The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

所有的动物都按照实验动物的护理和使用2013版的指南治疗，研究协会在视觉与眼科（ARVO）机构在眼科和视觉研究的用途声明，并作为批准的机构动物护理和使用委员会为西北大学。 注意：下面的过程可以完全与一个操作者来完成;然而，可以更有效地与两个操作员进行的任务分割相应。 1.准备激光和预激光站<ol><li>位置激光器和裂隙灯它可以很容易地访问。打开激光，并设置到预先确定的参数 （例如为75μm斑点尺寸，100毫瓦功率，100毫秒持续时间）。 注意：确保操作人员佩戴所有的动物和激光安全个人防护装备和相关的激光安全标志显示在操作室之外。 <ol><li>使用任何动物实验之前，要使用校正，非实验小鼠理想的参数。激光参数将取决于所用的激光器。理想的参数是由最低的激光功率设置，始终会导致“泡沫”的形成正确的时候集中定义。例如，见病变，适当的“泡沫”的形成视频。 </li></ol></li><li>制备预激光站，使得麻醉，动物温暖，组织擦拭物，以及所有滴眼剂（托品，丁卡，以及人工泪液）​​容易伸手可及到操作者。 <ol><li>确保动物温暖被预热以纠正注射麻醉剂到第一鼠标以避免麻醉诱导的低温之前的温度（37℃）。 </li></ol></li><li>放置在温暖的鼠标的阶段，因此它可以保持热量和保持温暖，一旦激光过程开始。 </li></ol> 2.鼠标麻醉和预激光制备<ol><li>注射前荷兰国际集团麻醉，检查眼睛宏观，以确保它有没有畸形，或减少角膜透明度（ 如白内障）异常。 </li><li>称量鼠标。 </li><li>记录体重，性别，和动物的ID号。 </li><li>使用重量，计算麻醉剂的合适量基于由机构给予（ 如100毫克/千克氯胺酮盐酸盐，10毫克/千克甲苯噻嗪或三溴250毫克/千克的准则来使用;对于20克小鼠注入0.20 ml的甲苯噻嗪/氯胺酮鸡尾酒或0.25毫升三溴的）。有为了减少的数学错误中的1克增量每重量预先计算的剂量表。 </li><li>颈背鼠标和注入腹腔麻醉根据计算步骤2.4。 </li><li>将鼠标放在动物的温暖，等待鼠标被通过脚趾捏（约3-5分钟），检查踏板反射完全麻醉。 </li><li>卷起纸巾擦拭，并保护鼠标的鼻区，以防止aspirat离子液体的滚降。在其一侧滚动鼠标，并将盐酸丁卡因一滴（约30微升）到每只眼睛局部麻醉。等待2分钟的解决方案才能生效。 </li><li>用一滴局部托吡卡胺的瞳孔扩张，重复步骤2.7。另外，使用盐酸去氧肾上腺素（2.5％），用于扩张。 </li><li>等待2分钟的解决方案才能生效;在此期间，保持动物的温暖。 </li><li>经过适当的时间过去后，迅速将鼠标鼠标舞台放在裂隙灯的下巴休息阶段。 </li><li>打开裂隙灯到最低光亮度和检查瞳孔扩张的程度。如果学生没有充分扩张（约2.5-3毫米），返回鼠标动物温暖和等待。另外，管理另一滴托吡卡胺。一旦眼睛充分扩张，进入激光治疗。 </li></ol> 3.激光治疗注意：确保其他的persons在房间里戴防护眼罩时远离激光保护裂隙灯目镜<ol><li>调整的放置鼠标在鼠标阶段，因此，它是理想的位置为视神经可视化（参见3.1.2）。 <ol><li>定向鼠标在其支架中，使其位于水平，垂直于裂隙灯光束，用头在一侧和尾部在其他。理想的鼠标放置会使视神经视觉一次盖玻片应用于容易得多。 </li><li>稍微转动鼠标所以它是在一个〜170°角与头部更接近激光操作者。 </li><li>确保小鼠是尽可能接近到裂隙灯，但仍然在一个位置，它是稳定的并且其中所述操作者的手将有足够的空间用于精细操作。 </li></ol></li><li>之后，鼠标的理想位置，放置的人造泪液一滴在25毫米×25毫米的玻璃盖玻片。 <ol><li>将一滴人造泪液鼠标的OPPosite的眼睛 - 这将确保眼睛的滋润，有助于延缓白内障的形成。 </li></ol></li><li>用拇指和指针手指之间盖玻片的角落;位置，以使该玻璃被挤压两根手指的尖端之间。 </li><li>轻轻地缠绕在动物的尸体，其余三个手指的支持和手部稳定。位置手使玻璃盖玻片可以容易地放置在鼠标的眼球。 <ol><li>确保手腕稳定在一个坚固的表面，以减少手震。 </li></ol></li><li>一旦获得稳定的位置，小心地按玻璃盖玻片（用一滴人工泪液仍然坚持）到鼠标的眼睛。 <ol><li>确保盖玻片被定位为垂直尽可能激光束，以防止激光束散射或反射。盖玻片用作隐形眼镜变平角膜。 </li></ol></li><li>看看通过裂隙灯和自由之手拨动集中UNTIL视网膜可以可视化。视网膜将具有浅黄色/红色取决于可视的位置，不同的，红色船只将可见。 </li><li>慢慢地，小心地操纵鼠标的头部和/或盖玻片，直到可视化的视觉神经。视神经将黄色与多支来自它的辐射。 </li><li>一旦运营商已经确认视神经的可视化，打开激光聚焦光束。 </li><li>一旦激光束已经接通，操纵激光聚焦束以期望的位置（约1盘直径从视神经）。 </li><li>聚焦激光束在眼底的视网膜色素上皮。正确的焦点是通过具有锐利和最清晰的激光束来实现。如果瞄准光束看起来呈椭圆形或失焦，拨动裂隙灯焦点或重新定位玻璃盖玻片。 </li><li>一旦瞄准光束聚焦在视网膜色素上皮，启动用激光的脚触发激光施用。 <ol><li>一定要避免视网膜血管，防止眼内，他morrhage。 </li></ol></li><li>观看的气泡的激光施用后立即出现。激光照射的轮廓应该是明确的，不以任何方式朦胧。 <ol><li>如果激光照射不会导致泡沫的形成，或冲击的区域看起来朦朦胧胧（混浊外观上采用不明确的圆形边框与一个成功的影响，明确，清晰的边界），或出血激光给药后看到，不包括这些病变，供日后分析。 </li></ol></li><li>重复步骤3.10-3.12对于所有所需的CNV的位置（通常在3，6，9，及左右视神经12点钟的位置）。如果激光导入装置被应用在从视神经大致相同的距离，再聚焦没有必要。