Name: in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy
Uploaded: 2017-11-11T14:00:00.000+00:00
Duration: 6 min 19 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

这项工作得到了瑞士国家科学基金会 (SNSF; #320030_156019) 的资助。作者获得德国海德堡工程有限公司的非金融支持。

在所有过程中, balb/c/c 的成年雄性和雌性小鼠在 "Cx3cr1" 的启动子下表达 gfp, 使用 24 。根据《关于在眼科和视力研究中使用动物的帕特声明》对小鼠进行了治疗, 所有的程序都是瑞士政府根据《联邦瑞士动物福利条例》批准的。通过皮下注射盐酸 medetomidine (0.75 毫克/千克) 和氯胺酮 (45 毫克/千克) 麻醉小鼠。通过监测呼吸速率和动物和 #39 对尾部捏的反射, 确定了适当的麻醉。在实验结束时, 小鼠被安乐死, CO 2 吸入. 
 注意: 尽可能快地执行每个成像会话 (最多20分钟), 因为麻醉后白内障形成可能会妨碍视网膜可视化 30 . 
 1. 系统配置 <ol> <li> 打开计算机. </li>
	<li> 打开电源。消息和 #34; 启动采集模块和 #34; 将出现在设备的控制面板屏幕上. </li>
	<li> 双击软件的桌面快捷方式来操作软件. </li>
	<li> 在 "数据库" 视图中, 单击 "#34"; 添加患者和 #34; 图标. </li>
	<li> 在弹出窗口中, 插入所有必需的信息 (姓氏、名字、标题、出生日期、性别和患者 ID), 然后单击并 #34; 好的 #34。在弹出窗口设置的角膜曲率为2毫米, 并点击和 #34; 好的 #34;... </li>
	<li> 在标准30和 #176 前放置一个78D 标准的眼科非接触式狭缝灯透镜, 并将其安全地固定在磁带上. </li>
	<li> 确保设备左侧的滤镜拉杆位于指示和 #34 上; A 和 #34; 允许 IR + OCT 和 AF 成像 ( 图 1 ). </li>
</ol> 2。鼠标准备 <ol> <li> 使用 medetomidine 盐酸盐剂量为0.75 毫克/千克和氯胺酮剂量为45毫克/千克的磷酸盐缓冲盐水 (PBS, pH 7.4) 准备麻醉解决方案。因此, 对于一个20克的老鼠, 混合15和 #181; 盐酸 medetomidine 与18和 #181; l 的氯胺酮到最后的体积50和 #181; 我在 PBS将解决方案存储在4和 #176; C 最多为一周. </li>
	<li> 抓住鼠标的颈背和应用一滴卡 0.5% + 肾上腺素盐酸盐2.5% 在每只眼睛, 以达到瞳孔扩张. </li>
	<li> 将鼠标保持克制, 并将胰岛素注射器附在30G 针上, 注入50和 #181; 麻醉液的 L将鼠标放回放在加热垫上方的笼子里。等待3-5 分钟, 直到鼠标完全镇静。为了评估麻醉深度, 捏一下老鼠的尾巴。用棉签轻轻触摸鼠标角膜, 检查角膜反射。如果没有观察到阳性反射, 请继续进行成像. 
	注意: 监测麻醉期间的呼吸率是很重要的。如果老鼠没有完全镇静, 呼吸的速率将会在痛苦的刺激下增加. </li>
	<li> 在每只眼睛上使用 2% hydroxypropylmethylcellulose 滴的小鼠角膜. </li>
</ol> 3。SD OCT <ol> <li> 将温度约32和 #176 的变暖补丁放在设备的下巴休息的 custom-made 平台上 ( 图 1 ). </li>
	<li> 若要图像右眼, 请将鼠标放在平台的左部分 ( 图 1 )。确保鼠标的右轨道朝向镜头, 其主体位于平台的左侧. </li>
	<li> 在右眼上应用一滴 hydroxypropylmethylcellulose, 并小心地将 +4 屈光度刚性透气接触镜置于其上 (球形电源:-25.00 至 +25.00 屈光度)。将隐形眼镜储存在平衡盐溶液 (BSS) 中。要抓住镜片, 使用塑料或硅钳来避免损坏. </li>
	<li> 按下控制面板显示的右上角的黄色框以启动采集模块 (绿色方框在 图 2A )。黄色框将变成绿色, 控制面板菜单将出现在屏幕上 ( 图 2A ). </li>
	<li> 要获取 B 扫描, 请在控制面板上选择 "IR + OCT" 选项 ( 图 2A )。选中的设置在 "控制面板" 中以蓝色突出显示. </li>
	<li> 选择和 #34; 视网膜和 #34; #34; 应用和结构; #34; 在软件中, 使用设备上的微将镜头移向鼠标眼睛 ( 图 1 )。在聚焦到视网膜之前, 检查指示和 #34; OD 和 #34; 在屏幕的左下部分选择。该软件根据目标的位置自动识别左 (OS) 和右 (OD) 眼. 
	注意: 如果目标定位在平台的中间, 屏幕底部将出现一条错误消息。在这种情况下, 稍微重新鼠标, 直到软件识别正确的眼睛. </li>
	<li> 使用焦点旋钮, 将焦点放在视网膜上, 直到在计算机屏幕左侧的眼底图像中清晰地看到大血管。通过向左或向右转动微来移动照相机, 将相机按该方向移动, 或顺时针或逆时针, 分别将摄像机上下移动. 
	注意: 在平台上重新定位鼠标或将镜头向上或向下移动, 以实现红外图像中的视觉神经头的首选定位. </li>
	<li> 逆时针旋转灵敏度旋钮以减少或顺时针方向增加眼底图像的亮度。当实现最佳聚焦时, 屏幕右侧将出现一个 SD OCT B 扫描. 
	注意: 如果无法通过调整焦点来可视化 B 扫描, 请按键盘上的组合 Ctrl + Alt + Shift + O。在弹出窗口中, 调整参考臂的值, 直到 B 扫描出现在屏幕上. </li>
