This work was supported by the National Institutes of Health (HL114669). CM is a recipient of a postdoctoral research fellowship grant awarded by the Myocarditis Foundation, NJ.

ETHICS STATEMENT:
All animal procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the University of Nebraska-Lincoln, Lincoln, NE.
1. Induction of Experimental Autoimmune Myocarditis
<ol>
	<li>Prepare the peptide solution by dissolving Myhc-&#945; 334-352 in 1x phosphate-buffered saline to a final concentration of 2 mg/1.5 ml.</li>
	<li>Prepare the pertussis toxin (PT) by adding 1 m...

<ol>
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L., 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=Myocarditis-inducing+epitope+of+myosin+binds+constitutively+and+stably+to+I-Ak+on+antigen-presenting+cells+in+the+heart.">Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.</a> J Exp Med. 182 (5), 1291-1300 (1995).</li><li>Gangaplara, 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=Coxsackievirus+B3+infection+leads+to+the+generation+of+cardiac+myosin+heavy+chain-alpha-reactive+CD4+T+cells+in+A/J+mice.">Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-alpha-reactive CD4 T cells in A/J mice.</a> Clin Immunol. 144 (3), 237-249 (2012).</li><li>Huber, S. A., Lodge, P. 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No conflicts of interests were declared.

This study describes the MRM procedure and its utility as a non-invasive tool to ascertain cardiac abnormalities in mice affected with autoimmune myocarditis. Since the histologic features of EAM resemble postinfectious myocarditis of humans, mouse models are commonly employed to delineate the immune mechanisms of myocardial injuries23-25. However, the animals affected with myocarditis appear clinically normal, and the diagnosis is made based on histology at the termination of experiments7. The anim...

Heart failure is the leading cause of deaths, and myocarditis is one predominant cause of heart failure in young adolescents1. Most patients affected with myocarditis remain asymptomatic and the disease is spontaneously resolved2. However, 10-20% of those affected can develop chronic disease, leading to dilated cardiomyopathy (DCM)3. Various animal models have been developed to study the immune pathogenesis of myocarditis. The disease can be induced in myocarditis-susceptible A/J and Balb/c mice by immunizing the animals with cardiac myosin heavy chain (Myhc)-&#945; or its immunodominant peptide fragments or by infecting with pathogens like coxsackievirus B3 4-9. This study involves Myhc-&#945; 334-352-induced myocarditis in A/J mice. Despite showing myocardial infiltrations, the myocarditis-affected animals appear clinically normal; diagnosis is based on histological evaluation of hearts for inflammation7 and echocardiography10.
Magnetic resonance microscopy (MRM) is a commonly used method to obtain cardiovascular imaging with high resolution three-dimensional planes, permitting assessment of functional details to the level of minute blood vessels (up to 10 &#956;m diameter), but this level of resolving power is not achievable with the routine magnetic resonance imaging (MRI) scanning procedure, in which, the resolution is generally obtained up to 1 mm11-14. MRM offers an advantage as it permits acquisition of high-resolution images and also to derive performance parameters in the early time points of disease process14. Clinically, MRM imaging has been extensively applied to study the functional parameters of diseased heart, lung or brain15-17. In this study, the use of an MRM technique as a non-invasive tool to ascertain cardiac abnormalities in A/J mice affected with autoimmune myocarditis is shown. Specifically, the MRM imaging allows quantification of functional parameters such as left ventricular (LV) end-diastolic volume and ejection fraction (EF) with reasonable accuracy18. The definitions of respective parameters are: LV end diastolic volume, volume of blood in the left ventricle at the end of diastolic cycle, and ejection fraction, stroke volume/end-diastolic volume. The data analysis is performed using the freely available Segment software developed for processing DICOM-compliant cardiovascular images acquired by magnetic resonance scanners19. The data revealed an increase in thickness of the LV wall in the myocarditic animals, corresponding to a decrease in LV end-diastolic volume, stroke volume and ejection fraction, as compared with these functional parameters in healthy mice.

