走査レーザー検眼鏡(SLO)によるラット網膜神経線維層(RNFL)の観察と定量性の検討Brown NorwayラットのRNFLは、SLOによって明瞭に観察された。SLOの共焦点口径を最小としてRNFLが観察される屈折値の幅(ΔF)を決定した。視神経挫滅モデル(片眼の視神経を眼窩内で30秒間挫滅)では、挫滅後2週目からΔFは有意に減少し、虚血再灌流モデル(片眼の前房に45分間160mmHgを負荷)では虚血後1週目からΔFは有意に減少した。対照眼のΔFは不変であった。また、両モデルにおいてSLO撮影後に網膜組織切片を作成しRNFLの厚み(NFLT)を定量したところ、SLOでのΔFと網膜組織切片でのNFLTは有意に相関した。したがって、SLOはラットRNFLを生体内で定量的に観察するのに有用である。SLOによるラツト網膜神経節細胞体(RGC)の観察と定量性の検討上丘に注入されたDiA(4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine)によって逆行染色されたRGCは、SLO(フルオレセイン蛍光眼底造影用フィルター使用)によって明瞭に観察された。1眼1ヶ所の任意の設定部位においてSLOにて蛍光(+)細胞数を経時的に計測したところ、視神経挫滅後1週目から有意に細胞数は減少した。視神経挫滅前のSLO画像を白黒反転し、挫滅後のSLO画像と重ね合わせることによって新たに生じた蛍光点は、isolectin B4蛍光染色によってミクログリアに一致することが判明した。したがって、視神経挫滅後にSLOにおいて新たに生じた蛍光点を差し引いて蛍光(+)細胞数を定量したところ、網膜伸展標本でのRGC数とよく一致した。したがって、SLOはラットRGCを生体内で定量的に観察するのに有用である。1) In vivo imaging and quantitative evaluation of rat retinal nerve fiber layer (RNFL) by scanning laser ophthalmoscopy (SLO)Methods: Fundus images of both eyes were recorded over time by SLO using an argon blue laser (488 nm) in unilateral optic nerve crush or ischemia-reperfusion model. The focused plane was sequentially moved by changing the refractive values in the SLO setting. The range of refractive values (AF) in which RNFL reflex was clearly observed was determined. The RNFL thickness in retinal sections was measured and compared to the DF value. Results: The QF value was unchanged 1 week after the crush, but then decreased significantly after the second week, while it decreased significantly from the 1st week after the ischemia-reperfusion. The OF value correlated significantly with the histologically determined RNFL thickness.2) In vivo imaging and counting of rat retinal ganglion cells (RGCs) by SLOMethods: RGCs of Brown Norway rats were retrogradely labeled bilaterally with the fluorescent dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine (DiA). RGCs were imaged in vivo with a SLO using an argon blue laser and optical filter sets for fluorescein angiography, before and 1, 2, and 4 weeks after the crush. Fluorescent cells were also counted on retinal flatmounts. 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 by SLO. RGC survival decreased gradually after the 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 by retinal flatmounts.3) Conclusions: The SLO is useful for in vivo imaging and quantitative evaluation of rat RNFL and RGCs, and therefore may be a valuable tool for monitoring RNFL and RGC changes over time in various rat models of RGC damage.研究課題/領域番号:17591825, 研究期間(年度):2005-200