Name: oxygen-induced retinopathy model for ischemic retinal diseases in rodents
Uploaded: 2020-09-16T14:30:00.000+00:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents at JoVE.com

我们感谢玛丽安·卡尔斯伯格、安妮·玛丽·哈帕涅米、佩维·帕塔宁和安妮·坎库宁提供出色的技术支持。这项工作由芬兰科学院、皮维基和萨卡里·索尔伯格基金会、坦佩雷结核病基金会、芬兰医学基金会、皮尔坎马医院地区研究基金会和坦佩雷大学医院研究基金资助。

此处描述的议定书已得到芬兰国家动物伦理委员会的批准（协议编号ESAVI/9520/2020和ESAVI/6421/04.10.07/2017）。
1. 实验动物和鼠标OIR模型感应
注:使用时间适应的动物，例如常用的C57BL/6J小鼠，让幼崽在同一天出生。使用培养水坝，例如129株（129S1/SvImJ或129S3/SvIM）哺乳水坝，在诱发超氧期间和之后给幼崽喂奶。或者，确保有额外的哺乳水坝可用的情况下，护理水坝需要更换由于疲惫。使用 C57BL/6J 小鼠/水坝时，将每个大坝的垃圾大小限制为 6-7 幼崽（如果垃圾大于幼崽往往限制体重增加）16。
<ol><li>记录动物在超氧诱导前后的重量，以及牺牲时的体重。</li>
	<li>确保笼子底部有足够的食物，这样水坝就很容易获得食物。</li>
	<li>在不使用过滤系统时，将带有颜色指示器的苏打石灰添加到腔室底部，以吸收多余的 CO2。</li>
	<li>监测室内的湿度和温度，并将湿度保持在 40% 到 65% 之间。如果需要，通过在房间底部放置带水的菜肴（例如培养皿），增加室内的湿度。</li>
	<li>校准 O2 传感器与 100% O2 和正常的房间空气。</li>
	<li>将 P7 小鼠放入一个腔室，并将 O2 级别设置为 75%。将老鼠留在室内5天，直到P12。避免在超氧感应期间打开腔室。检查 O2 气缸的气体压力，并在需要时更换气缸。在上岗期间监测动物。</li>
	<li>把老鼠笼子从房间里拿出来，称重所有的幼崽。根据体重对幼崽进行分组，使每个实验组在幼崽中的体重分布相似。</li>
</ol>2. 实验动物和大鼠OIR模型诱导（使用半封闭系统）
注:使用时间适应的动物让幼崽在同一天出生。对于大鼠OIR，使用增加的垃圾大小，约18幼崽/大坝，以获得足够的NV感应在老鼠模型。从几个垃圾池幼崽获得足够的幼崽到每个垃圾。
<ol><li>记录动物在上岗前后的重量，以及牺牲时的体重。</li>
	<li>确保笼子底部有足够的食物，这样水坝就很容易获得食物。</li>
	<li>在不使用过滤系统时，将带有颜色指示器的苏打石灰添加到腔室底部，以吸收多余的 CO2。</li>
	<li>监测室内的湿度和温度。通过在腔底添加二氧化硅凝胶来吸收额外的湿度（由多个数量的老鼠产生）。</li>
	<li>用 100% N2 和普通房间空气校准 O2 传感器。</li>
	<li>将大鼠放入P0室（出生后几个小时）。将 O2 级别设置为 50%，并将 O2 气缸连接到腔室 24 小时。之后，将设置切换到 10% O2， 并将氮气 （N2） 圆柱体连接到腔室 24 小时。继续 24 小时骑自行车在 50% 和 10% O2 水平之间 14 天。</li>
