We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

כל הנהלים המיושמים במחקר זה עולה בקנה אחד עם האגודה לחקר משפט חזון עיניים עבור שימוש בבעלי חי עיניים וחזון המחקר אושרו על ידי ועדת הטיפול בבעלי החיים המוסדיים השתמש בבית החולים האוניברסיטאי הלאומיים בטייוואן. 1. ניתוק, הכנת התרבות ראשית ולאחר Immunostaining של השור CECs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> רוכשת עיני שור טריות מטבחיים מקומי. </li><li style=";text-align:right;direction:rtl"> לחטא את העיניים בתוך 10% w / v פתרון povidone יוד במשך 3 דקות. לשטוף אותם עם תמיסת מלח פוספט שנאגר (PBS). </li><li style=";text-align:right;direction:rtl"> קציר את הכפתור הקרני עם אזמל ומספריים בתנאים סטריליים. מקלף את הממברנה של Descemet עם מלקחיים תחת מיקרוסקופ לנתח (לציין כי במחקר זה, בעיני השור היו enucleated ידי מטבחיים מקומי, ולכן הליך המחקר הראשון היה חיטוי בעיני preenucleated במעבדה). </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה של Descemet ב 1 מ&quot;ל של לנסותpsin על 37 מעלות צלזיוס למשך 30 דקות. אסוף את CECs שור על ידי צנטריפוגה ב 112 XG במשך 5 דקות. Resuspend תאי 1 מיליליטר של מדיום אפיתל הורמונלי השלים (שם) המכיל כמויות שווות של מדיום הנשר HEPES שנאגר השונה של Dulbecco והבינוני F12 של חם, בתוספת 5% בסרום שור עוברי, sulfoxide דימתיל 0.5%, 2 ng / ml של אפידרמיס אדם גורם הגדילה, 5 מ&quot;ג / מ&quot;ל ​​של אינסולין, 5 מ&quot;ג / מ&quot;ל ​​של transferrin, 5 ng / ml של סלניום, 1 nmol / L של רעלן הכולרה, 50 מ&quot;ג / מ&quot;ל ​​של גנטמיצין, ו -1.25 מ&quot;ג / מ&quot;ל ​​של B. amphotericin </li><li style=";text-align:right;direction:rtl"> זרעים תאים (כ 1 x 10 5 תאים לכל עין) לתוך צלחת 6 ס&quot;מ. תרבות אותם שם. דגירת הצלחת ב 37 מעלות צלזיוס אווירה של 5% CO 2 באוויר. שינוי התרבות בינוני כל 3 ימים. </li><li style=";text-align:right;direction:rtl"> כאשר התאים מגיעים למפגש, לשטוף אותם PBS דגירה אותם ב 1 מ&quot;ל של טריפסין על 37 מעלות צלזיוס למשך 5 דקות. אסוף אותם על ידי צנטריפוגה ב 112 XG במשך 5 דקות. Re- להשעות את התא גלולה ב 1 מ&quot;ל של שם טוב. <ol&gt; <li style=";text-align:right;direction:rtl"> ספירת התאים ב hemocytometer. זרעים התאים בשקופיות כיסוי בצפיפות של 1 x 10 4 לכל היטב צלחת 24 גם, והתרבות התאים שם טוב. על חקירת השפעת EnMT-הדיכוי של marimastat, דגירת התאים שם טוב עם 10 מיקרומטר של marimastat הוסיפו לתוך מדיום התרבות, ולשנות את מדיום התרבות כל 3 ימים. </li></ol></li><li style=";text-align:right;direction:rtl"> תקן את התאים בנקודת זמן מצוינת עם 250 μl של paraformaldehyde 4%, pH 7.4, למשך 30 דקות בטמפרטורת חדר. Permeabilize עם 250 μl של 0.5% Triton X-100 במשך 5 דקות. חסום עם אלבומין בסרום שור 10% למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> דגירת התאים עם נוגדנים ראשוניים נגד-קטנין ביתא הפעיל (ABC), חילזון, חילזון הלילה ב 4 ° C.. הדילול של הנוגדנים העיקריים בשימוש הוא 1: 200. הנוגדנים הם מדולל diluent נוגדן מורכב של 10 מ&quot;מ PBS, 1% w / v אלבומין בסרום שור, ו 0.09% w / v אזיד הנתרן. </li><li style=";text-align:right;direction:rtl"> שוטפים את התאים פעמיים עם PBS במשך 15 דקות, דגירהמ &#39;עם נוגדנים משני מצומדות פלואוריד אלקסה (1: 100 ב diluent נוגדן) בטמפרטורת החדר למשך שעה 1. </li><li style=";text-align:right;direction:rtl"> Counterstain בגרעין התא על ידי כיסוי התאים עם 2 מיקרוגרם / מיליליטר של 4 &#39;, 6-diamidino-2-phenylindole, לשטוף את התאים פעמים עם PBS במשך 15 דקות, ו הר איתם נגד דהיית פתרון גובר. </li><li style=";text-align:right;direction:rtl"> להשיג תמונות immunofluorescent על אורכי גל עירור של 405 ו 488 ננומטר באמצעות מיקרוסקופ confocal סריקת לייזר עם 20X ההגדלה אובייקטיבי. </li></ol> 2. מודל עכברוש קרנית האנדותל