Name: partial heterotopic hindlimb transplantation model in rats
Uploaded: 2021-06-09T14:45:00
Description: Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

وقد دعم هذا العمل مكتب مساعد وزير الدفاع للشؤون الصحية من خلال برنامج البحوث الطبية الموجه من الكونغرس بموجب الجائزة رقم W81XWH-17-1-0680. الآراء والتفسيرات والاستنتاجات والتوصيات هي آراء المؤلفين ولا تؤيدها بالضرورة وزارة الدفاع.

وقد تلقت جميع الحيوانات رعاية إنسانية وفقا لدليل المعاهد القومية للصحة لرعاية واستخدام المختبر. وافقت اللجنة المؤسسية لرعاية الحيوانات واستخدامها (IACUC-protocol 2017N000184) ومكتب مراجعة رعاية الحيوانات واستخدامها (ACURO) على جميع بروتوكولات الحيوانات. استخدمت الفئران لويس الذكور الأصيلة (250-400 غرام) ...

<ol>
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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=A+modified+model+of+hindlimb+osteomyocutaneous+flap+for+the+study+of+tolerance+to+composite+tissue+allografts.">A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.</a> Microsurgery. 27 (7), 630-636 (2007).</li><li>Arav, A., Friedman, O., Natan, Y., Gur, E., Shani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hindlimb+cryopreservation+and+transplantation:+a+step+toward+"organ+banking".">Rat hindlimb cryopreservation and transplantation: a step toward "organ banking".</a> American Journal of Transplantation. 17 (11), 2820-2828 (2017).</li><li>Gok, E., 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=Development+of+an+ex-situ+limb+perfusion+system+for+a+rodent+model.">Development of an ex-situ limb perfusion system for a rodent model.</a> ASAIO Journal. 65 (2), 167-172 (2019).</li><li>Gok, E., Rojas-Pena, A., Bartlett, R. H., Ozer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+skeletal+muscle+metabolomic+changes+associated+with+static+cold+storage.">Rodent skeletal muscle metabolomic changes associated with static cold storage.</a> Transplantation Proceedings. 51 (3), 979-986 (2019).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Research. Part C, Embryo Today. 96 (1), 39-50 (2012).</li><li>Fleissig, Y., 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=Modified+heterotopic+hindlimb+osteomyocutaneous+flap+model+in+the+rat+for+translational+vascularized+composite+allotransplantation+research.">Modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research.</a> Journal of Visualized Experiments: JoVE. (146), e59458 (2019).</li><li>Ulusal, A. E., Ulusal, B. G., Hung, L. M., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+hindlimb+allotransplantation+in+rats:+an+alternative+model+for+immunological+research+in+composite-tissue+allotransplantation.">Heterotopic hindlimb allotransplantation in rats: an alternative model for immunological research in composite-tissue allotransplantation.</a> Microsurgery. 25 (5), 410-414 (2005).</li><li>Jang, Y., Park, Y. E., Yun, C. W., Kim, D. H., Chung, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vest-collar+as+a+rodent+collar+to+prevent+licking+and+scratching+during+experiments.">The vest-collar as a rodent collar to prevent licking and scratching during experiments.</a> Lab Anim. 50 (4), 296-304 (2016).</li><li>Kern, B., 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=A+novel+rodent+orthotopic+forelimb+transplantation+model+that+allows+for+reliable+assessment+of+functional+recovery+resulting+from+nerve+regeneration.">A novel rodent orthotopic forelimb transplantation model that allows for reliable assessment of functional recovery resulting from nerve regeneration.</a> American Journal of Transplantation. 17 (3), 622-634 (2017).</li><li>Perez-Abadia, 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=Low-dose+immunosuppression+in+a+rat+hind-limb+transplantation+model.">Low-dose immunosuppression in a rat hind-limb transplantation model.</a> Transplant International. 16 (12), 835-842 (2003).</li><li>Sucher, 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> Journal of Visualized Experiments. (41), e2022 (2010).</li><li>Fleissig, Y. Y., Beare, J. E., LeBlanc, A. J., Kaufman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+rat+hind+limb+transplant+as+an+experimental+model+of+vascularized+composite+allotransplantation:+Approaches+and+advantages.">Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.</a> SAGE Open Medicine. 8, 2050312120968721 (2020).</li><li>Lindboe, C. F., Presthus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+denervation,+immobilization+and+cachexia+on+fibre+size+in+the+anterior+tibial+muscle+of+the+rat.">Effects of denervation, immobilization and cachexia on fibre size in the anterior tibial muscle of the rat.</a> Acta Neuropathologica. 66 (1), 42-51 (1985).</li><li>Nazzal, J. A., Johnson, T. S., Gordon, C. R., Randolph, M. A., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+limb+allotransplantation+model+to+study+skin+rejection+in+the+rat.">Heterotopic limb allotransplantation model to study skin rejection in the rat.</a> Microsurgery. 24 (6), 448-453 (2004).</li></ol>

