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.

NOTA: Tutti gli esperimenti del mouse sono stati esaminati e approvati dalla cura degli animali e Istituzionale Usa Comitati presso l&#39;Università del Texas Health Science Center a San Antonio (protocollo # 13032-34-01C). La cura degli animali e protocolli sperimentali aderito Diritto Pubblico 89-544 (Animal Welfare Act) e successivi emendamenti, Servizi pubblici Salute linee guida, e la Guida per la cura e l&#39;uso di animali da laboratorio (US Department of Health &amp; Human Services). 1. Infezione <ol><li> Ottenere topi BALB / c di entrambi i sessi di età compresa tra 10-12 settimane di età tra. GLI APPUNTI. pneumoniae sierotipo 4 ceppo TIGR4 è un isolato clinico virulenta che è stato sequenziato 16. </li><li> Grow TIGR4 in Todd-Hewitt brodo a 37 ° C in 5% CO 2 fino a raggiungere una densità ottica (OD) 620 0,5 (corrispondente a 1,0 x 10 8 unità formanti colonie [CFU] / ml). Pellet cultura pneumococcica mediante centrifugazione per 10 min a 3, 500 xg e rimuovere il supernatante utilizzando una linea del vuoto. Sospendere e diluire batteri con PBS sterile (PBS) ad una concentrazione finale di 1,0 x 10 4 CFU / ml. </li><li> Usando una camera di induzione, anestetizzare topi con 2,5% isoflurano vaporizzato in ossigeno. Confermare stato anestetizzato dal pizzico punta dolce con le pinzette smussate. <ol><li> Tenendo il mouse anestetizzato dal suo nuca e verticale con una mano, iniettare ciascun topo per via intraperitoneale (ip) con 100 microlitri della S. sospensione pneumoniae usando una siringa con un ago calibro 27-30 (corrispondente ad una dose di 1,0 x 10 3 CFU). Posizionare la parte posteriore del mouse nella sua gabbia. I topi in genere risvegliano 30-40 secondi dopo l&#39;iniezione. </li></ol></li><li> Lasciare che l&#39;infezione di procedere per almeno 24 (presto) o 30 (avanzato) h. <ol><li> Euthanize i topi da CO 2 asfissia. Eseguire dislocazione cervicale per garantire il mouse è deceduto. L&#39;eutanasia è ulteriormente confermata dalla rimozionedel cuore nel passo 1.6. </li></ol></li><li> Disinfettare la coda con un batuffolo imbevuto di alcool e tagliare una sezione di 2-3 mm. Raccogliere 2 ml di sangue e serialmente diluire il sangue 10 volte in PBS contenente eparina sodica 1 U / ml cinque volte. Diluizioni seriali piastra su un piatto trittico di agar sangue soia e incubare una notte a 37 ° C in 5% CO 2. Il giorno successivo estrapolare dalla colonia conta il livello di batteriemia. </li><li> Immobilizzare il mouse in posizione supina su una piattaforma chirurgica. Spruzzare il petto con il 70% di etanolo e asciugare. Utilizzando forbici chirurgiche e pinze aprire la cavità toracica, rimuovere la gabbia toracica, e transetto il diaframma per esporre il cuore ei polmoni. Utilizzando forbici tagliare i vasi sanguigni collegati al cuore e alle accise delicatamente cuore con cura per evitare lividi con una pinza. </li><li> Sciacquare il cuore con PBS e poi mettere in campioni di tessuto cassette di raccolta di tali sezioni coronali che sarebbero stati ottenuti durante il sezionamento dei tessuti. Per embedding di paraffina, plaCe le cassette in soluzione al 10% di formalina tamponata-e il giorno successivo invio per paraffina. <ol><li> In alternativa freeze flash utilizzando ottobre Compound entro un cryomold che è stato immesso sul ghiaccio secco. </li></ol></li></ol> 2. Visualizzazione cardiache lesioni e pneumococchi all&#39;interno lesioni <ol><li> Ridurre le sezioni cardiache in paraffina tale che i 4 camere del cuore sono visibili in ogni sezione di tessuto. Tagliare vetrini a spessore di 5 micron. </li><li> Deparaffinare e macchia diapositive con ematossilina e eosina (H &amp; E) usando metodi standard 17. </li><li> Vista H &amp; E macchiato sezioni cardiache utilizzando un microscopio ottico a bassa (100X, 200X) e alta (immersione in olio: 400X, 1,000X) ingrandimenti. Caratterizzare cuori per la presenza di microlesioni nel miocardio. Qui, l&#39;acquisizione di H &amp; E le immagini utilizzando un microscopio Zeiss Axioskop 2 dotato di 100X 1.3 numerica obiettivo dell&#39;apertura Plan-Neofluar. NOTA: nelle fasi iniziali l cardiacoesions sono discriminati per il loro aspetto colorato differenziale e in alcuni casi la presenza di cellule immunitarie (monociti e granulociti) che sembrano essere monociti. In lesioni avanzate, in