Name: a rabbit model of durable transgene expression in jugular vein to common carotid artery interposition grafts
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Description: Watch this Scientific Journal Video about A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts at JoVE.com

כל הפרוטוקולים בעלי חיים ולימודי אושרו על ידי אוניברסיטת וושינגטון למשרד של צער.
1. מבצע קדם (עבור כל ניתוחים)
<ol><li>עזים ומתנגד הארנבת עם 30 מ"ג/ק"ג קטמין ו 1.5 mg/kg חריגות השירותים הווטרינריים בזריקה תוך שרירית (IM) בשרירים paraspinous. הערה: מזון ומים הם לא מוגבל לפני הניתוח.</li>
	<li>בזמ?...

<ol>
	<li>Libby, P., Bornfeldt, K. E., Tall, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis:+Successes,+Surprises,+and+Future+Challenges.">Atherosclerosis: Successes, Surprises, and Future Challenges.</a> Circ Res. 118, 531-534 (2016).</li><li>Fowkes, F. G., 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=Comparison+of+global+estimates+of+prevalence+and+risk+factors+for+peripheral+artery+disease+in+2000+and+2010:+a+systematic+review+and+analysis.">Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis.</a> Lancet. 382, 1329-1340 (2013).</li><li>Mohr, F. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+saphenous+vein+graft+patency.">Understanding saphenous vein graft patency.</a> Circulation. 124 (3), 273-275 (2011).</li><li>Owens, C. D., Ho, K. J., Conte, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+extremity+vein+graft+failure:+a+translational+approach.">Lower extremity vein graft failure: a translational approach.</a> Vasc Med. 13, 63-74 (2008).</li><li>Robertson, K. E., McDonald, R. A., Oldroyd, K. G., Nicklin, S. A., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+coronary+in-stent+restenosis+and+vein+graft+failure:+does+vascular+gene+therapy+have+a+role.">Prevention of coronary in-stent restenosis and vein graft failure: does vascular gene therapy have a role.</a> Pharmacol Ther. 136, 23-34 (2012).</li><li>Yla-Herttuala, S., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Gene+Therapy:+Past,+Present,+and+Future.">Cardiovascular Gene Therapy: Past, Present, and Future.</a> Mol Ther. 25, 1095-1106 (2017).</li><li>Schwartz, L. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+adenoviral+gene+transfer+to+early+venous+bypass+grafts:+comparison+with+native+vessels.">Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.</a> Cardiovasc Res. 35, 505-513 (1997).</li><li>Flynn, R., 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=Expression+of+apolipoprotein+A-I+in+rabbit+carotid+endothelium+protects+against+atherosclerosis.">Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis.</a> Mol Ther. 19, 1833-1841 (2011).</li><li>George, S. 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השלבים הקריטיים של פרוטוקול זה כוללים את הניהול של הרדמה, נוגד קרישה, ניתוח מניפולציה של הווריד של עורק/הושתל, בגרימת מדידות של הווריד של המושתל. ניהול נכון של הרדמה היא קריטית במודל זה מרובים הישרדות ניתוח הכולל שתי פעולות ארוכות יחסית (בדרך כלל 3-3.5 h על הווריד הדו-צדדיים הרכבה ו- 1.5-2.5 h עבור ...