	<li>在研究期间监测动物的气体消耗和健康。在 50/10% O2 之间的变化期间打开房间，并在需要时添加更多的食物和水。在诱导过程中将动物的笼子改为清洁笼子。</li>
	<li>把老鼠笼从房间里拿出来，称重所有的幼崽。根据体重对幼崽进行分组，使每个实验组在幼崽中具有相似的体重分布。</li>
</ol>3. 药物管理（可选）
注:OIR 中常用的药物管理路线是通过静脉注射治疗 （ivt），在 P12-P14 为小鼠，在 P14 为大鼠。根据实验设置确定治疗日。当实验中使用多只幼崽时，将治疗组从所有垃圾中分离出动物。最好将药物注射到一只眼睛，并保持反向眼睛作为控制。
<ol><li>称量动物，并在尾巴和/或耳朵上留下识别标记。</li>
	<li>麻醉动物要么注射麻醉（例如氯胺酮和美多米丁的混合物，30毫克/公斤和0.4毫克/公斤的小鼠）或与吸入麻醉（异丙酮在2-3.5%异丙酮和200-350 mL/分钟气流）。捏脚趾检查麻醉深度。治疗期间将动物放在加热垫上。</li>
	<li>对于局部麻醉，请将一滴镇痛药涂在眼睑上。在执行 ivt 之前，用钳子小心地打开眼睑，小鼠和大鼠在 P14 周围睁开眼睛。在角膜上涂抹一滴镇痛药（例如盐酸氧蛋白）。</li>
	<li>在进行 ivt 注射之前，先涂抹一滴碘。</li>
	<li>对于 ivt 注射，请使用带有 33-34 G 针头的玻璃注射器。按下眼睑，用钳子抓眼球。使注射后侧的跛行，大约在45° 角针指向视神经。</li>
	<li>避免向内部空间注入超过 1.0 μL。注射药物后保持针头到位30s，以避免注射溶液倒流。</li>
	<li>切除针头后，检查眼睛（例如，用眼镜）是否出现任何并发症，如出血或视网膜损伤。注射后在角膜上涂抹抗生素软膏。 注:小鼠的ivt注射量应为0.5-1.0μL。</li>
	<li>逆转麻醉（例如，用+2拮抗剂进行美多米丁（2.5毫克/千克），将小狗送回笼子。通常把垃圾存放到研究结束。</li>
</ol>4. 体内成像和电视网膜成像（可选）
<ol><li>如有需要，在随访期间对活体动物进行体内成像，以记录在血管反应期间视网膜发生的变化。例如，执行荧光素血管造影术 （FA） 或扫描激光结肠显微镜19以可视化血管（图 4）。使用光谱域光学相干断层扫描 （SD-OCT） 在体内可视化视网膜层 （图 4）。</li>
	<li>如有需要，使用电视网膜成像（ERG）（图5）调查OIR诱导后不同视网膜细胞群的功能变化。</li>
</ol>5. 视网膜平坐架的组织收集和准备
注:根据所需的研究假设收集组织。对于小鼠，收集样本，例如在P12（研究高氧化期后的血管消灭）或在缺氧期（P13-P17）。在 P17（最常见的取样时间点）收集鼠标 OIR 样本，以检测 NV 量的峰值。在大鼠OIR中，在P18-P21采集样本，以观察最高数量的NV（图3）。
<ol><li>取样前称量动物。</li>
	<li>为了给视网膜血管贴上标签，深度麻醉动物可以用FITC-德克斯特兰进行心电图的注入。（或者，稍后用异黄素涂污视网膜平面坐骑）。</li>
	<li>牺牲动物使用过量的麻醉药物（例如氯胺酮和美多米丁的混合物，300毫克/公斤和4毫克/公斤的小鼠）或二氧化碳 吸入。</li>
	<li>用弯曲的钳子抓住眼球后面，切开眼睛周围的组织，把眼睛从轨道上抬起来，来收集动物的眼睛。</li>
	<li>将眼球孵化在新鲜制作的、过滤的 4% 甲醛（磷酸盐缓冲盐水中，PBS）中，1-4 小时。取出固定剂，用PBS洗眼球3×10分钟。立即解剖视网膜，或在+4°C时将其存储在PBS中。 警告:甲醛是有毒的吸入，接触皮肤，如果吞咽。请在使用安全数据表之前阅读。 注意:如果完成整个眼球的横截面，在取样或组织处理的任何阶段不要对眼球施加压力，以避免视网膜分离。</li>