وقد وصفت نماذج زرع الأطراف التقويمية في القوارض في الأدب15،16،17؛ ومع ذلك ، فإنها تتطلب إصلاح الأعصاب ، وإعادة ربط العضلات ، و التمثيل العظمي المثالي لعظم الفخذ ، والتي يمكن أن تكون خطوة صعبة للغاية. وترتبط هذه النماذج أيضا مع ارتفاع معدل ال...

على مدى العقدين الماضيين، تطورت VCA كعلاج ثوري للمرضى الذين يعانون من تشوه شديد بما في ذلك الوجه1،بتر الأطراف العليا2،القضيب3،وغيرها من عيوب الأنسجة المعقدة4،5. ومع ذلك، فإن عواقب كبت المناعة مدى الحياة لا تزال تعيق تطبيق أوسع لهذه العمليات الجراحية الترميمية المعقدة. البحوث الأساسية أمر بالغ الأهمية لتحسين استراتيجيات مكافحة الرفض. زيادة الوقت الحفاظ على VCA أمر ضروري أيضا لتحسين الخدمات اللوجستية زرع وزيادة تجمع المانحين (كما يجب على المتبرعين VCA تلبية معايير أكثر من المتبرعين بالأعضاء الصلبة، بما في ذلك لون البشرة، وحجم التشريح، والجنس). في هذا السياق ، تستخدم عمليات زرع أطراف الفئران على نطاق واسع في الدراسات حول الرفض المناعي ل allografts6،7، بروتوكولات تحريض التسامح الجديدة8، ودراسات الحفظ9،10،11. وبالتالي، فإن نماذج VCA هذه هي عنصر أساسي لإتقان أبحاث VCA التحويلية.
وقد وصفت اللوحات العظمية في الأدب كنماذج موثوقة لدراسة VCA في الفئران8،12،13،14. على الرغم من أن عمليات زرع الأطراف الكاملة التقويمية تسمح بتقييم طويل الأجل لوظيفة الكسب غير المشروع ، إلا أنه إجراء يستغرق وقتا طويلا مرتبطا بارتفاع معدلات الاعتلال والوفيات بعد الجراحة14. وعلى النقيض من ذلك، فإن نماذج زرع الأطراف غير النظائرية غير وظيفية، ولكنها تمكن من إجراء دراسات قابلة للاستنساخ على VCA. يمكن توقع نتائج ما بعد الجراحة بشكل موثوق قبل بدء دراسة زرع الفئران VCA. تفيد هذه الدراسة عن نموذج جزئي لزرع اللوحات العظمية النظيرية في الفئران يتضمن نتائج ومضاعفات محتملة متكررة يمكن أن تنشأ داخل الجراحة وبعد الجراحة خلال فترة متابعة مدتها ثلاثة أسابيع.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 GA angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture Black monofilament non absorbable suture</td>
			<td>Ethicon</td>
			<td>1667</td>
			<td>Used to suture the E-collar to the back of the neck</td>
		</tr>
		<tr>
			<td>4-0 suture Coated Vicryl Plus Antibacterial</td>
			<td>Ethicon</td>
			<td>VCP496</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polyamide Monofilament USP 10-0, 4 mm 3/8c</td>
			<td>AROSurgical</td>
			<td>T04A10N07-13</td>
			<td>Used to perform the microvascular anastomoses</td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern</td>
			<td>ASSI</td>
			<td>ASSI.SAS15RVL</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Micro Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>18040-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Elizabethan collar</td>
			<td>Braintree Scientific</td>
			<td>EC-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2)</td>
			<td>ASSI</td>
			<td>ASSI.JFL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>Fine science tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Lone Star Elastic Stays</td>
			<td>CooperSurgical</td>
			<td>3314-8G</td>
			<td>Used to retract the inguinal ligament for femoral vessels dissection</td>
		</tr>
		<tr>
			<td>Lone Star Self-Retaining Retractors</td>
			<td>CooperSurgical</td>
			<td>3301G</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Mosquito Hemostats</td>
			<td>Fine science tools (FST)</td>
			<td>13010-12</td>
			<td>Used to retract the inguinal fat pad distally</td>
		</tr>
		<tr>
			<td>Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock</td>
			<td>ASSI</td>
			<td>ASSI.B1582</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c</td>
			<td>Ethilon</td>
			<td>2808G</td>
			<td>Used to ligate collateral branches on the femoral vessels</td>
		</tr>
		<tr>
			<td>Offset Bone Nippers</td>
			<td>Fine science tools (FST)</td>
			<td>16101-10</td>
			<td></td>
		</tr>
		<tr>
			<td>S&#38;T Vascular Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>00398-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Walton scissors</td>
			<td>Fine science tools (FST)</td>
			<td>14077-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial heterotopic hindlimb transplantation model in rats

