作者要感谢 Yuliya Naumchuk (芝加哥大学) 和 Agne žiniauskaitė (Experimentica Ltd.) 的优秀技术和录像支持。Dr. 卡洛特尼斯卡娅的研究项目是由 Dr. 的约翰 P 和苔蕾丝马尔卡希在美国芝加哥大学的眼科教授的支持。

所有的动物都是按照帕特声明, 为使用动物在眼科和视力研究和欧共体指令 86/609/EEC 动物实验, 使用的协议批准和监测的动物实验委员会芬兰。
1. 激光诱导小鼠 CNV 模型5
<ol><li>从宏观上检查动物的眼睛是否有异常。</li>
	<li>称量鼠标。</li>
	<li>计算和准备适当数量的麻醉剂使用, 根据动物的重量,例如medetomidine (1 毫克/千克), 氯胺酮 (75 毫克/千克) 和蒸馏水 (0.9% NaCl 溶液) 的比例为 1:1. 5:2. 5, 或氯胺酮 (40-75 毫克/千克),嗪 (5 毫克/千克), 蒸馏水 (0.9% 氯化钠溶液) 的比率为 1:2. 5:1;对于20克老鼠, 注入0.1 毫升的混合物。</li>
	<li>注射麻醉腹腔。</li>
	<li>把老鼠放回笼子里, 等到动物被麻醉。确认鼠标没有踏板反射就能正确麻醉。</li>
	<li>确保使用激光安全个人防护设备。</li>
	<li>打开一个狭缝灯和 532 nm 二极管激光器。</li>
	<li>将鼠标从笼子里取出, 放在暖气垫上。</li>
	<li>应用一滴卡进行瞳孔扩张。等待3-5 分钟的全 (3 毫米) 瞳孔扩张。</li>
	<li>将鼠标放在狭缝灯的舞台上。</li>
	<li>将一滴眼科液体凝胶放在片上, 扁平角膜。</li>
	<li>用中心的视神经头定位鼠标的眼睛。</li>
	<li>将激光功率设置为100兆瓦, 持续时间为100毫秒, 光斑尺寸为50µm。</li>
	<li>将激光束聚焦于视网膜色素上皮 (RPE)。</li>
	<li>通过避免视网膜血管理想地在 4, 8, 和12点周围的视神经, 分别做三激光射击入一只眼睛。在所有激光照射后检查眼底, 因为没有视网膜出血。对侧眼作为非激光的控制。</li>
	<li>丢弃片并将鼠标放回加热垫上。</li>
	<li>用一滴 PEG 凝胶滴在两只眼睛上。</li>
</ol>2. SD-OCT 6,7
<ol><li>将鼠标放置到啮齿动物的对齐阶段, 并固定头部。</li>
	<li>将 SD-OCT 系统 (例如, Bioptigen/莱卡 Envisu R2200) 的镜头对准, 以使用 X 和 Y 级控制器对活体进行成像。</li>
	<li>执行 sd oct 扫描以验证赫膜的断裂: 一旦 sd oct 扫描整个眼睛, 在激光站点上手动移动参考线。赫膜的断裂应在激光区域中清晰可见 (请参见图 1)。</li>
</ol>3. 荧光素血管造影7,8,9
<ol><li>取出鼠标与持有人, 并将其放在 FA 系统 (例如, 海德堡 Spectralis HRA2)。</li>
	<li>聚焦在眼睛眼底的激光烧伤区域, 用红外线反射模式与视神经的头部在观察窗口中间。</li>
	<li>注射0.1 毫升的5% 荧光素钠盐为20克鼠皮下或腹腔。</li>
	<li>关注脉络膜水平。</li>
	<li>从脉络膜的焦点水平进行图像。</li>
	<li>重新聚焦在视网膜水平上, 并采取图像。</li>
	<li>等待三十年代, 重复步骤 3.4-3.6。</li>
	<li>将鼠标从支架上取出, 并将其放在加热垫上。</li>
	<li>反向麻醉的2拮抗剂的 medetomidine, 唑 (0.5 毫克/千克, ip), 或等待动物恢复从麻醉。</li>
	<li>在后续的天数5、10和14中, 在麻醉动物中重复在体内 SD OCT 和 FA 成像。</li>
</ol>4. CNV 分级
<ol><li>赫在0天激光后立即采取的 OCT 图像和脉络膜 FA 图像的损伤等级如下: 0-赫的膜未损坏 1-赫膜的成功损坏</li>
	<li>通过比较一系列视网膜 FA 图像中荧光素信号的动态观察, 发现有渗漏的激光斑点 CNV 的存在, 如下所示: 0-视网膜的正常出现 0.5-渗漏的微弱的染色 1.0-漏的 CNV 区域 注: 使用 oct 成像, 以进一步确认 CNV 或可疑 FA 中存在的 intraretinal 流体在 OCT 图像将建议 CNV 评分。</li>
</ol>5. 视网膜厚度测量
<ol><li>使用自动分割软件进行视网膜厚度测量。确保视网膜总厚度被认为是从神经纤维层到 rpe (健康测量点) 的所有层的厚度, 或者是连接 rpe (激光站点) 周围的假想线 (也请参见图 7)。</li>
</ol>