	<li>准备视网膜平面安装，以量化NV的量和AVA的大小。或者，处理眼球/视网膜以进行组织学或RNA或蛋白质分析。使用微剪刀和钳子在立体显微镜下解剖视网膜。<ol>
<li>将眼球放在PBS中，使其保持湿润，用针（23G）刺穿边缘的眼球，并用弯曲的微剪刀切开眼球，以去除虹膜和角膜。</li>
		<li>小心地将剪刀尖放在硬皮和视网膜之间，并将硬皮切向视神经。对眼球的另一侧也这样做，并小心地切割/撕裂硬盘，直到视网膜杯暴露。将镜头从视网膜杯中取出，并将 PBS 添加到杯子中。</li>
		<li>在不损坏视网膜的情况下清除所有透明质容器、残体和碎屑。通过将PBS添加到视网膜杯中来洗净视网膜。用直微剪刀对网膜进行四个切口（12点、3点、6点和9点），以制作花状结构。可选地，在安装样品之前用手术刀片进行切割。使用柔软的画笔将网膜提升到良好的板材上进行染色。</li>
	</ol>
</li>
	<li>使用同位素 B4标记视网膜血管，该血管会弄脏内皮细胞的表面（如果动物没有注入 FITC-dextran）。在阻塞缓冲区（10% NGS = TBS 中 0.5% 特里顿）中孵化视网膜 1 小时，在 TBS 中用 1% NGS = 0.1% 三顿洗涤 10 分钟。将视网膜与荧光染料结合的异丙汀 B 4（5-10μg/ml）在 TBS 中孵育 1% NGS = 0.1% 特里顿过夜，在 +4 °C 下，同时防止光线照射。 注:如果需要，使用特定抗体标记其他细胞，如炎症细胞和小细胞。</li>
	<li>用 TBS 中的 1% NGS = 0.1% 三顿洗净视网膜 3x 10 分钟，并在微观滑梯上抬起视网膜，内视网膜朝上。使用软画笔小心地展开网膜，并清除任何剩余的透明质容器或碎屑。将安装介质添加到盖滑，并将其放置在网膜顶部。将视网膜存储在+4°C，并防止光线照射。</li>
</ol>6. 平坐架分析
<ol><li>使用具有 10 倍目标的荧光显微镜拍摄视网膜平面坐骑的图像。专注于表面血管丛和前视神经血管化。制作磁贴扫描图像以捕获整个视网膜并合并磁贴扫描</li>
	<li>通过使用图像处理程序测量 AVA、NV 面积和总视网膜区域来量化图像（见 材料表）。<ol>
<li>使用免费手绘工具绘制 AVA 和总视网膜区域，然后使用选择工具选择神经区。该软件测量对像素感兴趣的区域，AVA 和 NV 区域（以像素表示）可用于计算它们相对于视网膜总面积的百分比。此外，一些软件工具可用于量化NV。 注:最近，采用深度学习神经网络对OIR图像关键值进行量化的开源、全自动化程序已经推出，为视网膜AVA和NV（例如https://github.com/uw-biomedical-ml/oir/tree/bf75f9346064f1425b8b9408ab792b1531a86c64）20、21的可重复量化提供了可靠的工具。</li>
	</ol>
</li>
	<li>如果使用抗体对单个细胞群进行免疫组织化学检测，则可在需要时用手或自动图像分析系统从视网膜平面安装中量化染色细胞的数量（如微胶质、图2B）。</li>
</ol>7. 统计
<ol><li>根据学生 t 测试或单向 ANOVA 分析通常分发的数据，然后酌情进行邓内特或图基的多次比较测试。使用非参数测试，如曼-惠特尼U测试或克鲁斯卡尔瓦利斯测试的非正常分布数据。考虑在 P < 0.05 级别具有统计显著性的差异。</li>
</ol>

