Name: glaucoma-inducing procedure in an in vivo rat model and whole-mount retina preparation
Uploaded: 2016-03-12T14:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation at JoVE.com

C. Linn is supported by an NIH grant (NIH NEI EY022795).

使用受试动物的所有程序已经按照动物护理和使用委员会（IACUC）在西密歇根大学研究所的标准。 1.动物<ol><li>使用雄性和雌性大鼠3个月大了这项研究。 </li><li>保持12小时光/暗周期动物自由获取食物和水。 </li></ol> 2. KAX鸡尾酒动物麻醉的制备<ol><li>在5ml氯胺酮（100毫克/毫升）与1ml乙酰丙嗪（10毫克/毫升）和3毫升蒸馏水溶解50mg的甲苯噻嗪（20毫克/毫升）的。调匀。 </li><li>用注射器过滤消毒并存储该溶液到10ml血清瓶中。 </li></ol> 3.注射KAX <ol><li>称取动物（G），并返回到笼子里，直到准备注射。 </li><li>注入0.1毫升KAX / 100g的动物体重腹膜内，使用具有28克针1毫升胰岛素注射器。 </li><li>允许动物变得不省人事。捏脚和尾巴检查见长。 </li><li>保留所有动物安全实验室进行手术的时间。 </li><li>手术后，替代动物进入它们的笼子，并在RT保持舒适的意识，直到重新获得。只有回到动物动物设施当动物苏醒并恢复正常的行为。 </li></ol> 4.准备手术以及微大会<ol><li>使无菌2M的NaCl溶液。 </li><li>使用微电极拉出器（ 图1C）到拉一0.86毫米内径重抛光标准和薄壁的硼硅管成两个细的锥形玻璃微针（ 图1D，图1E）。 </li><li>从与用回填注射器针和1ml注射器（ 图1B）的2M食盐先前步骤回填一个微型针。从电极的尖端挖掘出气泡。 </li><li>填写第二1ml注射器用2M氯化钠。连接一个18G的针头，然后安装聚乙烯管（ 图1A）的长度（约10英寸）。使用针筒柱塞通过针来填充用生理盐水聚乙烯管。 </li><li>当两个微针和导管填充有盐水，仔细连接两个。消除任何空气中（ 图2）在它们之间的连接。 </li><li>刮得很轻轻地靠着一门课程纸巾的晶粒细锥微针的尖端。 </li><li>轻轻推动注射器柱塞，直到液体的微细流可以在纸巾中可以看出检查微针的电阻。的液体流应不大于0.5mm宽。 </li></ol> 5.编写动物<ol><li>应用1 - 2滴局部麻醉剂至角膜（丙美卡因盐酸眼用溶液，USP，0.5％）。等到没有眼反射发生。 </li><li>修剪用剪刀晶须。 </li><li>小号aturate与优碘溶液和周围的实验眼拭子区棉尖涂药。 </li><li>使用显微镜，重视止血钳将上下眼睑鼓起眼睛，露出巩膜静脉和限制眼球运动。 （ 图3，箭头） </li></ol> 6.青光眼诱导盐水注射<ol><li>当制备微针组装和动物，开始注射。 </li><li>当动物被证实是没有反应英尺/尾捏，小心地在到静脉（ 图3，白色箭头）10和20度之间的低角度来刺破用微针的巩膜静脉。一个成功的穿刺到静脉可知当血液进入微针（ 图3，黑色箭头）的前端。 </li><li>慢慢地，手动注入约50微升生理盐水静脉。这大约需要10秒。静脉将灼白盐循环救援人员到场啊脉管（ 图4，箭头）。一些地区可能维持一个血红色的外观（ 图4，箭头）。 <ol><li>执行第二注射到静脉，相对的第一部位，以确保对完整视网膜神经节细胞层彻底视网膜损伤。 注意:在几分钟之内，应该看到，通过眼睛的虹膜独特混浊外观的盐循环通过血管系统。 </li></ol></li><li>离开对面眼未处理用作内部对照。 </li></ol> 7.动物恢复<ol><li>取出止血。 </li><li>用棉签三联抗生素软膏（杆菌肽锌，硫酸新霉素，polymysin乙硫酸盐）适用于止血和注射部位夹住的部位。眼部周围组织损伤不会出现使用止血。 </li><li> &gt;将麻醉动物在笼子上的循环温水毯上一个耳鼻喉科体温过低。不时观察动物直至意识和正常行为的恢复。运输清醒动物回到动物殖民地。动物留在殖民地，直到牺牲的时间。 </li></ol> 8.