Name: puncture-induced iris neovascularization as a mouse model of rubeosis iridis
Uploaded: 2018-03-08T15:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

作者感谢 Linnea Tankred 和戴安娜 Rydholm 的畜牧业。

BALB/c 的两个性别的老鼠幼崽是按照在眼科和视力研究中使用动物的声明使用的, 这些议定书是由斯德哥尔摩伦理动物研究委员会批准的。老鼠被安置在凋落物中, 连同哺乳母亲, 每天有12小时/夜间循环, 免费获得食物和水, 每天都进行监测。
注: 在手术过程中, 小鼠在麻醉下使用挥发性异氟醚。眼部药膏在眼部手术中不鼓励, 因为它们可能会干扰治疗和使用的物质。如有必要, 为防止干眼, 可应用一滴无菌生理盐水。虽然葡萄膜黑色素穿刺是自我愈合, 在葡萄膜黑色素穿刺期间采取护理, 以确保不育的手术器械。术后治疗包括经皮下生理盐水溶出, 及盐酸丁卡因眼部局部用药镇痛。幼崽被允许恢复到胸骨卧床在一个暖气垫, 然后返回到护理母亲, 在一个干净的笼子。与同样的哺乳母亲保持了垃圾, 以避免压力。
1. 麻醉
<ol><li>准备麻醉感应室, 管理异氟醚麻醉, 并确保小鼠面罩也被耦合。 注: 执行所有动物实验与所有适用的道德许可协议。</li>
	<li>在大气空气中预先填充3-4% 挥发性异氟醚的感应腔。 注: 根据道德许可, 除异氟醚以外的其他麻醉物质可以使用。应在通风的工作台或通风罩上进行挥发性麻醉程序。</li>
	<li>在感应室放置一个产后 (P) 12.5 天的 BALB/c 小鼠幼犬。确认麻醉器具已准备好将异氟醚送到感应室。 注意: 老鼠幼崽是脆弱的, 最好处理的颈背。将幼崽放在一起作为垃圾或与哺乳母亲避免压力。</li>
	<li>让幼犬完全麻醉。</li>
	<li>轻轻地, 拿起小狗的颈背。 注: 保持感应室在任何时候关闭, 以安全和稳定的感应。</li>
	<li>把鼠标转移到老鼠面具上。移动鼠标时, 一定要将感应腔内的麻醉流转换为面罩。</li>
	<li>执行脚趾捏, 以确认麻醉。</li>
</ol>2. 外科立体镜穿刺程序
<ol><li>将鼠标定位在侧卧床位置。确认掩模定位良好, 使鼠标眼睛在立体镜的视野中。</li>
	<li>轻轻旋转鼠标头, 使眼睛朝向立体镜。 注意: 眼睛的清晰焦点应该在立体镜下实现。对于定位, 建议放大 16X, 而40X 应用于执行手术程序。如果道德许可允许, 修剪胡须的尖端可以促进眼睛的可视化。</li>
	<li>使用小栓钳, 小心地突出幼犬的眼睛, 施加向下的压力, 眼睑背部和腹部的眼睛。</li>
	<li>保持一个温柔, 但坚定的持有小狗的眼睛盖子, 用小型镊子。 注意: 推荐用非显性手进行眼突出和盖子, 因为主导手应执行穿刺程序。</li>
	<li>使用立体镜定位角膜缘。白化 BALB/c 的角膜缘可以很容易地识别由圆形血管丛后角膜。</li>
	<li>用0.25 毫米直径 (30 克) 斜面针, 执行小葡萄膜黑色素穿刺, 靠近 uvea 的后角膜缘限制。只使用针的尖端, 不超过一半的斜角 (相当于0.5 毫米), 刺穿 uvea。如果正确执行, 葡萄膜黑色素穿孔是自密封的, 但人工注射的研究物质可以通过相同的穿刺伤口管理。 注: 如有小鼠眼解剖学经验, 葡萄膜黑色素穿刺在睫状体与锯缘之间执行。在执行葡萄膜黑色素穿刺时必须小心, 以避免晶状体破裂。</li>
	<li>执行第二个葡萄膜黑色素穿刺在对面的眼睛从第一个穿刺地点。优化穿刺的距离和位置;考虑12和6点穿孔, 12 点的背部尽可能。</li>
	<li>每四天重复一次葡萄膜黑色素穿刺程序, 直到 P24.5。在每一个连续的葡萄膜黑色素穿刺前, 监视动物的状态特别注意在葡萄膜黑色素穿刺中由于晶状体损伤而出现的外伤性白内障, 或明显减少的眼压。目的是执行重复穿刺在相同的位置, 如前穿刺的最佳效果。</li>
</ol>3. 无创体内监测
<ol><li>在每个实验日的穿刺或注射前, 麻醉鼠标进行上述操作。</li>
	<li>用鼠标在侧卧床位置, 轻轻地凸出眼睛。</li>
	<li>以虹膜血管为中心, 以手术立体镜。</li>
	<li>拍一张虹膜脉片的照片, 相机安装在手术立体镜上。 注意: 虽然没有必要, 诱导减数分裂有助于规范虹膜区域的每一个动物。在选择减数分裂诱导剂时考虑道德许可。仔细的聚焦虹膜血管将有助于进一步定量分析。建议使用40X 级。</li>
