Name: optical coherence tomography: imaging mouse retinal ganglion cells in vivo
Uploaded: 2017-09-22T13:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

这项工作由 Inserm, 大学蒙彼利埃, 视网膜法国, 联合国家 des Aveugles et Déficients Visuels (UNADEV), 协会综合征钨, 基金会倾吐 la 研究 Médicale, 法兰西基金会和卓越实验室支持EpiGenMed 计划。

《实验议定书》获得了国家 #233 和 #233;d icale (Inserm; 研究);蒙彼利埃, 法国), 符合欧洲的指示, 并符合帕特声明的使用动物在眼科研究。它是根据《动物实验 (CEEALR; nuCEEA-LR-12123) 》中的《伦理道德的一致性的协议进行的. 
 1. 设备设置和预成像准备 注意: 在这里, OCT 是利用光谱域 (SD) 眼科成像系统 ( 图 1A ) 在小鼠视网膜上进行的。SD OCT 设备包括一个基地和动物成像坐骑 (AIM) 与啮齿动物对齐阶段 (RAS) ( 图 1B )。该底座包括计算机、oct 引擎、SD oct 探头和鼠标专用镜头。探头安装在目标上, 其中包括 Z 转换器。该 RAS 用于鼠标定位感谢表与 X 和 Y 翻译, 磁带可以旋转和旋转, 以及可移动的叮咬酒吧与鼻带。制造商提供的软件允许对 OCT 文件进行获取和分析, 尽管后者也可以通过开源图像处理程序完成. 
<ol> <li> 将探测器牢牢地放置在目标中. </li>
	<li> 将鼠标特定的镜头连接到探测器. </li>
	<li> 调整特定镜头的参考臂设置 (此处为 086, 位置为 964). </li>
	<li> 确保鼠标特定的镜头离卡带足够远, 将咬杆与鼻带连接到卡带上. 
	注: 镜头与卡带之间的距离可与 Z 型转换器螺钉一起调整, 位于目标背面。鼻带是一个商业上可用的弹性带, 其张力应根据鼠标大小调整. </li>
	<li> 打开 "电源 (购物车右下角)" 和 "计算机". </li>
	<li> 若要启动图像处理程序, 请双击屏幕上相应的快捷方式. 
	注: 这取决于透镜的类型;这里, 老鼠视网膜. </li>
	<li> 创建新的临床研究并添加所需的协议。否则, 使用现有的临床研究和/或协议. 
	<ol> <li> 通过选择和 #34 添加新的研究; 研究名称和 #34;; #34; ID 和 #34;; #34; 处理臂规格和 #34; (这里, #34; 未和 #34;); 预定义和 #34; 扫描协议和 #34; 如果它们存在. </li>
	</ol> </li> <li> 选择 #34; 考官和 #34; 在和 #34; 临床研究和 #34; 部分. 
	注意: 对于一个新的 #34; 考官和 #34;, 去和 #34; 设置考官和 #38; 医生和 #34; 定义它. </li>
	<li> 通过单击添加患者, 在患者/考试部分添加新病人; 进入 #34; 身份证和 #34;, #34; 姓名和 #34;、#34; 姓氏和 #34;; #34; 性与 #34; #34;d 吃了生与 #34; (可选). 
	注意: 在测试每只鼠标之前, 如果更方便的话。请确保该 ID 不长于10字符。对于鼠标, 两个眼睛的折射误差为 0, 轴向长度为 23.0. </li>
	<li> 单击和 #34; 添加考试和 #34; 选择所需的和 #34;P rotocol 和 #34; 通过添加和 #34;P 重置扫描和 #34; 从列表中, 从将首先测量的眼睛开始 (这里, 右眼), 或通过自定义扫描. </li>
	<li> 若要自定义扫描, 请使用现有的扫描作为模板并对其进行编辑, 或从头开始通过和 #34 创建它; 添加自定义扫描和 #34; 选项。将所有扫描添加到列表后, 使用 "#34" 定义新协议; 输入和 #34; 新协议名称和 #34; 选项 (OS:26 险恶, 左眼;26: 德克斯特, 右眼). 
	注意: 在这里, 协议涉及三扫描: (i) 径向扫描, 具有1.4 毫米直径, 0 毫米水平和垂直偏移, 1000 行 a 扫描/b 扫描, 100 b 扫描/卷, 1 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷;(二) 矩形扫描, 长度和宽度为1.4 毫米, 0 和 #176; 角度, 0 毫米水平和垂直偏移, 1000 行 a 扫描/b 扫描, 100 B 扫描, 1 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷;和 (iii) 环形扫描, 最大直径为0.4 毫米和0.6 毫米, 0 毫米水平和垂直偏移, 1000 行扫描/b 扫描, 3 b 扫描/卷, 48 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷. </li>
</ol> 2。鼠标准备 <ol> <li> 眼球扩张和固定 <ol> 在数据采集前至少15分钟, 将10% 肾上腺素滴眼液注入老鼠眼中, 除去多余的, 并注入0.5% 卡滴眼液. <li>
		注: 第一次扩张可以做一次对所有的老鼠, 将在会议上进行测试。确保液体在室温下. </li>
		<li> 在诱导全麻 (步骤 2.2) 后, 立即管理0.4% 盐酸因滴眼液, 使其保持在3秒的位置以麻醉和固定眼睛。然后, 擦掉掉滴, 再重复10% 肾上腺素和0.5% 卡的灌注, 以确定眼睛是否有很好的扩张. 
		注意: 确保液体在室温下, 并且老鼠不会吞下因. </li>
	</ol> </li> <li> 全麻 <ol> <li> 在盐水中制备氯胺酮 (20 毫克/毫升) 和嗪 (1.17 毫克/毫升) 的麻醉液。存储在4和 #176; C 最多2周. </li>
		<li> 大约5分钟前测试, 腹腔注射 5-10 和 #181; L 的麻醉液每1克的身体质量, 取决于年龄和大小的鼠标. 
		注意: 幼鼠和/或瘦老鼠需要较少的麻醉剂, 少花点时间麻醉, 但也能更快地苏醒。这里, 8 和 #181; 升/克 (160 毫克/千克氯胺酮, 9.33 毫克/千克的嗪). </li>
		<li> (可选) 用粘稠滴眼药水或眼部凝胶润滑眼睛, 以避免角膜干燥. </li>
	</ol> </li> </ol> 3。鼠标定位 <ol> <li> 用粘性 glycol-based 眼药水润滑眼睛以提供角膜水化. 
	注意: 如果在考试中眼睛看起来很干燥, 请重新申请眼药水. </li>
	<li> 将鼠标包裹在一张外科纱布上以保持体温. </li>
	<li> 使用海绵或棉芯, 在每只眼睛上应用一层薄薄的眼部凝胶0.3% 甲, 以减少光的折射, 避免混浊, 并确保角膜水化。这样做的时候, 把睫毛和胡须移到一边. </li>
	<li> 将鼠标置于盒式磁带中, 并将其头部伸直并向前指向. </li>
	<li> 轻轻打开咬杆钳, 将杆放在嘴里; 用鼻带固定位置. 
	注意: 确保咬杆位于卡带的中间. </li>
	<li> 如果右 (左) 眼正在测试, 请在右侧 (左) 侧放置一个棉卷. </li>
	<li> 同时在鼠标特定的镜头上保持夹紧的瞄准头, 通过逆时针旋转 Z 转换器螺钉将其对准眼睛. </li>
	<li> 通过转动和旋转卡带, 并通过转动咬杆和 X 转换器螺钉, 预先设定鼠标的位置; 目标是让右眼直视镜头, 从而使眼睛和镜片的光轴对准. 
	注意: 如果鼠标太高或过低, 应首先使用 Y 型转换器螺钉. </li>
	<li> 选择 #34 中的第一个扫描; 考试和 #34; 单击 #34; 开始瞄准和 #34; 并对视网膜成像进行进一步的人工调整. 
	<ol> <li> 使用 Z 转换器螺钉, 在左侧面板上垂直移动视网膜 ( 图 2 , 水平 B 扫描对齐), 并水平在 r 上可选面板 ( 图 2 , 垂直 B 扫描对齐). </li>
		<li> 旋转盒式磁带, 通过将 ONH 向上或向下移动, 使 ONH 位于右侧面板的中间。使用咬杆螺钉在右侧面板上伸直视网膜。旋转卡带, 将 ONH 放置在左侧面板的中间. </li>
		<li> 使用 X 转换器螺钉在左侧面板上水平视网膜。记住每个调制器的主要功能, 进一步调整视网膜的位置以集中 ONH. 
		注: 如有必要, 使用 Y 型转换器螺钉在右侧面板上上下移动 ONH。该位置可以在扫描之间的任何时间被细化. </li>
	</ol> </li> </ol> 4。ONH 和视网膜的 SD oct 成像 <ol> <li> 一旦内容具有调整, 请单击 #34; 启动快照和 #34; 开始 sd oct 扫描. 
	<ol> <li> 如果不需要3D 成像, 请取消选中 OCU 选项. </li>
	</ol> </li> <li> 保存扫描和报表. </li>
	<li> 继续进行下一次扫描. </li>
	<li> 图像的第二眼, 在缩回镜头后, 相应地打开卡带, 重复步骤 3.6-4.3. </li>
</ol> 5。购置完成 <ol> <li> 当采集完成时, 将鼠标从卡带上取出, 用0.3% 甲的眼部凝胶涂抹在每只眼睛上, 并将鼠标放在加热板上唤醒. </li>
	<li> 上次购置后, 关闭软件并关闭计算机和 OCT 计算机 (电源按钮). </li>
	<li> 用消毒剂清洗卡带. </li>
</ol> 6。分析 <ol> <li> 用于视网膜层厚度测量, 请使用制造商提供的自动分割软件. 
	<ol> <li> 单击 和 #34;P atient 和 #34; , 选择所需的 和 #34; 考试和 #34; 然后单击 和 #34; 查看考试和 #34; 选项. 
		<li> 通过右键单击所需扫描的文件夹图标, 将所需的 oct 数据加载到 oct 软件中. </li>
		<li> 右键单击 B 扫描; 通过启用10测量卡尺来配置卡尺; 并输入它们的名称、角度和颜色. </li>
		<li> 通过右键单击 B 扫描来选择所需的卡尺; 将其相应放置到视网膜上进行测量. 