作者玛丽亚·维赫图帕博士、尼娜·约斯凯莱宁、马克·塞拉达-吉梅内斯博士和鲁比娜·塔帕是实验有限公司的员工。
作者Giedrius Kalesnykas博士是一名雇员（总裁兼首席执行官），也是实验有限公司的股东，该公司提供合同研究服务，使用本文中使用的学前OIR模型。
特罗·约尔维宁，医学博士，博士，汉内勒·乌西塔洛-约尔维宁，博士，没有什么可透露的。

疾病表型的严重程度取决于小鼠和大鼠OIR模型23中的菌株甚至供应商。这表明病理学发展存在广泛的基因变异性。一般来说，色素啮齿动物比白化病的啮齿动物发展更严重的表型。例如，白化病BALB/c的视网膜血管在多发性缺氧后迅速重新血管化，24日不会发展为NV。同样，在大鼠中，色素棕色挪威大鼠表现出比白化病更严重的病理学（SD）大鼠25。</s...

需要可靠和可重复的实验模型来研究血管原性眼病背后的病理学，并开发新的治疗这些破坏性疾病的方法。病理血管生成是湿年龄相关的黄斑变性（AMD）和许多缺血性视网膜疾病的标志，其中包括早产（ROP）、增殖性糖尿病视网膜病变（PDR）和视网膜静脉闭塞（RVO）1，2，3，4。人类和啮齿动物视网膜遵循类似的发展模式，因为人类和啮齿动物视网膜都是最后被血管化的组织之一。在视网膜血管完全发育之前，视网膜从透明体血管中接收其营养供应，当视网膜血管开始发育1、2时，直肠血管会退步。在人类中，视网膜血管发育在出生前完成，而在啮齿动物中，视网膜血管的生长发生在出生后。由于视网膜血管发育发生在啮齿动物产后，它提供了一个理想的模型系统来研究血管生成2，3。新生啮齿动物有一个血管性网膜，逐渐发展，直到到第三产后第4周结束时完成血管网膜发育。新生儿小鼠的血管生长是塑料的，它们在高氧刺激5期间会经历回归。
ROP是西方国家儿童失明的主要原因，因为它影响近70%的出生体重在1250克6，7以下的早产儿。ROP 发生在早产儿中，这些婴儿在视网膜血管完成正常生长之前出生。ROP进展分两个阶段:在第一阶段，早产延迟视网膜血管生长，在第二阶段之后，发育中的视网膜未完成血管化导致缺氧，从而诱发血管生长因子的表达，刺激新的和异常的血管生长8。OIR模型是研究ROP和其他缺血性视网膜病理学以及测试新药候选者2、3、9的一个广泛使用的模型。它被广泛认为是进行眼部和非眼部疾病潜在抗心原药物概念验证研究的可重复模型。两种啮齿动物模型，即小鼠和大鼠OIR在诱导模型和疾病表型上有所不同。大鼠模型更准确地模仿ROP表型，但小鼠模型为视网膜神经化（NV）提供了一个更坚固、更快、更可重复的模型。在鼠标模型中，NV 发展到中央视网膜。这种病理读出对于许多缺血性视网膜病变（如 PDR、RV 和渗出性 AMD）以及癌症等非眼性血管病的药理疗效研究非常重要。此外，基因操纵（转基因和淘汰）小鼠的可用性使小鼠OIR模型成为更受欢迎的选择。然而，无论是小鼠还是大鼠OIR模型都没有产生视网膜纤维化，这在人类疾病中是典型的。
认识到高氧水平有助于在20世纪50年代发展ROP10，11导致动物模型的发展。关于氧气对视网膜血管影响的第一批研究是在1950年12月12日、13日、14日进行的，直到20世纪90年代，OIR模型还进行了许多改进。史密斯等人在1994年的研究为目前的小鼠OIR模型设定了一个标准，该模型将透明质病与视网膜病变15分开。康纳等人广泛采用的血管抹杀和病理NV量化方法（2009年）进一步增加了其受欢迎程度16.在这个模型中，小鼠被放置在75%的氧气（O2）在P7 5天，然后5天在正常条件下。从 P7 到 P12 的超氧会导致视网膜血管在中央视网膜中退步。回到正常状态后，血管视网膜变得缺氧（图1A）。由于血管中央视网膜的缺氧刺激，一些视网膜血管发芽向视网膜，形成前视网膜NV，称为前视网膜塔夫茨2，3。这些塔夫特是不成熟的，而且透水性很强。NV 峰值量在 P17，之后会退步。视网膜完全重新血管化，NV由P23-P25（图2A）2，3完全回归。
大鼠OIR模型（使用不同级别的O2）在20世纪90年代首次描述，显示不同的O2水平在80%和40%导致更明显的NV比低于80%O2恒定接触17。后来发现间歇性缺氧模型，其中O2循环从超氧（50%）到缺氧（10-12%），导致更多的NV比80/40%O2模型18。在50/10%的模型中，大鼠幼崽在24小时内接触50%，随后在10%O2中接触24小时。这些周期一直持续到P14，当老鼠幼崽回到正常状态（图1B）。与人类ROP患者一样，在大鼠模型中，由于视网膜血管丛不成熟，血管区域发展到视网膜外围（图3）。
在这两种型号中，通常量化的主要参数是 AVA 和 NV 的大小。这些参数通常从视网膜平面坐骑中分析，其中内皮细胞被标记为4，16。以前，通过计数血管或血管细胞核延伸至内限膜上方的静脉，从视网膜横截面评估了视网膜NV的量。这种方法的主要局限性是无法量化 AVA。

