Bu çalışma ABD Ordusu Grant (W81XWH-08-1-0701) ve Carle Vakfı Hastanesi&#39;ne Kardeşliği tarafından finanse edildi.

Prosedür IACUC protokolü numarası 13302 ile tam uyumluluk içindedir ve hayvan kaynaklarının bölünmesi tarafından onaylandı. 1. Enstrüman Hazırlık <ol><li> Sterilite sağlamak için cerrahi işlem öncesinde kullanılan tüm olmayan tek kullanımlık aletleri otoklavlayın. </li><li> Kullanımlar arasında bir cam boncuk sterilizatör bir oturumda birden çok kez kullanılan aletleri sterilize edin. </li></ol> 2. Hidrojel Hazırlık <ol><li> Çözülme ve hidrojel yapının oluşturulması için en az 24 saat önce plaka kök hücreler. </li><li> Önceki yayınlarda 8,18 detaylı bir şekilde tarif edilmiş bir stereolitografi aparatı kullanılarak cerrahi (ya da benzer bir alet), bir gün önce, yamalar hazırlayın. </li><li> Hidrojel oluşturmak için, ilk olarak istenen yama bir bilgisayar destekli tasarım (CAD) modeli oluşturmak ve stereolitografik biçimi (STL) tasarımını ihracat. </li><li> Dissolvin bir ön jel çözeltisi oluşturmakSteril 1x PBS g poli (etilen glikol) dimetakrilat (PEGDMA)% 20 ağırlık / hacim solüsyonu elde etmek için. Aynı zamanda, foto-başlatıcı 1- [4- (2-hidroksi-etoksi) -fenil] -2-hidroksi-2-metil-1-propan-1-on, DMSO içinde çözülür. / 0.5 hacimce% nihai olarak PEGDMA çözeltisine foto-başlatıcı ekleyin. Doğrudan çapraz bağlama öncesi hücrelerin istenen yoğunlukta 100 ml. Not: hücre / ml önerilir 6 10 x -2.0 5 10 x 2.0 arasında bir hücre yoğunluğuna. </li><li> SLA platformun ortasında bir çanak içine önceden jel çözümü Pipet ve önceden yüklenmiş STL formatlı tasarımı ile SLA yazılımını çalıştırın. </li><li> Aletinden yamalar çıkarın ve Dulbecco modifiye edilmiş Eagle ortamı,% 5 fetal sığır serumu (FBS) ile desteklenmiş (DMEM), 100 U / ml penisilin hidrojeller göre O / N inkübe ve hücreler, ayarlamak için 100 mg / ml streptomisin izin vermek için yama içinde çevreye. </li></ol> 3. Hayvan Hazırlama ve Sözlü intubatiüzerinde <ol><li> Onların kafes arkadaşları ile onların tımar alışkanlıkları ve etkileşimleri belirlenen hayvan, sağlık durumunun iyi olduğundan emin olmak için prosedür öncesinde her fare değerlendirin. Not: Bayan 8-10 haftalık C57BL / 6J fareler bu işlem için kullanılan, ancak, herhangi bir fare yaş ve tipi kullanılabilir. </li><li> Bir anestezik odasına hayvanları yerleştirin ve 1 L / dk O 2 desteği ile% 5 isofluorane maruz. Ayak tutam refleks ile anestezi düzeyini izleyin. </li><li> Hayvan düzgün uyuşturulduktan sonra, tartmak ve entübasyon standı üzerine koyun. </li><li> Hayvanın göğüs boşluğuna doğru bir ışık kaynağı yönlendirin ve ses tellerini laringeal yüzeyini geri çekin ve ortaya çıkarmak için forseps