טרשת העורקים היא מחלה דלקתית כרונית שבה הצטברות שומנים בדם ודלקת בדופן כלי הדם להוביל היצרות של כלי השיט לומן, התקפי לב, שבץ, אובדן גפיים1,2. התערבויות מלעורית (למשל., אנגיופלסטיקה ולא סטנט) וטיפול רפואי (למשל., סטטינים antiplatelet סוכנים) טיפולים שימושי עבור טרשת עורקים; עם זאת, לעתים קרובות הם יעילים בטיפול חמור מחלה חסימתית שני ב circulations כלילית ואת היקפי. מעקף השתלת עור, באמצעות מקטעים וריד autogenous, נשאר הליך נפוץ לטיפול בחולים עם חמור, מפוזר כלילית והיקפי מחלת כלי דם3,4. עם זאת, הווריד שתלי להציב שני עוצר ויש circulations היקפי המסכן המחירים patency לטווח ארוך. במחזור כלילית, כ- 10-20% של וריד שתלי הם occluded-1 שנה ו- 50% הם occluded 10 שנים5,6. במחזור היקפיים, וריד שתל כשל תעריפי 30-50%-5 שנים7.
ריפוי גנטי היא גישה אטרקטיבית למניעת כשל שתל הווריד. כי זה יכול לספק מוצר גנים טיפוליים בדיוק באתר של המחלה. בהתאם לכך, מחקרים פרה רבים בדקו וריד שתל ג'ין טיפול8,9. עם זאת, בעיקרו של דבר כל מחקרים אלו בחנו את היעילות בגיל מוקדם זמן נקודות (2-12 שבועות)10,11,12,13,14,15, 16 , 17. אנו מודעים אין ראיות כי התערבויות ריפוי גנטי יכול לספק עמיד (שנים) הגנה מפני המנוח כשל שתל הווריד הנובעת בדרך כלל היפרפלזיה של טרשת עורקים neointimal4. פיתחנו שיטה מאפשרת ביטוי transgene עמיד בורידים המושתל, ובכך מאפשר בדיקה של ריפוי גנטי התערבויות בנקודות זמן מאוחר, כמו גם מוקדם. כדי להשיג את הביטוי transgene עמיד, השיטה משלבת HDAd וקטורים אסטרטגיה התמרה חושית מושהית. וקטורים HDAd לספק ביטוי transgene ממושך בשל העדר גנים ויראלי, מניעת זיהוי (ולא דחייה) של תאים transduced על ידי המערכת החיסונית18,19,20, 21. מושהית התמרה חושית (שבוצעו 28 ימים לאחר הצבת שתל) מונע איבוד תאים transduced במהלך תהליך arterialization המתרחש מוקדם לאחר grafting22.
שיטות אחרות להשיג transgene טיפולית ביטוי בקיר שתל הווריד להסתמך על התמרה חושית של השתל וריד בזמנו של שתל השמה10,11,12,15,16 ,17. כאשר נמדד באופן סדרתי, transgene הביטוי תוך שימוש בגישה זו יורדים במהירות לאחר התמרה חושית22,23. בהתאם לכך, מחקרים באמצעות גישה זו לא בחנו את היעילות מעבר 12 שבועות לאחר וריד משתילים, עם ביותר לא הערכת יעילותם מעבר 4 שבועות. לעומת זאת, השיטה שלנו משיגה שתל הווריד ביטוי transgene stably נמשך לפחות 24 שבועות מבוסס על מחקרים דומים שבוצעו בסביר-העורקים ממשיך22,יותר24. אנחנו מודעים ללא אחרים וריד שתל ג'ין טיפול התערבות שמקבל ביטוי transgene יציבה משך זמן זה.
השתמשנו דגם ארנב לפתח את השיטה שלנו. אחרים השתמשו מכרסמים, ארנבות, או חיות גדולות כדי לבדוק את הווריד להשתיל ג'ין טיפול10,11,12,15,16,17,25, 26. לעומת מודלים מכרסמים, ארנבונים יקרים יותר, והם כפופים דרישות רגולטוריות מחמירים יותר. עם זאת, בהשוואה חיות גדולות (למשל., חזירים, כלבים), ארנבים הם הרבה פחות יקרים לרכוש בית הרבה יותר קל להתמודד. יתר על כן, הארנב כלי דומה כלי האנושי מבחינה פיזיולוגית27, הם מספיק גדולים כי הם יכולים לשמש לבדיקת התערבויות מלעורית28,29, הם מספקים מספיק רקמה את זה נקודות קצה מרובות (למשל., היסטולוגיה, חלבון, RNA) יכולים להיבדק באמצעות כלי דם בודדת הדגימה22,30. בנוסף, כאשר הארנבים עם שתלי העורק ניזונים מדיאטה עתירת שומן, הם מפתחים וריד שתל טרשת עורקים31,32, אשר היא גורם שכיח של עורקים מעקף לווריד שתל כשל4,5 . אלה שתלי העורק ארנב טרשת עורקים יכול לשמש מצע לבדיקת טיפול גנטי התערבויות נמסרות בשיטה זו. פרוטוקול שסופקו יכול לעזור חוקרים לשלוט טכנית הכישורים הדרושים כדי להשיג transgene עמיד ביטוי שתלי העורק ארנב.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Disposables</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:207px;">2x for vein graft surgery; 2x for gene transfer&#160; surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">1 mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:207px;">2x for vein graft surgery, 5x for gene transfer surgery</td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:215px;">19G needle</td>
			<td rowspan="2">Becton Dickinson</td>
			<td rowspan="2">305187</td>
			<td rowspan="2" style="width:207px;">Gene transfer surgery</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">20 mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Catheters, 24Ga x 3/4&#8221;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:207px;">Gene transfer surgery and for 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Gauze 4&#8221; x 4&#8221;</td>
			<td>Dynarex</td>
			<td style="width:119px;">3242</td>
			<td style="width:207px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8703P</td>
			<td style="width:207px;">Vein graft surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:207px;">Gene transfer surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:207px;">For the placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>5098916106</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Lidocaine, 20 mg/mL</td>
			<td>Pfizer</td>
			<td>409427702</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Marcaine 0.5%</td>
			<td>Pfizer</td>
			<td>409161050</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:207px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Buprenorphine HCl, 0.3 mg/mL</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Papaverine (3.5 mg/ml)</td>
			<td>American Reagent Inc.</td>
			<td>NDC 0517-4002-25</td>
			<td style="width:207px;">Diluted from 30mg/mL stock; Use 1 mL maximum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Fentanyl patch, 25 mcg/h</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">&#160;Viral vector</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surgical Instruments</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7900</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6828</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:119px;">8-14-IMC</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6412</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5943</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6514</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:119px;"></td>
			<td style="width:207px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6496</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:119px;">1001-81M</td>
			<td style="width:207px;">5 cm length</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:215px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5210</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Dumont #3 forceps Inox tip size .17 &#215; .10 mm</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5042</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8 mm tip</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5280</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7110</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7111</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7117</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7305</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-8162</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">11092-12</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">11006-12</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:119px;">HPAC-1; R-F11</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Bair hugger warming unit</td>
			<td>3M</td>
			<td style="width:119px;">Model 505</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:119px;">Vet IV 2.2</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:119px;">Surgivet Advisor</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:119px;">MV-9</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Surgical microscope</td>
			<td style="width:167px;">D.F. Vasconcellos</td>
			<td style="width:119px;">M900</td>
			<td style="width:207px;">25X magnification for vein graft surgery; 16X magnification for gene transfer surgery</td>
		</tr>
	</tbody>
</table>

