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>

<ol>
	<li>Madeira, M. H., Boia, R., Santos, P. F., Ambrosio, A. F., Santiago, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia-mediated+neuroinflammation+to+retinal+degenerative+diseases.">Contribution of microglia-mediated neuroinflammation to retinal degenerative diseases.</a> Mediators Inflamm. , 673090 (2015).</li><li>Ng, T. F., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+migration+of+retinal+microglia+into+the+subretinal+space.">Light-induced migration of retinal microglia into the subretinal space.</a> Invest Ophthalmol Vis Sci. 42 (13), 3301-3310 (2001).</li><li>Langmann, T. <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+in+retinal+degeneration.">Microglia activation in retinal degeneration.</a> J Leukoc Biol. 81 (6), 1345-1351 (2007).</li><li>Joly, 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=Cooperative+phagocytes:+resident+microglia+and+bone+marrow+immigrants+remove+dead+photoreceptors+in+retinal+lesions.">Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.</a> Am J Pathol. 174 (6), 2310-2323 (2009).</li><li>Arroba, A. I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+IGF-I+neuroprotection+in+the+rd10+mouse+model+of+retinitis+pigmentosa.">Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52 (12), 9124-9130 (2011).</li><li>Zhang, C., Lam, T. T., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+populations+of+microglia/macrophages+in+the+retina+and+their+activation+after+retinal+ischemia+and+reperfusion+injury.">Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury.</a> Exp Eye Res. 81 (6), 700-709 (2005).</li><li>Geng, 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=Optical+properties+of+the+mouse+eye.">Optical properties of the mouse eye.</a> Biomed Opt Express. 2 (4), 717-738 (2011).</li><li>Lozano, D. C., Twa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rat+schematic+eye+from+in+vivo+biometry+and+the+correction+of+lateral+magnification+in+SD-OCT+imaging.">Development of a rat schematic eye from in vivo biometry and the correction of lateral magnification in SD-OCT imaging.</a> Invest Ophthalmol Vis Sci. 54 (9), 6446-6455 (2013).</li><li>Vaz-Pereira, 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=Optical+Coherence+Tomography+Features+Of+Active+And+Inactive+Retinal+Neovascularization+In+Proliferative+Diabetic+Retinopathy.">Optical Coherence Tomography Features Of Active And Inactive Retinal Neovascularization In Proliferative Diabetic Retinopathy.</a> Retina. 36 (6), 1132-1142 (2016).</li><li>Kokona, D., Haner, N. U., Ebneter, A., 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=Imaging+of+macrophage+dynamics+with+optical+coherence+tomography+in+anterior+ischemic+optic+neuropathy.">Imaging of macrophage dynamics with optical coherence tomography in anterior ischemic optic neuropathy.</a> Exp Eye Res. , (2016).</li><li>Makiyama, 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=Macular+cone+abnormalities+in+retinitis+pigmentosa+with+preserved+central+vision+using+adaptive+optics+scanning+laser+ophthalmoscopy.">Macular cone abnormalities in retinitis pigmentosa with preserved central vision using adaptive optics scanning laser ophthalmoscopy.</a> PLoS One. 8 (11), e79447 (2013).</li><li>Paques, M., 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=High+resolution+fundus+imaging+by+confocal+scanning+laser+ophthalmoscopy+in+the+mouse.">High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse.</a> Vision Res. 46 (8-9), 1336-1345 (2006).</li><li>Joshi, R., 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=Spontaneously+occurring+fundus+findings+observed+using+confocal+scanning+laser+ophthalmoscopy+in+wild+type+Sprague+Dawley+rats.">Spontaneously occurring fundus findings observed using confocal scanning laser ophthalmoscopy in wild type Sprague Dawley rats.</a> Regul Toxicol Pharmacol. 77, 160-166 (2016).</li><li>Muraoka, 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=Real-time+imaging+of+rabbit+retina+with+retinal+degeneration+by+using+spectral-domain+optical+coherence+tomography.">Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography.</a> PLoS One. 7 (4), e36135 (2012).</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>Bell, B. 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+vasculature+of+adult+zebrafish:+in+vivo+imaging+using+confocal+scanning+laser+ophthalmoscopy.">Retinal vasculature of adult zebrafish: in vivo imaging using confocal scanning laser ophthalmoscopy.</a> Exp Eye Res. 129, 107-118 (2014).</li><li>Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-domain+optical+coherence+tomography+as+a+noninvasive+method+to+assess+damaged+and+regenerating+adult+zebrafish+retinas.">Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas.</a> Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).</li><li>Huber, 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=Spectral+domain+optical+coherence+tomography+in+mouse+models+of+retinal+degeneration.">Spectral domain optical coherence tomography in mouse models of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 50 (12), 5888-5895 (2009).</li><li>Dysli, C., Enzmann, V., Sznitman, R., 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=Quantitative+Analysis+of+Mouse+Retinal+Layers+Using+Automated+Segmentation+of+Spectral+Domain+Optical+Coherence+Tomography+Images.">Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.</a> Transl Vis Sci Technol. 4 (4), 9 (2015).</li><li>Sim, D. 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=A+simple+method+for+in+vivo+labelling+of+infiltrating+leukocytes+in+the+mouse+retina+using+indocyanine+green+dye.">A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.</a> Dis Model Mech. 8 (11), 1479-1487 (2015).</li><li>Bosco, A., Romero, C. O., Ambati, B. K., Vetter, M. 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. 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. 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=A+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., 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=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&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; 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&#39;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&#223;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 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			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>&#945;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

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

科研

医学

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

多普勒光学相干断层扫描视网膜循环

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...