大鼠视网膜缺血再灌注损伤后脑源性神经生长因子对细胞凋亡及caspase-2和caspase-3表达的影响

目的:探讨caspase-2和caspase-3在大鼠视网膜缺血再灌注损伤中的表达与细胞凋亡的关系及脑源性神经生长因子对其的影响及对视网膜的保护作用。方法:实验于2007-02/2007-07在青岛大学医学院附属医院中心实验室完成。前房加压法制作大鼠视网膜缺血再灌注损伤模型,28只大鼠随机分为正常组和手术组,其中手术组大鼠左眼为缺血再灌注组,右眼为治疗组(BDNF玻璃体腔注射),手术组又按照再灌注后不同时间段分为1,6,12,24,48,72h组。光学显微镜观察并计数视网膜神经节细胞的数量。应用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法(TUNEL)检测视网膜神经节细胞凋亡、免疫组织化学法(SABC)和酶联免疫吸附实验(ELISA)检测视网膜组织中caspase-2和caspase-3的表达情况。结果:正常视网膜未见凋亡细胞表达,缺血后6～24h可见大量凋亡细胞表达,48h开始下降。凋亡细胞在缺血后24h达到高峰,caspase-2缺血6h后逐渐增加,24h达高峰,然后在48至72h下降。caspase-3表达改变与caspase-2改变基本一致。BDNF治疗组各观察指标表达变化规律与缺血组基本一致,但能明显抑制凋亡细胞的表达,同时使caspase-2和caspase-3的表达降低。结论:视网膜缺血再灌注损伤诱导了神经节细胞的凋亡;BDNF可抑制caspase-2和caspase-3的表达,减少神经节细胞凋亡,对视网膜缺血再灌注损伤有治疗作用。

Effects of brain-derived neurotrophic factor on the expression of caspase-2 and caspase-3 and cell apoptosis in retinal ischemia/reperfusion injury

AIM:To explore the relationship between the expression of caspase-2 and caspase-3 and the apoptosis in retinal ischemia/reperfusion(I/R) injury of rats,as well as the therapeutic effects of brain derived neurotrophic factor(BDNF) on the ischemic and reperfused retina.METHODS:This experiment was conducted at the laboratory of Affiliated Hospital of Qingdao University Medical College from February 2007 to July 2007.The models of retinal ischemia/reperfusion injury were made by transiently elevating intraocular pressure.A total of 28 rats were divided into Normal and Operative Groups.Operative group was divided into six subgroups.In each subgroup there were four rats.The left eyes of rats were used for I/R and the right eyes were used for intravitreal injection of brain-derived neurotrophic factor(BDNF) as treatment group.After reperfusion we divided our subgroups according to the reperfusion time as 1,6,12,24,48,72 hours.The retinal ganglion cell number was counted by using optic microscope(BX-51,Olympus) .Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling(TUNEL) method,and the expression of caspase-2,caspase-3 was studied by enzyme linked immunosorbent assay(ELISA) and strept avidin-biotin complex(SABC) immunohistochemistry.RESULTS:No positive apoptotic cells were observed in the normal rats' retinae,but there were a significant number of positive apoptosis cells in 6-24 hours after transient ischemia followed by a decrease at 48 hours.The number of apoptotic cells reached a maximum at 24 hours after ischemia.The expression of caspase-2 gradually increased as early as at 6 hours,reached a peak at 24 hours,then decreased between 48 and 72 hours.Similarly,caspase-3 has the same rule with caspsae-2 in the time courses of expression in retinal tissues.BDNF administered before reperfusion inhibited the expression of apoptosis and ameliorated the retinal tissue damage.It also decreased caspase-2 and caspase-3 expression in ischemic/reperfused retina.CONCLUSION:Retinal ischemia-reperfusion can induce apoptosis of cells in the retina.BDNF rescues retinal ganglion cells(RGCs) from retinal ischemia/reperfusion injury through down-regulation of cell apoptosis and caspase-2 and caspase-3 expression.BDNF have a neuroprotective effect on retina.