分析Klotho突變小鼠作為老年性黃斑部病變的動物模型

全球8.7％的失明是由老年性黃斑部病變(AMD)引起，且在人口老化及3C產品大量普遍過度使用下，罹病率將大幅上升，是急需解決之問題。目前可以於短期內連續觀察早期及晚期AMD病理變化特徵的動物模型非常有限，且等待衰老需要大量時間，是研究上的一大障礙，因此有必要創造新的能夠模仿人體AMD且快速病理發生的動物研究模式。先前研究顯示Klotho蛋白量減少會縮短小鼠的壽命。Klotho加速老化小鼠在第4週開始有老化現象，然而是否可作為AMD動物模式，目前並無相關研究。因此本研究以六週和八週三種不同基因型Kl+/+、Kl+/-以及Kl-/-的老鼠做為研究對象，探討Klotho缺失對視網膜之影響。以H&E染色及觀察脂褐素(lipofuscin)自體螢光後發現六週和八週Kl-/-小鼠視網膜產生色素上皮細胞(RPE)基底沉積物增加、RPE細胞排列不均勻以及網膜節細胞層(GCL)細胞密度降低，以及脂褐素堆積增加等現象。而以免疫組織化學(IHC)染色觀察視網膜之Opsin以及Calbidin表現，發現錐狀細胞以及無軸突細胞和水平細胞的數量在Kl-/-的老鼠中有下降趨勢。最後，透過偵測瞳孔縮放對視網膜進行功能性分析後，發現Kl+/-之小鼠的瞳孔最大直徑以及收縮所需時間皆有增加的趨勢。綜上所述，本研究顯示Kl-/-小鼠表現出與乾性AMD一致的病理變化，具有成為研究乾性AMD之動物實驗模式之潛力。

AMD (age-related macular degeneration) prevails globally and is responsible for 8.7％ total blindness worldwide. With global population ageing and increasing popular use of 3C display products, its prevalence will be largely increased and a solution for this problem is in urgent need. However, there has been scarce animal models to provide early and late AMD pathological signs in a short time. Researchers will have to wait for a long time to examine the animal ageing processes, which is a major obstacle for ageing research. Therefore, to develop a rapid AMD animal model that displays the whole gamut of pathogenesis is warranted. Formerly, Klotho expression is found to affect life span by showing shorter life span under the influence of diminished Klotho expression. Although Klotho mutant mice accelerate ageing process from week 4 of age, whether the mice are suitable for use as an AMD study model remains to be confirmed. Thus, this study investigated and compared the retinal changes among Kl+/+, Kl+/-, and Kl-/- genotypes at their 6 weeks and 8 weeks of age. H&E stain and lipofuscin auto-fluorescence photography showed increased accumulation of cellular debris at the basal retinal pigmented epithelium (RPE), dis-arrangement of RPE cells, reduction of ganglion cell density at the ganglion cell layer (GCL), and increased lipofuscin accumulation in the homozygous mutant retinas, compared with heterozygous and wild-type littermates. Immnuohistochemistry (IHC) detection with opsin and calbidin antibodies showed reduced cones photoreceptor cells, as well as amacrine and horizontal cells in the mutant retinas. Pupil reflex tests were performed to reflect the functional aspect of vision and the results were compared between the aged Kl+/+K and Kl+/- mice. A longer time for the Kl+/- to reach maximal pupil diameter and to retract to minimal diameter was found. To conclude, the results of this study indicated that Kl-/- retinal pathological characteristics are representative of dry AMD, supporting the potential use of Klotho mutant mice as a suitable animal model for dry AMD studies in the future.