This work was supported by grant 13IRG14560023 from the American Heart Association and NIH grants AI078972 and HL108054 to CJO. Support for AOB was from the NIH National Center for Advancing Translational Sciences NIH ULTR001120, and F31 A110417701.

NOTE: All mouse experiments were reviewed and approved by the Institutional Animal Care and Use Committees at The University of Texas Health Science Center at San Antonio (protocol #13032-34-01C). Animal care and experimental protocols adhered to Public Law 89-544 (Animal Welfare Act) and its amendments, Public Health Services guidelines, and the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health &#38; Human Services).
1. Infection
<ol>
	<li>Obtain BALB/c mice of both sexes between the ages of 10-12 weeks of age. 
	NOTE: S. pneumoniae serotype 4 strain TIGR4 is a virulent clinical isolate that has been sequenced16.</li>
	<li>Grow TIGR4 in Todd-Hewitt broth at 37 &#176;C in 5% CO2 until reaching an optical density (OD)620 of 0.5 (corresponding to 1.0 x 108 colony forming units [CFU]/ml). Pellet the pneumococcal culture by centrifugation for 10 min at 3,500 x g and remove the supernatant using a vacuum line. Suspend and dilute bacteria with sterile phosphate-buffered saline (PBS) to a final concentration of 1.0 x 104 CFU/ml.</li>
	<li>Using an induction chamber, anesthetize mice with 2.5% vaporized isoflurane in oxygen. Confirm anesthetized state by gentle toe pinch with blunt tweezers.
	<ol>
		<li>Holding the anesthetized mouse by its scruff and upright with one hand, inject each mouse intraperitoneally (i.p.) with 100 &#181;l of the S. pneumoniae suspension using a syringe with a 27-30 gauge needle (corresponding to a challenge dose of 1.0 x 103 CFU). Place the mouse back in its cage. Mice typically awaken 30-40 sec after injection.</li>
	</ol>
	</li>
	<li>Allow the infection to proceed for at least 24 (early) or 30 (advanced) hr.
	<ol>
		<li>Euthanize the mice by CO2 asphyxiation. Perform cervical dislocation to ensure mouse has deceased. Euthanasia is further confirmed by removal of the heart in Step 1.6.</li>
	</ol>
	</li>
	<li>Disinfect the tail with an alcohol swab and snip a 2-3 mm section. Collect 2 &#181;l of blood and serially dilute the blood 10-fold in PBS containing sodium heparin 1 U/ml five times. Plate serial dilutions on a tryptic soy blood agar plate and incubate overnight at 37 &#176;C in 5% CO2. The next day extrapolate from colony counts the level of bacteremia.</li>
	<li>Immobilize the mouse in a supine position on a surgical platform. Spray the chest with 70% ethanol and pat dry. Using surgical scissors and forceps open the chest cavity, remove the rib cage, and transect the diaphragm to expose the heart and lungs. Using scissors cut the blood vessels connected to the heart and gently excise the heart with care to avoid bruising with forceps.</li>
	<li>Rinse the heart with PBS and then place into tissue specimen collection cassettes such that coronal sections would be obtained during tissue sectioning. For paraffin embedding, place the cassettes in 10% buffered-formalin solution and the next day send for paraffin embedding.
	<ol>
		<li>Alternatively flash freeze using O.C.T. Compound within a cryomold that has been placed on dry ice.</li>
	</ol>
	</li>
</ol>
2. Visualization of Cardiac Lesions and Pneumococci within Lesions
<ol>
	<li>Cut down paraffin-embedded heart sections such that the 4 chambers of the heart are visible in each tissue section. Cut slides at thickness of 5 &#181;m.</li>
	<li>Deparaffinize and stain slides with hematoxylin &#38; eosin (H&#38;E) using standard methods17.</li>
	<li>View H&#38;E stained cardiac sections using a light microscope at low (100X, 200X) and high (oil immersion: 400X, 1,000X) magnifications. Characterize hearts for the presence of microlesions in the myocardium. Here, acquire H&#38;E images using a Zeiss Axioskop 2 microscope equipped with a 100X 1.3 numerical aperture Plan-NEOFLUAR objective. 
