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>

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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。

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			<td height="21" style="height:21px;width:216px;">Trephine 1.5 mm</td>
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			<td height="41" style="height:41px;width:216px;">Tricaine Hydrochloride 0.5%</td>
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			<td height="21" style="height:21px;width:216px;">7-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
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			<td height="41" style="height:41px;width:216px;">2.5% Phenylephrine HCl</td>
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			<td height="41" style="height:41px;width:216px;">1% Tropicamide</td>
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			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
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			<td height="21" style="height:21px;width:216px;">33 gauge needle</td>
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			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
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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

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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.