Cryoinjury הזרקת Intracameral <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים 12 שבועות בן חולדות Sprague-Dawley זכר עם זריקה תוך שרירית של 2% xylazine (5.6 מ&quot;ג / ק&quot;ג) ו tiletamine בתוספת zolazepam (18 מ&quot;ג / ק&quot;ג). בעדינות לצבוט את העור של החיות כדי לאשר הרדמה תקינה בהעדר מתעוות עור. </li><li style=";text-align:right;direction:rtl"> להחדיר טיפה אחת של hydrochloride proparacaine 0.5% אל עינו הימנית של כל העכברים כדי למזער את הכאב העין ואת רפלקס מצמוץ. לְהַחדִירמשחת טטרציקלין לעין השמאל כדי למנוע יובש קרני. </li><li style=";text-align:right;direction:rtl"> מגניב בדיקת נירוסטה (קוטר = 3 מ&quot;מ) בחנקן נוזלי. החלת את חללית הנירוסטה אל הקרנית המרכזית של עין ימין למשך 30 שניות. לעתים קרובות להנחיל PBS על עינו הימנית במהלך ההליך כדי למנוע יובש קרני. </li><li style=";text-align:right;direction:rtl"> להחדיר 0.1% אטרופין סולפט גנטמיצין 0.3% מייד לאחר cryoinjury ופעם יומי כדי להקל על כאב עיני נובע התכווצות ריסים ולמנוע זיהום. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר ההליך, לשמור על החולדות לחמם באמצעות מנורת חום, ולבחון החלים כל 15 דקות אחרי ההרדמה עד שהם להחזיר שליטה מוטורית. בנוסף, החל משח טטרציקלין לעין הזכות למנוע יובש קרני במהלך תקופת ההחלמה. </li></ol></li><li style=";text-align:right;direction:rtl"> חזור על cryoinjury הקרנית במשך 3 ימים רצופים. </li><li style=";text-align:right;direction:rtl"> עבור אספקת marimastat או bFGF לתוך החדר הקדמי של עיני החולדה, להרדים החולדות כפי שתואר לעיל. להחדיר טיפה אחתשל hydrochloride proparacaine 0.5% אל תוך עינו הימנית כדי למזער את כאב עין ואת רפלקס המצמוץ. </li><li style=";text-align:right;direction:rtl"> להשקות את פני השטח של העין עם PBS סטרילית. בצע ניקור הלשכה הקדמית תחת מיקרוסקופ ההפעלה על ידי החדרת מחט 30 G מצורף מזרק 1 מ&quot;ל על הקרנית ברור paralimbal במטוס מעל ובמקביל הקשתית. </li><li style=";text-align:right;direction:rtl"> סובבו את המחט שפוע למעלה, ומעט לדכא את הפצע בקרנית לנקז קצת הומור מימית ולהפחית את הלחץ התוך עיני. להזריק 0.02 מ&quot;ל של התרופה intracamerally. בעדינות לדחוס את דרכי מחט עם קצה כותנה בזמן נסיגת המחט. </li><li style=";text-align:right;direction:rtl"> צלם את שלהוציא בנקודת זמן מצוינת מתחת למיקרוסקופ ההפעלה. </li></ol> 3. קצירי העכברוש קרני לחצן ו Immunostaining <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להרדים את החולדות, למקם אותם בתא המתת חסד להשרות 100% CO 2 בקצב מילוי של 10-30% מנפח התא לדקה. שמור עירוי CO 2 עבורדקה נוספת אחרי חוסר נשימת צבע עיניים דהוי. </li><li style=";text-align:right;direction:rtl"> לחדור לעין עכברוש בבית לימבוס עם להב חד. חותך את הקרניות במספריים קרניים לאורך לימבוס. שטח את הקרניות בשקופית. ודא חתכים רדיאליים נוספים אם הקרניות להתכרבל. </li><li style=";text-align:right;direction:rtl"> תקן את קרניות עם 250 μl של paraformaldehyde 4%, pH 7.4, למשך 30 דקות בטמפרטורת החדר. Permeabilize עם 250 μl של 0.5% Triton X-100 במשך 5 דקות, ולחסום עם אלבומין בסרום שור 10% למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> דגירה קרנית עם נוגדנים ראשוניים נגד ABC הלילה ב 4 ° C עם דילול של 1: 200 ב diluent נוגדן. שטפו את קרניות פעמיים עם PBS במשך 15 דקות, דגירה אותם עם נוגדנים משני (1: 100 ב diluent נוגדן) בטמפרטורת החדר למשך שעה 1. Counterstain בגרעין התא על ידי כיסוי תאים עם 2 מיקרוגרם / מ&quot;ל ​​של 4 &#39;, 6-diamidino-2-phenylindole, לשטוף את התאים פעמיים עם PBS במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> הר הקרני בתמיסה נגד דהייה גוברת. אובטיין התמונות immunofluorescent באורכי גל עירור של 405 ו 561 ננומטר באמצעות מיקרוסקופ confocal סריקת לייזר ב 20X ההגדלה אובייקטיבי. </li></ol>