然而，由于在小鼠眼和小的变化的强曲率中可能存在的视网膜上，再聚焦光束可以是连续激光施用之间必要的。 </li><li>记录在笔记本上的位置和电子邮件的结果ACH激光照射管理，结果管理的每个镜头的眼睛（成功，朦朦胧胧，出血等 ）。一定要放置在激光待机模式时不使用。 </li><li>重复3.1-3.14为鼠标的另一只眼，如果需要的话，用另一只手进行稳定和一个新盖玻片。 </li><li>毕竟所需的激光枪施用，关闭激光器和裂隙灯。 </li><li>丢弃盖玻片和地点鼠标回暖从麻醉中苏醒。宏观检查眼睛的任何伤害，并把一滴人工泪液解决方案，以保持眼睛水分，并可能防止未来白内障的发展。一旦小鼠从麻醉中恢复，回到笼子里。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td></td><td>阿佛丁</td><td>阿佛丁</td><td>阿佛丁</td><td> XYL / KET </td><td> XYL / KET </td></tr><tr><td> 鼠标重量（g） </td><td> 剂量（毫克/千克） </td><tD&gt; 溶液浓度（毫克/毫升） </td><td> 麻醉剂剂量（毫升） </td><td> 剂量（毫克/千克） </td><td> 麻醉剂剂量（毫升） </td></tr><tr><td> 15 </td><td> 250 </td><td> 20 </td><td> 0.1875 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.15 </td></tr><tr><td> 16 </td><td> 250 </td><td> 20 </td><td> 0.2 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.16 </td></tr><tr><td> 17 </td><td> 250 </td><td> 20 </td><td> 0.2125 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.17 </td></tr><tr><td> 18 </td><td> 250 </td><td> 20 </td><td> 0.225 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.18 </td></tr><tr><td> 19 </td><td> 250 </td><td> 20 </td><td> 0.2375 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.19 </td></tr><tr><td> 20 </td><td> 250 </td><td> 20 </td><td> 0.25</td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.2 </td></tr><tr><td> 21 </td><td> 250 </td><td> 20 </td><td> 0.2625 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.21 </td></tr><tr><td> 22 </td><td> 250 </td><td> 20 </td><td> 0.275 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.22 </td></tr><tr><td> 23 </td><td> 250 </td><td> 20 </td><td> 0.2875 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.23 </td></tr><tr><td> 24 </td><td> 250 </td><td> 20 </td><td> 0.3 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.24 </td></tr><tr><td> 25 </td><td> 250 </td><td> 20 </td><td> 0.3125 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.25 </td></tr><tr><td> 26 </td><td> 250 </td><td> 20 </td><td> 0.325 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.26 </td></tr><tr><td> 27 </td><td> 250 </td><tD&gt; 20 </td><td> 0.3375 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.27 </td></tr><tr><td> 28 </td><td> 250 </td><td> 20 </td><td> 0.35 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.28 </td></tr><tr><td> 29 </td><td> 250 </td><td> 20 </td><td> 0.3625 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.29 </td></tr><tr><td>三十</td><td> 250 </td><td> 20 </td><td> 0.375 </td><td> 100毫克/千克氯胺酮; 10mg / kg的甲苯噻嗪</td><td> 0.3 </td></tr></tbody></table> 表1：XyIKet剂量图表。