particolare quelli visti a 30 ore, le cellule immunitarie sono lesioni vacuolo come assenti e grandi sono stati osservati nel loro posto. È importante notare che, mentre afflusso cellule immunitarie durante le prime fasi di sviluppo lesione cardiaca può verificarsi non risulta essere un requisito. </li></ol> 3. immunofluorescente Microscopia per pneumococchi all&#39;interno microlesioni <ol><li> Cuore Sezione in cryomolds con microtomo ad uno spessore di 5 sezioni micron e posto su vetrino carica positiva. Consenti sezioni cardiache decongelamento e aria secca. Fissare sezioni per 10 min a 10% formalina tamponata neutra (pH 6.8-7.2 a 25 ° C). </li><li> Lavare slitte (3x) in PBS per 5 min, permeabilize per 15 min in 0,2% Triton X-100 in PBS, poi lavare di nuovo in PBS. </li><li> Secti tessuto Block<html> su su vetrini con siero di capra 10% in PBS per 1 ora. </li><li> Sciacquare i vetrini con PBS, coperchio e incubare sezioni cardiache con coniglio anti-pneumococco sierotipo 4 antisiero a 1: 1000 in PBS con siero di capra 10% per 2 ore a 37 ° C. NOTA: Per i controlli negativi investigatori dovrebbero utilizzare antisiero di coniglio ingenuo </li><li> Dopo lavaggio con PBS, coperchio e incubare sezioni con 10% di siero di capra-PBS con capra anti-coniglio FITC anticorpo coniugato a 1: 2.000 per 30 min a 37 ° C. </li><li> Seguendo le istruzioni del produttore, utilizzare DAPI (4 &#39;, 6 Diamidino-2-fenilindolo) a 5 mg / ml per la visualizzazione dei nuclei eucariotiche. Lavare e montare sezioni di tessuto con FluorSave. </li><li> Acquisire le immagini fluorescenti utilizzando un sistema di microscopio confocale sia a bassa che ad alta ingrandimento. Regolare l&#39;apertura numerica di conseguenza per ottenere risultati ottimali. Qui, acquisire immagini fluorescenti utilizzando un sistema confocale con un obiettivo 60X apertura numerica 1.42. </li></ol>&quot;&gt; 4. Antibiotica Salvataggio di Mice Septic <ol><li> Inizia con i topi infettati con ip 1.0 x 10 3 CFU di TIGR4 come precedentemente indicato al punto 1. </li><li> Con inizio alle 30 ore dopo l&#39;infezione, amministrare grado farmaceutico ampicillina ip (80 mg / kg di peso corporeo) in 100 ml di soluzione salina ogni 12 ore per 36 ore. Raccogliere i cuori al punto desiderato e di processo come descritto al punto 1.6 per paraffina e il tessuto, il sezionamento. </li></ol> 5. deposizione di collagene Detection <ol><li> Utilizzando xilene e lavaggi alcol classificati con acqua, Deparaffinare e idratare le sezioni di tessuto deionizzata utilizzando metodi standard 17. Trattare sezioni con acquosa 0,2% di acido fosfomolibdico per 5 minuti e risciacquare con acqua deionizzata per 5 min. </li><li> Sezioni Macchia con 0,1% di Sirius Red in acido picrico per 2 ore a temperatura ambiente. </li><li> Lavare le sezioni in 0,01 N acido cloridrico per 3 minuti e risciacquare con etanolo al 70% per 1 min. </li><li> DisidratazioneTE sezioni con l&#39;identico ma l&#39;ordine di lavaggi a quello delineato in 5.1 invertire, disidratare le sezioni cardiache in aumento e graduale delle concentrazioni di etanolo poi xilene. </li><li> Sezioni di Monte di tessuto con una soluzione di montaggio per la valutazione microscopica. </li><li> Per identificare le regioni di deposizione di collagene, esaminare sezioni di tessuto colorate per le zone di profonda colorazione lettura in condizioni di scarsa e alto ingrandimento con un microscopio ottico. Ogni area all&#39;interno dei ventricoli del cuore che è il rosso è un settore in cui il collagene è stato depositato. Aree senza questa macchia rosso sono siti di tessuto sano privi deposizione di collagene. Qui, acquisire Picro Sirius Red immagini colorate utilizzando un microscopio equipaggiato con un obiettivo apertura numerica 20X 0.5. NOTA: Le macchie rosse Atrium in tessuti sani. </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 questo rapporto, un metodo altamente riproducibile per indurre S. pneumoniae mediata microlesioni cardiaci nei topi durante IPD e le tecniche per la loro visualizzazione è dimostrata. I topi sono infettati con una dose di batteri che supera la soglia del sistema immunitario di attestazione, portando a batteriemia alta qualità, simile a quella che si verifica durante la sepsi umana, e conduce ad eventuali traslocazione batterica nel miocardio. Per ragioni ancora sconosciute, S. pneumoniae nel cuore...