<table><tbody><tr><td>33 gauge, Small Hub RN Needle</td><td>Hamilton Company</td><td>7803-05, 10mm, 25&amp;deg;, PS4</td><td>For intravitreal injection</td></tr><tr><td>Adobe Photoshop</td><td>Adobe Inc.</td><td></td><td>For image analysis</td></tr><tr><td>Air pump air100</td><td>Eheim GmbH &amp;amp; Co. KG.</td><td>143207</td><td>For inhalation anaesthesia</td></tr><tr><td>Anaesthesia unit 410 AP</td><td>Univentor Ltd.</td><td>2360309</td><td>For inhalation anaesthesia</td></tr><tr><td>AnalaR NORMAPUR Soda lime</td><td>VWR International Ltd</td><td>22666.362</td><td>For CO2 control during model induction</td></tr><tr><td>Attane Vet 1000 mg/g</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 17 05 79</td><td>For inhalation anaesthesia</td></tr><tr><td>Brush</td><td></td><td></td><td>For preparation of flat mounts</td></tr><tr><td>Carbon dioxide gas</td><td></td><td></td><td>For sacrifice</td></tr><tr><td>Celeris D430 ERG system</td><td>Diagnosys LLC</td><td>121</td><td>For &lt;em&gt;in vivo&lt;/em&gt; ERG</td></tr><tr><td>Cell culture dishes</td><td>Greiner Bio-One International GmbH</td><td>664 160</td><td>For preparation of flat mounts</td></tr><tr><td>Cepetor Vet 1 mg/mL</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 08 78 96</td><td>For anaesthesia</td></tr><tr><td>Cover slips</td><td>Thermo Fisher Scientific</td><td>15165452</td><td>For preparation of flat mounts</td></tr><tr><td>O2 Controlled InVivo Cabinet, Aninal Filtrarion System and Dehumidifier</td><td>Coy Laboratory Products</td><td></td><td>Closed system for disease model induction, optional for semi-closed system</td></tr><tr><td>E702 O2 sensor</td><td>BioSphenix, Ltd.</td><td>E207, 1801901</td><td>For oxygen level measurement</td></tr><tr><td>Envisu R2200 Spectral Domain Optical Coherence Tomograph (SD-OCT)</td><td>Bioptigen, Inc.</td><td>BPN000668</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Eye spears</td><td>Beaver-Visitec International, Inc.</td><td>0008685</td><td>For intravitreal injection and &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Flexilux 600LL Cold light source</td><td>Mikron</td><td>11140</td><td>For intravitreal injection or tissue collection</td></tr><tr><td>Fluorescein sodium salt</td><td>Merck KGaA</td><td>F6377-100G</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Gas Exhaust unit (+Double 3-way valve, mouse and rat face masks, UNOsorb filter)</td><td>UNO Roestvaststaal BV</td><td>GEX 17015249</td><td>For inhalation anaesthesia</td></tr><tr><td>Glass syringe, Model 65 RN</td><td>Hamilton Company</td><td>7633-01</td><td>For intravitreal injection</td></tr><tr><td>HRA2 Retina angiograph (FA)</td><td>Heidelberg Engineering GmbH</td><td>Spec-KT-05488</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Isolectin GS-IB4, Alexa Fluor 488 Conjugate</td><td>Thermo Fisher Scientific</td><td>I21411</td><td>For labeling retinal vasculature on flat mounts</td></tr><tr><td>Ketaminol Vet 50 mg/mL</td><td>Intervet International B.V.