Organisciak D T, Li M, Darrow R M, Farber D B
Wright State University Petticrew Research Laboratory Department of Biochemistry and Molecular Biology USA Ohio, Dayton, OH 45435, USA.
Curr Eye Res. 1999 Aug;19(2):188-96. doi: 10.1076/ceyr.19.2.188.5333.
To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats.
Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels.
At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats.
The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.
确定遗传背景和光照饲养条件对幼龄RCS大鼠强光介导的视网膜变性的影响。
将rdy基因纯合或杂合的白化大鼠在昏暗的循环光照下繁殖并出生。在出生后第7天（P7），将它们转移到黑暗环境中，并维持在那里，直到在P18或P25暴露于强光（绿光）下。其他大鼠则留在昏暗的循环光照环境中。在P11至P40之间的不同时间处死大鼠，以测定视紫红质和光感受器细胞DNA水平，通过蛋白质免疫印迹法分析视网膜S抗原（抑制蛋白）和α-转导蛋白，或通过核酸印迹分析法分析它们各自的mRNA水平。
在P18时，未暴露于强光的黑暗饲养的纯合RCS大鼠及其表型正常的杂合对应物具有几乎相等的视紫红质水平和光感受器细胞DNA。与各自未暴露的对照组相比，在这个年龄暴露于8小时连续光或3小时间歇光下，不会导致视紫红质或视网膜DNA的损失。在P25时，视紫红质水平高于P18，而光感受器细胞DNA与较年幼的大鼠基本相同。然而，在P25暴露于强光会导致视紫红质和光感受器细胞DNA的大量损失，并且纯合大鼠的损失比杂合动物更大。在昏暗循环光照下饲养的P25大鼠的光损伤与在黑暗中饲养的纯合大鼠基本相同，但在杂合动物中未发现损伤。通过蛋白质免疫印迹分析，视网膜中的α-转导蛋白水平在黑暗中随时间增加，而视网膜S抗原水平在P15 - P35期间要么保持不变要么下降。对于处于循环光照环境中的大鼠，在所有年龄段S抗原表达均大于α-转导蛋白。对这两种蛋白质的mRNA进行核酸印迹分析，其模式通常与蛋白质免疫印迹分析所见一致。在两种光照饲养条件下的两种类型大鼠中，S抗原mRNA的表达年龄更早且水平更高于α-转导蛋白。在杂合和纯合大鼠中，S抗原的峰值表达最常出现在P18。
P18和P25大鼠中S抗原和α-转导蛋白的相对表达与其在P18时对视网膜光损伤的相对抗性及其在P25时增强的易感性相关。当用光感受器细胞DNA来估计视网膜光损伤程度时，rdy基因纯合的大鼠也比杂合动物表现出更多的损伤。