تمثل عمليات زراعة الأوعية الدموية المركبة (VCA) خيار إعادة الإعمار الأكثر تقدما للمرضى الذين لا يعانون من إمكانيات جراحية تلقائية بعد عيب معقد في الأنسجة. لقد غيرت عمليات زرع الوجه واليد حياة المرضى المشوهين، مما منحهم جهازا اجتماعيا جماليا وعمليا جديدا. على الرغم من النتائج الواعدة ، لا يزال أداء VCA ضعيفا بسبب الأمراض المرضية المثبطة للمناعة مدى الحياة والمضاعفات المعدية. الجرذ هو نموذج حيواني مثالي للدراسات الحية التي تحقق في المسارات المناعية وآليات رفض الكسب غير المشروع. وتستخدم الفئران أيضا على نطاق واسع في تقنيات جديدة للحفاظ على الكسب غير المشروع الأنسجة المركبة، بما في ذلك التغلغل ودراسات الحفظ بالتبريد. يجب أن تكون النماذج المستخدمة ل VCA في الفئران قابلة للاستنساخ وموثوقة وفعالة مع انخفاض المراضة والوفيات بعد الجراحة. إجراءات زرع الأطراف النظائرية تفي بهذه المعايير وهي أسهل في الأداء من عمليات زرع الأطراف التقويمية. يتطلب اتقان نماذج جراحة القوارض الدقيقة خبرة قوية في الجراحة المجهرية ورعاية الحيوانات. هنا يتم الإبلاغ عن نموذج موثوق به وقابل للاستنساخ لزرع رفرف العظام غير النظائري الجزئي في الفئران، والنتائج بعد الجراحة، ووسائل الوقاية من المضاعفات المحتملة.

في هذه الدراسة التي أجريت على مشغل واحد، تم إجراء 30 عملية زرع أطراف غير نظائرية سينجينية. تم تعريف النجاح في اليوم 21 بعد الجراحة على أنه عدم وجود فشل VCA أو مضاعفات تتطلب القتل الرحيم. يتم تمثيل التطور الطبيعي للكسب غير المشروع في الشكل 3. وكان متوسط المدة لشراء أطراف جزئية وت?...

Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Partial Heterotopic Hindlimb Transplantation Model in Rats

تقدم هذه الورقة بروتوكولا جزئيا لزرع اللوحات العظمية النظيرية في الفئران ونتائجه المحتملة في المتابعة في منتصف المدة.

نموذج زرع الخلايا الهنترونظيرية الجزئية في الفئران

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

نحن نقدم نموذجا جراحيا موثوقا وقابلا للاستنساخ لزرع الأطراف الخلفية غير النظيرية في الفئران. هذه التقنية مثالية للدراسات قصيرة الأجل حول الحفظ أو الرفض المناعي في VCA. سيسمح الوصف خطوة بخطوة للمتدربين بإتقان الجراحة بسرعة.للبدء، قم بعمل شق محيطي على الجلد فوق الكاحل في الثلث القاصد من الساق. هيكل عظمي وكى الشريان السابهين والفرع النهائي للشريان البوبلايتال باستخدام ملقط ثنائي القطب. الكي وقطع gastrocnemius، سوليوس، الساق الأمامي، والعضلة ذات الرأسين femoris العضلات حتى يتم الكشف عن العظام الظنبوبية.جعل شق 2.5 سم في تجعد الإربية الحق وتشريح لوحة الدهون الأربية. استخدام الأسماك هوك retractor لفهم الرباط الأربي. عقد لوحة الدهون الأربية distally مع ملقط لقط وتراجع لوحة الدهون لفضح الأوعية الفخذية.تشريح الأوعية الفخذية، وتفرد فروع ميرفي، وligate مع العلاقات النايلون 8/0. Heparinize الجرذ المانحة عن طريق حقن الهيبارين في الوريد الظهر القضيب باستخدام إبرة قياس 27.5. أكمل شق الجلد حول الورك.استخدام ملقط ثنائي القطب لكي العضلة ذات الرأسين femoris وglutes السطحية العضلات، ثم الكي وقطع العصب الوركي في منتصف طول عظم الفخذ. فضح عظم الفخذ بشكل قريب على مستوى قمة الفخذ الخلفية وligate الأوعية الفخذية مع ربطات النايلون على مستوى الرباط الأربي. إجراء عملية استئصال الشرايين على الشريان الفخذي أسفل الرباط بقليل وتمدد لإدخال الأوعية الدموية قياس 24.الكي وقطع العضلات المتبقية تحت باديكل لفضح الجانب الأمامي من عظم الفخذ. استخدام قطع العظام لقطع الساق وكيلا وقطع عظم الفخذ كما distally ممكن. قم بغسل الجزء الخلفي الجزئي مع ملليلترين من محلول الهيبارين المالح للحصول على تدفق وريدي واضح.ضع الأطراف الخلفية على الثلج في شاش معقم حتى نقل الأوعية الدموية الدقيقة. تشريح لوحة الدهون الأربية واتكاء لوحة الدهون الأربية distally لفضح الأوعية الفخذية. استخدام هوك لسحب الرباط الأربي واللقط ملقط لعقد لوحة الدهون الأربية distally.تشريح الأوعية الفخذية، وتفرد فروع ميرفي، وligate مع العلاقات النايلون كما هو موضح. Ligate كلا السفن فوق الأوعية epigastric باستخدام العلاقات النايلون. ضع المشابك التقريبية بشكل تقريبي ، ووسع عدسة السفينة ، وشطفها بمحلول ملحي الهيبارين.إجراء شق على الجناح الأيسر فوق الورك وإنشاء جيب تحت الجلد مع نفق تحت الجلد إلى تجعد الأربية. ضع الجزء القريب من الطرف الجزئي ووسادة الدهون الأربية عبر النفق تحت الجلد لنقل الأوعية الدموية الدقيقة. إجراء anastomoses الوريدية والشريانية باستخدام الغرز النايلون.إزالة المشابك تقريبا على حد سواء ومراقبة إعادة الأوعية الدموية من الطرف. إجراء اختبار الحلب على كل من السفن لتقييم patency من كل التخدير. إجراء شق الجلد الطولي على الجانب الوسيط من الطرف المزروع وإدراج الكسب غير المشروع.