我们感谢实验室的成员，为帮助这些细胞培养特征，蒂娜杰弗里Adijanto班松，李蓉，秦环，从小张，赵菁，康妮志，Awais齐亚，纳塔利娅Strunnikova。特别感谢赵菁，康妮志，他们在帮助维持细胞培养的大量库存和Tina班松。 这项工作是由美国国立卫生院内研究计划研究院的支持。

1。人类胎儿组织所有的人体组织的相关研究如下的“赫尔辛基宣言”和美国国立卫生研究院的机构审查委员会的信条。胎儿的眼睛是由一个独立的皮条客，先进的生物科技资源（ABR，阿拉米达，CA）获得的，随意放置在RPMI - 1640含管（ABR提供）的媒体，捐助者在16至22周妊娠，装在冰，交付隔夜优先递送服务。组织仍然可行眼球摘除后长达48小时。 2。细胞培养液 MEM -α修饰培养基（​​Sigma - Aldrich公司）作为基介质准备5％和15％的含血清培养的视网膜色素上皮细胞（RPE细胞培养基;下面的表1）媒体。 <table border="1"><tbody><tr><td>名称</td><td>西格玛</td><td> Gibco公司</td><td>金额</td><td>存储</td></tr><tr><td>纪念，阿尔法修改</td><td> M - 4526 </td><td></td><td> 500毫升</td><td> 4 ° C </td></tr><tr><td> N1的补充</td><td> N - 6530 </td><td></td><td> 5毫升</td><td> 4 ° C </td></tr><tr><td>青霉素链霉素</td><td></td><td> 15140-148 </td><td> 5毫升</td><td> -20℃ </td></tr><tr><td> GlutaMax - 我</td><td></td><td> 35050 </td><td> 5毫升</td><td> -20℃ </td></tr><tr><td>非必需氨基酸</td><td> M - 7145 </td><td></td><td> 5毫升</td><td> 4 ° C </td></tr><tr><td> THT * </td><td></td><td></td><td></td><td> -80℃ </td></tr><tr><td>牛磺酸</td><td> T - 0625 </td><td></td><td> 125毫克</td><td></td></tr><tr><td>氢化可的松</td><td> H - 0396 10 </td><td></td><td> 10微克</td><td></td></tr><tr><td>三碘thyronin </td><td> T - 5516 </td><td></td><td> 0.0065微克</td><td></td></tr><tr><td>小牛血清** </td><td></td><td></td><td> 5％或15％ </td><td> -80℃ </td></tr></tbody></table> 表1。人胚RPE中等组件500毫升培养基的制备 * THT是由溶解在1 1.5毫升PBS中期之前，牛磺酸氢化可的松三碘- thyronin。多个等份并保存于-80 ° C至简化培养基中培养的准备。 **胎牛血清未获得来自Sigma - Aldrich公司或Gibco公司。 胎牛血清培养基制备中的应用是来自亚特兰大的生物制品（诺克罗斯，GA）获得。每一瓶血清热灭活（56℃为1小时），使用前。为了确保细胞培养的一致性，从同一批次血清大量购买，并保存在-20℃，直到它们被用于媒体准备。 24 - 或12 - 孔板是生长在人烟稀少的从一个特定的批次hfRPE细胞种子细胞在含有20％胎牛血清的不同批次的媒体的几个星期。最经典的RPE细胞形态（鹅卵石），增长最快的melanated细胞，可用于细胞培养表明适当的血清很多。 培养液中还包含：N1补充（Sigma - Aldrich公司）1:100毫升/毫升，GlutaMax /青霉素，链霉素1:100毫升/毫升（GIBCO），和不必要的氨基酸液（Sigma - Aldrich公司）1:100毫升/毫升。此外，氢化可的松（20微克/升），牛磺酸（250 mg / L的）和三碘thyronin（0.013微克/升）（THT）提前准备这三个组成部分溶解在PBS到终浓度为1:500 （毫升/毫升）。 THT等分储存在80 °彗星，直到添加到视网膜色素上皮介质。 3。细胞培养在收到完整的地球抗生素antimycotic溶液冲洗（稀释到10倍;。猫没有15240-096; Invitrogen公司）加庆大霉素（1毫克/毫升）为3〜5分钟（图1）。 <img src="/files/ftp_upload/2032/2032fig1.jpg" alt="图1" /> 图1。