a rabbit model of durable transgene expression in jugular vein to common carotid artery interposition grafts

ניתוח מעקפים שתל הווריד הוא טיפול נפוץ מחלת עורקים ריטלין; עם זאת, הצלחה לטווח ארוך הוא מוגבל על ידי שתל כשל פקקת, היפרפלזיה intimal וכתוצאה טרשת עורקים. מטרת מאמר זה היא להדגים שיטה עבור הצבת interposition ורידים דו-צדדית שתלי ארנב, ואז transducing את השתלים עם וקטור העברת גנים שמקבל ביטוי transgene עמיד. השיטה מאפשרת החקירה של התפקיד הביולוגי של הגנים שלהם מוצרי חלבון הומאוסטזיס שתל הווריד נורמלי. זה גם מאפשר בדיקה של transgenes על הפעילות שעלולים למנוע כשל שתל הווריד, למשל., אם הביטוי של transgene ומונע גידול neointimal, מפחית דלקת כלי הדם או מפחית טרשת עורקים בארנבים האכיל עם דיאטה עתירת שומן. במהלך ניתוח הישרדות הראשונית, פילוחי ימינה ושמאלה חיצוני ומוחלף טוחנות, להציב מבקיעים כמו הפוכה הקצה לצד נפוצות העורק interposition שתלים. במהלך ניתוח הישרדות נוסף, ביצע 28 ימים לאחר מכן, כל אחד השתלים הוא מבודד זרימת הדם עם תפסים, לומן (דרך arteriotomy) מלאים פתרון שמכיל וקטור (HDAd) adenoviral תלויי-עוזר. לאחר דגירה 20-מין, הפתרון וקטור הוא aspirated arteriotomy יתוקן, זרימה משוחזר. הורידים נקצרים בנקודות זמן מוכתב על ידי בודדים בפרוטוקולים ניסיוניים. העיכוב 28 יום בין הצבת שתל את התמרה חושית יש צורך להבטיח את העיבוד של השתל וריד אל הדם העורקי. הסתגלות זו מונע אובדן מהיר של הביטוי transgene המתרחשת אצל שתלי העורק transduced לפני או מיד לאחר הרכבה. השיטה היא ייחודית ביכולתה כדי להשיג את הביטוי transgene עמיד, יציב בורידים המושתל. לעומת דגמים אחרים של שתל הווריד חיה גדולה, ארנבים יש יתרונות הטיפול זולה וקלה. לעומת מודלים שתל הווריד מכרסמים, ארנבונים יש כלי דם גדולים יותר, קל יותר לתמרן, המספקים שפע רקמות לצורך ניתוח.

יש לאמת את מיומנות טכנית של מפעיל חדש לפני האופרטור באפשרותך להשתמש בשיטה זו כדי ליצור נתוני הניסוי. אבן הדרך הראשונה זה מפעיל חדש חייב להשיג הוא patency שתל הווריד עקבי אחרי הניתוח הראשוני הרכבה וריד והן את הניתוח מושהית-התמרה חושית עוקבות. Patency מעל 90% לאחר כל אחד הניתוחים רצ...

Watch this Scientific Journal Video about A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts at JoVE.com

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

דגם ארנב של ביטוי Transgene עמיד ומוחלף כדי שתלי Interposition נפוצים של העורק הראשי

שיטה זו מתארת את המיקום של שתלי העורק interposition בארנבים, את התמרה חושית של השתלים, ההישג של הביטוי transgene עמיד. דבר זה מאפשר החקירה של תפקידים פיזיולוגיים ופתולוגיים של transgenes ומוצרים שלהם חלבון בורידים המושתל, ובדיקות של ג'ין טיפולים למחלות שתל הווריד.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