<ol>
	<li>Maurice, D. 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+location+of+the+fluid+pump+in+the+cornea.">The location of the fluid pump in the cornea.</a> J Physiol. 221 (1), 43-54 (1972).</li><li>Joyce, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+capacity+of+the+corneal+endothelium.">Proliferative capacity of the corneal endothelium.</a> Progress in Retinal and Eye Research. 22 (3), 359-389 (2003).</li><li>Morishige, N., Sonoda, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bullous+keratopathy+as+a+progressive+disease:+evidence+from+clinical+and+laboratory+imaging+studies.">Bullous keratopathy as a progressive disease: evidence from clinical and laboratory imaging studies.</a> Cornea. 32, 77-83 (2013).</li><li>Bates, A. K., Cheng, H., Hiorns, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudophakic+bullous+keratopathy:+relationship+with+endothelial+cell+density+and+use+of+a+predictive+cell+loss+model.+A+preliminary+report.">Pseudophakic bullous keratopathy: relationship with endothelial cell density and use of a predictive cell loss model. A preliminary report.</a> Curr Eye Res. 5 (5), 363-366 (1986).</li><li>Wilson, S. E., Lloyd, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+its+receptor,+basic+fibroblast+growth+factor,+transforming+growth+factor+beta-1,+and+interleukin-1+alpha+messenger+RNA+production+in+human+corneal+endothelial+cells.">Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells.</a> Invest Ophthalmol Vis Sci. 32 (10), 2747-2756 (1991).</li><li>Chen, K. H., Harris, D. L., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta2+in+aqueous+humor+suppresses+S-phase+entry+in+cultured+corneal+endothelial+cells.">TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.</a> Invest Ophthalmol Vis Sci. 40 (11), 2513-2519 (1999).</li><li>Senoo, T., Obara, Y., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDTA:+a+promoter+of+proliferation+in+human+corneal+endothelium.">EDTA: a promoter of proliferation in human corneal endothelium.</a> Invest Ophthalmol Vis Sci. 41 (10), 2930-2935 (2000).</li><li>Yue, B. Y., Sugar, J., Gilboy, J. E., Elvart, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+corneal+endothelial+cells+in+culture.">Growth of human corneal endothelial cells in culture.</a> Invest Ophthalmol Vis Sci. 30 (2), 248-253 (1989).</li><li>Peh, G. S., Beuerman, R. W., Colman, A., Tan, D. T., 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=Human+corneal+endothelial+cell+expansion+for+corneal+endothelium+transplantation:+an+overview.">Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview.</a> Transplantation. 91 (8), 811-819 (2011).</li><li>Zhu, C., Rawe, I., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+expression+in+human+corneal+endothelial+cells+cultured+from+young+and+older+donors.">Differential protein expression in human corneal endothelial cells cultured from young and older donors.</a> Mol Vis. 14, 1805-1814 (2008).</li><li>Ko, M. K., Kay, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+role+of+FGF-2+on+type+I+collagen+expression+during+endothelial+mesenchymal+transformation.">Regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation.</a> Invest Ophthalmol Vis Sci. 46 (12), 4495-4503 (2005).</li><li>Lee, H. T., Kay, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2+induced+reorganization+and+disruption+of+actin+cytoskeleton+through+PI+3-kinase,+Rho,+and+Cdc42+in+corneal+endothelial+cells.">FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.