (5), 287-292 (2013).</li><li>Huffman, M. D., Curci, J. A., Moore, G., Kerns, D. B., Starcher, B. C., Thompson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+importance+of+connective+tissue+repair+during+the+development+of+experimental+abdominal+aortic+aneurysms.">Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms.</a> Surgery. 128 (3), 429-438 (2000).</li><li>Wu, D., Shen, Y. H., Russel, L., Coselii, J. S., LeMaire, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+thoracic+aortic+dissection.">Molecular mechanisms of thoracic aortic dissection.</a> Journal of Surgical Research. 184 (2), 907-924 (2013).</li><li>Bruel, A., Ortoft, G., Oxlund, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+cross-links+in+collagen+is+associated+with+reduced+stiffness+of+the+aorta+in+young+rats.">Inhibition of cross-links in collagen is associated with reduced stiffness of the aorta in young rats.</a> Atherosclerosis. 140 (1), 135-145 (1998).</li><li>Martinez-Revelles, S., 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=Lysyl+oxidase+induces+vascular+oxidative+stress+and+contributes+to+arterial+stiffness+and+abnormal+elastin+structure+in+hypertension:+Role+of+p38MAPK.">Lysyl oxidase induces vascular oxidative stress and contributes to arterial stiffness and abnormal elastin structure in hypertension: Role of p38MAPK.</a> Antioxidants &amp; Redox Signaling. 27 (7), 379-397 (2017).</li><li>Kumar, D., Trent, M. B., Boor, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allylamine+and+beta-aminopropionitrile+induced+aortic+medial+necrosis:+Mechanisms+of+synergism.">Allylamine and beta-aminopropionitrile induced aortic medial necrosis: Mechanisms of synergism.</a> Toxicology. 125 (2-3), 107-115 (1998).</li><li>Ren, W., 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=&#946;-Aminopropionitrile+monofumarate+induces+thoracic+aortic+dissection+in+C57BL/6+mice.">&#946;-Aminopropionitrile monofumarate induces thoracic aortic dissection in C57BL/6 mice.</a> Scientific Reports. 6, 28149 (2016).</li><li>Kanematsu, Y., 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=Pharmacologically-induced+thoracic+and+abdominal+aortic+aneurysms+in+mice.">Pharmacologically-induced thoracic and abdominal aortic aneurysms in mice.</a> Hypertension. 55 (5), 1267-1274 (2010).</li><li>Weyers, J. J., Carlson, D. D., Murry, C. E., Schwartz, S. M., Mahoney, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+Perfusion+and+Filling+of+Mouse+Coronary+Vasculature+as+Preparation+for+Micro+Computed+Tomography+Imaging.">Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging.</a> J Vis Exp. (60), e3740 (2012).</li><li>Cortell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+rubber+for+renal+tubular+injection.">Silicone rubber for renal tubular injection.</a> Journal of Applied Physics. 26 (1), 158-159 (1969).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 282 (5), R1267-R1279 (2002).</li><li>Marxen, 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=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Medical Physics. 31 (2), 305-313 (2004).</li><li>Yang, J., Yu, L. X., Rennie, M. Y., Sled, J. G., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+hemodynamic+analysis+of+vascular+trees.">Comparative structural and hemodynamic analysis of vascular trees.</a> American Journal of Physiology - Heart and Circulatory Physiology. 298 (4), H1249-H1259 (2010).</li><li>Jia, L. X., 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=Mechanical+stretch-induced+endoplasmic+reticulum+stress,+apoptosis,+and+inflammation+contribute+to+thoracic+aortic+aneurysm+and+dissection.">Mechanical stretch-induced endoplasmic reticulum stress, apoptosis, and inflammation contribute to thoracic aortic aneurysm and dissection.</a> The Journal of Pathology. 236 (3), 373-383 (2015).</li><li>Kurihara, T., 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=Neutrophil-derived+matrix+metalloproteinase+9+triggers+acute+aortic+dissection.">Neutrophil-derived matrix metalloproteinase 9 triggers acute aortic dissection.</a> Circulation. 126 (25), 3070-3080 (2012).</li><li>Dillavou, E. D., Buck, D. G., Muluk, S. C., Makaroun, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+versus+three-dimensional+CT+scan+for+aortic+measurement.">Two-dimensional versus three-dimensional CT scan for aortic measurement.</a> Journal of Endovascular Therapy. 10 (3), 531-538 (2003).</li><li>Muratoglu, S. C., 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=LRP1+protects+the+vasculature+by+regulating+levels+of+connective+tissue+growth+factor+and+HtrA1.">LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.</a> Arterioclerosis, Thrombosis, and Vascular Biology. 33, 2137-2146 (2013).</li><li>Badea, C. T., Dragova, M., Holdsworth, D. W., Johnson, G. 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Mikro-CT billeddannelse kan bruges til at give meget detaljerede og tre-dimensionelle rekonstruktioner af vaskulære patologi i dyremodeller. Gennem brug af intravaskulære kontrast-forbedret medier, kan ikke forbedret blødt væv, såsom lumen af et blodkar, skelnes fra dem, der styrke. Mens laser Doppler, microangiography, magnetisk resonans angiografi, histologi med fluorescens, eller to-foton mikroskopi kan bruges til at vurdere vaskulære senge, de typisk fokuserer på et begrænset område af undersøgelse og/eller...