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作者 Symantas Ragauskas, 博士是 Experimentica Ltd. 的一名雇员 (研究科学家) 和股东, 提供合同研究服务, 采用了本文所用的临床前 CNV 模型。
作者 Eva Kielczewski 是一个员工 (研究应用工程师, oct) 的徕卡系统, 生产的 SD oct 在本文中使用。
作者约瑟夫万斯是一个雇员 (NA oct 销售总监) 的徕卡微系统, 生产的 SD oct 体系在本文中使用。约瑟夫. 万斯也是 Spective 的总裁兼董事总经理。
作者西蒙卡洛特尼斯卡娅博士是 Experimentica Ltd. 的顾问首席科学官和股东, 这是一家提供合同研究服务的临床前合同研究机构, incl. 了本文所用的临床前 CNV 模型。西蒙卡洛特尼斯卡娅, 博士也是 & p 科学, LLC, 生命科学咨询公司的首席执行官, 并担任 Dr. 约翰 p 和苔蕾丝 e. 马尔卡希在 Stritch 医学院芝加哥大学的眼科教授。这项安排的条款已根据其利益冲突政策, 由美国芝加哥大学学院审查和批准。
作者 Giedrius Kalesnykas, 博士是 Experimentica Ltd. 的一名雇员 (CEO) 和股东, 提供合同研究服务, 采用本文所用的前 CNV 模型。

多模态成像为 CNV 病理评价提供了有价值的工具。在这里, 我们提出了一个成像协议, 包括 FA, SD OCT, 自动分割的快速, 重现性, 可靠的评价 CNV 病理。经激光管理后, 赫膜断裂。此外, 在这个阶段使用 SD OCT 也可以立即可视化可能的 intraretinal 和视网膜出血, 这可能混淆结果的解释。根据 FA 图像的荧光素信号对视网膜渗漏进行分级。使用 SD OCT 提供了更详细的描述 CNV 病理。此外, 纵向 SD OCT 分析在不同的时间点在整个随访期强调的时间差异的病理, 将仍然难以捉摸, 如果依赖于 FA 单独。
用自动分割法测量视网膜总厚度。人工测量 CNV 诱导的视网膜厚度。对脉络膜 flatmount 的组织学评价进行了验证, 用图像分析软件图像 J 对新生血管面积进行了测量。
可视轴的正确透明度对于所提出的协议的成功执行至关重要。角膜干燥和白内障的形成是诊断的主要因素。因此, 一旦鼠标被麻醉, 眼睛应该不断水合与人工泪液或凝胶保持适当的角膜水化。建议的协议应在10分钟内优先进行麻醉诱导。延长麻醉时间可能导致白内障的形成, 并防止在体内成像。
所描述的协议仅限于基于视网膜水平血管渗漏的 CNV 进展的观察分级。可以使用海德堡 Spectralis 软件增加对视网膜渗漏的定量评估, 允许对泄漏区域进行划定, 并提供有关区域的定量数据。此外, 苏莱曼和同事 (2015) 最近提出了一个计算 CNV 体积从在体内获取 OCT 图像使用椭球方法10。椭圆形模型最有可能引入偏倚, 以估计病变体积的 CNV 在大多数情况下有一个不规则的形状。然而, 从 OCT 图像的共焦分析和建议的椭球量化的体积测量之间的高度相关性, 提供了证据证明该方法是定量评估 CNV 卷的重要工具10.
最后, 我们认为, 不同的在体内成像模式的组合, 以及自动分割和组织学分析, 提供了可重复和可靠的评估 CNV 病理学临床前研究。该方法对于概念治疗干预研究的证明尤其有用。