	<li> 在屏幕右下角, 从 "图案" 菜单中选择 "单个扫描" (在 "图 2B" ) 中的 "单行" . </li>
	<li> 将设备的微 ( 图 1 ) 向左、向右、向前或向后打开 (以该方向打开照相机), 以确保 B 扫描位于 SD OCT 扫描窗口的顶部和底部角之间. </li>
	<li> 将自动实时 (ART) 值设置为至少9以获得高质量的图像. 
	注意: ART 通过平均连续扫描来提高图像质量。#34; 艺术与 #34; 价值; 高信噪比和图像质量。然而, 通过增加 #34; 艺术和 #34; 价值, 获取时间也增加. </li>
	<li> 按 #34; 获取和 #34; 按钮 ( 图 2 ) 在控制面板屏幕上并获取图像. </li>
	<li> 重新定位鼠标以图像左眼并重复步骤 3.2-3.12. </li>
	<li> 用塑料/硅钳取出隐形眼镜, 并将其放入 BSS 中. </li>
	<li> 用一滴新的 hydroxypropylmethylcellulose 将老鼠角膜水合, 然后用纸巾除去多余的部分. </li>
	<li> 通过逆时针旋转来移除标准30和 #176; 光学. </li>
	<li> 安装55和 #176; 镜头并对每只眼睛重复步骤 3.4-3.12. </li>
</ol> 4。自动荧光成像 <ol> <li> 不在平台上移动鼠标, 请在控制面板上选择 IR. </li>
	<li> 聚焦旋钮, 聚焦于大的视网膜血管. </li>
	<li> 在控制面板上选择 AF. </li>
	<li> 逆时针旋转灵敏度旋钮以减少或顺时针方向, 以增加图像的亮度. </li>
	<li> 按下敏感旋钮并设置 #34; 艺术和 #34; 值为67或更高, 以获得高质量的图像. </li>
	<li> 当集和 #34; 艺术和 #34; 价值已经达到, 新闻和 #34; 获取和 #34; 在控制面板上获取图像。再次按下灵敏度旋钮停止平均。调整焦点, 以可视化不同的视网膜层. </li>
	<li> 删除55和 #176; #160; 镜头并安装宽字段102和 #176; #160; 镜头。按照步骤4.1 和 #8211; 4.6 为每个鼠标和每只鼠标的眼睛. </li>
	<li> 单击并 #8220; 保存图像和 #8221; 在窗口的左上边框, 然后单击和 #8220; 退出和 #8221;。要保存计算机中的图像, 请通过双击鼠标名称从屏幕右侧的列表中选择鼠标。分别双击每个图像, 然后单击软盘图标。将图像另存为所需文件夹中的. tif、. bmp、. jpg 或. png。退出软件 #8220; 文件和 #8221; #8220; 退出和 #8221;. </li>
</ol> 5。麻醉逆转 <ol> <li> 用一滴 hydroxypropylmethylcellulose 将鼠标的眼睛与热垫上的鼠标返回. </li>
	<li> 以2.25 毫克/千克的剂量制备唑溶液, 以逆转盐酸 medetomidine 的镇静效果。对于20克鼠标, 添加9和 #181; atipamezol 在最后的150和 #181 的数量; l PBS。将解决方案存储在4和 #176; C 最多为一周. </li>
	麻醉注射后最小值 (步骤 2.2), 注入 150 #181; 唑溶液皮下注射. </li>
	<li> 监视鼠标直到麻醉恢复。当鼠标完全恢复 (唑注射后5-10 分钟) 把它放回笼子里。恢复是指动物的能力, 在一侧奠定了它的身体时, 重拾步行活动, 并响应环境刺激反应. </li>
</ol> 6。人工视网膜厚度测量从 SD oct 图像 <ol> <li> 通过双击该动物的名称打开 oct 扫描. </li>
	<li> 打开用30和 #176 获得的 B 扫描; 或55和 #176; 镜头. </li>
	<li> 选择和 #34; 厚度剖面和 #34;. </li>
	<li> 按 #34; 编辑图层分割和 #34; 图标 ( 图 2B )。该软件自动识别内限和基膜. </li>
	<li> 如有必要, 手动更正内限和基底膜的位置。为此, 请选择要从屏幕左侧修改的图层, 然后选取红色圆圈选项 ( 图 2B )。按下鼠标按钮移动圆圈以修改该行. </li>
	<li> 修改行以正确定位相应图层。单击并 #34; 保存和关闭 #34; 退出窗口. 
	注: 由于脉络膜反射率的原因, 该软件可能不正确地识别出底座。因此, 最好在进行视网膜厚度测量之前, 手动定义它. </li>
	<li> 选择和 #34; 视网膜和 #34; 下 #34; 图层和 #34; 选项。屏幕底部会出现视网膜厚度图. </li>
	<li> 单击图上或 B 扫描上的不同位置可查看所选位置的视网膜厚度 (显示在和 #181; m). </li>
	<li> 测量所需距离视神经头的视网膜厚度并在电子表格中复制值. </li>
	<li> 对30和 #176 中的每个鼠标重复步骤 5.1-5.9; 55 和 #176; 镜头. </li>