孵育后，抗生素两次中等的HBSS或PBS冲洗掉。一个在当时的眼球转移到10厘米的培养皿Sylgard - 184（WPI），涂层和27G针头担保。使用皆伐板载刀（爱尔康）切口通过巩膜睫状体（从眼球赤道前表面的距离的1 / 3）以下。这个切口是用来启动一个圆形切割拆除眼前部分。采用的是同一个刀片锯齿（FST）的一个碳化钨涂层弧形光圈剪刀剪开的。眼球前部的搬迁之前，降息是通过玻璃体，以避免分离的Retina从后极部视网膜色素上皮。眼的前部被删除后，后极是在5％dispase，我的解决方案（2 U /毫升，猫没有04942086001;罗氏诊断，印第安纳波利斯，在。）孵育40-60分钟的血清培养基在37 ° C - 5％的CO 2。 dispase治疗后，后极硅填充（Sylgard 184，道康宁，美联，心肌梗死）的培养皿转移到的HBSS到象限或大块足够的扁平化组织解剖。然后用镊子轻轻除去视网膜。单细胞的RPE层成片剥落，直接收集到冷的胰蛋白酶- EDTA（GIBCO，＃25200-056）解决方案。后的RPE收集，组织中的胰蛋白酶- EDTA管密封，转移到为10-15分钟的水洗澡，在37 ° C。孵育10分钟后，管大力动摇，分成小簇的RPE。如果分离不彻底，管放入水浴，再过5分钟。胰蛋白酶- EDTA的潜伏期后，联合国可能溶解混合细胞群管检查。任何观察到的集群都将被删除，用细尖玻璃巴斯德吸管。纺（1.4临床离心机转速为4分钟）后，hfRPE细胞重新悬浮在15％的视网膜色素上皮媒体，然后把Primaria瓶（例如：猫没有08-772-45;费舍尔科学，宾夕法尼亚州匹兹堡。 ）。这种介质更换后1天用5％的血清中含有的RPE中等，并随后进行了更改，每2至3天。 3〜4周后，细胞融合和统一色素。然后，他们在0.25％胰蛋白酶- EDTA为10至15分钟胰酶消化，重悬在15％血清中含有色素上皮细胞培养液中，每口井在150到200K细胞（Transwell小，康宁中光学到清晰的细胞培养插入种子，康宁，NY），使用12毫米直径的插入，0.4微米的毛孔，聚酯膜（例如：猫，Fisher Scientific则没有07-200-161）。播种前，与人类细胞外基质（在150μL的HBSS，每孔10微克，猫没有354237;。BD公司，新泽西州富兰克林湖，）涂井，并在2小时的引擎盖UV光固化。在某些情况下，胰蛋白酶消化过程重复第二次，收集细胞，没有分离后的第一个胰蛋白酶。相同的协议（不包括涂层与ECM）是用于培养细胞的烧瓶，生成细胞的P1人口。这些细胞进行实验，当他们≥200Ω•2厘米的一个总的组织性和均匀色素。 <a href="http://www.jove.com/files/ftp_upload/2032/2032fig1full.jpg">请点击这里为一个较大的数字</a> 1 。 4。一步步过程<ol><li>准备了多种解决方案（每个4口井需要眼睛）12孔板： <ol class="lalpha"><li>添加10X抗生素antimycotic解决方案 - 1井/眼</li><li>添加冲洗PBS / HBSS解决方案 - 2井/眼</li><li>添加dispase解决方案 - 1井/眼</li></ol></li><li>解开5个固定针和填充的HBSS，准备解剖​​培养皿</li><li>拆开的眼睛和抗生素antimycotic的解决方案，为3-5分钟（在第1步准备） </li><li>在两口井（在第1步准备），用PBS / HBSS冲洗眼睛，然后将它们传输到解剖盘。 </li><li>修剪过多的眼部周围的肌肉和结缔组织</li><li>在解剖盘对准角膜面临的方式使用27G的针头安全眼（牵制硅基地）。 </li><li>使用板载刀下面的角膜切口，圆形切割将开始</li><li>使用虹膜剪刀，减少眼部周围，通过玻璃体切割和解除眼的前部，然后使用相同的剪刀。 注意：在步骤3至8，避免任何过大的机械压力眼球。 </li><li> dispase解决方案转移到开放眼睛（在第1步准备） </li><li>眼罩孵育40-60分钟，在37℃，5％的CO 2 </li><li>替换在解剖与新鲜菜之一的HBSS解决方案。 </li><li> dispase解决方案传输眼睛解剖盘。 </li><li>在培养皿中的位置眼（眼球杯朝上）和安全两个27G的针头。 </li><li>轻轻抬起部分分离视网膜和视网膜剪刀切断视神经视网膜。丢弃视网膜。 </li><li>使用虹膜剪一个切口从外围对视神经的眼。 </li><li>使用所有5个27G的针头很好地拉长眼RPE层扁平化。 </li><li>使用视网膜剪刀，使视神经周围的圆形切割分离挂职视神经RPE层。 注意：步骤13-17可以做低倍率或无立体显微镜</li><li>调整体视显微镜250倍的放大倍率或以上，并找到密切沿视神经，虹膜剪刀切割边缘的RPE表。 </li><li>利用两个钳单独的视网膜色素上皮布鲁赫的脉络膜组织层膜。它可能需要几个试图找到沿边缘区视网膜色素上皮及脉络膜是最弱的。 </li><li>进入寒冷的胰蛋白酶- EDTA溶液放置的RPE张在15毫升管</li><li>后的RPE收集到水浴组织中的胰蛋白酶- EDTA为10 - 15分钟，管帽和转让，在37 ° C。 </li><li>孵育10分钟后，大力摇晃15毫升管，分成小簇的RPE。如果分离是不完整的，放入水浴管另一个5分钟。 </li><li>联合国可能溶解混合细胞群的检查管。用细尖玻璃巴斯德吸管，应删除任何观察到的集群。 </li><li>离心（1.4临床离心机转速为4分钟）hfRPE细胞，去除上清液，重新悬浮细胞在15％的视网膜色素上皮媒体（9毫升总） </li><li> Primaria烧瓶中加入2 ml新鲜的15％的视网膜色素上皮媒体，放入孵化器的烧瓶中，直到第二天（37℃，5％CO 2）放入3 ml的细胞悬液</li></ol> 5。代表性的成果 hfRPE细胞培养的功能验证在以前的实验中，我们已经使用了这些原代培养，以确定质膜转运蛋白的表达，多极化和功能，并确定具体的信号转导通路，调节视网膜色素上皮功能1-11的这些特点，为他们积累提供属性，可应用于设置一个验证每个细胞的产生。总结如下实验确定的蛋白质，确定跨上皮流体输送和完整性的paracellular途径。这些在体外实验中已证实在动物模型的视网膜重新复位6。 <img src="/files/ftp_upload/2032/2032fig2.jpg" alt="图2" /> 图2。本地化hfRPE细胞CFTR的 左上面板：CFTR的检测中使用膜富集提取的hfRPE细胞。男，分子量标记; 1巷 ，主要成熟（C波段）和不成熟（带A和B）中心较低的面板：CFTR的免疫荧光定位（绿色标签）右上角的面板 ：扩大交叉通过的 Z平面显示剖面视图。通过Z轴的最大强度投影。 ZO - 1（红色标记）作为一个紧密连接的标记划定的RPE心尖和基底双方的供应。 DAPI染色（蓝色）标签的原子核靠近基底膜。 ZO - 1的显示为黄色/橙色，因为它的红色标签重叠CFTR的绿色荧光。 <img src="/files/ftp_upload/2032/2032fig3.jpg" alt="图3" /> 图3。 γ干扰素的诱导生理变化，在 hfRPE答：IFNγ基础浴除了增加整个小学，培养hfRPE细胞单层的跨上皮流体输送（J V）J V是作为一个在顶部跟踪功能的时间和净液绘制。吸收（根尖基础浴）表示正面的价值观;跨上皮电位（TEP）和总的组织电阻（R T）绘制的时间在底部的痕迹功能。乙：基础浴抑制CFTRinh - 172（5微米）的γ干扰素刺激J v增加。 下面是一个动物模型实验，在体外研究结果证实hfRPE的例子。在这个实验中，人为地造成视网膜脱离除了IFNG外部眼球表面（图4，A，B）后显着降低。这种效应可部分阻止：（1）除营阻滞剂（图4D）;（2）完全的JAK +营阻滞剂后除了封锁。下面的图片是使用华侨城扫描仪获得。 <img src="/files/ftp_upload/2032/2032fig4.jpg" alt="图4" /> 图4。 体内实验的程序， 这些在体内视网膜脱离的实验 。 以前 6详细描述。在这些实验中，视网膜脱离0.5-3μL修改PBS液注射到视网膜下腔（SRS），单独或与相结合的JAK - STAT和PKA通路抑制剂在大鼠眼球。每次实验后，视网膜脱离创造一个初步控制在40-70分钟时间。在此期间，大疱体积的变化率测量，以确保气泡稳定。光学相干断层扫描成像（应用物理研究所，俄罗斯科学院，下诺夫哥罗德，俄罗斯）是用来衡量SRS体积变化的时间过程。 重新附件卷的时间当然UME变化是衡量光学相干断层扫描（OCT）。 OCT图像显示在支队尺寸变化（箭头在A，B和D）的IFNγ除了40-70分钟的前表面，从4个不同的实验（公元在左边的面板）。 A和B显示，IFNγ的增加其控制率的合资企业（≈2μL•厘米•HR -1）2至14和12μL•厘米2•HR - 1，分别为。除了​​JAK - STAT通路和PKA抑制剂，γ干扰素（C和D） -引起的吸收率显着降低为0.2和7.9μL•2•HR - 1厘米，分别。箭头指示的气泡的边界内虚线包围的区域，开始卷比较。图：右侧顶部面板摘要测量JV率从几个实验;中间面板电影（ <a href="http://www.jove.com/files/ftp_upload/2032/Bleb%205.wmv">点击这里</a> ）;下部面板- 3D B.蓝色的伪彩色总结实验的部分表示在t = 0的空间支队程度和40分钟后INFγ此外。 所有的动物实验与研究，在视觉与眼科声明协会进行遵守。该协议是由美国国立卫生研究院的动物护理和使用委员会批准。