		注意: 对于以 ONH 为中心的扫描, 在它的每一侧设置5卡尺, 彼此等距。对于 peripapillary 分析, 请确保卡尺没有放置太远的 ONH。对于径向扫描, 分析 OCT 软件选择的每扫描10图片。在 和 #34; 报告和 #34; 文件夹中可以找到它们的编号. </li>
		<li> 通过右键单击 B 扫描并单击 并 #34 将结果保存在电子表格软件程序中进行分析; 保存结果和 #34; 
		注意: 结果可以在与扫描数据相同的文件夹中找到. </li>
	</ol> </li> <li> 也可以使用自制的卡尺宏, #34; MRI 视网膜工具和 #34, 为开源图像处理程序开发 (参见材料表). 
	<ol> <li> 确保 #34; MRI 视网膜工具和 #34; 和 #34; 修改多边形剖面工具和 #34; 宏处于活动状态。加载图像。单击 m 按钮开始测量. 
		注意: 宏自动在 ONH 的两侧创建一个0.2 毫米长的测量盒。每个卡带包含5测量点, 功能作为卡尺。它们的横向位置是不可更改的, 并为径向扫描中的 peripapilla 定制。盒式磁带的水平位置是预先定义的, 用来测量纤维/协变厚度, 但可以很容易地修改以测量 GC 复杂层。如果扫描质量较差, 则需要调整水平位置, 或者需要从分析中排除图片. </li>
		<li> 对于水平调整, 单击 "e" 按钮。在新开的 #34; ROI 管理器和 #34; 窗口中, 选择第一个卡带. </li>
		<li> 单击蓝色多边形按钮, 然后通过单击图片中测量图层的边框来调整第一个卡带的位置. </li>
		<li> 对第二个卡带进行重复, 首先在 #34 中选择它; ROI 管理器和 #34; 窗口, 然后单击相应的图像。点击 r 按钮测量。查看 #34 中的结果; 测量和 #34; 窗口. 
		注意: 结果可以随时复制到电子表格软件程序中。它们包含了正确的卡带 (r), 左卡式 (l) 和总计: Intden-综合密度在卡带内的以下值;面积: 测量面积, 以 mm 2 ;长度的卡尺内的卡带, 在毫米;平均值: 在卡带内的平均信号强度;和 Std: 信号平均强度的标准误差. </li>
		<li> 继续执行下一图像. </li>
		<li> 进一步分析电子表格软件程序中的数据. 
		注意: 要查找层的平均厚度, 请取十 Len 值. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Drexler, W., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-of-the-art+retinal+optical+coherence+tomography.">State-of-the-art retinal optical coherence tomography.</a> Prog Retin Eye Res. 27 (1), 45-88 (2008).</li><li>Grenier, J., 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=WFS1+in+Optic+Neuropathies:+Mutation+Findings+in+Nonsyndromic+Optic+Atrophy+and+Assessment+of+Clinical+Severity.">WFS1 in Optic Neuropathies: Mutation Findings in Nonsyndromic Optic Atrophy and Assessment of Clinical Severity.</a> Ophthalmology. 123 (9), 1989-1998 (2016).</li><li>Zmyslowska, A., 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=Retinal+thinning+as+a+marker+of+disease+progression+in+patients+with+Wolfram+syndrome.">Retinal thinning as a marker of disease progression in patients with Wolfram syndrome.</a> Diabetes Care. 38 (3), e36-e37 (2015).</li><li>Fischer, M. D., 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=Noninvasive,+in+vivo+assessment+of+mouse+retinal+structure+using+optical+coherence+tomography.">Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.</a> PLoS One. 4 (10), e7507 (2009).