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+as+an+Independent+Risk+Factor+for+Severity+of+Experimental+Choroidal+Neovascularization.">Age as an Independent Risk Factor for Severity of Experimental Choroidal Neovascularization.</a> IOVS. 43, 1567-1573 (2002).</li><li>Zhu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+and+Optimization+of+Standards+for+a+Preclinical+Animal+Test+Model+of+Laser+Induced+Choroidal+Neovascularization.">Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization.</a> PLoS ONE. 9, e94743 (2014).</li><li>Weinstock, M., Stewart, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+in+rodents+of+reversible+drug+induced+opacities+of+the+lens.">Occurrence in rodents of reversible drug induced opacities of the lens.</a> British J Ophthalmol. 45, 408-414 (1961).</li><li>Ridder III, W. H., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+Cataract+Development+in+Anesthetized+Mice.">Causes of Cataract Development in Anesthetized Mice.</a> Exp Eye Res. 75, 365-370 (2002).</li></ol>

The authors declare they have no competing financial interests.

有成功的激光诱导后会影响激光传输和由此产生的CNV病变的发展多因素。这些因素应控制为与标准化，以便能有最可靠的结果。最相关的这些因素中的鼠标选择（基因型，年龄和性别），麻醉剂的选择，以及激光设置。 使用的特定小鼠模型可以对CNV的发展过程中一个显著效果。最广​​泛使用的基因型是C57BL / 6小鼠。从该动物得到的供应商可以影响所得CNV大小。普尔等人。?...