This method can answer key questions about the pathogenesis of vein graft failure. For example, it could identify mediators of graft failure and test strategies aimed at preventing graft failure. The main advantage of this technique is its ability to achieve durable, stable transgene expression in the vein graft endothelium.After anesthetizing a rabbit according to the text protocol, shave the animal from the sternal notch to the edge of the mandible. Upon completion of the surgical setup use electrocautery to cut the skin along the midline between the sternal notch and the mandible, approximately seven to nine centimeters in length. Then attach towel clamps to hold open the incision.Bluntly dissect under the fascia along the entire midline. Then use electrocautery to cut through the dissected fascial layer along the midline. Next, beginning on the right side, dissect between the sternal hyoid muscle overlying the trachea and the sternoephalic muscle to expose the common carotid artery.Then carefully dissect a four to five centimeter segment of the common carotid artery and its larger branches free from surrounding tissues. Use 5-0 silk sutures to ligate the larger branches of the common carotid artery before cutting each branch distal to the suture. After repeating the dissection on the left common carotid, use tissue holding forceps to temporarily close the subcutaneous tissue layer.Then dissect the superficial fascia from the skin on the right side until the external jugular vein is exposed. Dissect a four to five centimeter segment of the external jugular vein and free its branches from the surrounding tissue. Use 5-0 silk sutures to ligate the branches, then cut the branches.Next ligate the isolated vein at both ends, cut the cranial end, and flush the lumen with heparinized saline. To carry out vein grafting, use a vascular clip to clamp the right common carotid artery at the cranial end of the isolated segment. Then place a vascular clip on the caudal end.Now perform a caudal arteriotomy just cranial to the caudal clamp equal in length to the diameter of the cranial end of the vein segment. Then insert an IV catheter connected to a syringe of heparinized normal saline through the caudal arteriotomy and flush the carotid artery lumen. The fluid will exit via the arteriotomy.Returning to the jugular vein, excise the vein and lay it next to the carotic artery. Using single stitches of 7-0 polypropylene suture the caudal end of the caudal arteriotomy to the cranial end of the vein segment. Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the caudal arteriotomy to the cranial end of the vein segment.Place a third stitch at a point equidistant from the first two stitches on the lateral side of the anastomosis. Then place a fourth stitch on the medial side of the anastomosis equidistant from the first two stitches and across from the third stitch. Place four additional stitches between each of stitches one through four.Perform the cranial arteriotomy on the caudal side of the distal clip. The distance between the cranial end of the cranial arteriotomy and the caudal end of the caudal arteriotomy should be the same as the length of the vein segment. The length of the cranial arteriotomy is equal to the diameter of the caudal end of the vein segment.With the vein extended cranially from the anastomosis along the carotid, ensure the vein is not twisted. Then use heparinized normal saline to flush the carotid artery and vein via the cranial arteriotomy. Next place a first stitch to suture the caudal end of the cranial arteriotomy to the caudal end of the vein segment.Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the cranial arteriotomy to the caudal end of the vein segment. Complete the anastomosis as demonstrated earlier in the video. Temporarily release the cranial clip to allow the vein graft to fill with blood and apply gentle pressure with dry gauze to stop any bleeding.Repeat this step until the anastomosis no longer bleeds. Then remove both the cranial and caudal clips. Use gauze to stop any additional bleeding at the anastomoses.Use a 3-0 silk suture to ligate both ends of the common carotid artery segment between the anastomoses. Then divide the carotid halfway between the ligatures. Brisk pulsations should be seen in the vein graft.Repeat the vein graft placement on the left side. With a 5-0 PGA suture, close the subcutaneous tissue with a continuous pattern. Then use a 3-0 PGA suture to close the skin with a intradermal pattern and bury the knots on both ends.Carry out post operative care and transcutaneous ultrasound according to the text protocol. To perform gene transfer surgery, carefully isolate the right vein graft including 1.5 to two centimeters of the carotid