</li><li>Grieve, K., Thouvenin, O., Sengupta, A., Borderie, V. M., Paques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+the+Retina+With+Full-Field+Optical+Coherence+Tomography.">Appearance of the Retina With Full-Field Optical Coherence Tomography.</a> Invest Ophthalmol Vis Sci. 57 (9), OCT96-OCT104 (2016).</li><li>Chang, B., 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=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42 (4), 517-525 (2002).</li><li>Mustafa, S., Pandit, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+diagnosis+and+management+of+optic+neuropathy.">Approach to diagnosis and management of optic neuropathy.</a> Neurol India. 62 (6), 599-605 (2014).</li><li>Sarzi, E., 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=The+human+OPA1delTTAG+mutation+induces+premature+age-related+systemic+neurodegeneration+in+mouse.">The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.</a> Brain. 135 (Pt 12), 3599-3613 (2012).</li><li>Sarzi, E., 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=Increased+steroidogenesis+promotes+early-onset+and+severe+vision+loss+in+females+with+OPA1+dominant+optic+atrophy.">Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.</a> Hum Mol Genet. 25 (12), 2539-2551 (2016).</li><li>Delettre-Cribaillet, C., Hamel, C. P., Lenaers, G., Pagon, R. A., 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=Optic+Atrophy+Type+1.">Optic Atrophy Type 1.</a> Gene Reviews. 7 (2007), (2007).</li><li>Lenaers, 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=Dominant+optic+atrophy.">Dominant optic atrophy.</a> Orphanet J Rare Dis. 7 (46), (2012).</li><li>Delettre, C., 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=Nuclear+gene+OPA1,+encoding+a+mitochondrial+dynamin-related+protein,+is+mutated+in+dominant+optic+atrophy.">Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.</a> Nat Genet. 26 (2), 207-210 (2000).</li><li>Liu, 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=Monitoring+retinal+morphologic+and+functional+changes+in+mice+following+optic+nerve+crush.">Monitoring retinal morphologic and functional changes in mice following optic nerve crush.</a> Invest Ophthalmol Vis Sci. 55 (6), 3766-3774 (2014).</li></ol>