年龄相关性黄斑变性（AMD）是失明的个体50 1-3岁以上的主要原因之一。 AMD可分为两种形式：萎缩性（“干”）AMD和新生血管（“湿”），AMD。前者的特征在于视网膜色素上皮细胞（RPE），脉络膜，和光感受器地理萎缩，而后者的特征在于侵入从脉络膜异常血管的插入外视网膜层造成渗漏，出血和纤维化，最终导致失明1,2。这两种形式中，新生血管性AMD占绝大多数视力减退1。幸运的是，这种形式有许多有效的药物管理选项，而它的萎缩对应目前还没有成熟的医学治疗3。此外，由于新生血管形态已经很容易地重新投降的动物模型，它已经被越来越广泛地接触到基本的AMD研究探索潜在的病理机制，以便开发新型疗法4。 由Ryan等人开发的实验性脉络膜新生血管形成（CNV）的第一动物模型。在非人灵长类5。布鲁赫膜通过激光光凝该模型诱发破裂，这引起导致血管生成类似于见于新生血管性AMD的局部炎症反应。血管生成后的激光诱导的组织病理学进展，发现以模仿新生血管性AMD，这证实了模型的有效性6。非人灵长类提供最相似解剖人类，但不幸的是，维护费用昂贵，不能轻易遗传操作，并且有疾病进展7的慢时间过程。与此相反，啮齿动物模型是更经济有效的维护，可以遗传操作相对容易，并具有更快的凑疾病进展的RSE（实验可以在周的时间尺度来进行与个月）。这些实验应该只在有色啮齿类动物中进行，因为它是非常困难的白化动物来可视化。 鼠标激光诱导CNV模型，首先由坎波基亚罗集团在90年代末10开发，已经成长为在大多数最近的研究11-16的占主导地位的动物模型。由于CNV的复杂，目前还不清楚发病机理，激光模式已在湿性AMD的研究，从学习驾驶血管生成来评估新的治疗模式为未来的人类使用的分子机制各个方面的应用。例如，Sakurai等人和埃斯皮诺萨，Heidmann 等 。使用的激光模型，探讨巨噬细胞对CNV的使用转基因小鼠和药理耗尽处理15，16中的发展的影响。吉亚尼等 。和Hoerster 等。用的光学相干断层扫描（OCT），以图像的激光诱导的CNV在努力表征CNV的进展和比较组织病理学结果以看到的OCT成像12,17的调查结果。最后，涉及玻璃体内注射的抗血管生成剂的研究已被用作先决条件人体试验并分别在显影的第一代中新生血管性AMD今天10,18,19的管理中使用的抗VEGF剂是至关重要的。 替代模式的实验CNV利用手术的方法，诱导CNV。此过程涉及注射促血管生成物质 （如重组病毒载体过量表达血管内皮生长因子，视网膜下注射RPE细胞和/或聚苯乙烯珠），以模仿见于新生血管性AMD的增加的VEGF表达，以引起血管生成8,20的目标。然而，这种方法产生新血管形成的显着较低的发生率;这些研究显示，在CNVC57 / BL6小鼠中发生注射抗小鼠8,14的相同应变出现在激光光凝法的〜70％的成功率的31％。由于这些原因，并给予使用啮齿类动物对非人类灵长类动物的优势，激光诱导CNV的小鼠模型已成为CNV的标准动物模型对于大多数新生血管性AMD的研究实验8。 鼠标的眼睛是微乎其微的，细腻的组织一起工作。眼可视化的视网膜机动是困难的，需要大量的练习，直到掌握为止。这个任务是由一个事实，即它必须与显性和非优势手学习复杂。此外，以显现视网膜所需的精细动作已经被学习之后，双手和脚踏板操作激光之间的协调是重要的。在本文中，我们试图以蒸馏学习所有参与的激光诱导的CNV进程内的物理操纵的挑战edure成一个指南，将帮助运营商实现快速成功，这种模式。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

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.

CNV病变的定量可以通过使用免疫荧光染色以标记CNV血管平置式脉络膜分析来执行。组织制备的两个最频繁使用的方​​法是FITC-葡聚糖标记，立即动物牺牲，或与内皮细胞标记物验免疫染色之前经由灌注完成。这两种方法都已经详细13,14,21先前描述;图每1 和 2示出的例子，分别共聚焦显微镜图像采集，或者区域（2-尺寸）或音量后（3-尺寸）可被计算和...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

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) ...

a-mouse-model-for-laser-induced-choroidal-neovascularization

Research

JoVE Journal

Medicine

17.4K 次观看.  Northwestern University Feinberg School of Medicine. 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.

视频: A Mouse Model for Laser-induced Choroidal Neovascularization

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

科研

医学

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

按时间当然吲哚菁绿血管造影检测脉络膜血管系统异常在年龄相关性黄斑变性的小鼠模型

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

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

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

一种监测人牙龈移植新生血管的新方法

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...