The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

注意:在此过程中使用的所有动物都按照研究协会在视觉与眼科语句在眼科和视觉研究用动物，以及订定圣路易斯大学动物监督委员会的指导方针进行处理。 注:所有手术器械和解决方案之前进行消毒手术，以限制眼睛微生物感染。应当指出的是，虽然动物做这个过程遇到一些痛苦，我们不使用止痛剂。这样做的理由是因为所有的止痛药是消炎和自角膜移植反应涉及炎症，使用抗炎药物会损害我们确定参与角膜移植失败哪些因素的能力。 1.麻醉<ol style="margin-left: 40px;"><li>氯胺酮（86腹腔注射将在全身麻醉供体和受体的小鼠。98毫克/公斤）和甲苯噻嗪（13.04毫克/千克）。 </li><li>保持麻醉状态下的受体小鼠在整个这通常需要30分钟到一个小时的过程。因此，不断地监视鼠标苏醒的迹象。 </li><li>申请puralube软膏，不会接受手术后的动物被麻醉，以防止干眼。 </li></ol> 2.角膜移植术<ol style="margin-left: 40px;"><li>获得捐赠的角膜按钮。 <ol><li>一旦动物完全麻醉，达到充分散瞳由一对夫妇1％托吡卡胺和2.5％盐酸去氧肾上腺素的眼药水管理。 </li><li>放置供体动物的头部水平在电路板上放置在一个坚固的可移动的支持。固定头的整个颈部带的条带，以保证眼睛处于整个操作的水平位置。 </li><li>使用2毫米直径的环钻，其前端被染成用甲基蓝，出行角膜中央部位移植。 </li><li>用锋利的刀片，穿透角膜与注入healon进入前房加深它减少损坏施主内皮和底层透镜的机会。 </li><li>切除供体移植物与vannas剪刀，并放入含有Hanks平衡盐溶液，直到使用一盘菜。 </li><li>后供体移植物已被移除，通过安乐死二氧化碳吸入供体小鼠。 </li></ol></li><li>准备移植床。 <ol><li>如在2.1.1中描述过2.1.2收件人重复相同的步骤。 </li><li>使用1.5毫米直径的环钻，勾勒出收件人移植部位。 </li><li>用锋利的刀片，穿透角膜与注入healon进入前房加深它来减少损伤的机会底层透镜。 </li><li>使用vannas剪刀取下收件人概述角膜中央按钮并丢弃。 </li></ol></li><li>缝合接枝<ol><li>将供体角膜在移植床收件人的角膜。确保有足够的healon是在角膜供体，以保护捐赠者的内皮细胞通过镜头直接接触伤害。 </li><li>使用超细尖microforceps，放置11-0尼龙缝线咬头到捐助方，通过与全层到收件人的面为90％深度的捐助，然后打结。 </li><li>一旦角膜被固定到位，执行midcardinal间断缝合，使得所述角膜有8至10个总缝线和供体角膜牢固地排队与和连接到接收方的角膜移植物床。 </li></ol></li><li>深化前房<ol><li>通过注入的HBSS或气泡进入前房加深前房并轻轻检查角膜移植物的完整性对于泄漏用纤维素海绵。 注意:如果前房不能改革则是C的概率高ataract这将使得移植角膜的未来的评估很困难，也将有可能导致供体角膜内皮功能紊乱，因此移植物衰竭。 </li></ol></li><li>终期评估<ol><li>观察的眼睛，以确定瞳孔是圆的，前房深度是正常的。 注意:如果瞳孔不圆这表明缝合虹膜过程中损坏，因此，移植被认为是一个技术故障。 </li><li>应用抗生素软膏的眼睛。可选:合上眼睑用7-0丝线缝合。 </li><li>观察小鼠直到它们完全清醒，然后分别容纳他们最少两天后手术。 </li></ol></li></ol> 3.拆线<ol style="margin-left: 40px;"><li>在盖体缝合用于这些情况下，麻醉上述老鼠和盖子取下缝合处48小时。 </li><li>麻醉小鼠术后7天。取出suturES确保角膜移植。一旦缝线被删除，动物已经完全觉醒，将其放回笼子。 </li></ol> 4.临床评价<ol style="margin-left: 40px;"><li>检查眼适应症手术并发症，包括，白内障（晶状体混浊），前房出血（血液中的前房）的前房，它是适当的深度，或角膜的显著不透明度不。想想那些表现出这些复杂的"妥协"，并通过二氧化碳吸入安乐死他们。 <ol><li>执行对未麻醉的小鼠的所有考试。按住鼠标，用一只手从而抑制鼠标，这样另一方面可以proptose眼睛，使眼睛更好的视野。一旦观测完成后，返回到动物的笼子。 </li></ol></li><li>有一个观察者不熟悉治疗组评估所移植的角膜每周2至3次，角膜移植的迹象排斥或角膜移植失败。请使用手术显微镜或水平裂隙灯生物显微镜对这些意见。 <ol><li>评估用于使用比例为0〜5的不透明度的尺度定义如下每角膜: <ol><li>分配给0分给那些有混浊的迹象眼角膜。 </li><li>指定1分给那些表现出最小的浅表角膜混浊。 </li><li>分配给2分，以用于显示轻度和更深的不透明性，但潜在的瞳孔和虹膜仍是有迹可寻的眼角膜。 </li><li>分配3分至角膜中显示基质的不透明度，其特征在于所述虹膜不能看到详细除瞳孔边缘的。 </li><li>分配的4分至角膜中显示致密的基质混浊并且如果没有下面的结构能够被观看。 </li><li>分配给5分，以角膜中显示完整的不透明性和密集的间质水肿，瞳孔与虹膜完全遮蔽。 </li></ol></li><li><html>还评估每个角膜供使用的1〜8的等级规模要做到这一点的血管浸润（新血管形成）的程度，查看角膜作为组成4相等象限并确定在每一个象限中具有的血管的量分数，范围从0（无船），以2象限广泛的血管。添加单独得分从各象限来计算最终新生血管得分。 </li><li>归类为急性排斥，如果他们有连续两次观测的比分为3点的时间最多5周的眼角膜。 </li><li>分类小鼠的角膜是透明的5个星期，但发展混浊，有时&gt;45天植入后，用连续两个时间点分3，具有经过后期长期的角膜移植排斥反应。采用Kaplan-Meier生存曲线分析移植物的存活。 </li></ol></li></ol> 5.操纵的模型在左:40PX;"&gt; <li>制备单细胞脾。 <ol><li>制备单细胞，首先安乐死的供体小鼠。然后，取出脾脏。 </li><li>将脾脏在细胞过滤并破坏其与由3毫升注射器的注射器柱塞。 </li><li>在10毫升Hanks平衡盐溶液中洗涤细胞并重新悬浮。 </li><li>删除10微升细胞悬液，加入10微升0.4％台盼蓝混匀。补充一点，对血球和细胞计数在中央格。细胞在管的数量是细胞数目×10 4×2（稀释因子中的台盼蓝）×10（在管体积）。 </li></ol></li><li>注射进入前房。 <ol><li>麻醉小鼠，如前所述。 </li><li>执行使用解剖显微镜注射。对于每个房内注射，使用10 6脾细胞中0.005毫升容积并配有一个33克针一0.25毫升微升注射器。 注意:其他操作该模型可通过治疗动物与充当要么拮抗剂或激动剂，以确定特定因素可能起以下原位角膜移植术中的作用的试剂进行。 </li></ol></li></ol>

