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 replicate undeterred by the host immune response. This is the subject of ongoing investigation and this publication may facilitate other investigators in their efforts to participate.
H&#38;E staining allows for microlesion visualization using standard light microscopy since cardiomyocytes stain pink to red, whereas cardiac microlesions and the bacteria within stain blue to deep purple. This sharp contrast enables easy detection of the microlesions at later stages of infection when the lesions are larger. In order to verify the presence of S. pneumoniae within the cardiac microlesions, immunofluorescent microscopy using antiserum against serotype 4 capsular polysaccharide that is carried by TIGR4 was performed. Importantly, the specific anti-capsular antibody that should be used depends on the serotype of the pneumococcal strain that is used for experimental challenge. Alternatively, investigators can also use commercially available monoclonal antibodies against pneumolysin, the pore-forming toxin of S. pneumoniae, or that detect components of the pneumococcal cell wall (i.e., TEPC-15). While these antibodies detect pneumococci within the heart, past experience suggests that antibodies against capsular polysaccharide provide the most compelling images7.
S. pneumoniae mediated cardiac microlesion formation requires the interaction of the pneumococcal adhesin Choline binding protein A with the host protein Laminin receptor for bacterial adhesion to the vascular endothelial cells and interaction of bacteria cell wall phosphorylcholine with host Platelet-activating factor receptor for bacterial transmigration through the cell7,8. It is therefore important to note that microlesion formation is tightly linked to infectious burden and time, which increases the frequency and duration of these interactions and allows time for the bacteria to replicate and visible lesions to develop. We have previously determined that an i.p. infectious dose greater than 1.0 x 103 CFU of S. pneumoniae strain TIGR4 results in too rapid death of the mouse and as a result poor lesion formation. Below this infectious dose spontaneous clearance occurs in some mice and in others delayed onset of severe disease is observed. Yet for mice challenged with 1.0 x 103 CFU of TIGR4 between 24 and 36 hr, lesions rapidly accumulate in number and grow in size continuously until the mouse becomes moribund. Thus, the infectious dose of the infecting strain needs to be titrated to replicate a similar state of disease with a moribund state developing in no less than 30 hpi. Importantly, we have repeatedly observed cardiac lesion formation in mice that were infected intranasal with 1.0 x 107 CFU and intratracheally with 1.0 x 105 CFU of TIGR4. Following these challenge routes development of severe disease occurred at later time points. We also observed microlesion formation with other virulent strains of S. pneumoniae, such as serotype 2 isolate D39, albeit at different frequencies. Thus, lesions are not a result of the i.p. infection route or specific to TIGR4, but instead tied to duration and severity of the disease episode. Herein we advise to infect mice i.p. since this challenge route offers the highest degree of reproducibility. If a different strain of S. pneumoniae is used or a different infectious route is desired, investigators should aim for bacteremia at levels &#62;105 CFU ml of blood for at least 24 hr.
Antimicrobial intervention allows the study of events that occur during convalescence, modeling a human in the Intensive Care Unit setting. It is worth noting that not all mice survive despite antimicrobial treatment and confirmed bacterial clearance. Investigators should expect survival rates between 50-60% and as such should adjust the cohort size accordingly to deal with the loss of animals. Regular monitoring of animals after 24 hr is highly recommended to ensure compliance with Institutional approved animal protocols. Picro Sirius Red staining is a simple and standard technique used to visualize collagen deposition. Herein, it is demonstrated how this stain can be used to examine cardiac remodeling in the ventricles of the heart following a traumatic infectious episode. Picro Sirius Red was chosen over other commonly used techniques to visualize collagen deposition, such as &#8220;Masson&#8217;s Trichrome Stain&#8221;, because of the sharp contrast it offered between the deposited collagen and surrounding tissue. This has proven to be tremendously useful when assessing the extent of myocardial collagen deposition following antibiotic in a quantitative fashion using software such as ImageJ.

