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.

NOT: Tüm fare deneyleri gözden ve San Antonio Texas Sağlık Bilimleri Merkezi Üniversitesi&#39;nde (protokol # 13032-34-01C) Kurumsal Hayvan Bakım ve Kullanım Komiteleri tarafından kabul edildi. Hayvan bakımı ve Kamu Hukuku 89-544 (Hayvan Refahı Yasası) ve değişiklikleri, Halk Sağlığı Hizmetleri kurallar ve Laboratuvar Hayvanları Bakımı ve Kullanımı Kılavuzu (Sağlık ve İnsan Hizmetleri Bakanlığı&#39;nın) yapıştırılır deney protokolleri. 1. Enfeksiyon <ol><li> Yaş 10-12 haftalık yaş arası her iki cinsiyetten, BALB / c fareleri elde edilir. NOT: S. pneumoniae serotip 4 suşu TIGR4 16 sıralandı edilmiş bir şiddetli klinik izolat olduğunu. </li><li> Optik yoğunluk (OD) 0.5 620 ulaşana kadar% 5 CO2 içinde 37 ° C &#39;de, Todd-Hewitt lapası içindeki TIGR4 büyütün (1.0 x 10 8 koloni oluşturan birim [CFU] / ml&#39;ye karşılık gelir). 3 ile 10 dakika boyunca santrifüj ile pnömokokal kültür Pelet500 xg ve bir vakum hattı kullanarak süpernatant kaldırmak. Askıda ve 1.0 x 10 4 CFU / ml&#39;lik bir son konsantrasyona değin steril fosfat-tamponlu tuzlu su (PBS) ile bakteri seyreltin. </li><li> Bir indüksiyon odasını kullanarak, oksijen% 2.5 buharlaşmış izofluran ile fareler uyutmak. Künt cımbız ile nazik ayak tutam tarafından anestezi durumunu onaylayın. <ol><li> Bir yandan ile ense anestezi fare tutarak, dik, S. 100 ul her bir fare karın içinden (ip) enjekte (1.0 x 10 3 CFU yükleme dozuna tekabül eden), 27-30 ölçü iğne ile bir şırınga kullanılarak pneumoniae süspansiyon. Kendi kafeste fare geri yerleştirin. Fareler genellikle enjeksiyondan sonra 30-40 sn uyandırmak. </li></ol></li><li> Enfeksiyon en az (erken) 24 veya 30 (gelişmiş) saat devam izin verin. <ol><li> CO 2 boğularak fareler Euthanize. Fareyi sağlamak için servikal dislokasyon yapın vefat etti. Ötenazi ayrıca uzaklaştırılarak onaylanırAdım 1.6 kalbin. </li></ol></li><li> Alkollü bir bezle kuyruk dezenfekte ve 2-3 mm&#39;lik bölümü makasla kesme. Kan, 2 ul toplayın ve seri olarak, sodyum heparin, 1 U / mL, beş kez içeren PBS içinde kan 10 kat seyreltilir. Plaka seri bir triptik soya kan agar plakası üzerinde seyreltme ve koloni ertesi gün tahmin etmek bakteremi seviyesini önemli olan CO 2.