ويدعم هذا العمل RO1 DK064938 (لTH).

<ol>
	<li>Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+as+a+treatment+for+diabetes.">Islet transplantation as a treatment for diabetes.</a> J Am Optom Assoc. 69, 727-732 (1998).</li><li>Gangemi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+for+brittle+type+1+diabetes:+the+UIC+protocol.">Islet transplantation for brittle type 1 diabetes: the UIC protocol.</a> Am J Transplant. 8, 10-1111 (2008).</li><li>Zmuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+ATF3,+an+adaptive-response+gene,+protects+islets+and+ameliorates+inflammation+in+a+syngenic+transplantation+model.">Deficiency of ATF3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngenic transplantation model.</a> Diabetologia. , (2010).</li><li>Bottino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+Islets+to+Isolation+Stress+and+the+Effect+of+Antioxidant+Treatment.">Response of Human Islets to Isolation Stress and the Effect of Antioxidant Treatment.</a> Diabetes. 53, 2559-2568 (2004).</li><li>Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+islet+cell+culture+in+vitro+at+24+degrees+C+on+graft+survival+and+MHC+antigen+expression.">The effect of islet cell culture in vitro at 24 degrees C on graft survival and MHC antigen expression.</a> Transplantation. 49, 272-277 (1990).</li><li>Terasaka, R., Lacy, P. E., Hauptfeld, V., Bucy, R. P., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cyclosporin-A,+low-temperature+culture,+and+anti-Ia+antibodies+on+prevention+of+rejection+of+rat+islet+allografts.">The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.</a> Diabetes. 35, 83-88 (1986).</li><li>Ricordi, C., Lacy, P. E., Sterbenz, K., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+culture+of+human+islets+or+in+vivo+treatment+with+L3T4+antibody+produces+a+marked+prolongation+of+islet+human-to-mouse+xenograft+survival.">Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.</a> Proc Natl Acad Sci U S A. 84, 8080-8084 (1987).</li><li>Schnedl, W. J., Ferber, S., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STZ+transport+and+cytotoxicity.+Specific+enhancement+in+GLUT2-expressing+cells.">STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.</a> Diabetes. 43, 1326-1333 (1994).</li><li>Eckhard, M., Brandhorst, D., Brandhorst, H., Brendel, M. D., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+in+osmolality+and+range+of+density+of+a+continuous+ficoll-sodium-diatrizoate+gradient+for+isopycnic+purification+of+isolated+human+islets.">Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.</a> Transplantation Proceedings. 36, 2849-2854 (2004).</li><li>Gores, P. F., Rabe, F., Sutherland, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+survival+of+intraportal+versus+subrenal+capsular+transplanted+islet+allografts.">Prolonged survival of intraportal versus subrenal capsular transplanted islet allografts.</a> Transplantation. 43, 747-749 (1987).</li><li>Nasr, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+Immune+Privilege+Promotes+Transplantation+Tolerance+by+Altering+the+Balance+between+Memory+and+Regulatory+T+Cells.">Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells.</a> J Immunol. 174, 6161-6168 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95, 1449-1461 (2008).</li><li>Inoue, M., Maeno, T., Hatchell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+allografted+pancreatic+islets+in+the+subretinal+space+in+rats.">Survival of allografted pancreatic islets in the subretinal space in rats.</a> Ophthalmic Res. 35, 48-53 (2003).</li></ol>

الإعلان عن أي تضارب في المصالح.