	NOTE: At early stages cardiac lesions are discriminated by their differential colored appearance and in some instances the presence of immune cells (monocytes and granulocytes) what appear to be monocytes. In advanced lesions, particularly those seen at 30 hr, immune cells are absent and large vacuole-like lesions are observed in their place. It is important to note that while immune cell influx during the early stages of cardiac lesion development can occur it does not appear to be a requirement.</li>
</ol>
3. Immunofluorescent Microscopy for Pneumococci within Microlesions
<ol>
	<li>Section heart in cryomolds with microtome to a thickness of 5 &#956;m and place sections onto positively charged glass slides. Allow frozen cardiac sections to thaw and air dry. Fix sections for 10 min in 10% neutral buffered formalin (pH 6.8-7.2 at 25 &#176;C).</li>
	<li>Wash slides (3x) in PBS for 5 min, permeabilize for 15 min in 0.2% Triton X-100 in PBS, then wash again in PBS.</li>
	<li>Block tissue section on slides with 10% goat serum in PBS for 1 hour.</li>
	<li>Rinse slides with PBS, cover and incubate cardiac sections with rabbit anti-serotype 4 pneumococcus antiserum at 1:1,000 in PBS with 10% goat serum for 2 hr at 37 &#176;C. 
	NOTE: For negative controls investigators should use naive rabbit antiserum</li>
	<li>After washing with PBS, cover and incubate sections with 10% goat serum-PBS with goat anti-rabbit FITC conjugated antibody at 1:2,000 for 30 min at 37 &#176;C.</li>
	<li>Following the manufacturer&#8217;s instruction, use DAPI (4&#39;,6- Diamidino-2-Phenylindole) at 5 mg/ml for visualization of eukaryotic nuclei. Wash and mount tissue sections with FluorSave.</li>
	<li>Acquire fluorescent images using a confocal microscope system at both low and high magnification. Adjust the numerical aperture accordingly to obtain optimal results. Here, acquire fluorescent images using a confocal system with a 60X 1.42 numerical aperture objective.</li>
</ol>
4. Antibiotic Rescue of Septic Mice
<ol>
	<li>Begin with mice infected i.p. with 1.0 x 103 CFU of TIGR4 as previously indicated in step 1.</li>
	<li>Beginning at 30 hr post-infection, administer pharmaceutical grade&#160;ampicillin i.p. (80 mg/kg body weight) in 100 &#181;l saline every 12 hr for 36 hr. Collect hearts at the desired time point and process as described in step 1.6 for paraffin embedding and tissue, sectioning.</li>
</ol>
5. Collagen Deposition Detection
<ol>
	<li>Using xylene and graded alcohol washes with deionized water, deparaffinize and hydrate tissue sections using standard methods17. Treat sections with aqueous 0.2% phosphomolybdic acid for 5 min and rinse with deionized water for 5 min.</li>
	<li>Stain sections with 0.1% Sirius Red in picric acid for 2 hr at room temperature.</li>
	<li>Wash sections in 0.01 N hydrochloric acid for 3 min and rinse using 70% ethanol for 1 min.</li>
	<li>Dehydrate the sections using the identical but reverse order of washes to that outlined in 5.1, dehydrate cardiac sections in increasing and graded concentrations of ethanol then xylene.</li>
	<li>Mount tissue sections with a mounting solution for microscopic evaluation.</li>
	<li>To identify regions of collagen deposition, examine stained tissue sections for areas of deep read staining under low and high magnification using a light microscope. Any area within the ventricles of the heart that is red is an area where collagen has been deposited. Areas without this red stain are sites of healthy tissue lacking collagen deposition. Herein, acquire Picro Sirius Red stained images using a microscope equipped with a 20X 0.5 numerical aperture objective. 
	NOTE: The atrium stains red in healthy tissues.</li>
</ol>

