不同吸氧浓度和时间在新生小鼠视网膜病发病中的作用

Role of different oxygen concentration and different period of oxygen exposure in pathogenesis of retinopathy in neonatal mice

目的评价不同吸氧浓度(fraction of inhaled oxygen,FiO_2)及吸氧时间在新生小鼠视网膜病中的致病作用。方法选择7日龄(P7)新生 C57BL/6J 小鼠204只,分为8组:组1～组3(每组均为24只)分别吸30%氧气5、7、9 d,组4～组6(每组均为24只)分别吸50%氧气5、7、9 d,组7(n=30)吸75%氧气5 d,组8(n=30)对照组在空气中生长。采用视网膜铺片法经 ADP 酶染色,观察吸氧后视网膜血管形态的动态改变;视网膜切片经常规 HE 染色,计数平均每只小鼠每张切片上突破视网膜内界膜的新生血管内皮细胞核数目,以定量反映视网膜血管的增生情况。结果 (1)视网膜铺片结果:①FiO_2为30%:吸氧5 d 或7 d 后视网膜血管形态全部正常;吸氧9d 后深层血管网轻度阻塞,出氧箱2 d 后迅速恢复正常,至 P21发育成熟。②FiO_2为50%:吸氧5 d 后视网膜血管较明显收缩,视网膜中央较大区域无血管灌注,出氧箱2 d 后出现较多新生血管,但出氧箱5 d 后血管形态基本恢复正常;随着吸氧时间的延长,视网膜血管异常程度逐渐减轻,恢复更快。③FiO_2为75%:吸氧5 d后视网膜血管明显收缩,视网膜中央大片区域无血管灌注,出氧箱2 d 后出现新生血管,P17～P21达到高峰;延长吸氧时间后母、幼鼠死亡率明显上升。(2)视网膜切片结果:仅组7(FiO_275%×5d)新生血管内皮细胞核数目达(41.9±2.8)个,与其余各组(均<1个)差异有显著统计学意义(P<0.0001),其余各组间差异均无统计学意义。结论吸氧浓度和持续时间可影响视网膜血管的发育,长时间高浓度吸氧导致不可逆的血管增生性改变,临床上应尽量避免。

Objective To evaluate the role of different oxygen concentration （FiO2） and different period of oxygen exposure on oxygen-induced retinopathy （OIR） in neonatal mice and to provide evidences for proper clinical oxygen therapy. Methods Two hundred and four 7-day-old （P7） C57BL/6J mice were exposed to different FiO2 30%, 50% and 75% for 5, 7 and 9 days. The mice were divided into eight groups： groups 1-3 （n = 24 in each） were exposed to 30% oxygen for 5, 7 and 9 days, respectively; groups 4-6 （n = 24 in each） were exposed to 50% oxygen for 5, 7 and 9 days, respectively; group 7 （n = 30） was exposed to 75% hyperoxia for 5 days; group 8 （n = 30） was exposed to room air. Proliferative neovascular responses were estimated by observing vascular patterns in adenosine diphosphate-ase （ADPase） stained retina fiat- mounts and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections. Results （1） Vascular patterns in retina fiat-mounts： a） When FiO2 was 30%, the entire vascular pattern was completely normal after 5 and 7 days exposure; although the deep vascular system seemed slightly constricted after 9 days exposure, it recovered 2 days later and matured at P21. b） When FiO2 was 50%, after 5 days exposure （group 4）, the larger vessels constricted and central perfuaion decreased moderately; after exposing to room air for 2 days, neovascularization was seen; however, the entire vascular pattern was almost normal at P17. After 7 days of exposure to 50% O2 （group 5）, the vascular pattern recovered a bit, seemed to be better than that of group 4; after 9 days of exposure to 50% O2 （group 6）, only slight constriction could be seen and it disappeared 2 days later and all vessels matured later, c） When FiO2 was 75%, after 5 days exposure to hyperoxia, the larger vessels became tortuous and constricted, central perfusion became decreased obviously; after exposing to room air for 2 days, neovascularization was seen; and this response was maximal at P17-P21. However, the mortality of nurser mice and pups increased dramatically when the duration of hyperoxia was prolonged. （2）Quantitative results in cross-sections： neovascular nuclei extending into the vitreous reached （41.9±2.8） per section in 75% oxygen group, while less than 1 in every other groups, which was statistically different （P 〈 0.0001）. Conclusions FiO2 and the duration of hyperoxia cold affect retinal vascular development. Low and moderate FiO2 could induce reversible vessel changes, while high FiO2 induced irreversible changes which should be avoided in clinic.