<ol>
	<li>Farooq, A. V., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+epithelial+and+stromal+keratitis:+an+epidemiologic+update.">Herpes simplex epithelial and stromal keratitis: an epidemiologic update.</a> Surv. Ophthalmol. , 448-462 (2012).</li><li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+and+diseases+of+the+ocular+surface+and.">Age-related changes and diseases of the ocular surface and.</a> Invest. Opthlamol. Vis. Sci. 54, 48-53 (2013).</li><li>Edwards, 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=Indications+for+corneal+transplantation+in+New+Zealand:+1991-1999.">Indications for corneal transplantation in New Zealand: 1991-1999.</a> Cornea. 21, 152-155 (2002).</li><li>Thompson, R. W., Price, M. O., Bowers, P. J., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+after+penetrating+keratoplasty.">Long-term survival after penetrating keratoplasty.</a> Ophthalmol. 110, 1396-1402 (2003).</li><li>Williams, K. A., Roder, D., Esterman, A., Muehlberg, S. M., Coster, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+predictive+of+corneal+graft+survival.+Report+form+the+Australian+Corneal+Graft+Registry.">Factors predictive of corneal graft survival. Report form the Australian Corneal Graft Registry.</a> Ophthalmology. 99, 403-414 (1992).</li><li>Larkin, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+allograft+rejection.">Corneal allograft rejection.</a> Br. J. Ophthalmol. 78, 649-652 (1994).</li><li>Boisjoly, H. 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=Risk+factors+of+corneal+graft+failure.">Risk factors of corneal graft failure.</a> Ophthalmol. 100, 1728-1735 (1993).</li><li>Sugar, 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=Recipient+Risk+Factors+for+Graft+Failure+in+the+Cornea+Donor+Study.">Recipient Risk Factors for Graft Failure in the Cornea Donor Study.</a> Ophthalmol. 116, 1023-1028 (2009).</li><li>Namba, K., Kitaichi, N., Nishida, T., Taylor, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+regulatory+T+cells+by+the+immunomodulating+cytokines+alpha-melanocyte-stimulating+hormone+and+transforming+growth+factor-beta2.">Induction of regulatory T cells by the immunomodulating cytokines alpha-melanocyte-stimulating hormone and transforming growth factor-beta2.</a> J. Leukoc. Biol. 72, 946-952 (2002).</li><li>Taylor, A. W., Yee, D. G., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+nitric+oxide+generated+by+inflammatory+macrophages+by+calcitonin+gene-related+peptide+in+aqueous+humor.">Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor.</a> Invest. Ophthalmol. Vis. Sci. 39, 1372-1378 (1998).</li><li>Wilbanks, G. A., Mammolenti, M., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+induction+of+anterior+chamber-associated+immune+deviation+(ACAID).+III.+Induction+of+ACAID+depends+upon+intraocular+transforming+growth+factor-beta.">Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta.</a> Eur. J. Immunol. 22, 165-173 (1992).</li><li>Volpert, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducer-stimulated+Fas+targets+activated+endothelium+for+destruction+by+anti-angiogenic+thrombospondin-1+and+pigment+epithelium-derived+factor.">Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor.</a> Nat. Med. , 8-349 (2002).</li><li>Apte, R. S., Sinha, D., Mayhew, E., Wistow, G. J., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+role+of+macrophage+migration+inhibitory+factor+in+inhibiting+NK+cell+activity+and+preserving+immune+privilege.">Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege.</a> J. Immunol. 160, 5693-5696 (1998).</li><li>Kennedy, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+production+of+interleukin-1+receptor+antagonist+peptides+in+normal+human+cornea.">Novel production of interleukin-1 receptor antagonist peptides in normal human cornea.</a> J. Clin. Invest. 95, 82-88 (1995).</li><li>Shimmura-Tomita, M., Wang, M., Taniguchi, H., Akiba, H., Yagita, H., Hori, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-9-mediated+protection+from+allo-specific+T+cells+as+a+mechanism+of+immune+privilege+of+corneal+allografts.">Galectin-9-mediated protection from allo-specific T cells as a mechanism of immune privilege of corneal allografts.</a> PLoS One. 8, (2013).</li><li>Goldberg, M. F., Ferguson, T. A., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cellular+adhesion+molecules+in+inflamed+human+corneas.">Detection of cellular adhesion molecules in inflamed human corneas.</a> Ophthalmol. 101, 161-168 (1994).