	<li> 使用所需软件执行统计分析. </li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+retinal+microglial+activation+during+neurodegeneration:+confocal+ophthalmoscopic+imaging+and+cell+morphometry+in+mouse+glaucoma.">In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma.</a> J Vis Exp. (99), e52731 (2015).</li><li>Acton, J. H., Cubbidge, R. P., King, H., Galsworthy, P., Gibson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+detection+in+retro-mode+imaging+by+a+scanning+laser+ophthalmoscope.">Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope.</a> Acta Ophthalmol. 89 (5), e404-e411 (2011).</li><li>Greenstein, V. 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=Structural+and+functional+changes+associated+with+normal+and+abnormal+fundus+autofluorescence+in+patients+with+retinitis+pigmentosa.">Structural and functional changes associated with normal and abnormal fundus autofluorescence in patients with retinitis pigmentosa.</a> Retina. 32 (2), 349-357 (2012).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Wang, X., 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=Requirement+for+Microglia+for+the+Maintenance+of+Synaptic+Function+and+Integrity+in+the+Mature+Retina.">Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.</a> J Neurosci. 36 (9), 2827-2842 (2016).</li><li>Ebneter, A., Casson, R. J., Wood, J. P., Chidlow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+the+visual+pathway+in+experimental+glaucoma:+spatiotemporal+characterization+and+correlation+with+axonal+injury.">Microglial activation in the visual pathway in experimental glaucoma: spatiotemporal characterization and correlation with axonal injury.</a> Invest Ophthalmol Vis Sci. 51 (12), 6448-6460 (2010).</li><li>Ebneter, A., Kokona, D., Schneider, N., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Activation+and+Recruitment+of+Circulating+Macrophages+During+Ischemic+Experimental+Branch+Retinal+Vein+Occlusion.">Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.</a> Invest Ophthalmol Vis Sci. 58 (2), 944-953 (2017).</li><li>Lin, Y. L., Potter-Baker, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Effect+of+Oral+CSF-1R+Kinase+Inhibitor+on+Retinal+Microglia+Revealed+by+In+Vivo+Scanning+Laser+Ophthalmoscopy.">Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy.</a> Transl Vis Sci Technol. 6 (2), 10 (2017).</li><li>Gabriele, M. L., 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, S., 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. K., 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=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. K., 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=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. H., 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

