Objective: To study the relationship between the apoptosis of retinal neurons and changes of manganese superoxide dismutase (MnSOD) activity and mRNA expression in early diabetic rats.
Methods: Controlled experimental study. Seventy two male 8-week-aged SD rats were divided into control group and diabetic mellitus group. Each group were subdivided into 4, 8 and 12 weeks groups (each group, n = 12). The diabetic group rats were injected with a single dose of streptozotocin (60 mg/kg) to induce the diabetic model, the control group rats were raised without any intervention. Apoptosis of retinal neurons was detected by TdT-mediated dUTP nick end label (TUNEL) assay. The protein expression of caspase-3 was detected by immunohistochemistry. Xanthine oxidase method was used to measure the activity of MnSOD and copper-znic superoxide dismutase (Cu-ZnSOD). MnSOD mRNA, Cu-ZnSODmRNA and caspase-3 mRNA levels were determined by SYBR Green Realtime PCR Master Mix. ANOVA was used to test the comparisons of the apoptosis ratio of retinal ganglion cells, the levels of caspase-3 mRNA, and the activity and mRNA levels of Cu-ZnSOD and MnSOD in the control groups and the diabetic groups, and LSD was used to test multiple comparisons.
Results: (1) The difference of the retinal ganglion cells apoptosis ratio in the control groups and the diabetic groups had statistical significance at 4, 8, 12 weeks (F = 19.5412, P < 0.05). There were no apoptosis neurons in control groups' retina at 4, 8, 12 weeks. Apoptosis of the retinal neurons occurred 4 weeks after the onset of diabetes. The apoptosis ratio of retinal ganglion cells in rats that had diabetes for 8 and 12 weeks was (5.7 +/- 3.9)% and (11.8 +/- 5.1)%, respectively, and was significantly higher than that of age-matched control groups (8 and 12 weeks: P = 0.000). (2) Caspase-3 protein expression was not observed in the control rats' retina at 4, 8, 12 weeks. Positive staining of caspase-3 occurred 4 weeks after the onset of diabetes, and enhanced at 8 and 12 weeks. The difference of caspase-3 mRNA levels in the control groups and the diabetic groups had statistical significance at 4, 8, 12 weeks (F = 105.175, P < 0.05). In control rats at 4, 8 and 12 weeks, caspase-3 mRNA levels were 1.649 +/- 0.586, 1.526 +/- 0.486, 1.614 +/- 0.296, respectively. Caspase-3 mRNA levels in diabetic rats that had diabetes for 4, 8 and 12 weeks were 5.672 +/- 1.193, 12.566 +/- 2.272, 14.297 +/- 2.11, respectively, which were greater than that in the control groups (4, 8 and 12 weeks: P = 0.000). (3) The difference of the activity and mRNA levels of MnSOD and Cu-ZnSOD in the control groups and the diabetic groups had statistical significance at 4, 8, 12 weeks (MnSOD: activity: F = 19.709, P < 0.05, mRNA: F = 93.352, P < 0.05; Cu-ZnSOD: activity: F = 16.708, P < 0.05, mRNA: F = 16.332, P < 0.05). In the control groups at 4, 8 and 12 weeks, the activity of MnSOD was (47.118 +/- 5.018), (46.033 +/- 6.835) and (45.813 +/- 6.859) U/mg, respectively; and MnSOD mRNA levels were 0.973 +/- 0.123, 0.974 +/- 0.085 and 0.994 +/- 0.074, respectively. The activity of Cu-ZnSOD was (113.884 +/- 9.07), (112.301 +/- 5.24) and (117.52 +/- 7.982) U/mg, respectively; and Cu-ZnSOD mRNA levels of were 1.067 +/- 0.109, 1.055 +/- 0.119, 1.092 +/- 0.180, respectively. In the rats that had diabetes for 4, 8 and 12 weeks, the activity of MnSOD was (33.863 +/- 6.909), (22.877 +/- 7.875) and (20.034 +/- 6.796) U/mg, respectively; and MnSOD mRNA levels were 0.627 +/- 0.083, 0.333 +/- 0.080, 0.256 +/- 0.057, respectively; these data were less than those in the age-matched control groups (activity: 4 weeks: P = 0.002, 8 and 12 weeks: P = 0.000; mRNA: 4, 8 and 12 weeks: P = 0.000). The activity (109.793 +/- 7.468) U/mg and mRNA level (0.976 +/- 0.108) of Cu-ZnSOD in rats had diabetes for 4 weeks showed no significant difference as compared to age-matched control group (activity: P = 0.426; mRNA: P = 0.172). In diabetic rats at 8 and 12 weeks, the activity of Cu-ZnSOD was (98.588 +/- 9.212) and (78.168 +/- 12.180) U/mg, respectively; Cu-ZnSOD mRNA levels were 0.829 +/- 0.048 and 0.621 +/- 0.033, respectively; these data were less than those in the age-matched control group (activity: 8 weeks: P = 0.011, 12 weeks: P = 0.000; mRNA: 8 weeks: P = 0.001, 12 weeks: P = 0.000). The changes of MnSOD occurred as early as 4 weeks after the onset of diabetes, while the changes of the Cu-ZnSOD occurred later, mainly at 8 and 12 weeks after the onset of diabetes.
Conclusions: Apoptosis of the retinal neurons in early diabetic rats may correlate with the decline of the activity and mRNA expression of MnSOD.