作者没有什么可透露的。

OCT 系统是一种非侵入性的活体成像方法, 它提供了高分辨率的视网膜横断面扫描。因此, 它的主要优势是它的详细分析的潜力, 与极好的机会, 转置协议常规适用于人类的鼠标模型。
在 Opa1delTTAG突变小鼠的例子中, SD OCT 结果显示了纤维和 GC 复合层厚度的增加, 这使得对 DOA 病理生理学的进一步探索9。这是不可能完全通过组织学分析。相比...

OCT 是一种诊断工具, 有助于检查视网膜结构1, 包括视神经头 (ONH)。多年来, 它已经成为人类疾病进展的可靠指标2,3, 以及啮齿动物4,5。它使用干涉法来创建2µm 轴向分辨率的视网膜层的横断面图像。最内层是视网膜神经纤维层 (纤维), 含有研资局轴突, 其次是神经节细胞层 (协鑫), 其中大部分为研资机构。其次是内部状层 (彩光), 在那里, 王国的树突满足两极, 水平, 和突细胞轴突。这些, 连同水平的细胞, 形成内部核层 (INL), 并且他们的突起连接与感光轴突在外部状层 (组织)。其次是外核层 (只), 与感光细胞体, 并通过外部限制膜 (OLM) 与感光层分离, 也称为内段/外段 (是/OS) 层。最后, 在小鼠视网膜的最后可见层是视网膜色素上皮 (RPE) 和脉络膜 (C)。单独的纤维通常太薄, 不能用老鼠来测量;因此, 分析纤维/协鑫是最好的4,5。另一个可能性是 GC 复合层, 它包含了后者, 除了光子, 使它更厚, 从而更容易测量 OCT 扫描4。因此, OCT 可以提供洞察的病理状态的视网膜, 如在 OPNs。
另一种方法是, 用剖检组织学来分析小鼠视网膜的厚度。然而, 这项技术面临的限制与组织收集, 固定, 切割, 染色, 安装,等因此, 一些缺陷, 如微妙的厚度变化, 无法检测到。最后, 由于同一鼠标不能在多个时间点测试, 所以每项研究的动物数量大大增加, 不像 OCT。所有的侵入、高分辨率、重复的可能性、时间的监控以及 OCT 技术的易用性使其成为视网膜疾病研究的首选方法。
小鼠模型用于识别基因缺陷并阐明 retinopathies6的分子机制。OPN 是一种视网膜病变, 对视神经 (ON) 有很大的损害, 由大约120万王国的神经轴突构成。OPN 可以集中在上, 也可以是次要的其他疾病, 先天或不7, 导致视觉领域的损失, 后来, 失明。OPN 的特征性状为研资损失和损害, 可在人类 OCT 中观察到为纤维和协变细化2,3。同时, OPN 的病理生理学尚不清楚, 因此需要对小鼠视网膜进行检测。
这份手稿描述了视网膜层厚度的成像和量化, 使用 Opa1delTTAG鼠标线8,9的例子, 一个显性光学萎缩 (DOA)10的模型。评估研资局的病理生理学、放射、矩形和环形扫描的数量。这是通过 OCT 软件提供的标准卡尺或为开源图像处理程序开发的自制宏完成的。标准卡尺是难以操作, 往往比纤维/协进会厚, 而自制卡钳是易于使用, 重现性, 更精确。该宏在 peripapillary 区域的 ONH 的两侧对自动检测到的层进行了测量, 在5点和固定位置上。所提出的协议的目的是描述 OCT 扫描获得指定视网膜定位, 重点是视网膜。

<table><tbody><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Opa1&lt;em&gt;&lt;sup&gt;delTTAG&lt;/sup&gt;&lt;/em&gt; mouse</td><td>Institute for Neurosciences in Montpellier, INSERM UMR 1051, France</td><td>-</td><td>Opa1 knock-in mice carrying&amp;nbsp; OPA1 c.2708_2711delTTAG mutation on C57Bl6/J background</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EnVisu R2200 SD-OCT Imaging System</td><td>Bioptigen, Leica Microsystems, Germany</td><td>-</td><td>Spectral-Domain Optic Coherence Tomography system</td></tr><tr><td>EnVisu R2200 SD-OCT Imaging System Software</td><td>Bioptigen, Leica Microsystems, Germany</td><td>-</td><td>Software for OCT acquisition and analysis</td></tr><tr><td>ImageJ 1.48v</td><td>Wayne Rasband, National Institutes of Health, USA</td><td>-</td><td>Software for analysis, requires downloading and installing two hommade macros: http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Retina_Tool</td></tr><tr><td>Self-regulating heating plate</td><td>Bioseb, France</td><td>BIO-062</td><td>Protection against hypothermia</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nose Band</td><td>-</td><td>-</td><td>Elastic band</td></tr><tr><td>Gauze pads 3&quot; x 3&quot;</td><td>Curad, USA</td><td>CUR20434ERB</td><td>Protection against hypothermia</td></tr><tr><td>Dual Ended Cotton tip applicator</td><td>Essence of Beauty, CVS Health Corporation, USA</td><td>-</td><td>Gel application</td></tr><tr><td>Cotton Twists</td><td>CentraVet, France</td><td>T.7979C.CS</td><td>Mouse positioning</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents and Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N&amp;eacute;osyn&amp;eacute;phrine Faure 10%</td><td>Laboratoires Europhtha, Monaco</td><td>-</td><td>Eye dilatation</td></tr><tr><td>Mydriaticum 0.5%</td><td>Laboratoires Th&amp;eacute;a, France</td><td>3397908</td><td>Eye dilatation</td></tr><tr><td>Cebesine 0.4%</td><td>Laboratoire Chauvin, Bausch&amp;amp;Lomb, France</td><td>3192342</td><td>Local anesthesia</td></tr><tr><td>Imalgene 1000</td><td>Merial, France/CentraVet, France</td><td>IMA004</td><td>General anesthesia</td></tr><tr><td>Rompun</td><td>Bayer Healthcare, Germany/CentraVet, France</td><td>ROM001</td><td>General anesthesia, analgesia, muscle relaxation</td></tr><tr><td>NaCl 0.9%</td><td>Laboratoire Osalia, France&amp;nbsp;</td><td>103697114</td><td>Physiological serum</td></tr><tr><td>Systene Ultra</td><td>Alcon, Novartis, USA</td><td>-</td><td>Hydration of eyes</td></tr><tr><td>GenTeal&#039;</td><td>Alcon, Novartis, USA</td><td>-</td><td>Ophtalmic gel to minimize light refraction and opacities</td></tr><tr><td>Aniospray Surf 29</td><td>Laboratoires Anios, France</td><td>59844</td><td>Desinfectant</td></tr></tbody></table>