% 5, 37 ° C&#39;de gece boyunca inkübe edilir. </li><li> Bir cerrahi platformda bir yatar pozisyonda fare hareketsiz. % 70 etanol ve pat kuru ile göğüs püskürtün. Cerrahi makas ve forseps göğüs boşluğu açmak kullanarak, göğüs kafesini çıkarmak ve kalp ve akciğerler maruz diyaframı transect. Kullanımı makas kalbe bağlı kan damarlarını kesmek ve yavaşça forseps ile morarma önlemek için dikkatle kalp tüketim. </li><li> PBS ile kalp durulayın ve ardından koronal kesitler doku kesit sırasında elde olacağı gibi doku örnek toplama kasetleri içine yerleştirin. Parafin gömme, pla içinparafin gömme için göndermek% 10 tamponlu formalin çözeltisi ve ertesi gün kasetler ce. <ol><li> Alternatif kuru buz konulmuş bir cryomold içinde Ekim Bileşik kullanarak flaş dondurma. </li></ol></li></ol> Kardiyak Lezyonların ve Lezyonlarının içinde pnömokok 2. Görselleştirme <ol><li> Kalbin 4 odaları her doku bölümünde görünür şekilde parafin kalp bölümleri azaltın. 5 mikron kalınlığında slaytlar kesin. </li><li> Standart yöntemler kullanılarak 17 hematoksilen eosin ve (H &amp; E) ile Deparaffinize ve leke slaytlar. </li><li> Büyütme: Görünüm H &amp; E düşük (100X, 200X) ve yüksek (400X, 1,000X yağ daldırma) bir ışık mikroskobu kullanılarak kalp bölümleri boyandı. Miyokardiyumda microlesions varlığı için kalpleri karakterize etmektedir. Burada, bir 100X 1.3 Sayısal açıklık Planı-Neofluar amacı ile donatılmış bir Zeiss Axioskop 2 mikroskop kullanılarak, H &amp; E görüntüleri kazanır. NOT: En erken kardiyak l aşamalarıesions bağışıklık hücrelerine monositler gibi görünen (monositler ve granülositler) varlığında ayırıcı renkli görünüm ve bazı durumlarda ayrımcılık yapılmaktadır. Özellikle 30 saatte görülen ilerlemiş lezyonlar, ise, bağışıklık hücreleri yok ve büyük vakuol benzeri lezyonlar onların yerine gözlenmektedir vardır. Kalp lezyon gelişiminin erken evrelerinde bağışıklık hücre akını oluşabilir ederken bir gereklilik olarak görünmüyor unutmayın önemlidir. </li></ol> Microlesions içinde pnömokokki, 3. immünoflüoresan mikroskopi <ol><li> Pozitif yüklü cam slaytlar üzerine 5 um ve yer bölümleri bir kalınlığa mikrotom ile cryomolds Bölüm kalp. Dondurulmuş kalp bölümler çözülme ve hava kuru izin ver. (25 ° C&#39;de, pH 6.8-7.2) 10 dakika,% 10 nötr tamponlu formalin için bölümler düzeltildi. </li><li> Daha sonra PBS içinde tekrar yıkama, PBS içinde% 0.2 Triton X-100 içinde, 15 dakika boyunca geçirgenliği, 5 dakika boyunca PBS içinde slaytlar (3x) yıkanır. </li><li> Blok doku secti<html> 1 saat boyunca PBS içinde% 10 keçi serumu ile slaytlar üzerine. </li><li> PBS, kapaklı slaytlar durulayın ve 1 tavşan anti-serotip 4 pnömokok antiserumu ile kardiyak bölümleri inkübe: 1000 PBS içerisinde% 10 keçi serumu ile 2 st için 37 ° C &#39;de. NOT: Negatif kontroller için araştırmacılar naif