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y(2)+receptor+agonist+INS37217+stimulates+RPE+fluid+transport+in+vitro+and+retinal+reattachment+in+rat.">The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.</a> Invest Ophthalmol Vis Sci. 43, 3555-3566 (2002).</li><li>Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204/211+alters+epithelial+physiology.">MicroRNA-204/211 alters epithelial physiology.</a> FASEB J. 24, (2010).</li><li>Adijanto, J., Banzon, T., Jalickee, S., Wang, N. S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2-induced+ion+and+fluid+transport+in+human+retinal+pigment+epithelium.">CO2-induced ion and fluid transport in human retinal pigment epithelium.</a> J Gen Physiol. 133, 603-622 (2009).</li><li>Li, R., Maminishkis, A., Zahn, G., Vossmeyer, D., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+mediates+attachment,+migration,+and+proliferation+in+human+retinal+pigment+epithelium:+relevance+for+proliferative+retinal+disease.">Integrin alpha5beta1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.</a> Invest Ophthalmol Vis Sci. 50, 5988-5996 (2009).</li><li>Peterson, W. M., Meggyesy, C., Yu, K., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+activates+calcium+signaling,+ion,+and+fluid+transport+in+retinal+pigment+epithelium.">Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium.</a> J Neurosci. 17, 2324-2337 (1997).</li><li>Voloboueva, L. A., Liu, J., Suh, J. H., Ames, B. N., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R)-alpha-lipoic+acid+protects+retinal+pigment+epithelial+cells+from+oxidative+damage.">R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage.</a> Invest Ophthalmol Vis Sci. 46, 4302-4310 (2005).</li></ol>