第一次成功的尝试模仿人类 CNV 在啮齿目动物的病理证实了近三年前与氪激光在长埃文斯鼠1。此后, 氪激光被用来打破赫的细胞膜在最流行的鼠标应变, C57BL/6J2,3,4。用 FA 和组织学染色验证了 CNV 诱导的成功率。快速发展的无创成像模式, 如 OCT, 促进了啮齿动物的临床前模型领域的增长。在同一眼的多个时间点, 监测视网膜形态变化的能力大大降低了动物的使用, 提高了实验研究的效率。组织学评价 CNV 病变是相当直接的, 并要求标签的异常血管生长周围的激光管理, 图像采集, 和面积/体积估计使用图像分析软件。相比之下,在体内成像模式引入了对 CNV 病理及其解释的更复杂的分析。
在这里, 我们提出了一个简单的和相对快速的方法来评分的诱导, 进展, 和回归的 CNV 使用 FA, SD OCT, 和自动分割方法的小鼠激光诱导 CNV 模型。

<table><tbody><tr><td>Medetomidine (commercial name Domitor)</td><td>Orion</td><td>Vnr 01 56 02</td><td>Anesthesia</td></tr><tr><td>Ketamine</td><td>Intervet</td><td>Vnr 51 14 85</td><td>Anesthesia</td></tr><tr><td>0,9% NaCl</td><td>B Braun</td><td>357 0340</td><td>Anesthesia</td></tr><tr><td>Xylazine (commercial name Rompun vet)</td><td>Bayer</td><td>vnr 14 89 99</td><td>Anesthesia</td></tr><tr><td>Tropicamide</td><td>Santen</td><td>Vnr 04 12 36</td><td>Mydriatic agent</td></tr><tr><td>Viscotears</td><td>Alcon</td><td>Vnr 44 54 81</td><td>Lubricant</td></tr><tr><td>Systane</td><td>Alcon</td><td>&amp;nbsp;-</td><td>Lubricant</td></tr><tr><td>5% Fluorescein sodium salt</td><td>Sigma Aldrich</td><td>F6377-100G</td><td>Fluoresent agent</td></tr><tr><td>Atipamezole (commercial name Antisedan)</td><td>Orion</td><td>Vnr 47 19 53</td><td>Anesthesia</td></tr></tbody></table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