في المقاطع المذكورة أعلاه ، وصفت لنا خطوات تفصيلية من أجل عزل وزرع البنكرياس الجزر الماوس. هنا نسلط الضوء بإيجاز الخطوات التي تعتبر بالغة الأهمية لنجاح الإجراءات. 1. جزيرة العزلة الخطوات الرئيسية هي تحديد أمثل الوقت الهضم الأنزيمي (1.4) ، وتحديد المواقع المشبك مرقئ على مدى الاثنى عشر لفتح القناة الصفراوية المشتركة (2.2.3) ، cannulating القناة الصفراوية المشتركة (2.3) ، والهز قوية في البنكرياس بعد ال 37 خلاصة درجة مئوية (3.2) ، وإزالة التلوث الخلايا الإفرازية (3.3.7 و 3.3.16). هذه الخطوات لها تأثير كبير على المحصول وجودة ونقاء من الجزر. ولعل الخطوة الأكثر تحديا تقنيا هو إقناء ؛ إدخال القنية من القناة الصفراوية المشتركة. في الفئران كثيرة ، يمكن أن تكون القناة الصفراوية المشتركة من الصعب تصور ذلك بسبب اللفافة المحيطة بها. ولذلك ، فمن الشائع أن محاولات يقني ؛ يدخل القنية القناة الصفراوية المشتركة نتيجة تغلغل في نسيج اللفافة فقط. في هذه الحالات ، ويبدأ Liberase لملء الأنسجة المحيطة الضام ، ولا يروي البنكرياس. إذا لوحظ هذا ، ووقف تدفق Liberase وإعادة الإبرة بحيث تخترق تجويف القناة الصفراوية. مع الممارسة ، فمن الممكن التعرف بسرعة على الموقف الأمثل لوضع الإبرة واستكمال إقناء ؛ إدخال القنية دون الإضرار قناة قابلة للتفتيت. 2. جزيرة زرع الخطوات الحاسمة هي تحريض STZ من مرض السكري (5.1) ، والتنفيذ الفعال من الجزر في الحقيبة تحت المحفظة (5.3.14-5.3.16). STZ التماثلية الجلوكوز هو تحدث بصورة طبيعية وغير انتقائي السامة للخلايا إفراز الإنسولين β خلايا الجزر 9. ومع ذلك ، هذا يحدث السمية الانتقائية في مجموعة تركيز ضيق نسبيا. ويتجلى ذلك من خلال حقيقة أن جرعات عالية من STZ يمكن أن يؤدي إلى الفتك (2-3 أيام) السريع في غياب ارتفاع السكر في الدم. ولذلك ، ينبغي توخي الحذر لتحديد الجرعة المثالية من STZ للسلالة وعمر الفئران المستخدمة قبل الشروع في أي تجارب على نطاق واسع. التسليم المادي من الجزر في الحقيبة تحت المحفظة هو أيضا خطوة رئيسية. لتحقيق نتائج متسقة ، لا بد من تقليل الخسائر في الجزر خلال إيصالها. وهذا يمكن أن يحدث إذا كانت هناك عيوب او الدموع في كبسولة الكلى تغطي الحقيبة أو إذا كان حجم حقن كبير جدا. ويمكن تجنب الأضرار التي لحقت كبسولة من خلال التلاعب طيف ودقيق ، وباستخدام الزجاج السلس التحقيق لإعداد الحقيبة تحت المحفظة. بالإضافة إلى ذلك ، يمكن تصغير حجم الحقن مع إزالة دقيق لطاف في الخطوة 5.3.6.3. إذا تمت إزالة طاف وصولا الى مستوى الجزر الرسوبية ، يوجد عادة أكثر من مساحة كافية في الحقيبة تحت المحفظة إلى مكان 400-500 الجزر. ومع ذلك ، إذا كان حجم الحقن كبير جدا ، والجزر هي أكثر عرضة للتسرب من الحقيبة أثناء عملية الزرع ، المساس استنساخ التجربة. ضمن هذا البروتوكول ، فمن الممكن تعديل بعض الخطوات وتحقيق النتائج لا تزال مرضية. وتشمل هذه ما يلي : (أ) استخدام انزيم مختلفة لهضم البنكرياس ، (ب) طريقة مختلفة لايصال لانزيم البنكرياس ، (ج) استخدام التدرج المستمر (FSD) Ficoll - الصوديوم ودياتريزوات بدلا من خطوة واحدة Histopaque1077 الكثافة ، و (د) زرع جزيرة إلى أماكن أخرى من subcapsule الكلى. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في هذا البروتوكول ، ويستخدم لإضعاف Liberase الاتصالات خلية خلية داخل البنكرياس. Liberase هي في جوهرها مزيج من تخصيبه بدرجة collagenases التي أثبتت مفيدة في الإفراج عن الجزر من البنكرياس. ومع ذلك ، أقل collagenases المنقى قادرون على تحقيق نتيجة مماثلة. لذلك ، فإنه من الممكن استخدام مختلف الاستعدادات انزيم المتاحة تجاريا لهضم البنكرياس ، طالما هو الأمثل لحالة الهضم لتحقيق جودة عالية جزيرة والمحصول والنقاء. </li><li style=";text-align:right;direction:rtl"> ومن الممكن أيضا لهضم البنكرياس دون نضح الأقنية من الانزيم. تسليم Liberase عبر القناة الصفراوية المشتركة يسمح التعرض القصوى من المساحة السطحية للانزيم البنكرياس. هذه النتائج في عملية الهضم أكثر من البنكرياس والافراج عن أكبر الجزر سليمة. ومع ذلك ، يمكن استخدام خطوات أخرى لزيادة المساحة السطحية للالبنكرياس لهذا الأنزيم. على سبيل المثال ، يمكن استخدام تنميق البنكرياس قبل تفرخ في Liberase مباشرة أو عن طريق الحقن من خلال الكبسولة Liberase البنكرياس لعزل الجزر. ومع ذلك ، لا توجد طريقة أخرى لتقديم Liberase فعالة كما هو الارواء عن طريق القناة الصفراوية المشتركة. لذا ، ينبغي أن تكون محفوظة الحقن المباشر تنميق أو كملاذ أخير ، في حالة تلف القناة الصفراوية المشتركة وperfusable لا. </li><li style=";text-align:right;direction:rtl"> طريقة بديلة لفصل الجزر من الخلايا الإفرازية ينطوي علىاستخدام التدرج بدلا من FSD Histopaque1077 10. فصل كفاءة من الجزر من الخلايا الإفرازية في هذا البروتوكول هو في جزء منه نتيجة لكثافة الفرق بين أنواع الخلايا اثنين. Histopaque1077 لديها كثافة من 1.0771 غ / مل عند 25 درجة مئوية. عند هذه الدرجة ، Histopaque هو أكثر كثافة من الجزر ولكن أقل كثافة من الخلايا الإفرازية. لذا ، في إطار قوة الطرد المركزي ، Histopaque1077 الكريات الخلايا الإفرازية ، بينما رفع الجزر إلى السطح. من حيث المبدأ ، يمكن استخدام أي مادة أخرى ذات الكثافة التي يمكن أن الجزر منفصلة من الخلايا الإفرازية في هذه الخطوة والتدرج FSD هي واحدة من هذه المواد. </li><li style=";text-align:right;direction:rtl"> مع الاحترام لموقع لزرع الجزر ، الكبسولة الكلى المستخدمة في هذا البروتوكول هو واحد من المواقع القليلة ممكن. في حين أن subcapsule الكلى هو الموقع الأكثر شيوعا من زرع في الفئران ، تم العثور على مواقع أخرى لزرع تكون فعالة وأنها تشمل الوريد البابي الكبدي ، والفضاء تحت الشبكية والخصية ، وسادة بربخي الدهون والطحال والبنكرياس وحتى 11-14. كل من هذه المواقع ومزاياها والتحديات. ولذلك ، ينبغي تحديد اختيار موقع لزرع جزيرة من الأسئلة التي وجهت والظروف المحددة للتجارب. </li></ol> ويعتقد أن الاستراتيجيات التي يمكن التغلب على القيود المفروضة على زرع جزيرة ستعزز بشكل كبير قدرته العلاجية. لذا ، فإن استخدام نموذج الفئران على النحو الذي فصله هذا البروتوكول يوفر نهج جذابة لتحديد مثل هذه الاستراتيجيات.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI 1640</td>
<td>Reagent</td>
<td>Gibco</td>
<td>31800-022</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TL</td>
<td>Reagent</td>
<td>Roche</td>
<td>05401020001</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Histopaque1077</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>10771</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Citrate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S4641</td>
<td>&nbsp;</td>
<tr>
<td>Streptozotocin</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S-0130</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A144-212</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>Reagent</td>
<td>Vedco</td>
<td>ISOSOL</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillary Tubes</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22362574</td>
<td>&nbsp;</td>
<tr>
<td>Insulin Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>329461</td>
<td>&nbsp;</td>
<tr>
<td>27 Gauge Syringe Needle</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton Syringe #1702</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14813133</td>
<td>&nbsp;</td>
<tr>
<td>10cm Petri Plates</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>0875712</td>
<td>&nbsp;</td>
<tr>
<td>6cm Petri Plates</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>07770212</td>
<td>&nbsp;</td>
<tr>
<td>6-0 Suture</td>
<td>Equipment</td>
<td>Ethicon</td>
<td>8726H</td>
<td>&nbsp;</td>
<tr>
<td>PE 50 Flexible Tubing</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>NC9083963</td>
<td>&nbsp;</td>
<tr>
<td>5ml Disposable Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>309603</td>
<td>&nbsp;</td>
<tr>
<td>50ml Conical Tube</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15ml Conical Tube</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>352097</td>
<td>&nbsp;</td>
<tr>
<td>0.1mm Nylon Mesh</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22363549</td>
<td>&nbsp;</td>
<tr>
<td>0.419mm Wire Mesh</td>
<td>Equipment</td>
<td>Newark Wire Cloth Company</td>
<td>0300241</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath Incubator</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Equipment</td>
<td>Zeiss</td>
<td>Stemi SV6</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>Equipment</td>
<td>Nikon</td>
<td>TMS</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Incubator</td>
<td>Equipment</td>
<td>Napco</td>
<td>6300</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Scissors</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS14393</td>
<td>&nbsp;</td>
<tr>
<td>McPherson-Vannas Scissors</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS500260</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS15915</td>
<td>&nbsp;</td>
<tr>
<td>Hemostatic Forceps</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS 500451</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane Nebulizer</td>
<td>Equipment</td>
<td>Smiths Medical Surgivet</td>
<td>ISOTech 4 OHMEDA</td>
<td>&nbsp;</td>
<tr>
<td>Swing Bucket Centrifuge</td>
<td>Equipment</td>
<td>Dupont &#x2013; Sorvall</td>
<td>RT6000D</td>
<td>&nbsp;</td>
<tr>
<td>Swing Bucket Rotor</td>
<td>Equipment</td>
<td>Dupont &#x2013; Sorvall</td>
<td>H1000B</td>
<td>&nbsp;</td>
<tr>
<td>pH Meter</td>
<td>Equipment</td>
<td>Mettler Toledo/ Fisher Scientific</td>
<td>09313509</td>
<td>&nbsp;</td>
<tr>
<td>Gel Loading Tips</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>05408151</td>
<td>&nbsp;</td>
<tr>
<td>p200 Tips</td>
<td>Equipment</td>
<td>USA Scientific</td>
<td>111-0730</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a method for murine islet isolation and subcapsular kidney transplantation