kullanabilir. Dikkatle ses tellerinin arasında 20 G angiocath rehberlik ve sorunsuz trakea içine yerleştirin. Kateter küçük hayvan ventilatöre bağlı bir kez göğüs boşluğunun mekanik hareketi ile uygun ekleme algılar. </li><li> Inci havalandırma ayarlarını yapınüreticinin yönergelerine göre hayvanın e ağırlığı. </li><li> Entübe sonra hipotermi önlemek için ısıtma pedi üzerinde yatar pozisyonda hayvan yerleştirin. Isıtma yastığı 40 ° C&#39;yi aşmaması emin olun. Gerekirse, yanık şansını en aza indirmek için hayvan ve ısıtma yastığı arasında bir bariyer yerleştirin. </li><li> Bir epilasyon kremi kullanılarak cerrahi bölgeden saç çıkarın. Doğru sterilizasyon sağlamak için Betadin ve% 75 etanol 3 alternatif scrubs tarafından siteyi sterilize edin. Sadece cerrahi siteyi açığa ve işlem sırasında kuruluğunu önlemek için gözleri veteriner merhem yerleştirmek için bir örtü yerleştirin. </li></ol> 4. Cerrahi Prosedür <ol><li> torakotomi nedenle, sternum uzunluğu çalışan sternum sol, bir cilt kesisi yaklaşık 1 cm yapmak, hayvanın sol tarafında yapılır. </li><li> Iki adımda yatan göğüs kafesi arasında kas tabakası ayrı forseps kullanın. Bir Deline&#39;a gözlemleyinhayvan pektoralis majör kaslarını gösteren ating hattı. Altta yatan dış eğik kas ayırmak ve mediale geri, biraz bu kası kaldırın. Ardından, aynı şekilde yatan göğüs kafesi dış oblik kas özgür ve ikinci, üçüncü ve dördüncü kaburga bir unobscured görünüm sağlayan, yanal geri çekin. </li><li> Üçüncü ve dördüncü kaburga arasındaki torakotomi gerçekleştirin. Yavaşça dördüncü kaburga kaldırın ve üçüncü ve dördüncü kaburgalar arasındaki göğüs boşluğunu açmak için bir cauterizer kullanın. Ayrıca boşluğu açmak ve kalp maruz Ekartörleri yerleştirin. </li><li> Kalbin ince perikard rüptürü için forseps kullanabilir. </li><li> Bir 8-0 monofilament naylon sütür ile sol koroner arter Arter. Doğrudan sol atrium alt ucu altında, yaklaşık 4 mm kalbin apeks gelen dikiş yerleştirin. Ventriküler miyokard bir blanching ve Sutu sonra sol atrium boyutunda bir artış ile doğru dikiş yerleştirme belirleyinYeniden bağladı edilmiştir. </li></ol> 5. Patch Yerleştirme ve Tutkal <ol><li> Kullanılana kadar CO2 şartları, 37 ° C&#39;de tutun yamalar ve% 5. </li><li> Yavaşça düz uçlu spatula ile yama kaldırın ve yavaşça infarktüsü yerinde, kalbin bir yüzeye yerleştirin. Not: Sol ventrikül kaymasını önlemek için, yama hafifçe spatula ya da forseps bir çift ucu kullanarak kişiyi koruyarak yerinde tutulabilir. </li><li> Trombin, fibrinojenin: 1 oranını 4 karıştırılarak tutkal hazırlayın. Bu hazırlık başladıktan sonra genellikle 1 dakika içinde, kalınlaştırmak başlayana kadar tekrarlanan pipetleme çözüm karıştırın. </li><li> Çözelti