tavşan antiserumunu kullanmanız gerekir </li><li> 37 ° C&#39;de 30 dakika süre ile 2000: 1 bir keçi anti-tavşan FITC ile konjüge edilmiş antikor ile% 10 keçi serumu-PBS ile bölümlere PBS, kapak ile yıkanıp inkübe sonra. </li><li> Üreticinin talimatlarına sonra ökaryotik çekirdeklerin görünüm için, 5 mg / ml DAPI (4 &#39;, 6-diamidino-2-fenilindol) kullanın. Yıkayın ve FluorSave ile doku bölümleri monte edin. </li><li> Düşük ve yüksek hem büyütmede bir konfokal mikroskop sistemi kullanılarak floresan görüntü elde. En iyi sonuçları elde etmek için buna göre sayısal diyafram ayarlayın. Burada, bir 60X 1.42 sayısal diyafram amacı ile bir konfokal sistemi kullanılarak floresan görüntüler elde. </li></ol>Septik Fareler &quot;&gt; 4. Antibiyotik Kurtarma <ol><li> Daha önce 1. adımda belirtildiği gibi TIGR4 1.0 x 10 3 CFU ile fareler enfekte ip ile başlayın. </li><li> 30 saat sonra enfeksiyondan itibaren, 36 saat süre ile 100 ul tuzlu su içinde her 12 saat farmasötik dereceli ampisilin ip (80 mg / kg vücut ağırlığı) uygulamaktadır. Parafine ve doku, kesit olarak, adım 1.6 de tarif edildiği gibi arzu edilen bir zaman noktasında ve süreç kalpleri toplayın. </li></ol> 5. Kollajen Biriktirme Algılama <ol><li> Standart yöntemler kullanılarak 17 deiyonize su, deparaffinize ve hidrat doku bölümleri ile ksilen ve kademeli alkol yıkar kullanma. 5 dakika boyunca, sulu% 0.2 fosfomolibdik asit ile bölümleri tedavi ve 5 dakika boyunca iyonu giderilmiş su ile durulama. </li><li> Oda sıcaklığında 2 saat süre ile pikrik asit içinde% 0.1 Sirius Kırmızı leke bölümleri. </li><li> 3 dakika boyunca 0.01 N hidroklorik asit bölümleri yıkayın ve 1 dakika boyunca% 70 etanol ile yıkayın. </li><li> Dehydraaynı kullanarak bölümleri te ancak 5.1 de tarif edilene yıkama sırasını tersine çevirmek, ksilen artan kardiyak bölümleri kurutmak ve daha sonra etanol konsantrasyonlarını kademeli. </li><li> Mikroskopik değerlendirme için bir montaj çözümü Mount doku bölümleri. </li><li> Kollajen birikimi bölgeleri belirlemek için, bir ışık mikroskobu kullanılarak düşük ve yüksek büyütme altında derin okuma boyama alanlarında lekeli doku bölümleri inceleyin. Kırmızı kalp ventriküller içinde herhangi bir alan kollajen tevdi edilmiş bir alandır. Bu kırmızı leke olmadan Alanları kollajen birikimi eksik sağlıklı doku sitelerdir. Burada, bir 20X 0.5 sayısal diyafram objektif ile donatılmış bir mikroskop kullanılarak Picro Sirius Kırmızı lekeli görüntüler elde. NOT: Sağlıklı dokularda kırmızı atrium lekeleri. </li></ol>

