Salinas-Navarro M, Jiménez-López M, Valiente-Soriano F J, Alarcón-Martínez L, Avilés-Trigueros M, Mayor S, Holmes T, Lund R D, Villegas-Pérez M P, Vidal-Sanz M
Laboratorio de Oftalmología Experimental, Facultad de Medicina, Universidad de Murcia, E-30100 Murcia, Spain.
Vision Res. 2009 Mar;49(6):637-47. doi: 10.1016/j.visres.2009.01.010. Epub 2009 Jan 29.
In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus((R)) (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina.
The mean total numbers of RGCs labeled from the ON, in Swiss (49,493+/-3936; n=18) or C57 mice (42,658+/-1540; n=10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733+/-3954; n=43) or C57 mice (41,192+/-2821; n=42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.
在成年瑞士白化小鼠和C57有色小鼠中，通过将逆行运输的神经元示踪剂应用于双侧视神经（ON）或上丘（SCi）来识别视网膜神经节细胞（RGCs）。经过组织学处理后，将视网膜制成整装片，在配备有由商业计算机图像分析系统Image-Pro Plus((R))（IPP）控制的电动载物台的荧光显微镜下进行检查和拍照。使用Scope-Pro插件5.0的IPP将视网膜成像为一系列24位彩色图像（每个视网膜140帧），并在需要时将图像拼接以创建视网膜整装片的高分辨率合成图像。单个图像也通过IPP中编写的特定宏程序进行处理，这些宏程序应用一系列滤镜和变换以分离单个细胞进行自动计数。细胞计数随后转移到电子表格中进行统计分析，并用于生成每个视网膜的RGC密度图。
从瑞士小鼠（49,493±3936；n = 18）或C57小鼠（42,658±1540；n = 10）的视神经标记的RGC平均总数略高于从瑞士小鼠（48,733±3954；n = 43）或C57小鼠（41,192±2821；n = 42）的上丘标记的RGC平均数。RGC分布于整个视网膜，密度图显示在视神经盘附近的视网膜上部有一个水平区域，RGC密度最高。总之，小鼠RGC群体可以自动计数，其置信水平与手动计数相当。RGC的分布呈现出一种类似于水平视觉条纹的区域特化形式。