viskozitesi istenen seviyeye ulaştığında, hızla yama yüzeyine yaklaşık 10 ml aktarın. Not: fibrin yapıştırıcı pıhtılaşma zamanı hızlıdır, tutkal verimli transferi için küçük bir fırsat penceresi sağlar. </li></ol> 6. Dikiş <ol><li&gt; 6-0 monofilament naylon dikiş kullanarak tüm kapanış sütür gerçekleştirin. </li><li> 3-4 ayrı kesintiye dikişlerle kaburga katmanı kapatın. </li><li> Interkostal katmanın tam kapanmasından önce, kapatma işlemi tamamlandıktan sonra göğüs boşluğuna tahliye ve uygun intraplevral basınç yeniden kurmak için kesi içine PE-10 kanül yerleştirin. </li><li> 3-4 ayrı kesintiye dikişlerle göğüs kasları kapatın. </li><li> Sürekli dikiş ile cilt katmanı mühür. </li><li> Tam kapatılması sonrasında, kanül sonuna 1.0 ml şırınga takmak ve göğüs boşluğunu tahliye için kullanabilirsiniz. </li><li> Dikiş sitesini güçlendirmek için kesi sitesine doku yapıştırıcısı sürün. </li></ol> 7. Post-cerrahi tedavi <ol><li> Fareler buprenorfin (0.05-1.0 mg / kg) ve deri altından hemen önce canlanma koroner ligasyon ve en az 20 dakika, aşağıdaki karprofen (2.2 mg / kg) enjeksiyonu ver. Her iki ilaç da ameliyattan sonra 6-8 saat yönetmeVe sonra ağrı ve sıkıntı kontrolü üç gün süreyle günde iki kez vermek. </li><li> Sürekli ilk 4 saat aşağıdaki ameliyat için bilinçli ve her saatte kadar hayvanları izlemek. Analjezik ikinci bir dozunu vermek için ikinci bir sağlık kontrolü ameliyat (2-4 saat sonra) gecesini gerçekleştirin. </li><li> Hayvanlar kararlı görünen kadar prosedürü izleyerek ikinci günden sonra her gün hayvanları izleyin. </li></ol> Kalp Fonksiyonu ve Histoloji 8. Analiz <ol><li> MI Aşağıdaki hasarın boyutunu belirlemek için prosedür izlenerek fareler 4 hafta echocardiographs gerçekleştirin. </li><li> Inhalasyon anestezi indüksiyonu için bir anestezi odasının içinde% 4-5 izofluran ile fareler anestezi ve bakım anestezi için yüz maskesi aracılığıyla daha sonra% 2 izofluran. </li><li> Bir tüy dökücü krem ​​ile görüntüleme alanını temizleyin ve kalpte bir 2B M-mod ekokardiyografi tarama elde etmek için küçük bir hayvan ultrason kullanımı ve fonksiyonel parametreler asso kayıtonunla anılmaktadır. </li><li> Daha sonra 24 saat boyunca bir% 10 formalin çözeltisi içinde hayvanlar ve yerden kalpleri toplamak, bir karbon diyoksit odasında fareler öldürülür. 24 saat sonra,% 70 etanol kalpleri aktarmak ve doku işlenene kadar 4 ° C&#39;de tutun. </li><li> Kalpleri işlemek ve parafin bloklar onları gömmek. </li><li> Apeks başlayan ve atrium biten, kalbin ventriküler bölgesi boyunca 5 mm bölümleri kesin. </li><li> Leke Masson trikrom ve hematoksilen ve standart protokoller kullanılarak eozin leke hem dilimleri kesitli. </li></ol>