</ol>4. 术后护理
<ol><li>从啮齿目动物面具中取出 BALB 幼犬, 然后将其轻轻地转移到一个用手术垫分层的加热垫上。</li>
	<li>应用一滴1% 丁卡因被刺穿的眼睛。 注意: 根据道德许可, 还可以使用其他止痛方法。</li>
	<li>用皮下注射200µL 的无菌生理盐水溶液对动物进行水合。</li>
	<li>在干净的老鼠笼子里把幼崽还给哺乳妈妈。</li>
	<li>监视恢复过程。幼崽应该能够 deambulate 和返回到垃圾。</li>
</ol>5. 眼球摘除术
<ol><li>宫颈脱位弄死动物。在立体镜下进行摘除和解剖, 以更好地可视化程序。 注: 可使用不同的安乐死程序。建议严格遵守道德许可。</li>
	<li>把眼睑拉开, 以改善眼睛的接触。</li>
	<li>用弯曲的波恩眼剪刀做一个小切口 (大约 0.5 cm) 在眼睛盖子的眼角附近。</li>
	<li>使用暴露的眼周空间, 在轨道内的地球后面插入微栓钳。</li>
	<li>抓住眼睛周围的组织, 轻轻地向上拉。避免抓住眼球, 使用靠近眼窝的周围组织。</li>
	<li>在眼眶内插入弯曲的波恩眼剪刀。</li>
	<li>切开周围的组织直到眼睛从插座中释放出来。 注意: 这种解剖技术保持眼部解剖和好处后分析。</li>
	<li>继续从眼睛中取出多余的组织。去除多余的组织可以在修复后进行, 如果首选。 注: BALB 小鼠黑色素缺乏, 因此, 为了增加对比度, 建议采用深色背景来促进解剖程序。</li>
	<li>在充满无菌磷酸盐缓冲生理盐水 (PBS) 的培养皿中简单冲洗全眼。</li>
	<li>将眼睛转移到含有1.5 毫升4% 缓冲福尔马林溶液的2毫升管上。</li>
	<li>在室温下固定眼睛6小时。 注意: 固定能赋予组织的刚性, 有利于虹膜的解剖。固定时间应测试和调整不同的应用。</li>
</ol>6. 立体镜下虹膜解剖程序
<ol><li>用1.5 毫升的塑料巴斯德吸管 (或类似的) 从管子中取出固定液, 用新鲜的 PBS 冲洗眼睛三次。</li>
	<li>将固定的眼睛放在平滑的干燥表面 (解剖支架) 上, 前面的腔朝向向上。</li>
	<li>用斜面30克针, 在角膜缘后执行一个入口点。</li>
	<li>用小栓钳握住前段, 将 Clayman Vannas 直剪刀的一尖端插入先前创建的开口。 注: 推荐用非显性手控制钳, 并采用具有主导性的剪刀。</li>
	<li>当用栓钳转动眼睛时, 在角膜缘周围切开以去除眼睛的后段。 注意: 执行部分切口将允许剪刀的内尖端继续在组织中, 并大大促进过程。</li>
	<li>将前段朝向向下, 露出晶状体。</li>
	<li>用小钳抓住它, 然后向上拉, 小心地取下镜片。 注: 斜面针可用于穿刺和拉镜头。</li>
	<li>将前段与角膜朝向下, 垂直于视野的切口定位。</li>
	<li>用小栓钳轻轻抓住睫状体后部的组织。</li>
	<li>用 Clayman-Vannas 直剪刀, 进行修剪刚刚前的睫状体, 以消除小梁网和隔离虹膜。确认小梁网被移除;虹膜应该在角膜内移动。</li>
	<li>小心转移前眼罩与小栓钳, 持有角膜, 到一个96井板包含200µL 的 PBS。</li>
	<li>继续保持在井中的角膜和解剖虹膜通过冲洗与 PBS 与一个吸管。 注意: 如果虹膜仍然附着在角膜上, 一些小梁网可能存在, 而30克针的斜角可以用来帮助取代虹膜。</li>
	<li>用小栓钳将角膜从96井中取出, 并将孤立的虹膜放在井中进行进一步分析。</li>
	<li>将游离漂浮的虹膜样品存放在摄氏4摄氏度。 注意: 尽管是固定的, 但不建议长期存储虹膜自由浮动样品。</li>
</ol>7. 可能的实验性读出
<ol><li>为了可视化解剖虹膜中的血管, 对血小板内皮细胞黏附分子 (PECAM)-1 或 isolectin B4 等标记物采用自由浮动全组免疫组织化学染色方法。或者, 使用全身灌注血管染色, 如荧光标记的高分子量 dextrans。</li>
	<li>使用体内无创图像执行在硅片平面安装和量化完整的虹膜血管与适当的成像软件。 注意: 使用密度测量法或与血管分析软件9可以量化血管内的体内和体外。</li>
	<li>处理整个眼球的核酸和蛋白质提取, 其次是 PCR 或免疫印迹法方法, 进行分子分析的穿刺眼。 注意: 组织的分子分析最好用非固定组织进行, 但非固定虹膜的解剖是非常具有挑战性的, 而整个眼睛的使用在以前的研究中呈现出统计学意义的结果8。</li>
</ol>