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The authors declare no conflict of interest in this study.

Bu raporda, son derece tekrarlanabilir bir yöntem S. ikna etmek pneumoniae IPD sırasında farelerde kalp microlesions aracılı ve görselleştirme teknikleri gösterilmiştir. Fareler, insan sepsiste oluşur ve miyokarda nihai bakteriyel translokasyon neden benzer yüksek dereceli bakteremi, yol temizlenmesi için bağışıklık sisteminin eşiği aşması bakterilerin bir dozu ile enfekte edilir. S., henüz bilinmeyen nedenlerden dolayı merkezinde pneumoniae vücut bağışıkl?...

Toplum kökenli pnömoni (TKP) için yetişkinlerin Yatış mortalite 1-4 katkıda istenmeyen kardiyak olaylar için belgelenmiş bir risk taşır. Corrales-Medine ve ark tarafından yapılan yeni bir çalışmada. Kardiyak komplikasyonlar ve / veya sorumlu pnömoni ile ilişkili ölümlerin 3% 27 için ilişkili bulunmuştur. Streptococcus pneumoniae (pnömokok), TKP&#39;nin ve sepsis 5 en yaygın nedeni, doğrudan başvuran erişkin hastaların 6 gibi birçok 19 olarak% olumsuz kardiyak olaylar ile ilişkili olmuştur. Pnömoni ile ilişkili advers kardiyak olaylar sıklığını 6 azalan yeni veya kötüleşen konjestif kalp yetmezliği, aritmi ve miyokard infarktüsü sayılabilir. Brown ve arkadaşları tarafından yapılan bir PLoS patojenler makalede., S. pneumoniae myocardiu içine kardiyak vasküler endotelyum, giriş ile translokasyon yeteneğine sahip olduğu bulunmuşturm ve invaziv pnömokokal hastalığın (IPD) 7 sırasında ventrikül bakteri dolu ayrık olmayan pürülan mikroskobik lezyonlar (microlesions) oluşumu. Kardiyak microlesion oluşumunun kanıtı pnömokok enfeksiyonuna yenik olan insan olmayan primatlar ve bireylerden kardiyak örnekleri gözlenmiştir. Benzer şekilde, deneysel olarak enfekte olmuş fareler, yeniden üretilebilir kalp microlesions geliştirdi. Farelerde, microlesion büyüklüğü ve sayısı olumlu süresi ve bakteriyemi, serumlar kardiyak troponin düzeyleri şiddeti ve anormal kalp elektrofizyoloji ile korelasyon. Kalbine bakteriyel translokasyon, kan-beyin engeli ve pnömokok menenjit, bir laminin reseptörü ve Trombosit vasküler endotel hücrelerinin, yani kolin bağlanma proteini A aracılı istilası karşısında pnömokokun translokasyon için sorumlu olan mekanizmalar aracılığıyla meydana geldiği bulunmuştur harekete geçirme faktörü reseptörü bağımlı bir şekilde 8. Microlesiyon oluşumu, kardiyomiyositlerde 7 öldürdüğü bulunmuştur pnömokok gözenek oluşturucu toksin pnömolizin gerekiyor. Pnömokok kalp microlesions Staphylococcus aureus gibi diğer gram pozitif bakterilerin neden olduğu iltihaplı yumuşak doku ve kardiyak abseler farklıdır. Bu, nötrofillerin ve fibrin oluşumu, 9,10 ile çevrili bir bakteri, tek odakları ile karakterize edilir. Microlesions S. tarafından oluşturulan pneumoniae etkilenen bir kalp boyunca dağılmış, boyut olarak daha küçüktür, ve immün hücre infiltrasyonu yoksundur. Enfeksiyonun erken aşamalarında, microlesions S. neden pneumoniae kardiyomiyopati patolojik bulguların anımsatan hasarlı veya iltihaplı doku alanlar olarak görünür. Bazı monositler, ancak kendi varlığını kısa ömürlü ve lezyonlar hızla aynı anda GR devam ederken görünüm nekrotik hale gelir ve bakteri dolu, bu süre içinde görülebilirhayvan veya antimikrobiyal müdahale ölümüne kadar boyutu ow. Önemli bir şekilde, antibiyotik müdahale 3 gün içinde, aşırı bağışık hücre içi filtratı önceki lezyon bölgesinde görülmektedir ve bu sağlam kollajen birikimi eşlik eder. Benzer kalp biçimlenme kardiyak fonksiyon 11-15 sonuçlarını kalıcı birlikte enfarktüsü aşağıdaki meydana bildirilmiştir. Böylece, microlesions hastalık bölüm hayatta nekahet bireylerde kalp-ilişkili mortalite insidansında artış IPD&#39;ye sırasında ve muhtemelen ortaya istenmeyen kardiyak olaylar için potansiyel bir açıklama vardır. Burada, kullanıcı enfeksiyonun erken ve geç aşamalarında IPD ve kardiyak lezyon oluşumu ve kardiyak microlesions görselleştirme deneysel fare modelinde sağlanır. antimikrobiyal müdahale ile kaydedilmiş olan hayvanlarda kollajen birikimi tespiti için protokol gösterilmiştir. Bu makalenin amacı f olduğunuBu önemli ve yeni pnömokok patoloji diğer araştırmacıların araştırma acilitate.