منذ بداية العمل الرائد الذي Ballinger Reckard ومما يدل على أن زرع جزيرات لانجرهانز في القوارض السكري يمكن تطبيع مستويات السكر في الدم ، وقد اقترح أن تكون جزيرة زرع علاج محتمل لمرض السكري نوع 1 ، 1،2. في الآونة الأخيرة ، عززت التقدم في زرع المزيد من جزيرة الإنسان هذا الرأي 1،3. ومع ذلك ، واثنين من القيود الرئيسية منع زرع جزيرة من كونها حقيقة واقعة على نطاق واسع السريرية : (أ) شرط لأعداد كبيرة من الجزر لكل مريض ، الأمر الذي يقلل كثيرا من عدد من المستفيدين المحتملين ، و (ب) الحاجة إلى كبت المناعة الثقيلة ، الأمر الذي يؤثر بشكل كبير السكان للأطفال المرضى بسبب تعرضهم لكبت المناعة على المدى الطويل. الاستراتيجيات التي يمكن التغلب على هذه القيود لديها القدرة على تعزيز المرافق العلاجية لزرع جزيرة. جزيرة زرع كبسولة تحت الماوس الكلى هو النموذج المقبول على نطاق واسع لتحقيق استراتيجيات مختلفة لتحسين جزيرة الزرع. هذه التجربة يتطلب عزل من الجزر ذات جودة عالية وزرع جزيرات إلى المستلمين السكري. كل من الإجراءات الجراحية التي تتطلب خطوات يمكن أن يكون أظهر بشكل أفضل عن طريق الفيديو من قبل النص. هنا ، ونحن الوثيقة الخطوات التفصيلية لهذه الإجراءات من قبل كل من الفيديو وبروتوكول مكتوب. نحن أيضا مناقشة لفترة وجيزة زرع نماذج مختلفة : ، خيفي مسانج ، مسانج المناعة الذاتية ، وخيفي الذاتية.

Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

وقد اقترح زرع الجزر المعزولة لتكون المعاملة المحتملة للاصابة بداء السكري من النوع 1. نحن هنا وصف الأسلوب لعزل الجزر من بنكرياسات الماوس وزرع لهم في الفضاء تحت المحفظة في الكلى.

وهناك طريقة لعزل جزيرة الفئران تحت المحفظة وزراعة الكلى

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

a-method-for-murine-islet-isolation-subcapsular-kidney

Nanyang Technological University

Research

JoVE Journal

Medicine

63.3K Views.  The Ohio State University. وقد اقترح زرع الجزر المعزولة لتكون المعاملة المحتملة للاصابة بداء السكري من النوع 1. نحن هنا وصف الأسلوب لعزل الجزر من بنكرياسات الماوس وزرع لهم في الفضاء تحت المحفظة في الكلى.

Video: A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

تطوير النزلات الشعبية مسد في نموذج الفئران من زرع الرئة Orthotopic

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

الفئران عنق الرحم القلب زرع نموذج باستخدام أزرار وتقنيات التعديل

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

الفئران غيري مكاني القلب زرع تقنية

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

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.

الفئران زرع القرنية: نموذج لدراسة الشكل الأكثر شيوعا من الصلبة زراعة الأعضاء

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

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

الماوس زرع الكلى: نماذج من طعم خيفي رفض

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

الكلى الفئران زرع تقنية

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

سوي الحرارة<em&gt; السابقين فيفو</em&gt; الإرواء الكلى للحفاظ على الكلى الطعوم قبل الزرع

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

طريقة عزل رواية الصف سريرية للكلى البشرية ارتشاح الخلايا اللحمية

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

زرع الكلي الفئران العظام: نهج الجراحية الجديدة والمبسطة

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

التطهير البصري والتصوير من أنسجة الكلى المسماة بالمناعة

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...