作者没有什么可透露的。

本篇文章展示了一个协议为获取视网膜 B 扫描和成像的 gfp 阳性小胶质细胞分布在老鼠视网膜在同一成像会议。SD OCT 和斯洛伐克越来越多地用于视网膜疾病的动物模型, 提供信息的视网膜改变的时间10,14,17,18,21。使用此协议, Cx3cr1gfp/gfp 或Cx3cr1gfp/+ </em...

在实验性眼科, 视网膜病理学的检查通常使用组织学技术进行评价。然而, 组织学要求动物 euthanization, 并可能导致改变的实际性质的组织。SD OCT 和斯洛伐克是常规用于临床眼科诊断目的和监测的一些视网膜疾病, 如糖尿病性黄斑水肿9, 前缺血性视神经病变10, 或视网膜色素变性11.SD OCT 和斯洛伐克是非侵入性的技术, 产生高分辨率的图像的视网膜, 这是可视化通过扩张瞳孔不进一步干预。SD OCT 提供视网膜结构和视网膜厚度的信息, 通过收集后向散射数据来创建视网膜的横断面图像, 而斯洛伐克收集荧光数据以产生视网膜的立体高对比度图像。现在, 这两种技术都越来越多地用于实验性眼科使用小鼠12,13,14,15 (甚至斑马鱼16,17), 可以提供定性和定量信息12、17、18、19、20、21。
内源性荧光如 lipofuscins 的积累或视网膜疣的形成可通过斯洛伐克作为自动荧光信号进行可视化。该功能使斯洛伐克成为诊断和监测视网膜疾病, 如年龄相关的黄斑变性或视网膜色素变性的有价值的技术22,23。在实验性眼科, 自动荧光成像 (AF) 可用于检测特定的细胞类型在记者的鼠标线。例如, 在Cx3cr124的启动子下, 杂表达 gfp 的小鼠对在正常视网膜中胶质细胞的可视化以及对胶质细胞/巨噬生物的研究是有利的。视网膜疾病的动态21。小胶质细胞是视网膜的常驻巨噬生物, 在损伤的组织稳态和组织修复中起关键作用1,25,26。小胶质细胞活化在视网膜已报告视网膜损伤, 缺血, 和变性, 提示在视网膜疾病的这些单元的作用2,3,4,5,6。
本议定书的目的是描述一个相对简单的方法, 视网膜成像和测量视网膜厚度使用 SD OCT, 并可视化 gfp 阳性小胶质细胞在Cx3cr1gfp/gfp 鼠标视网膜使用斯洛伐克 (海德堡 Spectralis HRA + OCT 系统)。本协议可用于各种小鼠行中健康或病变视网膜的成像和厚度测量。此外, 还可以使用斯洛伐克21对视网膜小胶质细胞数和微胶质细胞的活化进行形态学分析。小胶质细胞与中枢神经系统 (CNS) 的退行性疾病有关, 包括视网膜27,28,29。因此, 结合本协议中使用的两种方法, 可以使小胶质细胞分布与视网膜变性的相关性, 从而有助于监测疾病的严重性或治疗方法的有效性在体内。