<ol>
	<li>Corrales-Medina, V. F., 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=Cardiac+complications+in+patients+with+community-acquired+pneumonia:+incidence,+timing,+risk+factors,+and+association+with+short-term+mortality.">Cardiac complications in patients with community-acquired pneumonia: incidence, timing, risk factors, and association with short-term mortality.</a> Circulation. 125, 773-781 (2012).</li><li>Corrales-Medina, V. F., 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=Acute+bacterial+pneumonia+is+associated+with+the+occurrence+of+acute+coronary+syndromes.">Acute bacterial pneumonia is associated with the occurrence of acute coronary syndromes.</a> Medicine (Baltimore). 88, 154-159 (2009).</li><li>Corrales-Medina, V. F., 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=Cardiac+complications+in+patients+with+community-acquired+pneumonia:+a+systematic+review+and+meta-analysis+of+observational+studies.">Cardiac complications in patients with community-acquired pneumonia: a systematic review and meta-analysis of observational studies.</a> PLoS Med. 8, e1001048 (2011).</li><li>Corrales-Medina, V. F., 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=Risk+stratification+for+cardiac+complications+in+patients+hospitalized+for+community-acquired+pneumonia.+Mayo+Clin.">Risk stratification for cardiac complications in patients hospitalized for community-acquired pneumonia. Mayo Clin.</a> Proc. 89, 60-68 (2014).</li><li>Kumar, 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=Detection+of+11+common+viral+and+bacterial+pathogens+causing+community-acquired+pneumonia+or+sepsis+in+asymptomatic+patients+by+using+a+multiplex+reverse+transcription-PCR+assay+with+manual+(enzyme+hybridization)+or+automated+(electronic+microarray)+detection.">Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection.</a> Journal Of Clinical Microbiology. 46, 3063-3072 (2008).</li><li>Musher, D. M., Rueda, A. M., Kaka, A. S., Mapara, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+pneumococcal+pneumonia+and+acute+cardiac+events.">The association between pneumococcal pneumonia and acute cardiac events.</a> Clin Infect Dis. 45, 158-165 (2007).</li><li>Brown, A. O., 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=Streptococcus+pneumoniae+translocates+into+the+myocardium+and+forms+unique+microlesions+that+disrupt+cardiac+function.">Streptococcus pneumoniae translocates into the myocardium and forms unique microlesions that disrupt cardiac function.</a> PLoS Pathogens. In press, (2014).</li><li>Orihuela, C. J., 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=Laminin+receptor+initiates+bacterial+contact+with+the+blood+brain+barrier+in+experimental+meningitis+models.">Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models.</a> The Journal of Clinical Investigation. 119, 1638-1646 (2009).</li><li>Flick, M. J., 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=Genetic+elimination+of+the+binding+motif+on+fibrinogen+for+the+S.+aureus+virulence+factor+ClfA+improves+host+survival+in+septicemia.">Genetic elimination of the binding motif on fibrinogen for the S. aureus virulence factor ClfA improves host survival in septicemia.</a> Blood. 121, 1783-1794 (2013).</li><li>Cheng, A. G., DeDent, A. C., Schneewind, O., Missiakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+play+in+four+acts:+Staphylococcus+aureus+abscess+formation.">A play in four acts: Staphylococcus aureus abscess formation.</a> Trends Microbiol. 19, 225-232 (2011).</li><li>Arenal, 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=Do+the+spatial+characteristics+of+myocardial+scar+tissue+determine+the+risk+of+ventricular+arrhythmias.">Do the spatial characteristics of myocardial scar tissue determine the risk of ventricular arrhythmias.</a> Cardiovasc Res. 94, 324-332 (2012).</li><li>Deneke, 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=Human+histopathology+of+electroanatomic+mapping+after+cooled-tip+radiofrequency+ablation+to+treat+ventricular+tachycardia+in+remote+myocardial+infarction.">Human histopathology of electroanatomic mapping after cooled-tip radiofrequency ablation to treat ventricular tachycardia in remote myocardial infarction.</a> J Cardiovasc Electrophysiol. 16, 1246-1251 (2005).</li><li>Bakker, J. 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=Reentry+as+a+cause+of+ventricular+tachycardia+in+patients+with+chronic+ischemic+heart+disease:+electrophysiologic+and+anatomic+correlation.">Reentry as a cause of ventricular tachycardia in patients with chronic ischemic heart disease: electrophysiologic and anatomic correlation.</a> Circulation. 77, 589-606 (1988).</li><li>Verma, 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=Relationship+between+successful+ablation+sites+and+the+scar+border+zone+defined+by+substrate+mapping+for+ventricular+tachycardia+post-myocardial+infarction.">Relationship between successful ablation sites and the scar border zone defined by substrate mapping for ventricular tachycardia post-myocardial infarction.</a> J Cardiovasc Electrophysiol. 16, 465-471 (2005).</li><li>Wu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+risk+for+ventricular+tachyarrhythmias+and+sudden+cardiac+death:+is+there+a+role+for+cardiac+MRI.">Assessing risk for ventricular tachyarrhythmias and sudden cardiac death: is there a role for cardiac MRI.</a> Circ Cardiovasc Imaging. 5, 2-5 (2012).</li><li>Tettelin, H., 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=Complete+genome+sequence+of+a+virulent+isolate+of+Streptococcus+pneumoniae.">Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.</a> Science. 293, 498-506 (2001).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> CSH Protoc. 2008, (2008).</li></ol>