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			<td height="42" style="height:42px;width:308px;">O.C.T Compound&#160;</td>
			<td style="width:175px;">Tissue-Tek (Sakura Finetek)&#160;</td>
			<td style="width:119px;">4583</td>
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			<td height="21" style="height:21px;">Triton X-100&#160;</td>
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			<td>9002-93-1</td>
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		<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

Bakteriyemi sırasında Streptococcus pneumoniae miyokard içine vasküler endotel boyunca transloke ve ayrık bakteri dolu mikroskobik lezyonlar nedeniyle bağışıklık hücrelerini infiltre yokluğu dikkat çekiyor (microlesions) oluşturabilir. Nedeniyle kardiyotoksik ürünlerin onların serbest bırakılması, S. microlesions içinde pneumoniae sıklıkla erişkinlerde fulminate invazif pnömokok hastalığı sırasında gözlenen kalp yetmezliğine katkıda bulunduğu düşünülmektedir. Burada 30 saat içinde kalp microlesion oluşumunu tekrarlanabilir neden deneysel fare enfeksiyonu için bir protokol gösterilmiştir. Öğretim Hematoksilin ve lekeli kalp bölümleri ve erken ve geç microlesions arasındaki morfolojik farklılıkları eozin içinde microlesion tanımlama sağlanır vurgulanır. Öğretim S. doğrulanması için bir protokol sağlanır Pnömokok kapsüler polisakkarit ve karşı antikorlar kullanılarak microlesions içinde pneumoniaeimmunofluorescent mikroskopi. Son, enfekte fare ve algılama ve nekahet farelerin kalplerinde skar oluşumu değerlendirilmesi için kurtarır antibiyotik müdahale için bir protokol sağlanır. Birlikte, bu protokoller S. maruz kalma sonucu pnömokok kalp işgali, kardiyomiyosit ölümü, kardiyak remodeling altında yatan moleküler mekanizmaların soruşturma kolaylaştıracaktır pneumoniae ve kalp pneumococci için bağışıklık tepkisi.

H &amp; E ile Microlesions görselleştirme Kardiyak microlesions Şekil 1. TIGR4 ile ip meydan aşağıdaki İPH farelerin kalp bölümleri lekeli H &amp; E ventrikül gözlenen hematoksilen daha fazla sayıda 24 ve 30 saat sonrası enfeksiyon (hpi) arasındaki bu lezyonların boyutunda artış gösteriyor edildi lezyon içinde görünür -stained (yani, mor-mavi) pnömokok. Microlesions bir vakuol-benzeri morfolojisi, daha yoğun eosin (kırmızı), lezyo...

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.

Görselleştirme<em&gt; Streptococcus pneumoniae</em&gt; Kalp Microlesions ve sonraki Kardiyak Tadilat içinde

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 Görüntüleme.  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.

Kardiyak Stres Testi Dobutamin İndüklemiş ve Farelerde Kalp Kateterizasyonu tarafından İzlenen

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

Tıp

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.

Doğrudan Basıncı İzleme Doğru Cryoballoon Ablasyon sırasında Pulmoner Ven Tıkanıklığı tahmin

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.

Sıçanlarda Karın Aort Konstrüksiyon sayesinde Kardiyak Yaptırma Bir Model

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.

Hazırlık ve<em&gt; İn Vitro</em&gt; Mıknatıslanmış karakterizasyonu Endotel Hücreleri miR-modifiye

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.

Şüpheli kardiyak Trombülerin değerlendirilmesi için kardiyak manyetik rezonans: konvansiyonel ve gelişmekte olan teknikler

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.

Murine Kardiyak Perisitlerin İzolasyon ve Safifikasyonu

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.

Bozulmamış Bir Perikard İskemik Kemirgen Modeli

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.

Açık Kalp Cerrahisinin Stereo 3D Görselleştirilmesi için İdeal Yapılandırmayı Belirlemek için Kamera Konumlarının Robotlu Testi

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.

Dolaşım Ölümü Sonrası Büyük Hayvan Bağış Modeli ve Kardiyak Değerlendirme için Norannemik Bölgesel Perfüzyon

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.

Kemirgen Miyositlerde Kardiyak Kasılma Disfonksiyonu ve Ca2+ Geçicilerinin Analizi

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