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 bot...

<ol>
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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.

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			<td>A9393</td>
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			<td height="21" style="height:21px;">phosphomolybdic acid 0.2%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-01</td>
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			<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>
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			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
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			<td height="21" style="height:21px;width:308px;">Paralube Vet Ointment</td>
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			<td height="21" style="height:21px;width:308px;">Hematoxylin and Eosin Staining Kit</td>
			<td style="width:175px;">unspeciified&#160;</td>
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			<td>Standard</td>
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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 vistas.  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. 