<table><tbody><tr><td>Spectralis Imaging system (HRA+OCT)</td><td>Heidelberg Engineering, Germany</td><td>N/A</td><td>ophthalmic imaging platform system</td></tr><tr><td>Heidelberg Eye Explorer</td><td>Heidelberg Engineering, Germany</td><td>N/A</td><td>Version 1.9.13.0</td></tr><tr><td>78D standard ophthalmic non-contact slit lamp lens</td><td>Volk Optical Inc., Ohio, USA</td><td>V78C</td><td></td></tr><tr><td>Spectralis wide angle 55&amp;deg; lens</td><td>Heidelberg Engineering, Germany</td><td>50897-002</td><td></td></tr><tr><td>ultra widefield 102&amp;deg; lens</td><td>Heidelberg Engineering, Germany</td><td>50117-001</td><td></td></tr><tr><td>medetomidine hydrochloride 1 mg/mL (Domitor)</td><td>Provet AG, Lyssach, Switzerland</td><td>Swissmedic Nr. 50&#039;590 - ATCvet: QN05CM91</td><td>anesthetic/analgesic</td></tr><tr><td>ketamine 50mg/ml (Ketalar)</td><td>Parke-Davis, Zurich, Switzerland</td><td>72276388</td><td>anesthetic</td></tr><tr><td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td><td>ISPI, Bern, Switzerland</td><td>N/A</td><td>pupil dilation</td></tr><tr><td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td><td>B. Braun Mesungen AG, Carl-Braun-Stra&amp;szlig;e, Germany</td><td>9151125</td><td></td></tr><tr><td>hydroxypropylmethylcellulose (Methocel 2%)</td><td>OmniVision, Neuhausen, Switzerland</td><td>N/A</td><td></td></tr><tr><td>+4 dpt rigid gas permeable contact lens</td><td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td><td>N/A</td><td>Base Curve: 7.20 to 8.40 mm&lt;br/&gt; Diameter: 9.00 / 9.60 / 10.20 mm&lt;br/&gt; Power: -25.00 to +25.00 Diopters</td></tr><tr><td>balanced salt solution (BSS)</td><td>Inselspital, Bern, Switzerland</td><td>N/A</td><td></td></tr><tr><td>silicon forceps</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>atipamezole 5 mg/mL (Antisedan)</td><td>Provet AG, Lyssach, Switzerland</td><td>N/A</td><td>&amp;alpha;2 adrenergic receptor antagonist</td></tr><tr><td>GraphPad Prism 7</td><td>GraphPad Software, Inc, San Diego, CA, USA</td><td>N/A</td><td>statistical analysis software</td></tr></tbody></table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

光谱领域光学相干层析成像 (SD OCT) 和扫描激光眼底 (斯洛伐克) 广泛应用于实验眼科。在本协议中, 在Cx3cr1的启动子 (balb/c/c-Cx3cr1gfp/gfp) 下, 使用表达绿色荧光蛋白 (gfp) 的小鼠在视网膜中对胶质细胞进行了体内的图像。小胶质细胞是视网膜的常驻巨噬, 并有牵连在几个视网膜疾病1,2,3,4,5,6。该协议提供了一个详细的方法来生成视网膜 B 扫描, 与 SD OCT, 和成像的小胶质细胞分布在Cx3cr1gfp/gfp 鼠与斯洛伐克在体内, 使用眼科成像平台系统。该协议可用于几个记者鼠标线。但是, 这里提出的协议有一些限制。首先, 斯洛伐克和 SD OCT, 当用于高分辨率模式, 收集数据具有较高的轴向分辨率, 但横向分辨率较低 (3.5 µm 和6µm, 分别)。此外, 斯洛伐克的焦点和饱和度高度依赖于参数的选择和眼睛的正确对准。此外, 使用设计的人的病人在小鼠是具有挑战性的, 由于更高的总光功率的鼠标眼睛相比, 人眼;这可能导致侧向放大率不准确7, 这也取决于鼠标镜头在其他方面的放大倍数。然而, 尽管轴向扫描位置取决于横向放大率, 轴向 SD OCT 测量是准确的8。

使用这里提供的协议, SD OCT 扫描和斯洛伐克图像是从Cx3cr1gfp/gfp 鼠标在同一成像会话中获得的。图 3包括具有代表性的 SD OCT 单扫描, 它获得了30°或55°透镜 (图 3A) 和具有55°或102°透镜的具有代表性的斯洛伐克图像, 其中 gfp 阳性小胶质细胞是可视化的。与55°透镜相比, 在30°的 SD OCT 扫描中观察到脉络膜的高反射率?...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

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

本协议描述了如何利用眼科成像平台系统, 在小啮齿动物中利用光谱域光学相干层析成像和扫描激光眼底等高分辨率成像技术, 获取有关视网膜厚度和胶质细胞分布, 分别。

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 ...

in-vivo-imaging-cx3cr1gfpgfp-reporter-mice-with-spectral-domain

Research

JoVE Journal

Medicine

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

10.5K 次观看.  Bern University Hospital and University of Bern. 本协议描述了如何利用眼科成像平台系统, 在小啮齿动物中利用光谱域光学相干层析成像和扫描激光眼底等高分辨率成像技术, 获取有关视网膜厚度和胶质细胞分布, 分别。

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

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

科研

医学

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

生物工程

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

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 ...

神经科学

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 ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

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 ...

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.

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

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

摘要

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此视频中的章节

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

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

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

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

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

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

摘要

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此视频中的章节

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

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

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 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

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

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

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像