</li><li>Stuart, P. M., Griffith, T. S., Usui, N., Pepose, J. S., Yu, X., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD95+ligand+(FasL)-induced+apoptosis+is+necessary+for+corneal+allograft+survival.">CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival.</a> J Clin Invest. 99, 396-402 (1997).</li><li>Yamagami, S., 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=Role+of+Fas-Fas+ligand+interactions+in+the+immunorejection+of+allogeneic+mouse+corneal+transplants.">Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.</a> Transplantation. 64, 1107-1111 (1997).</li><li>Stuart, P. M., Pan, F., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fas/Fas+ligand+interactions+regulate+neovascularization+in+the+cornea.">Fas/Fas ligand interactions regulate neovascularization in the cornea.</a> Invest. Ophthalmmol. Vis. Sci. 44, 93-98 (2003).</li><li>Stuart, P. M., Yin, X. T., Pan, F., Haskova, Z., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+matrix+metalloproteinases+activity+prolong+corneal+allograft+acceptance+by+increasing+FasL+expression.">Inhibitors of matrix metalloproteinases activity prolong corneal allograft acceptance by increasing FasL expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1169-1173 (2004).</li><li>Joo, C. -. K., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+mediated+responses+in+a+murine+model+of+orthotopic+corneal+transplantation.">T-cell mediated responses in a murine model of orthotopic corneal transplantation.</a> Invest.Ophthalmol. Vis. Sci. 36, 1530-1540 (1995).</li><li>Sonoda, Y., Sano, Y., Ksander, B., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell-mediated+immune+responses+elicited+by+orthotopic+corneal+allografts+in+mice.">Characterization of cell-mediated immune responses elicited by orthotopic corneal allografts in mice.</a> Invest. Ophthalmol. Vis. Sci. 36, 427-434 (1995).</li><li>Sano, Y., Osawa, H., Sotozono, C., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+expression+during+orthotopiccorneal+allograft+rejection+in+mice.">Cytokine expression during orthotopiccorneal allograft rejection in mice.</a> Invest. Ophthalmol. Vis. Sci. 39, 1953-1957 (1998).</li><li>Haskova, Z., Usui, N., Ferguson, T. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cells+are+critical+in+corneal+but+not+skin+allograft+rejection.">CD4+ T cells are critical in corneal but not skin allograft rejection.</a> Transplantation. 69, 483-488 (2000).</li><li>Tan, 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=Immunological+disruption+of+antiangiogenic+signals+by+recruited+allospecific+T+cells+leads+to+corneal+allograft+rejection.">Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.</a> J. Immunol. 188, 5962-5969 (2012).</li><li>Dana, M. R., Yamada, J., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+interleukin-1+receptor+antagonist+promotes+corneal+transplant+survival.">Topical interleukin-1 receptor antagonist promotes corneal transplant survival.</a> Transplantation. 63, 1501-1507 (1997).</li><li>Cunnusamy, K., Chen, P. W., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17A-dependent+CD4+CD25++regulatory+T+cells+promote+immune+privilege+of+corneal+allografts.">IL-17A-dependent CD4+CD25+ regulatory T cells promote immune privilege of corneal allografts.</a> J. Immunol. 186, 6737-6745 (2011).</li><li>Fu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+depletion+as+a+mechanism+for+the+immune+privilege+of+corneal+allografts.">Arginine depletion as a mechanism for the immune privilege of corneal allografts.</a> Eur. J. Immunol. 41, 2997-3005 (2011).</li><li>Medina, C. A., Rowe, A. M., Yun, H., Knickelbein, J. E., Lathrop, K. L., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azithromycin+treatment+increases+survival+of+high-risk+corneal+allotransplants.Cornea.">Azithromycin treatment increases survival of high-risk corneal allotransplants.Cornea.</a> , 32-658 (2013).</li><li>Cho, Y. K., Zhang, X., Uehara, H., Young, J. R., Archer, B., Ambati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Endothelial+Growth+Factor+Receptor+1+morpholino+increases+graft+survival+in+a+murine+penetrating+keratoplasty.">Vascular Endothelial Growth Factor Receptor 1 morpholino increases graft survival in a murine penetrating keratoplasty.</a> Invest. Ophthalmol. Vis. Sci. 53, 8458-8471 (2012).</li><li>Kim, H. K., Choi, J. A., Uehara, H., Zhang, X., Ambati, B. K., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+graft+rejection.">Corneal graft rejection.</a> Surv. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