إزالة الجلد الزائد من الكسب غير المشروع وإغلاق الجرح مع الغرز منفصلة وخياطة تشغيل باستخدام خياطة قابلة للامتصاص 4/0. خياطة معا منصات الدهون الأربية من الطرف المزروع والمتلقي باستخدام اثنين من الغرز قابلة للامتصاص منفصلة وإغلاق تجعد الأربية بعد الفحص الأخير من الحشرات الأوعية الدقيقة. تم رصد تطور نموذج الأطراف الخلفية غير النظيرية حتى نهاية الدراسة.لوحظت إعادة نمو الشعر خلال الأسبوع الأول بعد الجراحة ، وظهر التراجع الجلدي بعد أسبوعين. يمكن أن يحدث فشل التكسير المركب الوعائي خلال الأسبوع الأول بعد الجراحة بسبب تجلط الأوعية الدموية الدقيقة. كان التجلط الوريدي هو سبب القتل الرحيم المبكر في 20٪ من الحالات، وكلها حدثت قبل اليوم الخامس بعد الجراحة.وبدا الجلد الأزرق وأصبح أكثر قتامة كل يوم. تشويه الذات هو مصدر قلق خطير في الطعوم لا معنى لها وغالبا ما يحدث بين اليوم الثاني والسابع بعد الجراحة. إذا اقتصرت على أقل من ثلث سطح الكسب غير المشروع وتتعلق فقط الجلد، يمكن مناقشة debridement الجراحية وخياطة باستخدام الغرز غير قابلة للامتصاص مع الطبيب البيطري الموظفين.تعتمد الوقاية من تشويه الذات على استخدام طوق إلكتروني مخيط في الرقبة حتى اليوم السابع بعد الجراحة وتنظيف أي دم أو قشرة على الجروح الجراحية للحيوان. الأوتوفاجيا الشديدة من طبقات متعددة من الكسب غير المشروع يؤدي إلى القتل الرحيم للحيوان. ظهرت الخراجات الجلدية بعد اليوم الرابع عشر بعد الجراحة ، وأحيانا مع مركز جلدي قبل الناسور.من المهم التأكد من أن وضع الكسب غير المشروع لن يمنع الحيوان من أن يكون متنقلا ، وأن البديكل ليس مشدودا أو ضيقا أثناء فترة الكسب غير المشروع. أجرى فريقنا دراسات الحفظ في الأطراف المشتراة قبل الزرع. وقد تم تقييم آثار فترات مختلفة من التخزين البارد الساكن وكذلك بروتوكولات مختلفة لتشويش الأطراف.

partial-heterotopic-hindlimb-transplantation-model-in-rats

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

الفئران نموذج تفعيل CD40 - B من الخلايا

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

مثلي هند زرع الأطراف الاصطناعية في الفئران

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

في الجسم الحي أسلوب التصوير لتميز الالتهاب الحاد والمزمن

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

على المدى الطويل المزمن<em&gt; الزائفة الزنجارية</em&gt; عدوى مجرى الهواء في الفئران

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

في المختبر و في فيفو التقييم تي وب وخلايا النقوي النشاط القمعية واستجابات خلطيه من المتلقين زرع

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

نموذج استرداد تجاوز القلبي الرئوي دون نقل الدم أو وكلاء إينوتروبيك في الفئران

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

نموذج مناعي لزرع خلايا القلب الهيترولوجي وخلايا عضلة القلب في الفئران

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

تحسين تقنية الكفة لزراعة القلب مورين

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

إجراءات زرع نخاع العظم في الفئران لدراسة الهيماتوبويز الكلونية

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

تنقية وتوسيع الخلايا T القاتل الطبيعي الثابت الماوس في المختبر وفي دراسات في الجسم الحي

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...