artery adjacent to the graft on both the cranial and caudal sides. Repeat the dissection on the left side in the same manner.Use normal saline to fill the surgical wound cavity on the right side to enable the transmission of sound waves. Connect a two millimeter perivascular flow probe to a volume flow meter and immerse the probe in the saline in the wound cavity. Set the flow rate to zero.Place the flow probe around the carotid artery either caudal or cranial to the vein graft. Then use an electronic data acquisition system to record data from the flow probe. After injecting heparin into the rabbit via the the IV catheter and flushing it with saline, use a vascular clip to clamp the common carotid artery at the cranial end of the vein graft.And then apply a second clip to the carotid approximately 10 millimeters caudal to the caudal anastomosis to leave room for the arteriotomy. Then place two silk ties loosely around the carotid artery just caudal to the vein graft. Next using a previously prepared bent 21 gauge needle, puncture the carotid caudal to the graft and just cranial to the caudal vascular clip.Advance the needle into the lumen back and forth twice to ensure that the lumen is clear. Then carefully withdraw the needle. After preparing a syringe and IV catheter with DMEM according to the text protocol, insert it into the common carotid arteriotomy up to the bend point and wash the vein graft lumen by filling it with 0.5 milliliters of DMEM two times.Remove the DMEM from the graft by using a gloved finger to lightly press at the cranial end of the graft. Then carefully slide the finger along the graft toward the caudal end of the graft to flush the luminal contents out via the arteriotomy. Remove the DMEM syringe from the catheter leaving the catheter in the vessel.Connect the syringe containing the virus solution making sure that no air enters the catheter. Move the loosely knotted silk ties down the artery until they are around the catheter tip but do not tighten them. Infuse 0.03 milliliters of virus solution to push the remaining DMEM out of the catheter.Then using a finger, remove all fluid from the vein graft lumen pressing gently from cranial to caudal. Tighten the two ties around the artery and catheter tip to seal the lumen. Then infuse the virus solution until the vein is distended.Gently lay the syringe down on a nest of gauze. After 20 minutes, use an empty syringe to gently aspirate the virus containing solution from the graft until the vessel collapses. Remove the syringe leaving the catheter in place.Then cut and untie the silk sutures and withdraw the catheter from the vessel. Now use 7-0 polypropylene suture in an X pattern to close the arteriotomy. Then gently pull the suture ends and tie them together with two square knots.After restoring blood flow in the right vein graft as described in the text protocol, repeat the virus infusion on the left side. To harvest the transduced grafts, dissect free the vein grafts and common carotid arteries and make flow measurements on both the left and right sides as demonstrated earlier. Use 3-0 silk suture to ligate the right common carotid artery cranial to the graft.Then use 3-0 silk suture to ligate the carotid caudal to the graft. Excise the right vein graft by dividing the adjacent carotid artery between each of the ligations and the adjacent anastomosis. Then remove the vein graft from the rabbit and use saline to flush the lumen.The first milestone that a new operator must achieve is consistent vein graft patency after both the initial vein grafting surgery and the subsequent delayed transduction surgery. When a vein graft is patent, the ultrasound exam will reveal a large blood vessel with brisk caudal to cranial blood flow. The grafts were harvested three days after the transduction, cut into sections and stained with X-gal.En-face imaging of the luminal surfaces showed vein grafts with efficient transduction and poor transduction. As shown in this plot, the levels of beta galactosidase mRNA in vein grafts transduced by the new operator, were not significantly different from the levels of beta galactosidase mRNA in vein grafts transduced by an experienced operator. When performing this protocol, it is important to remember to reverse the vein graft cranial to caudal before contracting the anastomosis.This is necessary to avoid obstruction of blood flow by venous bolus. Once mastered these surgeries can be completed in about three hours for the vein grafting procedure, two hours for the vein graft gene transfer procedure and 45 minutes for the harvest procedure. Don't forget that because this protocol requires working with viral vectors and sharp objects, proper biosafety and sharps procedures must be followed.

