In vivo imaging and counting of rat retinal ganglion cells using a scanning laser ophthalmoscope
In vivo imaging and counting of rat retinal ganglion cells using a scanning laser ophthalmoscope
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DOI：
10.1167/iovs.05-0708
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发表时间：
2006-07-01
影响因子：
4.4
通讯作者：
Sugiyama, Kazuhisa
中科院分区：
文献类型：
--
作者：
Higashide, Tomomi;Kawaguchi, Ichiro;Sugiyama, Kazuhisa
目的。为了确定扫描激光检眼镜是否可用于活体成像和计数大鼠视网膜神经节细胞(RGC)。方法：用荧光染料4-(4-(dihexadecylamino)styryl)-N-methylpyridinium碘(DIA)逆行标记双侧大鼠视网膜神经节细胞。眼眶内用夹子夹住单侧视神经。分别在粉碎前、粉碎后1周、2周和4周，用氩蓝激光(488 Nm)SLO和荧光血管造影仪对视网膜神经节细胞进行活体成像。在基础状态和挤压后1、2、4周，对视网膜平铺片进行荧光细胞计数。执行图像叠加分析以检查随时间变化的SLO图像中的细胞位置。用凝集素组织化学方法检测小胶质细胞与图像叠加分析检测到的新的Dia荧光的关系。结果：在SLO的活体内可见荧光的RGC。RGC存活率在挤压后逐渐下降。在视神经夹伤后的视网膜，图像叠加分析检测到新出现的Dia荧光对应于与视网膜平台上的RGCs形态不同的荧光细胞，并主要与凝集素染色的小胶质细胞突起共存。结论：SLO可用于大鼠RGC的活体成像，因此可能是监测各种RGC损伤模型中RGC随时间变化的有价值的工具。
PURPOSE. To determine whether a scanning laser ophthalmoscope (SLO) is useful for in vivo imaging and counting of rat retinal ganglion cells (RGCs).METHODS. RGCs of Brown Norway rats were retrogradely labeled bilaterally with the fluorescent dye 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine (DiA). The unilateral optic nerve was crushed intraorbitally with a clip. RGCs were imaged in vivo with an SLO with an argon blue laser (488 nm) and optical filter sets for fluorescein angiography, before and 1, 2, and 4 weeks after the crush. Fluorescent cells were also counted in retinal flatmounts at baseline and 1, 2, and 4 weeks after the crush. An image overlay analysis was performed to check cell positions in the SLO images over time. Lectin histochemical analysis was performed to determine the relationship of microglia to the newly emerged DiA fluorescence detected by image overlay analysis after the optic nerve crush.RESULTS. Fluorescent RGCs were visible in vivo with an SLO. RGC survival decreased gradually after the crush. In the retina after the optic nerve crush, newly emerged DiA fluorescence detected by image overlay analysis corresponded to fluorescent cells morphologically different from RGCs in the retinal flatmount and was colocalized mostly with lectin-stained microglial processes. RGC counts by SLO were comparable to those in retinal flatmounts.CONCLUSIONS. The SLO is useful for in vivo imaging of rat RGCs and therefore may be a valuable tool for monitoring RGC changes over time in various rat models of RGC damage.