Ricovero di adulti per polmonite acquisita in comunità (CAP) comporta un rischio documentato per eventi avversi cardiaci che contribuisce alla mortalità 1-4. In un recente studio condotto da Corrales-Medina et al. Sono risultati associati con e / o responsabile del 27% dei decessi polmonite associata 3 complicazioni cardiache. Streptococcus pneumoniae (pneumococco), la causa più comune di PAC e sepsi 5, è stata direttamente associata ad eventi avversi cardiaci a ben il 19% dei pazienti adulti ricoverati 6. Eventi cardiaci avversi associati con polmonite includono insufficienza nuovo o peggiorata cardiaco congestizio, aritmie, infarto del miocardio e in ordine decrescente di frequenza 6. In un recente articolo PLoS Pathogens da Brown et al., S. pneumoniae è risultata capace di traslocazione attraverso l&#39;endotelio vascolare cardiaca, l&#39;ingresso nel myocardium, e la formazione di discrete lesioni microscopiche non purulente (microlesioni) pieni di batteri nei ventricoli durante la malattia pneumococcica invasiva (IPD) 7. Prove di formazione microlesion cardiaca è stata osservata in campioni cardiaci dai primati non umani e gli individui che avevano ceduto alle infezioni da pneumococco. Allo stesso modo, i topi infettati sperimentalmente riproducibile sviluppato microlesioni cardiaci. Nei topi, le dimensioni e il numero microlesion stata positivamente correlata con la durata e la gravità di batteriemia, livelli di troponina cardiaca nel siero, e aberrante elettrofisiologia cardiaca. Traslocazione batterica nel cuore è stato trovato per avvenire attraverso gli stessi meccanismi responsabili traslocazione del pneumococco attraverso la barriera emato-encefalica e lo sviluppo della meningite pneumococcica, vale a dire, la proteina-Colina legame Un&#39;invasione mediata delle cellule endoteliali vascolari in un recettore laminina e piastrine modo dipendente recettore del fattore di -activating 8. Microlesformazione di ioni richiesto anche il pneumococco formano pori tossina pneumolysin che è stato trovato per uccidere cardiomiociti 7. Microlesioni cardiaci pneumococco sono distinti dal purulenta dei tessuti molli e ascessi cardiaci che sono causate da altri batteri Gram-positivi, tra cui Staphylococcus aureus. Questi sono caratterizzati da un unico foci di batteri circondato da neutrofili e deposizione di fibrina 9,10. Microlesioni formata da S. pneumoniae sono di dimensioni più piccole, distribuiti su tutto il cuore colpito, e la mancanza di infiltrati di cellule immunitarie. Durante le prime fasi dell&#39;infezione, microlesioni causate da S. pneumoniae appaiono come aree di tessuto danneggiato o infiammato ricorda i segni patologici di cardiomiopatia. Alcuni monociti possono essere osservati durante questo tempo, ma la loro presenza è di breve durata e lesioni rapidamente diventa necrotico nell&#39;aspetto e riempito con batteri contemporaneamente continuando a grow dimensioni fino alla morte dell&#39;animale o interventi antimicrobica. È importante sottolineare che, entro 3 giorni di intervento antibiotico, profusa infiltrazione di cellule immunitarie si osserva presso l&#39;ex sede della lesione e questo è accompagnato da una robusta deposizione di collagene. Simile rimodellamento cardiaco è stato riscontrato dopo infarto insieme con conseguenze permanenti sulla funzione cardiaca 11-15. Così, microlesioni sono una potenziale spiegazione per gli eventi avversi cardiaci che si verificano durante IPD e forse la maggiore incidenza di mortalità cardiaca correlata in individui di convalescenza che sono sopravvissuti l&#39;episodio di malattia. Qui, l&#39;istruzione è disponibile sul modello sperimentale murino di IPD e formazione della lesione cardiaca e la visualizzazione delle microlesioni cardiaci a precoci e tardive fasi dell&#39;infezione. Il protocollo per la rilevazione di deposizione di collagene in animali che sono stati salvati da un intervento antimicrobico è dimostrata. L&#39;obiettivo di questo articolo è quello di facilitate ricerca di altri investigatori su questo importante e nuova patologia pneumococcica.