在目前的实验中，我们描述了我们先前公布的技术，旨在简化产生一致的hfRPE一个较高的数字每眼细胞的原代培养所需的多个步骤3的其他修改。在多种生理和分子生物学实验中的每个改变原来的程序，经过严格的测试，以确保变更没有引入文物，并不断被许多其他实验室用这些细胞进行测试。最后，进行了实验动物模型，在体外实验结果比较类似的扰动。

仪器需要清扫协议<ol><li>立体显微镜 - 任何需要放大解剖X250 </li><li>解剖菜 - 如金布尔100 x 20毫米＃23062（每清扫一）任何来源</li><li> Sylgard 184 - WPI的猫。 ＃SYLG184 * *为了准备解剖盘后续Sylgard 184包括混合指示，倒入培养皿混合液体弹性体，形成5 - 8MM层。允许弹性治愈至少24小时。菜固化的弹性体，可以使用70％乙醇和重复使用多次（100倍）的消毒</li><li> 27 G针头从屋宇署PrecissionGlide 1 ¼长度（每清扫5）任何来源</li><li> 1X的HBSS溶液中含有钙，镁等盐类 - GIBCO猫＃14025500毫升（好多个解剖）。 </li><li>虹膜剪刀硬质合金锯齿刀片，弧形 - 福斯特猫＃14559-11（一）。 </li><li>视网膜剪刀 - Katena猫＃K4 - 5300（一）。 </li><li>钳 - 如杜蒙镊子与WPI的猫打磨提示＃5＃500085（2件）。 </li><li>板载刀 - 爱尔康，皆伐1毫米，双斜面，角度猫＃8065921540。 </li><li>离心管15毫升 - （每眼）任何来源</li><li>巴斯德玻璃吸管 - （每清扫一）任何来源</li><li>图版12（每夹层）任何来源</li><li> Primaria25厘米2烧瓶- （每眼2-3）任何来源</li></ol>

experimental models for study of retinal pigment epithelial physiology and pathophysiology

我们已经开发出可以产生大量的初级人类胚胎视网膜色素上皮（hfRPE），母语的人RPE的特点的形态，生理和遗传，文化融合的单层细胞培养过程。这些hfRPE细胞培养表现出沉重的色素沉着，电子显微镜显示广泛的顶膜微绒毛。交界各种紧密连接蛋白的免疫荧光标记复合物。这些容易复制的原代培养的上皮极性和功能酷似以前学过本土的RPE细胞，包括人类的哺乳动物模型。这些结果是延长治疗干预在几个人的眼睛疾病的动物模型的发展。我们把重点放在消除异常液体积聚在视网膜或视网膜下腔的战略。视网膜细胞外空间分隔的感光细胞外段的视网膜色素上皮的顶膜和视网膜附件和维护整个主机的RPE /视网膜之间的相互作用的关键。

Watch this Scientific Journal Video about Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology at JoVE.com

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

我们提供了一个汇合单层培养的人胚胎视网膜色素上皮细胞（hfRPE）细胞重现的方法，表现出形态，生理，极性，和成人自然组织的蛋白质和基因表达模式。这项工作已经延伸到一些眼部疾病的动物模型。

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

experimental-models-for-study-retinal-pigment-epithelial-physiology

Research

JoVE Journal

Neuroscience

19.1K 次观看.  National Institutes of Health. 我们提供了一个汇合单层培养的人胚胎视网膜色素上皮细胞（hfRPE）细胞重现的方法，表现出形态，生理，极性，和成人自然组织的蛋白质和基因表达模式。这项工作已经延伸到一些眼部疾病的动物模型。

视频: Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

科研

神经科学

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

实验模型研究酸性后处理对脑缺血的神经保护作用

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

培养的原代鼠 RPE 的表征和形态动力学

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

作者聚焦：从成年小鼠中有效分离原代视网膜色素上皮细胞用于视网膜病变研究

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...