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作者没有什么可透露的。

在本协议中, 提出了一种新的葡萄膜黑色素穿刺诱导虹膜血管反应的方法。穿刺触发伤口愈合机制和促进血管反应在虹膜10,11。这与眼部病理一致, 如民主共和国和 NVG, 在虹膜虹膜的临床情况下, 眼睛后段视网膜血管生成反应加剧, 虹膜的血管化增加12 ,13。
角膜是缺血性的, 并且具有特殊的?...

虹膜, 连同睫状体和脉络膜, 包括 uvea, 这是最具血管的眼睛组织。虹膜血管是维持眼睛前腔内稳态的必要条件。由于动脉和静脉之间有大量的吻合口连接, 虹膜血管不仅为虹膜本身提供养分和氧气供应, 而且对眼睛的整个前段1。
新血管的形成, 或预先存在的血管生成, 是生理过程的基础, 如发展和伤口愈合2。血管内皮生长因子 (VEGF) 和纤溶酶原激活剂抑制剂 (排) 等多种典型因素对血管生成有很好的调节作用, 并有多个炎症因子, 这些因素的不平衡会导致病理血管生成3。
在眼中, 新生血管是引起视力威胁的疾病的原因, 例如增生性糖尿病视网膜病变 (NVG) 和新生血管性青光眼。在这些眼部疾病中, 病灶新生血管通常位于视网膜组织中, 但眼后和前眼室炎症和血管生成因子的不平衡与虹膜虹膜有关,临床术语虹膜病理血管生成 4.这些病理表明, 成人虹膜的能力, 以接受血管生成。小鼠出生后眼血管发育不成熟, 产后持续成熟。这种发展的特点被利用在老鼠模型的氧诱导视网膜病变, 一个模型, 密切模仿的临床条件的早产儿视网膜病变5。此外, 血管生成和炎症在创伤愈合机制中起关键作用6, 伤口愈合本身也与血管生成模型7有关。
在本研究中, 我们描述了穿刺诱导的虹膜新生血管模型。葡萄膜黑色素穿刺在边缘附近进行, 通过触发创面愈合系统诱导虹膜新生血管。由于角膜的透明性, 虹膜血管可以通过无创方法在体内进行简单的活体分析。穿孔的眼睛呈现增加的血管床在虹膜, 这是与增加的纤溶酶激活和炎症标记8。该模型具有很大的潜力, 作为一种新的工具来研究血管新生和筛选血管生成化合物, 并允许直接在体内可视化血管形成过程。