动物牺牲和视网膜去除<ol><li>一个月之后，以诱发青光眼的过程中，动物用CO 2窒息和二次胸穿安乐死。 <ol><li>放置在腔室中的动物，并把盖子上的安全。 </li><li>打开的 CO 2和气体调节阀，以允许20％体积/分钟的 CO 2的位移 氧气在该腔室。 </li><li>允许四到5分钟的动物过期。 </li><li>关闭两个阀门。 </li><li>从腔室中取出的动物和用无菌解剖刀执行辅助胸椎穿刺。 </li></ol></li><li>安乐死后，用手术刀切在眼眶周围的眼睛，贝结缔组织NG小心不要切入眼球本身。 </li><li>小心使用曲边剪刀剪断视神经和任何剩余的组织中提取完整的眼球。将提取的眼球无菌60毫米×15毫米培养皿一次性含有新鲜的PBS菜。 </li><li>使从眼球的眼罩。要做到这一点，请用手术刀小切口正好后方虹膜和巩膜之间的边界。按照眼部周围的小弹簧剪刀圆周此切口从眼球取出角膜半球。连接到视神经半球保持。 </li><li>查找安乐死的动物眼罩内的非常薄的粉红/米色视网膜。握住钝镊子视网膜的色素层，以稳定眼罩。使用另一对闭合钳非常轻轻挑逗整个完整的视网膜落睛的背面。避免捏，拉，或揪着直接视网膜。 </li><li>用小弹簧剪刀剪开区域，视神经仍连接到视网膜。 </li><li>一定要切掉从视网膜任何残留色素上皮或巩膜组织。 </li><li>使用移液管，分离的视网膜轻轻转移到涂覆的含有新鲜的PBS35毫米×10mm的皮氏培养皿一干净SYLGARD。 </li></ol> 9.整个安装视网膜准备<ol><li>一旦在Sylgard菜，使用镊子和一个仙人掌针引脚视网膜到位。保持视网膜神经节细胞层朝上和视神经下来。视网膜的半球形状，即使固定后显着。视网膜的曲率将卷曲朝向天花板时视网膜神经节细胞层是在所希望的方向。 </li><li>用小剪刀到视网膜切成四个象限，使得四叶草从视神经头散热的形状。 </li><li>针附加仙人掌针的视网膜象限使视网膜尽可能平坦无覆盖UT拉伸（ 图5）。 </li><li>修复在Sylgard菜的固定视网膜用3毫升的4％多聚甲醛O / N在室温。 </li></ol>视网膜10.抗体染色注意:与第一和第二抗体染色的固定视网膜中视网膜观看神经元（ 图6）。 <ol><li>漂洗固定的，平面安装视网膜2分钟3次，每次在PBS中。 </li><li>透视网膜，用1％的Triton X-100用在PBS中的1％胎牛血清60分钟。 </li><li>漂洗视网膜三次，每次2分钟，在PBS中。 </li><li>用0.1％的Triton X-100的PBS，每次洗涤5分钟漂洗两次。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>与在RT 1％的Triton X-100和1％胎牛血清的PBS中孵育45分钟。 </li><li>用0.1％的Triton X-100的PBS，每次洗涤5分钟漂洗两次。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>孵育在PBS中的3毫升1-％胎牛血清每个视网膜用纯化的小鼠抗大鼠CD90 /小鼠CD90.1（1:300稀释）O / N在室温。 </li><li>用0.1％的Triton X-100冲洗视网膜一旦在PBS 5分钟。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>继发的Alexa Fluor 594山羊抗小鼠IgG孵育在3ml的PBS（无FBS）的各视网膜（1:300）O / N在室温。 </li><li>用PBS洗涤宽松视网膜。 </li><li>使用解剖显微镜，小心地从视网膜移除仙人掌针。 </li><li>轻轻地用移液管转移到视网膜显微镜载玻片。一定要保持朝向天花板视网膜神经节细胞层的方向。视网膜的半球形状，即使固定后显着。视网膜的曲率将卷曲朝向天花板时视网膜神经节细胞层是在所希望的方向。 </li><li>吸收任何过量的PBS与的Kimwipe或其它这类吸湿材料。要小心，不要吸收视网膜。 </li><li>加入5滴½甘油和重量上午½PBSounting媒体。 </li><li>盖上盖玻片视网膜，避免产生气泡。 </li><li>安全使用盖玻片透明指甲油，胶水或其他粘合剂。 </li></ol>

The authors have no conflicting or competing interests to disclose.