激光诱导脉络膜新生血管 (CNV) 是模拟年龄相关性黄斑变性 (AMD) 的湿型的一个良好建立的模型。在本议定书中, 我们的目标是引导读者不只是通过技术考虑产生激光诱导的损害, 以触发新生血管的过程, 而是侧重于强大的信息, 可以获得从多式联运纵向在体内的整个随访期内的成像。
激光诱导的小鼠 CNV 模型是由二极管激光管理产生的。多模态在体内成像技术被用来监测 CNV 的诱导, 进展和回归。首先, 在激光后立即进行光谱域光学相干层析成像 (SD OCT) 来验证赫膜的断裂。随后使用荧光素血管造影 (FA) 进行的活体成像证实了赫膜从脉络膜水平获得的序列图像的成功损伤。CNV 增殖和回归的纵向后续在天 5, 10 和14以后使用了 SD OCT 和 FA 激光执行。提出了从 FA 图像中 CNV leasions 的简单可靠的分级方法。自动分割的测量总视网膜厚度, 结合手动口径应用测量视网膜厚度的 CNV 地点, 允许无偏见的评价存在水肿。最后, 利用 isolectin GS-IB4 染色对脉络膜 flatmounts 进行了 CNV 的组织学验证。染色是值, isolectin 阳性面积计算 ImageJ。
该协议特别适用于需要高通量样的 CNV 病理筛查的治疗研究, 因为它可以快速、多式、可靠地分类 CNV 病理和视网膜水肿。此外, 高分辨率 SD OCT 可以记录其他病理特征, 如视网膜或 intraretinal 液的积累。但是, 此方法不提供从 SD OCT 图像中自动进行 CNV 卷分析的可能性, 必须手动执行。

激光后立即出现气泡或视网膜出血并不总是可见的。因此, SD OCT 对于验证赫膜的损伤尤为重要。图 1显示了激光管理后在不同时间点的 OCT 成像的示例。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> 图 1: 眼睛眼底的 OCT (VIP 图像) 显示在白色、绿色和红色圆圈中勾勒出的三激光区域.OCT B 扫描图像之前采取的激光 (基线), 立即激光后, 以验证赫的膜断裂 (0 天, 箭头点的损害网站), 和 5, 10 和14天后, 激光管理。从白色 (第一排图像) 中所勾画的区域可以看出, CNV 在以后的 timepoints 中没有发展。在绿色和红色概述的区域开发了 CNV, 在跟随的天5被发现了。然而, 在10和14天 timepoints, 这些 CNV 病变退化。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图2和 3显示了使用 FA 的串行成像, 它证实了赫的细胞膜在0天的所有三个斑点中的 C57BL/6jRj 老鼠的成功损伤。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56173/56173fig2.jpg"> 图 2: 连续 FA 成像每年二十年代 (图像1到 18) 在脉络膜水平后立即激光管理.图像中的白色箭头1指向激光站点, 显示荧光素在以后的 timepoints (图18中为白色箭头)。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56173/56173fig3.jpg"> 图 3: 连续 FA 成像每年二十年代 (图像1到 18) 在视网膜水平后立即激光管理.CNV 地区, 有荧光素渗漏和评分 1 (漏 CNV) 是指出的白色箭头的图像18。注意在 FA 成像 (图8和11中的白色箭头) 期间, timecourse 的强度, 以及荧光素阳性区的增加。图18中白色轮廓的两个区域被分级为有微弱的 FA 信号 (等级 0.5)。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
除了 CNV 病理学的分级外, SD OCT 还可以在病变部位显示更多的信息,例如, 如, 视网膜液的存在, 水肿, 和 CNV 回归。图 4显示了激光诱导的 CNV 在小鼠体内的主要病理特征。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56173/56173fig4.jpg"> 图 4: CNV 病理学的光谱域光学相干断层成像.SD OCT 提供了一个详细的 CNV 病理在视网膜组织, 从这些代表性的图像可以看到疤痕组织, CNV 形成, 和液体的积累。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
黄斑水肿是人体内湿型 AMD 的主要病理标志之一。在激光诱导的 CNV 模型中, 可以使用自动分割来评估视网膜厚度。人工测量选定的激光的地点是需要测量视网膜厚度在 CNV。图 5显示了自动分段后生成的报告的示例。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56173/56173fig5.jpg"> 图 5: 视网膜厚度的定量.自动分割法测量视网膜厚度<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
使用自动分割是一个快速的方法来提供视网膜厚度的概述 (表 1)。图 6A和6B分别显示了来自健康视网膜区域和 CNV 病理的视网膜区域自动分割的典型例子。尽管在区分个别视网膜层方面存在微小的不精确性, 但总的来说, 该软件能可靠地识别色素小鼠的视网膜总厚度。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56173/56173fig6.jpg"> 图 6: 视网膜层的自动分割.自动分割健康的视网膜区域 (A) 和包含 CNV 的视网膜区域 (在图像B中为星号)。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了评估激光地区的视网膜厚度, 每个激光区域都是人工测量的: 视网膜总厚度被认为是所有层的厚度, 从神经纤维层到连接 RPE 周围的假想线损坏 (图 7和表 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>地区</td>
			<td>5天</td>
			<td>10天</td>
			<td>14天</td>
		</tr>
<tr>
<td>总视网膜厚度, μ m</td>
			<td>218±7。8</td>
			<td>220±7。2</td>
			<td>221±9。8</td>
		</tr>
<tr>
<td>激光面积1</td>
			<td>200</td>
			<td>204</td>
			<td>214</td>
		</tr>
<tr>
<td>激光面积2</td>
			<td>226</td>
			<td>217</td>
			<td>220</td>
		</tr>
<tr>
<td>激光面积3</td>
			<td>222</td>
			<td>223</td>
			<td>227</td>
		</tr>
<tr>
<td colspan="4">数据显示为 mean±SD</td>
		</tr>
</tbody></table>表1。总视网膜厚度和视网膜厚度在 CNV 站点在14天的后续, 由自动分割使用 inVivoDiver 软件 (v 3.0.8) 确定。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56173/56173fig7.jpg"> 图 7: 人工测量激光区视网膜厚度与 CNV 病理.黄线表明, 在激光管理的地方, 视网膜的总厚度从神经纤维层到假想的 RPE 层 (黑线)。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
组织学上, CNV 病变的确认使用 isolectin GS-IB4 标记 (图 8A)。图像分析软件图像 J 被用来计算 CNV 病变面积 (图 8B)。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56173/56173fig8.jpg"> 图 8: 组织学分析.脉络膜 flatmount CNV 病变的组织学染色 (绿色、 A) 可以使用图像 J (B) 中的阈值进行量化。A的刻度线为50μ m。<a href="//cloudflare.jove.com/files/ftp_upload/56173/56173fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