</a> Mol Vis. 9, 624-634 (2003).</li><li>Pipparelli, 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=ROCK+Inhibitor+Enhances+Adhesion+and+Wound+Healing+of+Human+Corneal+Endothelial+Cells.">ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.</a> PloS one. 8 (4), e62095 (2013).</li><li>Roy, O., Leclerc, V. B., Bourget, J. M., Theriault, M., Proulx, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+process+of+corneal+endothelial+morphological+change+in+vitro.">Understanding the process of corneal endothelial morphological change in vitro.</a> Invest Ophthalmol Vis Sci. 56 (2), 1228-1237 (2015).</li><li>Jakobiec, F. A., Bhat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrocorneal+membranes:+a+comparative+immunohistochemical+analysis+of+keratocytic,+endothelial,+and+epithelial+origins.">Retrocorneal membranes: a comparative immunohistochemical analysis of keratocytic, endothelial, and epithelial origins.</a> Am J Ophthalmol. 150 (2), 230-242 (2010).</li><li>Okumura, 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=Involvement+of+ZEB1+and+Snail1+in+excessive+production+of+extracellular+matrix+in+Fuchs+endothelial+corneal+dystrophy.">Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy.</a> Lab Invest. 95 (11), 1291-1304 (2015).</li><li>Okumura, 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=Enhancement+on+primate+corneal+endothelial+cell+survival+in+vitro+by+a+ROCK+inhibitor.">Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.</a> Invest Ophthalmol Vis Sci. 50 (8), 3680-3687 (2009).</li><li>Zhu, Y. T., Chen, H. C., Chen, S. Y., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+p120+catenin+unlocks+mitotic+block+of+contact-inhibited+human+corneal+endothelial+monolayers+without+disrupting+adherent+junctions.">Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.</a> J Cell Sci. 125 (15), 3636-3648 (2012).</li><li>Ho, W. 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=Inhibition+of+Matrix+Metalloproteinase+Activity+Reverses+Corneal+Endothelial-Mesenchymal+Transition.">Inhibition of Matrix Metalloproteinase Activity Reverses Corneal Endothelial-Mesenchymal Transition.</a> Am J Pathol. 185 (8), 2158-2167 (2015).</li><li>Kay, E. D., Cheung, C. C., Jester, J. V., Nimni, M. E., Smith, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+collagen+and+fibronectin+synthesis+by+retrocorneal+fibrous+membrane.">Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane.</a> Invest Ophthalmol Vis Sci. 22 (2), 200-212 (1982).</li><li>Li, C., 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=Notch+Signal+Regulates+Corneal+Endothelial-to-Mesenchymal+Transition.">Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition.</a> Am J Pathol. 183 (3), 786-795 (2013).</li><li>Okumura, 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=The+ROCK+Inhibitor+Eye+Drop+Accelerates+Corneal+Endothelium+Wound+Healing.">The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing.</a> Invest Ophthalmol Vis Sci. 54 (4), 2493-2502 (2013).</li><li>Mannermaa, E., Vellonen, K. S., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transport+in+corneal+epithelium+and+blood-retina+barrier:+emerging+role+of+transporters+in+ocular+pharmacokinetics.">Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics.</a> Adv Drug Deliv Rev. 58 (11), 1136-1163 (2006).</li></ol>