<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:175px;">Fisher Scientific (Acros)</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Normal Goat Serum</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:119px;">ab7481</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DAPI</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fluorsave</td>
			<td style="width:175px;">Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>S70104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ampicillin</td>
			<td style="width:175px;">Sigma</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<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>
		<tr height="21">
			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
</table>

visualization of streptococcus pneumoniae within cardiac microlesions and subsequent cardiac remodeling

Durante batteriemia Streptococcus pneumoniae può traslocare attraverso l&#39;endotelio vascolare nel miocardio e formare batteri-riempite discrete lesioni microscopiche (microlesioni) che sono notevoli per l&#39;assenza di infiltrarsi cellule immunitarie. Grazie alla loro rilascio di prodotti cardiotossici, S. pneumoniae entro microlesioni si pensa di contribuire allo scompenso cardiaco che è spesso osservato durante fulminato malattia pneumococcica invasiva negli adulti. Qui è dimostrato un protocollo per infezione sperimentale del mouse che porta alla formazione di riproducibili microlesion cardiaco entro 30 ore. L&#39;istruzione è fornita sull&#39;identificazione microlesion in ematossilina e eosina sezioni cuore colorato e le distinzioni morfologiche tra inizio e fine microlesioni sono evidenziati. L&#39;istruzione è fornita su un protocollo per la verifica di S. pneumoniae entro microlesioni utilizzando anticorpi contro pneumococco polisaccaride della capsula emicroscopia a immunofluorescenza. Infine, è previsto un protocollo di intervento antibiotico che salva i topi infetti e per l&#39;individuazione e la valutazione di formazione della cicatrice nel cuore dei topi convalescente. Insieme, questi protocolli faciliteranno lo studio dei meccanismi molecolari alla base dell&#39;invasione cardiaco pneumococco, la morte dei cardiomiociti, rimodellamento cardiaco a seguito di esposizione a S. pneumoniae, e la risposta immunitaria al pneumococchi nel cuore.

Visualizzazione di microlesioni da H &amp; E Microlesioni cardiaci sono stati osservati nei ventricoli di H &amp; E macchiato sezioni cardiache di topo con IPD seguenti la loro sfida ip con TIGR4. Figura 1 dimostra l&#39;aumento delle dimensioni di queste lesioni tra 24 e 30 ore dopo l&#39;infezione (HPI), con maggior numero di ematossilina -stained (cioè, viola-blu) pneumococchi visibile all&#39;interno della lesione. Microlesioni sono stati caratterizzat...

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.

Visualizzazione<em&gt; Streptococcus pneumoniae</em&gt; Entro cardiache microlesioni e successive rimodellamento cardiaco

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

visualization-streptococcus-pneumoniae-within-cardiac-microlesions

Research

JoVE Journal

Medicine

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

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.

Stress Test cardiaca indotta da dobutamina e monitorato da cateterizzazione cardiaca nei topi

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

Ricerca

Medicina

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.

Pressure Monitoring diretto predice accuratamente occlusione della vena polmonare durante l&#39;ablazione Cryoballoon

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.

Un modello di rimodellamento cardiaco Attraverso Costrizione del Aorta addominale 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 ...

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.

preparazione e<em&gt; In Vitro</em&gt; Caratterizzazione di magnetizzato miR-modificato cellule endoteliali

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.

Risonanza magnetica cardiaca per la valutazione di un trombo cardiaco sospetto: tecniche convenzionali ed emergenti

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.

Isolamento e purificazione dei Periciti Cardiaci Murine

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

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.

Un modello di roditore ischemico del pericardio intatto

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.

Test robotizzati delle posizioni della telecamera per determinare la configurazione ideale per la visualizzazione 3D stereo della chirurgia a cuore aperto

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

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

Modello di donazione su animali di grandi dimensioni dopo morte circolatoria e perfusione regionale normothermica per la valutazione cardiaca

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Analisi della disfunzione contrattile cardiaca e dei transitori Ca2+ nei miociti dei roditori

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...