</td><td>vnr 51 14 85</td><td>For anaesthesia</td></tr><tr><td>Medicinal Oxygen gas</td><td></td><td></td><td>For disease model induction</td></tr><tr><td>Mice C57BL/6JRj</td><td>Janvier Labs</td><td></td><td>Also other strains possible</td></tr><tr><td>Microscope slides</td><td>Thermo Fisher Scientific</td><td>J1800AMNZ</td><td>For preparation of flat mounts</td></tr><tr><td>Minims Povidone Iodine 5% (unit)</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>vnr 24 11 304</td><td>For intravitreal injection</td></tr><tr><td>Nitrogen gas</td><td></td><td></td><td>For disease model induction (rat)</td></tr><tr><td>Oftan Chlora 10 mg/g</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 01 11</td><td>For intravitreal injection</td></tr><tr><td>Oftan Metaoksedrin 100 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 03 43</td><td>For &lt;em&gt;in vivo&lt;/em&gt; ERG</td></tr><tr><td>Oftan Obucain 4 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 03 50</td><td>For intravitreal injection</td></tr><tr><td>Oftan Tropicamid 5 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 04 12 36</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>ProOx Model 110 O2 controller and animal chamber</td><td>BioSphenix, Ltd.</td><td>803</td><td>For disease model induction, semi-closed system, optional for closed system</td></tr><tr><td>ProOx Model P360 O2 controller and animal chamber</td><td>BioSphenix, Ltd.</td><td>538</td><td>For disease model induction, semi-closed system, optional for closed system</td></tr><tr><td>Rats CD(SD)</td><td>Charles River Laboratories</td><td></td><td>Also other strains possible</td></tr><tr><td>Revertor 5 mg/mL</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 13 04 97</td><td>For anaesthesia reversal</td></tr><tr><td>Silica gel</td><td></td><td></td><td>For humidity control during model induction</td></tr><tr><td>Systane Ultra 10ml</td><td>Alcon</td><td>Tamro 2050250</td><td>For hydration of the eye</td></tr><tr><td>Systane Ultra unit 0.7ml</td><td>Alcon</td><td>Tamro 2064871</td><td>For hydration of the eye</td></tr><tr><td>Transfer pipette</td><td>Thermo Fisher Scientific</td><td>1343-9108</td><td>For preparation of flat mounts</td></tr><tr><td>VENTI-Line VL 180 PRIME Drying oven</td><td>VWR</td><td>VL180S 170301</td><td>For drying silica gel</td></tr><tr><td>VisiScope SZT350 Stereomicroscope</td><td>VWR</td><td>481067</td><td>For intravitreal injection or tissue collection</td></tr></tbody></table>