The authors declare no conflict of interest in this study.

In this report, a highly reproducible method to induce S. pneumoniae-mediated cardiac microlesions in mice during IPD and the techniques for their visualization is demonstrated. Mice are infected with a dose of bacteria that surpasses the immune system&#8217;s threshold for clearance, leading to high-grade bacteremia, similar to that which occurs during human sepsis, and leads to eventual bacterial translocation into the myocardium. For yet unknown reasons, S. pneumoniae in the heart is able to replicat...

Hospitalization of adults for community-acquired pneumonia (CAP) carries a documented risk for adverse cardiac events that contributes to mortality1-4. In a recent study by Corrales-Medina et al. cardiac complications were found to be associated with and/or responsible for 27% of pneumonia-associated deaths3. Streptococcus pneumoniae (the pneumococcus), the most common cause of CAP and sepsis5, has been directly associated with adverse cardiac events in as many as 19% of admitted adult patients6. Adverse cardiac events associated with pneumonia include new or worsened congestive heart failure, arrhythmias, and myocardial infarction in order of decreasing frequency6.
In a recent PLoS Pathogens article by Brown et al., S. pneumoniae was found to be capable of translocation through the cardiac vascular endothelium, entry into the myocardium, and formation of discrete non-purulent microscopic lesions (microlesions) filled with bacteria in the ventricles during invasive pneumococcal disease (IPD)7. Evidence of cardiac microlesion formation was observed in cardiac samples from non-human primates and individuals who had succumbed to pneumococcal infection. Likewise, experimentally infected mice reproducibly developed cardiac microlesions. In mice, microlesion size and number was positively correlated with the duration and severity of bacteremia, levels of cardiac troponin in sera, and aberrant cardiac electrophysiology. Bacterial translocation into the heart was found to occur through the same mechanisms responsible for translocation of the pneumococcus across the blood-brain barrier and development of pneumococcal meningitis, i.e., Choline-binding protein A mediated invasion of vascular endothelial cells in a Laminin receptor and Platelet-activating factor receptor dependent manner8. Microlesion formation also required the pneumococcal pore-forming toxin pneumolysin that was found to kill cardiomyocytes7.
Pneumococcal cardiac microlesions are distinct from the purulent soft-tissue and cardiac abscesses that are caused by other Gram-positive bacteria including Staphylococcus aureus. These are characterized by a single foci of bacteria surrounded by neutrophils&#160;and fibrin deposition9,10. Microlesions formed by S. pneumoniae are smaller in size, distributed throughout an affected heart, and lack immune cell infiltrates. During the early stages of infection, microlesions caused by S. pneumoniae appear as areas of damaged or inflamed tissue reminiscent of the pathological signs of cardiomyopathy. Some monocytes&#160;may be observed during this time, however their presence is short lived and lesions rapidly become necrotic in appearance and filled with bacteria while simultaneously continuing to grow in size until the death of the animal or antimicrobial intervention. Importantly, within 3 days of antibiotic intervention, profuse immune cell infiltration is observed at the former lesion site and this is accompanied by robust collagen deposition. Similar cardiac remodeling has been reported to occur following infarction along with lasting consequences on cardiac function11-15. Thus, microlesions are a potential explanation for the adverse cardiac events that occur during IPD and possibly the increased incidence of cardiac-related mortality in convalescent individuals who have survived the disease episode.
Herein, instruction is provided on the experimental mouse model of IPD and cardiac lesion formation and visualization of cardiac microlesions at early and late stages of infection. The protocol for detection of collagen deposition in animals that have been saved by antimicrobial intervention is demonstrated. The goal of this article is to facilitate the research of other investigators on this important and novel pneumococcal pathology.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Todd-Hewitt broth&#160;</td>
			<td style="width:175px;">Neogen</td>
			<td>7161A</td>
			<td></td>
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			<td height="42" style="height:42px;width:308px;">O.C.T Compound&#160;</td>
			<td style="width:175px;">Tissue-Tek (Sakura Finetek)&#160;</td>
			<td style="width:119px;">4583</td>