<table border="0" cellpadding="0" cellspacing="0" width="1185">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:509px;">Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:145px;"></td>
			<td style="width:167px;">Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CFA</td>
			<td style="width:364px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:145px;">5881</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MTB&#160; H37Rv extract&#160;</td>
			<td>Difco Laboratories, Detroit, MI</td>
			<td>231141</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PT</td>
			<td>List Biologicals Laboratories, Campbell, CA</td>
			<td>181</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1x PBS&#160;</td>
			<td>Corning, Manassas, VA</td>
			<td>21-040-CV</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane&#160;</td>
			<td>Piramal Healthcare, Mumbai, India</td>
			<td>NDC66794-013-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female A/J mice&#160;</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 1 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309628</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 3 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 18 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 27 1/2 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stopcock&#160;</td>
			<td>Smiths Medical ASD, Inc. Dublin, OH</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerlix gauze bandage rolls&#160;</td>
			<td>Covidien, Mansfield, MA</td>
			<td>6720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes</td>
			<td>Kimberly-Clark Professional, Roswell, GA</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protouch Stockinette&#160;</td>
			<td>Medline Industries, Mundelein, IL</td>
			<td>30-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical scissors and forceps</td>
			<td style="width:364px;">INOX tool Corporation</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro oven</td>
			<td style="width:364px;">GE Healthcare,&#160;</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ThermiPAQ hot and cold therapy system&#160;</td>
			<td>Theramics Corporation, Springfield, IL</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reptile heating lamp&#160;</td>
			<td>Energy Savers Unlimited, Inc. Carson, CA</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Transpore tapes&#160;</td>
			<td>Target Corporation, MN</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Up and Up Polymyxin B sulfate/Bacitracin/Neomycin sulfate antibiotic ointment</td>
			<td>Target Corporation, MN</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">North Safety DeciDamp-2PVC foam ear plugs</td>
			<td style="width:364px;">North Safety Products, Smithfield, RI</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton tipped applicator, 6&#8217;&#8217; wooden stem&#160;</td>
			<td>Jorgensen Laboratories, Inc. Loveland, CO</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia induction chamber&#160;</td>
			<td>Summit Anesthesia Solutions, Ann Harbor, MI</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Summit Anesthesia Support system for regulating flow of anesthesia&#160;</td>
			<td>Summit Anesthesia Solutions, Ann Harbor, MI</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specially designed animal holder</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bickford&#160;Omnicon F/Air&#160;anesthesia gas filter unit&#160;</td>
			<td>A.M. Bickford, Inc. Wales Center, NY</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulse-oximeter module, MR compatible small animal monitoring and gating system&#160;</td>
			<td>Small Animal Instruments, Inc. Stony Brook, NY</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen cylinder&#160;</td>
			<td>Matheson-Tri Gas, North-Central Zone, Lincoln, NE</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gas regulator&#160;</td>
			<td>Western Medica, West Lake, OH</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Signal breaking module, MR compatible small animal monitoring and gating system</td>
			<td>Small Animal Instruments, Inc. Stony Brook, NY</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.4 T (400 MHZ) 89 mm vertical core bore MR scanner&#160;</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-cm millipede micro-imaging RF coil&#160;</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAM PC monitor&#160;</td>
			<td>Small Animal Instruments, Inc. Stony Brook, NY</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quantitative Medical Image analysis software&#160;</td>
			<td colspan="3">http://segment.heiberg.se;&#160; Segment v1.8 R1430,&#160; Medviso, Oresunds region, Sweden</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab software&#160;</td>
			<td>The Mathworks, Inc.&#160; Natick, MA</td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer-Unix operating system</td>
			<td style="width:364px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

noninvasive assessment of cardiac abnormalities in experimental autoimmune myocarditis by magnetic resonance microscopy imaging in the mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

In this report, the utility of MRM technique as a non-invasive modality to determine the structural and functional changes in the hearts of animals affected with EAM is shown. Myocarditis was induced in A/J mice by immunizing the animals with Myhc-&#945; 334-352 in CFA7, and the animals were subjected to MRM experiments on day 21 post-immunization. The MRM imaging was performed on live animals under isoflurane anesthesia at 9.4 T (400 MHz for protons) using an 89&#160;mm vertical-bore magnet equipped with trip...

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Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

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9.8K vues.  University of Nebraska-Lincoln. This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.