<ol>
	<li>Kolk, M. V., 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=LAD-ligation:+a+murine+model+of+myocardial+infarction.">LAD-ligation: a murine model of myocardial infarction.</a> Journal of visualized experiments : JoVE. , (2009).</li><li>Li, Y., Yao, Y., Sheng, Z., Yang, Y., Ma, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-modal+tracking+of+transplanted+mesenchymal+stem+cells+after+myocardial+infarction.">Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction.</a> International journal of nanomedicine. 6, 815-823 (2011).</li><li>Nagaya, N., 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=Intravenous+administration+of+mesenchymal+stem+cells+improves+cardiac+function+in+rats+with+acute+myocardial+infarction+through+angiogenesis+and+myogenesis.">Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis.</a> American journal of physiology. Heart and circulatory physiology. 287, H2670-H2676 (2004).</li><li>Wang, J. S., Shum-Tim, D., Chedrawy, E., Chiu, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+delivery+of+marrow+stromal+cells+for+myocardial+regeneration:+pathophysiologic+and+therapeutic+implications.">The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications.</a> The Journal of thoracic and cardiovascular surgery. 122, 699-705 (2001).</li><li>Cashman, T. J., Gouon-Evans, V., Costa, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+cardiac+therapy:+practical+challenges+and+potential+mechanisms.">Mesenchymal stem cells for cardiac therapy: practical challenges and potential mechanisms.</a> Stem cell reviews. 9, 254-265 (2013).</li><li>Sutton, M. G., Sharpe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+remodeling+after+myocardial+infarction:+pathophysiology+and+therapy.">Left ventricular remodeling after myocardial infarction: pathophysiology and therapy.</a> Circulation. 101, 2981-2988 (2000).</li><li>Smolina, K., Wright, F. L., Rayner, M., Goldacre, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+recurrence+after+acute+myocardial+infarction+in+England,+2004+to+2010.">Long-term survival and recurrence after acute myocardial infarction in England, 2004 to 2010.</a> Circulation. Cardiovascular quality and outcomes. 5, 532-540 (2012).</li><li>Chan, V., Zorlutuna, P., Jeong, J. H., Kong, H., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+photopatterning+of+hydrogels+using+stereolithography+for+long-term+cell+encapsulation.">Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation.</a> Lab on a chip. 10, 2062-2070 (2010).</li><li>Kai, D., 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=Stem+cell-loaded+nanofibrous+patch+promotes+the+regeneration+of+infarcted+myocardium+with+functional+improvement+in+rat+model.">Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model.</a> Acta biomaterialia. 10, 2727-2738 (2014).</li><li>Serpooshan, V., 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=The+effect+of+bioengineered+acellular+collagen+patch+on+cardiac+remodeling+and+ventricular+function+post+myocardial+infarction.">The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction.</a> Biomaterials. 34, 9048-9055 (2013).</li><li>Cavallo, L. M., Solari, D., Somma, T., Savic, D., Cappabianca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Awake+Endoscope-Guided+Sealant+Technique+with+Fibrin+Glue+in+the+Treatment+of+Postoperative+Cerebrospinal+Fluid+Leak+After+Extended+Transsphenoidal+Surgery:+Technical+Note.">The Awake Endoscope-Guided Sealant Technique with Fibrin Glue in the Treatment of Postoperative Cerebrospinal Fluid Leak After Extended Transsphenoidal Surgery: Technical Note.</a> World neurosurgery. , (2013).</li><li>Chung, H. W., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+for+Gundersen+flap+surgery.">Fibrin glue for Gundersen flap surgery.</a> Clinical Ophthalmology. 7, 479-484 (2013).</li><li>Dunn, C. J., Goa, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+sealant:+a+review+of+its+use+in+surgery+and+endoscopy.">Fibrin sealant: a review of its use in surgery and endoscopy.</a> Drugs. 58, 863-886 (1999).</li><li>Chi, N. 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=Cardiac+repair+achieved+by+bone+marrow+mesenchymal+stem+cells/silk+fibroin/hyaluronic+acid+patches+in+a+rat+of+myocardial+infarction+model.">Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model.</a> Biomaterials. 33, 5541-5551 (2012).</li><li>Erb, M. A., Claus, T., Hartrumpf, M., Bachmann, S., Albes, J. 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+use+of+Tachosil+surgical+patch+or+fibrin+glue+in+coronary+artery+surgery+does+not+affect+quality+of+anastomosis+or+provoke+postoperative+adhesions+in+pigs.">The use of Tachosil surgical patch or fibrin glue in coronary artery surgery does not affect quality of anastomosis or provoke postoperative adhesions in pigs.</a> European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 36, 703-707 (2009).</li><li>Okada, K., 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=Surgical+treatment+for+rupture+of+left+ventricular+free+wall+after+acute+myocardial+infarction.">Surgical treatment for rupture of left ventricular free wall after acute myocardial infarction.</a> Interactive cardiovascular and thoracic surgery. 4, 203-206 (2005).</li><li>Simpson, D., Liu, H., Fan, T. H., Nerem, R., Dudley, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue+engineering+approach+to+progenitor+cell+delivery+results+in+significant+cell+engraftment+and+improved+myocardial+remodeling.">A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling.</a> Stem Cells. 25, 2350-2357 (2007).</li><li>Jeong, J. 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=Living'+microvascular+stamp+for+patterning+of+functional+neovessels;+orchestrated+control+of+matrix+property+and+geometry.">Living' microvascular stamp for patterning of functional neovessels; orchestrated control of matrix property and geometry.</a> Advanced Materials. 24, 58-63 (2012).</li><li>Wu, X., Ren, J., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+the+cell-delivery+vehicle+for+mesenchymal+stromal+cells+in+regenerative+medicine.">Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.</a> Cytotherapy. 14, 555-562 (2012).</li></ol>