Forekomsten af aorta dissektion er 3 tilfælde per 100.000 per år1. Aorta dissektion og aneurysmal sygdomme tegner sig for over 10.000 dødsfald i USA hvert år, tegner sig for 1-2% af alle dødsfald i vestlige lande2. Aorta dissektion er initieret af en rive i intima lag af fartøjet med udbredelsen af blod gennem lag af aorta væggen under fysiologisk pres. Forhøjede patientens puls pres er forbundet med en øget forekomst af dissektion og komplikationer. Øget væg shear stress er forbundet med aortavæggen udvidelsen fører til en aneurisme dannelse3,4. Konsekvenserne af aortadissektion omfatter okklusion af blodgennemstrømningen til fjerntliggende organer, herunder hjerne, nyrer, tarme, og lemmer, dannelse af kronisk aneurismer, brud eller død5,6,7.
I øjeblikket er de biokemiske og cellulære processer i indledningen og progression af aortaaneurismer og dissektioner stadig dårligt forstået. Reproducerbare dyremodeller for aorta aneurisme og dissektion er nøglen til at forstå deres patofysiologi. Β-aminopropionitrile (BAPN) er en lysyl oxidase hæmmer, som forhindrer cross-linking af elastin og kollagen, og har vist sig at markant ændrer strukturen af fartøjet væg ekstracellulære matrix og dens biomekaniske integritet6 8. Rotter behandlet med BAPN har været udnyttet som en fælles dyremodel af aorta aneurisme og dissektion9,10.
Vaskulære billeddiagnostiske modaliteter er medvirkende til at identificere vaskulære patologi, bekræfter fartøjets passage og evaluere orgel perfusion. For nylig, mikro-beregnet tomografi (mikro-CT) er blevet udnyttet til at studere Vaskulaturen af mus og ligeledes store dyr. I modsætning til knoglen er den aksiale billeddannelse af blodkar ved computertomografi begrænset, da intraluminal blod er i sagens natur relativt radiolucent. Når de kombineres med intravaskulær kontraststoffer, dog muliggør mikro-CT detaljeret tre-dimensionel rekonstruktioner af animalske vasculatures til studiet af makro-anatomiske vaskulære patologi11.
Den valgte kontraststof er (Se Tabel af materialer) en røntgenfast silikonegummi, der indeholder bly chromat og bly sulfat. Ved perfusion i tilstedeværelse af en katalysator, det hurtigt hærder til at danne en afstøbning af Vaskulaturen med minimale ændringer i den makro-anatomiske arkitektur af fartøjer, gør Vaskulaturen meget røntgenfast i modsætning til baggrunden væv når undersøgt vurdere. Denne kontraststof er fordelagtig, fordi det er let at håndtere og undgår væv nedbrydning og fartøj tab på grund af brud ofte forbundet med vaskulære støbt korrosion. Som det hærder med minimal krympning12, fartøjer ryddet af blod fortsat patent og giver mulighed for en præcis vurdering af dyrs makro-Vaskulaturen i ikke-overlevelse eksperimenter. Tidligere arbejde har med held brugt røntgenfast silikone gummi-kontrast i en række dyreforsøg. Specifikt, har anvendelighed i at visualisere koronar, glomerulær, placenta, og cerebral oplag11,12,13,14,15 været vist. I dette papir detalje vi metode af åbne venstre ventrikel punktering for det intravaskulære perfusion af blyholdige røntgenfast silikonegummi kvantitativt kendetegner BAPN-induceret aorta patologi i en musemodel af mikro-CT.