<table><tbody><tr><td>Bonn eye scissors</td><td>Bausch &amp;amp; Lomb</td><td>23060</td><td></td></tr><tr><td>Clayman-Vannas curved scissors</td><td>Bausch &amp;amp; Lomb</td><td>E3383 C</td><td></td></tr><tr><td>Clayman-Vannas straight scissors</td><td>Bausch &amp;amp; Lomb</td><td>E3383 S</td><td></td></tr><tr><td>Objective adapter for camera</td><td>Handcrafted</td><td>N/A</td><td>Or any system that allows adapting a camera to the microscope</td></tr><tr><td>Heating Pad 100-110 watts</td><td>Non Applicable</td><td>N/A</td><td>Available in pet/veterinarian stores</td></tr><tr><td>Hypodermic 30g beveled needle</td><td>KDM GmBH germany</td><td>911914</td><td></td></tr><tr><td>Iphone 4S</td><td>Apple</td><td>Non Applicable</td><td>Or other high resolution image acquistion device</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>KDG 9623</td><td></td></tr><tr><td>McPherson tying forceps</td><td>Bausch &amp;amp; Lomb</td><td>E1815 S</td><td></td></tr><tr><td>Micro tying forceps</td><td>Bausch &amp;amp; Lomb</td><td>63140</td><td></td></tr><tr><td>Minims tetracaine hydrochloride</td><td>Bausch &amp;amp; Lomb</td><td>N/A</td><td>1 % (w/v) Eye Drops</td></tr><tr><td>Neutral-buffered formalin</td><td>Bioreagens</td><td>0018-40</td><td></td></tr><tr><td>Normal saline solution</td><td>Fresenius Kabi</td><td>210352</td><td>0.9 % (w/v) NaCl in injectable water</td></tr><tr><td>Phosphate-buffered saline</td><td>ThermoFisher Scientific</td><td>10010023</td><td>Balanced and buffered PBS pH 7.4</td></tr><tr><td>Petri dish 10 cm</td><td>Starstedt</td><td>83.3902</td><td></td></tr><tr><td>Petri dish 3 cm</td><td>Starstedt</td><td>83.3900</td><td></td></tr><tr><td>Safe Seal Tube 2.0 mL</td><td>Starstedt</td><td>72.685.200</td><td>Or any eppendorf style tubes</td></tr><tr><td>TC plate 96-well</td><td>Starstedt</td><td>83.3924</td><td></td></tr><tr><td>Transfer pipette 3.5 mL</td><td>Starstedt</td><td>86.1171</td><td>Or any other Pasteur pipette style</td></tr><tr><td>Univentor 400 anesthesia unit</td><td>Univentor Limited</td><td>N/A</td><td>Or equivalent flow regulator with induction chamber and mask for volatile anesthesia</td></tr><tr><td>Wild M650 surgical microscope</td><td>Wild Heerbrugg</td><td>N/A</td><td>Or other surgical or magnifying stereoscope</td></tr></tbody></table>

puncture-induced iris neovascularization as a mouse model of rubeosis iridis

我们描述了穿刺诱导的虹膜新生血管模型作为无创性血管生成评价的一般模型。该模型还与靶向新生血管性青光眼有关, 这是糖尿病视网膜病变的一种视力威胁并发症。该方法以 BALB 小鼠的一系列自密封葡萄膜黑色素穿刺诱导虹膜血管反应为基础, 利用小鼠眼血管的产后成熟。老鼠幼崽从产后12.5 葡萄膜黑色素穿刺, 当幼崽自然睁开眼睛, 直到产后24.5 天。由于角膜的透明度, 虹膜血管可以通过无创的体内方法很容易地通过时间进行分析。此外, BALB 小鼠的半透明虹膜可 flatmounted 为详细的 immunohistologic 分析和最小的非特异背景染色。在该模型中, 血管生成主要由炎症和纤溶酶激活系统驱动。穿刺诱导模型是诱发小鼠虹膜新生血管的第一种, 具有直接无创性在体内分析血管生成过程的优势。此外, 该模型可与血管生成调节物质结合, 突出了其在血管新生研究中的潜力, 其中以体内为视角。

白化 BALB/c 小鼠幼崽在 P12.5 受到葡萄膜黑色素穿刺, 重复每第四天 (实验日 0, 4, 8, 12), 直到 P24.5。在 P27.5, 老鼠被安乐死和虹膜仔细解剖 (实验日 15)。在每一个实验日的每一个穿刺系列中, 用一台附有手术立体镜的照相机拍摄了老鼠眼的照片, 以评估虹膜血管反应的无创性评价。葡萄膜黑色素穿刺通过触发伤口愈合系统(图 1) 引起虹膜的血管反应。由于?...

Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

虹膜新生血管是缺血性视网膜疾病的常见并发症, 可能导致视力威胁的新生血管性青光眼。在这里, 我们描述了诱导实验性虹膜新生血管的小鼠协议, 可用于无创性评价血管生成调节物质。

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

puncture-induced-iris-neovascularization-as-mouse-model-rubeosis

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.8K 次观看.  Karolinska Institutet. 虹膜新生血管是缺血性视网膜疾病的常见并发症, 可能导致视力威胁的新生血管性青光眼。在这里, 我们描述了诱导实验性虹膜新生血管的小鼠协议, 可用于无创性评价血管生成调节物质。

视频: Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

小鼠模型的激光诱导脉络膜新生血管

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

科研

医学

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

神经科学

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 ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

优化视网膜静脉遮挡小鼠模型以限制变异性

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

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 ...

<meta charset="utf8"></head><body>用于研究视网膜病变的视网膜缺血再灌注损伤小鼠模型

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

<meta charset="utf8"></head><body>小鼠眼模型中视网膜缺血再灌注损伤的诱导

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

小鼠眼模型中视网膜缺血再灌注损伤的诱导

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

摘要

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此视频中的章节

小鼠模型的激光诱导脉络膜新生血管

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

优化视网膜静脉遮挡小鼠模型以限制变异性

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

小鼠眼模型中视网膜缺血再灌注损伤的诱导

小鼠眼模型中视网膜缺血再灌注损伤的诱导