The authors declare that they have no competing financial interests.

Fare MI modeline Bu yaklaşımla, diğer kemirgen MI teknikleri ile ilişkili olmayan miyokard alanlara zarar en aza indiren bir sistem geliştirdik. Bu alanlar trakeostomi yol açtığı hasarı, kas tabakasının kesilmesi, ve göğüs boşluğunu maruz kaburga kırılması sayılabilir. Biz bu değişikliklerin cerrahi işlem sırasında olduğu gibi, kaburga ve kas tabakaları dahil, önemli yapıların çok sağlam tutmak için alınan bakım genel cerrahi son...

Bir miyokard enfarktüsü (MI) büyük bir koroner arter tıkanmasına neden olduğu kalp bölgeye kan kesintisiz olarak tanımlanır. Bir MI kaynaklanan hasar nedeniyle ya da daha spesifik olarak, sol ventrikül düzgün yenmek için kalp yeteneğini azaltır fonksiyonel olmayan skar dokusu içine canlı kalp dokusunun yeniden, etmektir. Bu vuruş hacmi olarak bilinir, her kalp atışı, ve ejeksiyon fraksiyonu 6 olarak bilinen her kalp atışında kalp dışarı pompalanır, bir kan yüzdesi, gövdeye teslim edilebilir kan hacminde bir azalma ile sonuçlanır. Bunlar, diğer azalmış fonksiyonları ile birlikte, yeterli işlevini korumak için kalbin geri kalanı üzerinde gerginlik artar. Ikinci bir kalp krizi, bireylerin 7 yaklaşık% 10&#39;unda görülen bir fenomen neden sık sık bu artış suş çok şiddetli olabilir. Tıbbi uygulamalar tedavisinde geliştiğini ikenBir MI hemen sonrasında, hiçbir teknik, yavaş, durdurmak veya doku yeniden olumsuz yan etkilerini tersine çevirmek için geliştirilmiştir. Kök hücre terapileri da umut verici etkisine karşın, bununla birlikte, böyle bir tedavi için, olası bir yol olarak ortaya çıkmıştır kök hücreler klinik başarılı kanıtlanmamıştır. Onların eksikliklerini bir teori yararlı hücreler olumlu sonuçlar üretmek için 5 yeterince uzun enfarktüsü yerinde kalmasını sağlamak için yetersizliğidir. Bu sadece enfarktüs sitesi içine enjekte edilir hücrelerin en fazla% 24 hayatta kaldı ve zarar görmüş Alanı 1 gün sonrası teslimat 2 de kaldığı gösterilmiştir. Hücre tutma, bu konuyu ele için olası bir ihtimal hücreleri veya hasarlı siteye teslim edilebilir terapötikleri ya saklanması biyouyumlu hidrojel sistemleri geliştirmektir. Bu protokolde seçim hidrojel nedeniyle hücre encapsulation pr önceki kullanımına bir poli (etilen glikol) dimetakrilattırocedures Bununla birlikte, kapsülleme yeteneğine sahip herhangi bir hidrojel 8 kullanılabilir. yaralanmanın sitesine yama dağıtım hücreleri yatan miyokard yararlı faktörler sağlayabilir süreyi artırmak, uzun bir zaman süresi boyunca hücreden doku teması sağlar. Yama yaklaşımına bir darboğaz kalp yüzeyine yapışan yama zorluğudur. Birçok grup teknikleri, kalp yüzeyine 9,10 için yapı kravat basit bir dikiş olmanın en yaygın çeşitli aracılığıyla bunun üstesinden gelmiş. Bu yapı, daha sert bir malzemeden imal edildiği durumlarda, bir dizi başarılı olduğu kanıtlanmıştır, ancak, yüksek su konsantrasyonu ve yama yapısının hassas doğası, bir hidrojel sistemi üzerinde çalışırken başarısız olmuştur. Bunun üstesinden gelmek için, biz pıhtı oluşumu kimyası taklit eden bir fibrin yapıştırıcı dış yapıştırıcı sistemini kullanmışlardır. Fibrin yapıştırıcı inc, çok sayıda tıbbi ameliyatlarında kullanılmaktadırBir yara dolgu 11-13 olarak ürünün biyouyumluluk vurgulayarak, dura gözyaşları, bronşiyal fistül ve kornea nakli luding. Buna ek olarak fibrin Bununla birlikte, kalp yama için bir yapışma yapıştırıcı olarak kullanımı yaygın olarak 14-17 kullanılmaz sol ventrikül kopmalar ve koroner arter by-pass ameliyatları cerrahi tedavisi de dahil olmak üzere kalp amacıyla, çeşitli kullanılmıştır. Kalp etkileşim yama sağlamak için uygun bir yapıştırma sisteminin sağlanması, harici kalp yama dışında doğrudan yerleştirilebilir, bir biyo-uyumlu yapıştırıcı Trombin ve fibrinojen sonuçları basit bir formülasyon.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