这个协议描述诱导在体内大鼠模型青光眼样病症的方法。此过程使用高渗盐水的注射诱导疤痕在小梁网29，32。发展疤痕组织闭塞房水的流出这增加前房压力。随着流出和压力降低建立，通过弹性悬挂韧带镜头推回到玻璃体腔。玻璃体然后施加压力到视网膜损害脆弱的视网膜神经细胞。使用此过程我们的结果表明，视网膜神经节细胞数开始以2周1个月后的过程神经节细胞损失显著损?...

青光眼是一组影响的神经元中的视网膜，具体而言，视网膜神经节细胞1-2眼疾。这些细胞的轴突收敛到成为视神经承载可视信息，其中视觉感知的大脑。因此损害视网膜神经节细胞和其轴突导致视觉缺陷。 与青光眼疾病相关的主要特征是RGC变性和死亡，增加的眼内压（IOP），并视盘拔罐和萎缩。这些特点导致视野丧失或完全，不可逆转的失明。目前，青光眼全球7000万人造成3失明。因此，它是世界上第三大致盲4的原因。 RGC死亡的青光眼的确切机制尚不清楚。许多研究已经完成，以解开这个谜。然而，已经知道的是，青光眼的主要危险因素之一是增加我Ñ​​眼压由于房水（AH）在眼睛的前房的不规则循环。 AH充当眼睛的缺血性前房无色透明的替代血液。它滋养周围的细胞，分泌删除废物从代谢过程，运输的神经递质，并允许在病理状态1的药物和眼内炎症细胞的循环。 房水流通的维护包括睫状体和小梁网。房水由睫状体产生的。然后，它流入前房维持眼组织的总体健康状况。 75 - 房水外流的80％时，流体通过在睫状肌海绵组织的三层过滤正在积极通过非颜料睫状体上皮分泌的。穿过小梁网并通过施累姆氏管其制止所述流体排出独立实体进入血液系统5。其余20 -流出的25％绕过小梁网和通过超滤和扩散通过uveo巩膜途径被动分泌。这一途径似乎是相对独立的眼压1。 当房水的生产和流出都失去平衡，压力增大眼内。如上所述，这种增加的眼内压是在青光眼的发展的主要危险因素。这种压力导致在眼睛的回视网膜神经元的复杂的层的损伤。损伤视神经的视网膜神经节细胞轴突导致大脑不再接收准确的视觉信息。其结果是，视觉的知觉丧失，并且可以发生完全失明。 到目前为止，还没有治愈青光眼。不同的治疗方法存在的主要目的是降低眼压。这些措施包括外用药物类，如β1的肾上腺素能受体阻断剂，或局部前列腺素类似物。 β阻滞剂通过减少房水的产生7的降低眼内压。前列腺素的作用，通过增加房水8-14外流以减少眼压。 α-肾上腺素能激动剂和碳酸酐酶抑制剂也可作为治疗的辅助方法。阿尔法肾上腺素受体 ​​激动剂通过葡萄膜巩膜途径15-17加大流出。碳酸酐酶抑制剂，通过抑制酶的18降低生产AH的。更侵入性程序也被用于治疗青光眼。激光小梁成形术是用于增加房水19的流出。另一个手术疗法，称为小梁切除术，创建了一个另类的排水现场时，传统的小梁途径被阻断20-21过滤啊。 这些治疗方案已经知道EFFectively眼压降低。然而，青光眼患者的高达40％的正常显示眼压水平表明需要更完整的治疗方法。22,23此外，在青光眼看到视网膜神经节细胞的死亡是不可逆的，一旦开始，目前的治疗不停止疾病的进展24-28。这强调了靶向神经元本身的生存有效的神经保护治疗的需要。青光眼模型的发展是这一发展的关键。 在这项研究中，我们证明使用最初由莫里森29中概述的改性过程诱导成年龙Evans大鼠青光眼样作用的方法。在此过程中，2M的高渗盐水注射到巩膜静脉丛被瘢痕组织引起青光眼样的条件，以减少在小梁网，导致增加的眼内压和视网膜神经节细胞的w的显著损失水状液流出ithin程序30-31的一个月。青光眼诱导程序，如这里描述的，可能是关键解锁在青光眼治疗的新发展。