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><tbody><tr><td>Todd-Hewitt broth&amp;nbsp;</td><td>Neogen</td><td>7161A</td><td></td></tr><tr><td>O.C.T Compound&amp;nbsp;</td><td>Tissue-Tek (Sakura Finetek)&amp;nbsp;</td><td>4583</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific (Acros)</td><td>9002-93-1</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Abcam</td><td>ab7481</td><td></td></tr><tr><td>anti-serotype 4 pneumococcus antiserum&amp;nbsp;</td><td>Statens Serum Institut</td><td>16747</td><td></td></tr><tr><td>goat anti-rabbit FITC conjugated antibody&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>111-096-144</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Fluorsave</td><td>Millipore</td><td>345789</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>S70104</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9393</td><td></td></tr><tr><td>phosphomolybdic acid 0.2%&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>26357-01</td><td></td></tr><tr><td>0.1% Sirius Red in picric acid&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>26357-02</td><td></td></tr><tr><td>0.01 N hydrochloric acid&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>26357-03</td><td></td></tr><tr><td>Tissue Tack Microscope Slides</td><td>Polysciences, Inc</td><td>24216</td><td></td></tr><tr><td>Paralube Vet Ointment</td><td>Dechra</td><td>12920060</td><td></td></tr><tr><td>Hematoxylin and Eosin Staining Kit</td><td>unspeciified&amp;nbsp;</td><td></td><td>Standard</td></tr></tbody></table>

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, denser eosin (red) staining of cardiomyocytes in the cells immediately adjacent to the lesion site, diplococci within the lesions, and a general absence of immune cells. Approximately half the lesions were adjacent to visible blood vessels. Between 24 and 30 hpi microlesions became more numerous and substantially larger. At 30 hpi extracellular bacteria could often be seen streaming from a lesion between cells.
Visualization of S. pneumoniae Using Immunofluorescent Microscopy
To visualize and confirm that S. pneumoniae were indeed within lesions, immunofluorescent staining for serotype 4 capsular polysaccharide that is produced by TIGR4 (Figure 2) was used. Images captured showed the presence of tightly packed and dense aggregates of S. pneumoniae within microlesion sites.
Evidence of Cardiac Scarring
Picro Sirius Red staining was performed on cardiac sections from mice that had been rescued with ampicillin and uninfected controls. In antibiotic rescued convalescent mice, there was a dramatic increase in the presence and intensity of the Picro Sirius Red stain with bands streaming from what appear to be former lesion sites in the ventricles (Figure 3). In contrast, the ventricles in control sections were unstained indicating that collagen deposition was absent (not shown).
Together these results demonstrate the ability to visualize cardiac microlesions in mice with severe IPD and to access resultant scar formation in the ventricles using standard histological procedures. Importantly, cardiac microlesions are invariably linked to high titers of S. pneumoniae and time needed to mediate their formation.
<img alt="Figure 1" src="/files/ftp_upload/52590/52590fig1.jpg" /> 
Figure 1: Detection of microlesions in H&#38;E stained cardiac sections. Representative images (1,000X) of cardiac microlesions observed at 24 hor and 30 hr post infection. Black scale bars correspond to 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52590/52590fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52590/52590fig2.jpg" /> 
Figure 2: Immunofluorescent detection of S. pneumoniae within cardiac microlesions. Immunofluorescent microscopy using antibody against serotype 4 capsular polysaccharide (green) confirmed the presence of S. pneumoniae within cardiac microlesions. The nuclei were stained blue with DAPI. White scale bar correspond to 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52590/52590fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52590/52590fig3.jpg" /> 
Figure 3: Collagen deposition at a former cardiac microlesion site in a convalescent mouse. Representative image (200X) showing a Picro Sirius Red stained cardiac microlesion in the ventricle of a mouse 5 days after antibiotic intervention for invasive pneumococcal disease. Red indicates collagen deposition. Black scale bar correspond to 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52590/52590fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

9.0K 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

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Biology

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Revealing Murine Pulmonary Veins Myocardial Architecture

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

Author Spotlight: Developing a Translational Model for Atrial Fibrillation Research Across Species 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Samenvatting

Meer video's verkennen