Cerrahi işlem sırasında, sol koroner arter ligasyonu bir Tıkalı arterin alt beyazlatma işaretlenmiş tarafından tespit edilebilir. Düğüm atarak önce bir test olarak, dikiş uygun yerde olup olmadığını kontrol etmek için kısa bir süre sıkılır olabilir. Ayrıca, düzgün yenmek için sol atrium yeteneği neredeyse anlık azalma arter sonuçlarının tıkanması beri, sol atrium sisteminde kan geri akışının cevaben büyütmek olacaktır. M-mod ekokardiyografi ölçümle...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Bir Hidrojel Construct ve Fare Miyokard Enfarktüsü Modeli Therapeutics sunun için Tutkal Yaklaşım Fibrin tabanlı.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

a-hydrogel-construct-fibrin-based-glue-approach-to-deliver

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.6K Görüntüleme.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Video: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Sıçanlarda Akut Miyokard Enfarktüsü

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Tıp

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Enfarktüsü bir murin Model Koroner Arter ligasyon ve intramiyokardiyal Enjeksiyon

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Miyokard İskemi ve Reperfüzyon bir Mürin Kapalı göğüs Modeli

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Domuzlar miyokard enfarktüsü ve Fonksiyonel Sonuç Değerlendirmesi

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Akut Miyokard İnfarktüsü bir Domuz Modeli İlköğretim Sonuç Değerlendirmesi

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Yenidoğan Farelerde Miyokard Enfarktüsü, Kalp Rejenerasyon Bir Model

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Sol Anterior İnen Koroner Arterin Kalıcı Ligasyonu ile Murine Miyokard Enfarktüsü Modeli

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Farede Miyokard Enfarktüsü Çalışması İçin Kriyoyaralanma Modeli

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Kök Hücre Tedavisi Için Post-miyokard Enfarktüsü Kalp Yetmezliği Domuz Modeli Oluşturma

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine Modelinde Miyokard İnfarktüsünden Sonra MSC Yüklemeli Enjekte Edilebilir Hidrojellerin intramiyokardiyal Transplantasyonu

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...