<table><tbody><tr><td>Xylazine hydrochloride, Minimum 99%</td><td>Sigma, Life Science</td><td>X1251-1G</td><td></td></tr><tr><td>Ketamine hydrochloride injection, USP, 100mg/ml&amp;nbsp;</td><td>Putney, Inc</td><td>NDC 26637-411-01</td><td>10 ml bottle</td></tr><tr><td>Acepromazine Maleate, 10mg/ml</td><td>Phoenix Pharmaceutical, Inc</td><td>NDC 57319-447-04, 670008L-03-0408</td><td>50 ml bottle</td></tr><tr><td>Serum bottle, 10 ml</td><td>VWR</td><td>16171319</td><td>Borosilicate glass</td></tr><tr><td>1 ml insulin syringe&amp;nbsp;</td><td>VWR</td><td>BD329410</td><td>28 G needle&amp;nbsp;</td></tr><tr><td>Sodium chloride</td><td>Sigma&amp;nbsp;</td><td>S7653</td><td>2 M Solution&amp;nbsp;</td></tr><tr><td>Microelectrode Puller&amp;nbsp;</td><td>Narishige Group</td><td>PP-830</td><td></td></tr><tr><td>Heavy Polished Standard and Thin Walled Borosilicate Tubing&amp;nbsp;</td><td>Sutter Instruments</td><td>B150-86-10HP</td><td>without filament, 0.86 mm</td></tr><tr><td>Microfil syringe needle for filling micropipettes</td><td>World Precision Instruments, Inc</td><td>MF28G</td><td></td></tr><tr><td>18 gauge Luer-Lock needle</td><td>Fisher Scientific</td><td>1130421</td><td>Syringe needle</td></tr><tr><td>Flexible Polyethylene Tubing</td><td>Fisher Scientific</td><td>22046941</td><td>0.034 inch diameter, approximately 10 inches&amp;nbsp;</td></tr><tr><td>Proparacaine Hydrochloride Opthalmic Solution, USP, 0.5%</td><td>Akorn, Inc</td><td>NDC 17478-263-12</td><td>15 ml&amp;nbsp; sterile bottle&amp;nbsp;</td></tr><tr><td>Curved Scissors</td><td>Fine Science Tools</td><td>14061-11</td><td></td></tr><tr><td>Microscope</td><td>Leica&amp;nbsp;</td><td>StereoZoom 4</td><td></td></tr><tr><td>Hemostat Clamp&amp;nbsp;</td><td>Fine Science Tools</td><td>1310912</td><td>curved edge</td></tr><tr><td>Triple Antibiotic Ointment&amp;nbsp;</td><td>Fisher Scientific</td><td>NC0664481</td><td></td></tr><tr><td>Scalpel handle</td><td>Fine Science Tools&amp;nbsp;</td><td>10004-13</td><td></td></tr><tr><td>Scalpel blade #11</td><td>Fine Science Tools&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>60 mm x 15 mm Disposable Petri Dish</td><td>VWR</td><td>351007</td><td></td></tr><tr><td>Phosphate Buffered Saline 10x Concentrate</td><td>Sigma, Life Science&amp;nbsp;</td><td>P7059-1L</td><td>1x dilution&amp;nbsp;</td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools&amp;nbsp;</td><td>15009-08</td><td></td></tr><tr><td>Forceps (2), Dumont # 5</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>3 ml Transfer Pipets, polyethylene, non sterile</td><td>BD Biosciences</td><td>357524 or 52947-948</td><td>1 and 2 ml graduations</td></tr><tr><td>35 mm x 10 mm Easy Grip Petri Dish&amp;nbsp;</td><td>BD Biosciences</td><td>351008</td><td></td></tr><tr><td>Sylgard 184</td><td>VWR</td><td>102092-312</td><td></td></tr><tr><td>Cactus Needles</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMD Millipore&amp;nbsp;</td><td>PX0055-3 or 818715.0100</td><td>Made into a 4% solution&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T9284-100 ml</td><td>Made into both a 1% and 0.1% solution&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlanta Biological</td><td>S11150</td><td>500 ml</td></tr><tr><td>Purified Mouse Anti-Rat CD90/mouse CD90.1</td><td>BD Pharmingen</td><td>Cat 554892</td><td>1:300 dilution&amp;nbsp;</td></tr><tr><td>Alexa Fluor 594 goat anti-mouse&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>A11005</td><td>1:300 dilution&amp;nbsp;</td></tr><tr><td>Microscope Slides</td><td>Corning&amp;nbsp;</td><td>2948-75x25</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>G5516-100 ml&amp;nbsp;</td><td>50% glycerol to 50% PBS, by weight&amp;nbsp;</td></tr><tr><td>Coverglass&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>2975-225</td><td>Thickness 1 22 x 50 mm&amp;nbsp;</td></tr><tr><td>Confocal Microscope</td><td>Nikon&amp;nbsp;</td><td>C2 Eclipse Ti</td><td></td></tr></tbody></table>