这里描述角膜移植的小鼠模型使研究人员研究人类角膜移植排斥反应的模型，该模型预测的因素是什么，最好既排斥15,17,18,20，26-30和角膜的验收21-25关联移植。不象人类角膜移植，其中给予患者或者局部或全身激素治疗，以任一治疗或预防排斥31，该模型通常用于确定那些是相关的同种异体移植排斥反应在不存在这样的治疗的因素。除了一款型号为急性角膜移植排斥反应?...

角膜移植是最成功的和常见的类型移植在人执行的一个。为什么进行该手术的原因是损伤，感染性疾病1，或其他形式的非感染性角膜病2的结果。从美国眼库协会的数据显示，46000在2011年进行（见网站:restoresight.org/eye_banks/eye_banks.html）。其成功的标志是有一年不合格率同种异体角膜移植物的范围从10％至15％，并在5年的成功是超过70％3-8。因为许多研究已经显示，角膜移植的成功是直接关系到一个事实，即眼睛的免疫特权部位。负责角膜状态作为免疫特权部位因素包括缺乏既血管和淋巴管在角膜中，相对缺乏抗原呈递细胞的因子，由角膜该suppres产生免疫效应器funtions 9-15，MHC的低表达抗原16，和FasL 17-20的表达。 然而，尽管这些因素诱发这些移植成功的，它们经历排斥3-7。因此，了解这些调解这种排斥反应的机制，以及测试各种治疗，以防止排斥反应是至关重要的。为此，我们在这里描述角膜移植是已经使用了超过20年，研究角膜移植在受控的实验环境的小鼠模型。自移植反应涉及协同工作的许多不同的因素，这将最终确定移植的组织是否失败或成功，则它不可能了解这些因素中的任何体外模型中的重要性。因此，使用完整的动物研究是必需的，以确定哪些因素是重要的成功或者failu重新移植组织。 而其它种类的动物被用来研究角膜移植中，当相比于使用其他种类的鼠模型中具有几个优点。第一个是表示某些转基因的或已被基因靶向至缺乏特异性免疫因子，其功能在移植可以更好地研究了表达小鼠的许多菌株的存在。此外，还有许多试剂（包括重组因子和抗体是中和因子）是特定于小鼠，并且不为其他许多种动物的存在。由于这些因素的存在，该模型已被广泛用于鉴定参与急性角膜移植反应15，17,18,20 -29有关因素。此外，有许多参与角膜移植的因素也是已知的是功能在其他组织中的移植。

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

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.

已用于角膜移植的小鼠模型中超过20年，成功刻画两个角膜移植排斥反应19-23和角膜移植验收13，15,16,18，24-27机制。该模型被用于建立在角膜移植接受FasL表达的重要性，在该动物缺乏FasL的不能够接受角膜移植物15。它也被用来表明，血管内皮生长因子受体1啉治疗显著增加角膜移植存活28。在一个非常最近的一份报告此模型来之前，角膜移植测试是否小鼠的预处理与...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

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

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.

小鼠角膜移植模型，研究实体器官移植的最常见形式

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

murine-corneal-transplantation-model-to-study-most-common-form-solid

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.6K 次观看.  Saint Louis University. 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.

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

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.

鼠肾移植:移植排斥反应模型

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

科研

医学

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.

小鼠肾脏移植技术

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

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

免疫与感染

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型:有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

符合当代无菌技术、麻醉和镇痛标准的改良小鼠异位心脏移植方案

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

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

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

使用分裂的角质按钮的孔膜内皮器官培养模型

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

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

使用24小时器官保存和连续遥测的猪模型中正交肾自动移植

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.