oxygen-induced retinopathy model for ischemic retinal diseases in rodents

缺血性视网膜病变的常用模型之一是氧诱发的视网膜病变 （OIR） 模型。在这里，我们描述了OIR模型诱导的详细协议，以及它在小鼠和大鼠身上的读数。视网膜神经血管化通过让啮齿动物幼崽暴露在高氧（小鼠）或交替的超氧和缺氧水平（大鼠）中诱导。这些模型的主要读数是视网膜内血管 （NV） 和血管 （AVA） 区域的大小。这种前体模型可用于评估潜在抗血管生成药物的疗效，或使用基因操纵的动物解决特定基因在视网膜血管生成中的作用。该模型在 OIR 感应中具有一些应变和供应商特定的变化，在设计实验时应考虑这些变化。

模型的主要结果是血管表型:AVA的大小和NV的量。在小鼠OIR模型中，血管抹杀发生在中央视网膜（图2A），而在大鼠模型中，它在外围发育，即类似于人类ROP22（图3A）。这是因为当小鼠暴露于高氧时，表面血管丛已经发育，而在大鼠模型中，网膜在OIR诱导（P0）时是血管性的。前视网膜血管化在血管区域附近发展，即小鼠的中央视网膜?...

Watch this Scientific Journal Video about Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents at JoVE.com

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

氧气诱发的视网膜病变 （OIR） 可用于模拟缺血性视网膜疾病，如早熟和增殖性糖尿病视网膜病变的视网膜病变，并作为概念验证研究的模型，用于评估神经血管疾病的抗角原药物。OIR 在可量化的网膜中诱导强健和可重复的神经血管化。

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 ...

oxygen-induced-retinopathy-model-for-ischemic-retinal-diseases

Research

JoVE Journal

Medicine

8.3K 次观看.  Tampere University & Tampere University Hospital. 氧气诱发的视网膜病变 （OIR） 可用于模拟缺血性视网膜疾病，如早熟和增殖性糖尿病视网膜病变的视网膜病变，并作为概念验证研究的模型，用于评估神经血管疾病的抗角原药物。OIR 在可量化的网膜中诱导强健和可重复的神经血管化。

视频: Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

科研

神经科学

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学诱导后部缺血性视神经病变的大鼠模型

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

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, ...

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 ...

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 ...

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, ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

监测氧诱导视网膜病变小鼠模型中视网膜血管的动态生长

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

生物学

<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, ...

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

摘要

探索更多视频

此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

光化学诱导后部缺血性视神经病变的大鼠模型

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

非动脉炎性前部缺血性视神经病变的啮齿动物模型（rNAION）

优化视网膜静脉遮挡小鼠模型以限制变异性

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

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

体内小鼠视网膜血管损伤读数以促进可重复性

监测氧诱导视网膜病变小鼠模型中视网膜血管的动态生长

小鼠眼模型中视网膜缺血再灌注损伤的诱导

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

摘要

探索更多视频

此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

光化学诱导后部缺血性视神经病变的大鼠模型

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

非动脉炎性前部缺血性视神经病变的啮齿动物模型（rNAION）

优化视网膜静脉遮挡小鼠模型以限制变异性

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

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

体内 小鼠视网膜血管损伤读数以促进可重复性

监测氧诱导视网膜病变小鼠模型中视网膜血管的动态生长

小鼠眼模型中视网膜缺血再灌注损伤的诱导

体内小鼠视网膜血管损伤读数以促进可重复性