			<td></td>
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			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:175px;">Fisher Scientific (Acros)</td>
			<td>9002-93-1</td>
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			<td style="width:175px;">Abcam</td>
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			<td height="21" style="height:21px;">anti-serotype 4 pneumococcus antiserum&#160;</td>
			<td>Statens Serum Institut</td>
			<td style="width:119px;">16747</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit FITC conjugated antibody&#160;</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-096-144</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">DAPI</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
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			<td height="21" style="height:21px;width:308px;">Fluorsave</td>
			<td style="width:175px;">Millipore</td>
			<td>345789</td>
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			<td height="21" style="height:21px;width:308px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>S70104</td>
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			<td height="21" style="height:21px;width:308px;">Ampicillin</td>
			<td style="width:175px;">Sigma</td>
			<td>A9393</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">phosphomolybdic acid 0.2%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">0.1% Sirius Red in picric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-02</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-03</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">Tissue Tack Microscope Slides</td>
			<td style="width:175px;">Polysciences, Inc</td>
			<td style="width:119px;">24216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Paralube Vet Ointment</td>
			<td style="width:175px;">Dechra</td>
			<td>12920060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Hematoxylin and Eosin Staining Kit</td>
			<td style="width:175px;">unspeciified&#160;</td>
			<td style="width:119px;"></td>
			<td>Standard</td>
		</tr>
	</tbody>
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visualization of streptococcus pneumoniae within cardiac microlesions and subsequent cardiac remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Microlesions by H&#38;E
Cardiac microlesions were observed in the ventricles of H&#38;E stained cardiac sections from mice with IPD following their i.p. challenge with TIGR4. Figure 1 demonstrates the increase in size of these lesions between 24 and 30 hr post-infection (hpi), with greater number of hematoxylin-stained (i.e., purple-blue) pneumococci visible within the lesion. Microlesions were characterized by a vacuole-like morphology, dens...

Watch this Scientific Journal Video about Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling at JoVE.com

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

visualization-streptococcus-pneumoniae-within-cardiac-microlesions

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8.9K Views.  The University of Texas Health Science Center at San Antonio. Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals. 

Video: Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Robotized Testing of Camera Positions to Determine Ideal Configuration for Stereo 3D Visualization of Open-Heart Surgery

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room and the surgical field is restricted. It is a sterile environment, and the physical space is crowded with surgical staff and technical equipment. In this setting, unobscured capture and realistic reproduction of the surgical procedures are difficult. This paper presents a method for rapid and reliable data collection of stereoscopic 3D videos at different camera baseline distances and distances of convergence. To collect test data with minimum interference during surgery, with high precision and repeatability, the cameras were attached to each hand of a dual-arm robot. The robot was ceiling-mounted in the operating room. It was programmed to perform a timed sequence of synchronized camera movements stepping through a range of test positions with baseline distance between 50-240 mm at incremental steps of 10 mm, and at two convergence distances of 1100 mm and 1400 mm. Surgery was paused to allow 40 consecutive 5-s video samples. A total of 10 surgical scenarios were recorded.

The human depth perception of 3D stereo videos depends on the camera separation, point of convergence, distance to, and familiarity of the object. This paper presents a robotized method for rapid and reliable test data collection during live open-heart surgery to determine the ideal camera configuration.

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...