<table>
	<tbody>
		<tr>
			<td>Microfil</td>
			<td>Flow Tech, Inc</td>
			<td>MV-122</td>
			<td>We use yellow, a different color can be ordered as desired. Kit includes MV-Compound, MV-Diluent, and MV-Curing Agent.</td>
		</tr>
		<tr>
			<td>Heparin (1000 U/mL)</td>
			<td>Sagent Pharmaceuticals</td>
			<td>25021-400-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vet One, MWI</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropionitrile fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A3134</td>
			<td></td>
		</tr>
		<tr>
			<td>Single syringe pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td></td>
		</tr>
		<tr>
			<td>27-gauge scalp vein set needle</td>
			<td>Exel Int</td>
			<td>26709</td>
			<td>27G x 3/4&#34;, 12&#34; tube</td>
		</tr>
		<tr>
			<td>Inveon Micro-CT scanner</td>
			<td>Siemens Medical Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osirix MD</td>
			<td>Pimxmeo SARL</td>
			<td></td>
			<td>Version 8.0.2</td>
		</tr>
		<tr>
			<td>Inveon Research Workplace</td>
			<td>Siemens Medical Solutions</td>
			<td></td>
			<td>Version 4.2</td>
		</tr>
		<tr>
			<td>Rodent Chow</td>
			<td>Harlan Teklad</td>
			<td>2018sx</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative micro-ct analysis of aortopathy in a mouse model of &#946;-aminopropionitrile-induced aortic aneurysm and dissection

Aorta aneurisme og dissektion er forbundet med betydelig morbiditet og dødelighed i befolkningen og kan være meget dødbringende. Mens dyremodeller for aorta sygdom findes, har i vivo billeddannelse af Vaskulaturen været begrænset. I de seneste år, mikro-edb tomografi (mikro-CT) fremstod som en foretrukne modalitet for imaging både store og små fartøjer både i vivo og ex vivo. I forbindelse med en metode af vaskulære støbning, har vi med succes brugt micro-CT til at karakterisere frekvens og distribution af aorta patologi i β-aminopropionitrile-behandlede C57/Bl6 mus. Tekniske begrænsninger af denne metode omfatter variationer i kvaliteten af perfusion indført af dårlig dyr forberedelse, anvendelsen af passende metoder til fartøjets størrelse kvantificering og overlevelsesevne af denne procedure. I denne artikel beskrives en metode for det intravaskulære perfusion af blyholdige røntgenfast silikonegummi til kvantitative karakterisering af aortopathy i en musemodel af aneurisme og dissektion. Ud over at visualisere aorta patologi, kan denne metode anvendes til at undersøge andre vaskulære senge i vivo eller vaskulær senge fjernet post mortem.

For at evaluere denne protokol, 20 mandlige voksne mus, blandet baggrund som tidligere beskrevet19 og 20-30 ugens i alder, med eller uden BAPN behandling, var perfunderet med en bly-baserede røntgenfast silikonegummi (Se tabel af materialer ) ved hjælp af den protokol, der er beskrevet ovenfor. De gennemgik mikro-CT-scanning på den følgende dag (figur 1 og figur 2). D...

Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ÃŽÂ²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-aminopropionitrile-induced Aortic Aneurysm and Dissection

Kvantitative mikro-CT-analyse af Aortopathy i en musemodel af β-aminopropionitrile-induceret aorta aneurisme og dissektion

I denne artikel beskrives en detaljeret metode til ved hjælp af en røntgenfast bly-baseret silikonegummi til perfuse murine Vaskulaturen for aorta diameteren kvantificering i en musemodel af aorta aneurisme og dissektion.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

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Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

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