glaucoma-inducing procedure in an in vivo rat model and whole-mount retina preparation

青光眼是一种影响视网膜神经节细胞（RGC）的中枢神经系统的疾病。研资局轴突组成视神经进行视觉输入到大脑的视觉感受。损害视网膜神经节细胞和它们的轴突导致视力丧失和/或失明。尽管青光眼的具体原因不明，为疾病的主要危险因素之一是升高的眼内压。在动物模型青光眼诱导过程是一个有价值的工具，研究人员在研究RGC死亡的机制。这样的信息可导致可能在预防视力丧失的有助于有效的神经保护治疗的发展。本文中的协议描述诱导的青光眼的方法-像，其中50微升2M的高渗盐水注入巩膜静脉丛在体内大鼠模型的条件。血管的热烫表示成功注入。此过程会导致视网膜神经节细胞的损失来模拟青光眼。一个月以下注射后，将动物处死，眼睛被除去。接着，角膜，晶状体和玻璃体被除去以使一个眼罩。视网膜然后从眼睛的后部剥离和钉扎到使用仙人掌针SYLGARD菜肴。在这一点上，在视网膜的神经元可染色进行分析。从这个实验结果表明，相对于内部控制时，视网膜神经节细胞的约25％是在过程的一个月内丢失。这个过程允许在体内大鼠青光眼模型的视网膜神经节细胞死亡的定量分析。

本节说明用于诱导在体内大鼠青光眼模型青光眼样条件的装置组件和过程。我们表明用于执行高渗盐水注射导致眼内压增加的个体的工具和设备。我们展示了注入以其特有的漂白效果和前房导致的浑浊外观巩膜静脉丛。我们还描述了视网膜去除和平面安装丢失RGC的分析过程。最后，我们显示在视网膜神经节细胞存活的注射的效果。作为视网膜神经节细胞的分布是在?...

Watch this Scientific Journal Video about Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation at JoVE.com

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

glaucoma-inducing-procedure-an-vivo-rat-model-whole-mount-retina

Research

JoVE Journal

Medicine

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

13.0K 次观看.  Western Michigan University. Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis. 

视频: Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

科研

神经科学

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

生物工程

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

A磁条微珠缺血模型，以诱导小鼠高眼压，青光眼相关

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

医学

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

非动脉炎性前部缺血性视神经病变的啮齿动物模型（rNAION）

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

大鼠模型中的视网膜病理生理学评估

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

视网膜低温切片、全支架和低渗隔离血管毛细血管微血管组织免疫组化的制备

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

生物学

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

经结膜入路注射大鼠视神经

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

Source: Gossman, C. A. et al. <a href="https://app.jove.com/v/53831">Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation.</a> J. Vis. Exp. (2016)
This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm&#39;s canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

一种高渗盐水注射技术诱导大鼠青光眼

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

青光眼诱导在程序

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