Research

JoVE Journal

Medicine

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

אימון טכניקה מתוחכמת מייקרו: התערבותם של שתלי וריד צוואר חיצוניים בעורק התרדמה המשותף בחולדות

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

ספיגה דו צדדי משותף עורק תרדמה כגירוי נפשית מראש מספיק כדי לגרום סובלנות איסכמי מוקדמת לאיסכמיה מוחית מיקוד

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

למבוגרים עכבר ורידי יתר לחץ דם דגם: עורק תרדמה משותף לחיצוני הצוואר וריד השקה.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

צנתור של עורק התרדמה וצוואר הווריד לבצע צעדים המודינמית, חליטות ודגימת דם במודל עכברוש מודע

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

חליטות עורק הראשי לpharmacokinetic וpharmacodynamic ניתוח של taxanes בעכברים

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Performing מוחות תיכונים דיסטלי קבוע עם ספיגה עורקת Common הראש אצל חולדות בנים לחקר קורטיקלי איסכמיה עם נכות קבועה זה

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

וריד ביון דגם: מודל מתאים ללמוד patency מעקף

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

אין ויוו העברה גנטית כדי אנדותל העורק נפוצות הארנב

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

שלב ניגודיות תהודה מגנטית בעורק החולדה נפוצים

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

פקקת הברזל ותחמוצת כלוריד-induced הכלבי העורק הראשי: דגם בעל חיים גדול של פגיעה בכלי הדם

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...