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&#176;, 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 &#38; 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 in vivo 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 in vivo imaging</td>
		</tr>
		<tr>
			<td>Eye spears</td>
			<td>Beaver-Visitec International, Inc.</td>
			<td>0008685</td>
			<td>For intravitreal injection and in vivo 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 in vivo 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 in vivo 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 &#38; 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 in vivo 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 in vivo 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

8.2K 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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

科研

医学

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

一个生产缺血性损伤模型的简化技术

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

远程肢体缺血预适应:在啮齿动物的保护技术

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

定量自体荧光眼底视网膜疾病的评价

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

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. ...