Authors: Lyu Hang, Wang Qichang, Tang Luosheng
To investigate the protective effect of intravitreal injection of nerve growth factor (NGF) on apoptosis of retinal ganglion cells (RGCs) in early diabetic rats and its possible mechanism.
SD rats were divided into normal control group, diabetic model group, phosphate buffer solution (PBS) group and NGF group according to random number table method, with each group contain 6 rats.The rats in diabetic model group, PBS group and NGF group were injected intraperitoneally with streptozotocin (STZ) to establish diabetic rat model.After 4 weeks of modeling, 2 μl PBS and 2 μl NGF (0.5 μg/μl) were injected into the vitreous cavity in PBS group and NGF group, respectively.The right eyes served as the experimental eyes, inject once a week for 4 weeks.After 4 weeks of injection, the retina microangiopathy of each group was observed by Phoenix Micron Ⅳ small animal retinal imaging system.After high dose anesthesia, the eyeball of one rat from each group was taken to prepare ultrathin sections and observed by transmission electron microscopy.Five rat eyeballs were taken to prepare retinal paraffin sections from each groups.The RGC apoptotic index was observed by TUNEL method.The expressions of RGC bcl-2 protein and bax protein were analyzed by immunohistochemistry.This study was approved by the Experimental Animal Ethics Committee of Central South University, and the experimental procedures were in accordance with the National Institutes of Health(NIH) guidelines for the Care and Use of Laboratory, and follow the 3R principle.
The weight of the diabetic model rats was significantly lower than that of the normal group, and the intake of water and food were significantly increased, the urine volume was also in creased.The body weight and blood glucose level of the rats were significantly different among different groups after 4 weeks of modeling (F=202.352, 148.444, both at P<0.001). Fundus photography of early diabetic rats showed no obvious diabetic retinal microangiopathy.The numbers of RGC in the diabetic model group and PBS group were significantly lower than that in the normal control group under the transmission electron microscope.The membrane shrinkage, cytoplasmic condensation, organelle edema, chromatin peripheral collection were obvious and the cell apoptosis number was increased.The RGC lesions in the NGF group were lighter than those in the diabetic model group and PBS group.The apoptotic indexs in the normal control group, diabetic model group, PBS group and NGF group were (3.88±1.28)%, (92.56±1.58)%, (92.64±2.30)% and (59.34±3.89)%, respectively, the overall difference of apoptotic index between the four groups was statistically significant (F=854.554, P<0.001); the RGC apoptotic index of the diabetic model group was significantly higher than that of the normal control group.The RGC apoptotic index of the NGF group was significantly lower than that of the diabetic model group and PBS group (both at P<0.05). The expression levels of bcl-2 protein in normal control group, diabetic model group, PBS group and NGF group were 38.11±1.01, 22.38±3.90, 23.04±3.14 and 84.69±1.45, respectively, with a significant difference among the groups (F=366.206, P<0.001). The expression levels of bax protein in normal control group, diabetes model group, PBS group and NGF group were 4.22±1.89, 56.59±6.67, 56.30±8.51 and 26.19±2.44, respectively, with a significant difference among the groups (F=61.435, P<0.001). The expression of bcl-2 protein in the diabetic model group was significantly lower than that in the normal control group (P<0.05), and the expression of bax protein was significantly increased (P<0.05). The expression of bcl-2 protein in NGF group was significantly higher and the expression of bax protein was significantly lower than those in the PBS group and diabetic model group (all at P<0.05).
The ultrastructural damage of RGCs has occurred in early diabetic rats without diabetic retinal microangiopathy.Intravitreal injection of NGF may produce retinal neuroprotective effects by inhibiting apoptosis of RGCs in diabetic rats.