The authors have no competing financial interests to declare.

CECs ידוע נטייתן לעבור EnMT במהלך התפשטות תאים. כדי לפתח אסטרטגיות דיכוי תהליך EnMT למטרות טיפוליות, הבנה מעמיקה של מנגנון EnMT הכרחית. תיארנו 2 דגמים לחקור EnMT, כלומר CEC שור במודל תרבות במבחנה דגם cryoinjury האנדותל עכברוש הקרנית. התוצאות שלנו הוכיחו את תהליך EnMT בשני המוד?...

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table><tbody><tr><td>trypsin</td><td>ThermoFisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium and Ham&#039;s F12 medium</td><td>ThermoFisher Scientific</td><td>11330</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher Scientific</td><td>26140-079</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>human epidermal growth factor</td><td>ThermoFisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>insulin, transferrin, selenium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>cholera toxin</td><td>Sigma</td><td>C8052-1MG</td><td></td></tr><tr><td>gentamicin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>amphotericin B</td><td>ThermoFisher Scientific</td><td>15290-026</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>111219</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>marimastat</td><td>Sigma</td><td>M2699-25MG</td><td></td></tr><tr><td>anti-active beta-catenin antibody</td><td>Millpore</td><td>05-665</td><td></td></tr><tr><td>anti-snail antibody</td><td>Santa cruz</td><td>sc28199</td><td></td></tr><tr><td>anti-slug antibody</td><td>Santa cruz</td><td>sc15391</td><td></td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>for staining of ABC of bovine CECs</td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11003</td><td>for staining of ABC of rat corneal endothelium</td></tr><tr><td>goat anti-rabbit IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11008</td><td>for staining of snail and slug of bovine CECs</td></tr><tr><td>antibody diluent</td><td>Genemed Biotechnologies</td><td>10-0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>laser scanning confocal microscope</td><td>ZEISS</td><td>LSM510</td><td></td></tr><tr><td>xylazine&amp;nbsp;</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>tiletamine plus zolazepam</td><td>Virbac</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>proparacaine hydrochloride ophthalmic solution</td><td>Alcon</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>0.1% atropine</td><td>Wu-Fu Laboratories Co., Ltd</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>0.3% gentamicin sulfate</td><td>Sinphar Group</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>basic fibroblast growth factor</td><td>ThermoFisher Scientific</td><td>PHG0024</td><td>clinical drug&amp;nbsp;</td></tr></tbody></table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

לאחר בידוד של CECs שור, התאים היו בתרבית במבחנה. איור 1 מציג את התמונות בניגוד שלב של CECs שור. צורת המשושה של התאים בנקודת המפגש ציינה כי התאים לא היו מזוהמים על ידי פיברובלסטים סטרומה הקרניים במהלך בידוד תא. איור 2 מתארים את immunostaini...

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

תרבות עיקרית של תאי אנדותל קרניים שור שמשה לחקור את המנגנון של מעבר אנדותל-mesenchymal הקרני. יתר על כן, מודל cryoinjury האנדותל עכברוש הקרני שמש להפגין מעבר אנדותל-mesenchymal קרני in vivo.

במבחנה מודלים Vivo ללמוד קרנית האנדותל-mesenchymal מעבר

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Research

JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K Views.  Far Eastern Memorial Hospital.  תרבות עיקרית של תאי אנדותל קרניים שור שמשה לחקור את המנגנון של מעבר אנדותל-mesenchymal הקרני. יתר על כן, מודל cryoinjury האנדותל עכברוש הקרני שמש להפגין מעבר אנדותל-mesenchymal קרני in vivo. 

Video: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

הנדסת רקמות הקרנית: במבחנה מודל של אינטראקציות סטרומה-עצב של הקרנית האנושית

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

השתלת קרנית עכברית: דגם לחקר הצורה נפוצה ביותר של Solid השתלות איברים

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Medicine

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

קרנית Micropocket Assay: דגם של אנגיוגנזה בעין העכבר

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

צמיחת הקרנית של האדם והכבשים שכבות של תאי-על ביומטריה

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Bioengineering

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex-Vivo איברים הקרנית מודל תרבות עבור ריפוי פצעים מחקרים

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

מודל של הקרנית של הקרניות הפותל באמצעות לחצני הקרנית מפוצלת

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

פיתוח של מודל פולשני, בסיוע לייזר של הקרנית הפסד של תא אנדותל

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

מודלים של בעלי חיים של Ex Vivo ו-In Vivo לפציעות מכניות וכימיות של אפיתל הקרנית

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

אנליזה מולקולרית של אנדותל-mesenchymal מעבר המושרה על ידי הפיכת גורם גידול-β איתות

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Biology

ב- Vivo השתלת לב לחקר המעבר האנדותלי למזנכימלי

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

מחבר זרקור: מודל השתלת לב עכברוש הטרוטופי בילוד לחקר המעבר האנדותלי למזנכימלי  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

מודל השתלת לב חולדה הטרוטופי בילוד לחקר המעבר מאנדותל למזנכימלי

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...