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

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

在这里, 我们介绍了纵向的在体内成像在激光诱导的小鼠脉络膜新生血管形态学变化中的作用。

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

in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

9.1K 次观看.  Experimentica Ltd.. 在这里, 我们介绍了纵向的在体内成像在激光诱导的小鼠脉络膜新生血管形态学变化中的作用。

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

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

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

通过光学相干断层扫描 在 小鼠脉络膜中植入和评估黑色素瘤

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

癌症研究

A Mouse Model for Laser-induced Choroidal Neovascularization

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

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

小鼠模型的激光诱导脉络膜新生血管

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

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

生物工程

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

免疫与感染

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

神经科学

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 ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

<meta charset="utf8"></head><body>图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

摘要

探索更多视频

此视频中的章节

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

通过光学相干断层扫描在小鼠脉络膜中植入和评估黑色素瘤

小鼠模型的激光诱导脉络膜新生血管

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

在体内图像的Cx3cr1^gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

小鼠视网膜的经瞳双光子体内成像

体内小鼠视网膜血管损伤读数以促进可重复性

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

摘要

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此视频中的章节

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

通过光学相干断层扫描 在 小鼠脉络膜中植入和评估黑色素瘤

小鼠模型的激光诱导脉络膜新生血管

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

小鼠视网膜的经瞳双光子体内成像

体内 小鼠视网膜血管损伤读数以促进可重复性

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

通过光学相干断层扫描在小鼠脉络膜中植入和评估黑色素瘤

在体内图像的Cx3cr1^gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

体内小鼠视网膜血管损伤读数以促进可重复性