optical coherence tomography: imaging mouse retinal ganglion cells in vivo

视网膜结构改变是眼科疾病的常见表现。光学相干断层扫描 (OCT) 使他们的识别在体内-迅速, 反复, 并在高分辨率。该协议描述 OCT 成像在鼠标视网膜作为一个强大的工具, 研究光学神经病 (OPN)。OCT 系统是一个 interferometry-based, 非侵入性的替代一般的验尸组织学化验。它提供了一个快速和准确的评估视网膜厚度, 使可能跟踪变化, 如视网膜变薄或增厚。我们以 Opa1delTTAG鼠标线为例介绍了成像过程和分析。提出了三种扫描方式, 采用两种定量方法: 标准和自制卡尺。后者是最好的使用在 peripapillary 视网膜的径向扫描;更精确, 更适合分析更薄的结构。这里描述的所有方法都是为视网膜神经节细胞 (王国) 设计的, 但很容易适应其他细胞的数量。总之, OCT 是有效的小鼠模型分, 并有可能用于可靠的评价治疗干预。

SD OCT 技术使视网膜的想象和厚度分析可以与组织学相媲美, 但速度更快且更详细 (图 3)。正如野生 C57Bl/6 小鼠所提出的, 即使 SD OCT 扫描 (图 3A, 右) 的质量不如视网膜横断面 (图 3A, 左) 的图像好, 它可视化更多的层 (例如, OLM)。此外, 它只需要大约40分钟, 包括小鼠准备, 与几天或几周的组织学分析。最?...

Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

这篇手稿描述了一个协议为在体内成像的小鼠视网膜与高分辨率光谱领域光学相干断层扫描 (SD OCT)。它的重点是视网膜神经节细胞 (王国) 在 peripapillary 地区, 有几个扫描和量化的方法描述。

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

optical-coherence-tomography-imaging-mouse-retinal-ganglion-cells

Research

JoVE Journal

Neuroscience

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

19.1K 次观看.  INSERM U1051, Institut of Neurosciences of Montpellier. 这篇手稿描述了一个协议为在体内成像的小鼠视网膜与高分辨率光谱领域光学相干断层扫描 (SD OCT)。它的重点是视网膜神经节细胞 (王国) 在 peripapillary 地区, 有几个扫描和量化的方法描述。

视频: Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

科研

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 ...

医学

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

神经科学

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 ...

小鼠视网膜 OCT 纤维图与共聚焦图像的比对

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

作者聚焦：用于改善青光眼诊断和治疗的体内和离体视网膜成像的进展 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

光学相干断层扫描在视网膜病变小鼠模型中的应用

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

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.

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.

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

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

通过共聚焦扫描激光检眼镜对具有定制腺相关病毒血清型的视网膜细胞进行纵向体内成像

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

摘要

探索更多视频

此视频中的章节

使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估

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

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

小鼠视网膜的经瞳双光子体内成像

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

光学相干断层扫描在视网膜病变小鼠模型中的应用

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

通过共聚焦扫描激光检眼镜对具有定制腺相关病毒血清型的视网膜细胞进行纵向体内成像